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IDR 2000 Vol.2. No.3. (www.idreview.co.uk)

grow as a major field of research, not only out offundamental interest but also for the transduction of genesin gene therapy. Another field of major interest was thestudy of mechanisms of viral replication that is unique toparvoviruses. This meeting also underscored the greatinterest in parvoviruses as vectors for gene therapy. Theseviruses have unique properties that make them suchattractive vectors. Milestones in vector development, vectortargeting and their use in gene therapy were reported.Last but not least, many new aspects of B19 humanparvovirus in various syndromes were presented.

The organization of this meeting was made possible,among others, by the generous support from the Ministèrede la Recherche, de la Science et de la Technologie(Québec) and Targeted Genetics Corporation (Seattle,Washington). In particular, they made it possible for manyyoung parvovirologists to attend these stimulatingpresentations and it was gratifying that the next generationwas so well represented.

Finally, I would like to express my gratitude to all whohave made this meeting such a success, Drs S Cotmore,C Parrish and P Tattersall for general advice, the LocalOrganizing Committee, the Scientific Committee, theChairs of the Sessions, the collaborators from mylaboratory for running the meeting, and the participantsfor their input.

Proceedings of the VIIIth Parvovirus Workshop

Infect Dis Rev 2000;2(3):135-177.

VIIIth Parvovirus Workshop

Mont Tremblant, Quebec, CanadaJune 28th - July 2nd, 2000.

INTRODUCTION

The VIIIth Parvovirus Workshop was recently held in theMont Tremblant Conference Center in Québec (Canada)under the auspices of the Centre de microbiologie etbiotechnologie (Institut National de RechercheScientifique-Institut Armand-Frappier, Université duQuébec) with participants from over 20 countries. PreviousWorkshops, after an unofficial precursor at Cold SpringHarbor (USA), were in Grangeneuve (Switzerland, 1985),Oxford (UK, 1987), Jerusalem (Israel, 1989), Elsinore(Denmark, 1991), Crystal River (USA, 1993), Montpellier(France, 1995), and Heidelberg (Germany, 1997). TheIXth Parvovirus Workshop will be held in 2002 in Italy andthe contact email address for that meeting [email protected].

The very high quality of the communications underscoredthe dynamics of the parvovirus field, in fundamental virusresearch, in pathogenesis, diagnosis and prevention,and particularly in vector development and gene therapy.Highlighting particularly stimulating presentations wouldnot do justice to the others. The paradigm that the viralcapsid has a passive role to protect the viral genome andis prodded along by cellular proteins to enter the cell andto decapsidate to present the DNA was challenged by anumber of presentations. The emerging picture is that ofa very dynamic particle with enzymatic activity that playsan active role in a temporal order during viral entry. Viralentry, which up till recently was a grey area, is expected to

Peter TijssenINRS-Institut Armand-Frappier

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The Infectious Disease Review - microbes of man, animals and the environment 2000 Vol.2. No.3.

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ABSTRACTS

1. CAPSID STRUCTURE AND DYNAMICS

Towards the atomic structure of the Adeno-Associated Virus 2 capsid

Qing Xie, Joan Hare, Thayumanasamy Somasundaram,Smita Bhatia, Shawn Clark, Connie Alford, Pam Pruett,

Michael S Chapman.Department of Chemistry and Institute of Molecular Biophysics,

Florida State University, Tallahassee, FL 32306-4380, USA.

Most of the prerequisites for an atomic structure of AAV-2have been completed. Samples have been prepared inmulti-milligram quantities and at high concentration.Conditions have been found for making crystals, and cryo-freezing them to extend their life in the x-ray beam fromminutes to hours. Diffraction data have been collected atthe Cornell Synchrotron. About ¾ million diffraction spotshave been processed with average error of 16%. Thedata are 80% complete to 3.5 Å, less so beyond. With theicosahedral symmetry, this should be ample.

The diffraction shows 3-fold symmetry, but rotationfunctions show that icosahedral 3-folds are tilted 4º fromthe diffraction 3-fold. Packing analysis shows 3 particlesper asymmetric unit related by a non-crystallographic 3-fold screw axis. Approximate particle positions have beendetermined from a native Patterson. Refinement of thepositions is the final step before cycles of phase andimage enhancement that should yield an interpretableimage of the atomic structure.

Structural determinants of tissue tropism and in vivopathogenicity for the murine parvovirus Minute Virus

of MiceMavis Agbandje-McKenna,1 Maria Kontou,1,2 Antonio L

Llamas-Saiz,3 Concepción Foces-Foces,3 Eva Hernando,4

José M Almendral,4 Mari-Paz Rubio,4 Robert McKenna.1

1Department of Biochemistry and Molecular Biology, College ofMedicine, University of Florida, Gainesville, FL. 32610-0245,

USA; 2Department of Biological Sciences, University ofWarwick, Coventry, CV4 7AL, UK; 3Departamento de

Cristalografia, Instituto de Quimica-Fisica “Rocasolano”, CSIC,Serrano 119, 28006 Madrid, Spain; 4Centro de BiologiaMolecular “Severo Ochoa” (UAM-CSIC), UniversidadAutónoma de Madrid, Cantoblanco, Madrid, Spain.

The high resolution structure of native full and emptycapsids of the immunosuppressive strain of the murineparvovirus, minute virus of mice (MVMi), has beendetermined by X-ray crystallography. Analysis of the MVMistructure indicated that amino acids which differ betweenMVMi and the fibrotropic prototype strain of MVM, MVMp,and have been identified, by molecular genetics, as beingimportant for host tropism are clustered on or near thecapsid surface, possibly forming a surface feature usedfor the recognition of a tropism determining intracellularreceptor engaged after the initial cellular entry step.

We have determined the structures of baculovirusexpressed virus-like particles (VLPs) of MVMp containingonly the VP2 major capsid protein as well as native emptyMVMp particles to 3.25Å and 3.75 Å resolution, respectively.A comparison of the MVMp VLPs and native empty capsidsprovided a structural verification of reports that thecharacteristics of some parvoviral particles produced ina heterologous system are indistinguishable from nativecapsids produced in host systems. And further supportsthe observation that the major capsid protein ofparvoviruses is sufficient to form native-like particles. Wehave also refined the MVMi and MVMp capsid structures,to enable a detailed analysis of structural contributionsto their pronounced differences in in vitro tissue tropismand in vivo pathogenicity, despite their high sequencehomology, differing by only 14 amino acids in VP2. Whilethe capsid structures of these two murine parvovirusstrains are almost identical, capsid surface variationsoccur at or near the allotropic determinant residues ofMVM and both intra- and inter-subunit interactions nearthese regions are altered. The exact role played by theobserved structural differences during the course of MVMinfection and disease pathogenesis requires furtherexploration.

Conformational transitions in the MVM virion thatallow egress of the VP1 specific peptide and viral

genomeSusan F Cotmore, Anthony M D’Abramo Jr.,

Glen A Farr, Peter Tattersall.Yale School of Medicine, New Haven, CT 06510, USA.

The N-terminus of MVM VP1 is sequestered within thecapsid shell. Genetic data suggests that this sequenceis essential for entry into host cells, and, since it containspotent nuclear localization sequences, it is likely tobecome extruded during cell entry and may guide thegenome to replication centres in the nucleus. The MVMcoat is quite rugged, and physical conditions encounteredduring cell entry, such as exposure to acidic pH,proteases, divalent cation fluxes or ionic shock, fail toinduce particle disassembly or exposure of the VP1specific region (VP1SR). Limited heating inducesconformational rearrangements in the intact virion whichcause the VP1SR to be exposed irreversibly and renderthe genome available for exogenous polymerase.Cellular factors encountered during entry may trigger asimilar transition, so we have used replication-competentcell extracts to uncoat and replicate virion DNA in vitro. Inthe absence of NS1, the product of this reaction is acovalently-closed, unit-length duplex molecule whichremains tightly associated, via its left-end hairpin, to theparticle, which itself remains intact with its VPSRsexposed. We have used gradient centrifugation andsynthetic oligonucleotides to explore the disposition ofthe genome in transitioned particles, cell fractionation todetermine the nature of the uncoating factor(s), and invitro assays to examine distinct steps in the entry pathway.Surrounding each 5-fold symmetry axis in the virion arecylindrical projections containing pores that directly

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connect the virion core to the particle exterior. Only VP2 N-termini are known to project through these pores, butthey represent a strucure which could expand to allowegress of VP1SR and the viral DNA. The leu residue atVP2 172 forms a constriction at the base of this pore, andthe effects of mutating this residue, on virion stability anduncoating, will be discussed.

Capsids of most parvoviruses possess aphospholipase A2 activity

Zoltán Zádori, Marie-Claude Lacoste, Marc Allaire,Jozsef Szelei, Yi Li, Peter Tijssen.

Centre de microbiologie et biotechnologie, INRS-InstitutArmand-Frappier, Université du Québec, Laval, QC, Canada

The VP1 of most parvoviruses (30/34), with notableexceptions of ADV and Brevidensoviruses, contains anevolutionary highly conserved sequence in its unique part(VP1up). Sequence alignments of thirty parvovirus VP1uprevealed that within this region 9 amino acids are fullyconserved with a significant conservation of others. Fiveout of the nine fully conserved amino acids were alsoconserved in different secreted phospholipase A2(sPLA2) enzymes, particularly within the catalytic site.Despite the lack of the characteristic disulfide bondsfound in sPLA2, VP1up from B19, GmDNV and PPV provedto have a Ca-dependent phospholipase A2 activity whenassayed with radioactively labeled E. coli membranephospholipids, or with the mixed micelles assay. Thecatalytic activity depended on the substrate, the assayconditions and the origin of the PLA2.

A structural model of the vPLA2 permitted the selection ofsite-specific mutations of the PPV VP1up, that affectedthe amino acids of the putative active center (H41A, D42N,D63N,A), the Ca-binding loop (P21L,R,W) andmembrane-binding helix (K88R). These reduced theenzyme activity or even abolished it. Furthermore, the viralinfectivity was reduced to a degree that was proportionalto the decrease in PLA2 activity of the various mutants.Phospholipase inhibitors significantly reduced both theactivity of the viral PLA2s and the infectivity of PPV.

Our results demonstrated that VP1ups from mostparvovirus capsids have phospholipase A2 enzymeactivity that is indispensable for a successful viralinfection. The central role of PLA2 in autoimmunediseases, such as lupus and arthritis, and the presenceof this enzyme in parvovirus capsids lends credence tothe hypothesis that these viruses are involved in theseautoimmune diseases.

Calcium and Transferrin Receptor Binding by Canineand Feline Parvoviruses

Benoît Hébert,1 Alan A Simpson,1 Veda Chandrasekar,1 JohnSL Parker,2 Gail M Sullivan,2 Colin R Parrish,2 Michael G

Rossmann.1

1Department of Biological Sciences, Purdue University, WestLafayette, IN 47907-1392, USA; 2James A Baker Institute of

Animal Health, College of Veterinary Medicine, CornellUniversity, Ithaca, NY 14853, USA.

Canine parvovirus (CPV) emerged in 1978 as a hostrange variant of feline panleukopenia virus (FPV). Thischange of host was mediated by the mutation of fiveresidues on the surface of the capsid. CPV and FPV entercells by endocytosis and can be taken up by many non-permissive cell lines, showing that their host range andtissue specificity are largely determined by eventsoccurring after cell entry. We have determined thestructures of a variety of strains of CPV and FPV at variouspH values and in the presence or absence of Ca2+. Thelargest structural difference was found to occur in a flexiblesurface loop, consisting of residues 359-375 of the capsidprotein. This loop binds a Ca2+ ion in FPV and is adjacentto a double Ca2+ binding site, both in CPV and FPV.Residues within the loop and those associated with thedouble Ca2+ binding site were found to be essential forvirus infectivity. The residues involved in the double Ca2+

binding site are conserved only in FPV and CPV.

Our results show that the loop conformation and theassociated Ca2+ binding are influenced by the Ca2+ ionconcentration, as well as pH. These changes arecorrelated with the ability of the virus to haemagglutinateerythrocytes. The co-localization of haemagglutinatingactivity and host range determinants on the virus surfaceimplies that these properties may be functionally linked.We speculate that the flexible loop and surroundingregions are involved in binding an as yet unidentified hostmolecule and that this interaction influences host range.

Finally, we present preliminary cryoEM results from ourstudies of canine parvovirus and the transferrin receptor.Although we have yet to obtain stable complexes, ourdata suggests that interaction destabilizes the viruscapsid.

Homology model of the viral phospholipase A2 fromParvovirus

Marc Allaire, Zoltán Zádori, Peter Tijssen.Centre de recherche en biotechnologie et microbiologie, INRS

Institut Armand-Frappier, Laval, Québec, Canada.

Amino acid sequence comparisons of the unique aminoextension of VP1 revealed conserved motifs reminiscentof the catalytic residues of the secreted phospholipaseA2 (sPLA2). It was shown that this unique region of VP1does contain PLA2 activity and is essential for the infectivityof the virus. A homology model of the VP1 viral PLA2(vPLA2) was built with the two known structural classes(group I/II and group III) of the sPLA2. The vPLA2 wouldpossess amino and carboxy ends similar to the group III


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sPLA2 with an internal region comparable to the group I/II sPLA2. The predicted group III-type carboxy helix knownas the amphiphatic helix would define the hydrophobicchannel for binding the substrate. The vPLA2 would becontained within an about 90 residues-long domainlacking both disulfide bridges and the common beta-wingloop. The homology model of the vPLA2 helped inidentifying the essential catalytic residues which were inturn confirmed by site-directed mutagenesis. The vPLA2model from Parvoviruses could have arisen by a horizontalgene transfer between the two structural classes ofsPLA2.

2. EVOLUTION AND TROPISM

Multiple modes of variation and selection explain theevolution of canine parvovirus

Uwe Truyen,1* Ariane Steinel,1 Allen Gruenberg,2 James FEvermann,3 Moritz van Vuuren,4 Walter Hermanns,5 Colin R

Parrish.2

1Institute for Medical Microbiology, Infectious and EpidemicDiseases, Ludwig Maximilians University, Veterinaer Str. 13,

80539 Munich, Germany; 2James A Baker Institute, College ofVeterinary Medicine, Cornell University, Ithaca, NY 14853,USA; 3Washington Animal Disease Diagnostic Laboratory,

College of Veterinary Medicine, Washington State University,Pullman, WA 99164, USA; 4Department of Veterinary Tropical

Diseases, Faculty of Veterinary Science, University ofPretoria, Private Bag X04, Onderstepoort, 0110, South Africa;5Institute for Animal Pathology, Ludwig Maximilians University,

Veterinaer Str. 13, 80539 Munich, Germany.

Canine parvovirus (CPV) was first recognized in 1978when it spread around the world in a few months. Wehave examined the variation and evolution of the virussince 1978, and show that this is characterized primarilyby selection for three mutations or groups of mutations,which spread around the world during three clearlydefined sweeps. The first was seen as the completeglobal replacement between 1979 and 1980 of the 1978virus by a genetically variant virus that contained 4 non-synonymous and 1 synonymous substitutions in itsgenome, and those affected antigenicity and host range.A second sweep occurring around 1984 was due to asingle non-synonymous substitution which altered anantigenic site of the virus, and that now is found in 30 to80% of viruses in different parts of the world. A third sweepwas of a single non-synonymous substitution whichbecame widespread in the USA during 1990, was presentin New Zealand,Taiwan and Japan by 1992, and becamecommon in Germany during 1993. That mutation wasnot detected in Southern African viruses in 1997 or 1998.Both mutations of aa297 and aa426 appear to have arisenmore than once, and represent convergent evolution, sothat calculated phylogenies do not represent the trueevolution of the viruses.

Ubiquitous human adeno-associated virus type 2autonomously replicates in differentiating

keratinocytes of a normal skin modelCraig Meyers,1 Michael Mane,2 Natalia Kokorina,2 N Samina

Alam,1 Paul L Hermonat.2

1Department of Microbiology and Immunology, ThePennsylvania State University College of Medicine, Hershey,PA. 17033, USA; 2Department of Obstetrics and Gynecology,University of Arkansas for Medical Sciences, Little Rock, AR.

72205, USA.

Since its discovery in 1966 adeno-associated virus type2 (AAV) has been described as a helper-dependentparvovirus. However, in this study we demonstrate thatAAV undergoes its complete life cycle, devoid of helperviruses or genotoxic agents, in the organotypic epithelialraft tissue culture system, a model of normal skin. AAVprogeny production directly correlated with epithelialdifferentiation as non-differentiating keratinocytes weredefective for this activity. Large nuclear virus arrays ofparticles of approximately 26 nanometers in size(parvovirus size) were observed in the granular layers ofthe raft epithelium by electron microscopy. Additionally,dosage-dependent histologic changes, some of whichmight be interpreted as cytopathology, were induced inthe AAV-infected epithelial tissues. These data suggesta new biological model for AAV; that AAV is an epithelial-tropic autonomous parvovirus which can alter normalsquamous differentiation.

Different patterns of restriction to B19 parvovirusreplication in human erythro- and megakaryoblastoid

cell linesGiorgio Gallinella, Elisabetta Manaresi, Elisa Zuffi, Simona

Venturoli, Laura Bonsi, Gian Paolo Bagnara, Monica Musiani,Marialuisa Zerbini.

Department of Clinical and Experimental Medicine, Division ofMicrobiology, University of Bologna, Via Massarenti 9, I-40138

Bologna, Italy.

B19 parvovirus can replicate in erythroid progenitor cellsfrom bone marrow and fetal liver, and in a small numberof erythroblastoid and megakaryoblastoid cell lines. Weanalyzed the susceptibility and permissiveness to B19virus infection of different cell lines (UT7, TF-1, M07, andB1647) and compared them to human bone marrow cells.Following in vitro infection, B19-specific nucleic acidswere characterized with respect to the genome replicativeintermediates and to the transcription patterns.

While all cell lines tested proved to be susceptible to B19virus infection, two different patterns of restriction toreplication were observed. In UT7 cells, the viral single-stranded (ss) DNA was converted to double-stranded(ds) replicative intermediates identical to those found inbone marrow cells, characterized by either continuous ornicked terminal hairpins. A full pattern of viral transcripts,similar to bone marrow cells, was observed. However,replication and transcription was restricted to a smallsubset of cells, and production of capsid proteins wasnot detected. In TF-1, M07 and B1647 cells the viral ssDNA

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was not converted to dsDNA replicative intermediates,and consequently, no transcription of the viral genomeoccurred.

In conclusion, in vitro infection of megakaryoblastoid celllines provided evidence for the presence of two differentlevels of restriction to B19 virus replication. A first level ofrestriction occurs related to the uncoating of the viralgenome and/or conversion of the viral ssDNA intoreplicating dsDNA, while a second level of restrictionoccurs related to the production of viral capsid proteins.

Infectious Hypodermal Hematopoietic Necrosis Virus(IHHNV) of shrimp is related to mosquito

brevidensoviruses.H Shike,1 AK Dhar,2 KR Klimpel,2 C Shimizu,1

JC Burns,1 M Bergoin.3

1Dept. of Pediatrics, UCSD School of Medicine, La Jolla, CA92093-0830, USA; 2Supershrimp Inc., National City, CA 91950,

USA, 3Laboratoire de Pathologie Comparée, UniversitéMontpellier II, France.

IHHNV is associated with impaired growth and cuticledeformities in cultured penaied shrimp, but detailedmolecular analysis of its genome has not been previouslyreported. IHHNV infection of wild Penaeus stylirostriscaught in 1998 in the Gulf of California was confirmed byPCR with commercially available primers (DiagXoticsInc.). Quantitative PCR with new primers designed fromthe amplicon sequence demonstrated 3x109 viralgenomes/g of shrimp tissue. Virions were purified fromsix frozen cephalothoraces by CsCl gradient. The buoyantdensity (1.45 g/ml) was determined by PCR screening offractions. Virion DNA was extracted and viral sequence(3873 bp) was obtained by inverse PCR. The 56 basesand the 28 bases at each extremity share identicalsequence and can fold into a typical hairpin structure.Three large open reading frames (ORFs) are all on thecomplementary (plus) strand. The left, mid and right ORFshave potential coding capacities of 666 aa (76 kDa), 363aa (42 kDa), and 329 aa (37.5 kDa), respectively. The leftORF most likely encodes NS1 since it contains aconserved NTP-binding domain and shares ~25% aasequence homology with the NS1 of mosquitobrevidensoviruses. The overall genomic organization issimilar to that of Aedes aegypti and Aedes albopictusDNVs and differs from Bombyx mori DNV(BmDNV, genusIteravirus) and Junonia coenia DNV (JcDNV, genusDensovirus).

Host range and antigenicity of Canine Parvovirusmutants

Karsten Hueffer, Colin R Parrish.James A Baker Institute, College of Veterinary Medicine,

Cornell University, Ithaca NY USA 14853.

Canine parvovirus (CPV) emerged in the caninepopulation in the mid 1970s. It is 99% identical to thefeline parvovirus (FPV) in both the DNA and proteinsequences. However those two viruses show distinct

phenotypes in terms of host range in vivo and in vitro, inhaemagglutination, and in antigenicity. Some of thesedifferences could be mapped to certain residues in theviral capsid, and Residues 93 (Lys in FPV, Asn in CPV)and 323 (Asp in FPV, Asn in CPV) were important indetermining the host range and antigenicity.

Two antigenic sites were defined (A: region around theresidue 93; B: around residue 300). Site A is composedof two loops which are very close in the tertiary structureof the capsid protein. In FPV two hydrogen bonds fromLys 93 to residues 225 and 227 connect the two loops. InCPV those hydrogen bonds are missing due to thechange of residue 93 from Lys in FPV to Asn in CPV.

In this study we tested viruses with mutations in the aboveregions for their antigenicity and for canine host range invitro. We found that the Lys in FPV at residue 93 could besubstituted by an Arg or a number of other residues, andthose mutant viruses gained the CPV host range (theability to replicate in dog cells) when residue 323 is alsochanged to an Asn (the CPV amino acid). These findingssupport the hypothesis that a lysine at residue 93 preventscanine cell infection. The antigenicity of the mutant viruseswas generally not changed when examined with fourdifferent monoclonal antibodies against the epitopes inthe antigenetic site A. We also found that a mutation fromAsn to Arg in a CPV background leads to a loss of theability to hemagglutinate feline red blood cells.

Genetic characterization of feline parvovirussequences from various carnivores

Ariane Steinel,1 Linda Munson,2

Moritz van Vuuren,3 Uwe Truyen.1

1Institute for Medical Microbiology, Infectious and EpidemicDiseases, Ludwig Maximilians University, Veterinaer Str. 13,

80539 Munich, Germany; 2Department of Pathology,Microbiology and Immunology, School of Veterinary Medicine,

University of California, Davis, California, USA; 3Department ofVeterinary Tropical Diseases, Faculty of Veterinary Science,University of Pretoria, Private Bag X04, Onderstepoort, 0110,

South Africa.

Infections with viruses of the feline parvovirus subgroupsuch as feline panleukopenia virus (FPV), mink enteritisvirus (MEV) and canine parvovirus (CPV-2) [together withits new antigenic types (CPV-2a, CPV-2b)] have beenreported from several wild carnivore species. To examinethe susceptibility of different species to the variousparvoviruses and their antigenic types, samples from wildcarnivores with acute parvovirus infections were collected.Viral DNA was amplified, and subsequently analysed,from faeces or formalin-fixed small intestines from anorphaned bat-eared fox (Otocyan megalotis), a free-ranging honey badger (Mellivora capensis), six captivecheetahs (Acinonyx jubatus), a captive Siberian tiger(Panthera tigris altaica) and a free-ranging African wildcat (Felis lybica). Parvovirus infection in bat-eared fox andhoney badger was demonstrated for the first time. FPV-sequences were detected in tissues of the African wildcat and in faeces of one cheetah and the honey badger,


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whereas CPV-2b sequences were found in five cheetahsand the bat-eared fox. The Siberian tiger (from a Germanzoo) was infected with a CPV-type 2a virus. Thisdistribution of feline parvovirus antigene types in captivelarge cats suggests an interspecies transmission fromdomestic dogs. CPV-2 sequences were not detected inany specimens and no sequences with featuresintermediate between FPV and CPV were found in any ofthe animals examined.

Relationship and genome organization of thedensoviruses from Casphalia extranea (CeDNV) and

Bombyx mori (BmDNV)Y Li,1 G Fédière,2 H Bando,3 P Tijssen.1

1INRS-Institut Armand-Frappier, Université du Québec, Laval,Canada; 2Laboratoire d’Entomovirologie, Faculté d’Agriculture-

Université du Caire, IRD-EGYPT; 3Faculty of Agriculture,Hokkaido University, Sapporo, Japan.

A densonucleosis virus, first isolated in Côte d’Ivoire asthe aetiologic agent of an infection of the oil palm pestCasphalia extranea and designated as CeDNV, contains4 capsid proteins with molecular masses of 82, 74, 54and 49 kDa and a single stranded linear DNA of about 5Kb. The viral DNA was isolated from CeDNV by standardmethod and cloned into a pBluescript vector and wassequenced. Analysis of DNA and protein sequencesshowed high homology with the BmDNV-1 with similarinverted terminal repeats of 225 nts of which the terminal153 forms a palindrome. Like in the case of BmDNV, theORFs are also located on the same strand but the ORForganization was very different. We have recentlyresequenced the published clone (Bando et al, 1987)and a new clone of BmDNV. The original clone showeddeletions, insertions, and inversions, whereas the newone has a similar genome organization as CeDNV. Tofurther address this question, the coding sequences offour overlapping polypeptides starting at four differentAUG codons and co-terminating at the stop codon of theputative capsid gene of CeDNV were inserted intobaculovirus transfer vector pFastbac to generaterecombinant capsids. In all cases, infection of insect High-Five cells by any of the four recombinant viruses resultedin the production of virus-like particles (VLPs) of 22-25nm in diameter. VP1 was less expressed than other VPs.VP4, the shortest polypeptide, appears to be sufficient forassembly of VLPs morphologically similar to thoseformed with two to four polypeptides. The mechanism oftranslation of structural proteins appears to be similar tothat of GmDNV and JcDNV, although the genomeorganization is different.

MVMp Persists in nonpermissive fischer ratfibroblasts by virus cell coevolution

Ayelet Keren-Naus, Roni Mintz, Jacov Tal.Department of Virology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.

Recent advances in the development of MVM as atransducing vector, and the ability of MVM to integrate site-

specifically emphasize the need to assess thepersistence and genetic stability of MVM in nonpermissivecells. Actively-dividing Fischer rat fibroblasts, line F-111that allow some virus replication yet are tightlynonpermissive to killing by MVMp, were infected at 10pfu/cell. A carrier state was established in which viralDNA replication, NS and VP protein synthesis andprogeny virus formation took place over a period of atleast 6 months. The virus replicated at low levels withintermittent peaks. The peaks were in correlation withincreased percentages of virus-replicating cells asdetermined by single-cell hybridization andimmunofluorescence, but were not accompanied bydetectable tissue destruction. Apoptotic ladders appearedoccasionally throughout the infection. Changes wereobserved in the virus and host cell population, resultingin the emergence of progeny virus with altered hostspecificities and sub-populations of cells that were nolonger permissive to MVM DNA replication. In contrast, inconfluent, non-dividing cell cultures, single stranded viralDNA and low levels of NS1 and VP proteins were detectedup to 3 weeks post-infection. Release of the cultures fromcontact inhibition to induce cell divisions at 1, 2 and 3weeks postinfection could revive active virus replicationin 25%, 8% and <1% of the cells, respectively, withoutdetectable destruction of the culture. No virus replicationcould be detected in non-dividing cells induced to celldivision after 3 weeks. These results reveal a basicdifference in the persistence pattern of MVMp in non-dividing and dividing cultures. While in non-dividing cellsthe virus seems to persist in form of single-stranded DNA,persistence in dividing nonpernissive cells seems to bepropagated by the continuous origination of virus variants(or host-range mutants) and susceptible cells in theculture.

Proposal for the new classification of parvovirusesZoltán Zádori, Yi Li, Peter Tijssen.

INRS-Institut Armand-Frappier, Laval, Québec, Canada.

Parvoviruses, according to the current ICTV taxonomy,are divided into two subfamilies; those of infectingvertebrates (Parvovirinae) and those infecting arthropods(Densovirinae). Within these subfamilies, viruses havebeen grouped into 3 genera based on their genomicproperties and phenotypic manifestations, such as host-range, tissue specificity, helper-virus dependency.

Phylogenetic analysis of the NS1 and VP1 proteins of 22representative vertebrate parvoviruses revealed 5 distinctgroups within the Parvovirinae subfamily, and this findingsuggests the formation of 5 genera - instead of the present3 - with MVM, ADV, BPV, AAV2, and B19 as prototypes.This suggestion for reclassification is reinforced by otherobservations such as differences in genome organizationand the presence of the phospholipase A2 motif (not inADV but present in other parvoviruses from vertebrates).The current classification of genera that comprise theDensovirinae subfamily should be reconsidered as well.Almost all of the proposed Contravirus members (13)

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belong to other genera. Eg. CeDNV belongs to Iteravirus,while AdDNV, DsDNV, PiDNV belong to Densovirus. Onthe basis of genomic organization and phylogeniccomparisons of 11 members of this subfamily wepropose the reorganization of the present classification.As with one of the parvovirus genera from vertebrates,one of the Densovirus genera (Brevidensovirus) lacksthe phospholipase A2 motif.

3. EARLY EVENTS IN INFECTION

Canine parvovirus (CPV) is trafficked to the recyclingendosome after virus entry in mink and feline cells

but not in canine cellsJohn SL Parker, Colin R Parrish.

James A. Baker Institute for Animal Health, College ofVeterinary Medicine, Cornell University, Ithaca, New York.

Canine parvovirus (CPV) is a small, non-enveloped virusthat is a host range variant of a virus that infected othercarnivores. Changes in the capsid protein control theability of the virus to infect canine cells. We have previouslyshown that CPV enters mink lung cells (Mv1Lu) by clathrin-mediated endocytosis. Following initial uptake into Mv1Lucells capsids are trafficked to a transferrin- (Tfn), andRab11-positive endocytic compartment in the perinuclearregion of the cell. In order to test if CPV is trafficked to lateendosomes in Mv1Lu cells, we stably transfected Mv1Lucells with an HA-epitope tagged Rab7 construct (Mv1Lu-Rab7 cells). Rab7 is a small GTP-binding protein thatregulates early-to-late endosomal trafficking, which islocated on the membrane of late endosomes. CPV didnot co-localize with Rab7 in the Mv1Lu-Rab7 cells after a2 h incubation at 37°C. Similarly, in feline CRFK cells,CPV co-localized with the Tfn receptor in a perinuclearvesicular compartment after a 2 h incubation at 37°C.The strong co-localization with Tfn and the Tfn receptorand the lack of co-localization with rab7 and other markersof late endosomes/lysosomes 2 h after virus uptakeindicates that CPV is trafficked to the perinuclear recyclingendosome in these cells. In contrast, in canine A72 cellsCPV co-localized with LGP, a marker for late endosomes2 h after uptake. In A72 cells stably transfected with thehuman Tfn receptor, virus and Tfn co-localized in only asmall proportion of endocytic vesicles. These resultsshow that there is a difference in the endosomal traffickingof CPV between canine and feline or mink cells.

Endocytic entry of Canine Parvovirus (CPV)Katja Sääjärvi, Sanna Suikkanen,Jonna Hirsimäki, Matti Vuento.

Department of Biological and Environmental Science,University of Jyväskylä, PO Box 35, 40351 Jyväskylä, Finland.

Immunoelectron microscopy showed CPV to be presentin coated pits and in endosomal vesicles pinching offfrom the plasma membrane. Infectious entry was

inhibited by cholesterol depletion as well as bymicroinjection of clathrin antibody. These results togetherwith work from other laboratories demonstrate theimportance of clathrin coated vesicles in CPV entry.Microinjection antibodies to beta-COP prevented infectionof host cells by CPV. Brefeldin A, which has a knowneffect of dissociating COP I protein from endosomalmembranes, was found to be a potent inhibitor ofinfectious entry of CPV when added before 2 h p.i. ThusCOP I seems to have a role in the early endocytic traffic ofCPV. Previous work had shown that endocytic entry ofCPV was dependent on intact microtubular network. Inline with these results, a spatial correlation of CPV-containing vesicles and microtubular structures wasobserved by using confocal immunofluorescence. Theintracellular distribution of CPV-containing vesicles wasdramatically altered (from perinuclear to peripheralcytoplasmic) when cells were microinjected with antibodyto dynein (a motor protein driving vesicles towardsnucleus) but not when microinjected with antibody tokinesin (a motor protein having a polarity opposite to thatof dynein).

The intracellular distribution of CPV antigens and CPVDNA during 0-8 h p.i. were studied by confocalimmunofluorescence microscopy and confocalfluorescent in situ hybridization (FISH), respectively, andfound to be essentially similar in A72 and NLFK cells.CPV antigens and CPV DNA were initially (0-30 min p.i.)detected in small peripheral vesicles and later (1-4 p.i.and thereafter) in larger perinuclear vesicles which oftenhad a polar location in respect to nucleus. There was nosignificant colocalization of CPV antigens during 0-8 hp.i. with the Golgi markers tgn38 and AP 1, lysosomalmarkers lamp1, lamp2, ci-mpr, or endosomal markerrab11. However, there was a profound colocalization ofCPV antigens with endocytosed WGA-FITC at 2-8 h.p.i.but not at 0-30 min p.i. The absence of WGA-FITC fromearly clathrin-coated endocytic vesicles was alsoconfirmed by electron microscopy. Colocalization of CPVantigens with WGA-FITC suggests that the bulk of capsidsreached so called multivesicular bodies in a destructiveendocytic pathway. The bulk of viral DNA also reachedthis location. It is interesting that both the clathrin-dependent pathway of CPV and the clathrin-independentpathway WGA-FITC converge at this point. This vesicularstructure was also reached by CPV antigens whenbafilomycin A (inhibitor of vacuolar ATPase) or someproton ionophores were present, while these compoundsstrongly inhibited CPV proliferation. It is thus possiblethat some pH-dependent functions of the observedperinuclear vesicles are involved in the release of viralDNA (or DNA-protein complexes) from endocyticcompartment for subsequent uptake into the nucleus.


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Recombinant Human Parvovirus B19 Vectors:Erythrocyte P Antigen is Necessary but not Sufficient

for Successful Transduction of Hematopoietic andNon-Hematopoietic Human Cells

KA Weigel-Kelley, MC Yoder, A Srivastava.Indiana University School of Medicine, Walther Oncology

Center, Walther Cancer Institute, Indianapolis, IN.

Parvovirus B19 is known to cause impairment oferythropoiesis in humans due to its lytic infection ofprogenitor cells in the erythroid lineage. The blood groupP antigen, known to be abundantly expressed on cells inthe erythroid lineage, has been reported to be the cellularreceptor for parvovirus B19. We have described thedevelopment of recombinant parvovirus B19 vectors withwhich high-efficiency, erythroid lineage-restrictedtransduction can be achieved (J. Virol., 72: 5224-5230,1998). However, a low-level transduction of non-erythroidcells could also be detected. Since P antigen is expressedin non-erythroid cells, and since additional clinicalpathological studies suggest the presence of parvovirusB19 in non-hematopoietic human cells, we re-evaluatedthe role of P antigen in parvovirus B19 vector binding andentry into cells. A recombinant vector containing thecytomegalovirus immediate-early promoter-drivenenhanced green fluorescent protein [EGFP] reporter genein an adeno-associated virus 2 (AAV) genome was usedto infect primary human low-density bone marrowmononuclear (LDBM) cells that had been separated intoerythroid and non-erythroid populations using glycophorinA+ conjugated beads. Consistent with our previousstudies, ~79% of cells in the erythroid fraction showedtransgene expression 48 hrs post-infection, comparedwith ~13% in the non-erythroid fraction. Cell surfaceexpression revealed that ~75% of glycophorin A+ and~31% of glycophorin A- cells were P antigen-positive. Twohuman erythroleukemia cell lines, HEL and K562, and ahuman promyelocytic leukemia cell line, HL-60, were alsoexamined for P antigen expression and binding and entryof recombinant parvovirus B19 vectors. HEL and K562cells showed intermediate levels, whereas HL-60 cellsdemonstrated high levels of expression of P antigen.However, the efficiency of binding of 35S-methioninelabeled vector to these cells did not correlate with Pantigen expression. Moreover, despite P antigen-positivityand efficient viral binding, HEL, K562, and HL-60 cellscould not be transduced with the recombinant vector.These studies were extended to include two primary celltypes, human umbilical vein endothelial cells (HUVEC)and normal human lung fibroblasts (NHLF). Low-levelsof P antigen expression could be detected in both celltypes. In addition, binding of the recombinant vectoroccurred in both cell types and was reduced by ~17% inHUVEC and ~48% in NHLF, respectively, uponcompetition with globoside, indicating the involvement ofP antigen in virus binding to these cells. In contrast to theestablished cell lines, these primary cells could beefficiently transduced with the recombinant vector. Takentogether, these data suggest that (i) P antigen isexpressed on a variety of cell types and is involved in

binding of parvovirus B19 to human cells, (ii) The level ofP antigen expression does not correlate with the efficiencyof viral binding, (iii) P antigen is necessary but not sufficientfor parvovirus B19 entry into cells, and (iv) Parvovirus B19vectors can be used to efficiently transduce HUVEC andNHLF in addition to erythroid cells in LDBM cells. Thesestudies further suggest the existence of a putative cellularco-receptor for efficient entry of parvovirus B19 into humancells.

Role of viral phospholipase A2 in porcine parvovirus(PPV) entry during infection.

Jozsef Szelei, Zoltan Zadori, Marc Allaire,Yi Li, Peter Tijssen.

Centre de microbiologie et biotechnologie, INRS-InstitutArmand-Frappier, Université du Québec, Laval, QC, Canada

In this study, the role of viral phospholipase A2 (vPLA2),using porcine parvovirus (PPV) as a model, wasinvestigated. Mutants that affected the vPLA2 catalyticactivity, as suggested by the predicted 3D structure, weregenerated. The mutants were transfected into PT cells toproduce mutant viruses. The concentration of full virusparticles (genome equivalents, GE) was determined bya MIMIC assay and the infectivity of the virus was identifiedby the number of infected PT cells usingimmunofluorescence 20 hours after the infection(fluorescent focus units). The infectivity of the mutantviruses decreased proportionately to the remaining vPLA2activity.

While, upon transfection, there was no significantdifference in the production of virions between the wild-type virus and the mutant genomes, the infectivity ofdifferent mutant viruses significantly decreasedcompared with the wild-type PPV demonstrating thatvPLA2 is required at some stage before replication. Inorder to identify this critical step, wt virus and its mutantswere both used for infection and the early events in theviral cycle, such as cell surface attachment, viral entry,and intracellular trafficking of PPV to the nucleus, werefollowed by immunofluorescence. Whereas both the wtand mutant incoming virus particles accumulated in aperinuclear region, new virus could be detected from 8hrs p.i. in the case of the wt but only from about 18 hrs forthe mutants with a significantly lower titer. Lysosomotropicagents, such as NH4Cl, exacerbated this effect andshowed that endocytosis was involved. These studiesshowed that vPLA2 mutants were impaired in tranferringtheir genome from the perinuclear localization to thenucleus.

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Cytoplasmic trafficking of Canine Parvovirus capsidsand the role of VP1 N-terminal sequences in cell

infection.Maija Vihinen-Ranta*, Wen Yuan,

Wendy Weichert, Colin R. Parrish.James A Baker Institute, College of Veterinary Medicine,

Cornell University, Ithaca NY USA 14853.

To begin a successful infection, viruses must enter thecytoplasm. Those viruses that replicate in the nucleusmust target their genome to that location. We examinedthe cytoplasmic transport of the canine parvovirus (CPV)capsid in productive infection by injecting two antibodiesrecognizing the intact CPV capsid into the cytoplasm ofcells, and by intra-cellular expression of variable domainsof a neutralizing antibody. The two antibodies tested andthe expressed scFv all efficiently blocked virus infection,probably by binding to virus particles while they were inthe cytoplasm and before entering the nucleus. Theinjected antibodies blocked most infections even wheninjected six hours after virus infection. In control studies,capsid antibodies did not interfere with CPV replicationwhen co-injected with an infectious plasmid clone of CPV.

Cytoplasmically injected full and empty capsids movedthrough the cytosol towards the nuclear membrane in aprocess that could be blocked by nocodazole treatmentof the cells. Nuclear transport of the capsids was slow,with significant levels of capsids being found in thenucleus only three to six hours after injection. The VP1-unique region of the parvoviruses is not required forcapsid assembly or for viral DNA packaging, but it isrequired for efficient cell infection by the virus. The N-terminus of the protein contains a classical basic nuclearlocalization sequence. N-terminal sequences of VP1became exposed on the exterior after treatment of thecapsids with heat (55-65°C), urea (3-4M) or with pHsabove 11 or below 4. The VP1 region was detected oninjected capsids within the cytoplasm or the nucleus 4 to8 hours after injection. A monoclonal antibody againstthe N-terminal residues 2-13 of VP1, and a rabbitpolyclonal against the entire VP1 unique region from MVMboth blocked virus infection when injected into thecytoplasm of the host cells.

Receptor binding by human parvovirus B19Bärbel Kaufmann,1,2 Ulrich Baxa,1 Susanne Modrow,2 Henrik C

Hansen,3 Ulf Nilsson,3 Robert Seckler.1

1Physical Biochemistry, Potsdam University, Germany;2Institute for Medical Microbiology, University of Regensburg,

Germany; 3Organic Chemistry II, Center for Chemistry andChemical Engineering, Lund University, Sweden.

Parvovirus B19 is an human pathogen virus with anextraordinary tropism for erythroid progenitor cells. Thesmall capsid (20 - 26 nm in diameter) is built of 60subunits in T=1 symmetry and consists of two structuralproteins, 95% viral protein 2 and 5% VP1.

In 1993 Brown and colleagues identified theglycosphingolipid globoside as the receptor for the virus.

Difference electron density maps from cryo-electronmicroscopy of empty VP2-shells with and withoutgloboside (Chipman et al., 1996) suggest that B19recognizes the receptor molecule presumably as a trimer.As the understanding of the interaction of B19 with itsputative receptor globoside is of basic interest and aprecondition for the development of reliable therapeuticswe are looking for a detailed biophysical and chemicalcharacterization of the binding.

For this reason we synthesized water-soluble mono- andoligomeric conjugates of globotetraose, the carbohydrateportion of the globoside. These derivatives were testedfor their capacities to inhibit haemagglutination inducedby recombinant VP2-capsids purified from insect cellsinfected with recombinant baculovirus.

Competition of globoside binding to VP2-capsids byglobotetraose derivatives was also tested in a solid phaseassay with 125I-labelled recombinant VP2-shells. SurfacePlasmon Resonance experiments were done in order todetermine a binding constant for globoside.

In a second approach, we are looking for other potentialreceptor molecules. We detected binding of radiolabelledcapsids to cellular surface proteins by a far western blotassay. Experiments to identify these proteins and to verifythe binding interaction in solution are in progress.

To address the role of the minor capsid protein VP1, weproduced heterogeneous capsids of varying VP2:VP1ratio by modulating the expression of both viral genes.Therefore the promoter regions of a dual expressionvector in the Baculo / Sf9 system were mutated.Furthermore, we varied harvesting time post infection,and analyzed capsid composition and properties byelectron microscopy and ultracentrifugation.

Regulation of NS1 functions in the course of a viralinfection: impact of mutagenesis at conserved PKC

phosphorylation sites on NS1 cytotoxic activitiesDaeffler L, Rommelaere J, Nüesch JPF.

Department of Applied Tumor Virology and INSERM U375,Deutsches Krebsforschungszentrum, D-69120 Heidelberg,

Germany.

The large non-structural protein of autonomousparvoviruses, NS1 is required for multiple functions duringa productive infection, suggesting a timely coordinatedregulation in order to achieve its distinct tasks. Cellularkinases may contribute to this tuning of NS1, as supportedby, the variation of the NS1 phosphopeptide pattern duringa viral life-cycle, the requirement of distinctphosphorylation events for NS1 replicative activities, andthe modulation of the NS1 biochemical profile accordingto its phosphorylation state. The PKC family of proteinkinases was found to be involved in the regulation of theNS1 replicative function. To determine whether additionalNS1 functions, in particular toxic activities leading to celllysis, are regulated by phosphorylation and to identify thecorresponding regulatory elements in NS1, mutant forms


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of NS1 were produced by mutagenesis directed atconserved PKC phosphorylation sites. These NS1mutants were analyzed for their biochemical profile, theircytopathic effects and their ability to induce cytolysis uponinfection with replication-active mutant viruses. SomeNS1 mutants proved to be competent for all functionsnecessary for progeny virus production, while beingendowed with a reduced toxicity. Although, these datasuggest that NS1 cytotoxicity is regulated by (a)phosphorylation event(s) that may occur late in infection.

Binding of the small nonstructural proteins of MVMpto CRM1: characterization of the NS2 nuclear export

signalU Bodendorf,1 V Eichwald,1 R Bischoff,2 M Fornerod,3

M Klein,1 J Rommelaere,1 N Salomé.1

1Dept of Applied Tumor Virology-Inserm U375/ATV F0100, and2Dept of Molecular Biology of Mitosis, DKFZ, Heidelberg;3Dept of Gene Expression, EMBL, Heidelberg, Germany.

We have previously reported that the nonstructuralproteins NS2 of MVMp are able to interact with the nuclearexport factor CRM1/Exportin 1. CRM1 functions as anessential nuclear export receptor for proteins carrying aleucine-rich nuclear export signal (NES) in a RanGTP-dependent manner. Using purified recombinant proteins,we showed by gel mobility assays that full-lenght NS2-P,-Y, and -L are able to form ternary complexes with bothCRM1 and RanGTP, and that these associations aresensitive to the cytotoxin leptomycin B (LMB). Usingvarious NS2 peptides in pull-down assays, we identifiedone candidate that efficiently binds CRM1. This peptide,which corresponds to the 79-96 amino acid region ofNS2, contains a sequence that shows a high similaritywith leucine-rich NESs. Moreover, this peptide is able tomutated NS2-NES sequences allow RanGTP hydrolysis,as demonstrated in vitro using RanGAP assays. Incontrast, mutated NS2-NES sequences allow RanGTPhydrolysis to various extents.

Altogether, our results show that MVMp NS2 proteinscontain a functional NES that leads to the formation of atrimeric complex involving both CRM1 and RanGTPcellular factors.

KIAA0176 a new Rep interacting proteinRalf Dubielzig,* Andrea Kern,

Florian Sonntag, Jürgen A. Kleinschmidt.Deutsches Krebsforschungszentrum,

Forschungsschwerpunkt Angewandte Tumorvirologie, ImNeuenheimer Feld 242, D-69120 Heidelberg, Germany,*Medigene AG, Lochhamerstrasse 11, D-82152 Planegg/

Martinsried, Germany

Using a yeast two hybrid library prepared from adenovirustype 5 infected 293 cells we isolated 8 cDNA clonesencoding two different but related proteins which showeda specific interaction with Rep78 and Rep68 in yeast.The cDNAs code for the theoretical protein KIAA0176 aswell as a potential gene product of gene dJ1170K4.1.

Homologous genes in mouse, fly and C. elegans aredescribed. The interaction with Rep proteins wasconfirmed in vitro via pull down assays. Expression of afull length flag-tagged KIAA protein by transienttransfection showed a predominant cytoplasmiclocalisation of the protein whose expression resulted insignificant cytoplasmic retention of coexpressed Repproteins. Antisera raised against various peptide-sequences of KIAA or dJ1170k4.1. recognized not onlythe anticipated 33kD polypeptide (KIAA) but also a numberof higher molecular weight polypeptides. The pattern ofthese polypeptides dramatically changed uponadenovirus infection and was reverted following AAVcoinfection. Infection of HeLa cells with adenovirusmutants showed that deletion of E2A (DBP) completelyabolished this effect, whereas deletion of E1B(p55) andE4orf6 only partially reverted this change in the polypeptidepattern. Immunofluorescence analysis showed that thenucleo/cytoplasmic distribution of these proteins wasaltered upon adenovirus infection to a perinuclearaccumulation. Coinfection with Ad5 and AAV-2 resultedin a reverted staining pattern. A significant number ofadenovirus infected cells, however, showed a strongoverexpression of KIAA proteins which was not revertedby AAV-2 coinfection. Taken together, these novel Repinteracting cellular proteins show strong changes in theirpolypeptide size, expression level and cellular localisationupon adenovirus infection which is at least partiallyinhibited by AAV-2 coinfection.

The transcription of the Acheta domesticusdensovirus (AdDNV) is unique among the densovirus

subfamilyY Li,1 FX Jousset,2 M Bergoin,2 P Tijssen.1

1Institute Armand-Frappier, Centre de Biotechnologie etMicrobiologie, H7V 4Z3 Laval, Canada; 2Unité de VirologieMoléculaire, Station de Recherches/Pathologie Comparée,

30380 Saint Christol-lez-Ales, France.

A small isometric nonenveloped virus was first identifiedas the etiologic agent of typical densonucleosis infectionin the cricket Acheta domesticus. This virus, designatedAdDNV, appeared to be highly specific. The virion is 22nm in diameter, its capsid contains 4 polypeptides andhas a single-stranded linear DNA of 5.4 kb. Western blotand Southern blot analyses failed to reveal any homologybetween AdDNV and other DNVs. Viral DNA from AdDNVwas cloned into pBluescript vector and sequenced in bothdirections. Although the AdDNV has been isolated froman insect belonging to the Orthoptera (ie. crickets) and itsgenome is considerably smaller than those of JcDNV,GmDNV, PiDNV from butterflies, the genome organizationresembles that of the latters. Similarly, the genome wastranscribed only from the 5' halves of the complementarystrands. No splicing has been found in densovirus.However, analyses of mRNAs isolated from infectedcrickets and from infected Bm36 insect cells by AdDNVvirus and those from the recombinant baculovirus intowhich the capsid gene of AdDNV was inserted showedthat two introns of 131 and 356 nt occurred within the

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capsid mRNA. This transcript splicing shifted the openreading frame of the ORF1 coding for the biggest capsidprotein VP1. The programmed translational frameshiftingmay be involved in the translation of capsid gene. Boththe mRNA splicing and the translational frameshiftingare the first events observed in densovirus and parvovirussubfamilies respectively.

4. DNA REPLICATION ORIGINS

Genomic and functional analysis of ParvoviralInitiation Factor: remarkable flexibility of the DNA

binding siteErik D Burnett, Christine M Ticknor, Susan F Cotmore,

Jesper Christensen, Peter Tattersall.Departments of Laboratory Medicine & Genetics, Yale

University School of Medicine, 333 Cedar Street, New Haven,CT 06510, USA.

The human site-specific DNA-binding protein that is arequired cofactor for DNA replication at the 3' end of MVMhas been shown to bind two ACGT motifs spaced fivebase pairs apart in the left end origin. This cofactor, termedthe parvovirus initiation factor (PIF), is a heteromericcomplex composed of two protein subunits designatedPIF96 and PIF79. Genomic analysis has shown that thePIF96 and PIF79 subunits are not linked in the humangenome, but are located at chromosome 1p33 and 20qter,respectively. The region of highest similarity between thetwo proteins, spans 3 exons with conserved internalboundaries, and contains a SAND domain. Recombinantsubunits can form both homo- and hetero-complexeswhen expressed in a baculovirus system. We haveconducted SELEX experiments to examine the optimalspacing and content of the DNA binding motif recognizedby the heterocomplex and the two homocomplexes. Theseexperiments indicate that the preferred half-site sequenceis ACGT, and the optimal spacing between these is sevennucleotides, although half-sites spaced at 5, 6 and 8nucleotides are selected with almost equivalent efficiency.The heterodimer and both homodimers bind similaroptimally configured sequence arrangements.

Mechanism of Rep-mediated AAV origin nickingJR Brister, N Muzyczka.

Department of Molecular Genetics and Microbiology andPowell Gene Therapy Center. University of Florida, Gainesville,

FL, USA.

The small single-stranded adeno-associated virus (AAV)genome is flanked by terminal hairpinned structures(TRs) that act as origins of DNA replication and viralpackaging signals. During viral DNA replication the AAVnon-structural Rep protein nicks the TRs at the terminalresolution site (trs) in an ATP-dependent, site-specific,strand specific manner. The trs endonuclease reactionrequires at least three TR sequence elements for efficient

cutting. These are the Rep binding element (RBE), ahairpin recognition element (RBE’), and a specificsequence at the trs. Here we determine the minimal trssequence necessary for Rep nicking, 3'-CCGGT/TG-5',and show that this base core sequence is required onlyon the nicked strand. We also identify a potential stem-loop structure at the trs. When we design a mutantsubstrate that fixes the trs stem-loop in the extruded form,Rep68 nicks this mutant better than wild type. Thissuggests that the stem-loop trs structure is a reactionintermediate and that extrusion of the trs cruciform is arate limiting step. Furthermore, Rep68 nicking of thismutant is completely ATP-independent, indicating that theRep transesterification reaction is not itself ATP-dependent. Rather, ATP is required for Rep DNA helicaseactivity, which in turn facilitates the formation of thecruciform substrate containing the trs. Thus, the trsendonuclease reaction consists of at least twobiochemical steps, cruciform extrusion andtransesterification. The first step requires the Rep bindingelement (RBE) that is upstream of the trs and the ATPdependent DNA helicase activity; the second requiresthe trs 7-base core sequence in an extruded stem-loopstructure and is ATP independent. We also investigatethe contribution of the RBE and RBE’ during this process.Our data indicate that Rep is tethered to the RBE in aspecific orientation during Rep trs nicking. This orientationappears to align Rep on the AAV TR allowing specificnucleotide contacts with the RBE’ and directing nickingto the trs. Accordingly, alterations in the polarity or positionof the RBE relative to the trs greatly inhibit Rep trs nicking,and substitutions or deletion of the RBE’ also reducenicking, although to a lesser extent. Together these resultssuggest a model of Rep interaction with the AAV TR duringorigin nicking in which an asymmetric interaction betweena Rep dimer and a tripartite cleavage signal that consistsof the RBE, RBE’ and the trs is essential forendonucleolytic activity.

A Rep recognition sequence is necessary but notsufficient for nicking of DNA by Adeno-Associated

Virus Type 2 Rep proteinsRoland A Owens, Jianwen Wu, Michael D Davis,

Thomasina L Bailey.Laboratory of Molecular and Cellular Biology, National Instituteof Diabetes and Digestive and Kidney Diseases, Bethesda,

Maryland 20892, USA.

The site-specific endonuclease activity of the Rep68 andRep78 (Rep68/78) proteins of adeno-associated virustype 2 (AAV) is involved in AAV replication and site-specificintegration. The endonuclease activity occurs withinGTTGG motifs 9-14 bases away from the imperfectrepeating ([GCTC]/[GAGC]) motifs which constitute thecore Rep68/78 binding sites, known as Rep recognitionsequences (RRSs). Both motifs are found within the AAVinverted terminal repeats (ITRs) and in the AAV preferredintegration locus on human chromosome 19 (AAVS1).Although a double-stranded, linear segment of DNA,containing an RRS and a cut site has previously been


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shown to function as a substrate for the Rep68/78endonuclease activity, it has never been formallydemonstrated that the RRS is necessary for this activity.We show here that mutation of the RRS, within such aDNA segment, abolished the ability of this substrate tobe cleaved by Rep78. We also performed endonucleaseassays on human genome-derived sequencescontaining RRSs and their flanking regions. With theexception of the AAVS1 fragment, the human RRS-containing sequences tested are not cleaved by Rep78.A computer-assisted search of the human genome hasidentified additional putative RRSs. None of these newRRS-containing sequences contains a consensusRep68/78 cleavage site, so they are unlikely to be high-frequency AAV integration targets.

Cleavage & ligation of single-stranded viral ori DNA bythe rep78 protein of Adeno-Associated Virus type 2

Richard H Smith, Robert M Kotin.Laboratory of Molecular Hematology, National Heart, Lung, and

Blood Institute, Bethesda, MD, USA.

Replicon-encoded initiators of rolling-circle replication(RCR) share conserved amino acid sequence motifs thatare directly associated with the ability of these proteins tomediate the cleavage and joining of appropriate single-stranded DNA substrates. The Rep78 protein of AAVcontains at least two of three evolutionarily conservedRCR-initiator protein motifs. According, we haveinvestigated the ability of AAV Rep78 to catalyze thecleavage and ligation of single-stranded DNA substratesderived from the AAV origin of replication. In an in vitrossDNA cleavage assay, purified maltose-binding protein(MBP)-Rep78 fusion protein cleaved single-stranded AAVori DNA predominantly at the terminal resolution site (trs),minor cleavage sites upstream of the trs site were alsoobserved. Cleavage of non-ori-derived substrates couldbe detected, but was much less efficient than with AAV oriprobes. Rep78 demonstrated a preference for cleavage5' of thymidine residues, and cleavage was strictlydependent upon the presence of a divalent cation cofactor.The cleavage of ssDNA, however, did not requirenucleoside triphosphate hydrolysis. Electrophoreticmobility shift assays demonstrated that binding of single-stranded oligodeoxynucleotide probes by MBP-Rep78was influenced by both probe length and the presence ofcis-acting sequence elements flanking the terminalresolution site. Site-directed mutagenesis was used toreplace specific tyrosine residues within the RCR ‘motif3’ equivalent of Rep78. Substitution of Tyr-156 withphenylalanine completely abolished the ability of MBP-Rep78 to cleave and ligate single-stranded DNAsubstrates, but not the ability to stably bind single-stranded DNA. It is postulated that the cleaving-joiningactivity of Rep78 may play a role in the resolution of AAVreplicative-form dimer (RFD) genomes. In addition, Rep-mediated intermolecular recombination may play a rolein targeted integration of AAV.

Amino-terminal domain exchange redirects origin-specific interactions of AAV Rep78 in vitro

Miran Yoon, Deborah H Smith, Peter Ward,R Michael Linden.

Institute for Gene Therapy and Molecular Medicine, Mount SinaiSchool of Medicine, New York, NY 10029, USA.

The unique ability of adeno-associated virus type 2 (AAV)to site-specifically integrate its genome into a definedsequence on human chromosome 19 (AAVs1) makes itof particular interest for use in targeted gene delivery. Theobjective underlying the study presented here is to provideevidence for the feasibility of re-targeting site-specificintegration into selected alternative loci within the humangenome. However, the mechanism underlying this viral-mediated phenomenon remains largely undefined.Current models purport that AAV DNA integration isinitiated through the interactions of the products of a singleviral open reading frame, rep, with sequences present inAAVS1 that resemble the minimal origin for AAV DNAreplication. In this study, we present a cell free systemdesigned to dissect the AAV Rep functions required totarget site-specific integration using functional chimericRep proteins derived from AAV Rep78 and Rep1 of theclosely related goose parvovirus (GPV). We show herethat amino-terminal domain exchange efficiently redirectsthe specificity of Rep to the minimal origin of DNAreplication as determined by DNA binding as well as cellfree DNA replication assays. Furthermore, we establishthat the amino-terminal 187 amino acids of Rep78/68constitute an additional catalytic domain of Rep sufficientto mediate site-specific endonuclease activity.

5. DEVELOPMENT OF VECTORS FOR GENE TRANSFER

Immunology of AAV as Vector for Human GeneTherapy

Yi Zhang, Naren Chirmule, Markus Hildinger, Maria Croyle,Guang-ping Gao, James M Wilson.

University of Pennsylvania, Philadelphia, PA, USA.

Vectors based on adeno-associated virus (AAV) are beingconsidered for human gene therapy. While mostexperiments have been based on AAV serotype 2, recentdata suggest that AAV serotypes 1, 4, 5 and 6 may alsobe useful. Critical to the eventual success of AAV vectorsin humans is an understanding of the immunologicconsequences of in vivo delivery of vector. We havecharacterized two general types of antigen specificresponses to AAV in a number of model systemsincluding mice, monkeys and humans. Several importantprinciples have emerged. AAV appears to avoid activationof T cells to non-secreted transgene products due in partto its rather weak tropism for antigen presenting cells(APCs), which may be inadvertently transduced during invivo vector administration. This form of immunologicignorance allows stable expression of antigenictransgenes. The basic immunology of this phenomenon

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and biologic settings where it may be compromised willbe presented. The other effector response is the activationof B cells to capsid proteins and formation of neutralizingantibodies that blocks vector uptake. We demonstratedthe surprising results that the qualitative nature of the Bcell response is dictated by the route of administration.Strategies to prevent it by modifications of the host (e.g.,transient systemic T cell inhibition) or the vector (e.g.,capsid modification with polyethylene glycol) will bediscussed.

Characterization of Recombinant AAV4 and AAV5 asVectors for Gene TransferJA Chiorini, Robert M Kotin,

Joseph Zabner, Beverly L Davidson.

Interest in the use of adeno-associated viruse type 2(AAV2) as a vector for gene transfer has increasedbecause of its ability to stably transduce cells in vivo. Atotal of six primate AAV serotypes have been identified ofwhich only AAV2 has been studied extensively as a vectorfor gene transfer. Recent data suggest that other AAVserotypes can also function as vectors for gene transfer.Divergence in the cap ORF of AAV4 and AAV5 anddifferences in sensitivity to competition with solubleheparin together with differences in transductionefficiencies in laboratory cell lines suggest that theseserotypes may exhibit distinct mechanisms oftransduction. We have tested the activity of recombinantAAV4 and AAV5 vectors in vivo and found differences inboth tissue tropism and transduction efficiency comparedto AAV2. Introduction of AAV5 into the lungs of mice showedimproved transduction of airway epithelia and alveolarcells. In vitro data suggest that the AAV5 transduction maybe occurring via the apical surface. Direct injection of AAV4or AAV5 into the brain was also examined. Whenintroduced into the ventricle all vectors showed a similarpattern of transduction although with varying efficiencies.When injected into the striatum, each of the serotypesdemonstrated distinct tropisms. These in vivo studiesconfirm the initial in vitro observations that each of theseserotypes utilizes a distinct mechanism of uptake duringtransduction and supports the use of AAV4 and AAV5 asvectors for gene transfer in both the lung and brain.

Self-Complementary rAAV (scAAV) gene deliveryvectors

Douglas M McCarty, R Jude Samulski.Gene Therapy Center, University of North Carolina, Chapel Hill,

NC 27514 USA.

One of the perceived limitations of recombinant adeno-associated virus (rAAV) gene delivery vectors has beenthe requirement for host cell directed synthesis of thecomplementary strand from the virion DNA template. Thisstep occurs with varying efficiency in different cell typesand can be induced through treatment with co-infectingadenovirus (Ad), UV irradiation, or other means of cell

stress. We have circumvented this problem by takingadvantage of the tendency of AAV to package dimeric DNAmolecules when the genome is half the length of the wtgenome. The replication model for AAV predicts that thedimeric genomes would be in the inverted repeatorientation, resulting in the packaging of a self-complementary DNA molecule. Because the genomeshould spontaneously re-anneal upon release from thecapsid, no host cell DNA synthesis is required fortransduction. We have generated a 2.3 kb CMV-GFPvector, separated dimeric and monomeric genome virionson CsCl gradients, and confirmed the predicted invertedrepeat structure. Comparing the transducing efficienciesin uninduced HeLa and 293 cells, the dimeric rAAV vectorsare 4 to 8-fold more efficient than the homologousmonomeric vectors. We have further characterized thescAAV vectors in terms of induction by Ad or UV, fate of thevirion DNA after infection, resistance to inhibitors of DNAsynthesis, and long term transducing potential. Theresults are consistent with the ideas that virion DNA isconverted to duplex prior to integration, and that therestrictions in host-cell directed complementary-strandsynthesis reflect limited processivity rather than initiation.The scAAV vectors have sufficient genetic capacity for anumber of potentially useful applications and offer avaluable tool for the study of the molecular mechanismsof rAAV transduction

MVM-based vectors for transducing human andmurine T cells

Gene A Palmer, Peter Tattersall.Departments of Laboratory Medicine and Genetics, YaleUniversity School of Medicine, New Haven, Connecticut,

06510 USA.

We have developed a series of capsid-replacement MVM-based vectors that significantly suppress the formationof replication-competent virus. The vector genome canaccommodate a transgene up to 2.2 kb in size and ispackaged in the MVMi coat allowing us to transducehuman as well as murine T cells. Many tumors evadeimmunosurveillance by downregulating expression ofcostimulatory molecules on their surface. EL4 is a murineT cell lymphoma that expresses no detectable B7.1, thecostimulatory ligand for CD28 on CTL. Our strategy wasto transduce EL4 in vivo by injecting tumor-bearing micewith recombinant oncotropic vector that will express highlevels of surface murine B7.1 under control of the lateviral P38 promoter. Results support the conclusion thata single injection of vector can transduce sufficientnumbers of EL4 to elicit a protective, tumor-specificimmune response with tumor-specific CTL memoryprotecting mice from subsequent challenge with 200times the lethal dose of EL4. This animal model for cancerimmunotherapy establishes an important clinicalapplication of parvovirus vectors as agents for genedelivery with great potential.


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AAV-Mediated gene transfer: Role of cellular FKBP52protein in transgene expression

KY Qing, J Hansen, A Srivastava.Indiana University School of Medicine, Walther Oncology

Center, Walther Cancer Institute, Indianapolis, IN

Adeno-associated virus 2 (AAV), a single-stranded DNA-containing, non-pathogenic human parvovirus, hasgained attention as a potentially useful vector for humangene therapy. However, the transduction efficiency of AAVvectors varies greatly in different cells and tissues in vitroand in vivo. We have documented that a cellular tyrosinephosphoprotein, designated the single-stranded D-sequence-binding protein (ssD-BP), plays a crucial rolein AAV-mediated transgene expression (Proc. Natl. Acad.Sci. USA, 94: 10879-10884, 1997). We have documenteda strong correlation between the phosphorylation stateof the ssD-BP and AAV transduction efficiency in vitro aswell as in vivo (J. Virol., 72: 1593-1599, 1998). We havealso established that the ssD-BP is phosphorylated bythe epidermal growth factor receptor protein tyrosinekinase (EGFR PTK) activity. The phosphorylated form, butnot the dephosphorylated form, of the ssD-BP preventsAAV second-strand DNA synthesis, and consequently,results in a significant inhibition in AAV-mediatedtransgene expression (J. Virol., 72: 9835-9843, 1998).We purified the ssD-BP to homogeneity from HeLa cells.The partial amino acid sequence of one of the peptides(24 amino acids) revealed 100% homology to a cellularprotein that binds the immunosuppressant drug FK506.This 52 kDa protein, termed FK506-binding protein(FKBP52), is an immunophilin. FKBP52 has also beenshown to be a chaperone protein. It is presentubiquitously, is phosphorylated, possesses a nucleotidebinding site, and localizes predominantly to the nucleus,at least three properties that are shared with the ssD-BP.A prokaryotic expression plasmid containing the humanFKBP52 gene was generated by PCR amplification froma cDNA library, and the purified protein was shown tointeract with the AAV single-stranded D-sequence probeby electrophoretic mobility-shift assays (EMSA). Thepurified FKBP52 could be phosphorylated both by caseinkinase II (CKII) and EGFR PTK. Furthermore, in in vitroDNA replication assays, the EGFR PTK-phosphorylatedFKBP52 inhibited the AAV second-strand DNA synthesisby greater than 90%. The CKII-phosphorylated FKBP52caused ~40% inhibition, whereas the dephosphorylatedFKBP52 had no effect on the AAV second strand DNAsynthesis. We also generated eukaryotic expressionplasmids containing the human FKBP52 gene and aselectable marker to obtain FKBP52 over-expressing celllines. The over-expressed FKBP52 in these cell lineswas present in predominantly the dephosphorylatedform, and upon transduction with a recombinant AAV-lacZvector, allowed a significant increase in AAV-mediatedtransgene expression. Taken together, these studiescorroborate that the cellular FKBP52 protein is a crucialdeterminant of AAV transduction efficiency.

In vivo multilineage hematopoietic reconstitution ofNOD/SCID mice with rAAV transduced human cord

blood CD34+CD38- cellsChristie Ann Wong, KK Wong Jr, Wei Li, Leah Tager,

Stephen J Forman, Saswati Chatterjee.Department of Virology and Hematology / Bone Marrow

Transplantation, City of Hope National Medical Center, Duarte,CA, USA.

Despite offering a valuable approach for gene therapy ofa number of human diseases, the efficient geneticmodification of human hematopoietic stem cells capableof long term engraftment remains elusive. In this studywe tested the potentials of rAAV vectors for the geneticmodification of primitive human hematopoietic stem/progenitor cells capable of long term in vivo multilineagereconstitution of immune deficient NOD/SCID mice.Cytofluorometrically sorted cord blood (CB) CD34+CD38-cells were transduced with a CsCl purified rAAV vector,vCWRHIVAPAP, encoding antisense RNA complementaryto the HIV_1 LTR at a particle MOI of 400 and infused intosublethally irradiated NOD/SCID mice. Transplanted cellswere shown to be free of contaminating lymphoid andmyeloid cells. Engraftment was evaluated from 8-24weeks post transplantation. Transplantation of 30,000 to70,000 CD34+CD38- CB cells resulted in 12_50%marrow engraftment as evidenced by the presence ofCD45+ human cells in NOD/SCID marrow. Similar levelsof engraftment were observed up to 24 weeks aftertransplantation indicating that human CB CD34+CD38-cells mediated long term reconstitution. Comparablelevels of engraftment were observed in mice transplantedwith either transduced or untransduced cells indicatingthat rAAV vectors did not compromise engraftment. Thepresence of rAAV sequences in the CD45+ fraction fromthe marrow of each mouse transplanted with transducedcells demonstrated that transduced cells were capableof hematopoietic engraftment. In order to determinewhether transduced CD34+CD38- cells coulddifferentiate in vivo into different hematopoietic lineages,human cells recovered from NOD/SCID marrow werefurther sorted into CD14+ myeloid cells and CD19+ Bcell populations. Amplification of rAAV sequences fromlineage_specific cells revealed the presence of rAAVvectors indicating that transduced CD34+CD38- CB cellswere capable of contributing to both lymphoid and myeloidlineages. Significantly the CD34+ human progenitor cellfractions recovered from the marrow of NOD/SCID miceat 8, 12 and 24 weeks post_transplantation alsocontained rAAV transduced cells. These results suggestthat rAAV transduced CD34+CD38- cells were capable ofeither surviving as or giving rise to hematopoieticprogenitors for extended periods in vivo. The presence ofvector specific signals in purified CD19+ splenic B cellsindicated that the differentiated progeny of rAAVtransduced CD34+CD38- cells were capable of normaltrafficking. Importantly analysis of rAAV sequences inCD34+ human clonogenic progenitor cells derived fromthe marrow at 12 weeks post_transplantation revealedthat 65_75% of myeloid colonies were derived from

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transduced CD34+CD38- cells, suggesting that rAAVtransduced progenitors retained their clonogenic andhematopoietic properties in vivo. Our results indicate thatrAAV transduced CD34+CD38- primitive humanhematopoietic stem/progenitor CB cells are capable oflong term multilineage hematopoietic engraftment in vivo.

AAV-mediated gene transfer: Effect on majorhistocompatibility complex class II gene expression

H-J Kwon, KA Weigel-Kelley, J Hansen,KY Qing, A Srivastava.

Indiana University School of Medicine, Walther OncologyCenter, Walther Cancer Institute, Indianapolis, IN, USA.

Adeno-associated virus 2 (AAV) vectors have gainedattention as a potentially useful alternative to the morecommonly used adenovirus- and retrovirus-basedvectors for human gene therapy. Interestingly, AAV vectorsseem to elicit little host immune response, the underlyingmolecular mechanisms of which remain unclear. Wehave identified a putative cellular protein, designated thedouble-stranded D-sequence-binding protein (dsD-BP),which interacts with the D-sequence in the viral invertedterminal repeats (ITRs) and plays a crucial role in AAVDNA replication and encapsidation (J. Virol., 70: 1668-1677, 1996). Since the AAV D-sequence shares asignificant homology with the X-box sequence in thepromoter sequence of the major histocompatibilitycomplex type II (MHC-II) genes, we reasoned that the AAVD-sequence might compete for the RFX transcriptionfactor required for MHC-II promoter function. Indeed, inelectrophoretic mobility-shift assays (EMSA), the dsD-BPwas shown to interact with synthetic oligonucleotidesspecific for both the AAV D-sequence and the X-boxsequence probes. Cross-competition experiments withthe two probes further suggested that the dsD-BP mightbe a putative RFX transcription factor. To directly test thehypothesis that the AAV D-sequence competes for theRFX transcription factor and therefore inhibits MHC-IIpromoter expression, a reporter construct containing thefirefly luciferase gene under the HLA DRA promoter wastransfected in HeLa and 293 cells, with and without theAAV D-sequence oligonucleotides, and the reporter geneactivity was determined 48 hrs post-transfection. The AAVD-sequence inhibited the reporter gene expression inHeLa and 293 cells by approximately 93% and 96%,respectively. No inhibition was detected when other non-specific synthetic oligonucleotides were used. Treatmentwith interferon-( (IFN-(), which is known to lead to activationof the DRA promoter in HeLa cells, resulted in enhancedtransgene expression which was also down-regulatedby the AAV D-sequence. These studies suggest that theD-sequence-mediated down-regulation of the MHC-IIgenes might be exploited by AAV to evade the host immuneresponse against recombinant AAV vectors.

Improvement of autonomous parvovirus-basedvectors by constructing pseudotypes and chimeric

vectorsC Wrzesinski, J Rommelaere, C Dinsart.

Applied Tumor Virology, Abt. F 0100 and INSERM U375,German Cancer Research Center,

Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

In our laboratory MVMp and H1-based vectors wereconstructed, in which VP-coding sequence is partiallydeleted and replaced by various transgenes. Throughcotransfection of the vector DNA and the helper plasmid,recombinant virus stocks are produced which contain0.01% replication-competent virus (RCV) as a result ofhomologous recombination. Our aim is to decrease thelevel of RCVs by reducing sequence homology betweenthe recombinant genome and the helper plasmid. Thiscan be achieved by constructing chimeras andpseudotypes between MVMp and H1. Such chimeras gaverise to virus titers that were not significantly different fromthose obtained with MVMp or H1, however, the fraction ofRCVs was considerably reduced (less than 0.0001%).Pseudotyping of H1 viral genomes with MVMp capsids(and vice versa) did not affect the viral titers and indeedreduced the level of RCVs in these stocks so that detectionwas only possible after two rounds of amplification.Testing of these pseudotype-constructs in murine A9 andhuman NB374K cells is now under way.

A potential parvovirus H-1-based vector for cancergene therapy

Huang Qingshan, Li Li, Gu Meigong, Guo Lanping,Luo Zuyu, Xie Yi.

School of Life Sciences, Fudan University, Shanghai 200433,China

Parvoviruses have potential for cancer gene therapy as avector of gene transfer. A two-plasmid system wasconstructed that package parvovirus H-1 recombinantsexpressing enhanced green fluorescent protein (GFP)reporter gene or wild type p53 cDNA. The vector uses H-1 NS-1 protein to drive expression of enhanced GFP orp53 under the control of the H-1 p38 promoter.

A point mutation was introduced within the initiator codonof VP gene by PCR to prevent frameshift of the transducedgenes but without effect on the codon of H-1 non-structureproteins. The overall genome size of recombinant viruseswas maintained in 5.18 kb for efficient packaging. H-1capsid proteins are supplied in trans by a helper plasmidwhich also contains a SV40 replicatin origin and a SV40polyA. The GFP-transducing virus and p53-transducingvirus are produced by trans-encapsidation.

Human hepatoma cell and liver cell lines were infectedwith the GFP-transducing virus and are observed byconfocal microscopy (LEICA TCS-NT). These cells areefficiently infected by H-1 recombinant viruses expressingthe GFP report gene. Titer of recombinant weredetermined by numbering cells with GFP on 36 h.p.i.


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with NBK cells. The recombinant H-1 virus containing wt-p53 cDNA (named pH1/p53) caused the hepatoma cellline QGY-7703 significant apoptosis at 36 h.p.i.. Theexperiments to assess in vitro infection efficiency of cellsfreshly excised human hepatoma tissue andtransplantable hepatoma model QGY-9204 are inprogress. *This project was supported by National NatureScience Foundation of China (39780027)

New MVM(p)-based vectors for cancer gene therapyClément Nathalie, EL Bakkouri Karim, Velu Thierry,

Brandenburger Annick.IBMM, IRIBHN, Université Libre de Bruxelles, campus

Gosselies, 6041 Gosselies, Belgium.

Considering the oncoselective replication of theautonomous parvovirus Minute Virus of Mice (MVM(p)), inaddition to its innocuousness in men, this virus waschosen as a backbone for the elaboration of vectors for atumor cell targeted interleukine-2 (IL2) gene transfer. MVM-IL2 vector stocks produced in the presence of a helperplasmid that contains the capsid coding sequences (VP),are routinely contaminated with wild-type (w.t) virus thatarises through homologous recombination. In order toavoid recombination, we have constructed new vector andhelper plasmids having reduced or no sequencehomology at the right of the transgene. These vectors arebased on the sequences of two different spontaneouslyoccurring defective particles of MVM(p). One of thesedefective particles lacks all the VP coding sequences,whereas the other conserves the right end of thesesequences. From the replication capacity of these twodefective particles we concluded that they conserve thecis-acting sequences required for viral replication. Thematched helper plasmids contain the capsid codingsequences under the control of either the parvoviral P38promotor (pP38-VP) or the SV40 promotor (pPSV40-VP).No wild-type virus was detected in stocks obtained bycotransfection of the new helpers with the first vector thatcompletely lacks VP sequences at the right of thetransgene. However, the second vector generates wild-type particles after cotransfection with either helper.

Comparison of the marker gene expressionmediated by AAV vectors carrying different

promotersSaburo Kashii, Li Dong Lan, Toshiyuki Sahara,

Katushiko Itoh*, Kazuhiko Ito.Department of Transfusion Medicine, *Clinical Molecular

Biology Kyoto University, Kyoto 6068507 Japan.

Hematopoietic stem cells are one of the potential targetsfor gene therapy of recombinant AAV vectors. However,the efficient gene transduction and stable expressionhave been limited due to the failure of its integration intogenome, or possibly due to the ineffective promoters intarget cells. By analyzing the expression of a markergene, mouse CD8a, we have evaluated the activities ofvarious promoters in recombinant AAV vectors in cell as

a transduced cell group. The recombinant viral vectorplasmids of pLINT, pLINC, pLINS or pLINF wereconstructed containing different promoters of TK, CMV,SV40, or SFFV (Retroviral LTR derived from Friend spleenfocus forming virus), respectively. All of these viral vectorscontained murine CD8a cDNA and a neo gene. Raji,K562 or 293 cells were infected with these recombinantviruses and selected with G418 for several weeks. Neoresistrant cells were analyzed on flowcytometer afterstaining with anti-murine CD8a monoclonal antibody.Those cells were also subject to Southern blot analysisto confirm the integration into the genome.

In K562 cells, the expression of CD8a by the SFFVpromoter (vLINF) was lower than SV or TK promoters(vLINS or vLINT, respectively), and the expression levelswere almost the same between these promoters in Rajicells. On the other hands, the promoter activity of SFFVLTR was about 5-fold higher than those of otherpromoters in 293 cells. These results indicated thattissue- or lineage specific optimization of the promotershould be needed for efficient gene expression by AAVvectors. Further analysis in other cell lines using therecombinant viral vectors are currently on going.

TBP-associated factorII110 binds the adeno-associated virus (AAV) Rep78 major regulatoryprotein in vivo and abrogates Rep78-mediated

inhibition of the AAV p5 promoterDeJin Zhan, AD Santin, Michael Mane, Paul L Hermonat.Department of Obstetrics and Gynecology, University ofArkansas for Medical Sciences, Little Rock, AR 72205

TBP associated factor p110 (TAFII110) is part of the TFIIDtranscripiton initiation complex which promotes RNApolymerase II-dependent transcription. TAFII110 is knownto bind a number of cellular factors including, but notlimited to, Sp1, and TAFII250. It is also known to bindseveral viral oncoproteins, including Humanpapillomavirus (HPV) type 16 E7, adenovirus E1A, andSV40 Large T Antigen. Thus, TAFII110 is involved in alattice of interactions, which although not presently fullyunderstood, ultimately regulates transcription. Adeno-associated virus (AAV) encoded Rep78 is a multifunctionalprotein which is required for the regulation of transcriptionfrom AAV promoters, AAV DNA replication, and for sitespecific integration of AAV into human chromosome 19.Recently, it has been demonstrated that Rep78 is able tobind to itself and other proteins, including cellulartranscription factors Sp1 and TBP. In studies presentedhere, it was observed that TAFII110 also interacted withRep78 in vitro and in vivo. Yeast GAL4 two hybrid cDNAanalysis suggested that the in vivo Rep78-TAFII110interaction was stronger, and thus possibly moresignificant, than the Rep78-Sp1 or Rep78-TBPinteractions. The Rep78 binding domain within TAFII110was found to reside within amino acids 309-603, usingamino- and carboxy- truncated versions of TAFII110. TheTAFII110 binding domain within Rep78 was found withinamino acids 121-370, using amino- and carboxy-

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truncated versions of Rep78 fused with MBP.Furthermore, Rep78 was found to inhibit AAV p5 promoteractivity in a dosage dependent manner in an in vitrotranscription system based on HeLa cell nuclear extracts.Using the series of truncated Rep78 proteins it was foundthat most of the Rep78 protein was required for theinhibition of p5. Only the extreme carboxy-terminus ofRep78 was dispensable for this activity. Finally, TAFII110was found to abrogate Rep78-mediated inhibition of p5activity in a dosage dependent manner. These data identifya new and likely important protein-protein interaction ofRep78 and suggest one possible mechanism by whichRep78-mediated negative regulation of transcription maybe altered in the cell.

Amplification of stably integrated rep/cap genes inthe production of AAV and the influence of inverted

terminal repeatsGuangping Gao,1 Ryan Engdahl,1 Sam Cho,1 Fengmin Lu,2

Jon Marsh,1 Bindu Joshi,1 Nancy Spinner,2 James M Wilson.1*1Institute for Human Gene Therapy and Departments of

Molecular and Cellular Engineering and of Medicine, and theWistar Institute, Philadelphia, PA 19104, USA;

2Department of Human Genetics, Children’s Hospital ofPhiladelphia, Philadelphia, PA 19104, USA.

Previously, we have reported that a temporal relationshipof infections between adenovirus helper and Ad-AAVhybrid was critical for AAV productivity in a vector productionsystem using a rep/cap cell line (called B50) with Ad-AAVhybrid shuttle vectors. A 24-hour pre-infection with Adhelper was essential. In an attempt to eliminate the Adhelper virus, replication competent Ad-AAV hybrids (rcAd-AAV), in which AAV vector genomes were cloned into theE3 region of a replication competent Ad genome, werecreated. However, efforts to optimize rcAd-AAV for AAV vectorproduction did not achieve an efficient AAV packaging. Asystematic study was carried out to understand generegulation of rep/cap gene expression in B50 cells and todevelop novel strategies for the high yield AAV vectorproduction. Our studies reveal that rep/cap geneamplification in B50 cells after adenovirus infection isessential for the high yield production of AAV vectors. Thisgene amplification process starts with the initial activationof integrated rep/cap genes in B50 cells, followed byexcision of P5-rep/cap genes from the B50 genome andby episomal amplification. However, the rep/cap geneamplification is inhibited by AAVITRs, most likely byblocking initiation of the gene amplification. Potentialmechanisms for the rep/cap gene amplification andinhibitory effect of AAVITRs are discussed.

Evaluation of the potential of Adeno-associated virustype 5 based recombinant vectors for gene therapy.M Hildinger, G-P Gao, G Kurtzman,* D Weiner, J-M Wilson.

Institute for Human Gene Therapy, 3604 Spruce Street,Philadelphia, PA and *Genovo, Inc., Sharon Hill, PA, USA.

Adeno-associated viruses (AAVs) are small, icosahedralparvoviruses that encapsidate about 4.7 kb of single-stranded DNA. Six primate serotypes have been reportedso far (AAV1 to AAV6). With the exception of AAV5, whichhas been isolated from a patient’s lesion, all other AAVserotypes have been discovered in cell culture. In addition,AAV5 is the only serotype which does not show cross-complementation with any other serotype; whereas thehomology between the other serotypes is in the range of75% to 82%, AAV5 reveals only about 50% homology,and infection with AAV5 cannot be inhibited by heparin.Therefore, another tissue tropism of AAV5 compared tothe other serotypes seems to be intriguing.

In order to evaluate the usefulness of AAV5 as an in vivogene transfer vector, we constructed AAV5 basedrecombinant vectors harboring the lacZ or EGFPtransgene. The recombinant vectors were injected intomurine lung, liver and muscle tissue with recombinantAAV2 as a control vector. Preliminary results suggest ahigher transduction efficiency of lung tissue by AAV5 thanby AAV2. In addition, AAV5 mediates stable, efficient andlong-term gene expression in muscle. Moreover,immunological studies revealed that there are nosignificant levels of neutralizing antibodies to AAV5 in thepopulation, and neutralizing antibodies to AAV2 do notcross-react with AAV5. These features make AAV5pseudotyped vectors an attractive tool for human genetherapy.

Development of a recombinant baculovirus-basedsystem for production of recombinant adeno-

associated virus (rAAV) vectorsLinda C Rogers, Haim Burstein.

Department of Reseach, Targeted Genetics Corporation,Seattle, WA USA.

Current methods for production of rAAV vectors requireeither (1) the time-consuming and labor-intensivedevelopment and characterization of a vector-specificstable cell line, or (2) transfection, which is cumbersome,expensive and of limited scalability. Delivery of the rAAVvector DNA to a universal packaging cell line via virusinfection rather than transfection would greatly reducethe time and improve scalability of rAAV production. Tothis end, we developed a recombinant baculovirus thatcontains a gene encoding luciferase flanked by the AAVITRs (BacAAV-luc). Co-infection of BacAAV-luc and Ad5into HeLa/C12 cells, which provide rep and cap, resultedin amplification, packaging and production of infectiousrecombinant AAV-luc vector. Infection of HeLa/C12 withBac-AAVluc alone neither amplified nor produced rAAV-luc vector, demonstrating that both steps were Ad5-dependent. Amplification was time and dose-dependent;


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rAAV-luc vector genomes were readily detectable within36 hours post-infection and gradually increased up to 96hours, the longest time point that was analyzed.Increasing the multiplicity of infection (MOI) of BacAAV-lucfrom 10 to 1000 corresponded with an increase in thepercentage of cells showing vector amplification (from12% to 26% respectively). Interestingly, rAAV-luc vectorproduction peaked at BacAAV-luc MOI of 100 anddecreased up to 10-fold at MOI of 1000. This system withfurther optimization is amenable to a scalable rAAVproduction.

6. DNA REPLICATION MECHANISMS

Binding of the major Non-Structural protein (NS1) ofMVM to the large subunit of human replication protein

A is involved in origin unwindingJesper Christensen.

Laboratory for Molecular Virology. Department of MedicalMicrobiology and Immunology. Copenhagen University, Panum

Institute, Blegdamsvej 3B, Copenhagen 2200 N, Denmark.

A cellular site-specific DNA binding factor, Pif, and NS1binds to the 3' minimal replication origin of MVM forminga ternary complex which effciently initiates replication bycatalyzing the nicking and covalent attachment of NS1. Ithas been suggested that an oligomer of NS1 includingthe covalently attached polypeptide organizes thereplication fork by catalyzing unwinding and recruitmentof the cellular DNA replication machinery for leadingstrand synthesis. To determine the extent of originunwinding mediated by NS1and Pif in the replicationinitiation process, the presence of single-stranded DNAwas determined by its sensitivity to KMnO4 oxidation. Thisanalysis revealed that a stretch of about 20 nucleotideson both DNA strands exhibited KMnO4 sensitivityindicating limited unwinding by NS1 alone. Addition ofrecombinant replication protein A (RPA)/human single-stranded DNA binding protein, but not E.coli single-stranded DNA binding protein (SSB), to the replicationinitiation reaction catalyzed extensive unwinding of theorigin. The specificity for RPA in the unwinding reactionindicated NS1:RPA protein interactions. Therefore, NS1was tested for binding to recombinant baculovirusexpressed two- or three subunit RPA complex or theindividual subunits expressed as maltose binding proteinin E.coli using an ELISA system. NS1 efficiently boundthe baculovirus expressed products, but only the largesubunit of E.coli expressed RPA showed NS1 binding.No NS1 interactions were observed with SSB or otherproteins included as controls. To further investigate theinteraction between NS1 and RPA, we are currentlyexpressing a number of NS1 deletion mutants to mapthe region in NS1 responsible for binding RPA. Theseresults will be reported.

Cellular factors contributing to the regulation of MVMNS1 activities

JPF Nüesch, S Lachmann, L Daeffler, J Rommelaere.Department of Tumor Virology and INSERM U375, Deutsches

Krebsforschungszentrum Heidelberg. Germany D-69120.

MVM NS1 is a mainly nuclear phosphoprotein, which isinvolved in multiple functions necessary for progeny virusproduction, ranging form viral DNA replication, promoterregulation to alterations of the host cell physiology leadingto cell death. The multiplicity of activities and the observedvariation of the NS1 phosphopeptide pattern in the courseof a viral life cycle suggested regulation of the polypeptideby phosphorylation. Indeed, the NS1 replicative functionsare dependent on phosphorylation by members of theprotein kinase C family, in particular atypical PKC8 in vitro.Moreover, the in vivo relevance of these kinases could beconfirmed by coexpression with a dominant-negativePKC8 mutant, resulting in a partially (de)phosphorylatedpolypeptide specifically lacking helicase function. Inaddition, moesin, an adapter protein mediating thespecificity of PKC phosphorylation, was identified withina cell fraction activating NS1 for replication, and thus mightcontribute to the interconnection between the PKCregulatory pathway(s) and virus replication. Furthermore,amino acid substitutions at distinct target PKCphosphorylation sites in NS1 were not only found toabolish initiation of viral DNA replication, but also to impairthe capacity of the viral product to induce morphologicalalterations on the host cell, suggesting that the cytotoxicfunctions of NS1 may also be regulated by (PKC)phosphorylation. Thus, due to their pivotal role in theregulation of NS1 functions, the PKC pathways seem toplay a central role in the regulation of parvoviruspropagation.

Biochemical characterization of Junonia coeniadensovirus (JcDNV) nonstructural protein NS-1

Chuantian Ding,1 Masashi Urabe,1 Max Bergoin,2

Robert M Kotin.1

1Laboratory of Molecular Hematology, NHLBI, NIH, Bethesda,MD, USA; 2Laboratoire de Pathologie Comparée, Université

Montpellier II, France.

The nonstructural (NS) replication initiator proteins of theParvoviridae are essential for parvoviral DNA replication,and play an important role in the regulation of viral geneexpression. In contrast to the nonstructural proteins ofthe Parvovirinae, the biochemical activities of thenonstructural proteins of the invertebrate Densovirinaehave not been well characterized. Because the NS proteinsequences between the vertebrate and non-vertebrateparvoviruses are divergent, analysis of the NS-1 proteinof Junonia coenia densovirus (JcDNV) should provideuseful information and insight into structure/functionrelationships for this class of proteins. The biochemicalactivities common to the parvovirus NS protein homologsinclude: specific binding to the viral origin of replication,strand-specific endonucleolytic cleavage of the replicativeintermediate duplex DNA, and helicase activity. Relative

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to the vertebrate Parvovirinae, the genome of JcDNV hasan unusual organization. The approximately 6000 ntgenome contains extended inverted terminal repeats (ITR)of approximately 540 nucleotides. Three opening readingframes (ORF) encode the nonstructural proteins NS-1,NS-2 and NS-3. ORF2 of JcDNV (nt 4662 to 3021) encodesthe NS-1 protein. JcDNV NS-1 contains two sequencemotifs in common with the vertebrate Parvovirinae: anNTP-binding domain and a motif common to rolling circlereplication initiator proteins. To determine the biochemicalproperties of the NS-1 protein, the JcDNV NS-1 openreading frame was cloned and expressed in Escherichiacoli as a fusion protein with maltose-binding protein(MBP). Recombinant MBP-NS-1 protein was isolated andanalyzed for the following activities: 1): Binding to JcDNVITR DNA; 2) DNA helicase activity; and 3) site-specificand strand-specific endonuclease activity that specificallycuts the JcDNV origin. These results provide a basis forcomparative analyses of the structure/functionrelationships among the parvovirus NS-1 equivalents.

Adeno-associated virus type2 contains a cis-actingelement in addition to the terminal repeats that is

required for efficient DNA replicationGreg Tullis,1 Tom Shenk.2

1Avigen, Inc. Alameda, CA, USA; 2Department of MolecularBiology, Princeton University, Princeton, NJ, USA

Recombinant AAV is produced by co-transfecting aplasmid containing the recombinant AAV genome alongwith a second plasmid expressing the AAV2 rep and capgenes and either infecting the cells with adenovirus ortransfecting the adenovirus helper genes on a thirdplasmid. This method is very inefficient relative wild-typeAAV2. It often produces 100-fold fewer virus particles percell than transfection of a plasmid containing a wild-typeAAV2 genome. We demonstrate that this difference isdue to the presence of a positive-acting, cis-elementpresent in wild-type AAV2 that is required for efficient DNAreplication. In addition, we demonstrate that a minimumgenome size of 3.5 kb or greater is required for the efficientproduction of single-stranded DNA. This defect in theaccumulation of single-stranded DNA was bestdemonstrated in deletion mutants that retain the DNAreplication element. These mutants accumulated double-stranded replicative forms equivalent to wild-type AAV2,but accumulated 7-fold less single-stranded DNA.

Characterization of Adenovirus-induced ITR-independent amplification of AAV rep-cap sequences:role of cellular polymerases and viral gene products

Jacques Tessier, Gilliane Chadeuf, Pascale Nony, HervéAvet-Loizeau*, Philippe Moullier and Anna Salvetti.

Laboratoire de Thérapie Génique & *Laboratoire d’Hématologieet Cytogénétique, CHU Hotel-Dieu, Nantes, France.

Development of stable packaging cell lines able to providein trans the Rep and Cap proteins is an attractivealternative to the co-transfection method generally usedto produce recombinant AAV (rAAV). Our laboratory has

isolated a Hela-derived stable cell clone harboring oneto two copies of an ITR-deleted AAV genome (HeRC32cells). Analysis of the HeRC32 cells indicated that, uponwild-type adenovirus infection, the integrated rep-capgenome underwent a dramatic amplification leading to a100-fold increase of the rep-cap copy number (Chadeufet al. 2000, J. Gene Med., in press). This increase in therep-cap templates number was correlated with high levelsof Rep and Cap proteins and efficient rAAV assembly. Adetailed study of this amplification phenomenon wasundertaken to define the factors involved. Our resultsindicated that rep-cap gene amplification was restrictedto Hela-derived cell backgrounds. Indeed, no evidence ofrep-cap amplification was found after adenovirus-infectionof other stable rep-cap cell clones derived from 293 andTE671 cells. However, adenovirus-induced amplificationwas seen in another HeLa-derived rep-cap cell clone -the B50 cell line - (Gao et al.1998, Hum. Gene Ther.,9:2353) indicating that this phenomenon was notrestricted to our HeRC32 cells. FISH and pulsed-field gelelectrophoresis analysis suggested that the amplifiedrep-cap sequences are found in an extra-chromosomalform including linear molecules of approximately 30-40kb in size. Additional studies were designed to define thecellular and viral factors involved in the amplificationprocess. In particular, thermosensitive adenoviral mutantsand DNA synthesis inhibitors were used to define therole of key adenoviral protein(s) and cellular polymerases,respectively. Also, stable Hela cell clones harboring aspecific deletion in the integrated rep-cap genome wereused to determine if amplification requires the presenceof AAV sequences and/or proteins. Preliminaryconclusions indicate that adenoviral factors together withRep proteins and cellular polymerases are necessary intrans for rep-cap amplification. The implication of thesefindings for the design of future generations of packagingcell lines will be discussed.

7. ONCOSUPPRESSION

Oncosuppressive effects of wild-type MVMp andrecombinant derivates in immunocompetent mice

Giese NA, Lang S, Raykov Z, Rommelaere J,Dinsart C, Cornelis JJ.

Applied Tumor Virology F0100 and INSERM U375, GermanCancer Research Center, Im Neuenheimer Feld 242, 69120

Heidelberg, Germany

The antineoplastic action of autonomous parvovirusesin vivo and its underlying mechanism are poorly describedand understood. We used several murine tumor systemsincluding melanoma B16.B78/H1 and metastatichemangiosarcoma H5V to analyze antitumor potential ofMVMp in immunocompetent mice. Infection of tumor cellsby MVMp prior to implantation into mice reduced theirtumorigenic potential: it delayed formation of melanomasB78 and, while not affecting the first phase of H5V tumorgrowth and regression, extended remission period and


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dramatically reduced metastasis, leading to prolongedsurvival of animals. Established H5V tumors respondedpoorly to multiple injections of MVMp, whereas B78melanomas ceased to grow or regressed during thetreatment. Antineoplatic effects were further improvedwhen MVMp transduced immunomodulating moleculessuch as IL-2 and IP-10. The examination of treated miceshowed that no adverse inflammatory reactions werecaused by application of MVMp as high as 1010 pfu/mouse. Although MVMp could enter all analyzed organs,viral gene expression was dose-, tissue- and tumormodel-dependent. The mechanism of anti-tumor activityof MVMp in relation to the strategies developed by thevirus in order to evade the immune system is currentlyunder investigation and will be discussed.

Binding of the human papillomavirus type 16 E7oncoprotein and the adeno-associated virus Rep78

major regulatory protein in vitro and in yeast, and thepotential for downstream effects

Paul L Hermonat,1 Alessandro D Santin,1 DeJin Zhan.1

Department of Obstetrics and Gynecology, University ofArkansas for Medical Sciences, 4301 West Markham St, Little

Rock, AR 72205, USA.

Both human papillomavirus (HPV) and adeno-associatedvirus (AAV) are common anogenital viruses and likely co-infect the epithelium in vivo. However, while HPVs arepositively associated with cervical cancer, AAV appearsto be negatively associated with it. In tissue culture AAVencoded Rep78, which is essential for AAV, inhibits geneexpression and oncogenic transformation by HPV-16/18and bovine papillomavirus type 1 (BPV-1). Here weobserved if the HPV-16 E7 oncoprotein might recognizeand bind Rep78. Further, upon finding Rep78-E7 binding,we investigated some of the potential downstream effectssuch an interaction might have. E7 is capable ofrecognizing a variety of proteins, including RB105, TBP,TAFII110, E2F, cyclins A and D, and c-jun . Some of theseinteractions are likely responsible for E7’s cancerpromoting activity. Rep78-E7 interaction was investigatedin vitro by West(far)-Western and affinity chromatographyanalysis, and in vivo by the yeast GAL4 two-hybrid cDNAassay. Mapping of the E7 binding domain within Rep78was carried out using a series of amino- and carboxy-truncated Rep78 proteins in a West(far)-Western assay.Downstream effects of the interaction were analyzed bycompetitive affinity chromatography (protein-protein) andcompetitive electrophoretic mobility shift assay (protein-DNA). E7 and Rep78 were found to interact both in vitroand in vivo, in all assays attempted. The E7 bindingdomain within Rep78 was found to reside within aminoacids 121-370. Regarding downstream effects of thisinteraction, Rep78 was found to mildly inhibit E7-TAFII110and E7-RB105 interaction in vitro, but had little affect onE7-TBP interaction. Finally, it was found that E7 was ableto affect Rep78’s interaction with AAV’s terminal repeat(TR) DNA in vitro, reducing the formation of the largestsized Rep78-TR complexes in a dosage dependentmanner. These data suggest that the Rep78-E7

interaction may have repercussions for both viruses. TheRep78-E7 interaction may be a second mechanism, inaddition to Rep78 regulation of the p97 promoter, by whichAAV inhibits HPV-16 oncogenic transformation. Thesedata also suggest that HPV-16 may affect the AAV lifecycle through altering Rep78-TR interaction.

Interaction of Parvovirus H1 and the tumorsuppressor ARF-p53 pathwayHaiyan Jiang, Frank McCormick.

University of California - San Francisco, Cancer ResearchInstitute, San Francisco, CA 94115, USA

The tumor suppressor pathway comprised of ARF andp53 guards the cell against various insults. Theexpression of ARF is activated by the mitogenicstimulations from oncogenes, which in turn, stabilizesp53 and promotes p53-dependent apoptosis or cell cyclearrest. Although the activation of p53 in response to theDNA damage does not require ARF, the presence of ARFsomehow augments and sustains the activity of p53against genotoxic stress. Considering the importance ofthe ARF-p53 pathway in cell defense, it is not surprisingto find that many DNA tumor viruses have evolved to directlyinactivate p53 so as to overcome the effect of prematureapoptosis or cell cycle arrest on viral replication. However,here we report a novel strategy that the autonomousparvovirus H1 adopts to interact with the ARF-p53 pathwayin the host cells. Rat parvovirus H1 preferentially infectsproliferating cells, as well as human transformed andtumor cells, though the mechanism for such specificity isnot entirely clear.

We found that H1 targets ARF for degradation in a numberof transformed/tumor human and rat cells. ARFdegradation is mediated by the proteasome, which canbe rescued by the proteasome inhibitor MG132. Thedegradation of ARF is not due to the cell cycle arrestinduced by H1 infection. However, in spite of thedegradation of ARF, H1 infection in permissive rat cellsresults in the accumulation of the nuclear p53. We alsofound that H1 displays in its natural host species apreference for cells spontaneously immortalized by lossof ARF or transformed by polyoma virus. In addition, p53null MEFs become highly susceptible to infection andkilling by mouse parvovirus MVMp, indicating that thenecessity to inactivate ARF and p53 may be instrumentalfor a productive parvovirus infection

Anti-tumour activity of recombinant parvovirus MVM/IL2, in immuno-competent mice

K El Bakkouri, N Clément, T Velu, A Brandenburger.IBMM-IRIBHN, Université Libre de Bruxelles, Campus

Gosselies. B-6041 Belgium.

Low titres and contamination by wild-type virus haverendered in vivo assessment of the anti-tumor effect ofrecombinant parvovirus MVM/IL2 impossible up to now.The new packaging cell line we have generated higher

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titres of recombinant virus after transfection compared topreviously described packaging cells and allows theiramplification by serial infections. These cells producethe MVM(p) capsid proteins from integrated sequences,thus minimizing recombination between vector and helperDNAs and greatly limiting the generation of wtMVM.

To set up an in vivo model for the evaluation of the anti-tumour activity of MVM/IL2, about 20 murine tumour celllines were tested as to their permissivity to MVM(p), i.e.their sensitivity to wild-type MVM(p) and their expressionof the transgene after infection with recombinant MVM.The K1735 melanoma cell line was retained for furthertesting, it is fairly resistant to the cytotoxic effect of wtMVM(about 70% survival at moi 5) and expresses high levelsof IL2 after infection with MVM/IL2. Syngeneic C3H/HeNmice were injected with 106 tumour cells, either non-infected or infected with wtMVM, MVM/IL2 or an MVM/IL2-frameshift mutant prior to injection. About 70% of miceinjected with MVM/IL2-infected cells remained tumour-free whereas all other mice developped a tumour. Infectionwith wtMVM however delayed the appearance of the tumourin most animals. Mice that had remained tumour-free fortwo months were protected against a challenge with 106non-infected cells.

Evidence for an AAV-2-induced cellular factor in AAV-mediated down-regulation of the human

papillomavirus type 18 (HPV-18) upstream regulatoryregion in vivo and in vitroC M Walz,* JR Schlehofer.

Deutsches Krebsforschungszentrum, AngewandteTumorvirologie, Abt. F0100, Im Neuenheimer Feld 242, D-69120

Heidelberg, Germany*Present address: Institute of Medical Psychology, MedicalFaculty, Otto-von-Guericke University Medical Faculty, D-

39120 Magdeburg, Germany.

Results from experiments in mice, transgenic for thedexamethasone-inducible promoter (UpstreamRegulatory Region, URR) of HPV type 18 controlling lacZhad shown that infection with AAV-2 and inoculation withempty AAV capsids down-regulates the dexamethasone-dependent expression of the transgene. Similarly, in vitroexperiments with BHK cells co-transfected with HPVconstructs and various AAV rep-gene expressionconstructs showed an efficient suppression of the HPV-URR-activity. To further analyze the mechanism of the AAV-2-mediated suppression of the dexamethasone-inducedHPV promoter activity, BHK cells transfected with a HPV-18-URR-lacZ with protein extracts obtained from tonguetissue from transgenic mice were: (i) infected with AAV,(ii) inoculated with AAV capsids or (iii) uninfected. Allanimals had been either treated or untreated withdexamethasone.

Extracts from tissue from AAV-infected animals stronglysuppressed the URR similar to the effect obtained withAAV-infection, albeit there was no indication for a possiblepresence of infectious AAV, AAV DNA or viral proteins inthe tongue extracts. Extracts from animals inoculated with

AAV capsids showed a much weaker effect compared toextracts from animal infected with infectious particles.Furthermore, treatment of the animals withdexamethasone did not alter the effect of the extracts onthe cell cultures. These data strongly indicate one or moresoluble cellular factors being induced in vivo after AAV-2-infection that are capable of replacing AAV-mediatedsuppression of the HPV URR. Preliminary results indicatethat the presumed factor might not only be induced in vivobut also in cell culture.

Antibodies against AAV-2 in sera of patients withHTLV-1-associated adult T-cell-leukemia lymphoma

and in sera of healthy HTLV-1 -carriersC M Walz,1* T Fukunaga,2 JR Schlehofer,1 Y Tanaka.3

1Deutsches Krebsforschungszentrum, AngewandteTumorvirologie, Abt. F0100, Im Neuenheimer Feld 242, D-69120

Heidelberg, Germany; 2Department of Virology, Faculty ofMedicine, University of the Ryukyus, Nishihara Okinawa,

Japan; 3Department of Infectious Disease and Immunology,Okinawa-Asia Research Center of Medical Science, Faculty of

Medicine, University of the Ryukyus, Nishihara Okinawa,Japan.

*Present address: Institute of Medical Psychology, MedicalFaculty, Otto-von-Guericke University Medical Faculty, D-

39120 Magdeburg, Germany

In view of the tumorsuppressive properties of AAV andfindings that in tumor patients antibodies against AAVwere found to be less prevalent, we analyzed the serologyof AAV-2 infection in patients with HTLV-1-associated adultT-cell leukemia lymphoma (ATLL) and healthy HTLV-1carriers from Okinawa, Japan.

The HTLV-1-associated adult T-cell leukemia (ATL) isendemic in Southern Japan and involves mainly CD4+-T-cells. About I Mio. people are infected with HTLV-1 inJapan: 35% in Okinawa, 10% in Kyushu Province, 01.2%in Mainland Japan. To a lesser extent, HTLV-1-infectionis found in the rest of the world, with an estimated numberof up to 10-20 Mio. infected people. The major mode ofinfection is transplacental passage of infected maternallymphocytes, however, perinatal transmission by infectedlymphocytes in breast milk also plays an important role.About 0.1% of HTLV-1-infected carriers develop ATL ataround 50 years of age. Patients have a median survivaltime of about I0 months.

In this study, sera from ATLL patients and age-matchedhealthy carriers were tested for the presence of anti-AAV-IgGs. As immunological controls, IgG titers against the(most prevalent) Epstein-Bar-virus EBNA1 antigen andwhole IgG titers were analyzed. The results revealedabout 80% anti-AAV-IgG positive sera in healthy carriers,whereas only 22 % of ATLL patients had serum antibodiesagainst AAV (p = 10-6). In patients lacking AAV-antibodies,there was no suppression of total IgG or EBNA1 antibodytiters. This indicates a specific correlation between AAVinfection and/or immune response and overt ATLL,suggesting that lack of AAV antibody prevalence might bea diagnostic marker for the development of ATLL.


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Alternatively, the data might point to a tumorsuppressiveactivity of AAV with regard to HTLV-1-induced malignancy,paralleling data of interaction of HPV and AAV.

The transcriptional profile in the parvovirus H1-sensitive liver cells

Huang Qingshan, Gu Meigang, Guo Lanping ,Li Yao, Xie Yi, Luo ZuYu.

School of Life Science, Fudan University , Shanghai, 200433,PR China.

To gain full understanding about antineoplasticmechanism of parvovirus, a complementary DNAmicroarray, representing 12800 different genes, was usedto compare the gene expression variance between a H1sensitive human liver cell line, L02, and its resistantderivative clone, L02R, which survived the infection of 10m.o.i. parvovirus H1. The prepared cDNAs from L02 andL02R were labled with fluorescent dyes, Cy5 and Cy3respectively, with which the mixed sample gave a red anda green image after hybridization. The two images weremerged into one for further analyses. The primitive datasuggest that the underlying mechanism of H1 sensitivityof L02 is involved with 164 up-regulated and 121 down-regulated genes, among which 81 are unknown ESTs. Itis interesting that a cluster of mitochondrion related genesare obviously up-regulated between 3.5-7.5 folds,including the cytochrome oxidase subunit and themitochondrial RNA encoding sequences which areprobably in relation with cell metabolism and a group ofcell skeleton related genes are greatly down-regulatedbetween 3.5-10 folds, such as fibronectin gene, keratinintermeiate filament gene and tropomyosins gene. Thesegives us a new view of H1 sensitive cells. Promisingly,further explorations of antineoplastic mechanism ofparvovirus will be directed by these microarray data andthe understanding of the ESTs. *This project wassupported by National Nature Science Foundation ofChina (39780027) and Shanghai Sciencefoundation(97ZA14005)

8. VIRAL MATURATION / APOPTOSIS

Efficient encapsidation of the AAV-2 genome ismediated by the DNA helicase activity of the AAV-2

Rep52 and Rep40 proteinsJason A King, Ralf Dubielzig,

Dirk Grimm, Jürgen A Kleinschmidt.Department of Tumour Virology at the German CancerResearch Centre, Im Neuenheimer Feld 242, D-69120

Heidelberg, Germany.

It has been previously reported that the small Rep proteins(Rep52 and Rep40) of AAV-2 are able to stabilize theaccumulation of single-stranded (ss) AAV-2 DNA(Chejanovsky & Carter 1989). We show that the increasedstabilization is mainly due to enhanced packaging of ss

genomes into protective capsids and that this is onlystimulated by the wild-type small Rep proteins and not byRep52/40 proteins containing a mutated helicase domain.The translation start site for the small Rep proteins wasmutated in pTAV2 (wild-type) to yield pTAV2-1. Virussupernatants generated by transfection of 293T cells witheither pTAV2 or pTAV2-1 and helper virus (Ad5) infectionwere analysed for infectious titer, capsid titer (ELISA) andDNA content of capsids. Although capsid titer and DNAreplication levels were very similar, the amount ofencapsidated DNA was significantly reduced for pTAV2-1, compared to pTAV2. Use of a capsid-specificimmunoprecipitation made possible the analysis of DNAmolecules associated with the capsids as well aspackaged within them. In the absence of small Reps(pTAV2-1) or the presence of small Rep proteinscontaining a mutated helicase domain (pTAV2-1 + Rep/52/40 mutants), full length ss genomes were found to bepresent on the capsid surface but not inside.Complementation of pTAV2-1 with wild-type Rep52/40resulted in a shift of full length genomes from “capsid-associated” to “encapsidated”, as represented by anincrease in the amount of DNase I protected full lengthgenomes. Having recently demonstrated that the smallRep proteins can be found in complexes with bothcapsids and large Rep proteins (Dubielzig et al. 1999),we propose that the small Rep proteins form part of apackaging complex between the capsid and large Rep-bound genome, most likely as multimers. With Rep52/40 immobilized in complexes on the capsid surface, thess genome is pushed through the complex into the emptycapsid. (Chejanovsky & Carter, Virology 1989;173:120-8;Dubielzig et al. J Virol 1999;73:8989-98).

Caspase-dependent apoptosis is required forpermissive infection of Aleutian mink disease

parvovirus (ADV) in vitroSonja M Best, James B Wolfinbarger, Marshall E Bloom.Lab. Persist. Viral Dis., NIAID, NIH, Rocky Mountain Labs.,

Hamilton, MT 59840 USA.

Apoptosis is important in viral pathogenesis, particularlyas a mechanism of immune evasion and viraldissemination. Apoptosis has been demonstrated in anumber of parvoviral infections, both in vitro and in vivo.We examined the role of apoptosis in permissive ADVinfection of synchronized Crandell Feline Kidney (CrFK)cells in vitro. By immunofluorescence, ADV-infected CrFKcells expressing NS-1 alone, or NS-1 plus capsid protein,but not capsid protein alone, stained positive for apoptosisby TUNEL reaction. This apoptosis appeared to be a directresult of virus replication because only virus-positive cellswere positive for TUNEL labeling and infection with UV-inactivated virus did not cause cell death. It is possiblethat TUNEL labeling may recognize free 3' ends of viralreplicative intermediates and label virus-infected cellsnot undergoing apoptosis. Hence, ADV-inducedapoptosis was confirmed by cell morphology andAnnexin-V staining. The level of apoptosis and theexpression of ADV proteins were greatly reduced by

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treatment of infected cells with broad spectrum andspecific caspase inhibitors, but not by inhibitors ofcaspase 1 or the c-myc protein. The titer of infectiousvirus produced from ADV-infected CrFK cells was reducedwhen cells were cultured with specific caspase inhibitors.These results suggested that ADV induces apoptosis ina permissive cell line by a caspase-dependentmechanism, and that apoptosis is required for permissiveADV infection in vitro.

The human adeno-asociated virus AAV type 2 (AAV2)increases cisplatin-induced apoptosis on HeLa cells:

role of the Viral capsid and the mitochondrialpotential

Valerie Duverger,1 Ute Sartorius,2 Petra Klein-Bauernschmitt,1

Peter H Krammer,2 Jörg R Schlehofer.11Tumorvirology Department, 2Immunogenetic Department,

German Cancer Research Center, 69120, Heidelberg,Germany.

The non-pathogenic human adeno-associated virus AAV-2 was shown to sensitize human cancer cells and tumorstowards the action of chemotherapeutic agents, such ascisplatin. Since chemotherapeutic drugs mainly involvethe induction of apoptosis, we investigated whether onepossible mechanism of AAV-mediated sensitization inHeLa cells results from an enhancement of cisplatin-induced apoptosis. Infection with AAV has no cytotoxiceffect by itself, but increases DNA fragmentation andPARP cleavage following exposure to cisplatin.Sensitization appears to relay on a reaction between acomponent of the viral capsid and the target cells, asempty or UV-inactivated AAV particles were still able toenhance cisplatin-induced DNA fragmentation. Theunderlying mechanism of AAV-mediated sensitization isnot associated with a modification in the expression ofCD95 ligand, CD95 receptor or other death receptors, asshown by RT-PCR and RNase protection assay. Incontrast, using a mitochondrial fluorescent dye JC-1 inflow cytometry, AAV infection further reduces themitochondrial transmembrane potential after treatmentwith cisplatin in a caspase-independent manner, thusdemonstrating that sensitization by AAV infection occursat the mitochondrial level. The results presented mayopen new perspectives for the use of AAV particles inimproving cancer therapy and also in constructing AAVvectors in gene therapy.

Adeno-Associated-Virus Type 2 Rep78 inducesapoptosis through caspase activation independently

of p53Michael Schmidt, Sandra Afione, Robert Kotin.

Laboratory of Molecular Hematology, National Heart, Lung, andBlood Institute, Bethesda, Maryland 20892, USA.

Adeno Associated Virus (AAV) Type 2 Rep78 is amultifunctional protein involved in the AAV DNA replication,integration and gene regulation. We have analyzed Repmediated cytotoxicity. We demonstrate that Rep78expression alone was sufficient to induce cell death and

disruption of the cell cycle. Cell death was mediated byapoptosis. Rep78 expression resulted in the activationof caspase 3, a terminal caspase directly involved in theexecution of cell death by proteolytically cleaving criticalcellular proteins. Among the targets of caspase-3 are thepoly(ADP-ribose) polymerase, lamins and actin. Rep78induced apoptosis was significantly inhibited by Z-DEVD-FMK, a caspase-3 inhibitor. Rep78 induced apoptosis inpluripotent human embryonal carcinoma NT-2 cells andalso in the p53-null promyelocytic human cell line HL-60,indicating that Rep78 triggers apoptosis by a p53-independent mechanism. Apoptosis was shown to bespecific for the G1 and early S phase of the cell cycle andwas associated with an accumulation of cells in G1.

9. CELL TARGETING/TROPISM IN GENE THERAPY

Mutational analysis of the AAV2 capsid gene andconstruction of vectors with altered tropismN Muzyczka, P Wu, W Xiao, T Conlon, J Hughes,

M Agbandje-McKenna, T Ferkol, T Flotte.Powell Gene Therapy Center, University of Florida, Gainesville,FL 32610; Department of Pediatrics, Washington University, St.

Louis, MO, USA.

To obtain a comprehensive genetic map of the AAV capsidgene, we have constructed 93 mutants at 59 differentpositions in the AAV capsid gene by site directedmutagenesis. Several types of mutants were studied,including epitope tag or ligand insertion mutants, alaninescanning mutants, and epitope substitution mutants.Analysis of these mutants revealed eight separatephenotypes. Infectious titers of the mutants revealed 4classes. Class 1 mutants were viable, class 2 werepartially defective, class 3 were temperature sensitive,and class 4 were noninfectious. Further analysisrevealed some of the defects in the class 2, 3 and 4mutants. Among the class 4 mutants, a subset ofmutations completely abolished capsid formation. Thesemutants were located predominantly, but not exclusively,in what are likely to be β-barrel structures in the capsidprotein VP3. Two of these mutants were insertions at theN and C termini of VP3, suggesting that both ends of VP3play a role that is important for capsid assembly orstability. Several class 2 and 3 mutants produced capsidsthat were unstable during purification of viral particles.One mutant, R432A, made only empty capsids,presumably due to a defect in packaging viral DNA.Additionally, five mutants were identified that weredefective in heparin binding, a step that is believed to beessential for viral entry. These were distributed into twoamino acid clusters in what is likely to be a cell surfaceloop in the capsid protein VP3. The first cluster spannedamino acids 509-522; the second was between aminoacids 561 and 591. In addition to the heparin bindingclusters, hemaglutinin epitope tag insertions identifiedseveral other regions that were on the surface of thecapsid. These included insertions at amino acids 1, 34,


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138, 266, 447, 591, and 664. Position 1 and 138 were theN termini of VP1 and VP2, respectively; position 34 wasexclusively in VP1; the remaining surface positions werelocated in putative loop regions of VP3. The remainingmutants, most of them partially defective, werepresumably defective in steps of viral entry that were nottested in the preliminary screening, including intracellulartrafficking, viral uncoating, or coreceptor binding. Finally,in vitro experiments showed that insertion of the serpinreceptor ligand in the N-terminal regions of VP1 or VP2can change the tropism of AAV. Our results provideinformation on AAV capsid functional domains and areuseful for future design of AAV vectors for targeting ofspecific tissues.

The identification of a non-heparin sulfate bindingadeno-associated virus by insertional mutagenesis

of the capsidJoseph E Rabinowitz, Lisa J Hanson, R Jude Samulski.Gene Therapy Center The University of North Carolina atChapel Hill, Chapel Hill, North Carolina 27599-7352, USA.

Linker insertion mutagenesis of the capsid codingdomain of AAV2 has been used to generate a number ofgenetically altered virions that contain 4-6 amino acid(AA) insertions that do not infect 293 or HeLa cells. Theability to package the recombinant genomes, asdetermined by dot blot hybridization, but not infect suggeststhat these insertions interfere with a step in the viralinfection pathway. The blocked steps may include binding,internalization, endosome escape, translocation to thenucleus, and or uncoating. These mutants have beenscreened by batch and column binding to heparinagarose. One of these mutants AAV2ins520 did not bindheparin agarose by either method. However, this virusappeared normal by electron microscopy, and containedthe three capsid subunits at the correct ratio asdetermined by Western blot. This virus represents thefirst description of a primary receptor binding mutant ofAAV2. Characterization of cell binding for this mutant by3H-thymidine labelling established that AAV2ins520 didnot bind HeLa cells in culture. Competition studiesdetermined that AAV2ins520 at a particle number of 105/cell did not substantially inhibit wild type virion infection ata particle number of 50/cell. To determine if the insertionof AAs’ ASIP at position 520 was a dominant mutation,helper plasmids containing either the coding sequencefor the wild type capsid (pACG2) or the insertion mutantwere co-transfected at different ratios into 293 cells. Theseexperiments revealed that between 75% wt/25% mutantand 50% wt/mutant there was a dramatic decrease in theresulting viruses ability to infect cells. This suggests thatthe AAV2 virion requires a significant number of heparinbinding epitopes on capsid subunits to efficiently bindcells. In an attempt to determine if the mutant AAV2ins520lost the ability to bind heparin as a result of a localinteraction, or a long range interaction deletion mutantswere developed that flanked the site of the insertion. Theresults of these experiments have helped localized theheparin binding epitope of AAV2.

Gene Array Analysis of Adeno-associated virusInfection in normal diploid cells

JL Stilwell,1,2 RJ Samulski.1,2

1Curriculum in Genetics and Molecular Biology; 2Gene TherapyCenter, University of North Carolina, Chapel Hill, NC 27599,

USA.

Adeno-associated virus type 2 (AAV) is a nonpathogenicmammalian virus with a single-stranded DNA genomeof 4,680 nucleotides. AAV has demonstrated anti-proliferative and anti-tumor properties with a number ofstudies implicating AAV gene expression as the cause.Similar effects upon infected cells have been observedwith recombinant AAV (rAAV) devoid of viral genes,suggesting an alternative mechanism involving cellularresponses to viral infection may be involved (Kube et al,J Virol 1997;71:7361-7371). A number of laboratories,including ours, have been working to understand themolecular mechanisms underlying these observationsmore thoroughly. Previous studies have only been ableto monitor a handful of genes at a time due to limitationsin available technologies and the majority of these studieswere carried out in immortalized cell lines. The recentintroduction of high-density microarrays has provided theability to monitor gene expression for large numbers ofgenes simultaneously. We have carried out a series ofexperiments using such large scale monitoring with high-density microarrays and normal diploid cells. Filter-basedgene arrays have allowed us to identify several mRNAsthat fluctuate during infection with wtAAV as compared tomRNA levels in mock-infected cells. Six of the genesidentified are related to cell cycle control, includingp21WAF, and two of the six have never been describedas being modulated upon infection with AAV. Somechanges in expression are observed as early as 4 hoursafter infection suggesting that viral gene expression maynot be responsible for this effect. These studies havebeen extended to GeneChip™ arrays, which allowmonitoring of even greater number of genes than thefilter-based arrays. Comparison of these analysis andimportance of the genes that are modulated after AAVinfection in relation to the cell cycle will be discussed.

Quick Method for Engineering and Screening EpitopeInsertion mutants in Adeno-Associated Virus Type 2

Virions for Surface DisplayLisa J Hanson, Joseph E Rabinowitz, R Jude Samulski.

The University of North Carolina at Chapel Hill, Gene TherapyCenter, Chapel Hill, North Carolina 27599-7352, USA.

Recombinant adeno-associated virus serotype 2 (rAAV2)is a small, nonpathogenic parvovirus that infects a broadrange of cell types. Although rAAV has many naturalproperties that make it useful as a gene therapy vector.However, for effective treatment in some disorders,specific cell types may need to be targeted. Identificationof the AAV2 receptor and co-receptors has provided anopportunity to develop targeting vectors with an alteredtropism. Previously, in our lab, a collection of AAV2insertion mutants, containing a unique restriction site in

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the capsid coding sequence, have shown that AAV virionscan tolerate amino acid inserts without disrupting capsidformation, packaging, and in some cases infectivity.Furthermore, these mutants validate the concept ofexpressing foreign epitopes on the surface of AAV2 virions.To investigate the insertion mutants further, specificepitopes generated from oligo sets have been clonedinto all three reading frames. Successful cloning eventsof the epitopes proved rare, in some cases and a large-scale screening process employing PCR was developed.PCR has facilitated the ability to identify positive clonesand begin virus production within 24 hr. Epitope insertionsinclude heparin sulfate, his tags, RGD, poly lysine, andbradykinin motifs. We have used Bradykinin mutantviruses in an ELISA to determine surface expression ofthe epitope on intact AAV2 virions. By incorporating PCRand ELISA, we can rapidly determine surface display ofthese foreign epitopes. Details of this approach will bediscussed.

AAV-mediated transduction of murine hematopoieticprogenitor cells in vitro and in vivo

MQ Tan, KY Qing, J Hansen, MC Yoder, A Srivastava.Indiana University School of Medicine, Walther Oncology

Center, Walther Cancer Institute, Wells Center for PediatricResearch, Indianapolis, IN, USA.

Although adeno-associated virus 2 (AAV) has gainedattention as a potentially useful alternative to the morecommonly used retrovirus- and adenovirus-basedvectors for human gene therapy, we have accumulatedevidence to suggest that efficient gene transfer andtransgene expression by AAV vectors require that thefollowing two obstacles be overcome. First, the targetcell must express the receptor and the co-receptor forAAV infection (J. Gen. Virol., 77: 1111-1122, 1996; NatureMed., 5: 71-77, 1999), and second, the cell must allow forviral second-strand DNA synthesis (Proc. Natl. Acad. Sci.USA, 94: 10879-10884, 1997; J. Virol., 72: 1593-1599,1998; J. Virol., 72: 9835-9843, 1998). We have alsoidentified a third obstacle that the cell must allow forefficient intracellular trafficking of AAV to the nucleus (J.Virol., 74: 992-996, 2000). Since those studies werecarried out with established cell lines, we examinedprimary murine hematopoietic cells to evaluate whetherimpaired intracellular trafficking of AAV vectors mightaccount for the observed low-efficiency of transduction ofthese cells. Purified murine Sca1+, lin- hematopoieticcells from C57Bl6 donor mice, with and without treatmentwith hydroxyurea (HU) [1 mg/g/mouse; intra-peritoneal(i.p.); 24 hrs prior to harvesting bone marrow cells], wereeither mock-infected or infected with a recombinant AAVvector containing the CMV promoter-driven enhancedgreen fluorescent protein (EGFP) reporter gene andtransplanted into lethally-irradiated congenic recipientmice. Twelve days post-transplantation, spleen cellcolonies (CFU-S) were enumerated and analyzed for thepresence of the transduced gene in single cell colonies.Both low and high Mr DNA samples were isolated fromthree CFU-S colonies each and analyzed on Southern

blots using EGFP DNA as a probe. A significanthybridization of the probe to the low Mr DNA was detectedin each of the colonies derived from vector-transducedcells. Since linear, monomeric duplex forms of the viralgenomes were not readily detectable, even in cellsobtained from animals with prior treatment with HU, inthe next set of experiments, CFU-S colonies from eachrecipient animal were pooled individually and nuclear andcytoplasmic fractions were prepared. Low Mr DNAsamples isolated from these fractions were analyzed onSouthern blots. A significant increase in the hybridizationintensity to the probe in the nuclear fraction as well asaugmentation in monomeric duplex forms of the viralgenome observed in cells in CFU-S colonies derived fromcells obtained from HU-treated animals suggested thatHU-treatment increases the efficiency of AAV traffickinginto the nucleus. This hypothesis was tested by usingNIH3T3 cells known to be impaired in efficient intracellulartrafficking of AAV. Cells were either mock-treated or treatedwith HU alone, tyrphostin 1 (Tyr1; known to promote theviral second-strand DNA synthesis) alone, or HU+Tyr1,followed by AAV-mediated transgene expressionanalyses. HU-treatment led to ~11-fold increase in thetransgene expression whereas Tyr1-treatment caused<1-fold increase. HU+Tyr1-treatment led to ~20-foldincrease. Southern blot analyses of nuclear andcytoplasmic fractions obtained following these treatmentsfurther corroborated the conclusion that HU-treatmentleads to a significant increase in intracellular traffickingof AAV vectors.

Transduction of motor neurons with adeno-associated virus (AAV) vectors requires the inclusion

of the cis-acting Woodchuck PosttranscriptionalRegulatory Element (WPRE)

Olivier ter Brake,1 Wim TJMC Hermens,1 Ewoud Moraal,1

Thomas J Hope,2 Joost Verhaagen.1

1Graduate School for Neurosciences. Netherlands Institute forBrain Research, Amsterdam; 2Infectious Disease Laboratory,

The Salk Institute for Biological Studies, La Jolla, CA, USA.

Among a number of neuronal cell types present in thebrain and spinal cord, motor neurons can be severelyaffected during the cause of many neurological diseasesor following trauma. Therefore motor neurons form animportant target for the delivery of therapeutic genes bymeans of AAV vectors.

We observed that in contrast to other neurons expressionof the transgene in motor neurons could not be detectedfollowing the injection of purified high titer AAV vectors.This observation suggested the incompetence of the AAVvector to enter motor neurons. Suprisingly however, wefound that injection of AAV vectors containing theWoodchuck Posttranscriptional Regulatory Element(WPRE) in the transgene expression construct resultedin abundant transduction of motor neurons and enhancedexpression of the transgene in other neurons. The WPREis a cis-acting element derived from Woodchuck Hepatitisvirus and it has been shown to enhance the translocation


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of intronless viral messenges from nucleus to cytoplasmindependent of the splicing machinery of the cell (Donelloet al., 1998, J. Virol. 72: 5085-5092). Since the AAV vectorconstructs that did not transduce the motor neuronscontain a SV40 intron sequence and therefore requiresplicing before transgene expression can occur wehypothesized that the failure to observe transduction isbased on an aberant splicing mechanism in motorneurons compared to other neuronal cell populations inthe brain and spinal cord.

In order to test this hypothesis localization of thetransgene message was studied by ISH experiments onrat brain and spinal cord injected with AAV vectorsharboring a SV40 intron sequence. Preliminary resultsshowed the presence of the transgene mRNA in thenucleus of motor neurons and the absence of the mRNAin the cytoplasm following injection of the SV40 intron-containing AAV vector. In contrast the transgene mRNA inneurons surrounding the motor neurons was localizedin the cytoplasm. Further experiment are currentlyperformed and will be presented.

We conclude that the WPRE is required in the AAV vectorconstruct to transduce motor neurons in the brain andspinal cord. In addition, inclusion of the WPRE in thevector construct enhances transgene expression inneurons in vivo in general.

rAAV transduction of quiescent CD34+++CD38-human hematopoietic progenitor cells residing in the

G0 phase of the cell cycleChristie Ann Wong, Wei Li, Stephen.J Forman,

KK Wong Jr, Saswati Chatterjee.Departments of Virology and Hematology/Bone Marrow

Transplantation. City of Hope National Medical Center, Duarte,CA, USA.

Genetic modification of hematopoietic stem cells (HSCs)is a promising approach for the treatment of congenitaland acquired human diseases. However, despiteintensive efforts, stable gene transfer to HSCs, which arebelieved to reside in a quiescent, mitotically dormantstate, has been difficult to achieve. In this study weevaluated the potentials of an rAAV vector to transduceand stably integrate in a homogeneous subpopulation ofmarrow derived, dormant CD34 cells residing in the G0phase of the cell cycle. Freshly isolated CD34+++ cellswere sorted by flow cytometry into specific subpopulationsbased upon (1) the CD38 expression or (2) cell cyclestatus as determined after live staining with Hoechst33342 (for DNA) and pyronin Y (for RNA). Cell cycleanalyses revealed that the majority (>96%) ofCD34+++CD38- cells also resided in G0, whileCD34+++CD38+ cells were primarily found to be in SG2M.Conversely, for CD34 cells initially isolated on the basisof their cell cycle status, those in G0 (2n DNA/low RNApopulation) were primarily CD38-, while cells in G1 wereprimarily CD38+. Membrane staining with PKH26showed that the vast majority, approximately >96% and80% of CD34+++CD38- cells remained non_divided on

days 2 and 7, respectively. The CD38- and G0subpopulations of CD34+++ cells were transducedovernight with a cesium chloride purified rAAV vectorencoding antisense RNA complementary to the HIV LTRat a particle MOI:1500 (infectious MOI:15). Gene transferfrequencies in primitive clonogenic cells were measuredby amplification of vector sequences from individualhematopoietic colonies derived from long term cultureinitiating cells (LTC-IC). Both the CD38- and the G0subpopulations of CD34+++ cells were highly enrichedfor extended clonogenic activity. In the absence ofselective pressure, 10-83% of colonies arising fromextended week 8 and 12 LTC-ICs from theCD34+++CD38- and 11-38% of LTC-ICs arising fromG0CD34 cells were found to contain vector-specificsignals. Metaphase FISH analyses revealed that 8-16%of CD34+++CD38—cells and 9-30% of the cells in G0 atthe time of transduction contained chromosome-associated integrated vector-specific signals. Sincestudies of cell division kinetics showed that >96% of cellshad remained non-divided when the cells were washedfollowing exposure to vector, these results suggest thatrAAV vectors are capable of transducing quiescentprimitive progenitor cells. Sequential analysis showedthat the integration levels remained comparable over theten week period of study, demonstrating stability ofintegration. Further analysis revealed that in addition tointegrated vector sequences, episomal vector genomeswere also detectable immediately after transduction.However these forms declined rapidly with cell division.Our results suggest that AAV vectors are capable of stabletransduction and integration in a discrete population ofprimitive CD34+++CD38- cells residing in G0 which havethus far been difficult to genetically modify.

Preparation of functional recombinant AAV-2 viruswith radioactive capsids to study vector tropism in

vivoE Lehtonen,1,2 F Lachapelle,3 F Jurysta,1 A Stathopoulos,1,2 ABaron-Van Evercooren,3 T Velu,2 M Levivier,1 L Tenenbaum.1,2

1Lab. Experimental Neurosurgery; 2IRIBHN, ULB-HôpitalErasme, Belgium; 3INSERM CJF 97/11 Myelin Pathologies, CHU

Pitié-Salpetrière, Paris.

We have previously observed that delivery of recombinantAAV-2 virus (rAAV) in the posterior part of the rat striatumresults in low and spatially limited local transduction butefficient and widespread transduction at distance fromthe injection site, in the globus pallidus (1). In order tocompare the spatial distribution of viral particles andtransduced cells, radioactive rAAV encoding greenfluorescent protein was prepared. A viral suspensiondevoid of contaminant wild-type AAV and adenovirus wasobtained by co-transfection of vector plasmid and a helperplasmid supplying adenoviral and AAV functionsnecessary for replication and encapsidation of the vector(2) in the presence of 35S-methionine. The virus waspurified using the iodixanol/heparin method (3). Thetransducing titer of the labelled virus was equivalent tothose routinely obtained with the same method. LabelledVP proteins were evidenced both by Western blotting and

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by autoradiography of polyacrilamide gels. Radioactivecapsids were evidenced by autoradiography of brainsections at different time points after infusion of the viralpreparation in the brain. The intensity of the signal wasmaximal at the delivery site and decreased with distance,suggesting that the observed preferential transduction ofthe globus pallidus does not reflect a preferential uptakeof the virus by neurons of this region. 35S labelling ofcapsids will be a useful tool to study the limiting steps inrAAV-mediated gene transfer in vivo. (1. Tenenbaum et al.(2000) NeuroReport 11:1-7; 2. Grimm et al. (1998) GeneTher 9:2745-2760; 3. Zolotukhin et al. (1999) Gene Ther6:973-985).

Building a chimeric shell: domain swapping betweenAAV serotypes

Joseph E Rabinowitz, Dawn Bowles, R Jude Samulski. Gene Therapy Center the University of North Carolina atChapel Hill, Chapel Hill, North Carolina 27599-7352, USA.

The primate adeno-associated virus serotypes AAV1-AAV6 represent a group of viruses some of which arequite unique from each other AAV4 and AAV5 while othersare more closely related AAV1, 2, 3 and 6. Up to 80% ofthe human population maybe seropositive for AAV2. In aneffort to design vectors suitable for re-administration itwill be important to determine which domains of theviruses are responsible for immunogenicity of theserotypes. So large domains were exchanged betweenserotypes. The capsid gene of AAV2, in the helper vectorpACG2, was digested with the enzymes Asp718 and BsiWI. These restriction enzymes provided a uniqueapproach of swapping loop domains between serotypes.A 904bp AAV2 fragment that contains the coding region ofVP3’s loop 2, 3, 4 domains was subsituted for AAV 3. Thecapsid gene of AAV3 was digested with the sameenzymes and a 907bp fragment was isolated and cloned.The resulting virions bind heparin agarose, infect HeLaand 293 cells, and are recognized by the B1 monoclonalantibody. The capsid gene of AAV4 was digested with thesame enzymes and a 928 bp fragment and similar loopsequences were swapped into AAV-2. This virus did notinfect HeLa cells. However, like AAV4 this virus infectedCOS7 cells at a low titer of 1 x 105 transducing units/mL.The VP subunits are not recognized by the AAV2monoclonal antibody B1. These results have suggestthat smaller domain swaps will reveal more exactinformation regarding the properties of the serotypesrequired for infection. Generation of a collection of theseswapped capsid domains is a first step in generatingaltered capsid that may contain unique infectivity andimmunogenicity.

Adeno-associated virus transduced GM-CSFsecreted from recombinant skin

PL Hermonat, Y Liu, M Mane.Department of Obstetrics and Gynecology, University ofArkansas for Medical Sciences, Little Rock, AR, USA.

Recently several groups have found that adeno-associated virus (AAV) is able infect the squamousepithelial tissues of the genital tract. This suggested thatAAV might be a good vector for skin gene therapy. In thisstudy we show that keratinocytes infected with an AAV/GM- CSF/Neo vector, are able to form a GM-CSF secretingskin using the organotypic epithelial raft culture system.When the rAAV virus stock was without wild type AAV themaximum production of GM-CSF was about 80 pg of GM-CSF per cm2 of skin over a 48 hour period, days 2-3 post-infection. However, when the rAAV virus stock containedwild type AAV the production of GM-CSF was much higherat about 130 pg per cm2 of skin. As GM-CSF has a halflife of 8 hours the actual secretion levels are likely muchhigher those measured (>400 pgs over 48 hours/cm2).These data suggest that AAV is appropriate for geneticallyaltering skin to secrete new proteins. Furthermore, theenhancement of activity by wild type AAV suggests thatsome form of complementation is taking place.

10. HOST IMMUNITY TO PARVOVIRUSES

Identification of capsid protein sequences ofAleutian Mink Disease parvovirus (ADV) that mediate

virus neutralization and antibody dependentenhancement of infection

Marshall E Bloom, Mavis Agbandje-McKenna, Stanley FHayes, Sonja Best, James B Wolfinbarger.

Laboratory for Persistent Viral Diseases, NIAID, NIH, RockyMountain Labs, Hamilton, MT 59840, USA.

Antiviral antibodies facilitate the immunopathogenesisof persistent ADV infections by mediating antibodydependent enhancement of infection (ADE) in K562 cells;however, antibody can effectively neutralize ADV in CrFKcell cultures. We studied this conundrum using polyclonaland monoclonal antibodies directed against syntheticADV VP2 capsid protein peptides. Antibodies against thepeptide VP2:428-446 both neutralized ADV infectivity forCrFK cells and also mediated ADE of K562 cells, as didanti-capsid antibodies. By immunoelectron microscopy(IEM), these same antibodies both decorated andaggregated capsids. In contrast, antibodies againstVP2:487-501 mediated ADE, but did not neutralizeinfectivity for CrFK cells; these antibodies decoratedcapsids to a lesser extent and failed to aggregate by IEM.Antibodies against VP2:455-470 failed to score in any ofthese assays. Modeling of these peptide sequences onthe cryo-EM structure of ADV suggested that capsiddeterminants of both neutralization and ADE mightoverlap and reside on the ridge between the canyonsurrounding the 5-fold axis and the 2-fold dimple.Furthermore, additional ADE determinants may belocated on the wall of the valley formed by protrusionsnear the 3-fold axis. Specific interactions between ADVand antibodies may be important in the unusualpathogenesis of ADV when compared to otherparvoviruses.


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Cellular Immune Response within the Placenta toHuman Parvovirus B19 Infection

JA Jordan. Magee-Womens Research Institute and the Department of

Pathology, University of Pittsburgh, Pittsburgh, PA 15213, USA.

Human parvovirus B19 can cause congenital infectionswith variable morbidity and mortality. While the role of thehumoral immune response to B19 infection has beenwell characterized, the role of the cellular immuneresponse is unclear. To begin to understand why variationexists in fetal outcomes in women who seroconvert toB19 during their pregnancies, we analyzed placentasfrom 4 groups of women for their immune cell content.Group 1 consisted of B19 IgM+ women who deliveredhealthy term infants. Group 2 included B19 IgM+ womenwhose pregnancies ended with poor outcomes;spontaneous abortion, fetal hydrops or fetal death. Group3 was comprised of pregnancies with hydropic fetusesdue to known, non-infectious etiologies, while Group 4contained women who delivered normal term infants.Tissue sections from each placenta were examined forthe types of immune cells present and for theinflammatory cytokine IL-2 using immunohistochemistry(IHC). B19 was detected in these same tissues usingPCR, DNA in situ and IHC. Maternal sera were analyzedfor B19 IgM and IgG using EIAs and for B19 DNA usingPCR. The results of this study revealed a statisticallysignificant increase in the number of CD3+ T cells foundwithin placental tissue sections from B19 infected casesfrom Groups 1 and 2 compared to placentas from Groups3 and 4, p<0.0001 (See Table). Additionally, positive IHCstaining for IL-2 was observed in every placenta examinedfrom Groups 1 and 2, while being entirely absent inplacentas from Groups 3 and 4. The location of B19, Tcells and IL-2 in Group 1 and 2 differed, with the formergroup having virus, T cells and IL-2 primarily on thematernal side of the placenta, while the latter had thesethings present mainly within fetal blood vessels withinthe placental villi. It is apparent from these results that aheightened cellular immune response existed on the fetalside of the placentas in those women with poor fetaloutcome as compared to those with good outcome. Wepostulate that a strong cellular immune response on thematernal side of the placenta may act to inhibit viraltransmission to the fetus.

Table: Comparison of CD3 and IL-2 IHC, and B19 DNAin situ staining in placental tissue sections.

Group Avg CD3+ Range CD3+ IL-2 /B19 location

1 (n=7) 7 2-8 Yes/maternal 2 (n=18) 10 2-20 Yes/fetal side 3 (n=20) 2 0-4 No 4 (n=8) 1 0-2 No

n; number of placentas, Avg CD3+; average number ofCD3+ T cells found in 10 random fields at 200X, RangeCD3+; range of the number of CD3+ T cells found in 10random fields at 200X.

Chronic human parvovirus B19 infection inrheumatic disease of childhood and adolescence

HW Lehmann, L Kühner, B Kochanowski,*KG Heide, RM Küster, Modrow S.*

Department of Pediatric Rheumatology, Rheumaklinik BadBramstedt, Postfach 1448, 24572 Bad Bramstedt, *Institut für Medizinische Mikrobiologie,Universität

Regensburg, Regensburg, Germany.

To find further evidence of parvovirus persistence inrheumatic diseases of childhood characterised byantibodies against the non-structural parvovirus proteinNS1 and to investigate the clinical course of the diseases,an extended study was performed. Methods: 48 childrenand adolescents (22 males, 26 females, all Caucasians)with joint complaints lasting longer than one year eitherfrom active arthritis (oligoarticular 24, polyarticular 18,systemic 1), arthralgias (3), juvenile systemic sclerosis(1), or juvenile dermatomyositis (1) were included in thestudy. Mean disease duration was 46 months. In nopatient antibodies to coxsackievirus, Epstein-Barr virus,ECHO-virus, borrelia, yersinia, campylobacter, chlamydia,salmonella, and streptolysin O were detectable and nonefulfilled the criteria of psoriatic arthritis. Laboratory markersof inflammation, specific IgM and IgG antibodies againstdifferent proteins of parvovirus B19 and detection of B19-genomes by PCR were investigated. The quantity ofarthritis and impaired joint function was compared topatients initial presentation. Disease relatedcomplications were recorded. Impairment of activities ofdaily living was assessed by the Childhood HealthAssessment Questionnaire (CHAQ), the Child HealthQuestionnaire (CHQ-PF50) and the KINDL test. Results:Nearly half of the patients showed laboratory signs ofchronic inflammation. 12 (25%) were ANA positive at theirinitial presentation. During follow up 4 patients becamenegative. One adolescent, suffering from systemicsclerosis, was ENA positive including Scl-70. In 45 seraIgM antibodies against parvoviral proteins and in 5 virusspecific DNA were detectable. The number of patientswith arthritical joints decreased from 44 at patients initialpresentation to 11 at the time of the study. Limited jointmotion without any further evidence of active arthritis wasfound in 16 individuals. 4 patients worsened, 27 improved,the others remained stable. 24 children were restrictedin their daily activities. The mean disability index was 0.21.All of the children suffered from pain, and nearly allfrequently used crutches. No patients developed uveitis.Hashimoto thyroiditis was seen in one. Conclusion:Parvovirus viremia usually reached a peak at days 8-9after infection and resolved after the development of anantibody response starting at day 10. The high humoralimmune response in our patients clearly indicates thatpersistent B19 parvovirus infection is not the result offailure to produce effective neutralising antibodies. Thedetection of viral DNA in sera of 5 patients years afterinfection, the presence of IgM antibodies against thestructural proteins in almost all patients and the presenceof IgG antibodies against the non-structural viral proteinNS1 indicates viral persistence. Rheumatic disease ofchildhood with persisting parvoviral infection in most

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instances takes a benign course unless its starts as alife threatening disorder. Childhood should be anunmolested life period. The occurrence of juvenilesystemic sclerosis, juvenile dermatomyositis andHashimoto thyroiditis, which are all rarely seen inchildren, in a relatively small series of patients and therestriction of half of the kids in their daily activitiesdemonstrated the substantial power of a persisting viralinfection to impair children’s health.

Differential IgM response to conformational andlinear epitopes of parvovirus B19 VP1 and VP2

structural proteins Elisabetta Manaresi, Elisa Zuffi, Giorgio Gallinella,

Giovanna Gentilomi, Marialuisa Zerbini, Monica Musiani. Department of Clinical and Experimental Medicine, Division of

Microbiology. University of Bologna. Via Massarenti 9.Bologna. Italy.

The immune response against B19 is mainly directedagainst the two viral capsid proteins VP1 and VP2. TheIgM immune response against conformational and linearepitopes of B19 structural proteins VP1 and VP2 wasexamined in serum samples of 189 subjects with asuspect B19 infection in order to analyse a possibletemporal relationship between the course of B19 infectionand the presence of epitope type-specific IgM and also toevaluate the the most suitable antigen to perform IgMdetection. The detection of IgM against conformationalepitopes was performed by two ELISA assays usingundenatured VP1 and VP2 recombinant antigensexpressed in baculovirus system, while the detection ofIgM against linear epitopes was performed by Westernblot assays using denatured VP1 and VP2 recombinantproteins expressed in E.coli.

IgM immune response against VP1 conformationalepitopes appeared dominant, being detected in the totalityof serum samples positive for specific IgM, while IgMagainst VP2 linear antigen were not very frequently found,being identified in less than half of the B19 IgM positivesera. In the analysis of B19 course of infection, in theactive phase of B19 infection (characterized by thepresence of B19 DNA, presence of any epitope-typespecificIgM with or without specific IgG), IgM against VP1conformational epitopes appeared in concomitance andwith the same frequency as IgM anti VP2 conformationalepitopes and IgM anti linear VP1 epitopes. IgM againstVP1 conformational epitopes were seen to be long lastingsince in the recent phase of infection (characterized bythe absence of B19 DNA, presence of specific IgM andIgG) they were still present when other specific IgM wereabsent. During the active phase of B19 infection, IgMagainst VP2 linear epitopes were less frequently foundthan other specific IgM and in the recent phase theyunderwent a rapid temporal diminution.

Our data demonstrate that a differential IgM response toconformational and linear epitopes of B19 VP1 and VP2takes place during the course of B19 infection moreover

our data demonstrate that a sensitive B19 IgM detectionneeds to be performed in diagnostic laboratories by ELISAassays using conformational B19 antigens and thatWestern blot assays can be used only as confirmatorytests using VP1 linear antigens.

Productive infection occurs in early pregnancy insimian parvovirus-inoculated fetuses from

seropositive mothersMG O’Sullivan, DA Feeney, AL Aber, JB Jarvis.

University of Minnesota College of Veterinary Medicine, St.Paul, MN, USA.

The cynomolgus monkey fetus infected with simianparvovirus is proving useful as an animal model forparvovirus B19 fetal infection, and studies have hithertobeen performed in fetuses whose mothers were SPV-seronegative. We examined the feasibility of infectingfetuses from SPV-seropositive females as this wouldfacilitate such studies. Our working hypothesis was thatinfection should occur in such fetuses, because(protective) antibody transfer from mother to fetus normallyoccurs late in gestation. Six cynomolgus monkey fetusesfrom SPV-seropositive mothers were inoculated withsimian parvovirus (SPV) prior to (gestation day [GD] ~ 59-62, n=2), or following (GD ~75-81, n=4) the developmentof immunocompetence at ~GD 70 (full-term is ~GD 165),respectively. SPV viremia, SPV-specific antibodies andhematological parameters were monitored in the fetuses,which were collected by Cesarean section on day 30 postinoculation. Infection was observed in both fetusesinoculated prior to immunocompetence, as detected bythe presence of viremia, a fetal antibody response andpresence of SPV DNA in several tissues. However, incontrast to our studies on fetuses from seronegativemothers, no evidence of clinical disease was present.Fetuses inoculated after onset of immunocompetencedid not become infected. The mechanisms responsiblefor resistance to infection later in gestation are unclearas antibody to SPV was not detected in these fetuses.

Direct ex vivo measurement of Cytotoxic TLymphocyte responses to human Parvovirus B19

T Tolfvenstam,1 A Oxenius,2 DA Price,2 BL Shacklett,3 HMLSpiegel,3 K Hedman,4 O Norbeck,1 M Levi,5 K Olsen,2 M

Kantzanou,2 DF Nixon,3 K Broliden,1 P Klenerman.2

1Department of Clinical Virology, Karolinska Institute, HuddingeUniversity Hospital, SE-141 86 Stockholm, Sweden; 2NuffieldDepartment of Medicine, John Radcliffe Hospital, Oxford, UK;

3Aaron Diamond AIDS Research Center, The RockefellerUniversity, New York, USA; 4Department of Virology, HaartmanInstitute, University of Helsiki, Helsinki, Finland; 5Department of

Virology, Swedish Institute of Infectious Disease Control,Stockholm, Sweden.

Parvovirus B19 (B19) is a common human pathogen,which usually causes a self-limiting illness (erythemainfectiousum or fifth disease). More severe syndromesinclude aplastic anemia in those with a rapid red cellturnover or in the immunosuppressed, fetal hydrops and


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loss, and arthralgia syndromes, which can progress tochronicity. Immunity to B19 has been generally regardedas mediated predominantly by antibody, and no previouscytotoxic T lymphocyte (CTL) responses have beenreported. We describe the mapping of the first parvovirusB19 derived CTL epitope. This HLA-B35 restricted epitope,(amino acid sequence “QPTRVDQKM”, positioned atamino acid number 391-9 of the B19 non structuralprotein), is commonly recognized by healthy B19seropositive, with CTL present at levels of up to 1/300CD8+ lymphocytes (i.e. at least as large as populationsof epstain barr virus-specific cells) as judged by ex vivotetramer and ELISpot staining. CD8+ T lymphocytemediated responses against parvovirus B19 are readilydetectable and may potentially play a role in immunityand immunopathology.

Serological survey of cynomolgus monkeys forsimian parvovirus

MG O’Sullivan, MP Murtaugh, AL Aber, TB Krueger,IH Suparto, D Sajuthi.

University of Minnesota College of Veterinary Medicine, St.Paul, MN, and Indonesian Primate Research Center, Institut

Pertanian Bogor, Bogor, Indonesia.

The purpose of this study was to determine theseroprevalence of simian parvovirus in differentcynomolgus monkey populations so as to assist inidentifying sources of seronegative monkeys, and to betterunderstand transmission patterns. Our workinghypothesis was that simian parvovirus would resemblehuman parvovirus B19 in these regards. IgG titers tosimian parvovirus were examined with a newly-developedELISA that utilizes the unique region of the SPV VP1 capsidprotein as capture antigen. Three diverse populationswere examined, i.e., middle-aged (>10-year old) wild-caught adult females imported ~ 4 months previously(n=55) from Indonesia, captive wild-caught monkeys atthe Indonesian Primate Center less than 2 years old(n=32) or greater than 5 years old (n=31), and Indonesianwild monkeys (n=36). The seroprevalence of SPV was6% in young captive animals less than 2 years old, butwas 71% in captive animals greater than 5 years old. Incontrast, the seroprevalence in wild monkeys was 36%,and was 45% in wild-caught monkeys recently importedfrom Indonesia. The seroprevalence pattern in the captivecolony monkeys resembles that for human parvovirusB19, in that seroprevalence increases markedly both inolder monkeys and in humans, respectively.Seroprevalence in wild monkeys is somewhat lower,possibly because of their free ranging activities and adecrease in close contact.

Serum cytokine responses in parvovirus B19-associated arthropathy: a Th1 or Th2 profile

William C Koch,1 Brian Barnstein,1 Frank T Saulsbury.2

1Pediatrics, Medical College of Virginia/Virginia CommonwealthUniversity, Richmond, VA, USA; 2Pediatrics, University ofVirginia Health Sciences Center, Charlottesville, VA, USA.

Objectives: To determine serum cytokine levels in patientswith parvovirus B19-associated arthropathy, characterizethem as predominantly Th1 or Th2 in nature, and comparethem to children with JRA. Background: Infection withparvovirus B19 (B19) causes erythema infectiosum (EI)and is an important cause of transient arthritis.Adolescents and adults who are infected with B19 maydevelop a chronic arthropathy which resembles juvenilerheumatoid arthritis (JRA) or rheumatoid arthritis (RA).Recent studies have examined the role of cytokines asmediators of joint inflammation and joint destruction inpatients with JRA and RA, but there is very little informationconcerning cytokine responses in patients with B19arthropathy. Methods: We measured levels of interleukin-2 (Il-2), Il-4, Il-6, Il-8, interferon-((IFN) and tumor necrosisfactor-α (TNF-α) by ELISA in stored serum from severalpatient groups: 1) 8 adults with B19 arthropathy. All hadrecent contact with a child with clinical EI, all were B19IgM-positive, and all had active arthritis involving >4 jointsat the time of sampling. 2) 5 children with systemic-onsetJRA. 3) 8 children with polyarticular JRA. 4) 8 childrenwith pauciarticular JRA. 5) 6 children with uncomplicatedEI (all B19 IgM-positive). 6) 10 healthy adults. Results:Serum levels of Il-4, Il-6 and TNF were consistently lowerin the B19 arthropathy patients than all groups of JRA orEI. Mean levels of Il-6 and TNF in the B19 group weresignificantly lower than those in the systemic JRA group(p<0.001) and the polyarticular JRA group (p<0.02). IFNwas detectable in only 2 of 43 sera tested.

Group No. IL-2 IL-4 IL-6 IL-8 TNF-α

1. (n=8) 5.5(0-26.5) 6(0-15) 1.4(0-2.4) 161(0-1215) 1.0(0-3)

2. (n=5) 6.6(0-33) 5(0-25) 23.5(1.2-40) 65(41-100) 7.1(4.2-9.7)

3. (n=8) 2.0(0-16) 24(0-140) 8.6(1-25.7) 54(16-165) 6.0(4.5-8.2)

4. (n=8) 17.8(0-53) 4(0-24) 3.4(0-6.6) 39(14-70) 5.9(4.7-6.8)

5. (n=6) 0 73(0-150) 1.4(0-3.4) 113(19-328) 4.2(1-8.8)

6. (n=10) 1.6(0-9.3) 4(0-12) 0.9(0-2.2) 233(5-1455) 1.1(0-2.1)

Group 1, B19 arthropathy; Group 2, systemic JRA; Group 3, polyarticular JRA; Group 4,pauciarticular JRA; Group 5, erythema infectiosum; Group 6, controls.All results are groups means (range) expressed in pg/ml.

Conclusions: We conclude that the serum cytokine profileof patients with B19 arthropathy is different from that ofchildren with JRA and from values reported in adults withRA. In fact, patients with B19 arthropathy demonstratevery little systemic inflammatory cytokine responses andcharacterization as Th1 or Th2 could not be determinedbased on serum cytokine levels.

11. INTEGRATION AND AAV HELPER FUNCTIONS

Donor requirements for MVM site-specificintegration

Roni Mintz, Michal Mincberg, Yael Mali,Ayelet Naus, Jacov Tal.

Department of Virology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.

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Minute virus of mice is capable of site-specific integrationinto target EBV-based (p220.2) episomes which containsequence motifs from its active origin of replication(Corsini et al., J. Virol. 71:9008-9015,1997). With the aimof simplifying the hybridization-based, episomalintegration assay, we modified the target episome to carrythe reporter gene β-galactosidase (β-gal). The rationalewas that any integration event within the β-gal openreading frames (ORF) would give rise to a readilydetectable white bacterial colony. In practice, however,we found that besides true integration events (dsetectedby hybridization), other ORF-disrupting events take placewithin the target. These are not integration events, theirfrequency is about 10 fold greater than the frequency oftrue integrants and they only occur in episomes carryingactive ori sequences, indicating their relevance toepisomal integration. This sensitive “blue/white” systemenabled the rescue, in bacteria, of recombinant targetepisomes (“integrants”) following MVM DNA transfectionand the study of the structural and functional requirementsfor MVM-based vectors. These requirements were studiedin vivo, by transfecting cells harboring target p220.2/MVMori/β-galactosidase with two types of donor DNAs: linearMVM-based vector DNA, and circular or linearizedconstructs based on the bacterial vector pACYC184 (NEBiolabs). This vector contains resistances to tetracyclin(Tc) and chloramphenicol (Cm), and it carries the originof replication of plasmid p15A, which enables it to co-exist in cells with pBR322-derived vectors such as p220.2.We will also report on our progress with an in vitrointegration system using similar donor and target DNAs.

Role of Adeno-associated virus Rep protein in viralsite-specific integration

Samuel M Young Jr., Doug McCarty, Natalya Degtyareva,Richard Jude Samulski.

UNC Gene Therapy Center and Lineberger Cancer CenterUNC-Chapel Hill, Chapel Hill, NC 27599, USA.

Adeno-associated virus (AAV) is the only known eukaryoticvirus capable of targeted integration in human cells. AAVintegrates preferentially (70%) into the chromosome (ch)19.3.13 qter. The nonstructural proteins of AAV-2, Rep78and Rep68, have been implicated as being essential forthis targeted integration. Rep78 and Rep68 aremultifunctional proteins with diverse biochemicalactivities, including site specific binding to the AAV andch-19 target sequences, single-stranded site-specificendonuclease, and helicase activity. A Rep binding andnicking site, normally present on the viral terminal repeatsand essential for AAV replication have been identified onch-19. The presence of these sequences has provided aworking model for AAV Rep mediated targeting in humancells. The exact role of how a viral replication functions intargeted integration is unclear. To better characterize therole of Rep in targeted integration, we cloned the AAV-2Rep68 protein into an E. coli overexpression system.Rep68 was purified to >95% and shown to retain allknown biochemical activities previously described for theviral gene product at 10-1000 fold more activity than

previously published purified Rep protein. Rep DNAbinding assays have determined that a minimum Repbinding element (RBE) constitutes 8 bp, which would bepresent 2 x10 5 times/genome. How Rep distinguishesthe Ch-19 site vs. other potential RBE was investigated.We established a Rep dependent filter binding assay, aswell as carried out electron microscopy (EM) analysis todetermine the characteristics of this protein/target DNAinteraction. Our results determined that Rep affinity forch-19 is not distinct compared to other RBE in the humangenome. In fact, the minimum-binding site (GAGYGAGC)competes with equal affinity for Rep binding. Moreimportantly, we show the first direct visualization of a Repcomplex bound to DNA through EM analysis anddemonstrated that only one Rep/DNA complex was foundon Ch-19. Rep DNA complexes involve a multimericprotein structure that spans about 60 bp.Immunoprecipitation of AAV latently infected cellsdetermined that sufficient Rep protein (1000-4000molecules) is expressed to interact with all potentialbinding sites (2 x105). Finally, we engineered mice tocarry a single 2.7 kb human ch-19 insertion containingthe AAV ch-19 target locus. Using cells derived from thesemice, we demonstrated that this sequence was sufficientfor site-specific recombination after infection withtransducing vectors expressing Rep. This result indicatesthat any host factors required for targeting are conservedbetween human and mouse. Furthermore, the humanch-19 cis-sequences and chromatin structure requiredfor site-specific recombination are contained within thisfragment. Overall, these results indicate that the specificityof targeted recombination to human ch-19 is not dictatedby differential Rep affinities for RBE sites. Instead,specificity is likely mediated by human ch-19 sequencesthat serve as a Rep protein-dependent origin of replication.These results imply viral targeting is initiated by Rep/Repinteractions followed by host enzymes resulting in site-specific recombination. These results and how they relateto a model for AAV site-specific recombination will bediscussed.

Site-specific integration by adeno-associated virus(AAV): minimal requirements for target site selection

Toni Cathomen, Matthew D Weitzman. Laboratory of Genetics, The Salk Institute for Biological

Studies, San Diego, USA.

AAV is unique among eukaryotic viruses in its ability tointegrate preferentially into a specific locus on humanchromosome 19 (the AAVS1 locus). Two elements of AAVare required for targeting integration: the viral Repproteins and the inverted terminal repeats (ITRs) whichcontain a specific Rep recognition sequence (RRS). Thesame recognition sequence was also found in the AAVS1locus and we previously showed that Rep can form abridge between the viral ITRs and the RRS in the pre-integration locus. In order to define the minimalrequirements for Rep to recognize its target site, wedeveloped one-hybrid assays in which DNA-proteininteractions are detected in vivo. Chimeric proteins


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consisting of the N-terminus of Rep fused to differentoligomerization motifs and a transcriptional activationdomain were analyzed for oligomerization, DNA bindingand activation of reporter gene expression. Expressionof reporter genes was driven from RRS motifs clonedupstream of minimal promoters and examined inmammalian cells and in Saccharomyces cerevisiae. Ourresults show for the first time that chimeric proteinscontaining the N-terminus of Rep are able to target theRRS in vitro and in vivo in the context of an artificialmultimer. We are currently examining the DNA sequencerequirements necessary for site-specific integration inan in vivo integration assay. Together, these studies willidentify the minimal viral elements mediating site-specificintegration and may allow this unique feature of AAV to beharnessed for gene delivery.

AAV site-specifically integrates into a muscle-specific DNA region

Nathalie Dutheil, Fabio Shi, Thierry Dupressoir,R Michael Linden.

Institute for Gene Therapy and Molecular Medicine, MountSinai School of Medicine, New York, NY 10029, USA.

AAV has evolved the strategy to establish latency by site-specifically integrating its genome into humanchromosome 19 at AAVS1. To assess whether the regionsurrounding AAVS1 might have contributed to theselection of the specific integration site, we haveinvestigated this locus. We show that AAVS1 is closelylinked to the slow skeletal troponin T gene, TNNT1, andto the cardiac troponin I gene, TNNI3, which have beenmapped previously to 19q13.4. In addition, wedemonstrate that site-specific integration can lead to theformation of TNNT1-AAV junctions and we show evidencefor TNNI3 discruption.

The question now arise whether muscle cells (skeletaland/or cardiac) represent a natural target tissue for latentAAV infection and whether the viral DNA integrationfrequency is correlated with the expression of TNNT1 and/or TNNI3.

Hghly Sensitive and Rapid Detection of AAV-MediatedSite-Specific Integration

D Wegner, S Weger, R Heilbronn. Inst. f. Infektionsmedizin, Abt. Virologie, UKBF, FU Berlin,

Hindenburgdamm 27, 12203 Berlin, Germany.

Adeno-associated virus type 2 integrates with highspecificity into the AAVS1-site of human chromosome 19(q13.3-qter). Integration kinetics and frequency are difficultto evaluate since AAV can integrate within in a range ofseveral 100bp around the Rep-binding site onchromosome 19. This leads to PCR products of varyingfragment lengths, which makes quantification difficult.Here we describe the development of a rapid andsensitive real-time PCR assay for AAV integration usingthe LightCycler technology with sequence-specificdetection probes. To quantify a pool of independent

integration events without clonal expansion of cells wedeveloped an inverse PCR strategy. Using this assay it ispossible to detect and quantify site-specific integration inhuman DNA within hours after AAV infection. At themoment we are able to detect less than 10 copies of acloned virus-cell junction on the background of 1:g humangenomic DNA.

Functional characterization of HSV-1 helper functionsfor AAV replication

Travis Stracker,1 Peter Ward,2 Sandra K Weller,3

Matthew D Weitzman.1

1The Salk Institute for Biological Studies and University ofCalifornia, San Diego, USA; 2Institute for Gene Therapy andMolecular Medicine, Mt. Sinai School of Medicine, New York;

3Department of Microbiology, University of Connecticut HealthCenter, Farmington, CT, USA.

Adeno-Associated Virus (AAV) replication is dependenton both cellular proteins and functions supplied by helperviruses such as adenovirus (Ad) or herpes simplex-1(HSV-1). The helper functions of Ad are well characterizedbut relatively little is known about the HSV-1 helperproteins. The minimal HSV-1 proteins needed to supportAAV replication are the helicase-primase complex UL5,UL8, and UL52 and the single-stranded DNA bindingprotein UL29. Using assays to examine individual stepsin the AAV lifecycle, we analyzed the functions of the HSV-1 helper proteins. Transfection of the AAV genome andexpression vectors for the four HSV-1 proteins wassufficient to establish replication centers for AAV in whichRep and UL29 co-localize. Transfections into culturedcells have shown that both the helicase-primase complexand UL29 were necessary for replication of AAVin vivo.However, we show that in in vitro replication assays UL29is sufficient to enhance AAV replication, while the additionof the helicase-primase complex has no effect. Wesuggest that UL29 plays an analogous role to the AdDBP by increasing the processivity of the polymerase.We propose that the helper activity of the helicase-primasecomplex in vivo is to modulate the function and sub-nuclear localization of UL29. To test this hypothesis, wehave screened a series of biochemically characterizedmutants in the helicase-primase complex to determinetheir effects on AAV replication. Functional characterizationof the HSV-1 helper functions will provide us with a betterunderstanding of the cellular environment that promotesproductive AAV replication.

An AAV rep and cap expressing herpes simplexvirus: colocalisation of AAV-rep and HSV-ICP8

R Heilbronn,1,2 A Krahn,1,2 C Schetter,2

M Engstler,3 S Weger,1,2 M Boshart.3

1Inst. f. Infektionsmedizin, Abt. Virologie, Freie UniversitätBerlin, 12203 Berlin, Germany; 2Max-Planck-Institut für

Biochemie, 82152 Martinsried, Germany; 3AG Mol. Zellbiologie,Inst. f. Molekularbiologie und Biochemie, Freie Universität

Berlin, 12203 Berlin, Germany.

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A system for the production of recombinant AAV vectorswas designed by combining herpes simplex virus helperfunctions with AAV rep and cap genes in a singlerecombinant herpesvirus (rHSV). This allows simpleinfection instead of laborious DNA transfection to deliverthe necessary components for packaging of rAAV. Therep- and cap-expressing herpesvirus was obtained afterseveral rounds of plaque purification and the integratedcassette was stably maintained without apparentreversion to wildtype. Productive infection of mammaliancells yielded high level expression of Rep and Cap withratios comparable to AAV wildtype. EM analysisdemonstrated formation of empty AAV capsids uponinfection with the recombinant HSV alone. HSV ICP8together with other components of the HSV replicationcomplex had been shown before to be required forproductive AAV replication (Weindler and Heilbronn, 1991,J. Virol. 65, 2476). We therefore assumed that Rep shouldcolocalize to HSV replication centers where ICP8 resides.3D-immunofluorescence analysis with digitaldeconvolution demonstrated a nuclear distributionpattern of AAV Rep different from that of the HSV replicationprotein ICP8. Further analysis showed that Rep changedits distribution pattern in the presence of AAV DNA andthen colocalized with ICP8. Our findings support a modelthat Rep and ICP8 directly interact on the AAV terminalrepeats which directs the HSV replication complex to theAAV template.

Human papillomavirus-(HPV)-provided helperfunctions for AAV-2 expression and replication

CM Walz,* M Ehrbar, JR Schlehofer. Deutsches Krebsforschungszentrum, Angewandte

Tumorvirologie, Abt. F0100, Im Neuenheimer Feld 242, D-69120Heidelberg, Germany;

*Present address: Institute of Medical Psychology, MedicalFaculty, Otto-von-Guericke University Medical Faculty, D-

39120 Magdeburg, Germany.

Infection with certain types of human papillomaviruses(HPV) is the major cause of cervical cancer, whereasinfection with AAV seems to be negatively correlated withthe development of this malignancy. Previously wereported the presence of infectious AAV-2 in cervicalbiopsies of women with HPV-related genital lesionsindicating a close interaction of both viruses probablyleading to AAV replication and production of infectiousprogeny particles in vivo. Moreover, cell cultureexperiments revealed that HPV type 16 (HPV-16) canprovide helper functions for AAV-2 replication.

To clarify the helper function of HPV for AAV geneexpression and replication, cells of the cervical carcinomalines HeLa (containing partial HPV-18 DNA), CaSKi(containing complete HPV-16 DNA) and C33A (devoid ofHPV DNA) were either infected with AAV-2 particles ortransfected with the full genomic DNA of AAV-2.Subsequently, cultures were transfected with the fullgenome of HPV-16 or with constructs carrying andexpressing either the E2 or the E1 gene of HPV-16. It

could be shown that complete genomic HPV-16 DNA isable to initiate AAV replication in all three cell lines.However, the sole E2-expression induced AAV-replicationonly in CaSKi and in HeLa cells but not in C33A cells. E1-expression did not provide this function.

In addition, in CaSKi cells (which carry the completegenome of HPV-16), infected with AAV or transfected withgenomic AAV DNA, expression of early AAV-rep-genescould be observed by immunofluorescence. This rep-gene expression was enhanced after transfection of HPV-16 E2 DNA.

These results indicate that E2 is not sufficient for inductionof AAV expression and replication. In addition, other HPVgenes seem to be required, presumably E6 and E7 whichare constitutively expressed in HeLa cells and in CaSKicells. Obviously, transfection of E2 into HeLa cellscompletes the functions required for AAV replication,whereas in C33A cells that do not contain HPVsequences, transfection of E2 alone is not sufficient forinduction of AAV expression. CaSKi cells contain multiplecopies of HPV-16 and express other early genes inaddition to E6 and E7 probably including E2, thus providingall necessary factors for initiation of AAV gene expression.In these cells addition of E2 increased the efficiency ofthis helper function leading to a stronger AAV geneexpression. These findings demonstrate that AAV-expression and -replication require -at least- the HPVgenes E6, E7 and E2.

AAV chromosome 19 site-specific recombinationdoes not required a Rep dependent origin of

replication on the AAV inverted terminal repeatsequence

Samuel M Young Jr, R Jude Samulski. Curriculum in Genetics and Molecular Biology and UNC Gene

Therapy Center, UNC-Chapel Hill, NC 27599, USA.

Adeno-associated virus (AAV) is the only known eukaryoticvirus capable of targeted integration in human cells. AnAAV Rep binding element (RBE) and terminal resolutionsite (trs) identical to the viral terminal repeats required forAAV DNA replication are located on ch-19. Both theseelements on ch-19 have been shown to be essential forviral targeting to this locus. To better characterize the roleof the AAV terminal repeat (ITR) cis-acting sequences inAAV targeted integration we tested a trs mutant incapableof supporting viral replication. Wild type and mutantsubstrates were assayed for targeted integration by co-transfection experiments in the presence or absence ofRep. Our results demonstrated that, in the presence ofRep78, both ITR substrates targeted to ch-19 with similarfrequency (wt 32% and mutant 47%). Molecularcharacterization of the mutant ITR integrants confirmedthe presence of the trs mutation in the majority of samplestested (67%). Unlike wild type rescue, complementationanalysis confirmed that the targeted mutant ITR wereunable to rescue and replicate. In addition, Rep78 inducedextensive rearrangement and amplification of ch-19


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sequences independent of targeted integration. Thesestudies demonstrate that nicking of the viral cis-acting trssequence is dispensable for site-specific recombinationand suggest that AAV targeting is mediated by Rep78/68dependent replication from the ch-19 origin. Thesestudies have significant impact towards theunderstanding of AAV site-specific recombination and thefuture development of targeting vectors.

12. PATHOGENESIS AND PERSISTENCE

Development of persistent rat virus infection and itsramifications for rat breeding colonies

Lisa Ball-Goodrich, Elizabeth Johnson,Frank Paturzo, Robert Jacoby.

Section of Comparative Medicine, Yale School of Medicine,New Haven, CT, USA.

Rat virus (RV) is a common parvovirus of laboratory ratswhich can cause disease, distort biological responsesthat rely on cell proliferation, and disrupt production inbreeding colonies. Persistence is an established featureof RV infection that has the potential to amplify interferencewith research. Our research examined the developmentof persistent infection in the presence or absence of hostimmunity. It also assessed whether viral persistenceoccurred after transplacental infection, and whetherpersistently infected dams transmitted RV to their progeny.Infection was assessed by virus isolation, serology,immunohistochemistry and in situ hybridization. An initialstudy compared the development of persistent RVinfection in RNU athymic and euthymic rats inoculatedwith the Umass strain of RV at six days of age. Tissuesfrom 4 to 6 rats of each phenotype were assessed duringacute and persistent infection. Selected tissues alsowere analyzed by Southern blot and serum was assayedfor RV antibody. Acute infection in both euthymic andathymic rats featured widespread dissemination of viruswhereas persistent infection occurred primarily in arteriesand arterioles. Vascular smooth muscle cells (SMC)emerged as the primary targets during persistent infection,and viral mRNA was detected in SMC indicating thatpersistent infection included virus replication. In addition,virus-positive pneumocytes and renal tubular epithelialcells also were detected through week 8 implying thatkidney and lung are sites for virus excretion duringpersistent infection. The prevalence of virus-positive cellsremained moderate to high among athymic rats through8 weeks, whereas hybridization signal decreased ineuthymic rats by 2 weeks, coincident with seroconversionand perivasculitis. Thus, host immunity reduced but didnot eliminate infection. To determine whether in uteroinfection with RV resulted in persistence of virus, acuteinfection was induced in Sprague-Dawley (SD) dams byoronasal inoculation of RV-UMass on gestation day 9.Nine of 12 progeny examined at 3 weeks were virus-positive and all had antibody to RV. No virus was detectedin progeny examined at 8 weeks (10 rats) or 16 weeks

(10 rats) post partum. However, 7 of 10 additional progenytransmitted infection to contact sentinels introduced at 9weeks, and 1 of 10 transmitted infection to a sentinelcagemate introduced at 15 weeks. These results indicatethat progeny of rats infected with RV during pregnancyare at risk for persistent infection of variable duration. Athird study determined whether persistently infected damstransmitted RV to either sire or progeny. Persistentinfection was induced in two day-old female SD rats byoronasal inoculation with RV-UMass . Between 7 and 9weeks of age, seropositive survivors were bred to RVseronegative males. Nine of 16 dams transmitted infectionto their breeding partners. Progeny were tested for virusin utero at gestation day 19, at birth, or at 3 weeks post-partum, and none were virus-positive. Maternal immunitylapsed in 10 progeny held for 16 weeks, and none ofthese animals subsequently transmitted infection tocontact sentinels or had detectable virus at necrposy.Some dams were tested for virus after their litters wereweaned, and all were virus-positive by virus isolation and/or in situ hybridization. These results indicate that progenyof persistently infected rats are protected from RVinfection, probably due to maternal immunity.

Regulation of the p6-promotor of parvovirus B19 bycellular proteins and the viral NS1-protein Ulla Raab,1 Birgit Bauer,1 Andreas Gigler,1 Karin

Beckenlehner,1 Sean Doyle,2 Hans Wolf,1 Susanne Modrow.1

1Institute for Medical Microbiology, University of Regensburg,Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany.2Biology Department, National University of Ireland, Maynooth,

Co. Kildare, Ireland.

Parvovirus B19 is the only known human pathogenicmember of this virus family causing erythema infectiosum,arthritis, hydrops fetalis in pregnant women and somefurther disease manifestation like anemia, thrombo- andneutrocytopenia. The molecular basis for the cell specifityof the infection causing such a variety of differentsymptoms is not understood. The synthesis of all viraltranscripts of identified so far are regulated by a singlepromoter at map unit 6 of the viral genome, the p6promoter. This promoter is highly active in various cellsand cell types. Its activity is, however, significantly higherin cells of the erythroid lineage and is in addition enhancedby the transactivating viral NS1-protein produced duringinfection.

To analyze cell specific influences on the p6 promoter weperformed gel retardation assays using nuclear extractsof different cell lines to identify transcription factors thatare involved in regulating the promoter activity. The cellularDNA-binding proteins were further characterized bycompetition by specific and unspecific oligonucleotidesand immunoshift assays in the presence of specificantibodies. Thereby we were able to demonstrate thebinding of the Oct-1-protein to an octamer motive withinthe p6 promoter and of the transcription factor Sp1 tothree GC-boxes. Preferential and specific interaction ofthe factor Sp3 to one of theses boxes was observedindicating that the ratio of Sp1:Sp3 may be involved in the

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regulation of the p6 promoter. Consensus sites for theregulatory YY1-protein to which the factor binds veryefficiently are located in three different regions.Furthermore the interaction of an EBS-factor to therespective Ets-site could be shown. It can be concludedthat multiple protein binding can be observed at the p6promoter region of parvovirus B19.

All identified binding motives within the p6 promoter arehighly conserved in any of the 19 virus isolates whichwere analyzed by DNA-sequencing indicating that theyare essential for the promoter activity. After the basicidentification of the DNA-binding factors from HeLa-cells,nuclear extracts from erythroleukemic K562 cells wereprepared. Here a more pronounced interaction of Sp3-proteins was observed together with additional factorsreflecting erythroid-specific influences on the viralpromoter activity.

We furthermore analyzed the activity of the viral NS1-protein to bind to the promoter-DNA and to the cellularpromoter-binding factors using supershift assays andcoimmunoprecipitation experiments. NS1-proteins wereproduced by in vitro translation. Alternatively NS1-proteinsynthesized by recombinant baculovirus was used.Beside interaction of the NS1-protein with varioussequence elements in the promoter binding to the Sp3-and Oct1-proteins was observed. It may be assumedthat these protein.

Mechanisms of Parvovirus B19 Persistence InPeripheral Blood Mononuclear Cells

Jennifer Lynne Reed, Bade Kucukcetin, Scott Koenig.MedImmune, Inc., Gaithersburg, Maryland, USA

Chronic detection of parvovirus B19 DNA has beenreported in bone marrow of symptomatic and healthypatients, and in inflammatory foci associated with arthritisand arteritis. The purpose of these studies was toinvestigate the mechanism of B19 persistence. PBMCsand immortalized cells of erythroid lineage, activated withphorbol ester and infected in vitro, produced transientspliced viral transcripts detectable by RT-PCR. Thesetranscripts were not detectable in the absence ofactivating agent. To distinguish among alternative formsof B19 DNA in these cells, an inverse PCR procedurewas employed to detect concatameric or integrated B19genomes. Inverse PCR products were amplified from invitro infections of unstimulated erythroid cell lines; similarproducts were also obtained from primary PBMCs ofhealthy blood donors, and from synovial tissue ofsymptomatic arthritis patients. Sequence analysis ofinverse PCR products revealed highly recombined viralDNA rarely juxtaposed with human chromosome. Ourobservations suggest an expanded tropism of parvovirusB19 and a non-lytic infection path which may contribute toin vivo viral persistence.

Infection prior to development ofimmunocompetence results in clinical disease in

simian parvovirus-inoculated fetuses MG O’Sullivan,1 DA Feeney,1 JB Jarvis,1 JC Veille,2

WA Block,2 NS Young,3 KE Brown.3

1University of Minnesota College of Veterinary Medicine, St.Paul, MN; 2Wake Forest University School of Medicine,

Winston-Salem, NC; 3National Heart, Lung and Blood Institute,Bethesda, MD, USA.

The consequences of fetal infection with parvovirus B19vary from inapparent infection to life-threatening hydropsfetalis. Using a monkey model of parvovirus fetal infection,we hypothesized that the timing of infection in relation toonset of fetal immunocompetence is critical for thedevelopment of clinical disease such as hydrops fetalis.Cynomolgus monkey fetuses were inoculated with simianparvovirus (SPV) prior to (gestation day [GD] ~ 49-63), orfollowing (GD ~80-109) the development ofimmunocompetence at ~GD 70 (full-term is ~GD 165),respectively. SPV viremia, SPV-specific antibodies andcomplete hematological profiles were monitored in thefetuses. Inoculation of fetuses after GD 80 (n = 5) resultedin viremia that was cleared, an IgG antibody response,normal hematologies and normal progression ofpregnancy with live births. Inoculation prior toimmunocompetence (n = 8) resulted in severe anemiathat frequently progressed to hydrops fetalis and/or death(6/8), transient hydrops that resolved (1/8), or normalprogression after a delayed onset of viremia (1/8). Threefetuses, inoculated on GD 49, 53 and 84, respectively,failed to become infected. Our findings indicate thatinfection prior to onset of immunocompetence is a keyfactor in producing hydrops fetalis or other adverseoutcomes.

Parvovirus B19-associated meningoencephalitisFaraj Barah, Pamela J Vallely, Paul E Klapper,

Malcolm L Chiswick,* Jonathan R Kerr. Department of Virology, University of Manchester and

*Neonatal Medical Unit, St Mary’s Hospital, Manchester, UK.

Neurological manifestations have been documented inassociation with erythema infectiosum both with andwithout proven B19 infection. To clarify the incidence andpathogenesis of B19 meningoencephalitis, weretrospectively examined 143 cerebrospinal fluid (CSF)samples (one per patient) received in our regional viruslaboratory from patients with aetiologically-undiagnosedmeningitis and/or encephalitis from 1995 - 1999 for thepresence of B19 DNA using nested PCR for two distinctregions of the B19 genome (NS-1 and VP-1 genes). Theage of these patients ranged from 1 day to 16 years,mean of 44 months. Of the total 143 patients, 61 werefemale, 77 were male, and in 5 the sex was unrecorded.

B19 DNA was detected in the CSF of 12 of 143 patients(8.4 %). The age of these ranged from 1 day to 15 years 7months, with a mean of 65 months. Clinical syndromesincluded meningitis, encephalitis, and


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meningoencephalitis. Associated abnormalities includedhepatitis (n=3), lymphadenopathy (n=1), familialerythrophagocytic lymphohistiocytosis (FEL) (n=1),Cockayne’s Syndrome / leukodystrophy (n=1), relapse ofcommon Acute Lymphoblastic Leukaemia (ALL) (n=1),and a karyotype of 47XXX (n=1). Regarding outcome, therewere 3 deaths, two cases of long-term mental retardation(one with additional personality change) and 7 caseswho completely recovered (one of these receivedintravenous immunoglobulin (IVIG)). In 9 of 12 cases,unextracted CSF was available. By Western blot, 2 of thesewere weakly anti-B19 IgM positive and the remainder werenegative; 7 were positive for anti-B19 IgG. In 4 of 12 cases,serum from around the time of meningoencephalitis wasavailable. Three of these 4 contained B19 DNA. ByWestern blot, all were positive for both anti-B19 IgM andIgG. The detection of B19DNA in the CSF suggests directinvasion of the central nervous system by the virus, andthe presence of specific serum / CSF IgM in 5 casesconfirmed recent B19 infection.

In conclusion, our results support a significant role forparvovirus B19 in neonatal, paediatric and adolescentmeningoencephalitis, which was associated with severemorbidity and mortality in 5 of 12 cases (42%). To ourknowledge, this is the first report of parvovirus B19meningoencephalitis from the UK, and we recommendthat B19 should be sought prospectively in neonatal,childhood and adolescent meningoencephalitis.

Parvovirus B19 infections in patientswith cutaneous vasculitis

KM Carlsen,1 L Danielsen,1 T Karlsmark,1 HK Thomsen,2

K Rosson,2 SF Sørensen.3

The Departments of 1Dermato-Venerology, 2Pathology and3Rheumatology, The National University Hospital of Bispebjerg,

Copenhagen, Denmark.

Background: Parvovirus B19 is a 5.4 kb single-strandedDNA virus that was first described in 1975. Since 1984 ithas been known as the etiology of “5th disease” witherythema infectiosum among other clinicalmanifestations as fetal hydrops, purpura, glove and socksyndrome, vasculitis or it may be subclinical.

Patients and methods: We investigated serum samplesfrom 15 patients, aged 16 to 87 years, admitted to theDermatology department due to cutaneous vasculitis inthe period July 1995 until February 2000. In this pilotstudy, serum samples were investigated for ParvovirusB19 IgM and IgG antibodies by a Parvovirus B19 IgM andIgG EIA. The majority of the patients admitted due tovasculitis were women (female:male = 11:4).

Results: Serum samples from 14 (93.3%) of the 16patients were found to be Parvovirus IgG positive, 13 ofthese (86.8%) were Parvovirus IgM negative and 1 (6.6%)had Parvovirus B19 IgM indicating an acute infection. Only1 (6.6%) patient had no antibodies. One of the patientswas found to be allergic to Penicillin, one had acuteHaemophilus infection, one had a chronic Hepatitis C

infection while the remaining 12 (80.0%) had vasculits ofunknown etiology.

Age range (years): 0-20 21-40 41-60 61-80 >80No. investigated: 2 1 4 4 4No. with IgG: 1 1 4 4 4

Discussion: Parvovirus Bl 9 IgG antibodies were detectedin 100% of the investigated patients, above 31 years ofage, in a period of 5 years. The high percentage withdetectable Parvovirus B19 IgG antibodies is due toincreasing exposure throughout life. Part of the reasonfor finding only one acute sporadic case with ParvovirusBl9 IgM antibodies could be that Parvovirus Bl9 infectionsusually are epidemic with approximately 3 years betweenoutbreaks. Only one epidemic period in 1997 wasrepresented during the study with 3 (20%) of the patientsaged 31-74 years, who were already parvovirus B19 IgGpositive due to earlier infection. Another reason couldalso be the fact that parvovirus B19 infection is a rareetiology to cutaneous vasculitis.

Fetal infection with parvovirus B19:infection time in gestation and prognosis

Tadasu Nunoue.Graduate School of Health and Nutrition Sciences, Nakamura

Gakuen University, Fukuoka, Japan.

Objectives: This study was undertaken to establish therelation of infection time in gestation of pregnant womenand fetal prognosis retrospectively.

Cases: 123 pregnant women (and their fetuses) whowere suspiciously infected because of abnormality orabnormal growth course in fetus. Methods: Materials werematernal serum, amniotic fluid, fetal ascites, cord blood,and fetal tissues autopsied. Test methods were PVB19-DNA by PCR, in situ hybridization in fetal tissue, and anti-PVB19 IgM and IgG by enzyme immunoassay wereapplied.

Results 1: Fifty seven out of 123 fetuses were confirmedwith PVB19 infection. Results 2: The time of infection wasclear in only 13 pregnant women, however, it wasundetermined in the rest 44 infected because ofsubclinical infection and unclear contact sources.

1) A fetus of 29 year-old pregnant woman whose infectionwas possibly at 2 gestation weeks (gw), showed hydropsat 23 gw and died at 28 gw. 2) A fetus of 28 year-oldpregnant woman infected at 15 gw resulted inspontaneous stillborn at 18 gw. Genomic DNA wasdiscovered in fetal tissues. 3) A fetus of 34 year-oldpregnant woman infected at 15 gw showed hydrops at21 gw, then died at 27 gw. 4) A fetus of 27 year-oldpregnant woman infected at 18 gw showed hydrops at26 gw and died at 32 gw. 5) Other 9 cases were born well.Time of infection of their mothers were 4, 5, 8, 11, 14, 17,22, 22, 32 gw. One of them had a low birth weight 1,998 gat 36 weeks, and his mother was infected at 4 weeks ofpregnancy. Genomic DNA were detected in all cord bloods

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except in the stillborn case. Suppl.) A fetus whose 33year-old mother was infected 3 weeks before pregnancywas grown well and born at 39 gw with 2960 g. Nogenomic DNA nor anti-PVB19 IgM was detected in thecord blood. They were positive in maternal serum at 3weeks before pregnancy.

Conclusion: PVB19 once infected persists in fetus in anytime from very early time of gestation to birth, and may kill30% of fetuses.

Abnormal Levels of liver enzymes associated with anasymptomatic parvovirus B19 infection

Marialuisa Zerbini, Simona Venturoli, Monica Cricca, *LuciaRinaldi, *Marcello Lanari, Monica Musiani.

Department of Clinical and Experimental Medicine, Division ofMicrobiology. University of Bologna. Via Massarenti 9.Bologna, Italy; *Pediatric and Neonatology Department,University of Bologna. Via Massarenti 9. Bologna, Italy.

Human parvovirus B19 infection shows a broad spectrumof clinical manifestation. Recently B19 has beenassociated with acute hepatitis, hepatic dysfunction andfulminant liver failure more commonly in children than inadults. Liver disease in B19 infection has been describedin addition to aplastic anemia, Erythema Infectiosum,arthralgia, aspecific symptoms and involvement of centralnervous system. We report a case of abnormal liver testsin an asymptomatic 15-month-old-infant (April 99)monitored since the birth, because of an HCV-positivemother, all the laboratory tests excluded a verticaltransmission of HCV infection.

On the check at 15 months the infant showed a significantraised in aspartate aminotransferase (AST, 486 U/L) andin alanine transferase (ALT, 819 U/L) in absence of clinicalsymptoms. At that time, metabolic causes and infectiousagents, such as HAV, HBV, HCV, Toxoplasma, HCMV,Enterovirus, HSV and EBV, were excluded while thediagnosis of parvovirus B19 infection was accomplishedwith the detection of B19 DNA in a serum sample by PCRand by the presence of specific Ig M and IgG.

Two months later (June 99) the infant had fever andfaringitis. B19 testing proved still positive for the presenceof DNA, IgM and IgG in serum samples; AST and ALTwere 139 U/L and 263 U/L respectively. In July 99 a serumsample proved to be B19 DNA and IgM negative, B19 IgGpositive, AST and ALT 67 U/L and 112 U/L respectively. InOctober all the laboratory tests were within normal limits.During all the period no hepatosplenomegaly wasapparent on physical examination or on ultrasonic study.Our observation suggests that B19 infection should beinvestigated in presence of abnormal liver tests even inabsence of clinical manifestations.

Prevalence of human Parvovirus B19 infection inintrauterine fetal death

O Norbeck,1 T Tolfvenstam,1 K Petersson,2

K Broliden,1 N Papadogiannakis.3

1Department of Clinical Virology, 2Department of Obstetricsand Gynecology, 3Department of Pathology, Karolinska

Institutet, Huddinge University Hospital, SE-141 86 Huddinge,Stockholm, Sweden.

In pregnant women with acute parvovirus B19 (B19)infection, the virus can be transmitted to the fetus. Fetalinfection can be either asymtomatic or cause fetalhydrops, spontaneous abortion or intrauterine fetal death(IUFD). The infection is well known to cause hydropsfetalis sometimes followed by fetal death in the secondtrimester. The presence of B19 in fetal tissues amongnon hydropic cases of IUFD has not been thoroughlyinvestigated, which is an important issue for primarypreventive measures and prioritising diagnosticinvestigations. In the present study we have examinedthe frequency of B19 DNA by polymerase chain reaction(PCR) and B19 viral proteins (VP) byimmunohistochemistry (IH) in fetal autopsy material andplacental tissues from a total of 114 pregnancies. Thehistopathology of the B19 DNA positive tissues was alsostudied in detail. The material was divided into threegroups: IUFD (n=48) as defined as death during or aftergestational week 22, spontaneous abortions (n=37)having a gestational age less than 22 weeks and caseswith known etiology (n=29) composed of legal abortionsbefore gestational week 22. There was a significantoverrepresentation of B19 DNA positive findings 7/48(14.6%) among the IUFD cases as compared to the othergroups (p<0.05). Two B19 DNA positive cases (5.4%)were found among the cases of spontaneous abortionsand no case was positive among the legal abortions.Additionally, among deliveries at term (n=50) no B19 DNApositive placental samples were found. PCR detection ofB19 is more sensitive than IH staining, as only three ofnine B19 DNA positive cases stained positive for B19 VP.No B19 DNA negative samples stained positive for B19VP by IH. Four cases exhibited hydrops fetalis in ourmaterial, one case was defined as IUFD andsubsequently found B19 PCR positive. The other threewere B19 PCR negative with one case in the spontaneousabortions group and the other two in the group of legalabortions. Our results suggest that the finding of B19infection among IUFD in late second and during the thirdtrimester is common. Furthermore, the finding of fetalhydrops in late pregnancy B19 associated fetal demiseis exceptional.

Prospective study on the clinical significance of AAVinfection in pregnancy

K Dietrich,1 GK Beisler,3 B Schlehofer,2 JR Schlehofer.1

1Angewandte Tumorvirologie, and 2AG Umweltepidemiologie,Deutsches Krebsforschungszentrum, Im Neuenheimer Feld

242, 69120 Heidelberg; 3Ärztehaus, Wildbader Str. 31, 75323Bad Wildbad-Calmbach, Germany.


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In a previous study we reported on the presence of AAVDNA and virions in amniotic fluid and trophoblast cells,indicating in utero infection with AAV (1). To further assessthe influence of AAV infection on the course of pregnancyand the developing embryo, we started a prospectivestudy with 400 pregnant women undergoingamniocentesis for medical reasons.

In this study, amniocentesis is carried out by agynecologist. Amnion fluid and serum is collected foranalysis of viral DNA (PCR analyses for AAV and itshelpers HPV and CMV) and of serum antibodies to AAV(ELISA), respectively. Clinical data on the pregnancy atthe time of amniocentesis are obtained with aquestionnaire. For assessment of the further course ofpregnancy and of perinatal conditions, women are givena questionnaire to be filled out at delivery by theobstetrician. In addition, a serum of mother and child(chord blood) is collected at this time. (Currently, theresponse rate at delivery is about 70%).

Data will be analyzed using standardized epidemiologicalmethods for cohort studies. Preliminary data of 86 women,who already gave birth, confirmed our previous findingsof a prevalence of AAV DNA in amnion fluid/cells of about38%. In addition, there was a higher rate of pre/peri-natalcomplications in mothers in whose amnion fluids AAVDNA had been detected (33% vs. 20% in non-infectedwomen), supporting the hints from our previous non-standardized study. Because of the standardized methodsand the collection of all pregnancy-related clinical data inaddition to the analysis of virus infection, this study willclarify whether AAV infection is correlated withcomplications during pregnancy.

Adeno-associated viruses (AAV) in semen samplesof infertile men

Kerstin Erles,1 Volker Rohde,2 Jörg R.Schlehofer.1

1Angewandte Tumorvirologie, DeutschesKrebsforschungszentrum, Im Neuenheimer Feld 242, 69120

Heidelberg,Germany; 2Urologische Universitätsklinik,Universität des Saarlandes, Homburg / Saar, Germany.

The helper virus-dependent human parvoviruses, adeno-associated viruses (AAV) are considered as non-pathogenic. However, experimental infection of pregnantmice with AAV led to fetal death and early abortion. Inhumans, persistent AAV infection was mainly found inthe female genital tract (including uterine epithelium,amniotic fluid, spontaneous abortion).

We assessed AAV (and helper virus) infection of the malegenital tract, by analyzing by PCR 94 semen samples forthe presence of DNA of AAV, papillomavirus (HPV), andcytomegalovirus (HCMV). In 73 infertile men with apathological spermiogram, AAV DNA was detected in 28ejaculates (38%). In contrast, only 1 sample of 21 personswith normal spermiogram contained AAV DNA (5%). Thirtysamples from infertile men (patients) and from 8 controls(fertile men) were separated in (i) spermatozoa, (ii) non

spermatozoal cells and (iii) cell free supernatant,revealing AAV DNA in 9/30 patients (AAV-DNA beingassociated with the spermatozoa fraction in 8 of the 9cases). HPV DNA (types 16, 18, 31, or 33) was detectedin semen of 13/73 patients (18%) and 4/21 controls (19%),HCMV DNA in 5 patients and 1 control. The data hint to asexual transmission of AAV and suggest a role of AAVinfection in the pathogenesis of male infertility.

Adeno-associated virus DNA in human gestationaltrophoblastic disease

Klaudia Kiehl,1 Jörg R Schlehofer,1

Edecio Armbruster-Moraes.2

1Angewandte Tumorvirologie, DeutschesKrebsforschungszentrum, Im Neuenheimer Feld 242, D-69120Heidelberg; Germany, 2Gynecology and Obstetrics Department,

Faculty of Medicine, University of Sao Paulo and HumanGenetics Section of the Butantan Institute, Sao Paulo, Brazil.

Previous studies had shown an association of infectionwith the human adeno-associated virus (AAV) withspontaneous abortion in early pregnancy. Furthermore,AAV DNA had been detected in cells of the humantrophoblast lines, Jeg-3, JAr, and BeWo, in trophoblastsfrom amnion fluids and in cells of the human amnionline, FL. Infectious AAV virions could be isolated fromamnion fluids.

To further analyze AAV infection during pregnancy, wetested material from gestational trophoblastic diseasefor the presence of AAV DNA. With 35 paraffin-embeddedsamples from patients from Brazil, including 17 hydatiformmoles, 5 choriocarcinomas, and 13 spontaneousabortions, PCR was performed to detect the presence ofAAV DNA.

In nested PCR analyses, AAV DNA was found in 16samples (7/17 hydatiform moles, 1/5 choriocarcinomas,8/13 miscarriage material). These findings confirm AAVinfection of embryo-derived tissue in humans and furthersuggest a role of AAV in miscarriage. It remains to bedetermined whether AAV (possibly together with helpervirus, [HPV, CMV] infection) is involved in the pathogenesisof gestational trophoblastic disease such as thedevelopment of hydatiform mole or choriocarcinoma.

13. GENE THERAPY

Treatment of experimental arthritis usingrecombinant AAV-TNFR:Fc vector gene therapy

Haim Burstein, Tony Stepan, James Chan, Sharon M Wahl. Department of Research, Targeted Genetics Corporation,Seattle, WA; National Institute of Dental and Craniofacial

Research, National Institutes of Health, Bethesda, MD USA.

Rheumatoid arthritis (RA) is a chronic inflammatoryimmune-mediated disease primarily manifested by

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chronic, erosive inflammation of the joints. Tumornecrosis factor-” (TNF-”) is a pleiotropic cytokine whichplays a key role in the pathophysiology of RA. Systemicbiweekly administration of tumor necrosis factor receptor-immunoglobulin Fc fusion protein (TNFR:Fc) to patientswith RA has resulted in improvement of joint symptoms.Recombinant adeno-associated virus (AAV)-basedTNFR:Fc gene therapy at infrequent intervals may be anattractive alternate modality for RA treatment. To this end,we developed recombinant AAV vectors, AAV-huTNFR:Fcand AAV-rTNFR:Fc, encoding the human and the ratTNFR:Fc fusion genes, respectively. Transduction of 293cells in vitro with either AAV-rTNFR:Fc or AAV-huTNFR:Fcvectors at multiplicities of infection (moi) ranging from 1to 104 resulted in secretion of a bioactive rat or humanTNFR:Fc fusion protein in a dose-dependent manner.The streptococcal cell wall (SCW)-induced arthritis modelin Lewis rats was employed to evaluate the effect of AAV-rTNFR:Fc vector administration on the development andseverity of arthritis on both the ipsilateral and thecontralateral joints. A single intra-articular (i.a.)administration of 2x1010 DNase I Resistant Particles(DRPs) of AAV-rTNFR:Fc vector into both rear ankle jointsresulted in significant reduction of hind paw swelling asmeasured by arthritis index scores. Moreover,administration of AAV-rTNFR:Fc to a single joint alsoresulted in significant reduction of paw swelling in thecontralateral joint. A single intramuscular (i.m.)administration of 1.2x1011 DRPs of AAV-rTNFR:Fc vectorresulted in a similar effect. Bioactive rat TNFR:Fc wasreadily detectable at day 33 in serum samples of ratsinjected i.m. with AAV-rTNFR:Fc. In contrast, rat TNFR:Fclevels in i.a.-injected rats were not significantly differentfrom control rats, suggesting that local joint administrationof AAV-rTNFR:Fc does not lead to significant systemicdistribution of this TNF-a antagonist. These resultsindicate that AAV-TNFR:Fc gene delivery may be a validapproach for the treatment of RA.

AAV delivered ribozymes for retinal genetherapy and gene discovery

William W Hauswirth, Quihong Li,Jianwen Liu, Alfred S Lewin.

Departments of Molecular Genetics and Ophthalmology, andGene Therapy Center, University of Florida, Gainesville, FL,

USA

We tested two hypotheses: 1. AAV-delivered ribozymesagainst a mutant allele in a somatic retinal tissue canreduce the corresponding mRNA level sufficiently torescue visual function in an animal model for autosomaldominant Retinitis Pigmentosa in which rod cellsprogressively die over an 8-12 month period after birth.2. A similarly designed and delivered ribozyme against awild type allele can create a “somatic knockdown” andlead to a retinal disease phenotype. Recombinant AAV-vectored ribozymes against a mutant P23H rod opsingene in transgenic rats preserved 30-80% of thephotoreceptors that would have been lost at 8 and 3months respectively. Rescue was confirmed functionally

by electroretinographic (ERG) analysis, cellularly bypreservation of photoreceptor morphology andmolecularly by specific reduction in mutant mRNA levels.To test the converse idea, that ribozymes targeted againstwild type alleles might also create retinal disease, weattempted to produce Retinitis Pigmentosa (RP)-like roddegeneration in rd/+ mice that have an apparently normalretina at all ages. In the homozygous condition, the rd/rdmouse is a classical model for recessive RP, losing allrods within a 1-2 months. At 4 and 8 months postinjection,we found 50%-80% fewer rod photoreceptors in ribozymetreated eyes relative to PBS treated contralateral controleyes. ERG analysis confirmed that a profound functionalvision deficit had also been created that paralleled theloss of rod cells in the treated eye. This AAV-ribozymetechnology can now be applied to a broad range oftherapeutic and functional genomic questions.

AAV-mediated gene delivery in vivoRO Snyder,1 BA Donahue,2 S Patel,2 T Nichols,3 M Read,3 KMBrinkhous,3 CH Miao,4 MA Kay,5 MS Szczypka,4 RJ Mandel,6

SE Leff,7 RD Palmiter,4 N Vincent-Lacaze,8 O Danos,8 MEslami,1 S Gangadharan,1 K Rhynhart,1 M Conte.1

1Harvard Medical School, 2Cell Genesys Inc., 3University ofNorth Carolina, 4University of Washington, 5Stanford University,

6University of Florida, 7Emory University, 8Genethon.

Recombinant adeno-associated viral (rAAV) vectors wereused to deliver therapeutic and marker genes into liver,muscle, brain, and vascular endothelium. rAAV vectorsexpressing the canine factor IX (cFIX) gene wereadiministered to the livers of hemophilia B dogs. FactorIX protein levels of 30- 100 ng/ml were detected in theserum of dogs following a single infusion of rAAV into theportal vein as measured by ELISA and by aPTT assays.Expression of cFIX has remained stable for at least 26months with a sustained reduction in the whole bloodclotting time (WBCT),

rAAV vectors encoding the human Tyrosine hydroxylaseand human GTP-cyclohydrolase I genes were injectedinto the striatum of DA-/- mice. Transduction of neuronsrestored feeding behavior and independence from dailyL-DOPA injections which has persisted for at least 14months.

Studies were carried out to compare the transduction ofrabbit jugular veins by rAd and rAAV vectors expressingmarker genes. Adenoviral vector transduction results in-robust but transient gene expression and induces anearly increase in monocyte adhesion to the vein surface.rAAV-mediated gene expression is delayed but persistsfor more than 90 days (the duration of the experiment),and transduction of the endotlelium does not result inmonocyte adhesion.

Molecular analyses were performed in liver and muscleto determine the status of vector genomes over time. Aconversion of input vector genomes to a double-strandedhigh molecular weight form is concomitant with the slowrise in gene expression during the first 3-5 weeks post-


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transduction observed in both of these tissues.Conversion to double-stranded monomer genome formsis not rate-limiting in murine muscle, with circularintermediates detected at early timepoints.

Efficient early and sustained AAV-mediatedtransduction of human and rat fetal brain tissue

L Tenenbaum,1,2 F Bonnaud,4 A Chtarto,1,2 C.Melas,1,2

G Saber, A Stathopoulos,1,2 F Rodesh,3 T Velu,2

M Peschanski,4 M Levivier.1

1Lab. Experimental Neurosurgery & 2IRIBHN; 3DeptGynecology, U.L.B.-Hôpital Erasme, Belgium; 4INSERM U421,

Créteil, France.

The success of transplantation of human fetalmesencephalic tissue into the putamen of patients withParkinson’s disease is still limited by the poor survival ofthe graft. In animal models, administration of the glial-derived neurotrophic factor (GDNF) results in increasedthe survival and reinnervation potential of the graft 1,2.Viral vectors might achieve local and sustained deliveryof GDNF at physiologically relevant doses.

Here, we report that an AAV vector can mediate theexpression of the EGFP reporter gene under the controlof a CMV promoter in organotypic cultures of freshlyexplanted solid fragments of human and rat fetalmesencephalic tissue as early as 3 days to at least 6weeks post-infection. These results suggest that AAVvectors expressing GDNF could be used to genetically-modify the fetal tissue prior to transplantation in order topromote graft survival and integration.

Vectors expressing rat or human GDNF under the controlof the constitutive CMV promoter or the tetracycline-responsive element are currently evaluated. (1.Rosenblad, et al. Neuroscience 1996;75:979-994; 2.Sinclair SR, et al. NeuroReport 1996;7:2547-2552).

Parvovirus vector tranduced MCP-3 reduces tumorgrowth rate in nude-mice and prevents tumor

formation in immunocompetent mice. K Wetzel,1 P Menten,2 J Van Damme,2 HJ Gröne,3 JJ

Cornelis,1 N Giese,1 J Rommelaere,1 C Dinsart.1

1Tumor Virology and INSERM U375 and 3Cellular and MolecularPathology, German Cancer Research Center, Im Neuenheimer

Feld 280, 69120 Heidelberg, Germany; 2Laboratory ofMolecular Immunology, Rega Institute, 3000 Leuven, Belgium.

The CC-chemokine MCP-3 (monocyte chemotacticprotein) interacts with a broad spectrum of target cellse.g. monocytes, NK cells, T-lymphocytes and dendriticcells. We constructed H-1- and MVMp-based parvoviralvectors by replacing the capsid protein genes by humanMCP-3 cDNA in order to investigate its oncosuppressivepotential. At low multiplicities of infection (MOI) hH1/MCP-3 and MVMp/MCP-3 virus infected HeLa or B78melanoma cells, respectively, secrete high amounts ofMCP-3 in the cell culture medium. When implanted inswiss CD1 nu/nu mice, tumor growth from hH1/MCP3-

infected HeLa cells (at a MOI of 1 replication unit/cell)was significantly retarded compared to tumors from hH1/GFP- or mock-infected cells. This effect was associatedwith a strong infiltration of macrophages and invation ofdendritic cells as well as NK cell activation. Inimmunocompetent C57BL6 mice, a completesuppression of tumor formation was observed afterimplantation of in vitro MVMp/MCP-3 infected B78melanoma cells at MOI of 10. In established B78melanoma tumors, repeated injections of MVMp/MCP-3induced tumor growth arrest. We are now analysing themechanism underlying the antitumor effect of MVMp/MCP-3 in this tumor model.

AAV-mediated transduction and erythroid lineage-restricted, long-term expression of a normal human

βββββ-globin gene in hematopoietic cells fromhomozygous βββββ-thalassemic mice in vivo

MQ Tan, KY Qing, MC Yoder, A Srivastava. Indiana University School of Medicine, Walther Oncology

Center, Walther Cancer Institute, Wells Center for PediatricResearch, Indianapolis, IN, USA.

Adeno-associated virus 2 (AAV), a non-pathogenic humanparvovirus, has gained attention as a potentially usefulvector for human gene therapy. We have reportedsuccessful transduction of a human hematopoietic cellline with recombinant AAV vectors containing humanglobin genes in vitro (J Exp Med 1994;179:733-738; GeneTher 1996;3:223-229). We have also reported successfultransduction of primary murine hematopoietic low-densitybone marrow mononuclear (LDBM) cells with arecombinant AAV vector containing the human β-globingene promoter-driven human (β-globin gene along withan upstream HS2 enhancer element from the β-globingene locus control region (LCR) and its long-termexpression in vivo (J Virol 1997;71:3098-3104). However,both the transduction efficiency and the transgeneexpression were less than 10%. We have now performedAAV-mediated stable transduction of primary murinehematopoietic stem cells and evaluated whether high-efficiency, long-term, erythroid lineage-restrictedexpression of a human β-globin gene in vivo can beachieved. Murine bone marrow-derived primitive Sca-1+,lin- hematopoietic stem cells from homozygous β-thalassemic mice were transduced ex vivo with arecombinant AAV vector containing a normal human β-globin gene promoter-driven human β-globin gene alongwith the entire mini-cassette (HS2+HS3+HS4) of the LCRelement followed by transplantation into low-doseirradiated C57BL/W41 anemic recipient mice. In the firstset, DNA samples from peripheral blood of 4 of 9, and inthe second set, 7 of 9 transplanted animals, were PCR-positive six months post-transplantation. Twelve monthspost-transplantation, recipient mice were sacrificed andbone marrow samples were analyzed by semi-quantitative PCR to document that the transductionefficiency ranged between 10-50% compared with themouse endogenous β-actin gene. Semi-quantitative RT-PCR analyses revealed that the efficiency of the human

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β-globin gene expression ranged between 25-35%compared with the mouse endogenous β-globin gene.Peripheral blood samples from all positive recipient micewere fractionated to obtain enriched populations ofgranulocytes, lymphocytes and erythrocytes. PCRanalyses revealed the presence of the human β-globingene sequences in granulocytes and lymphocytesindicating multi-lineage reconstitution. However, only theerythroid population was positive following RT-PCRanalyses suggesting lineage-restricted expression of thetransduced human β-globin gene. Southern blotanalyses of total genomic DNA samples isolated frombone marrow cells also documented proviral integration.Taken together, these results provide further support forthe potential use of recombinant AAV vectors in genetherapy of β-thalassemia and sickle-cell disease.

Triple transduction with Adeno-Associated Virusvectors expressing tyrosine hydroxylase, aromatic 1-amino acid decarboxylase, and GTP cyclohydrolase I

for gene therapy of Parkinson’s disease Yang Shen, Shin-ichi Muramatsu, Kunihiko Ikeguchi, Ken-ichi Fujimoto, Dong-Sheng Fan, Matsuo Ogawa, Hiroaki

Mizukami, Masashi Urabe, Akihiro Kume, Ikuko Nagatsu,Fumi Urano, Takahiko Suzuki, Hiroshi Ichinose, ToshiharuNagatsu, John Monahan, Imaharu Nakano, Keiya Ozawa.

Division of Genetic Therapeutics, Center for MolecularMedicine, and Department of Neurology, Jichi Medical School,

Tochigi, Japan; Department of Neurology, Third Hospital ofBeijing Medical University, Beijing, China; Department of

Anatomy, School of Medicine and Institute for ComprehensiveMedical Science, Fujita Health University, Aichi, Japan; Avigen,

Inc., Alameda, CA, USA

Parkinson’s disease (PD), a neurological disease suitedto gene therapy, is biochemically characterized by a severedecrease in the dopamine content of the striatum. Onecurrent strategy for gene therapy of PD is the localproduction of dopamine in the striatum achieved byinducing the expression of enzymes involved in thebiosynthetic pathway for dopamine. We previously showedthat the coexpression of tyrosine hydroxylase (TH) andaromatic l-amino acid decarboxylase (AADC) using twoseparate adeno-associated virus (AAV) vectors resultedin a more effective dopamine production and moreremarkable behavioral recovery in 6-hydroxydopamine-lesioned parkinsonian rats, than the expression of THalone. Not only TH and AADC but also tetrahydrobiopterin(BH4), a cofactor of TH, and GTP cyclohydrolase I (GCH),a rate-limiting enzyme for BH4 biosynthesis, levels arereduced in parkinsonian striatum. In the present study,we investigated whether transduction with separate AAVvectors expressing TH, AADC, and GCH was effective forPD. In vitro experiments showed that triple transductionwith AAV-TH, AAV-AADC, and AAV-GCH resulted in greaterdopamine production than double transduction with AAV-TH and AAV-AADC in 293 cells. Futhermore, the tripletransduction enhanced the BH4 and dopamineproduction in denervated striatum of parkinsonian ratsand improved the rotational behavior of the rats moreefficiently than did the double transduction. Behavioral

recovery persisted for at least one year after stereotaxicintrastriatal injection. These results suggest that GCHas well as TH and AADC is important for effective genetherapy of PD.

In vitro studies of a P210BCR_ABL fusion domaincandidate DNA vaccine targeted to human dendriticcells by an rAAV vector: Potential for development ofa vaccine against Chronic Myelogenous Leukemia

(CML) J Sun,1 S Chatterjee,2 SJ Forman,1 D Senitzser,1 I

Sniecinski,3 KK Wong Jr.1,2

Department of 1Hematology and Bone MarrowTransplantation, 2Virology, and 3Transfusion Medicine, City of

Hope National Medical Center, Duarte, CA 91010, USA.

Chronic myelogenous leukemia (CML) is characterizedby a t(9;22) translocation which results in the expressionof chimeric, fusion transcripts consisting of cellular bcrand abl genes. BCR_ABL fusion transcripts andcorresponding oncoproteins are both necessary foroncogenesis, and unique to the leukemic clones.Furthermore, of hematologic malignancies, the protectiverole of host immune responses has been mostconvincingly demonstrated for CML. Thus, methods toexploit and specifically enhance these responses couldreduce the risk of relapse following hematopoietic stemcell transplantation (HSCT) for CML. As a strategy toaugment anti_leukemic immune responses, wedeveloped a candidate DNA vaccine based onrecombinant adeno_associated virus (rAAV) vectorswhich targets the P210BCR_ABL fusion region. Wepreviously demonstrated that primary human dendriticcells (DCs), the most potent antigen presenting cellscurrently known, could be transduced with rAAV vectors.An 835 bp cDNA fragment containing the P210BCR_ABLb3a2 variant fusion domain and flanking sequences wasinserted into an rAAV vector under control of the RSVpromoter, yielding pCWRF8. 293 cells transfected withpCWRF8 expressed an appropriate transcript, as wellas the predicted 25 KDa truncated protein. Using aninstitutionally approved protocol, PBMCs from healthydonors were primed and restimulated in vitro withautologous DCs transduced with vCWRF8 (DC/F8),vCWRAP, an equivalent control vector encoding humanplacental alkaline phosphatase (hu_PLAP)(DC/AP), orpulsed with a 25_amino acid peptide (Pb3)corresponding to the p210 BCR_ABL fusion domain (DC/b3). No specific responses were generated using DC/AP, the negative transduction control. In contrast, DC/F8was capable of priming autologous T cells to anequivalent extent as DC/b3. Proliferative responses weredemonstrable in three donors with MHC alleles previouslydescribed in the literature as capable of responding tothe BCR_ABL fusion domain, but not in two donors withdivergent alleles. Furthermore, DC/F8 were also capableof presenting immunogenic epitopes present insequences flanking the BCR_ABL fusion domain. Allantigen specific T cell lines generated to date werephenotypically CD4+ and Th1. Importantly, several T cell


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lines demonstrating antigen specific (BCR_ABL b3a2subtype), MHC restricted cytotoxicity were identified. Acytotoxic T cell clone obtained from a representative cellline primed with DC/F8 recognized a cytotoxic epitopepresented by HLA_DR 15 (DRB1*1501 allele). Thus, theconstruct developed herein may serve as an importantcandidate for gene-based immunotherapy of CML.

14. PARVOVIRUSES IN BIOLOGICAL APPLICATIONS

Recombinant capsids of densovirus as a platformfor the presentation of an epitope of the respiratory

syncytial virus (RSV) Maud David, Yi Li, Michel Trudel, Peter Tijssen.

INRS-Institut Armand-Frappier, Laval, QC, Canada H7V 1B7.

Vaccination has been the most efficient method to stopdiseases from humans and animals. There is acontinuous need to develop safer vaccines. Presently,most of the vaccines in use are made with live orinactivated virus. However, insufficient attenuation orincomplete inactivation always constitutes a risk for thehealth of humans or animals. The recombinantbaculovirus has been used successfully to express thecapsid proteins that form the empty particle capable offorming virus-like particles (VLPs). The densovirusisolated from the larvae Galleria mellonella (GmDNV) isa small non-enveloped eucaryotic virus with a capsid of25 nm. The genome consists of an ambisense linearmonocatenar DNA of 6kb. DNVs, which infect onlyinvertebrate cells, are not pathogenic for the vertebrates.The expression of the viral protein VP4 of the GmDNV ininsect cells using the baculovirus system allows toproduce VLP form by the self-assembly of the VP4. TheseVLPs are used as platform for the presentation of anepitope of the respiratory syncytial virus to the immunesystem. The RSV is the most important causal agent ofthe viral disease of the inferior respiratory track innewborns and infants. The surface G viral glycoprotein,which is the major viral epitope, was inserted at a strategicplace on the capsid, the VP4 C-terminus. The immunogoldanalysis by using RSV specific antibody showed that theepitope of the RSV is located at the outside of the VLPs.These particles have the possibility to induce a very strongimmune response against the pathogen, the RSV.Protective assays are in progress.

Non-permissive infection and transfection ofvertebrate cells by the Mythimna loreyi densovirus

(MlDNV) Mohamed El-Far,1 Yi Li,1 Gilles Fédière,2

Said Abol-Ela,2 Peter Tijssen.1

1INRS-Institut Armand-Frappier, Laval, QC, Canada H7V 1B7; 2Laboratory of applied virology (IRD), Faculty of Agriculture,

Cairo University, Egypt.

The Mythimna loreyi densovirus (MlDNV) is an interestingand promising agent for insect pest control. As for allbiological control agents, it is essential to investigate itseffect on vertebrates before the field applications. As afirst step in MlDNV inoculation studies, four differentvertebrate cell lines (L cells of mouse, COS-7, 293, andPT cells) were used. These cell lines were infected withthe wild type MlDNV or transfected with its infectious clone(pMl28), respectively, the expression of viral protein wasthen tested by the immunofluoresence (IF) method. NoIF positive cells were observed using specific antibody tothe MlDNV capsid. However, the infection and transfectionof Ld652 insect cells by the same doses of both viralparticles and the infectious clone, showed strong positiveresults. The infected Ld652 cells, which lost theirfibroblast shape, rounded, and detached from the flaskwalls, showed clear cytopathic effects (CPE). While, thoseCPEs were not observed on the four tested vertebratecell lines. To establish whether the block in expression ofviral proteins in vertebrate cells was due to thetranscription or the translation level, Northern blots wereperformed with mRNAs from both vertebreate andinvertebrate cells after 4 days infection or transfection.Those results are contradictory to the findings of Kurtsaket al (1969) who reported that the infection of L cells ofmouse with Galleria mellonella densovirus (GmDNV),which shares 91% of homology with the MlDNV, showedCPE and positive in IF. Experiments to study whether theviral genome integrates into the host cell chromosomeare in progress.

Development of a new UVC irradiation method toimprove the safety of biological products potentially

contaminated by parvoviruses P Caillet-Fauquet,1 M Di Giambattista,2 M-L Draps,1 Th

Branckaert,2 R Tiebout,2 R Laub.2

1Laboratoire de Virologie Moléculaire, Faculté de Médecine,Université Libre de Bruxelles; 2Central Fractionation

Department, Red Cross, Brussels, Belgium.

Careful selection of blood donors and increased donationscreening have considerably increased the safety of bloodtransfusion and blood derivatives as regards viruses.Nevertheless, the risk of transmitting a viral diseaseremains. This applies particularly to non-envelopedviruses. There are well-documented cases oftransmission of HAV or parvovirus B19 throughadministration of solvent-detergent-treated or dry-heatedblood derivatives or recombinant products.

In this work, we 1) screened plasma pools for B19, usingPCR to detect B19-DNA and anti-B19 antibodies specificto capsid and NS1 viral epitopes; 2) examined, in spikedplasma concentrates, the antiviral efficacy of continuous-flow UVC in a newly designed irradiator.

First, using nested PCR, we recorded a significant B19-DNA-positive reaction for 55% of the plasma samplestested in 1998, in parallel with the presence of largeamounts of specific anti-B19 antibodies recognisingconformational and linear epitopes of the capsid and the

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NS1 peptide epitope. These results highlight thenecessity of developing new virus inactivation methodsto enhance the safety of biological products.

As B19 does not replicate in vitro, MVMp was chosen asa model for validation of parvivovirus inactivation methods.MVMp is very resistant to various inactivation treatments;pasteurisation or severe dry heat treatment of spiked factorVIII result in a reduction of only 3 log10 in virus infectivity.The reduction in infectivity achieved with gammairradiation is only 2 log10. All these treatments also resultin loss of protein activity. In contrast, UVC irradiation giveshigh reductions in infectivity (approximately 7log10) at lowirradiation doses preserving the biological andimmunological properties of the proteins.

A new apparatus for UVC continuous-flow irradiation wasdesigned for possible inclusion in the productionprocess. The proposed new method is safe and effective,completely traceable, easy to perform, reproducible, GMPcompliant, and preserves the integrity of the final product.Continuous-flow UVC irradiation successfully inactivatedMVMp in different spiked blood products (Factor VIII,albumin, fibrinogen, and immunoglobulin). The methodwas also used to enhance the safety of recombinantproteins.

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