Pathogenesis of Caprine ArthritisEncephalitis VirusCellular Localization of Viral Transcriptsin Tissues of Infected Goats
M. Christine Zink,*t Julie A. Yager,and Joseph D. Myers*From the Division ofComparative Medicine* and theDepartment ofPathology,t Johns Hopkins UniversitySchool ofMedicine, Baltimore, Maryland; and theDepartment ofPathology, Ontario Veterinary College,University ofGuelph, Ontario, Canada*
Pathologic specimens of 18 goats with classical le-sions ofcaprine arthritis-encephalitis (CAE) virusinfection were examined morphologically and byin situ hybridization using molecularly clonedCAEV deoxyribonucleic acid (DNA) to determinewhicb tissues and cells of naturally infected goatssupported virus replication. Large numbers ofcellswith viral transcripts were detected in inflamedbrain, spinal cord, lung, joints, and mammarygland. These cells were morphologically compatiblewith macrophages. Fewer cells with viral tran-scripts were seen in noninflamed tissues. ViralRNAwas identified in macrophagelike cells in lung,liver, spleen, and lymph nodes, in cells lining thevessels of brain and synovium, and in epithelialcells ofintestinal crypts, renal tubules, and thyroidfollicles. These data suggest that the cell tropism oflentiviruses may extend beyond the narrow bound-aries of lymphocytes and macrophages. (Am JPathol 1990, 136:843-854)
Lentiviruses are retroviruses that cause chronic diseasescharacterized by long incubation periods and insidious,slowly progressive clinical courses.1 Animal lentiviruseshave been known for several decades, but received rela-tively little attention until the 1980s, when the human im-munodeficiency viruses were identified as lentiviruses.23The lentiviruses compose a taxonomic group of patho-gens that include the human immunodeficiency viruses,HIV-1 and HIV-2,23 visna-maedi virus of sheep (visna),4caprine arthritis-encephalitis virus (CAEV),5 equine infec-tious anemia virus (EIAV),6 feline immunodeficiency virus
(FIV),7 bovine immunodeficiency virus (BIV)8 and immuno-deficiency viruses of Asian macaques9 and several spe-cies of African monkeys (SIV).10'11 Lentiviruses are knownfor their tropism for cells of monocyte-macrophage lin-eage, their dependence on exchange of body fluids fortransmission, and for causing persistent infection andchronic disease.
Caprine arthritis-encephalitis virus is widely dissemi-nated in the goat population in North America.12 Two ma-jor forms of disease due to CAEV have been recognized.Young goats (less than 1 year old) are afflicted with sub-acute multifocal, necrotizing encephalomyelitis, often ac-companied by diffuse interstitial pneumonia. In adults, dis-ease is generally manifest as chronic progressive arthritiswith proliferative lymphoplasmocytic synovitis.13 The le-sions of CAEV, particularly those of the central nervoussystem (CNS) and joint, are quite characteristic, and apresumptive diagnosis is usually made on the basis ofhistologic examination alone.
Epidemiologic evidence indicates that the virus istransmitted from an infected doe to its offspring by virus-infected cells in milk, although transmission among adultsis also thought to occur.12 Infected kids that do not de-velop clinical disease nevertheless remain infected for life.Some of these animals develop arthritis as adults.14 Fac-tors determining whether a persistently infected goat willdevelop disease of the juvenile or adult type are notknown.
The ability of lentiviruses to cause lifelong infectionsis due to their tropism for cells of the immune system,particularly cells of monocyte-macrophage lineage. Thetropism of a number of lentiviruses for these cells hasbeen confirmed in vivo, and macrophages containing viraltranscripts have been found in several tissues of naturalhosts. For example, HIV replicates in macrophages in the
Supported by NIH grants NS21916, NS12127, and RRO0130 and NSERCgrant OGP003107. Dr. Zink was supported by a Medical Research Councilof Canada Fellowship.
Accepted for publication November 27, 1989.Address reprint requests to Dr. M. C. Zink, Johns Hopkins University
School of Medicine, Division of Comparative Medicine, Retrovirus BiologyLaboratories, Traylor G-60, 720 Rutland Avenue, Baltimore, MD 21205.
843
844 Zink, Yager, and MyersAJPApril 1990, Vol. 136, No. 4
brain of humans with acquired immune deficiency syn-
drome (AIDS) dementia15 and alveolar macrophages are
the host cells for replication of visna-maedi in the lungs16and synovium'7 of sheep. In vitro studies on CAE virusreplication in macrophages have shown that expressionof the viral genome is dependent on the maturational stateof the cells; viral deoxyribonucleic acid (DNA) in mono-
cytes is not transcribed until the cells mature into macro-
phages.18 Restricted replication is a mechanism that al-lows the virus to remain undetected by other cells of theimmune system for long periods. That the immune cellsthemselves are host cells for virus replication probablycontributes to the inability of the hosts to clear the infec-tion.
In this study, we sought to determine which tissuesand cells of naturally infected goats supported virus repli-cation. Viral transcripts were detected in large numbersof cells that morphologically resembled macrophages ininflamed brain, spinal cord, lung, synovium, and mam-
mary gland. In addition, epithelial and cells in several unin-flamed organs also had viral transcripts. Viral replicationin cells other than macrophages suggests that CAEV hasa broader cell tropism than previously recognized. Wespeculate that these cells may play a role in the naturalhistory of the lentivirus in the host.
Methods
Animals
Pathologic specimens were obtained from the routinenecropsies of goats at the Ontario Veterinary College andthe Ontario Government Veterinary Laboratory ServicesBranch. Tissue sections from all goats with a presumptivediagnosis of caprine arthritis-encephalitis were examined(n = 18). Goats were included in this study if they hadmultifocal necrotizing encephalomyelitis, lymphocytic in-terstitial pneumonia, or chronic lymphoplasmacytic syno-
vitis. The study comprised 18 goats with CAEV; 8 were
kids and 10 were adults. The goats represented a varietyof breeds, including Toggenberg, Saanen, French Alpine,Nubian, and Angora. Two uninfected goats were used as
controls: one healthy kid goat and one adult goat, whichdied of parasitic pneumonia.
Preparation of Tissue Sectionsfor In Situ Hybridization
All tissues had been previously fixed in formaldehyde, de-hydrated, and paraffin-embedded. Tissue sections (6 M)were cut from all blocks, placed onto acid-treated, gelatin-coated glass slides, baked at 80°C overnight and depar-affinized with Histoclear (National Diagnostics, Manville,
NJ). The tissues were treated with 0.2 N HCI for 20 min-utes, rinsed in 2 X SSC (1 X SSC: 0.15 mol/l sodium chlo-ride, 0.015 mol/I sodium citrate), and digested for 15 min-utes with proteinase K (25 Ag/ml in 200 mM Tris-HCI, 40mmol/l CaCI2). Tissues then were washed in distilled wa-ter, acetylated with 0.13% acetic anhydride in 0.01 mol/ltriethanolamine, pH 8.0 for 10 minutes, dehydratedthrough a graded alcohol series, and air dried.
In Situ HybridizationWe used a molecular clone of caprine arthritis-encephali-tis virus DNA19 in a pBR322-derived vector, which con-sisted of the entire viral genome except 0.4 Kb at the 3'end of the env gene. The viral DNA was cleaved fromvector sequences with Hindlll. The resulting 9.0-Kbcloned CAEV-specific fragment was separated from thevector by electrophoresis in low melting point agaroseand purified by affinity chromatography (NACS Prepac,Bethesda Research Laboratories, Bethesda, MD) andethanol precipitation. The DNA was radiolabeled by nicktranslations' using [35S]dATP and [35S]dCTP (AmershamCorp., Arlington Heights, IL). DNAse concentration wastitrated to produce DNA fragments of approximately 40 to70 base pairs as previously described.21 Specific activi-ties of the radiolabeled DNA probes were greater than 7X 108 cpm/,ug.
The hybridization mixture consisted of radiolabeled vi-ral DNA at a concentration of 0.2 Ag/ml in a solution of50% formamide, 100 mg/ml dextran sulfate, 0.63 mol/lNaCI, 10 mmol/l Tris-HCI, 0.5 mmol/l Na ethylenediaminetetraacetic acid (EDTA) 0.02% polyvinyl pyrrolidone-40,0.02% Ficoll-400 1.5 mmol/l dithiothreitol (DTT), and 200A/ml sheared, denatured salmon sperm. Southern blot hy-bridization studies had shown that our purified clonedCAEV DNA insert was contaminated with plasmid se-quences at a concentration of 3 to 5%. We were con-cerned that this small amount of DNA would become la-beled in the nick translation reaction and would hybridizewith E. co/i sequences in tissues often contaminated withcoliforms (eg, intestine), thereby causing false-positivehybridization. Therefore, we routinely added a 1000-foldexcess of cold, nicked pBR322 to the hybridization mix-ture at a concentration of 10 Ag/ml to block hybridizationby any labeled contaminating plasmid sequences. Previ-ous experiments had shown that this technique waseffective in abrogating hybridization by radiolabeled plas-mid DNA in tissue sections.
The DNA in the hybridization mixture was denaturedby boiling and the tissue was overlaid with 30 Al of hybrid-ization solution, coverslipped, and incubated for 16 hoursat room temperature. After hybridization, the coverslipswere gently removed and the slides underwent four 30-minute washes in a solution of 1 X SSC, 0.13% Triton-X100, 1.25 mmol/l Na EDTA, and 1.25 mmol/l DTT. This
Tissue and Cell Tropism of CAE Virus 845AJPApril 1990, Vol. 136, No. 4
Table 1. Histologic Lesions and Presence of Viral Transcripts in Tissues ofCAEV-infectedJuvenile Goats
Age ClinicalID (mos) presentation Brain Spinal cord Lung Joints Other
1 1.5 Ataxia +/+ * +/+ -/+ NEt Bronchus-associatedlymphoid tissue
2 2.5 Ataxia +/+ +/+ +/+ NE Mycoplasma pneumoniae(M. ovipneumoniae)
3 3.5 Ataxia +/+ +/+ +/+ NE Mycoplasma pneumoniae(M. ovipneumoniae)
(Muellerius capillaris)First symbol designates presence or absence and extent of characteristic histological lesions of CAEV in the tissue; second symbol designates the
presence or absence of viral RNA by in situ hybridization.t NE, not examined.
was followed by a 20-minute rinse in 1 X SSC in 50%formamide at room temperature and a rinse in 1 X SSCfor 1 hour at room temperature. After a further 2-hour rinsein 0.6 mol/l NaCI, 10 mmol/l Tris-HCI, 1.0 mmol/l NaEDTA, and 1.25 mmol/l DTT at room temperature, thesections were dehydrated in graded alcohol solutionscontaining 0.3 mol/l ammonium acetate and air-dried.Slides were dipped in NTB3 autoradiographic emulsion(Eastman Kodak Co., Rochester, NY), air dried, and al-lowed to expose in a dark box for 2 to 5 days. After devel-opment of the emulsion, the sections were counter-stained with hematoxylin. Slides were examined by lightmicroscopy; the presence of viral RNA was indicated bysilver grains over cells.
Caprine arthritis-encephalitis virus-infected and unin-fected cultured goat synovial membrane cells and sec-
tions of spleen from uninfected and CAEV-infected goatswere used as positive and negative controls for every hy-
bridization reaction. In situ hybridization was also per-formed on multiple tissues from an uninfected kid goatand on tissues from an uninfected adult goat with chronicinterstitial pneumonia (due to a parasite, Muellerius capil-laris). Negative control studies were also performed us-
ing an irrelevant probe such as radiolabeled cloned hu-man cytomegalovirus DNA on goat tissue and using ra-
diolabeled cloned CAEV probes on tissues from otherspecies (eg, human).
Results
Clinicopathologic Correlation
Six of the eight infected kid goats studied (goats 1 through6) presented with classical signs of CAE with CNS dys-function, including ataxia, paresis, and progressive paral-
Table 2. Histologic Lesions and Presence ofViral Transcripts in Tissues ofCAEV-infectedAdult Goats
Age ClinicalID (yrs.) presentation Brain Spinal cord Lung Joints Other
9 7 Arthritis NEt NE +/+ * +/+ Lymphocytic enteritis,verminous pneumonia
11 3 Arthritis /+ -/+ +/+ Thyroid lymphocytic infiltrates12 6 Arthritis NE +/+ +/+ +/+ Verminous pneumonia13 3 Arthritis +/+ NE +/+ Lymphocytic pyelitis14 7 Scoliosis NE NE +/+ +/+ Lymphoplasmacytic enteritis
Verminous pneumonia15 3 Lymphadenitis NE NE +/+ Caseous lymphadenitis16 2 Ataxia / +/+ +/+ NE17 4 Pneumonia +/ NE +/+ -/- Lymphadenopathy, lymphocytic
mastitisVerminous pneumonia
18 2 Pneumonia NE NE +/+ NE MyocarditisPulmonary adenomatosis
First symbol designates presence or absence and extent of characteristic histologic lesions of CAEV in the tissue; second symbol designates thepresence or absence of viral RNA by in situ hybridization.
t NE, not examined.
846 Zink, Yager, and MyersAJPApril 1990, Vol. 136, No. 4
ysis (Table 1). On histologic examination, all of these ani-mals had multifocal areas of necrosis and malacia in boththe brain and spinal cord, accompanied by marked infil-tration of macrophages and lymphocytes. Five of thesekid goats had diffuse lymphoplasmacytic inflammation inthe periventricular white matter and/or the midbrain andfocal necrosis of the lateral white matter of the spinal cord.One kid had spinal cord involvement that was exclusivelypericanalicular. It was not possible to make an associationbetween location or severity of neurologic lesions andclinical findings because there was variability in the extentto which the brain was sampled at autopsy. Three of thekids presenting with neurologic dysfunction also hadchronic lymphocytic interstitial pneumonia.
Two animals less than 1 year of age (goats 7 and 8)did not present with classical signs of caprine encephalo-
i ni';l Figure 1. A: Spinal cord ofan affected kidgoat with loss ofarchitecture, and markedlymphocytic infiltration in Virchow-Robinspaces (arrows) and throughout the neu-ropil. Cells containing virus RNA can beseen (arrowheads) in areas of intense in-
t * flammation. (Goat5; X 60, H) Inset: Severalcells positive for CAE virus RNA (arrow-heads) adjacent to a neuronal cell body(n). (X240, H). B: Spinal cord from thesame goat hybridized with labeled cyto-
X w r- megalovirus (CMV) DNA (negative con-* . ~ trol) showing lack ofpositivity (X240, H).
myelitis. Neither of these goats had lesions typical of CAEin the brain or spinal cord. The incoordination of goat 7was a result of cerebellar dysplasia. Histologic examina-tion of the lungs from goats 7 and 8, however, revealedchronic lymphocytic interstitial pneumonia, typical ofCAEV infection. Thus, although disease in the young in-fected goats classically centered on neurologic tissue,pneumonia also occurred, either alone or as a complica-tion of CNS disease.
The major clinical manifestation of CAEV infection inadult goats was arthritis/synovitis. Seven of the 10 adultgoats examined (goats 9 through 15) had chronic arthritis(Table 2). Four arthritic goats (goats 9, 11, 12, and 14)also had chronic lymphocytic interstitial pneumonia andtwo had encephalitis (goat 13) or myelitis (goat 12). Threeadult goats (goats 16 through 18) did not have clinical or
Tissue and Cell Tropism of CAE Virus 847AJPApril 1990, VOL. 136, No. 4
A'-"~~~~~~A
44
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Figure 2. Cells positive for CAE virus RNAwere present within the lumens (arrow-bead) and on the endothelial surfaces (ar-rows) of vessels in inflamed CNS. (Goat 5;X340, H).
pathologic evidence of arthritis. One (goat 16) was ataxicand on postmortem examination was found to have se-vere necrotizing lymphocytic myelitis and interstitial pneu-monia. Thus, although this animal was 2 years old, it mani-fested clinical signs and pathologic changes of CAEV in-fection usually seen in kid goats. Two other adults (goats17 and 18) presented with clinical signs of pneumonia.On histologic examination, one of these animals (goat 17)also had periventricular lymphocytic cuffs in the brain.Thus, although CAEV classically causes arthritis in adultanimals, adult goats can have pneumonia and/or enceph-alomyelitis as in younger goats.
Other pathologic findings in adult goats included follic-ular hyperplasia of lymph nodes in goats 10 and 17 and
Figure 3. Noninflamed cerebellum hybrid-ized with labeled CAE virus DNA probeshowing occasional labeled cells in both ithe molecular (arrowhead) and granular +(arrow) layers (Goat 3; X200, H).
marked lymphocytic infiltration of other tissues such asintestine, kidney, lung, and thyroid in the absence of otherhistologic changes.
Pathologic Lesions and In Situ Hybridization
In this study, tissues that had classical lesions of caprinearthritis-encephalitis also were frequently positive by insitu hybridization (Tables 1 and 2). In some cases, therewere fewer viral RNA-positive cells than would be ex-pected from the severity of the histologic lesions. Thiswas particularly evident in sections of synovium and maybe a result of the activity of tissue RNAses during the inter-
IV 0
Ei w siQ t G t -', t s , -.eF~~~~~~f
848 Zink, Yager, and MyersAJPApril 1990, VoL 136, No. 4
Figure 4. Hybridization of sections ofnoninflamed brain with labeled CAE virusDNA showed grains overlying capillaries(arrows), suggesting either that vascularcells, such as endothelial cells orpericytes,contained virus RNA or that virus RNA-positive cells, such as macrophages, were adher-ent to the endothelium or passing throughthe vessel walls (Goat 11; X420, H).
Figure 5. A: Cells containing virus RNAwere present in areas ofperivascular (ar-rowhead) and peribronchial inflamma-tion and in cells at sites typically occupiedby alveolar macrophages (arrows). (Goat7; X 185, H) B: Lung sectionfrom the samegoat treated with labeled CMV DNA dem-onstrating lack ofpositivity in alveoli and inadjacentfocus ofinflammation (X380, H).
Tissue and Cell Tropism of CAE Virus 849AJPApril 1990, Vol. 136, No. 4
Figure 6. A: Spleen and other lymphoid or-gans commonly had virus RNA-positivecells in perifollicular zones (arrows).(Goat 7; X95, H) B: Spleen from the sameanimalprobed with labeled CMV DNA. Nopositive cells were seen (X 145, H).
val between death and tissue fixation. The viral RNA-posi-tive cells in areas of intense inflammation were large cellswith abundant cytoplasm and eccentric nuclei; morpho-logically, they resembled macrophages.
In affected CNS, viral RNA-positive cells were mostabundant in areas of inflammation and focal gliosis as wellas within and adjacent to perivascular cuffs (Figure 1).Commonly, blood vessels in affected brain and spinalcord contained cells that were strongly positive for viralRNA (Figure 2). These cells were present in the lumen andadherent to the vessel wall. Rare positive cells were alsoseen in the CNS of animals without lesions (goats 8, 10,and 1 1), in the molecular and granular layers of the cere-bellum (Figure 3), and in the thalamus, midbrain, and pi-neal gland. The morphology and location of these cellsand the absence of inflammation suggested that theywere either glia or small neurons. In several animals, hy-bridization occurred over capillaries in noninflamed CNS
(Figure 4), often adjacent to areas of inflammation. Suchpositivity may have indicated the presence of viral RNAin vascular endothelium, in pericytes, or in cells such asmacrophages that were adherent to the endothelium orpassing through the vessel. In the lung, virus RNA-positivecells were most commonly located in the peribronchialtissue, in foci of inflammation, and lining the interalveolarseptae in sites typically occupied by alveolar macro-phages (Figure 5). In synovium, as in the brain, viral RNAwas detected in macrophagelike cells in inflammatory foci.and in cells marginating in blood vessels and lining thevessel walls. Viral RNA was identified in cells surroundinglymphoid foci in the mammary gland.
Cells with viral RNA were commonly found in lymphoidtissues such as spleen (Figure 6) and lymph nodes in allanimals examined, even though these organs rarely hadevidence of histologic changes. In these tissues, positivecells were located in the zone immediately adjacent to
850 Zink, Yager, and MyersAJPApril 1990, Vol. 136, No. 4
Figure 7. Cells lining sinusoids in unin-flamed liver often had virus transcripts(arrows) (Goat 10; X380, H).
Figure 8. A: CAE virus transcripts were de-tected in thyroid follicular cells (arrows)and within theparenchyma ofuninflamedthyroid. (Goat 7; X320, H) B: Thyroid tissuefrom thesame goat hybridized with labeledCMVDNA demonstrating lack ofpositivityinfollicular epithelial cells (X380, H).
Tissue and Cell Tropism of CAE Virus 851AJPApril 1990, Vol. 136, No. 4
Figure 9. A: Cells with virus transcriptswere seen in renal tubular epithelium (ar-rows) in the absence of inflammation.(Goat 7; X400, H) B: Kidney section fromthe same goathybridized with labeled CMVDNA. No positive cells were identified.(X390, H).
I-_.%wI.., ' 1 s .'.
-4~~~~~~~~~~~~~~ ii:..
W .: : F * :1
Figure 10. In the small intestine, viral RNAwas identified in epithelial cells of thecrypts (arrows) (Goat 14; X425, H).
852 Zink, Yager, and MyersAJPApril 1990, Vol. 136, No. 4
lymphoid follicles and in the subcapsular sinus, locationswhere cells of macrophage lineage are found normally.22Positive cells were seen frequently in myelopoietic nestsin bone marrow.
Viral transcripts were often seen in noninflamed tis-sues, although in lower numbers than in inflamed tissues.For example, positive cells were consistently identified inthe liver in the absence of inflammation. These cells linedthe hepatic sinusoids and morphologically resembledKupffer cells (Figure 7).
A variety of endocrine organs including adrenal, thy-roid, and parathyroid occasionally harbored viral RNA-positive cells in the interstitium. In some cases, such as inthyroid, affected cells could be readily identified as theepithelial component (Figure 8). In the kidney, viral RNAwas present in tubular epithelial cells and in glomerulartufts, also in the absence of inflammation (Figure 9). Intes-tinal crypt epithelial cells were also positive in one animal(Figure 10). All tissues from uninfected goats that weretreated with labeled CAEV probes and from infectedgoats that were treated with labeled CMV probes werenegative.
Affected goats of both age groups had a variety ofconcurrent bacterial and parasitic infections, including C.pseudotuberculosis lymphadenitis (n = 2), Mycoplasmaovipneumoniae (n = 2), Muellerius capillaris pneumonia(n = 5), and intestinal coccidiosis (n = 1).
Discussion
This study of caprine arthritis-encephalitis virus in its natu-ral host confirmed the age-dependent nature of the dis-ease: arthritis in adults and encephalomyelitis in kids.However, strict adherence to this pattern was not alwaysfound. In inflamed organs, viral transcripts were most of-ten seen in cells morphologically compatible with macro-phages, the primary host cells for replication of the rumi-nant lentiviruses.16 Chemotactic factors produced in in-flamed tissue recruit macrophages (some of which maycarry the viral genome) to areas of inflammation inaffected brain, spinal cord, lung, synovium, and mam-mary gland. This would increase the virus load in inflamedtissues. The finding of cells with many viral transcriptsmarginating in vessels of encephalitic brain provided sup-port for such a mechanism.
Surprisingly, viral transcripts were also found, albeit infewer cells, in cells in a wide variety of noninflamed tis-sues. Whereas some of these infected cells appeared tobe of macrophage lineage (Kupffer cells in the liver, alveo-lar macrophages), infection was also apparent in othercell types, such as epithelial cells of the intestinal cryptsand renal tubules and in thyroid follicular epithelial cells. Anumber of recent studies provide evidence that the lentivi-ruses may replicate in a variety of cells other than macro-
phages and lymphocytes. Transgenic mice containingthe visna LTR (a conserved region of the genome contain-ing a viral promoter) linked to the chloramphenicol acetyltransferase (CAT) gene had CAT activity in brain and skinas well as in lymph node extracts and cultured peritonealand splenic macrophages, indicating that these tissuesare capable of supporting visna virus replication.23 In vivostudies on the cell tropism of HIV indicate that it may havea wide host cell range, replicating in endothelial cells ofthe brain24 and enterochromaffin cells of intestinalcrypts.25 Finally, epithelial cells that express the CD4 anti-gen are permissive for HIV replication.26
Although some virus RNA-positive cells were seen inalmost all tissues examined, inflamed target organs con-tained many more positive cells, suggesting an associa-tion between viral gene expression and inflammation. Fur-ther, the cells with viral transcripts in these inflamed areasappeared to be macrophages. The factors that influencethe development of inflammatory lesions in CAE arepoorly understood. However, in earlier studies wehad shown that a unique interferon is produced bylymphocytes co-cultivated with CAEV-infected macro-phages.27 Production of this cytokine is dependent onthe presentation of viral antigen by macrophages ex-pressing la antigen. The interferon causes further aug-mentation of expression of la.' This virus-positive and la-positive cell is thus in a state of constant antigen presenta-tion, a factor that probably perpetuates the inflammation.Lymphocytes trafficking through the CNS may encounterproductively infected, la-positive cells and initiate (or atleast augment) inflammation through the production andrelease of this cytokine.
Studies in our laboratory have shown that CAE viruscan pass directly from productively infected macro-phages to uninfected cells by fusion (Huso DL, unpub-lished data). To become infected via fusion, cells neednot have specific viral receptors. The flexibility of lentivi-ruses in infecting cells by both classical receptor-medi-ated endocytosis and by fusion may permit a wider hostcell tropism. The sporadic presence of viral transcripts inepithelial cells suggests that these cells may become in-fected by random fusion with infected macrophagespassing through the tissue.
The finding of many concurrent bacterial and parasiticinfections in these goats suggests that CAEV, like HIV,may compromise the immune system. Immunologic stud-ies have shown that visna-infected sheep in the terminalstages of disease have an inversion of T4/T8 lymphocyteratios in peripheral blood and affected joints.' It is thuspossible that, similar to AIDS in humans, diverse infectiousdiseases in CAEV-infected goats may result from com-promise of helper T-lymphocyte functions.
Caprine arthritis-encephalitis virus and HIV bear a num-ber of similarities. Both lentiviruses persist indefinitely andreplicate productively in macrophages, causing encepha-
Tissue and Cell Tropism of CAE Virus 853AJPApril 1990, Vol. 136, No. 4
lomyelitis, pneumonia, and lymphadenopathy. The dem-onstration of large numbers of viral transcripts in thesetissues suggests an association between viral gene ex-pression and the development of organ dysfunction. ThatCAEV apparently replicates in epithelial cells of a variety oftissues concurs with findings in HIV infections, and lendssupport to the concept that the cell tropism of the lentivi-ruses may extend beyond the narrow boundaries of lym-phocytes and macrophages.
References
1. Haase AT: The slow infection caused by visna virus. CurrTop Microbiol Immunol 1975, 72:101-156
2. Barre-Sinoussi F, Cherman JC, Rey F, Nugeyre MT, Cha-maret S, Gruest J, Dauguet C, Axler-Blin C, Brun-Vezinet F,Rouzioux C, Rosenbaum W, Montagnier L: Isolation of a T-lymphotropic retrovirus from a patient at risk for acquiredimmune deficiency syndrome (AIDS). Science 1983, 220:868-871
3. Clavel F, Guetard D, Brun-Vezinet F, Chamaret S, ReyM-A, Santos-Ferriera MO, Laurent AG, Dauguet C, KatlamaC, Rouzioux C, Klatzmann D, Champalimaud JL, Montag-nier L: Isolation of a new human retrovirus from West Africanpatients with AIDS. Science 1986, 233:343-346
7. Pedersen NC, Ho EW, Brown ML, Yamamoto JK: Isolationof a T-lymphotropic virus from domestic cats with an immu-nodeficiency-like syndrome. Science 1987, 235:790-793
8. Gonda MA, Braun MJ, Carter SG, Kost TA, Bess JW: Char-acterization and molecular cloning of a bovine lentivirus re-lated to human immunodeficiency virus. Nature 1987, 330:388-391
9. Daniel MD, Letvin NL, King NW, Kannagi M, Sehgal PK, HuntRD, Kanki PJ, Essex M, Desrosiers RC: Isolation of T-celltropic HTLV-111-like retrovirus from macaques. Science 1985,228:1201-1204
10. Murphy-Corb M, Martin LN, Rangan SRS, Baskin GB, Gor-mus BJ, Wolf RH, Andes WA, West M, Montelaro RC: Isola-tion of an HTLV-111-related retrovirus from macaques with sim-ian AIDS and its possible origin in asymptomatic manga-beys. Nature 1986, 321:435-437
11. Ohta Y, Masuda T, Tsujimoto H, Ishikawa K, Kodama T, Mor-ikawa S, Nakai M, Honjo S, Hayani M: Isolation of simianimmunodeficiency virus from African green monkeys andseroepidemiologic survey of the virus in various non-humanprimates. Int J Cancer 1988, 41:115-122
12. Crawford TB, Adams DS: Caprine arthritis-encephalitis: Clini-cal features and presence of antibody in selected goat pop-ulations. J Am Vet Med Assoc 1981,178:713-719
13. Cork LC, Hadlow WJ, Crawford TB, Gorham JR, Piper RC:Pathology of viral leukoencephalomyelitis of goats. ActaNeuropathol 1974, 29:281-292
14. Cork LC, Narayan 0: The pathogenesis of viral leu-koencephalomyelitis-arthritis of goats: I. Persistent viral in-fection with progressive pathologic changes. Lab Invest1980,42:596-602
15. Gabuzda DH, Ho DD, de la Monte SM, Hirsch MS, Rota TR,Sobel RA: Immunohistochemical identification of HTLV-lll an-tigen in brains of patients with AIDS. Ann Neurol 1986, 20:289-295
16. Gendelman HE, Narayan 0, Molineaux S, Clements JE,Ghotbi Z: Slow persistent replication of lentiviruses: Role oftissue macrophages and macrophage-precursors in bonemarrow. Proc Natl Acad Sci USA 1985, 82:7086-7090
17. Gendelman HE, Moench TR, Narayan 0, Griffin DE, Clem-ents JE: A double labeling technique for performing immu-munocytochemistry and in situ hybridization in virus infectedcell cultures and tissues. J Virol Methods 1985,11:93-103
18. Narayan 0, Kennedy-Stoskopf S, Sheffer D, Griffin DE,Clements JE: Activation of caprine arthritis-encephalitis virusexpression during maturation of monocytes to macro-phages. Infect Immun 1983, 41:67-73
19. Pyper JM, Clements JE, Molineaux SM, Narayan 0: Geneticvariation among lentiviruses: Homology between visna virusand caprine arthritis-encephalitis virus is confined to the 5'gag-pol region and a small portion of the env gene. J Virol1984,51:713-721
20. Rigby PW, Dickman M, Rhodes C, Berg P: Labelling deoxyri-bonucleic acid to high specific activity in vitro by nick transla-tion with DNA polymerase. J Mol Biol 1977,113:237-251
21. Moench TR, Gendelman HE, Clements JE, Narayan 0,Griffin DE: Efficiency of in situ hybridization as a function ofprobe size and fixation technique. J Virol Methods 1985,11:119-130
22. Sell S: Immunology, Immunopathology and Immunity. NewYork, Harper & Row, 1972, p 24
23. Small JA, Bieberich C, Ghotbi Z, Hess J, Scangos G, Clem-ents JE: The visna virus long terminal repeat directs expres-sion of a reporter gene in activated macrophages, lympho-cytes, and the central nervous systems of transgenic mice.J Virol 1989,63:1891-1896
24. Wiley CA, Schrer RD, Nelson JA, Lampert PW, Oldstone MB:Cellular localization of human immunodeficiency virus infectionwithin the brains of acquired immune deficiency syndrome pa-tients. Proc Natl Acad Sci USA 1986,83:7089-7093
25. Nelson JA, Wiley CA, Reynolds-Kohler C, Reese CE, Marga-retten W, Levy JA: Human immunodeficiency virus detectedin bowel epithelium from patients with gastrointestinal symp-toms. Lancet 1988,1:259-262
26. Chesebro B, Wehrly K: Development of a sensitive quantita-tive focal assay for human immunodeficiency virus infectiv-ity. J Virol 1988, 62:3779-3788
27. Narayan 0, Sheffer D, Clements JE, Tennekoon G: Restrictedreplication of lentiviruses: Visna viruses induce a unique inter-feron during interaction between lymphocytes and infectedmacrophages. J Exp Med 1985,162:1954-1969
28. Zink MC, Narayan 0: Lentivirus-induced interferon inhibitsmaturation and proliferation of monocytes and restricts the
854 Zink, Yager, and MyersAJPApril 1990, Vol. 136, No. 4
replication of caprine arthritis-encephalitis virus. J Virol 1989,63:2578-2584
29. Kennedy PGE, Narayan 0, Ghotbi Z, Hopkins J, GendelmanHE, Clements JE: Persistent expression of la antigen andviral genome in visna-maedi virus-induced inflammatorycells: Possible role of lentivirus-induced interferon. J ExpMed 1985,162:1970-1982
30. Kennedy-Stoskopf S, Zink MC, Narayan 0: Pathogenesis ofovine lentivirus-induced arthritis: Phenotypic evaluation of Tlymphocytes in synovial fluid, synovium, and peripheral cir-culation. Clin Immunol Immunopathol 1989, 52:323-330
Acknowledgments
The authors thank Dr. 0. Narayan, Dr. J. Clements, and Dr. K.Carbone, Departments of Comparative Medicine and Neurologyand Medicine, Johns Hopkins Medical Institutes, for scientific ad-vice and editorial assistance. Dr. T. Moench, Departments ofMedicine and Neurology, provided expert technical advice. Theauthors also thank the Department of Pathology, Ontario Veteri-nary College, and the Veterinary Laboratory Services Branch of theOntario Ministry of Agriculture and Food for pathologic specimens.
