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Retroviruses
October 5,6,7 2010
RetrovirusesViral RNA Viral DNA
(integrated into host genome)Viral mRNA,
genome
Reverse (retro) transfer of genetic information
Usually, well adapted to their hosts
Endogenous retroviruses
Retroviridae
Oncovirinae
Lentivirinae
Spumavirinae
Avian leukosis viruses
Feline, bovine, human immunodeficiency viruses
Equine infectious anemia virus
Feline leukemiavirus
“Foamy” agents
A retrovirus virionenvelope (ENV)
nucleocapsidp27 (GAG)
genome (2 copies)
RNA dep. DNA pol(POL)
p15e
gp70
The viral genome (oncornaviruses)
LTR LTRGAG POL ENV
Long terminalrepeat
Group specific antigens(nucleocapsid)
RNA dependant DNApolymerase
Envelope proteins(type-specific antigens)
All three genes - GAG, POL, ENV - required for replication
Replicative cyclereceptor
uncoating
RNA
ds DNA
proteinsmRNA
viral dsDNA integratedinto host genome
genomic RNA
blockedreceptors
assemblypro-virus
Interference
FeLV-A
FeLV-B
FeLV-C
blockedreceptors for FeLV-A
FeLV-A infectedcell
receptor forFeLV-B
receptor for FeLV-C
receptor for FeLV-A
PseudotypesFeLV-A
FeLV-B
P
P
How do oncoviruses cause cancer?
Peyton Rouse and the cancer causing chicken virus
Rockefeller University 1912
The Rouse sarcoma virus genome
LTR LTRGAG POL ENV SRC
LTR LTRGAG POL ENV
D. Stehelin, H.E. Varmus and J.M. Bishop1976
DNA related to the transforming gene of avian sarcoma virus is present in normal avian DNA
Nature 260:170-173
Retroviruses and cancerTransduction of oncogenes
Activation of oncogenes
Inactivation of tumour-suppressors
LTRGAG POL
LTRsrc
LTRGAG POL ENV
LTR myc
LTRGAG POL ENV
LTRRb Rb
Other transduced oncogenes1. Sis - platelet derived growth factor (PDGF) cDNA by wooly monkey or cat in simian sarcoma and feline sarcoma viruses
Cell transformed by SSV
PDGF receptor
Sis protein
proliferation
Other transduced oncogenes
2. Erb. Epidermal growth factor receptor transduced by avian erythroblastosis and fibrosarcoma causing viruses
PP
P
EGRF receptorvErb
kinase domain
stim. phosp.site
inhib. phos. site
ligand binding site
Feline leukemia viruses
• Most common infectious cause of death in cats
• Cytoproliferative and cytosuppressive diseases
• High mortality in persistently infected cats (>80% in 3 yrs)
Endogenous and Type A, B, C viruses
• Endogenous sequences• Group A
• ubiquitous, in all infectious samples, non-virulent
• Group B
• in 40% of infectious samples, endogenous X FeLV-A, virulent
• Group C
• in 1% of infectious samples. Derived by mutation from A
LTRGAG POL ENV
LTR
LTRGAG POL ENV
LTR
LTRGAG POL ENV
LTR
LTRGAG POL ENV
LTR
Type A
Type B
Type C
feline sarcoma and acute leukemia viruses
LTR LTRoncogene
GAG POL
• defective viruses that transduce cellular oncogenes
Pathogenesisinfection by oral route
infection by bite
replication in tonsils
replication in draining lymphnode
bone marrow thymus Peyer’s patches
protective immune response inadequate immune response
recovery latency viremia
Consequences of viremia
cytoproliferative cytosuppressive
lymphosarcomas
fibrosarcomas
leukemias
anemia pancytopenia
immunosuppressionsecondary infections
chronic stomatitisgingivitisnon-healing skin lesionsrespiratory infectionsHaemobartonella felis
thymic
multicentric
alimentary
Diagnostic tests
• Antigen capture ELISA “FeLuke test” (p27)• Fluorescent antibody test• Virus neutralization• PCR
Disease progression and diagnostic tests
infection by oral routeinfection by bite
replication in tonsils
replication in draining lymphnode
bone marrow thymus Peyer’s patches
protective immune response inadequate immune response
recovery latencyviremia
ELISA sometimes +
ELISA + FAT +
epitheial cells(shedding)PCR on bone marrow
Prevention of FeLV infection
• Management• Vaccines
Management• Multi cat households or breeders
– Can spread rapidly– to solve problem
• test• isolate + cats• retest
– + -> + PI– + -> - consider -– - -> + retest 12 weeks later
– to maintain• test and quarantine before new introductions
• single cat +– retest– inform owner of consequences
Vaccination
• Inactivated virus or subunits– FeLV-A
– FeLV-A, FeLV-B, FeLV-C
– FOCMA
• Adjuvants• Vectored• In practical terms only FeLV-A needed• Most vaccines have comparable efficacy
Evaluation of vaccine efficacy
• challenge systems• “preventable fraction”• factors to consider
– number in study– criteria for persistent infection– method of challenge– number of controls with persistent infection
http://www.inspection.gc.ca/english/anima/vetbio/prod/felchae.shtml
A,B
A,B,C
A,B
A,B,C
A,B
A,B,C
A,B,C
Considerations for vaccination
• risk?
• test before vaccination
• vaccine site sarcomas
Treatment
• Antivirals (experimental)• Immunostimulants (may not be effective)
Feline immunodeficiency virus (lentivirus)
• 1 to 5% of domestic cats infected (older male cats more likely)
• 5 sub-types with considerable antigenic variation• May lead to AIDS like disease• High rate of infection in some felids (lions,
cheetahs) with no obvious sickness
Pathogenesisbite
(virus shed in saliva)
virus infects B-cells,T-cells and macrophages
B-cell proliferation(swollen lymph nodes)
reversed CD4:CD8 ratiosimmunodeficiency
(opportunistic infections, B-cell lymphomas)
inapparent inmost cats
variabletime
Diagnostic test
• ELISA (antibody capture)– antibodies (most infected cats have low levels of
circulating antigen)
– maternal antibodies - false +ves
Cat retroviruses and endangered felids
Iberian Lynxhttp://www.lynxexsitu.es
PLoS One. 2009;4(3):e4744. Epub 2009 Mar 9.
Equine infectious anemia virusswamp fever
(equine lentivirus)
• Per-acute (rare)• Acute/chronic• Inapparent - Asymptomatic carriers
CFIA - EIA page (fact sheet and stats)
Transmission
• Blood (>million viruses/ml)
– Tack, instruments
– Biting insects• Virus in all secretions
– venereal
– Milk, colostrum
– In utero
horse fly deer fly
Venereal transmission
http://www.inspection.gc.ca/english/anima/heasan/disemala/equianem/equianemfse.shtml
Tashjian, 1984, JAVMA, 184:282Transmission by injection of semen from EIA+ and during natural breedingTransmission by injection of colostrum and natural feeding of foals(Insect transmission can not be ruled out)
Transmission by insects
fly feeds uninterrupted
virus is inactivated before next blood meal
feeding is interrupted
fly bites horse nearby(< 100m)
Pathogenesis (chronic infection)
Fever and viremia
antigenic change
infectious phases
virus multiplies inleukocytes
immunecomplexes
anemia
thrombocytopenia
Secondary effects of thrombocytopenia
petechial haemorrhagesedema
Slides: Hugh Townsend
Asymptomatic carriers
infectiousphase
stress
viremia
or steroids
Diagnostic Tests
• Agar gel immunodiffusion (AGID or Coggin’s test)
• ELISA kit (replaced AGID, August 2008)• Horse inoculation• Immuno-blot• cELISA
Coggin’s test
antigen
referencepositivesera
test samples
False reactions on Coggin’s test
• False positive– Foal with passive antibody (6 months)
• False negative– Recently infected horse
Excerpts from CFIA Disease Control Manual of Procedures, 1998 (section 7)
• Blood (no anticoagulants) collected by accredited vet and tested by accredited laboratory
• If accredited vet suspects exposure must inform District vet
• If sample positive, the animal, other in-contact susceptible animals and epidemiologically linked animals(30 days) re-bled and sent to The Retrovirology Centre of Expertise, PEI
Follow up after lab conformation of a Reactor
• Owner notified
• Premises declared infected, cold brand reactor (R)
• Clinical examination of all reactors and exposed animals
• Destruction order for animals showing clinical signs and asymptomatic reactors not isolated
• Compensation (up to $2,750)
• In contacts or animals pastured in contiguous pastures tested (30 days prior to testing)
• Owner’s responsibility and movement restrictions
Animal movement restrictions removed when:
• Reactor removed
• Remaining animals negative on tests 40 days apart– (first test after removal of reactor)
Conditions of isolation• Must be asymptomatic• Must not be used for breeding purposes• Premises inspected at least 4times/yr unannounced• Separated from other susceptible animals• Completely fly proof, vestibuled doors• Isolation notice posted on door• Exercise only in enclosed fly proof arena attached to stable• Exercise outside
– in enclosed area – if daily high <0oC.– No non-reactors within 200 yards
• All bandages, syringes etc. incinerated
Pregnant mares and their foals
• Remove foal from infected area (under license) not later than 6 months
• Moved to place where there are no other susceptible animals
• Tested after 40 days in isolation• If positive(maternal antibodies) retested in 4 months• If positive treat as a reactor.
CFIA - EIA control program
CFIA - EIA page (fact sheet and stats)