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

FIV vaccine

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)