Retroviridae

1. Retroviridae • Spherical enveloped virions with an icosahedral capsid • 80-100 nm in diameter • Diploid genome – 2 identical haploid molecules, noncovalently linked at their 5’ ends • Each haploid segment is a linear positive sense ss RNA – Does not serve as mRNA immediately after infection • Virions have REVERSE TRANSCRIPTASE – RNA dependent DNA polymerase • Which transcribes DNA from virion RNA • Virion DNA integrates into cellular chromosome as a PROVIRUS

2. Retroviridae • Replication takes place in the CYTOPLASM AND NUCLEUS • Virions bud from cytoplasmic membrane • NO INCLUSION BODIES • Some retroviruses are ONCOGENIC – many produce LATENT INFECTIONS • Retroviruses are inactivated by detergents, but are MORE RESISTENT TO UV LIGHT THAN OTHER VIRUSES • GENERA • Alpha – avian leukosis virus • Beta – mouse mammary tumor virus • Gamma – murine leukemia virus • Delta – bovine leukemia • Epsilon – walleye epidermal sarcoma virus • Lentivirus – HIV 1 and 2 • Spumavirus –Foamy viruses

3. Retroviridae • Arrangement of the Retroviral Genomes • All retroviruses have gag, pol, and env genes • Some acquire an oncogene, and are defective in their replication • Gag gene – group specific antigen – encodes the virion core/capsid proteins • Pol gene – polymerase – encodes the reverse transcriptase • Env gene – envelope – encodes the envelope glycoprotein peplomeres • Replication • Adsorption, penetration by fusion or receptor-mediated endocytosis, penetration and uncoating. • Viral RNA is released into the cytoplasm • Parental RNA is copied to single stranded DNA - RNA:DNA hybrid – by reverse transcriptase. Single stranded DNA is made DOUBLE STRANDED again by reverse transcriptase

4. Retroviridae • Double stranded DNA moves to the nucleus, becomes circularized, and then is integrated as a provirus at different sites in the cellular DNA • Provirus is replicated with host genome and is passed to daughter cells – type III spread • Needs host cell polymerases – provirus is a template for making mRNA for protein synthesis and positive sense RNA molecules which are encapsidated into progeny virions • Maturation of virions occurs by budding through plasma membrane

5. Retroviridae

6. Morphologic Classes of Extracellular Retrovirus Particles

7. Oncogenic Retroviruses • Major cause of leukemias, lymphomas, and sarcomas in many animal species • Malignant mesenchymal tumors are known as sarcomas • Malignant leukocyte tumors are lymphomas – if solid tumor ; leukemia if circulating cells are involved • Antibodies against epitopes during maturation are cytotoxic – ADCC and may prevent tumor formation • Acute- transforming viruses – • Viral genome has v-onc + • This is associated with deletions elsewhere in the genome – most v-onc+ viruses are replication defective, EXCEPT ROUS SARCOMA VIRUS

8. Oncogenic cont. • The viruses produce tumors shortly after infection in a high percentage of infected hosts – e.g. feline sarcoma virus • Chronic transforming viruses – • V-onc- viruses that cause cancers late after infection and in only a relatively low percentage of infected hosts

9. Bovine Leukemia/Enzootic bovine leukosis • Lymphosarcoma of the mammary gland • Highly fatal, systemic, malignant neoplasia of the reticuloendothelial system of cattle • Characterized by the development of aggregations of neoplastic lymphocytes - various organs • Worldwide • Etiologic agent – DELTARETROVIRUS – VONC- • Transmission – close, prolonged contact; primarily by transfer of blood lymphocytes between animals – found in blood, milk, tumor masses • Blood volumes capable of transmission = 0.1mL • Transfusions, trauma, needles, mechanical vectors - arthropods

10. Bovine Leukemia • Mediastinal lymph nodes associated with virus • Vertical transmission occurs in utero or through colostrum and milk, accounts for a small proportion of infections – less than 10% • Pathogenesis – B lymphocytes are the major targets • Four possible outcomes of infection • 1. Genetic resistance – no infection • 2. Asymptomatic infection – antibody production, seropositive 4-12 weeks later • 3. Permanent infection, persistent lymphocystosis – benign proliferation of B lymphocytes – 33% of cases • 4. Infected, seropositive animals – lymphosarcoma LESS THAN 1-2%; occurs in cattle between 4-8 years of age

11. Bovine Leukemia Clinical Signs, Dx, Prevention and Control • Clinical signs vary depending on organ/tissues affected • Weight loss, anorexia, decrease in milk production are common • Diagnosis – BLV infection must be distinguished from clinical disease? • Clinical hx and histopathologic examination of affected tissues • Serology – persistent infection leads to marked increase in antibody – detected easily • Agar gel immunodiffusion test – AGID, ELISA, Western Blot, IFA are used • Prevention and control – EBL can be eradicated from a herd by repeated serological testing of animals over 6 months of age at 2-3 months intervals

12. Sporadic Bovine Leukosis - SBL • SBL is a NONINFECTIOUS LYMPHOSARCOMA seen in cattle under 3 years of age • Etiologic agent is UNKNOWN • It occurs in three forms: • Calf form – less than six months old, multiple lymph node enlargement • Thymic form – between 6 months to 2 years of age – characterized by a swelling in the neck, causing bloat and edema • Cutaneous form – seen in cattle 1-3 years of age and characterized by the development of nodes and plaques in the skin. Uncommon

13. Feline Leukemia and Sarcoma • FeLV is a naturally occurring, contagiously transmitted virus associated with neoplastic and non-neoplastic disease • Profound anemia • Immunosuppression • Enteritis • Reproductive failure • Worldwide • Hosts – domestic and wild cats • Etiologic agent –gammaretrovirus • Noncytopathic and v-onc-

14. Feline Leukemia and Sarcoma • Neoplastic growth associated with feline sarcoma • Major viral proteins include: • P27, gp70 • P27 protein – structural component of the inner viral core; major FeLV virus – group specific antigen • Produced within leukocytes and platelets, excreted in saliva and tears, and found free in plasma • Detected by IFA and ELISA tests

15. Feline Leukemia and Sarcoma • Kidney lesions – Feline Sarcoma • Gp 70 protein – • Envelope protein responsible for viral attachment; based on differences in gp70 protein, there are 3 subgroups of FeLV • Neutralizing antibodies against gp70 proteins protect against viremia; they are subgroup specific • Subgroup A – in all viremic cats • Either alone or in combination with subgroup B and/or C. Although A can cause malignancy alone, in combination with B, may have a synergistic effect on oncogenicity

16. Feline Leukemia and Sarcoma • Mediastinal lesions – Feline Sarcoma • Only subgroup A is transmitted from cat to cat • B and C evolved by recombination between subgroup A and endogenous proviral sequences contained in normal feline DNA • Subgroup B – Not pathogenic, but may enhance A • Subgroup C – associated with nonregenerative anemia • p15E – enveloped protein associated with FeLV-induced immunosuppression. Suppresses lymphocyte blastogenesis – blocks the response of T cells to IL-1 and IL-2 and suppresses the response of cats to FOCMA

17. Feline Leukemia and Sarcoma • Ocular lesions – Feline Sarcoma • FeLV is extremely labile once outside cat – rapidly inactivated by household disinfectants • Dry environment – inactivated in 3-5 mins • Moist environment – may survive for 24-48 hours at room temp • Virus maintained in nature due to viremic cats living for several years with constant viral shedding. If all FeLV positive cats are removed from household, a new cat may be safely added after waiting 3-4 weeks

18. Feline Leukemia and Sarcoma • Transmission – prolonged, direct exposure is usually required for transmission. Viremic cats shed virus continuously • Occurs via saliva • Virus concentration is very high. Even though virus multiplication occurs in several tissues and organs, it is less likely to spread in feces, urine and fleas • Iatrogenic transmission – occurs via contaminated needles, instruments, fomites, or blood transfusion • In utero –infections occur but more kittens are infected when the queen licks them and nurses them • Pathogenesis – multiplies in T and B lymphocytes and myeloid cells

19. Feline Leukemia and Sarcoma • Pathogenesis – • Following oronasal exposure, the virus replicates in local lymphoid tissues • A low grade transient viremia occurs, spreading the virus to the spleen, lymph nodes, intestines and bone marrow – ELISA test is + • Infection of leukocyte and platelet precursors in the bone marrow and the subsequent release of infected cells into the circulation, results in a persistent secondary viremia. The IFA test is positive at this point • In persistently viremic cats, infection proceeds to extensive involvement of the bone marrow, pharynx, esophagus, stomach, bladder, respiratory tract, and salivary glands

20. Feline Leukemia and Sarcoma • FOCMA antigen – feline oncornavirus cell membrane antigen – This is a tumor-specific antigen present ONLY ON THE MEMBRANE OF CELLS TRANSFORMED BY EITHER FeLV or FeSV • FOCMA antibody lyses tumor cells via ADCC and complement activation. Thus, cats with high FOCMA antibodies are resistant to the development of leukemia and lymphoma, regardless of whether they are positive or negative for FeLV • FOCMA antibody does not neutralize virus, hence cats with increased FOCMA antibody titers may still be viremic and die of nonmalignant disease

21. Feline Leukemia and Sarcoma • Clinical and Pathological features – • IP – 3 months to 3 years • Within 6 weeks of infection, 1 of 3 host-virus relationships develops: • 1. Self limiting infection • 2. Persistent active infection • 3. Latent infections • Self-limiting infection – Majority of cats 92-96% • Develop neutralizing and FOCMA antibodies; no viremia and cat does not shed virus • Does not develop FeLV related disease, however, because a DNA copy of the FeLV genome stably integrates into the host cell chromosomal DNA, malignant transformation some time in the future cannot be excluded

22. Feline Leukemia and Sarcoma • Persistent active infection – • Persistent viremia, serum lacks neutralizing and FOCMA antibodies; terminates in FeLV-related disease • Immunosuppression – most common sequel to persistent FeLV viremia and accounts for most FeLV-related deaths • Latent infections – • Cat’s immune system may not overcome the infection, but not show clinical signs either. While virus is latent, the cat DOES NOT SHOW FELV SIGNS AND DOES NOT SHED VIRUS, so there is no risk of infection spread • Most remain asymptomatic, occasionally due to stress, disease, pregnancy or new household cat, they convert to PERSISTENT VIREMIA • Latent infections are detected following bone marrow culture and reactivation

23. Feline Leukemia and Sarcoma • Neoplastic Diseases • All hematopoietic cell lines are susceptible to transformation by the virus • Lymphoid and myeloid – granulocytic, erythroid and megakaryocytic types occur • Lymphosarcoma – malignant lymphoma – consists primarily of solid masses of proliferating lymphocytes. Accounts for 30% of all feline tumors. • Three major forms are described on the anatomic location of the tumors: • Multicentric – tumors are observed in multiple lymphoid and nonlymphoid organs and tissues. Predominantly, T cell tumor • Thymic/ mediastinal lymphosarcoma – kittens primarily – T cell tumor • Alimentary lymphosarcoma – older cats, lymphoid tissues of the GIT – GALT and/or mesenteric lymph nodes. B cell tumor • Cats are usually feline leukemia negative

24. Feline Leukemia and Sarcoma • Myeloproliferative disease – primary bone disorders characterized by abnormal proliferation of one or more hematopoietic cell lines. • The diseases are characterized by the presence of large numbers of neoplastic cells in the bone marrow, a nonregenerative anemia and immunosuppression • Specific types of myeloproliferative diseases: • Erythromyelosis – Erythroid cell lines • Granulocytic leukemia – granulocytic myeloid cell, usually a neutrophil • Erythroleukemia – both erythroid and granulocytic myeloid precursors become neoplastic • Myelofibrosis – proliferation of fibroblasts and cancellous bone resulting in medullary osteosclerosis and myelofibrosis

25. Nonneoplastic Diseases • Immunopathological diseases: • Immune complex diseases – Circulating immune complexes predispose cats to glomerulonephritis and polyarthritis • Immunosuppression - persistently viremic cats suffer from a severe lymphopenia – due to a loss of circulating T cells – neutropenia or both • Hypocomplementemia due to immune complex hypersensitivity; reactivation of latent infections; increased susceptibility to secondary infections. • Some commonly observed conditions include chronic gingivitis and stomatitis, feline infectious peritonitis, hemobartenollosis etc. • Reproductive disorders: infertility, resorption or abortion of fetuses, and endometritis have been observed in viremic queens • Most kittens born to viremic queens become viremic and die at an early age – FADING KITTEN SYNDROME – characterized by a failure to nurse, hypothermia, and thymic atrophy within the first two weeks of life

26. Diagnosis – Feline Leukemia… • Virus isolation is rarely attempted • FeLeuk test – detects the group specific p27 within the viral core • ELISA – tests for presence of circulating p27 antigen in plasma, whole blood, serum, saliva or tears • IFA test –tests for p27 antigen associated with WBCs and platelets in fixed blood smears – 97% of all IFA positive cats remain positive for life • Both ELISA and IFA detect viremia and a positive test only indicates increased risk of FeLV – related diseases • Control – FeLV positive cats can spread disease – they may or may not develop the disease themselves • IFA positive should be isolated or euthanized • Vaccination – two inactivated whole virus vaccines and a genetically engineered vaccine available – reduce incidence of disease by 80%

27. Feline Sarcoma - FeSV • FeSV is associated with multiple fibrosarcomas in young cats. The virus arises de novo as a result of recombinational event between FeLV and host cell chromosome – acute transforming viruses • FeSV is v-onc+, a defective virus • Replication requires a helper FeLV because a portion of its genetic information – env gene – is lost during recombinaion • ALL STRAINS CAUSING FIBROSARCOMAS ARE PSEUDOTYPES WITH ENVELOPE PROVIDED BY FELV. • Clincal Features – Fibrosarcomas express FOCMA antigens • They are locally invasive and metastasize to the lungs and other sites – • solitary fibrosarcomas in old cats older than 5 years of age – not caused by FeSV

28. Avian Leukosis • The viruses causing avian leukosis are endemic in all chicken populations worldwide. Incidence is 3-20% in most chicken populations. • Etiologic agent – Alpharetrovirus • Virus is v-onc- • Viruses are classified into 5 subgroups on the basis of their host range in chicken cells of differing genetic types and viral envelope antigens • Subgroups A and B – Most important in field outbreaks • Subgroups C and D – Recovered infrequently • Subgroup E – Genetically inherited – endogenous nononcogenic virus

29. Avian Leukosis – visceral lesions • Transmission – • congenitally via the egg – vertical – or horizontally with the first few days of life (less than five days) • The chicken becomes viremic for life because of the development of immunological tolerance. • The birds excrete virus in the saliva and feces • If horizontal transmission occurs beyond 5/6 days after hatching, the chicken develops neutralizing antibodies • The viremia is transient, and the chicken is unlikely to develop leukemia

30. Avian Leukosis • Pathogenesis – • The primary target cells are lymphoblasts with B lymphocyte markers in the bursa of Fabricius ; Bursectomy prevents the development of lymphoid leukosis • Clinical Features – Disease occurs sporadically in birds over 14 weeks of age • 1. Lymphoid leukosis – synonym – visceral lymphomatosis – big liver disease • Observed in chickens 14-30 weeks of age • Lymphoid cell infiltrations of various organs e.g. liver, spleen, kidney, bursa of Fabricius etc. • 2. Osteopetrosis – thick leg – characterized by a proliferation of the periosteal osteoblasts of the long bones of the limbs. Nonneoplastic, bilateral thickening • Diagnosis and control: • Flock history and tumors in multiple organs without nerve and ocular involvement; terminate breeder flocks

31. Genus Lentivirus – slow - FIV • Associated with chronic infections characterized by persistent viremia • Antigenic variation – a result of frequent random mutations – is commonly observed in lentivirus infections • Feline Immunodeficiency Virus • Etiologic agent – domestic cats and wild felidae – cheetahs • Four subgroups – based on enveloped proteins – Subgroups A,B,C,D • NO CROSS PROTECTION AMONG SUBGROUPS • Transmission – virus is shed mainly in the saliva. Infection is lifelong. • Via cat bites, disease most common in free-roaming cats and uncommon in closed purebred catteries • One bite sufficient to transmit the virus • In utero and postparturition to kittens via colostrum and milk

32. Ovine lentivirus • Lung lesions • Not needed for this course, but it is a virus

33. FIV Pathogenesis • Pathogenesis – • Hallmark of FIV is progressive disruption of normal immune function • Virus has tropism for helper T cells, brain macrophages, peritoneal macrophages, and astrocytes • Immunosuppresssion subjects the cat to opportunistic infections like Toxoplasmosis, Generalized demodectic mange, fungal diseases and FIP • Clinical Features -More common in cats 3-5 years of age • Acute stage – Fever, malaise, generalized lymphadenopathy, and complicating bacterial infections of the skin • Latent stage – Nonsymptomatic; may range from months to years • Terminal stage – variety of clinical signs that result from opportunistic bacterial and fungal infections e.g. chronic stomatitis and gingivitis, chronic respiratory disease, chronic diarrhea and wasting, dermatitis, neurologic signs

34. FIV Diagnosis • Diagnosis – • Detection of FIV specific antibodies in the blood using IFA, ELISA or Western blot • Antibodies appear in 2-4 weeks of infection and persist for life • If positive results are found in kittens less than 12 weeks old • Kittens must be retested after the age of 12 weeks to assure that antibodies detected were not maternally derived • Detection of FIV core antigen, p24, by ELISA or proviral DNA by PCR in peripheral blood leukocytes of infected cats • Control – Preventing contact between FIV negative and FIV positive cats • Many vaccines, but efficacy is in doubt • Cat to human transmission – virus is thought to be specie-specific. FIV antibodies have not been detected in people having FIV infected cats, or in people having inadvertently injected themselves with virus-containing materials

35. EIA • EIA in Equidae is characterized by a long relapsing illness after an initial acute attack • Etiologic agent – Equine lentivirus – one serotype • Transmission – occurs via transfer of blood cells from an infected horse • Mechanical transmission – by tabanids, stable flies, mosquitoes and probably Culicoides • Virus is infective for up to 30 minutes on a fly’s mouth parts • Vertical transmissions via placenta and through colostrum and milk

36. EIA – Swamp Fever • Ventral edema – chronic form • Pathogenesis – replicates in macrophages and lymphocytes • Lifelong, cell associated viremia develops in all infected horses • Persistent Ag-Ab complexes – immune complex hypersensitivity – result in damage to vascular endothelium – vasculitis – followed by inflammatory changes in parenchymatous organs – LIVER • Vasculitis in the CNS results in ataxia, spinal leptomeningitis and encephalomyelitis • Anemia – viral antigens adsorb to RBC, bind with EIA antibody, and trigger erythrophagocytosis by mononuclear phagocytes; complement-mediated hemolysis – type II hypersensitity

37. EIA diagram – pathogenesis cont. • Glomerulonephritis – • Results from immune complex hypersensitivity • Clinical Features – IP 2-43 weeks but can be up to 3 months • Acute – fever, severe anemia, jaundice, blood stained feces, tachypnea and petechial hemorrhages of the mucosae • Mortality rate is 80% • Subacute – moderate fever – recovery, may recur • Chronic – mild signs and failure to thrive and episodic persistent fever, cachexia and ventral edema

38. EIA – Coggins test • Agar gel immunodiffusion • Diagnosis – agar gel immunodiffusion test – AGID, Coggins test • Detects antibodies to the major group specific antigen of EIA virus • Results are valid for 1 year from the date the blood is collected • Detects all infected animals except those in early incubation period, the first 2-3 weeks after infection. A suspected horse should be retested in 4-6 weeks • Foals that have been nursed by infected dams may be temporarily positive, however, they should be negative by 6 months of age if not infected

39. EIA • Control – • Identifying positive horses by the Coggins test • Destruction – NOT MANDATORY – or isolation of positive reactors • Horses imported to the USA and some other countries are required to have a negative test certificate.

40. Caprine Arthritis- Encephalomyelitis - CAE • Swelling of the right stifle • CAE is characterized by polyarthritis- most common, mastitis, encephalomyelitis and interstitial pneumonia • Worldwide • 1981 survey – USA – 81% of goats are seropositive • Etiologic agent – Caprine lentivirus • Transmission – colostrum and milk from the doe to newborn • Pathogenesis – virus multiplies in monocytes and macrophages

41. Caprine Arthritis – Encephalomyelitis • Arthritis – The form seen most often clinically - seen in goats 12 months or older • It is characterized by bilateral or unilateral swelling of mostly carpal joints. Other joints such as the stifle, hock, fetlock, shoulder and vertebral joints, may affected • Pain and thickening of the joint capsules restrict the movement of the goat • Advanced cases are accompanied by emaciation, rough hair coat, and carpal hygroma

42. Caprine Arthritis – Encephalomyelitis • Encephalomyelitis – occurs most often in goats 1-5 months of age • Progressive leukoencephalomyelitis associated with ascending paralysis • Goats are afebrile, alert, and maintain a good appetite. • Severe signs upward deviation of the head, twisting of the neck, and paddling movements of the feet. • Irreversible signs • Interstitial pneumonia – Mostly in adults • It is insidious in onset and is progressive. There is a gradual weight loss and respiratory distress • Mastitis – presents as a diffuse swelling of the mammary gland with associated firmness – fibrosis • Diagnosis – clinical and serologic evaluation – detection of CAEV antibodies using agar gel immunodiffusion test –AGID, ELISA, IFA test

43. CAE • Control – • All kids must be immediately removed form the dam at birth and provided colostrum from known virus-free does • Colostrum from CAEV-positive does can be used if it is treated at 56 degrees C for 1 hour • Alternatively, kids can be reared on pasteurized milk • Test and removal – Goats are tested every six months and those testing positive are removed • A positive serologic result indicates a goat is infected for life and a potential continual source for transmission of the virus