Prospects in Innate Immune Responses as Potential Control Strategies against Non-Primate Lentiviruses.

Lentiviruses are infectious agents of a number of animal species, including sheep, goats, horses, monkeys, cows, and cats, in addition to humans. As in the human case, the host immune response fails to control the establishment of chronic persistent infection that finally leads to a specific disease development. Despite intensive research on the development of lentivirus vaccines, it is still not clear which immune responses can protect against infection. Viral mutations resulting in escape from T-cell or antibody-mediated responses are the basis of the immune failure to control the infection. The innate immune response provides the first line of defense against viral infections in an antigen-independent manner. Antiviral innate responses are conducted by dendritic cells, macrophages, and natural killer cells, often targeted by lentiviruses, and intrinsic antiviral mechanisms exerted by all cells. Intrinsic responses depend on the recognition of the viral pathogen-associated molecular patterns (PAMPs) by pathogen recognition receptors (PRRs), and the signaling cascades leading to an antiviral state by inducing the expression of antiviral proteins, including restriction factors. This review describes the latest advances on innate immunity related to the infection by animal lentiviruses, centered on small ruminant lentiviruses (SRLV), equine infectious anemia virus (EIAV), and feline (FIV) and bovine immunodeficiency viruses (BIV), specifically focusing on the antiviral role of the major restriction factors described thus far.

Microtubule-dependent retrograde transport of bovine immunodeficiency virus.

Microtubules are essential components of the cytoskeleton that participate in a variety of cellular processes such as cell division and migration. In addition, there is a growing body of evidence implicating a role for microtubules in intracellular viral transport. In this study, we found that pharmacological disruption of microtubules remarkably blocked bovine immunodeficiency virus (BIV) movement from the cell periphery to the perinuclear region, a process known as retrograde transport. A similar effect was observed by inhibiting function of the microtubule-associated motor protein dynein. By yeast two-hybrid assay, we found that the capsid protein (CA) of BIV interacted with the dynein light-chain component LC8. Immunoprecipitation and GST-pulldown assays further demonstrated an interaction between CA and LC8 in mammalian cells. In addition, our data revealed LC8 as a linker between BIV particles and microtubules. Retrograde transport of BIV was significantly inhibited by knockdown of LC8 expression. Our findings present the first evidence that incoming BIV particles employ host microtubule/dynein machinery for transport towards the perinuclear region. In addition, our data indicate that the LC8-CA interaction is a potential target for the design of antiviral strategies.

BTat, a trans-acting regulatory protein, contributes to bovine immunodeficiency virus-induced apoptosis.

Bovine immunodeficiency virus (BIV) is a member of the lentivirus subfamily of retroviruses highly related to human immunodeficiency virus in morphologic, antigenic and genomic features. BIV is known to induce chronic pathological changes in infected hosts, which are often associated with the development of immune-mediated lesions. However, the molecular events underlying the cytopathic effect of BIV remain poorly understood. In this study, BIV was found to induce apoptotic cell death, and a small trans-acting regulatory protein encoded by BIV, BTat, was found to participate in the pro-apoptotic action of BIV. Introduction of exogenous BTat to cells triggered apoptosis dramatically, as revealed by assays such as terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling, nuclear morphology analysis, flow cytometry, and cleavages of caspases and poly(ADP-ribose)polymerase. Interestingly, the pro-apoptotic effect of BTat was found to be mediated through its interaction with cellular microtubules and its interference with microtubule dynamics. These results provide the first evidence that induction of apoptosis may contribute to the cytopathic effect of BIV. In addition, these results uncover a novel role for BTat in regulating microtubule dynamics in addition to its conventional role in regulating gene transcription.

Natural bovine lentivirus type 1 infection in Holstein dairy cattle. II. Lymphoid tissue lesions.

Bovine immunodeficiency virus (BIV) in Holstein cows was associated with morphologic evidence of lymphoid organ deficiency. Cows were subjected to normal management practices including parturition and lactation without adverse environmental stresses. During the clinical disease process there was marked weight loss and wasting with frequent and severe concurrent infections. Lymphoid follicular hyperplasia and dysplasia in lymph nodes, and hypertrophy and hyperplasia in hemal lymph nodes were characteristics of the lymphoid tissues. Atrophy of lymphoid cell compartments with depletion of lymphocytes and a lymphocytic lymphoid folliculitis were components of the lymphoid system pathology. The nodal tissue lesions resembled those observed in feline, simian, and human lentiviral disease. A functional correlation with immune system deficiency was the development of multiple bacterial infections which failed to resolve after appropriate therapy. The BIV-associated disease syndrome in dairy cows may be useful as a model system for investigation of the pathogenesis of the lymphoid organ changes that occur in humans and animals with lentiviral infection.

Confirmation of vertical transmission of bovine immunodeficiency virus in naturally infected dairy cattle using the polymerase chain reaction.

The purpose of this study was to determine whether bovine immunodeficiency virus (BIV) is vertically transmitted in naturally infected dairy cattle. Twenty-two dam/calf pairs from a Mississippi Agriculture and Forestry Experiment Station dairy were the study group. Blood samples were collected following delivery of calves, the peripheral blood leukocytes were purified from these samples, and the leukocyte DNA was used in polymerase chain reactions targeting the pol gene region of the BIV provirus. Southern blotting and hybridization were used to confirm the BIV specificity of the amplified fragments. BIV provirus was detected in 14 of 22 calves (64%), demonstrating vertical transmission. Eight of the calves were disqualified from the final interpretation of transplacental transfer because they may have nursed their mothers prior to blood collection, allowing the possibility of lactogenic transfer of the virus. Transplacental transmission of BIV was identified in 6 of 22 calves (27%).

Tat protein expression in MDBK cells does not confer susceptibility to bovine immunodeficiency virus.

The ability of BIV strain R29 to infect bovine cell lines in the presence or absence of a functional lentiviral Tat protein is described. Jembrana disease virus (JDV) Tat protein was stably expressed in MDBK cells. No viral replication could be detected in this cell line after infection with BIV R29. Transfection of MDBK cells and MDBK Tat expressing cells with BIV R29 proviral DNA established that BIV R29 could not replicate in MDBK cells. Whether viral entry into MDBK cells is also a block to BIV R29 infection of MDBK cells has yet to be established.

Detection of genetic diversity among bovine immunodeficiency virus population by single-strand conformation polymorphism analysis.

Serial virus specimens rescued from rabbits, experimentally infected with bovine immunodeficiency (BIV) strain R29, were monitored for changes in quasispecies population, using the single-strand conformation polymorphism (SSCP) analysis. The generation of characteristic SSCP patterns enables the rapid differentiation of BIV variants derived from the conserved part on the env region of the BIV genome, reducing the need for expensive and time-consuming direct sequencing analyses. Our results showed genetic polymorphism among a number of sampled BIV population in experimentally infected rabbits. At least three SSCP patterns (BIV quasispecies) were detected. The SSCP analysis allows for an easy, sensitive, and rapid screening of genetic variants of the virus and the assessment of variation at a number of tissue target sites. These variations may relate to cell-type targets and/or disease progression, and could be significant to our understanding of lentiviral pathogenesis.

Bovine immunodeficiency virus and analogies with human immunodeficiency virus.

After describing the results of BIV research during the past years experimental data are presented which indicate that BIV does not cause any clinical symptoms after infection and that no correlation exists with the other widely spread retrovirus in the bovine, the bovine leukosis virus (BLV). Since contact obviously did not lead to a horizontal transmission it is suggested that transmission occurs, as in the cat, vertically from dam to offspring. It was also found that a long period of time after infection can elapse before antibodies against BIV can be detected. It is also quite clear that HIV and BIV do not have much in common except that both are lentiviruses.

Lymphadenopathy and non-suppurative meningo-encephalitis in calves experimentally infected with bovine immunodeficiency-like virus (FL112).

In an experiment on bovine immunodeficiency-like virus (BIV), the virological and serological aspects of which were reported in an earlier paper, three groups (A, B and C) of three calves were inoculated subcutaneously with a recently isolated strain (FL112). For group B and group C, the virus was suspended in milk, and for group C (controls) the viral suspension was subjected to pasteurization before inoculation. The calves were killed for necropsy 12 months later. Clinical assessment revealed subtle ataxia in two group A calves, which took the form of an intermittent "shifting" (from one leg to another) lameness, and palpable enlargement of the pre-scapular lymph nodes in one group B animal. At necropsy, haemal lymph nodes (0.1 to 0.5 cm in diameter), occurring singly, were observed in all animals. However, in groups A and B (but not C), enlarged haemal lymph nodes (< or = 2 cm in diameter) were also seen, occurring singly and in chains; and in one group A animal they occurred in grape-like clusters. In groups A and B (but not C), histopathological examination revealed generalized hyperplastic changes in lymph nodes, especially the haemal lymph nodes. This finding was particularly striking in the two clinically ataxic animals from group A, which also showed a non-suppurative meningo-encephalitis; the latter was possibly the cause of the subtle clinical signs. This study supports previous findings on lymphadenopathy resulting from experimental infection with BIV.

Infection of rabbits with R29 strain of bovine immunodeficiency virus: virulence, immunosuppression, and progressive mesenteric lymphadenopathy.

To assess the value of bovine immunodeficiency virus (BIV) infection as a model for human immunodeficiency virus (HIV) infection in man, we studied the impairment of certain immunologic functions in New Zealand white rabbits experimentally infected with an uncloned virulent isolate of the virus, BIV R29. Serum samples were tested by Western blot for the presence and persistence of antibody production. The T- and B-lymphocyte function was studied by evaluation of the blastogenic responsiveness to concanavalin A (Con A) and to dextran sulfate (DxS). All infected rabbits seroconverted to BIV antigens within 2 to 4 weeks postinfection (p.i.) The BIV was isolated from the peripheral blood lymphocytes (PBLs) of 13 of 17 rabbits (77%) early in the infection and also from 5 of 17 hyperplastic mesenteric lymph nodes (29%) and 10 of 17 spleens (59%) during the chronic stage of infection. Seven of 17 BIV-infected rabbits (41%) developed marked immunodepression 2 to 5 months p.i., and later, 5 exhibited a rapidly progressive disease with anorexia, weight loss, neurologic impairment, splenomegaly, and mesenteric lymphadenopathy. These data underline the value of the BIV model for studying HIV pathogenesis in vivo and the development of interventional strategies for AIDS.

Thermal inactivation of bovine immunodeficiency virus.

Cell-associated bovine immunodeficiency virus (BIV) and cell-free BIV were subjected to increasing temperatures, including pasteurization conditions. To determine the effect of heat treatment on BIV viability, reverse transcriptase activity and infectivity of the heat-treated virus were assessed. BIV was inactivated by heating to 47 degrees C for 30 min and by low- and high-temperature pasteurization conditions.

Encephalitis, lymphoid tissue depletion and secondary diseases associated with bovine immunodeficiency virus in a dairy herd.

Encephalitis, lymphoid tissue depletion and secondary infections occurred over a 5-yr-period in Holstein cows infected with bovine immunodeficiency virus (BIV). There were 59 cattle studied, the majority during 1991, when a severe environmental stress occurred, each with one or more primary causes of death, natural or by euthanasia, and most with several secondary diseases. The encephalitis was characterized by meningeal, perivascular and parenchymal infiltration with lymphocytes, occasional plasma cells and macrophages with perivascular edema in some cows. Affected areas included the cerebrum, cerebellum, and spinal cord with no particular distribution pattern recognized. The lymphoid depletion was primarily an absence of follicular development in nodes draining regions with secondary infections such as chronic mastitis and chronic suppurative pododermatitis. Paucity of lymphocytes in thymic-dependent regions of lymph nodes and the spleen suggested a primary depletion of T cells. Secondary infections were often multiple with each cow having several minor conditions, usually considered short-term and treatable. These included mastitis and pododermatitis, with many cows having non-responding abscesses, cellulitis and myositis attributed to injection site infections. A large number of the cattle had parturition difficulties such as dystocia, obturator paralysis, and metritis. Pulmonary, cardiovascular, and intestinal disease were recognized as both primary and secondary disease conditions. There was a high level of infection with bovine leukemia virus with 4 of the 59 cattle having lymphosarcoma. Under practical conditions, the infection with BIV has a different effect on the host than has been observed under experimental conditions. The presence of BIV combined with the stresses associated with parturition and a modern dairy production system were considered causal for the development of untreatable secondary diseases in immunocompromised cattle. The peak incidence in 1991 was attributed to increased environmental stress during renovation of the barn facility. During this time the cattle were kept on open pasture, exposed to an extremely wet winter, and spring weather conditions. The effect of co-infection with bovine leukemia virus, the influence of immunocompromise on the chronicity of mastitis, the relationship with laminitis and pododermatitis, and several questions related to viral transmission, complementarism with bovine leukemia virus, viral reactivation and immunoprophylaxis all remain as viable avenues for future investigations.

Histopathological changes in the lymphoid tissues of sheep exposed to the bovine immunodeficiency-like virus.

Six yearling sheep were inoculated intraperitoneally with peripheral blood from two sheep infected with the bovine immunodeficiency-like virus (BIV) strain R29. An additional five sheep served as sham-inoculated controls. Of the six sheep given BIV, five seroconverted, one of them remaining seropositive for the duration of the study. The polymerase chain reaction demonstrated BIV provirus in three of the five serologically positive animals. At necropsy, 1 year after inoculation, histological changes were found only in the lymphoid tissues. In sheep exposed to BIV, mild though significant increases were seen in the (1) number of splenic periarteriolar lymphoid sheaths, (2) number of secondary follicles in hilar and prescapular or popliteal lymph nodes, and (3) medullary sinus cellularity in prescapular and popliteal lymph nodes.

Acquired immune dysfunction in rabbits experimentally infected with an infectious molecular clone of the bovine immunodeficiency virus (BIV127).

To investigate the effect of bovine immunodeficiency virus (BIV) infection on the rabbit immune system, we studied the proliferative responses of peripheral blood lymphocytes (PBLs) of rabbits experimentally inoculated with BIV. All BIV127-inoculated rabbits seroconverted after 6 weeks and remained seropositive over a prolonged period of time. Assays for specific lymphocyte reactivity to concanavalin A (Con A) were performed monthly for over 1 year. One-hundred percent of infected rabbits developed abnormally low T cell responses, as measured by Con A stimulation. By 3 months postinoculation, the PBL response to Con A was diminished and remained depressed for 6 months. All animals were clinically asymptomatic within 14 months of BIV inoculation. By 15 and 16 months postinoculation, two of three infected rabbits exhibited recurrent lowering of the T cell responsiveness including a decrease in absolute PBL counts. One of these animals died unexpectedly. Our results further confirmed that a functional impairment of lymphocytes was induced early in the course of BIV infection, prior to clinical disease. These findings suggested that BIV infection may mimic asymptomatic infection of human immunodeficiency virus (HIV) and provided further evidence of the importance of BIV-induced disease in rabbits as a relevant model for the study of AIDS.

Infection and dysfunction of monocytes induced by experimental inoculation of calves with bovine immunodeficiency-like virus.

Three calves were experimentally inoculated with bovine immunodeficiency-like virus (BIV) to examine BIV pathogenesis. Inoculated calves produced specific antibody that could be detected from 3 to 5 weeks up to 1 year postinoculation (pi). Virus was isolated from peripheral blood mononuclear cells (PBMC) 3-4 weeks pi by syncytia assay. Thereafter, the virus could be continually isolated. BIV could be isolated from monocytes but not from T cells. Likewise, monocytes could be infected with BIV in vitro. Various monocyte functions of these BIV-infected calves and age-matched uninfected calves were tested; superoxide anion release, phagocytic activity, and chemotactic responsiveness of monocytes were depressed in BIV-infected calves compared with control calves. A slight delay in the humoral immune response against mouse serum protein was also evident. During the observation period of approximately 1 year, no significant clinical symptoms could be observed. One calf, however, was killed at 15 months pi. At the time of necropsy, BIV could be isolated from PBMC as well as from cells of the spleen, liver, and lymph nodes.

Characterization of early pathogenic effects after experimental infection of calves with bovine immunodeficiency-like virus.

The early pathogenic effects of bovine immunodeficiency-like virus (BIV) were studied in calves experimentally inoculated with BIV. All animals inoculated with BIV R29-infected cells seroconverted by 6 weeks postinoculation, and BIV was recoverable from each animal at 2 weeks postinoculation. However, levels of BIV replication in vivo appeared to be low. In situ hybridization studies indicated that during peak periods of viral replication in vivo, less than 0.03% of peripheral blood mononuclear cells were expressing detectable levels of viral RNA. Moreover, the levels of viral RNA in these cells in vivo were less than 1/10 the levels observed in persistently infected cells in vitro. BIV-inoculated calves had significantly higher numbers of circulating lymphocytes, and follicular hyperplasia was observed in lymph nodes, hemal nodes, and spleen. The histopathological changes observed in BIV-infected calves were similar to changes found early after infection with the immunosuppressive lentiviruses, including human immunodeficiency virus type 1.