Bovine Immunodeficiency-like Virus Research Articles

A sensitive luminescence syncytium induction assay (LuSIA) based on a reporter plasmid containing a mutation in the glucocorticoid response element in the long terminal repeat U3 region of bovine leukemia virus

BackgroundBovine leukemia virus (BLV) causes enzootic bovine leukosis, the most common neoplastic disease of cattle. Previously, we reported the luminescence syncytium induction assay (LuSIA), an assay for BLV infectivity based on CC81-BLU3G cells, which form syncytia expressing enhanced green fluorescent protein (EGFP) when co-cultured with BLV-infected cells. To develop a more sensitive LuSIA, we here focused on the glucocorticoid response element (GRE) within the U3 region of the BLV long terminal repeat (LTR).MethodsWe changed five nucleotide sites of the GRE in a pBLU3-EGFP reporter plasmid containing the BLV-LTR U3 region promoter by site-directed mutagenesis and we then constructed a new reporter plasmid (pBLU3GREM-EGFP) in which the EGFP reporter gene was expressed under control of the GRE-mutated LTR-U3 promoter. We also established a new CC81-derived reporter cell line harboring the GRE-mutated LTR-U3 promoter (CC81-GREMG). To evaluate the sensibility, the utility and the specificity of the LuSIA using CC81-GREMG, we co-cultured CC81-GREMG cells with BLV-persistently infected cells, free-viruses, white blood cells (WBCs) from BLV-infected cows, and bovine immunodeficiency-like virus (BIV)- and bovine foamy virus (BFV)-infected cells.ResultsWe successfully constructed a new reporter plasmid harboring a mutation in the GRE and established a new reporter cell line, CC81-GREMG; this line was stably transfected with pBLU3GREM-EGFP in which the EGFP gene is expressed under control of the GRE-mutated LTR-U3 promoter and enabled direct visualization of BLV infectivity. The new LuSIA protocol using CC81-GREMG cells measures cell-to-cell infectivity and cell-free infectivity of BLV more sensitively than previous protocol using CC81-BLU3G. Furthermore, it did not respond to BIV and BFV infections, indicating that the LuSIA based on CC81-GREMG is specific for BLV infectivity. Moreover, we confirmed the utility of a new LuSIA based on CC81-GREMG cells using white blood cells (WBCs) from BLV-infected cows. Finally, the assay was useful for assessing the activity of neutralizing antibodies in plasma collected from BLV-infected cows.ConclusionThe new LuSIA protocol is quantitative and more sensitive than the previous assay based on CC81-BLU3G cells and should facilitate development of several new BLV assays.

Cloning, expression, purification, crystallization and preliminary crystallographic study of the protein module (BIV2-Helix) in the fusion core of bovine immunodeficiency-like virus (BIV) gp40

The fusion core of bovine immunodeficiency virus (BIV) gp40 is proposed to be involved in membrane fusion. However, no crystal structures are yet available. A predicted protein module BIV2-Helix of BIVgp40 has been expressed in Escherichia coli and purified by chromatography. Recombinant BIV2-Helix was crystallized using the hanging-drop vapour-diffusion technique at 291 K. The crystals were grown in MPD and belonged to the primitive rhombohedral space group R3, with unit-cell parameters a = 39.17, b = 39.17, c = 295.05 A and two molecules per asymmetric unit. X-ray diffraction data were collected to 1.76 A in the home laboratory from a single crystal.

Recombinant Jembrana disease virus gag proteins identify several different antigenic domains but do not facilitate serological differentiation of JDV and nonpathogenic bovine lentiviruses

In Indonesia, it is suspected that there are two bovine lentiviruses circulating in the cattle population: a pathogenic Jembrana disease virus (JDV), and a nonpathogenic bovine immunodeficiency-like virus (BIV). Both viruses cross-react antigenically and cannot be differentiated by current serological tests using JDV antigens. To identify possible type-specific epitopes, a series of recombinant protein constructs including the matrix, capsid and nucleocapsid proteins were produced from JDV gag and the expressed proteins were tested by Western blot using JDV and BIV hyperimmune sera. JDV matrix and truncated capsid proteins were recognised by both JDV and BIV hyperimmune sera indicating that there were multiple cross-reactive epitopes present in JDV gag. At least three epitopic regions were identified in these constructs, including the major homology region, by monoclonal antibody binding studies. JDV nucleocapsid recombinant protein was not recognised by either JDV or BIV hyperimmune sera and none of the recombinant gag proteins were able to differentiate between JDV positive sera from Jembrana disease endemic and Jembrana disease-free areas. Additionally, a 40 amino acid recombinant subunit protein encompassing the region recently found to contain an epitope unique to BIV [Zheng, L., Zhang, S., Wood, C., Kapil, S., Wilcox, G.E., Loughin, T.A., Minocha, H.C., 2001. Differentiation of two bovine lentiviruses by a monoclonal antibody on the basis of epitope specificity. Clin. Diagn. Lab. Immunol. 8, 283–287] was tested but was not recognised by either JDV positive sera from Jembrana disease-endemic or Jembrana disease-free areas.

In vivo transcription of bovine leukemia virus and bovine immunodeficiency-like virus

Cellular tropism and transcription of bovine leukemia virus (BLV) and bovine immunodeficiency-like virus (BIV) were investigated using peripheral blood mononuclear cells (PBMC) collected from a cow infected with both viruses. Each PBMC subset, purified by magnetic cell sorting, was subjected to PCR and RT-PCR for detection of their integrated proviruses and transcript mRNAs. Both BLV and BIV genomes were detected by nested PCR in CD3 +, CD4 +, CD8 + and γδ T cells, B cells and monocytes. However, BLV tax transcription was only detected in B cells, and only B cells also formed BLV syncytia in CC81 cells. On the other hand, BIV transcript was detected in each subpopulation of PBMC. These results indicated that BLV can infect T cells and monocytes as well as B cells, but can be expressed by transcription only in B cells. In contrast, BIV can express its transcripts in all infected cells.

Clinical response and immunomodulation following experimental challenge of calves with type 2 noncytopathogenic bovine viral diarrhea virus.

Eight calves between 16 and 18 weeks of age that were seronegative to bovine viral diarrhea virus (BVDV), bovine leucosis virus and bovine immunodeficiency-like virus were infected (day 0) intranasally with the type 2 noncytopathogenic Canadian 24515 field isolate of BVDV in order to evaluate the effect of BVDV infection on certain clinical, hematological and immunological parameters. All virus-exposed animals developed fever and showed a significant (P < 0.05, 0.01 or 0.001) drop in the number of circulating leucocytes (neutrophils, lymphocytes and monocytes) by day 3 or 5 post-exposure (PE), which continued to the end of the experiment at day 12 PE. BVDV was consistently isolated from the peripheral blood buffy coat cells from day 5 PE, and also from selected tissues (spleen, thymus, mesenteric and submaxillary lymph nodes, small intestine, lungs and thyroid gland) that were collected at the time of euthanasia of the animals at day 12 PE. Diminished significant (P < 0.05) percentages of peripheral blood mononuclear cells (PBMCs) expressing at their surface either B7 and MHC II molecules were observed in virus-exposed calves at days 7, 10 and/or 12 PE, when compared to virus-nonexposed control calves (n = 5). However, no changes in the percentages of PBMCs expressing either B4 or MHC I molecules were observed throughout the experiment. Finally, a significant (P < 0.05 or 0.01) enhanced phagocytic capability of the PBMCs, as analyzed by flow cytometry, was observed in virus-exposed animals at days 3, 5, 7, 10 and 12 PE, when compared to control calves. These results demonstrated the virulence of the 24515 isolate of BVDV in 4 to 4.5 month-old calves, and suggest that type 2 BVDV infection in calves is associated with dysregulation of certain immunological functions.

A viral transmembrane recombinant protein-based enzyme-linked immunosorbent assay for the detection of bovine immunodeficiency virus infection

The expression of bovine immunodeficiency virus (BIV) truncated transmembrane envelope protein (designated hereafter tTM) in insect cells has been described previously (Abed, Y., St-Laurent, G., Zhang, H., Jacobs, R.M., Archambault, D., 1999. Development of a Western blot assay for detection of bovine immunodeficiency-like virus using capsid and transmembrane proteins expressed from recombinant baculovirus. Clin. Diagn. Lab. Immunol. 6, 168–172). In this study, a tTM-based enzyme-linked immunosorbent assay (ELISA) was developed for the serodetection of BIV infection. A total of 109 bovine sera including 86 BIV-negative and 23 BIV-positive serum samples were tested. The ELISA results were compared with those of three Western blot assays using, as test antigens, cell culture-derived whole virus proteins (WB1), and the tTM (WB2) and p26 (WB3) fusion proteins expressed from recombinant baculovirus in insect cells, respectively. The concordances of the ELISA results with those of the WB1, WB2, and WB3 were 97.2, 100 and 97.2%, respectively. The tTM protein-based ELISA and Western blot permitted the detection of BIV infection in cattle whose sera failed to react with the p26 fusion protein and the whole virus protein preparation. The tTM recombinant protein was also used to study the kinetics of appearance of antibodies against BIV transmembrane envelope protein in rabbits infected experimentally with BIV. Antibodies to tTM were detected at 28 days post-infection and persisted through the entire 36–39.5 months experimental time period. The results of this study showed that the tTM-ELISA might be useful for the serodetection of BIV-infected animals, and for basic studies on BIV replication life cycle.

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.

Investigation of the cellular tropism of bovine immunodeficiency-like virus

Bovine immunodeficiency-like virus (BIV) was first isolated from an animal showing transient leucocytosis, lymphadenopathy, lesions in the central nervous system and progressive weakness and emaciation. Similar signs are observed in other immunosuppressive lentiviral infections. BIV, like other lentiviruses, has been isolated from peripheral blood mononuclear cells and lymphoid tissue of infected animals. However, the in vivo cellular tropism of BIV remains unclear although initial studies indicate that BIV may be pantropic, infecting T cells, B cells and monocytes similar to some of the immunodeficiency-causing lentiviruses. PCR, Southern blot hybridisation, cell culture and reverse transcriptase assays were used to demonstrate the presence of BIV proviral DNA and the production of infectious virus in CD2 +, WC1 +, B cells and monocytes during the acute stages of infection. Western immunoblot assays were used to assess the development of antibody responses towards the virus.

PCR diagnosis of the bovine immunodeficiency-like virus.

AbstractThe bovine immunodeficiency-like virus (BIV), also known as the bovine lentivirus or the bovine immunodeficiency virus, causes a persistent viral infection of cattle, and is found in cattle populations around the world (1,2). A closely related lentivirus, Jembrana disease virus, causes an acute disease in Bos javanicus cattle, and while it also infects Bos taurus and Bos indicus cattle the infection is generally asymptomatic (3). Strong evidence has not been presented that links BIV to a specific cattle disease, but BIV has been associated with decreased milk yield and with a variety of disease conditions including clinical immunodeficiency, encephalitis, bovine paraplegic syndrome, skin infections, and emaciation (4-6).KeywordsPrime CombinationInfected CattleBovine Immunodeficiency VirusBlind PassageFetal Bovine LungThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Detection of bovine immunodeficiency-like virus infection in experimentally infected calves.

Detection of BIV virus infection by serological means, PCR and virus isolation in experimentally infected calves is described. Viral sequences were specifically detected by PCR in peripheral blood mononuclear cells (PBMCs), with primer systems located in the gag, pol and tat regions of the viral genome. An enzyme-linked oligosorbent assay (ELOSA) in microtiter plates is described, for the detection of PCR products, the sensitivity of which was shown to be comparable to that of membrane hybridization detection. Serological response of the animals against the BIV p26 protein was shown, using a recombinant fusion protein ((His)6p26) expressed in E. coli and purified by metal affinity chromatography, in ELISA and Western blot studies. The presence of infectious virus was demonstrated by its rescue, by virus isolation in cell cultures, from PBMCs during a one year follow-up.

Bovine immunodeficiency-like virus

Bioassay was used to determine whether bovine immuno-deficiency-like virus (BIV) in milk was inactivated by pasteurisation.&#x0D; &#x0D; Full article published in Veterinary Record (1997) 140, 275-277.

Longitudinal studies of immune function in cattle experimentally infected with bovine immunodeficiency-like virus and/or bovine leukemia virus.

The effects of single or dual infection with bovine immunodeficiency-like virus (BIV) and/or, bovine leukemia virus (BLV) on bovine immune function were examined over a 4 year period. Holstein calves were infected with BIV (four calves), BLV (five calves), BIV and BLV (five calves), or sham inoculated (three calves). Lymphocyte blastogenesis to mitogens, seven tests of neutrophil function, and mononuclear cell subset analysis by flow cytometry (BoCD4, BoCD8, BoCD2, BoWC1, sIgM+, and monocytes) were performed at regular intervals to 49 months post-infection. These data were analyzed for main effects of each virus and interaction as a 2 x 2 factorial. BIV infected cattle had lower neutrophil antibody-dependent cell-mediated cytotoxicity and iodination responses during 2 of the 4 years post-infection (P < 0.05). BIV infection was not associated with any long-term significant changes in lymphocyte blastogenesis to mitogens or changes in mononuclear cell subset numbers in blood. There was a tendency for animals infected with BIV alone to have decreased lymphocyte blastogenic responses to mitogens, but this was not statistically significant. BLV infection caused an increase in total mononuclear cells with no dramatic shift in the relative proportions of the various subsets. Co-infection with BIV and BLV did not consistently cause a different response than either virus did individually. One BIV infected animal died of non-BLV lymphosarcoma 7 months after infection. All other animals had no unusual clinical signs. In summary, infection with BIV caused a significant, temporary decrease in neutrophil function with no consistent statistically significant alteration in lymphocyte blastogenesis or mononuclear cell numbers during the first 4 years after infection. BLV infection caused an increase in lymphocyte numbers, and there appeared to be no synergism between the viruses.

Bovine immunodeficiency-like virus: inactivation in milk by pasteurisation

Bioassay was used to determine whether bovine immunodeficiency- like virus (BIV) in milk was inactivated by pasteurisation. Three groups of three calves were inoculated with virus (BIV isolate FL112), milk...

Size variation within the second hypervariable region of the surface envelope gene of the bovine lentivirus BIV in experimentally and naturally infected cattle.

The bovine lentivirus also known as the bovine immunodeficiency-like virus (BIV) has conserved and hypervariable regions in the surface envelope (SU) gene. Size variation between isolates can be as large as 200 bp, mostly occurring in the second hypervariable (V2) gene region of the SU gene. The V2 region was cloned and sequenced from both experimentally and naturally infected cattle. Temporal evaluation of provirus from an experimentally inoculated cow showed two different-sized variants that appeared over time. The variation appeared to result from a recombinational event resulting in an apparent direct repeat. Cloned proviral nucleotide sequence diversity increased over time. Virus that was cultured and then cloned and sequenced showed progressive change from the inoculum virus, but culturing reduced the diversity of the clones as compared with direct amplification of provirus from leukocyte samples from the cow. The quasispecies phenomenon was evident in clones sequenced from a cow naturally infected with BIV. Of 10 clones examined from the V2 region, 6 different-size clones were present with nine different patterns of sequence rearrangement. Sequence length of different clones varied by as much as 43 amino acids (aa), with 21- and 15-aa direct repeats accounting for most of the size variation. Similar to other lentiviruses, BIV appears to mutate rapidly, which may be important in viral persistence and pathogenesis.

Laboratory Diagnostic Tests for Retroviral Infections in Dairy and Beef Cattle

Detection of bovine retroviruses stretches our diagnostic creativity to its limits. The nucleic acid-based, PCR-amplified assays are finding increased clinical use as the veterinary and livestock industry seek earlier detection of infection for eventual corrective management decisions. We are evolving from a point of disease diagnosis by tumor identification through conventional histopathology, to molecular diagnostics for early identification of retroviral nucleic acid (provirus). The clinical use of antibody-based assays lies in the simplicity of testing large numbers of animals, the relative sensitivity of the assays, and the low cost of testing. Although the pathogenicity of bovine leukemia virus (BLV) for cattle has been well documented, the disease potential for bovine immunodeficiency-like virus (BIV) for cattle is still being determined. Nevertheless, pressure to test for retroviral infections of livestock and, when feasible, removal of these infected animals from the herd will be increased.

Bovine lentivirus induces early transient B-cell proliferation in experimentally inoculated cattle and appears to be pantropic.

Bovine immunodeficiency-like virus (BIV) was first isolated in 1972 (M. J. VanDerMaaten et al., J. Natl. Cancer Inst. 49:1649-1657, 1972). Much work has been done on the molecular characterization of BIV in studies using the original BIV R29 isolate; however, R29 is believed to be attenuated since it no longer causes either mononuclear cell number increases or detectable enlargement of lymphatic nodules in experimentally infected cattle. The host cell tropism and changes in host peripheral blood lymphocyte populations following infection with BIV are unknown. Recently, we isolated and characterized a field isolate of BIV, FL112 (D. L. Suarez et al., J. Virol. 67:5051-5055, 1993) that causes a transient, mononuclear cell lymphocytosis in experimentally infected cattle. In the present study, cattle were inoculated with BIV FL112, and data from flow cytometry showed that BIV causes a B-cell lymphocytosis with no consistent, significant changes in other mononuclear cell populations, including CD3+, CD4+, and CD8+ cells. Cell sorting and PCR amplification were used to show that BIV may be pantropic. Proviral DNA was present in CD3+, CD4+, CD8+, and B-cells, monocytes, and WC1 cells (gamma/delta T cells, null cells) by 3 to 6 days postinoculation and also at 2.5 years postinoculation.

Enhancement of monocyte migration and phagocytosis by the bovine immunodeficiency-like virus Gag proteins.

Supernatants from bovine immunodeficiency-like virus (BIV)-infected cells have been previously shown to affect monocyte random migration, chemotaxis, phagocytosis, and antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro. The experiments in this report demonstrate that the BIV Gag (core) proteins can enhance monocyte random migration, chemotaxis, and phagocytosis. Supernatants from BIV-infected cells contained 10-30 and 30-50 kDa proteins, which significantly (p < 0.05) increased monocyte chemotaxis. The 30-50 kDa protein(s) could be cleaved by limited proteolysis into 10-30 kDa active components. Affinity purification with monoclonal anti-p26 (capsid) antibodies yielded preparations that were active in the random migration, chemotaxis, and phagocytosis assays, but did not affect ADCC. Furthermore, activity of the affinity purified preparation could be specifically neutralized by hyperimmune rabbit serum against BIV Gag proteins. A recombinant Gag protein, consisting primarily of BIV p26, also enhanced monocyte random and chemotactic migration. It appears, therefore, that direct treatment with affinity-purified BIV Gag proteins or a recombinant Gag protein, is able to significantly affect the function of normal monocytes in vitro. Factors affecting monocyte migration and phagocytosis appear to be one or more breakdown products of the BIV Gag precursor, particularly those containing the p26 (capsid) protein.

Molecular biology of the feline immunodeficiency virus auxiliary genes.

Feline immunodeficiency virus (FIV) causes a slowly progressive multiorgan disease in infected cats and shows considerable pathogenic similarities to the human immuno-deficiency virus type 1 (HIV-1), the causative agent of AIDS (Miyazawa &amp; Mikami, 1993; Pedersen et al., 1987; Yamamoto et al., 1988). It is therefore considered a useful small-animal model for HIV-1-induced AIDS studies. FIV belongs to the family Retroviridae, a group of small, enveloped positive-strand RNA viruses. These viruses have an enzyme, reverse transcriptase, which enables them to replicate their RNA genome through a DNA intermediate (Coffin, 1992). FIV is a member of the genus Lentivirus and is closely related in biological characteristics and genome organization to other mammalian lentiviruses, including HIV, simian immunodeficiency virus (SIV), bovine immunodeficiency-like virus (BIV), equine infectious anaemia virus (EIAV), visna virus and caprine arthritis-encephalitis virus (CAEV) (Narayan &amp; Clements, 1990).

Lymphoproliferation assays in cattle naturally infected with bovine leukaemia virus (BLV) and bovine immunodeficiency-like virus (BIV).

Experiments were designed to evaluate the effect of BLV on mitogen-stimulated peripheral blood mononuclear cells (PBMC) from naturally infected cattle. BIV was also taken into consideration due to a recent report showing that in Costa Rica, most of the BLV-infected animals are also seropositive for BIV. The methodology was based on a non-radioactive technique to determine lymphoproliferation. A colorimetric assay using XTT (formazan salt) to measure cell multiplication was adapted for bovine PBMC. ELISA and Western blotting were used to determine the serologic status of the cattle. PCR was only available for BIV detection. Our results show clearly that, dually-infected cattle (BIV-BLV) have reduced lymphoproliferative responses to the mitogen Con A. Haematological abnormalities associated with viral infections were also observed, specially leukocytosis and lymphocytosis. Cows with lymphosarcomas are severely affected. The specific antibody response to different viral proteins could not be associated with the suppressive status of the animals. Due to the high rate of dual infections observed in Costa Rica, these results are not sufficient to clarify which virus is responsible for the suppressive activity, if one or both viruses are necessary, or if they act synergistically.

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.