Multicentric lymphoma in buffaloes in the Amazon region, Brazil.

BACKGROUND: The presence of lymphoma in buffaloes was first reported in India in the 1960s. The disease is similar to Enzootic Bovine Leucosis (EBL) caused by Bovine leukemia virus (BLV) in cattle; however, according to our results and those of other studies, the etiology of these lymphomas in buffalo do not appear to be associated with BLV. The objectives of this study are to describe four cases of the disease in buffaloes belonging to the same herd in the Amazon region of Brazil and to perform a clinical-anatomopathological, immunohistochemical, and etiological study of the lymphomas. RESULTS: Over a period of ten years, four buffaloes were observed presenting progressive weight loss, swelling of peripheral lymph nodes, and nodules in the subcutaneous tissue. Upon necropsy, whitish-colored tumor masses were observed in the form of nodules in the subcutaneous tissue, along with miliary nodules on the serosal surfaces of abdominal and thoracic organs and tumors in lymph nodes and other organs. Neoplastic lymphocyte proliferation was observed through histopathology. An immunohistochemical study revealed that the neoplasias were formed by proliferation of predominantly B lymphocytes. The presence of BLV genome was not detected in the lymphomas when using the real-time PCR technique, nor was it detected through immunohistochemical staining using monoclonal antibodies against two viral proteins. Bovine herpesvirus 6 was not detected in the tumors. However, Bovine immunodeficiency virus (BIV) was detected in samples of lymphoma and in the lymph nodes and kidneys of one of the animals. CONCLUSIONS: The occurrence of lymphoma in buffaloes is reported for the first time in Brazil and is characterized by B-cell multicentric lymphoma. The etiology of the disease does not appear to be associated with BLV; however, the detection of BIV in samples of lymphoma from one sick animal deserves further study, considering the oncogenic potential of this virus.

Molecular detection of bovine immunodeficiency virus in water buffaloes (Bubalus bubalis) from the Amazon region, Brazil.

Bovine immunodeficiency is a chronic progressive disease caused by a lentivirus that affects cattle and buffaloes. Although the infection has been described in cattle in some countries, including in Brazil, there are only two reports of infection in buffaloes: one in Pakistan and one in Cambodia. The aim of the present study was to survey the occurrence of bovine immunodeficiency virus (BIV) in water buffaloes from the Amazon region, Pará state, Brazil. BIV proviral DNA was surveyed in 607 whole blood samples of water buffaloes from 10 farms located in the state of Pará using semi-nested polymerase chain reaction (PCR) (PCR-SN) to amplify the pol region of the viral genome. Of the 607 samples tested, 27 (4.4 %) were positive for BIV proviral DNA. The amplified fragments were confirmed by sequence analysis after cloning and nucleotide sequencing. The sequence obtained had 99 % similarity to the reference strain (R-29). The present study provides important epidemiological data because BIV was detected for the first time in water buffaloes in Brazil. Further, the results suggest the possibility of the virus being a risk factor for herd health because it may be a potential causal agent of chronic disease and, also may be associated to other infectious diseases.

Serological survey for bovine immunodeficiency virus in dairy cattle from Poland.

A seroprevalence study of bovine immunodeficiency virus (BIV) was undertaken on 1,541 serum samples from Holstein cattle from 23 herds, located in different geographical regions of Poland. The analysis was performed using ELISA, with recombinant Gag protein of BIV as antigen. The average BIV prevalence was 4.9% in individual cattle, while the percentage of herds harboring at least one seropositive animal, was 82.6%. To demonstrate the correlation of BIV and bovine leukemia virus infection, all sera were analysed for BLV antibodies and there was only a slight association between both infections. Overall, these results show that BIV infection is present in dairy cattle in Poland at a prevalence rate found in other European countries.

Sequence analysis of mRNA transcripts encoding Jembrana disease virus Tat-1 in vivo.

Jembrana disease virus (JDV) is a lentivirus which replicates to very high titres in vivo and its Tat-1 protein has been shown to be a potent transactivator in vitro. Analysis of tat mRNA transcripts produced early in infection studies identified four predominant species which were generated by multiple splicing events. The use of a splice donor downstream of tat-1 was common indicating that a Tat-1 protein of 97 amino acids is expressed during the acute phase of Jembrana disease. The presence of an in-frame stop codon between tat-1 and tat-2 was identified in transcripts from three different strains of JDV confirming that expression of a single exon Tat-1 correlates with high viral titres in vivo. Sequence conservation in the regions of tat-1 that are critical for RNA binding and transcription activation in three different virus strains was high and the tat-2 sequences were completely conserved.

Development of a capsid based competitive inhibition enzyme-linked immunosorbent assay for detection of bovine immunodeficiency virus antibodies in cattle and buffalo serum.

The aim of this study was to develop a more specific and sensitive competitive inhibition ELISA (CI-ELISA) than the currently used indirect ELISA for detection of antibodies to bovine immunodeficiency virus (BIV) in cattle and buffaloes. Murine monoclonal antibodies (MAbs) were generated against a recombinant capsid (CA) protein of bovine immunodeficiency virus. Of the 13 anti-CA MAbs developed, MAb-9G10 was selected for CI-ELISA based on the maximum inhibition (98%) obtained with reference BIV antibody positive serum. Based on the distribution of percent inhibition of known negative sera (n=50), a cut-off value was set at 40% inhibition. The MAb-based CI-ELISA showed much higher agreement (concordance: 95.4%) than the indirect ELISA (concordance: 77.8%) with Western blot. Out of 672 sera of cattle and buffaloes tested by CI-ELISA from four states of India, 22% (113/516) of cattle and 19% (30/156) of buffalo were sero-positive for BIV with an overall seroprevalence of 21% (143/672) in India.

Evidence of bovine immunodeficiency virus (BIV) infection: serological survey in Argentina.

This is the first report of serological evidence for bovine immunodeficiency virus (BIV) infection in Argentina. The analysis was performed in 589 dairy bovine sera samples, applying indirect enzyme-linked immunosorbent assay (I-ELISA) using a synthetic antigen (transmembrane peptide, TM) and Immunofluorescent assay (IFA). In this study, 9 dairy herds from 4 Argentinian provinces were evaluated and 12% of the animals tested positive for BIV. Seven of the 9 herds tested were BIV seropositive and the percentage of BIV seropositive animals in the herds ranged from 2% to 42%. Direct detection of BIV provirus applying nested PCR was not conclusive. Antibody detection against bovine leukemia virus (BLV) in all sera was also performed applying immunodiffusion (ID) assay and 59% resulted seropositive. Statistical analysis of the results was carried out and possible evidence of association between BIV and BLV infection was considered. Future studies should be performed including local field isolates strains of BIV.

[Amplification of proviral DNA of bovine immunodeficiency virus in experimentally infected animals].

The primer set for detection of bovine immunodeficiency virus (BIV) proviral DNA, retrovirus of unknown pathology, by standard polymerase chain reaction was developed. Amplicon of the expected size was detected in DNA from peripheral blood mononuclear cells of seropositive experimentally BIV infected sheep and cattle. Primers targeted the short fragment of BIV pol gene. Sequences of BIV proviral DNA extracted from BIV infected cell culture as well as from experimentally infected cows were taken for targeted pol BI BPX gene locus. Problems of BIV detection from the clinical material of the experimentally and naturally infected animals are discussed.

Insights into the molecular and biological features of the dUTPase-related gene of bovine immunodeficiency virus.

This study was stimulated by our previous research of the dUTPase-related protein from bovine immunodeficiency virus (BIV) (Voronin et al., 2014). Despite the lack of detectable enzymatic BIV dUTPase activity (both of the recombinant protein and in virions), mutating the dUTPase gene was deleterious to viral production. However, cDNA synthesis and integration were apparently unaffected. Consequently, we have studied here two important issues. First, we showed that in cDNA produced by the dUTPase-mutated virions, the incidence of mutations was not higher than that found in wild-type BIV-infected cells. Second, single mutations, introduced in preserved dUTPase residues Asp48 and Asn57 (in the putative dUTPase active site or close to it), have led to abortive BIV infections (except for the conservative Asp48Glu mutation). Therefore, we postulate that the BIV dUTPase-related protein has a critical role in retroviral replication at steps that take place after viral cDNA synthesis and integration.

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.

[PCR-based detection of proviral DNA of bovine immunodeficiency virus].

A set of primers was developed to detect by polymerase chain reaction (PCR) the proviral DNA of bovine immunodeficiency virus (BIV). A short fragment of 101 bp BIV gene was selected as a target for primers; sequences of proviral DNA isolated from both a cell culture with BIV and from lymphocytes of an experimentally infected animal were known for the fragment. An amplicon of an expected size was detected by standard PCR in a transformed cell series of bovine testicles with Florida 112 BIV DNA, and in a plasmid DNA with a cloned proviral DNA of R29 BIV. Described in the paper are the results of a theoretical comparison of primers used in the detection of BIV by PCR. The presence of non-complementary nucleotides in the set of "primer-single stranded amplicon" was shown to bring about false positive results in the detection of BIV by PCR. No 1500 bp PCR product was detected after PCR with a synthesized pair of primers and with 100% homology for all known BIV isolates complementary to env gene. Finally, the issue of how to detectVIR in clinical samples obtained from experimentally and naturally infected is discussed.

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 gammadelta 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.

Loss of Gag-specific antibody reactivity in cattle experimentally infected with bovine immunodeficiency-like virus.

The development and persistence of virus-specific antibodies were investigated in eight cattle experimentally infected with the R29 isolate of bovine immunodeficiency-like virus (BIV). By 4 weeks postinoculation (p.i.), antibodies reactive to BIV gag- and env-encoded recombinant fusion proteins were detectable by immunoblotting in all animals. By 40 weeks p.i., seven of eight cattle had dramatically decreased Gag-specific antibodies, and anti-Gag reactivity remained very low or undetectable through 190 weeks p.i. Immunoprecipitation experiments revealed a similar loss of reactivity to nondenatured BIV Gag in these animals. In contrast, antibodies to a recombinant BIV Env protein were readily detectable throughout the study in all eight cattle. During the period of declining Gag antibody, infectious virus was recoverable from peripheral blood mononuclear cells of each animal. However, there was no evidence for sufficient amounts of BIV p26-containing immune complexes to explain the loss of anti-Gag reactivity. Interestingly, the single animal that maintained detectable anti-Gag reactivity throughout the study was repeatedly negative for virus recovery beyond 17 weeks p.i. All animals have remained clinically normal for over 4 years p.i., with no evidence of consistent changes in mononuclear cell subsets. These findings provide evidence that in BIV infection an early decline in Gag-specific antibody reactivity can occur without evidence of increasing viral replication or progression to overt clinical disease.

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.

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.

Evaluation of HIV type 1 western blot-indeterminate blood donors for the presence of human or bovine retroviruses.

From 1985 through 1990, 1100 of 500,000 human blood donations in Syracuse, New York were repeatedly reactive by ELISA for antibodies to the human immunodeficiency virus type 1 (HIV-1). Nine hundred of the ELISA-reactive samples were confirmed as negative by Western blot (WB), 40 were confirmed as positive, and the remaining 160 sera were indeterminate, reacting mainly with HIV-1 gag gene products. Twenty donors with the most reactive indeterminate WB were selected for follow-up studies. Four of these 20 donors admitted to retroviral risk factors and, interestingly, 12 (60%) had exposure to dairy cattle and drank unpasteurized milk. These 20 donors were analyzed over a 3-year period for the presence of the pathogenic human retroviruses HIV-1, HIV-2, human T cell lymphoma/leukemia virus types I and II (HTLV-I and HTLV-II), as well as bovine immunodeficiency virus (BIV) and leukemia virus (BLV). Retroviral analyses included serology, plasma antigen capture, virus culture, and the polymerase chain reaction. Only one donor seroconverted and was clearly infected with HIV-1. None of the other 19 donor serological reactivities to HIV-1 changed, nor were they positive for any of the above-mentioned retroviruses. Although we cannot ascertain whether these latter 19 HIV-1 WB-indeterminate donors were exposed to human or bovine retroviral proteins, it is unlikely that their HIV-1 seroreactivity was caused by infection with HIV-1, HIV-2, HTLV-I, HTLV-II, BLV, or BIV.

Detection of bovine immunodeficiency-like virus by the polymerase chain reaction.

This report describes a test for the bovine lentivirus, bovine immunodeficiency-like virus, in which polymerase chain reaction (PCR) technology is employed. Two pairs of oligonucleotide primers directed to sequences within the coding regions of the gag and pol genes generated the expected PCR products from molecular BIV clones and from DNA of BIV-infected cell cultures but not from DNA of uninfected cultures. Data indicating the specificity and sensitivity of these PCRs for BIV detection and the potential utility of this technology for diagnostic applications are presented.

Comparative evaluation of bovine immunodeficiency-like virus infection by reverse transcriptase and polymerase chain reaction.

Infection of embryonic bovine lung (EBL) cells by bovine immunodeficiency-like virus (BIV) were monitored by reverse transcriptase (RT), syncytia formation and polymerase chain reaction (PCR). Infection can be detected by PCR at 24 h while the presence of syncytia and RT were not detected until much later. The detection of BIV RT can be optimized by changing the pH and salt conditions. The enzyme is very sensitive to changes in pH but can tolerate a wider range of salt and MgCl2 concentrations. Infection of primary human cell cultures by BIV was monitored by both PCR and RT. No active infection of human cells were detectable.

In vitro detection of bovine immunodeficiency-like virus using monoclonal antibodies generated to a recombinant gag fusion protein.

An Escherichia coli recombinant fusion protein containing the major core protein of bovine immunodeficiency-like virus (BIV) was used to immunize mice for generation of monoclonal antibodies to BIV p26. Eight hybridomas specific for BIV p26 were identified and two antibodies, designated 104 and 142, were further characterized. Both 104 and 142 antibodies were isotyped as IgG1; they reacted specifically with both BIV p26 and the recombinant fusion protein in Western immunoblot analyses. However, the epitope specificity of the antibodies was different. Immunoperoxidase assays were used to determine if antibodies 104 and/or 142 could detect BIV replication in cell culture. Both antibodies were found to react with BIV-induced syncytia and individual BIV-infected cells. The antibodies were also used successfully in a focal immunoassay for quantitation of BIV-infected cells. These antibodies will provide valuable reagents for detection and quantitation of BIV replication in studies of viral pathogenesis and immunity.

Sensitive and specific detection of bovine immunodeficiency virus and bovine syncytial virus by 5' Taq nuclease assays with fluorescent 3' minor groove binder-DNA probes.

Sensitive assays are required to detect bovine retroviruses in donor cattle used for the in vivo preparation of Australian tick fever vaccines. 5' Taq nuclease assays using 3' minor groove binder DNA probes (TaqMan)MGB) were developed and compared to conventional PCR assays for the sensitive detection of bovine syncytial virus (BSV) and bovine immunodeficiency virus (BIV). Seven beef and dairy herds were screened to evaluate these tests. Comparative sensitivities of PCR tests were determined by testing log(10) dilutions of plasmids with inserts containing corresponding provirus sequences. Published PCR assays targeting BIV env sequences did not adequately amplify Australian BIV sequences. Pol sequences from Australian strains of BIV and BSV were used to design TaqMan MGB assays, which improved sensitivity 10-fold (BIV) and 100-fold (BSV), respectively, over conventional PCR tests. This is the first report of Australian sequences of BIV and BSV and the first 5' Taq nuclease assays described to detect these viruses. These methods could be applied to future studies requiring sensitive detection of these two bovine retroviruses.

Evidence for the presence of two bovine lentiviruses in the cattle population of Bali.

Antibodies directed against two bovine lentiviruses, Jembrana disease virus (JDV) and bovine immunodeficiency virus (BIV), were detected in Balinese cattle sera using two new enzyme-linked immunosorbent assays (ELISAs) based on the combination of capsid (CA) protein and transmembrane (TM) peptides derived from JDV or BIV sequences. Twenty eight of the 77 sera tested on the JDV ELISA showed anti-JDV antibodies with an unequal distribution of seropositive animals throughout the different districts of Bali. Furthermore, when 17 of the JDV positive sera were tested on Western blot, using the same JDV CA antigen, only 13 were judged positive confirming that the ELISA was a more sensitive technique for the detection of seropositive animals. Finally, 9 of the 49 JDV seronegative animals showed anti-BIV antibodies when tested on BIV-specific ELISA. These two ELISAs appeared to be highly sensitive for the detection of anti-JDV and anti-BIV antibodies. Moreover, for the first time, animals showing antibodies against BIV were identified on the main island of Bali and on the JDV-free Nusa Penida island.