Molecular detection of bovine immunodeficiency virus (BIV) in bovines from the state of Minas Gerais, Brazil / Detecção molecular do vírus da imunodeficiência bovina (BIV) em bovinos do estado de Minas Gerais, Brasil

O vírus da imunodeficiência bovina é o agente causador da imunodeficiência viral bovina que é conhecido por infectar bovinos em todo o mundo. Como em outras infecções por retrovírus, os hospedeiros desenvolvem uma infecção de longo prazo e a maioria dos animais infectados permanece assintomática. O objetivo deste estudo foi detectar DNA proviral BIV em amostras de sangue de bovinos e estimar a ocorrência de infecção no estado de Minas Gerais, Brasil. Amostras de sangue de 391 bovinos foram coletadas de duas regiões do estado, Zona da Mata e Central. O DNA proviral foi detectado por reação em cadeia da polimerase semi-nested (SN-PCR). Os resultados de SN-PCR indicaram uma ocorrência de BIV de 12,5% no estado. Os produtos amplificados foram confirmados como BIV por sequenciamento e a similaridade da sequência de nucleotídeos com a estirpe de referência (R-29) foi de 99%. Este é o primeiro estudo que relata a presença do BIV em Minas Gerais, Brasil. Os resultados indicam a necessidade de realizar um estudo detalhado sobre a prevalência da infecção por BIV no Brasil.(AU)

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.

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.

Bovine immunodeficiency virus and bovine leukemia virus and their mixed infection in Iranian Holstein cattle.

INTRODUCTION: Bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV) have worldwide distributions, but their prevalences in Iran are unknown. We investigated the presence of infections in Iranian Holstein cattle and determined changes in hematological values for infected animals. METHODOLOGY: Nested PCR was used on blood samples from 143 animals Holstein cattle to detect proviral BIV and BLV gag sequences. Flow cytometric analysis was performed using monoclonal antibodies (mAbs) against CD4, CD8, and CD21 bovine T lymphocyte subsets. RESULTS: Proviral BIV and BLV gag sequences were detected in 20.3% and 17% of the animals, respectively. BIV-BLV confection was also detected in 4.2% of the study population but this was not statistically significant. Flow cytometric analysis showed that both BIV-infected cows and non-infected ones had CD4/CD8 ratios of 2.45 and 1.43, respectively, and this difference was significant. BLV infected and non-infected animals had no significant differences in their CD4/CD8 ratio. In comparison to non-infected cattle, those with both BIV and BLV had a significant decrease in their CD4/CD8 ratios (1.5 % vs. 2.3; P = 0.01). CONCLUSION: This is the first report of BIV and BLV infections in Iran. We found no evidence that infection with one agent predisposed an animal to infection with the other. BIV infection may have a role in decreasing T CD8 counts, but this may depend on the genetics of the cattle and virus strains involved.

A quantitative assay for measuring of bovine immunodeficiency virus using a luciferase-based indicator cell line.

In order to quantitate the bovine immunodeficiency virus (BIV) infection in vitro, a BIV indicator cell line (BIVL) was established by transfecting baby hamster kidney cells with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the BIVL-based assay is 10 times more sensitive than the the CPE-based assay, and has similar sensitivity with the viral capsid protein Western blot assay. BIV indicator cell line could detect BIV infection specifically. Luciferase activity of BIV infected BIVL cells showed a time dependent manner, and 60 h post infection is the optimal time to detect BIV infection. Luciferase activity of BIVL cells correlates with the BIV capsid protein expression. Moreover, a linear relationship was found between MOI and the activated intensity of luciferase expression. In brief, the BIV indicator cell line is an easy, robust and quantitive method for monitoring BIV infection.

Establishment of an indicator cell line for monitoring bovine immunodeficiency virus infection and inhibitor susceptibility.

Indicator cell lines are useful biological tools for monitoring virus infection. In order to monitor infection with bovine immunodeficiency virus (BIV) in vitro, an indicator cell line derived from baby hamster kidney cells which contains integrated copies of an enhanced green fluorescent protein gene driven by the BIV long terminal repeat was constructed. The BIV indicator cell line, designated BIVE, can detect BIV infection more easily and effectively than the established method, which involves the observation of cell cytopathic effects. Furthermore, viral titration using an assay based on the indicator cells is 100 times more sensitive than the assay based on cytopathic effect. The finding that BIV can infect the hamster cell line expands the known host range of BIV in vitro. The BIV indicator cell line could also be used for the evaluation of the inhibitory effect of antiviral agents. The fusion inhibition effect of the heptad repeat 2 region of the BIV envelope protein could also be quantified.

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.

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.

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.

[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.

The bovine immunodeficiency virus: cloning of a tat/rev cDNA encoding a novel Tat protein with enhanced transactivation activity.

Previous studies have shown that BIV may encode two types of Tat proteins of 103 and 108 amino acids, respectively. Here, we report the characterization of a new BIV Tat protein (Tat236) derived from a tat/rev cDNA. The tat/rev cDNA was obtained by reverse transcription-PCR from RNA extracted from cells infected with BIV. BIV was rescued by cell co-cultivation from the spleen of rabbits exposed for 3 years to the R29 isolate of BIV. Sequence analysis indicated that BIV Tat236 contains the first 98 amino acids of Tat103 and the 3' end 138 amino acids of Rev. Reporter gene assays indicated that transactivation of BIV long terminal repeat (LTR) by Tat236 is higher than by the original BIV Tat proteins in several cell types. By using overlapping deletion mutants, evidence was given that the predicted basic domain of Rev within Tat236 plays a major role in the observed enhanced transactivation activity of the protein. However, the intact functional domain of the original BIV Tat is required for efficient transactivation. This is the first report of a hybrid Tat protein from BIV or any lentiviruses that shows higher transactivation than the original transactivator Tat proteins.

Evidence for bovine immunodeficiency virus infection in cattle in Zambia.

We report herein on the first evidence for the presence of bovine immunodeficiency virus (BIV) in Zambia. Serological surveillance of BIV and bovine leukemia virus (BLV) was conducted in traditional cattle herds in Zambia. Out of a total of 262 sera analyzed, 11.4% were found positive for anti-BIV p26 antibodies as determined by Western blot analysis, while 5.0% were positive for anti-BLV gp51 antibodies as detected by immunodiffusion test. Peripheral blood mononuclear cells from BIV seropositive cattle were found to have BIV-provirus DNA, as detected by nested polymerase chain reaction. A nucleotide sequence corresponding to a 298 bp fragment of the BIV pol gene was also analyzed. Amino acid sequences of these Zambian pol gene products showed 98.0 to 100% homology to the American strain BIV R29, 97.0 to 99.0% to Japanese BIV isolates, and divergence ranged from 0.0 to 2.0% among Zambian BIV isolates.

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.

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.

Estimation of sensitivity and specificity of two diagnostics tests for bovine immunodeficiency virus using Bayesian techniques.

The validation of assays for bovine immunodeficiency virus (BIV) in cattle is hampered by the absence of a gold standard. Two tests that often are used to detect BIV are the indirect fluorescent-antibody assay (IFA) and the nested-set polymerase chain-reaction assay (PCR). IFA detects an antibody response whereas PCR detects the provirus in white blood cells. Using Bayesian techniques performed simultaneously on animals from two different dairy herds, we estimated the performance of the IFA and PCR assays and infection prevalence. Bayesian techniques also were used to derive posterior distributions of sensitivities, specificities, and prevalences. The Bayesian estimates were IFA sensitivity=60%, IFA specificity=88%, PCR sensitivity=80%, PCR specificity=86%, Herd A prevalence=20%, and Herd B prevalence=71%. Although PCR was the more sensitive assay, substantial misclassification of infection would be expected in epidemiological studies of BIV regardless of which assay was used.

Evidence of bovine immunodeficiency virus in cattle in Turkey.

A seroepidemiological study of bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV) infections was conducted in four different cattle herds in Turkey. A total of 300 blood samples were analyzed and 12.3% were found to be positive for anti-BIV p26 antibodies by Western blot analysis and 1.6% positive for anti-BLV gp51 antibodies by an immunodiffusion test. BIV infection was confirmed with the detection of BIV-provirus DNA using the nested polymerase chain reaction. This is the first evidence for the presence of BIV in cattle in Turkey.

Seroprevalence of bovine immunodeficiency virus and bovine leukemia virus in dairy and beef cattle in hokkaido.

Serological survey of bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV) infection was conducted in dairy cattle from 10 different regions of Hokkaido, Japan. Among 390 cattle, 11.0% of cattle were BIV-seropositive and 3.3% were BLV-seropositive. Moreover, in two dairy farms, where bovine leukosis has been reported, prevalence of BIV infections were 6.4 and 9.1%, respectively. In contrast, among 150 beef cattle, 16.6% were BIV-seropositive while none was BLV-seropositive. Dual infections with BLV and BIV in dairy cattle were tested by using 107 BLV-seropositive sera, and 20 sera were found BIV-positive (18.7%). These results indicate that BIV infection was widespread in Hokkaido.