Molecular evidence for bovine immunodeficiency virus infection in Iranian sheep and cattle population

Bovine immunodeficiency virus (BIV), a member of the lentivirus subfamily of retroviridae, was originally isolated from a dairy cow with persistent lymphocytosis, lymphoid hyperplasia, and prevascular cuffing in the brain. Serological studies indicated that BIV has worldwide distribution, but its prevalence in Iran is not clearly known. In this investigation, we report the detection of proviral DNA sequence of BIV in 300 blood samples of cattle and 150 blood samples of sheep by polymerase chain reaction (PCR) using oligonucleotiode primers specific for the gag gene region of the virus. Blood samples of cattle and sheep were taken from Chaharmahal Bakhtiary province, Iran. According to the PCR results, infection rate in the cattle and sheep population were 60% and 30%, respectively. This is the first report for the presence of BIV in cattle and sheep population of Chaharmahal Bakhtiary province.     

Detection of bovine retrospumavirus by the polymerase chain reaction

A polymerase chain reaction (PCR) assay was developed for detection of bovine retrospumavirus (bovine syncytial virus; BSV) provirus DNA. Two different sets of oligonucleotide primers complementary to sequences located in the gag and the pol/env gene regions were used and compared for their ability to amplify the targeted BSV sequences by PCR. The results obtained from this study have shown that it is possible to amplify the BSV provirus DNA sequences not only from total DNA of BSV-infected cell cultures, but also from total DNA of various tissues and peripheral blood mononuclear cells (PBMCs) that were collected from two rabbits experimentally infected with BSV. Sensitivities of the PCR for amplification of BSV gag and pol/env nucleic acid sequences from cell culture total DNA were 10 ng and 10 pg of DNA, respectively, as determined by the analysis of the amplified PCR products on ethidium bromide-stained agarose gels. The specificity of the PCR for both primer sets tested was confirmed when the amplified cDNA products of the expected size reacted positively with the corresponding virus-specific digoxigenin-labeled cDNA probes in Southern blot chemiluminescent hybridization assays. No amplification was obtained when the BSV-specific primers were used in the PCR with DNA material specific to either bovine leukemia virus (BLV) or bovine immunodeficiency virus (BIV) provirus genomic DNA. No cross-hybridization was obtained when the BSV-specific cDNA probes were allowed to react with BLV or BIV provirus DNA. The PCR targeting the gag and pol/env gene regions of the BSV provirus genome may be an alternative to conventional methods for the confirmation of the presence of BSV in cell cultures used for virus isolation, and for the diagnosis of BSV infection from bovine peripheral blood leukocytes.     

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.     

Unique Epitope of Bovine Immunodeficiency Virus Gag Protein Spans the Cleavage Site between p16MA and p2L

Bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) are closely related bovine lentiviruses that are difficult to distinguish by presently available diagnostic methods. Recently, in our laboratory, a monoclonal antibody (MAb; MAb 10H1) against the BIV Gag protein identified a differential epitope, located at the 6.4-kDa N terminus of a 29-kDa Gag capsid protein, which was absent in JDV. To define the essential amino acids of the epitope, a series of primers within the 163 bp of DNA corresponding to the 6.4-kDa protein were designed. The full-length 163-bp DNA fragment and the smaller DNA fragments with deletions were amplified by PCR and then cloned into pQE32 vectors for protein expression studies. The expressed proteins were analyzed with MAb 10H1 by Western blotting. The differential epitope has been narrowed to a 26-amino-acid region (R121 to R146), which includes 6 residues of p16^MA (where MA represents the matrix protein) and 20 residues of p2L. A synthetic peptide corresponding to the putative 26-amino-acid epitope blocked MAb 10H1 binding to the expressed peptide. These experiments revealed that the epitope spans the cleavage site between p16^MA and p2L and presumably will be valuable in distinguishing the two viruses.     

Detection of SRV/D shedding in body fluids of cynomolgus macaques and comparison of partial gp70 sequences in SRV/D-T isolates

We previously reported the isolation of a novel subtype of SRV/D-Tsukuba (SRV/D-T) from two cynomolgus monkeys (Macaca facicularis) in the breeding colony of Tsukuba Primate Research Center (TPRC). We surveyed for SRV/D infection in the TPRC cynomolgus colony using SRV/D-specific PCR primer sets designed based on the entire gag region sequence. The only SRV/D subtype detected in the colony was SRV/D-T with a positive infection rate of 22.4% (n = 49). It has been reported that the mode of transmission of SRV/D is via contact with virus shed in the body fluids. In this report, to investigate the infection route of SRV/D-T in monkeys at TPRC, we performed virus isolation and PCR for detection of the SRV/D genome from peripheral blood mononuclear cells (PBMCs), plasma, saliva, urine, and feces. Virus isolation and PCR detection were positive in plasma, saliva, urine, and fecal samples from all monkeys on which virus was isolated from PBMCs. This suggests that the spread of SRV/D-T infection in TPRC is via contact with virus shed in saliva, urine, and/or feces. Also, comparison of sequences of gp70 on multiple SRV/D-T isolates revealed that there was little intra- and inter-monkey variation, suggesting that SRV/D-T is fairly stable.     

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.     

Development of a Western Blot Assay for Detection of Bovine Immunodeficiency-Like Virus Using Capsid and Transmembrane Envelope Proteins Expressed from Recombinant Baculovirus

A 120-amino-acid polypeptide selected from the transmembrane protein region (tTM) and the major capsid protein p26 of bovine immunodeficiency-like virus (BIV) were expressed as fusion proteins from recombinant baculoviruses. The antigenic reactivity of both recombinant fusion proteins was confirmed by Western blot with bovine and rabbit antisera to BIV. BIV-negative bovine sera and animal sera positive for bovine syncytial virus and bovine leukemia virus failed to recognize the recombinant fusion proteins, thereby showing the specificity of the BIV Western blot. One hundred and five bovine serum samples were tested for the presence of anti-BIV antibodies by the recombinant protein-based Western blot and a reference Western blot assay using cell culture-derived virions as test antigens. There was a 100% concordance when the p26 fusion protein was used in the Western blot. However, the Western blot using the tTM fusion protein as its test antigen identified four BIV-positive bovine sera which had tested negative in both the p26 recombinant-protein-based and the reference Western blot assays. This resulted in the lower concordance of 96.2% between the tTM-protein-based and reference Western blot assays. The results of this study showed that the recombinant p26 and tTM proteins can be used as test antigens for the serodetection of BIV-infection in animals.     

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.     

Unique epitope of bovine immunodeficiency virus gag protein spans the cleavage site between p16(MA) and p2L

Bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) are closely related bovine lentiviruses that are difficult to distinguish by presently available diagnostic methods. Recently, in our laboratory, a monoclonal antibody (MAb; MAb 10H1) against the BIV Gag protein identified a differential epitope, located at the 6.4-kDa N terminus of a 29-kDa Gag capsid protein, which was absent in JDV. To define the essential amino acids of the epitope, a series of primers within the 163 bp of DNA corresponding to the 6.4-kDa protein were designed. The full-length 163-bp DNA fragment and the smaller DNA fragments with deletions were amplified by PCR and then cloned into pQE32 vectors for protein expression studies. The expressed proteins were analyzed with MAb 10H1 by Western blotting. The differential epitope has been narrowed to a 26-amino-acid region (R121 to R146), which includes 6 residues of p16(MA) (where MA represents the matrix protein) and 20 residues of p2L. A synthetic peptide corresponding to the putative 26-amino-acid epitope blocked MAb 10H1 binding to the expressed peptide. These experiments revealed that the epitope spans the cleavage site between p16(MA) and p2L and presumably will be valuable in distinguishing the two viruses.     

Development of a Western blot assay for detection of bovine immunodeficiency-like virus using capsid and transmembrane envelope proteins expressed from recombinant baculovirus

A 120-amino-acid polypeptide selected from the transmembrane protein region (tTM) and the major capsid protein p26 of bovine immunodeficiency-like virus (BIV) were expressed as fusion proteins from recombinant baculoviruses. The antigenic reactivity of both recombinant fusion proteins was confirmed by Western blot with bovine and rabbit antisera to BIV. BIV-negative bovine sera and animal sera positive for bovine syncytial virus and bovine leukemia virus failed to recognize the recombinant fusion proteins, thereby showing the specificity of the BIV Western blot. One hundred and five bovine serum samples were tested for the presence of anti-BIV antibodies by the recombinant protein-based Western blot and a reference Western blot assay using cell culture-derived virions as test antigens. There was a 100% concordance when the p26 fusion protein was used in the Western blot. However, the Western blot using the tTM fusion protein as its test antigen identified four BIV-positive bovine sera which had tested negative in both the p26 recombinant-protein-based and the reference Western blot assays. This resulted in the lower concordance of 96.2% between the tTM-protein-based and reference Western blot assays. The results of this study showed that the recombinant p26 and tTM proteins can be used as test antigens for the serodetection of BIV-infection in animals.     

Investigation of vesicular rashes for HSV and VZV by PCR

Vesicular fluid from rashes of 132 patients was tested by a multiplex PCR shown to be specific for herpes simplex virus (HSV) type 1 and 2, and varicella zoster virus (VZV) genomic DNA. The results were compared with those obtained by examination by electron microscopy and virus isolation by cell culture. The PCR did not differentiate between HSV 1 and 2. By PCR, 64 HSV infections and 53 VZV infections were identified, with presumed 100% sensitivity and specificity. Fifteen specimens tested negative by PCR, electron microscopy, and virus isolation for herpes viruses. The sensitivities of virus isolation and electron microscopy for detection of herpes simplex virus were 56% and 80%. For varicella zoster virus, the sensitivities of virus isolation and electron microscopy were 47% and 60%. These data illustrate the advantage of rapid PCR diagnosis of herpes simplex virus and varicella zoster virus in vesicle fluids. J. Med. Virol. 54:155–157, 1998. © 1998 Wiley-Liss, Inc.     

Promoter activity in the 5′ flanking regions of the Bohle iridovirus ICP 18, ICP 46 and major capsid protein genes

Summary. Bohle iridovirus (BIV) belongs to the genus Ranavirus, of which Frog virus 3 (FV-3) is the type species. We are developing BIV as a recombinant viral delivery vector, and as a first step we located specific BIV promoter sequences to drive foreign gene expression in the recombinant virus. By comparison with FV-3 sequences, the genes encoding ICP 18 and ICP 46 in BIV were identified and sequenced. Putative promoter regions of these two early genes and of the major capsid protein (MCP) gene were identified, cloned into pSFM21, and luciferase production was then used to assess the promoter activity of these regions.     

Infection of macaques with an R5-tropic SHIV bearing a chimeric envelope carrying subtype E V3 loop among subtype B framework

Summary.  To establish simian/human immunodeficiency virus (SHIV) clones bearing a chimeric envelope carrying subtype E V3 loop among subtype B envelope, four subtype E V3 sequences were substituted into SHIV_MD14, a SHIV clone bearing an envelope derived from a CXCR4 (X4)/CCR5 (R5)-dual tropic subtype B HIV-1 strain. SHIV-TH09V3, an only V3-chimera clone capable of replicating in human and macaque peripheral blood mononuclear cells (PBMCs), was propagated in pig-tailed macaque PBMCs and in cynomolgus macaque splenic mononuclear cells. The propagated virus stocks were intravenously inoculated into respective macaque species. SHIV-TH09V3 infected both macaque species as shown by plasma RNA viremia, isolated viruses from PBMCs and plasma, and antibody production against viral proteins. To assess how the substituted V3 sequence affected coreceptor usage, SHIV-TH09V3 stocks propagated in vitro and after isolation from macaques were verified for their corecepor usage by GHOST cells assay. SHIV-TH09V3 maintained R5-tropic phenotype both in vitro and after isolation from macaques, in contrast to the X4/R5-dual tropic SHIV_MD14. This indicates the substituted V3 sequence among the backbone of SHIV_MD14 governs coreceptor usage. Future study of infecting macaques with SHIV-TH09V3 and SHIV_MD14 will focus on differences of the outcome caused by the different V3 sequences in connection with coreceptor usage. Received July 30, 2002; accepted November 13, 2002 Published online March 21, 2003     

Cloning of the Bovine Immunodeficiency Virus gag Gene and Development of a Recombinant-Protein-Based Enzyme-Linked Immunosorbent Assay

An enzyme-linked immunosorbent assay (ELISA) was established for the rapid detection of specific bovine immunodeficiency virus (BIV) antibodies in cattle, using recombinant Gag protein as an antigen. The gag coding region from BIV was cloned into an expression vector, pQE32, which expressed high levels of recombinant protein from Escherichia coli. The ELISA was standardized by a checkerboard titration against known BIV-positive and -negative sera from cattle and a monoclonal antibody to the Gag protein. A total of 139 cattle serum samples, from the diagnostic laboratory at Kansas State University, Manhattan, and from the Dairy Station, Louisiana State University, Baton Rouge, were compared by ELISA and immunoblot assays for the detection of BIV-specific antibodies. Of 26 cattle sera samples which tested positive using the immunoblot assay, 23 were positive by ELISA, thus establishing a strong correlation between the two tests. The sensitivity and specificity of ELISA relative to immunoblotting were 0.88 and 0.93, respectively. ELISA proved to be as specific as immunoblotting but was much less time-consuming and easier to perform.     

A Western blot assay for the detection of antibodies to bovine immunodeficiency-like virus in experimentally inoculated cattle,sheep, and goats

Summary A cocultivation method was used to establish a cytocidal bovine immunodeficiency-like virus (BIV) infection in primary fetal bovine lung (FBL) cell cultures. Cultures were monitored for virus production using radial immunodiffusion and agar gel immunodiffusion. Pelleted virus and detergent (CHAPS)-solubilized infected cell lysates from BIV-infected cell cultures were compared as sources of antigen for Western blots. Pelleted virus preparations from FBL-BIV cell cultures produced the best antigen for Western blot. Sheep and goats were inoculated with BIV and serum antibody responses were monitored up to 1 year post inoculation (PI). Sera from experimentally infected cattle, sheep, and goats reacted in Western blot assay with BIV viral induced polypeptides gp 110, p 72, p 55, p 50, gp 42, p 38, p 26, p 24, p 18, p 15, and p 13. Antibodies to p 26 were detected as early as 2 weeks PI in cattle, sheep, and goats. Antibodies to gp 110 were detected by 4 to 6 weeks PI in cattle, and by 9 months PI in sheep and goats. Antibodies to BIV proteins were still evident in cattle sera 2 1/2 years PI, and in sheep and goat sera 1 year PI.     

Development and evaluation of an antigen-capture ELISA for detection of the UL24 antigen of the duck enteritis virus, based on a polyclonal antibody against the UL24 expression protein

An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) method was developed for the efficient detection of the UL24 antigen of the duck enteritis virus (DEV) using polyclonal antibodies. Ducks and rabbits were immunized, respectively, with expressed UL24 recombinant protein. The IgG antibodies against UL24 from ducks and rabbits were purified and used as the capture antibodies. The specificity of the optimized AC-ELISA was evaluated by use of DEV, duck hepatitis virus (DHV), duck hepatitis B virus (DHBV), gosling plague virus (GPV), Riemerella anatipestifer (R.A.), Escherichia coli (E. coli), Pasteurella multocida (P.M.) and Salmonella Enteritidis (S.E.). Only DEV specimens yielded a specific and strong signal. The limit of the sensitivity of this method for the detection of DEV was 46 ng/100 μl. Compared with PCR and virus isolation, the rate of agreement for the detection of experimentally infected sera was 100%. A comparative test used on clinical specimens between the neutralization test and the AC-ELISA showed that the proportions of true positives and true negatives by the AC-ELISA were 0.90 and 0.67 respectively. These results indicated that the AC-ELISA approach is rapid, sensitive, and reliable for specific detection of DEV antigen.