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.     

Examination of whether persistently indeterminate human immunodeficiency virus type 1 Western immunoblot reactions are due to serological reactivity with bovine immunodeficiency-like virus.

The bovine lentivirus, known as bovine immunodeficiency-like virus (BIV), is genetically, structurally, and antigenically related to human immunodeficiency virus type 1 (HIV-1). It is not known whether sera from persons exposed to BIV proteins would show either positive or indeterminate reactivity on HIV-1 antibody tests. We used a BIV Western blot (immunoblot) analysis to examine human sera characterized as HIV-1 antibody positive, HIV-1 antibody negative, HIV-1 persistently indeterminate, HIV-1 p17 antibody positive only, HIV-1 p24 antibody positive only, human T-cell leukemia virus type 1 (HTLV-1) p19 antibody positive only, or HTLV-1 p24 antibody positive only. None of these sera were positive by Western blot to BIV-specific proteins. Many of these sera, however, displayed strong reactivities to bovine cell culture antigens on blots prepared from both mock-infected and BIV-infected cell cultures. The HIV-1 p17 and p24 antibody-positive and the HTLV-1 p19 and p24 antibody-positive sera were further examined by Western blot to bovine leukemia virus (BLV) and were found to be negative. We examined sera from laboratory personnel at risk for BIV exposure, including two laboratory workers who were exposed to BIV by accidental injection with BIV-infected cell culture material, and found no evidence of seroconversion to BIV-specific proteins. We tested 371 samples of fetal bovine sera, each sample representing serum pooled from one to three fetuses. All samples were negative by BIV Western blot. To date, we have not detected any human sera with antibody to BIV-specific proteins. Our data indicate that persistently indeterminate results on HIV-1 Western blot are not caused by a human antibody response to BIV proteins.     

A protein immunoblot test for detection of bovine leukemia virus p24 antibody in cattle and experimentally infected sheep

A protein immunoblot test for detecting antibody to the bovine leukemia virus p24 antigen is described. The test employs a crude antigen preparation derived from concentrated cell culture fluid, and an optimised biotin-streptavidin-peroxidase amplification system for immunodetection. The test is highly specific and is more sensitive than the gp51 agar gel immunodiffusion (AGID) test for detection of BLV antibody in cattle and experimentally infected sheep. In a selected set of 30 field sera from cattle which had given equivocal results in the gp51-AGID test, 21 were positive, 4 were negative, and 5 gave an uncertain result in the p24-immunoblot test.     

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.     

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.     

Bovine respiratory syncytial virus antibodies in non-bovine species

Summary To study the role of non-bovine species in the epidemiology of bovine respiratory syncytial virus (RSV) infections, sera obtained from 9 non-bovine animal species and from humans were examined for bovine RSV specific antibodies. Sera were mainly from animals and humans which had been in contact with cattle. Forty sera of each species were tested in an RSV specific whole virus ELISA as well as in a peptide based ELISA, that was developed to measure antibodies specific for bovine RSV. Antibodies directed against RSV were detected in over 50% of sera obtained from sheep, goat, cattle and human beings, and anti-RSV activity was also found in some roe and dogs and one horse. Antibodies to bovine RSV were found in sera of all tested cattle, 11 (27.5%) goats and in some other individual animals: 3 horses, 2 roe, 1 cat and 1 dog. These results indicate that of the investigated species, besides cattle only goats might play a role in the epidemiology of bovine RSV.     

Inhibition of bovine leukemia virus release by antiviral antibodies

Summary Peripheral blood lymphocytes from bovine leukemia virus (BLV) infected cattle were grownin vitro with serums from BLV-infected and uninfected cattle, sheep and rabbits. Only serums from infected animals which had antibody to gp 45/55, the major glycoprotein antigen, inhibited the release of virus from the cells. Although viral antigens could be detected in the cells themselves, none were detected in the supernatants of cultures grown with these serums. Normal serums and serums with antibody only to p23, the internal BLV antigen, did not inhibit virus release. To identify the factor responsible for virus release inhibition, 2 inhibitory serums were absorbed with either gp 45/55, p23 or tissue culture cells from BLV-infected and uninfected cell lines. The results indicated that antibody to gp 45/55 is the factor responsible for virus release inhibition.     

Expression of bovine immunodeficiency-like virus envelope glycoproteins by a recombinant baculovirus in insect cells.

The bovine immunodeficiency-like virus (BIV) env open reading frame (ORF) contains both sequences encoding env and sequences for exon 1 of the putative rev gene. Recombinant baculoviruses incorporating BIV env ORF sequences were constructed to characterize the expression, processing, and immunogenicity of products of the BIV env ORF in insect cells and to develop reagents to study native BIV Env glycoproteins. A recombinant baculovirus containing the entire env ORF synthesized a nonglycosylated, 20-kDa, BIV-specific protein, apparently unrelated to native BIV Env proteins. In contrast, a recombinant baculovirus containing a truncated env ORF in which the coding sequences for rev exon 1 were deleted synthesized three size classes of glycosylated proteins in insect cells related to the BIV Env precursor (gp145), surface (gp100), and transmembrane (gp45) glycoproteins observed in BIV-infected mammalian cells. Oligomers of recombinant BIV Env proteins also formed in these baculovirus-infected insect cells. Immunofluorescence staining of intact insect cells infected by the baculovirus expressing BIV Env with BIV-specific serum demonstrated that the recombinant Env glycoproteins were expressed on the cell surface. Antisera raised to recombinant Env glycoproteins immunoprecipitated native gp145, gp100, and gp45 in BIV-infected bovine cells similar to sera from animals naturally or experimentally infected with BIV.     

Characteristics of the major internal protein and RNA-dependent DNA polymerase of bovine leukaemia virus.

A virus designated bovine leukaemia virus (BLV), associated with leukaemia in cattle and previously demonstrated to induce the disease in sheep, was purified from chronically infected sheep cell cultures. Electrophoretic analysis showed a major protein of mol. wt. about 24,000 (p24) which reacted in gel diffusion and complement-fixation tests with sera from naturally infected cattle, experimentally infected sheep, and guinea pigs immunized with p24. BLV p24 has an isoelectric point of 8-6. Interspecies antigenic reactivities characteristic of mammalian Type C virus p30s were not detected in disrupted BLV or on p24. Sheep and guinea pig antisera to BLV, reactive with p24, also did not precipitate several Type C virus p30s in radioimmunoassays. BLV is also distinguished from Type C viruses and resembles mouse mammary tumour virus and Mason-Pfezer virus in having an RNA-dependent DNA polymerase which is preferentially active in the presence of Mg++ when synthetic templates are used. Along with previously published morphological data, the above indicates that BLV is not a Type C virus as classically defined. Four hundred and forty one human sera from cancer patients and matched controls were non-reactive with disruped BLV, BLV infected cells, and BLV p24 in complement-fixation tests.     

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.     

In animals infected by bovine leukemia virus (BLV) antibodies to envelope glycoprotein gp51 are directed against the carbohydrate moiety

The immunological reactivity of the surface glycoprotein gp51 of bovine leukemia virus was examined by radioimmunoassay with sera from infected animals and sera obtained from rabbits after injection of the purified antigen. The respective influence of the protein and carbohydrate portions of the glycoprotein on the antigenic reactivity was investigated by digestions with glycosidases and proteases. Digestion of gp51 with a mixture of glycosidases abolished the reactivity of the antigen with sera of infected cattle or sheep. In contrast, the reactivity of gp51 with monospecific rabbit antisera was only slightly modified by the glycosidase treatment. Digestion of the same antigen with proteinase K and Pronase completely eliminated its reactivity with monospecific rabbit antiserum; sera from infected cattle or sheep still precipitated 10 to 15% of the same ¹²⁵I-antigen in direct radioimmunoassay. These results strongly indicate that natural immunity against BLV gp51 depends upon an intact carbohydrate side chain. Following purification of the antigen, protein antigenic sites are uncovered and reacted against by the injected rabbit. Solid phase radioimmunoassay furthermore showed that the carbohydrate antigenic site is probably unique as opposed to the probably multiple protein antigenic determinants. Antibody to gp51 prepared in the rabbit by injection of intact BLV-infected bovine lymphocytes reacted, in all tests performed, in much the same way as natural antibody found in BLV-infected cattle or sheep.     

Isolation of a glycoprotein and two structural proteins of Maedi-Visna virus.

A viral glycoprotein of Maedi-Visna virus was purified by affinity chromatography, rate zonal centrifugation, and isoelectric focusing. Furthermore, two structural proteins of the virus were purified by isoelectric focusing and Sephadex gel filtration. In sodium dodecyl sulfate—polyacrylamide gel electrophoresis they were found to be homogeneous proteins with relative molecular weights of 15,000 (p15), 24,000 (p24), and 70,000 for the viral glycoprotein (gp70). In 71 out of 90 sera from sheep infected with Maedi-Visna, antibodies against gp70 were found, and only in five sera could antibodies be detected against p24 by agar gel immunodiffusion tests.     

Detection of antibodies against the core protein p24 of the bovine leukaemia virus in cattle for confirmatory serological testing

An electrophoretic immunoblotting technique which was developed recently was evaluated for the identification of serum antibodies against the bovine leukaemia virus core protein p24 by using 167 sera from a bovine leukaemia virus-negative herd, and 144 sera from herds naturally infected with the virus. The sensitivity of the immunoblot was 97.4%, relative to sera which were positive in the polymerase chain reaction and in a commercial EBL-ELISA. The specificity of the immunoblot was 99.4%, for the sera from a cattle herd in which all animals were negative by a commercial EBL-ELISA, and it was 96.7% relative to sera which were negative by the polymerase chain reaction and by the agar gel immunodiffusion test from bovine leukaemia virus-infected cattle herds. A p24-specific ELISA was developed, using a monoclonal anti-p24 antibody for coating microtitre plates, a crude antigen preparation, and a monoclonal anti-bovine IgG-horse radish peroxidase conjugate as components. All reagents were commercially available. While the p24-ELISA worked well with sera from serial bleeds from calves infected experimentally with the bovine leukaemia virus and its sensitivity with sera from the naturally-infected cattle was 96.5%, its specificity was relatively low at 85.0 or 53.3%, respectively for the two negative sera groups.     

Development of a syncytia inhibition assay for the detection of antibodies to bovine leukemia virus in naturally infected cattle; comparison with Western blot and agar gel immunodiffusion

A syncytia inhibition assay (SIA) for the detection of antibodies to bovine leukemia virus is described. This test involves specific antibody-mediated inhibition of BLV-induced cytopathic effects in an indicator cell line. A total of 300 sera were screened commercially by agar gel immunodiffusion (AGID) and were then screened by Western blot and SIA. The new assay system provided results which were comparable to Western blot and AGID. The results obtained suggest that SIA may be more sensitive than either of the other two assay systems examined for the determination of the infection status of cattle.     

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.     

Comparison of competitive and indirect enzyme-linked immunosorbent assays for detection of bluetongue virus antibodies in serum and whole blood.

An indirect (I) enzyme-linked immunosorbent assay (ELISA) and a competitive (C) ELISA, using a group-specific monoclonal antibody against bluetongue virus (BTV), are described for the detection of antibodies to BTV in cattle and sheep sera. The performance of these assays in detecting anti-BTV antibody in sequential serum samples and eluates from whole blood (WB) dried on filter paper from three calves and four sheep experimentally infected with type 10 BTV was evaluated. The C-ELISA was superior to the I-ELISA in the detection of anti-BTV antibody in the sera and WB samples from both cattle and sheep early after infection with BTV. BTV antibodies were demonstrable by C-ELISA in all the bovine and ovine sera and WB eluates by 9 days postinfection; whereas the I-ELISA results for sheep sera and WB eluates were similar, anti-BTV antibody was not detected in bovine serum and WB eluates until 26 and 14 days postinfection, respectively. While both ELISAs proved reliable, under the present test conditions involving detection of early postinfection reactions of experimentally infected animals, the C-ELISA was always as sensitive or more sensitive than the standard agar gel immunodiffusion test, the modified complement fixation test, and the plaque neutralization tests in the detection of anti-BTV antibodies. Unlike observations with the immunodiffusion test, no reaction was seen between BTV antigen and bovine epizootic hemorrhagic disease virus antiserum in either ELISA. The results suggest that either ELISA may be suitable for routine diagnostic testing and may have the potential to replace other tests for detection of anti-BTV group-specific antibodies and that the C-ELISA may have the most potential.     

The occurrence of human wart-virus antibodies in dogs,pigs and cattle

Summary Using the immunodiffusion method, antibodies against human wart-virus were detected in dog, pig and cattle sera but not in horse and reindeer sera. Antibodies were found in 25 per cent (28/114) of the dog sera, the prevalence of antibodies being fairly similar to that in children of the same age. By electron microscopy the antibodies in dog sera were shown to attach to and precipitate the human wart-virus particles, and in immunodiffusion the precipitation lines of human and dog sera were shown to be identical. Of the cattle sera studied, 10 per cent (10/97) and of the pig sera 13 per cent (5/40) gave a faint precipitation line, having identity with that given by the human sera, against human wart-virus. Conversely 18 per cent (9/50) of adult human sera contained precipitating antibodies against a bovine papilloma antigen, probably bovine papilloma virus.With 5 Figures     

Comparative analysis of Brucella serotype A and M and Yersinia enterocolitica O:9 polysaccharides for serological diagnosis of brucellosis in cattle, sheep, and goats.

Hapten polysaccharides of Brucella smooth M and A serotypes were prepared from Brucella sp. and Yersinia enterocolitica O:9 by previously described hydrolytic (O chain) or nonhydrolytic (native hapten [NH]) procedures. The purified polysaccharides differed only in the presence (O chain) or absence (NH) of lipopolysaccharide core sugars. The polysaccharides were compared by reverse radial immunodiffusion for the diagnosis of brucellosis in cattle (Brucella abortus biotype 1 [A serotype] and Brucella melitensis biotype 3 [AM serotype]), sheep (B. melitensis biotypes 1 [M serotype] and 3), and goats (B. melitensis biotype 1). The reverse radial immunodiffusion test with the NH from B. melitensis 16 M (serotype M) showed the highest sensitivity (89.6 to 97.3%), regardless of the host species and the serotype of the infecting Brucella sp. Y. enterocolitica O:9 NH (A serotype) was useful for diagnosing disease in cattle infected with B. abortus biotype 1, but not in cattle infected with B. melitensis biotype 3, sheep, or goats. The different results obtained with the serotype M and A polysaccharides and the sera from animals infected with M, A, and AM serotypes of Brucella spp. showed that in naturally infected animals, a large proportion of the antibodies are directed to or react with a previously defined common epitope(s) (J. T. Douglas and D. A. Palmer, J. Clin. Microbiol. 26:1353-1356, 1988) different from the A or M epitopes. By using the radial immunodiffusion test with B. melitensis 16M NH, it was possible to differentiate infected from vaccinated cattle, sheep, and goats with a sensitivity and specificity similar to that of the complement fixation test.     

Antibodies to common ovine and bovine C-type virus specific antigen in serum from sheep with spontaneous leukosis and from inoculated animals

C-type virus containing preparations from concanavalin stimulated sheep lymphocyte culture fluids were treated with ether, and used for antigen in immunodiffusion tests. 13 out of 14 sera from sheep with spontaneous lymphatic leukosis showed precipitation reaction with the antigen preparation. 3 out of 33 sera from clinically unsuspect sheep in the same multiple case herd also formed precipitin bands. 3 out of 37 sera from offspring of leukotic ewes, 3 out of 9 sera from sheep, inoculated as newborne lambs with leukotic material, and 1 out of 4 sera from inoculated goats precipitated in the same manner. Immunodiffusion tests with a similar antigen preparation from bovine lymphocyte culture fluid led to strong identical results. 162 sera from sheep out of leukosis free herds did not show precipitation reactions. These findings seem to demonstrate the presence of antibodies to common ether resistent ovine and bovine C-type virus specific antigen. Our investigations show a clear correlation between clinical findings in sheep leukosis, demonstration of C-type particles in lymphocyte cultures, and antibody reactions.