Jimmy Kwang

Detection of antibodies to ovine lentivirus using a recombinant antigen derived from the env gene

The Western blot assay was performed to characterize antibodies to the transmembrane glycoprotein (TGP) of ovine progressive pneumonia virus (OPPV) by using glutathione-S-transferase-TGP (GST-TGP) protein. The GST-TGP protein was efficiently expressed in Escherichia coli (E. coli) and was highly immunoreactive in the Western blot assay. This assay detected antibodies in 97% (103/106) of the sera from agarose gel immunodiffusion (AGID) positive OPP animals. Like human AIDS patients, antibodies to TGP appear to be one of the major serological markers in OPP infected animals.

Recombinant polypeptide from the gp48 region of the bovine viral diarrhea virus (BVDV) detects serum antibodies in vaccinated and infected cattle

To characterize the immune response of cattle to bovine viral diarrhea virus (BVDV) glycoprotein gp48, we have produced a large amount of recombinant glutathione-s-transferase-gp48 (GST-gp48) fusion protein in Escherichia coli. Antibodies to gp48 were present in cattle vaccinated with killed or modified-live virus vaccination, or following natural infection. These results were in agreement with results of serum neutralization (SN) test which detected gp53 of BVDV.

A comparison of whole virus and recombinant transmembrane ELISA and immunodiffusion for detection of ovine lentivirus antibodies in Italian sheep flocks

Sera from two sheep experimentally infected with ovine lentivirus (OLV) and from 186 sheep selected from flocks with known high or low prevalence of infection or on the basis of virological or histopathological examination were simultaneously tested by whole virus (WV) ELISA, recombinant transmembrane (r-TM) ELISA and AGID assay. Antigens for both the WV ELISA and AGID were prepared from an Italian field isolate; recombinant antigen was derived from the N′-terminal region of the transmembrane envelope protein of strain K1514. The WV ELISA detected the highest number of seropositives, followed by the r-TM ELISA and AGID test. The sensitivity and specificity of the r-TM ELISA relative to the WV ELISA were 0.66 and 0.95, respectively. Immunoblot analysis of 14 WV ELISA-positive and r-TM ELISA-negative sera showed that the major core protein was immunodominant on WV antigen. It is concluded that the r-TM ELISA was more sensitive than the AGID test but less sensitive that the WV ELISA, particularly for detecting antibodies in the early stages of infection.

Comparison of ovine lentivirus detection by conventional and recombinant serological methods.

Recombinant (r) transmembrane protein (TM), major capsid protein P25, and matrix protein P16 of ovine lentivirus (OLV) were used as solid phase antigens in enzyme-linked immunosorbent assays (ELISAs) for the detection of specific antibodies against OLV in sheep sera. Sensitivity, specificity, and agreement of these three recombinant assays were compared with each other and with two currently available conventional OLV serological assays, the agar gel immunodiffusion (AGID) test and a whole-virus (WV) ELISA. Field sera from a total of 412 Midwestern United States sheep were tested and compared by the five OLV detection methods, including visibly healthy sheep selected for public sale (Group A, n = 171), samples from a breeding flock of Finnsheep and Finn-cross ewes (Group B, n = 184) and moribund sheep with clinical signs associated with OLV (Group C, n = 57). The rTM ELISA was the most sensitive OLV detection assay, both overall and within each group. Sera from 48.1% (198/412) of field samples were rTM ELISA positive. By contrast, positive rates for the rP25, rP16, and WV ELISAs and AGID test were 34.2%, 32.3%, 36.9%, and 26.9%, respectively. The rTM ELISA reactivity was 36.8% for Group A sera, 50.0% for Group B sera, and 75.4% for Group C sera. Among the 21 Group C sheep possessing OLV lung lesions at necropsy, 20 (95.2%) were rTM ELISA positive. The greatest test agreement occurred between the rP25 and the rP16 ELISAs. The data suggest that the recombinant TM immunoassay is the most accurate and sensitive of the five methods evaluated for the detection of serum anti-OLV antibodies in sheep, both at the subclinical infection and overt clinical disease stages.

Effects of recombinant ovine interferon-τ on ovine lentivirus replication and progression of disease

The antiviral effects of recombinant ovine interferon-tau (roIFN-tau) were studied in 26 lambs inoculated with ovine lentivirus (OvLV) or mock-infected. Six of the OvLV-infected lambs and three of the mock-infected lambs were treated with 10(6) antiviral units (AVU) per kg roIFN-tau daily for 30 days starting at day 0 post-inoculation (p.i.) and twice a week thereafter (early treatment). Six of the OvLV-infected lambs and three of the mock-infected lambs were treated with 10(6) AVU/kg roIFN-tau daily for 30 days starting at day 150 p.i. and twice a week thereafter (late treatment). Six OvLV-infected and two mock-infected lambs were treated either early or late with placebo. Cell-associated viraemia was quantified by an end-point dilution method. The weekly antibody response against OvLV proteins was studied by ELISA. All experimental animals were killed at 27 weeks p.i. and histological sections of lung were scored for the degree of lymphoid interstitial pneumonia (LIP). A 90% reduction in OvLV titres was detected at 4 weeks post-treatment in lambs that received early roIFN-tau treatment (P<0.01). Differences in virus titres were also found at weeks 2 and 6 (P<0.05). Scores for LIP degree were higher in infected lambs treated with placebo or late roIFN-tau than in the mock-infected lambs or in the infected lambs that received early roIFN-tau (P<0.05). LIP scores were not different between mock-infected lambs and infected lambs that received early roIFN-tau. These results indicate that roIFN-tau curtails OvLV replication in vivo and reduces the likelihood of development of lentivirus-induced LIP when infected lambs are treated during the initial phases of OvLV infection.

Semiliki forest virus vector carrying the bovine viral diarrhea virus NS3 (p80) cDNA induced immune responses in mice and expressed BVDV protein in mammalian cells

Bovine viral diarrhea virus (BVDV) is a primary pathogen responsible for bovine enteric, respiratory and reproductive failure. A genetic region is encoding the p80 (NS3) of BVDV as the most conserved protein among Pestiviruses. BVDV infection in cattle induces NS3 specific lymphocyte proliferation and humoral responses. To generate a DNA vaccine against BVDV, the gene for BVDV-NADL NS3 was cloned into an eukaryotic expression vector of Semiliki Forest virus (pSFV-1). Quadriceps muscles of BALB/c mice were injected with recombinant DNA generated statistically significant cytotoxic T-lymphocyte activity (CTL) and cell mediated immune (CMI) responses against cytopathic and noncytopathic BVDV. Whereas, the BVDV-NS3 did not generate neutralizing antibodies against BVDVin mice. pSFV-1-NS3 DNA was subjected to in vitro transcription into mRNA. The mRNA was transfected into baby hamster kidney cells (BHK-21) and Madin-Darby bovine kidney cells (MDBK). The recombinant cells were used in the detection of DNA antigen responses by immunological assays. This report establishes the ability of BVDV-NS3 DNA inoculation to induce a strong cellular immune responses in mice.

Application of recombinant bovine viral diarrhea virus proteins in the diagnosis of bovine viral diarrhea infection in cattle

The National Animal Disease Laboratory (NADL) vaccine strain of bovine viral diarrhea virus (BVDV) genes for gp48 and p80 were expressed in Escherichia coli. The BVDV-NADL gene for gp62 was integrated into a baculovirus genome for expression in Spodoptera frugiperda (Sf-9) insect ovarian cells. The antigenicity of baculovirus expressed BVDV protein was detected by anti-BVDV specific antibodies in an enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent assay (IFA) and radio-immunoprecipitation (RIP). The recombinant proteins isolated from bacteria showed antigenic properties when analyzed by ELISA and immunoblotting using BVDV antibodies. The recombinant proteins were then used in ELISA or IFA to detect BVDV infection by testing 54 independent bovine serum samples. The baculovirus-expressed BVDV protein was used as an ELISA and IFA antigen, and the bacteria-expressed proteins were used as ELISA antigens. BVDV-NADL-infected Madin-Darby bovine kidney (MDBK) cell monolayers served as a control antigen. Statistical analysis showed a high degree of correlation between the reactivity of recombinants and natural antigens in ELISA using bovine sera. The results of ELISA or IFA proved there is a high degree of correlation with the virus neutralization. In the comparative ELISA assays, the insect-cell-mediated expression revealed greater specificity and sensitivity than the bacterial expression or the natural BVDV antigens produced by cell cultures.

Serological diagnosis of caprine lentivirus infection by recombinant immunoassays

Abstract Recombinant major core protein p25 (rP25) and transmembrane protein gp40 (rTM) of the ovine lentivirus (OLV) were used as immobilized antigens in enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against caprine arthritis-encephalitis virus (CAEV). The sensitivity and specificity of these assays were compared with an agar gel immunodiffusion (AGID) test and a whole-virus ELISA. The results showed that the rTM ELISA was more effective than rP25 ELISA or AGID test in identifying CAEV antibodies in goat populations. The rTM ELISA had similar sensitivity and specificity as the whole-virus ELISA, with an overall concordance of 87.5%. When data for rP25 and rTM were combined, the overall test agreement between whole-virus ELISA and combined recombinant ELISAs increased to 89.3%. The high quantity and purity characteristic of recombinant proteins should make them suitable as routine diagnostic antigens for CAEV and OLV serology.

Rick factors for seroprevalence of ovine lentivirus in breeding ewe flocks in Nebraska, USA

The prevalence of and risk factors for ovine lentivirus (OLV) infection in 1466 breeding ewes in nine US Meat Animal Research Center (MARC) flocks were determined using a recombinant transmembrane protein (PTM) enzyme-linked immunosorbent assay (ELISA) to detect serum anti-OLV antibodies and define infection. Based on multivariable logistic regression, confinement birth and rearing (odds ratio (OR) = 1.6), older weaning ages (OR = 1.1 week-1), and older age (OR = 1.3-2.5 year-1 beyond age 1 year) were significantly associated with higher OLV prevalence in ewes. Prevalence also varied significantly by flock, with Finnsheep and Texel ewes having the highest prevalences and Booroola Merino and Suffolk ewes having the lowest prevalences. These findings support the hypothesis that management control efforts should concentrate on events early in the life of sheep, as this period is associated with factors which can modulate the risk for OLV infection.

Ovine lentivirus antibody detection in serum, colostrum and milk using a recombinant transmembrane protein ELISA.

An enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against ovine lentivirus (OLV) in serum, colostrum, and milk from naturally infected sheep. The assay used OLV recombinant transmembrane envelope protein (rTM) as a test antigen. Matched serum/colostrum and serum/milk samples were collected at 24h, 4 weeks (mid-lactation), and 8 weeks (weaning) post-lambing. Among 129 paired samples collected at 24 h post-lambing, there was overall test agreement (concordance) of 82.9% and a kappa value of 0.658 between serum and colostrum rTM ELISA results. Among 130 mid-lactation samples, the milk ELISA had 100% specificity and 64.9% sensitivity relative to the serum ELISA, there was concordance of 79.2%, and a kappa value of 0.602. At mid-lactation, the serum agar gel immunodiffusion test had a sensitivity of 0.390 and 0.560 relative to the serum and milk rTM ELISAs, respectively. Matched serum and milk rTM ELISA results at weaning were very similar to those at mid-lactation. Finally, increased occurrence and severity of subclinical mastitis at weaning was found in ELISA-seropositive compared with ELISA-seronegative ewes. Both subclinical mastitis and ewe OLV infection had a negative impact on lamb growth and weaning weights. Compared with blood, colostrum and milk are easier and less expensive to sample and store. These results suggest that rTM ELISA testing of colostrum and milk could be used to supplement serologic testing in OLV screening or eradication programs.