[Show abstract][Hide abstract] ABSTRACT: Kataria, R.S., Tiwari, A.K., Butchaiah, G. and Rai, A. 2007. Sequence analysis of VP2 gene hyper variable region of a cell-culture adapted Indian classical infectious bursal disease virus of chicken. J. Appl. Anim. Res., 32: 49–54.An Indian classical infectious bursal disease virus (IBDV), adapted to grow in primary chicken embryo fibroblast culture, was characterized by RT-PCR/RFLP and sequence analysis of VP2 gene hyper variable region. The Indian classical virus displayed restriction profile similar to that already reported for attenuated IBDV strains. Sixteen nucleotide changes in Indian classical virus were revealed as compared to majority consensus with four unique changes 797A-T, 887C-A, 953A-C and 1017A-G. Deduced amino acid sequence analysis showed two unique changes 270A-K and 296I-V. Presence of amino acid changes 253Q-H, 279D-N and 284A-T, as well as 330S-R indicated this virus to have undergone attenuation after serial passaging in cell-culture. Phylogenetic analysis further confirmed this virus to be most closely related to attenuated strain, PBG-98. Antigenic index analysis and hydrophilicity plots clearly show differences in the peaks of cell culture adapted, classical and very virulent viruses. The present study confirmed that Indian very virulent viruses having not originated from Indian classical virus.
Preview · Article · Sep 2007 · Journal of Applied Animal Research
[Show abstract][Hide abstract] ABSTRACT: The present study was undertaken to antigenically characterize the buffalopox virus (BPV). Six monoclonal antibodies (MAbs) against the BP4 strain of BPV have been produced and characterized. All six MAbs appeared to be specific to BPV, as none of them showed cross-reactivity with other poxviruses in antigen capture ELISA. Only two MAbs (20AB8 and 20CD11) bound significantly with different BPV isolates in antigen capture ELISA, whereas the remaining four MAbs bound weakly with the BPV. In Western blot analysis with purified BPV-BP4, the rabbit hyperimmune serum against purified BPV-BP4 reacted with 15 immunodominant polypeptides (100 kDa to 25 kDa), whereas two MAbs (21CB6, 21DB11) reacted with 42 kDa and 45 kDa polypeptides, respectively. However, three MAbs (20AB8, 20CD11, 21CB5) reacted with three degraded polypeptides (100 kDa, 40 kDa and 87 kDa) of BPV-BP4. In radioimmunoprecipitation assay (RIPA) with the rabbit hyperimmune serum to BPV-BP4, three virus specific polypeptides (69 kDa, 34 kDa, 32 kDa) were recognized in BPV-BP4, whereas two polypeptides (69 kDa, 34 kDa) were recognized in other BPV isolates (BPV-Bly, BPV-Vij96, BPV-Vij97). In virus neutralization test, none of the six MAbs tested showed any significant neutralizing ability to infection with different BPV isolates. However, the hyperimmune serum showed weak neutralizing ability to BPV infection.
No preview · Article · Sep 2004 · Veterinary Research Communications
[Show abstract][Hide abstract] ABSTRACT: Infectious bursal disease (IBD) is a highly contagious, immuno-suppressive viral disease of young chickens. The disease, in susceptible birds, is characterized by whitish watery diarrhoea, soiled vent, ruffled feathers and retarded growth. Mortality with this disease may go up to 100% in susceptible flocks, inflicting heavy economic losses to the poultry industry. IBD virus (IBDV), which belongs to the genus Avibirnavirus of the family Birnaviridae, possesses double stranded bisegmented RNA genome. The large segment A encodes for the 110-kDa polyproteins NH2-VP2-VP4-VP3-COOH, which is auto-cleaved into individual mature proteins (Kibenge et al., 1988). The viral protein, VP2 bears major virus neutralizing epitopes in the central hypervariable region. The segment B encodes for multifunctional VP1 protein, having RNA polymerase activity (Azad et al., 1985). Detection of IBDV in clinical samples is conventionally carried out serologically by agar gel precipitation (AGPT) and enzyme linked immunosorbent assay (ELISA), using hyperimmune/antiserum (Nachimuthu et al., 1995; Patnayak et al., 1997), which besides being less sensitive and cumbersome, are often difficult to interpret. The nucleic acid based detection tests like RT-PCR and nucleic hybridization overcome these problems and have been used for the detection and differentiation of various IBD viruses (Lin et al., 1994; Kataria et al., 2000). We report here use of RT-PCR for the detection of IBD virus in clinical samples and the sensitivity of this technique in detecting IBDV RNA.
No preview · Article · Jan 2004 · The Indian veterinary journal
[Show abstract][Hide abstract] ABSTRACT: Infectious bursal disease (IBD) is a highly contagious immuno-suppressive viral disease of young chickens: Out of the two serotypes of IBD virus (IBDV) reported, only serotype1 viruses are pathogenic to chickens whereas, serotype2 viruses are apathogenic in nature. In India, after 1992 emergence of very virulent IBD viruses resulted in more than 70% mortality. We report here use of DIG-labelled probe prepared from RT-PCR amplified 552 bpVP2 gene sequence for the detection of IBDV in clinical samples.
No preview · Article · Jan 2004 · The Indian veterinary journal
[Show abstract][Hide abstract] ABSTRACT: A panel of monoclonal antibodies (mAbs) was generated against the RBOK strain of rinderpest virus (RPV). All of them bound to the N protein of RPV. The antigen capture ELISA using the mAbs could detect the virus in crude viral preparations. The mAb 12BF8.1.1 showed higher reactivity with cell-associated (CA) virus, whereas the mAbs 12AD10.1.1, 12BD7.1.1 and 12DG7.1.1 showed higher reactivity with extracellular virus (hereafter referred to as cell-free (CF) virus). The mAbs 12BF8.1.1 and 12AD10.1.1 could detect the virus in infected Vero cell culture supernatants (CCS) as early as 24 h post-cytopathic effect (CPE) initiation. Detergent treatment (Triton X-100) of RPV preparations enhanced the binding of the mAbs to the virus. All the seven mAbs showed specific fluorescence in virus-infected cell cultures. The immunofluorescence (IFA) using mAbs was found to be more sensitive and reliable than the immunoperoxidase test (IPT) for detection of rinderpest.
No preview · Article · Dec 2003 · Tropical Animal Health and Production
[Show abstract][Hide abstract] ABSTRACT: The polymorphism of the major histocompatibility complex (MHC) class II DRB gene of riverine buffalo (Bubalus bubalis) was studied. Second exon sequences from the buffalo DRB locus, homologous to the cattle DRB3 gene, were amplified and characterized. A combination of single strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) in a non-denaturing gel was used to identify new DRB second exon sequences. SSCP, HA and finally sequencing allowed the identification of 22 MHC-DRB exon 2 alleles from 25 unrelated Indian river buffalo. These are the first river buffalo DRB second exon sequences reported. A high degree of polymorphism in the sequences encoding the peptide binding regions was observed and some amino acid substitutions were found unique to the river buffalo.
[Show abstract][Hide abstract] ABSTRACT: A panel of 38 monoclonal antibodies (MAbs) that react with outer membrane proteins (OMPs) of Salmonella enteritidis was produced. On the basis of their binding pattern in ELISA, the MAbs were divided into three groups. The first group, consisting of 15 MAbs, was found to be Salmonella-specific as they did not cross-react with Escherichia coli or Pasteurella multocida. The second group of 15 MAbs cross-reacted with E. coli but not with P. multocida, reflecting the closer antigenic relationship of E. coli with Salmonella. The third group of 8 MAbs cross-reacted with both E. coli and P. multocida, indicating that the antigenic determinants identified by these MAbs are conserved in all the three genera.
The antigenic relationship of the Salmonella serovars (S. enteritidis, S. gallinarum, S. typhimurium, S. dublin, S. agona, S. indiana and S. choleraesuis) was studied using OMPs prepared from them and the anti-S. enteritidis MAbs. Three MAbs appeared to be specific for S. enteritidis as they did not cross-react with any of the other Salmonella serovars. Twelve of the 38 MAbs cross-reacted with all the serovars tested. Six of these were specific to the Salmonella genus as they did not cross-react with any of the other Gram-negative bacteria tested. The reactivity pattern of the other MAbs indicated that S. gallinarum was antigenically close to S. enteritidis, followed in order by S. dublin, S. agona, S. typhimurium and S. indiana, whereas S. choleraesuis seemed to be antigenically quite distant from S. enteritidis.
No preview · Article · May 2002 · Veterinary Research Communications
[Show abstract][Hide abstract] ABSTRACT: Extracts of four indigenous plants viz. Saraca indica (bark), Ocimum sanctum (leaves), Cynodon dactylon (stem and leaves) and Withania somnifera (whole plant powder), known to have anticancer activity, were assayed for their anti-telomerase activity using vero cell line-based non-radioactive TRAP assay system developed in our laboratory. Vero cells proved to be a suitable alternative to laboratory animals for screening of anticancer drugs including plant extracts. Saraca indica and Withania somnifera extracts were found to have cytotoxic effects on vero cells. Cytotoxic effects of Withania somnifera were more severe and morphological changes appeared as early as 24 hours post treatment. Cynodon dactylon and Ocimum sanctum did not have any cytotoxicity even up to 72 hours post treatment. Apoptotic changes were seen in in vero cells treated with Saraca indica, Withania somnifera and Cynodon dactylon. Out of the four plant extracts assayed, only lowest concentration (0.01 mg/ml) of Ocimum sanctum extract had progressive telomerase inhibitory effect with +1 level telomerase at 24 hours post treatment, ± level activity at 48 hours post treatment and no telomerase activity at 72 hours post treatment. Vero cell line-based assay system coupled with conventional polyacrylamide gels and non-radioactive TRAP assay proved to be an efficient in vitro assay system for screening of plant extracts for anti-telomerase activity.
No preview · Article · Jan 2002 · Physiology and Molecular Biology of Plants
[Show abstract][Hide abstract] ABSTRACT: Attempts have been made to characterize infectious bursal disease virus (IBDV) isolates collected from different parts of India during 1993 to 1999. Phylogenetic analysis was performed on a sequence generated by cycle sequencing comprising the variable region of the VP2 gene of 14 isolates. Indian IBDV isolates had divergence of 0.2 to 4.3% at nucleotide and 0 to 2.2% at amino acid levels among themselves. Nine nucleotide changes were found in Indian IBDV field isolates, resulting in the four specific amino acid changes at 222P-A, 256V-I, 294L-I and 299N-S, reported regularly in very virulent isolates. One of the Indian IBDV isolates, UP1/99, had change D to N at position 212 in the first hydrophilic region. The serine-rich heptapeptide sequence 'SWSASGS' was conserved in all the isolates. Phylogenetically, all Indian field isolates were found to be closely related to very virulent IBDV isolates from Europe, Japan, China and Israel.
[Show abstract][Hide abstract] ABSTRACT: A simple method for the rapid detection of rabies virus was developed employing a single-tube reverse transcription–polymerase chain reaction (RT-PCR). The method utilized a single buffer system for both RT and PCR and was performed without interruption as a single thermal cycling programme. Two primer sets within the genes coding for rabies nucleoprotein and glycoprotein were used to amplify a 533 bp and a 406 bp amplicon, respectively. The amplified products were detected with a challenge virus strain (CVS) of rabies. There was no amplified product with uninfected mouse or dog brain. The method was applied to detect rabies virus in 10 mouse inoculation test (MIT)-positive and three MIT-negative brain tissue samples. The amplified product was found only in the MIT-positive samples. The amplified product was confirmed by restriction endonuclease analysis using Hinf1. The results from RT-PCR correlated well with the results from MIT. This indicates that the single-tube RT-PCR may be a useful method for detecting rabies virus in brain tissue samples from suspected cases of rabies.
No preview · Article · Mar 2001 · Veterinary Research Communications
[Show abstract][Hide abstract] ABSTRACT: A DNA vaccine expressing glycoprotein C (gC) of bovine herpesvirus-1 (BHV-1) was evaluated for inducing immunity in bovines. The plasmid encoding gC of BHV-1 was injected six times intramuscularly or intradermally into calves at monthly intervals. After immunization by both routes neutralizing antibody and lymphoproliferative responses developed. The responses in the intradermally immunized calves were better than those in calves immunized intramuscularly. However, the intradermal (i.d.) route was found to be less efficacious when protection against BHV-1 challenge was compared. Following intranasal BHV-1 challenge, all immunized calves demonstrated a rise in IgG antibody titre on day 3, indicating an anamnestic response. The control non-immunized calf developed a neutralizing antibody response on day 7 post-challenge. The immunized calves showed a slight rise in temperature and mild clinical symptoms after challenge. The intramuscularly immunized calves showed earlier clearance of challenge virus compared with intradermally immunized calves. These results indicate that DNA immunization with gC could induce neutralizing antibody and lymphoproliferative responses with BHV-1 responsive memory B cells in bovines. However, the immunity developed was not sufficient to protect calves completely from BHV-1 challenge.
No preview · Article · Mar 2001 · Veterinary Microbiology
[Show abstract][Hide abstract] ABSTRACT: A highly reproducible, dominant, monomorphic fragment of 473 base pair (bp) amplified from the genome of Trypanosoma evansi by arbitrary primer-polymerase chain reaction (AP-PCR) was labelled with digoxigenin and investigated for its potential as DNA probe. Dot-blot hybridisation of total genomic DNA with the probe proved useful in detecting bubaline, cameline and equine strains of T. evansi down to 10 pg of parasite template DNA. No cross-hybridisation was seen with Babesia bigemina, Theileria annulata and the bubaline host DNA. This probe may facilitate laboratory identification of T. evansi in developing countries, without the inherent risk associated with radioisotopes.
No preview · Article · Feb 2001 · Acta Veterinaria Hungarica
[Show abstract][Hide abstract] ABSTRACT: The currently used Plowright's tissue culture rinderpest vaccine (RBOK strain) gives full protection and lifelong immunity, but it is highly thermolabile and requires maintenance of cold chain from vaccine production till delivery. Keeping in view the need for a thermostabile vaccine in tropical developing countries with limited refrigeration facilities, we passaged serially the RBOK strain of rinderpestvirus (RPV) at gradually elevated temperature up to 40 degrees C to obtain a thermoresistant RPV (TR-RPV) mutant. The thermoresistance (thermostability) and antigenicity of TR-RPV were compared with those of the vaccine virus by various methods, confirming the acquired properties. Thus, the infectivity titres of the TR-RPV mutant and vaccine virus were determined after incubation for various times at 37 degrees C. Regression analysis indicated that TR-RPV had a half-life of 1.81 hr and a degradation constant of 0.1656, while the parent vaccine virus had a half-life of 1.11 hr and a degradation constant of 0.2686. In capture ELISA with four different monoclonal antibodies (MAbs) to the N protein of RPV, TR-RPV showed a 10-fold higher reactivity with one MAb as compared to the vaccine virus. Although TR-RPV did react also with the other three MAbs, its reactivity was only 4-5 times higher than that of the vaccine virus. A treatment of the virus with Triton X-100 resulted in 2-4 times higher reactivity with the MAbs. The 35S-methionine-labeled vaccine virus-and TR-RPV-infected Vero cell lysates showed 6 polypeptide bands with identical pattern of migration in polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE). Radioimmunoprecipitation assay (RIPA) of the TR-RPV and vaccine virus with a rabbit anti-RPV immune serum (RHIS) and bovine anti-RPV hyperimmune serum (BHIS) showed the presence of four identical antigenic proteins, namely H, N, F and M, for both viruses. It can be concluded that TR-RPV has indeed retained the antigenic properties of the parental vaccine virus besides acquiring thermoresistance.
[Show abstract][Hide abstract] ABSTRACT: Five field isolates of Newcastle disease virus, including one from a pigeon from the Indian subcontinent, along with three vaccine strains have been characterized by sequence analysis of the fusion protein (F) gene in the region encoding the F 2 -F 1 cleavage site. Based on the amino acid sequence present at the cleavage site and on the percent divergence at nucleotide and amino acid levels, three field isolates could be classified as velogenic and two were of lentogenic pathotypes. The velogenic pathotypes had the sequence RRQK/RRF at the cleavage site, while the lentogenic strains had GRQA/GRL at the corresponding position.
[Show abstract][Hide abstract] ABSTRACT: Two different radio-labeled nucleic acid probes, prepared from reverse transcription-polymerase chain reaction (RT-PCR) amplified variable region of VP2 and VP1 gene sequences of a highly virulent infectious bursal disease virus (IBDV), were tested for their ability to detect field isolates of IBDV directly in clinical bursal tissue specimens and vaccine strains of IBDV in tissue cultures. The VP2 gene probe was able to detect both field isolates and vaccine strains of IBDV under high as well as low stringency while the VP1 gene probe could differentiate under high stringency field isolates from vaccine strains, hybridizing only with RNA of field isolates. The sensitivity of both the probes was found to be 4 ng of purified viral RNA.
[Show abstract][Hide abstract] ABSTRACT: The reverse transcription-polymerase chain reaction (RT-PCR) was standardized to amplify the VP-7 gene sequences of an Indian isolate of bluetongue virus serotype 23. Using two different sets of primers, a sequence of 1156 bp comprising the complete coding sequence of the VP-7 gene and its 770 bp internal sequence were amplified. The sensitivity of RT-PCR, using these two sets of primers individually was 40 pg and 4 pg, with the external and internal primers, respectively, whereas the nested PCR was 100-fold more sensitive than the single PCR with the external primers. Further, by restriction enzyme digestion of the 1156 bp amplicon, using CfoI, PstI and TaqI enzymes, the Indian isolate was found to be genetically different from isolates from the United States and Australia. RT-PCR and restriction enzyme digestion were applied to detect virus directly in blood samples taken from sheep suspected of bluetongue virus infection.
No preview · Article · Oct 2000 · Veterinary Research Communications
[Show abstract][Hide abstract] ABSTRACT: Genomic DNA was isolated from bovine milk somatic cells. Three milliliters (minipreps) and 30 ml (maxipreps) of milk samples were processed for isolation of genomic DNA. Use of this DNA for genotyping of cattle based on α-lactalbumin loci by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) indicated that milk somatic cells can be an easy and reliable source of DNA for genotyping of farm animals or any other enzymatic reactions.
Full-text · Article · Sep 2000 · The Indian journal of animal sciences
[Show abstract][Hide abstract] ABSTRACT: The technique of RT-PCR and restriction enzyme analysis was standardized to detect and differentiate Newcastle disease viruses. Digestion of RT-PCR-amplified, F gene sequences encoding for the cleavage activation sites of fusion protein with restriction enzymes AluI, BglI, HaeIII, HinfI, HhaI, RsaI, StyI and TaqI was carried out in order to characterize Newcastle disease viruses of varying pathogenicity. Restriction enzyme digestion of the amplicons by BglI and HhaI could group eight viruses, both field isolates and known vaccine strains, into lentogenic, mesogenic and velogenic pathotypes. By employing this technique directly on a clinical sample, Newcastle disease virus of the lentogenic pathotype could be detected.
Full-text · Article · Jun 2000 · Veterinary Research Communications