Bimalendu Mondal

Differentiation of Sheep Pox and Goat Poxviruses by Sequence Analysis and PCR-RFLP of P32 Gene

Sheep pox and Goat pox are highly contagious viral diseases of small ruminants. These diseases were earlier thought to be caused by a single species of virus, as they are serologically indistinguishable. P32, one of the major immunogenic genes of Capripoxvirus, was isolated and Sequenced from two Indian isolates of goat poxvirus (GPV) and a vaccine strain of sheep poxvirus (SPV). The sequences were compared with other P32 sequences of capripoxviruses available in the database. Sequence analysis revealed that sheep pox and goat poxviruses share 97.5 and 94.7% homology at nucleotide and amino acid level, respectively. A major difference between them is the presence of an additional aspartic acid at 55th position of P32 of sheep poxvirus that is absent in both goat poxvirus and lumpy skin disease virus. Further, six unique neutral nucleotide substitutions were observed at positions 77, 275, 403, 552, 867 and 964 in the sequence of goat poxvirus, which can be taken as GPV signature residues. Similar unique nucleotide signatures could be identified in SPV and LSDV sequences also. Phylogenetic analysis showed that members of the Capripoxvirus could be delineated into three distinct clusters of GPV, SPV and LSDV based on the P32 genomic sequence. Using this information, a PCR-RFLP method has been developed for unequivocal genomic differentiation of SPV and GPV.

Pox outbreaks in Sheep and Goats at Makhdoom (Uttar Pradesh), India: Evidence of Sheeppox Virus Infection in Goats

Sheeppox and goatpox outbreaks occur often in India incurring huge economic loss to the small ruminant industry. This paper describes two sheeppox outbreaks, of which one occurred in an organized sheep breeding farm at Makhdoom (Uttar Pradesh), India, during 2007 and another in goats at the Central Institute of Research on Goats, Makhdoom (Uttar Pradesh), India during 2008. In the first outbreak, a local Muzaffarnagari sheep breed was affected (n=477) with morbidity and mortality rates, respectively, of 100% and 53.9% accompanied by significant productivity losses. In the 2008 outbreaks, a small number of goats were affected without any mortality. The tissue and swabs collected from both the outbreaks were processed and inoculated onto Vero cells, and the causative agent of the outbreaks, capripox virus (CaPV), was isolated. The identity of the virus was confirmed as CaPV based on electron microscopy, experimental pathogenesis in sheep, capripox-specific conventional and real-time PCRs. Sequence analysis of the P32 envelope protein gene revealed that the causative agent of both outbreaks was confirmed as sheeppox virus (SPPV) implying SPPV infection not only in sheep but also goats in India.

Detection of Orf Virus from an Outbreak in Goats and Its Genetic Relation with Other Parapoxviruses

B. Mondal1, A.K. Bera1,2, M. Hosamani1, P.A. Tembhurne1,3 and S.K. Bandyopadhyay4 1Division of Virology, Indian Veterinary Research Institute, Mukteswar, Nainital, Uttaranchal 263138, India; 2Eastern Regional Station, Indian Veterinary Research Institute, Kolkata, India; 3Department of Animal Science, North Carolina State University, Raleigh, North Carolina, USA; 4Department of Animal Husbandry and Dairying, Ministry of Agriculture, Government of India, Krishi Bhaban, New Delhi, India ∗Correspondence: E-mail: bimalendu.m@email.com

Prokaryotic expression of truncated VP7 of bluetongue virus (BTV) and reactivity of the purified recombinant protein with all BTV type-specific sera.

Purification of bluetongue virus (BTV) group-specific VP7 protein, expressed in prokaryotic system as histidine-tagged fusion protein is described in the present study. The major antigenic portion of VP7 gene of BTV 23 was amplified from the extracted RNA by reverse transcription polymerase chain reaction and cloned. The recombinant expression construct (pET-VP7) was identified by the polymerase chain reaction and sequencing analysis. Expression of histidine-tagged fusion truncated VP7 protein with a molecular mass of 36 kDa was determined by Western blot analysis using anti-His antibody. The expressed VP7 was purified to near homogeneity by chromatography on nickel-agarose column as judged by sodium dodesyl sulfate-polyacrylamide gel electrophoresis analysis. The purified VP7 protein was recognized by antibody to BTV in Western blot analysis. The capability of the recombinant VP7 protein to differentiate hyperimmune serum of rabbit to BTV from normal rabbit serum was evident in the enzyme-linked immunosorbent assay (ELISA). The purified VP7 reacted well with the 24 BTV serotype-specific sera obtained from OIE Reference Laboratory on bluetongue. Our results indicated that the expressed VP7 protein could be used as antigen for development of antibody-capture ELISA for detection BTV group-specific antibodies. This recombinant protein may also be used as antigen in competitive ELISA format.

Evidence of mixed infection of peste des petits ruminants virus and bluetongue virus in a flock of goats as confirmed by detection of antigen, antibody and nucleic acid of both the viruses.

A mixed infection with peste des petits ruminants virus (PPRV) and bluetongue virus (BTV) occurred in goats which exhibited symptoms characteristic of PPR. A number of samples were collected from ailing or dead goats for labrotory diagnosis. Antibody to BTV and PPRV was detected in sera samples by competitive ELISA. No PPRV antigen was detected in tissue samples like lung and spleen, however, presence of PPRV antigen in some sera samples was confirmed by sandwich ELISA. All the blood samples collected from the ailing animals were found positive for BTV antigen by a sandwich ELISA. BTV- and PPRV nucleic acids were amplified from the pooled blood and tissue samples respectively by RT-PCR assays. The identity of the amplicons was confirmed by cloning and sequencing. All these tests confirm that the goats were infected with PPRV and BTV simultaneously. Isolation of viruses from the clinical samples is underway.

Inhibition of anatid herpes virus-1 replication by small interfering RNAs in cell culture system

Abstract RNA interference (RNAi) mediated by double stranded small interfering RNA (siRNA) is a novel mechanism of post-transcriptional gene silencing. It is projected as a potential tool to inhibit viral replication. In the present paper, we demonstrate the suppression of replication of an avian herpes virus (Anatid Herpes Virus-1, AHV-1) by siRNA mediated gene silencing in avian cells. The UL-6 gene of AHV-1 that codes for a protein involved in viral packaging was targeted. Both cocktail and unique siRNAs were attempted to evaluate the inhibitory potential of AHV-1 replication in duck embryo fibroblast (DEF) cell line. DEF cells were chemically transfected with different siRNAs in separate experiments followed by viral infection. The observed reduction in virus replication was evaluated by cytopathic effect, viral titration and quantitative real time PCR (QRT-PCR). Among the three siRNA targets used the unique siRNA UL-B sequence was found to be more potent in antiviral activity than the cocktail and UL6-A-siRNA sequences.

Molecular and phylogenetic characterization of multidrug resistant extended spectrum beta-lactamase producing Escherichia coli isolated from poultry and cattle in Odisha, India.

The present study was undertaken to determine the occurrence and characterization of extended spectrum beta-lactamase (ESBL) producing Escherichia coli isolated from cattle and poultry in Odisha, India. Of 316 E. coli isolated from 305 samples (170 fecal samples from poultry and 135 milk samples from cattle), a total of 18 E. coli isolates were confirmed as ESBL producers by combination disc method and ESBL E-test. The isolates were resistant to oxyimino cephalosporins and monobactam as revealed by disc diffusion assay and determination of minimum inhibitory concentration. Resistance against other antibiotics was frequently noted as well. Further, beta-lactamase genes viz., blaSHV, blaCTXM, blaTEM and blaampC were detected in 17, 13, 9 and 2 isolates, respectively in PCR. Of the 18 ESBL strains, 16 were positive for class I integron (int1), nine of them carried sulphonamide resistance gene (sul1) and one harbored quinolone resistance gene (qnrB). Virulence markers for extraintestinal pathogenic E. coli like astA, tsh and iucD were also present in 4, 3 and 3 isolates, respectively. All the PCR amplified products were cloned and subjected to sequencing for homology analysis and data were submitted to gene bank. Sequence analysis of the amplified variable regions of class 1 integron of four representative isolates revealed the presence of aadA2 and dfrA12 gene cassettes conferring resistance to aminoglycosides and trimethoprim, respectively. Most of the ESBL producing strains emerged as single lineage through phylogenetic analysis by RAPD and ERIC PCR. This is the first ever systemic study on multidrug resistant ESBL producing E. coli in food producing animals from India.

A polyclonal antibody-based sandwich ELISA for the detection of bluetongue virus in cell culture and blood of sheep infected experimentally.

A polyclonal antibody-based sandwich ELISA (s-ELISA) was developed for the detection of bluetongue viruses (BTV) in cell culture lysates and blood samples of sheep infected experimentally. Rabbit antiserum to purified BTV particles and guineapig antiserum to core particles were used as capture antibody and detection antibody respectively. The assay has detected several of the BTV serotypes isolated in India so far. Other common viruses of small ruminants did not cross-react in the assay. The analytical sensitivity of the assay was estimated to be between 10(2.4) and 10(2.6)TCID(50)/ml with different serotypes of BTV. The sensitivity was compared with that of the reverse transcription polymerase chain reaction (RT-PCR) and the latter was found to be at least 100 times more sensitive. In the infected sheep, BTV antigen(s) was detected in blood as early as on 5-day post-infection (dpi) till 35 dpi. The assay may be useful for testing large number of samples in a very short time.

Isolation and characterization of an Indian orf virus from goats.

Isolation and characterization of an orf virus has been described here. The virus was isolated from an outbreak of ‘scabby mouth’ in goats in Northern India. Viral morphology from the scab biopsy revealed typical ovoid‐shaped particles characteristic of Parapoxvirus. Virus was isolated from sonicated scab suspension and characterized by restriction enzyme (RE) analysis and sequencing of full‐length GM‐CSF‐ and interleukin‐2 inhibitory factor (GIF) gene. RE pattern of the virus did not show close resemblance to most of the orf viruses published earlier. However, it showed high sequence identity and closer phylogenetic relationship with previously published ORFV‐SA00 strain, as evident from the nucleotide and deduced amino acid sequence of GIF gene.

Apoptosis induced by peste des petits ruminants virus in goat peripheral blood mononuclear cells.

The ability of peste des petits ruminants virus (PPRV) to induce apoptosis in goat peripheral blood mononuclear cell (PBMC) culture was investigated. Goat PBMC were infected with PPRV and the infectivity was confirmed by cytopathic effect, demonstration of presence of infectious viral progeny and expression of viral antigens in the lymphocytes, cultured in vitro. Infected PBMC showed morphological features of apoptosis. DNA extracted from PPRV-infected cells displayed laddering pattern in agarose gel electrophoresis. Infected cells also showed significantly higher apoptotic indices measured by bisbenzimide staining than control cells. Electronmicrographs of PPRV-infected PBMC revealed features typical of apoptosis such as peripheral condensation of chromatin, blebbing of plasma membrane, fragmentation of nucleus and cell leading to formation of apoptotic bodies. Our results suggest that PPRV can induce apoptosis, in vitro, in goat lymphocytes.