Revanaiah Yogisharadhya

Zoonotic infections of buffalopox in India.

Four outbreaks of buffalopox in domestic buffaloes, with considerable mortality with high case fatality rates in young buffalo calves and high morbidity with significant productivity loss in terms of reduction in milk yield in adult animals along with severe zoonotic infection in milk attendants were recorded at various places in India, during 2006–2008. In buffaloes, the pox lesions were confined to udder and teats of the majority of the affected animals, and in few animals the lesions were appeared on the hindquarters, indicating generalized infection. The overall disease morbidity, mortality and case fatality rate were 6.8%, 0.7% and 11.4% respectively. Milkers developed pox‐like lesions on the hands, forearms and forehead accompanied by fever, axillary lymphadenopathy and general malaise. The causative agent of the outbreaks, buffalopox virus (BPXV), was confirmed upon virus isolation in cell culture, electron microscopy, A‐type inclusion (ATI) and ankyrin repeat protein (C18L) gene‐specific polymerase chain reactions (PCR). Further, sequence analysis of the BPXV isolates from human and buffalo showed more identity of ATI and C18L genes sequences with that of other orthopoxviruses at nucleotide and amino acid levels and confirmed a close relationship of BPXV with Vaccinia virus (VACV) or VACV‐like viruses. Considering the zoonotic impact and productivity losses of buffalopox infection, the control measures are imperative in curtailing economic and public health impact of the disease.

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.

Identification and phylogenetic analysis of orf viruses isolated from outbreaks in goats of Assam, a northeastern state of India

Two outbreaks of orf virus (ORFV) (a parapoxvirus) infection in goats, which occurred in Golaghat and Kamrup districts of Assam, a northeastern part of India, were investigated. The disease was diagnosed by standard virological and molecular techniques. The entire protein-coding region of B2L gene of two isolates were cloned and sequenced. Phylogenetic analysis based on B2L amino acid sequences showed that the ORFVs identified in these outbreaks were closely related to each other and both were closer to ORFV-Shahjahanpur 82/04 isolate from north India. The present study revealed that the precise characterization of the genomic region (B2L gene) might provide evidence for the genetic variation and movement of circulating ORFV strains in India.

TaqMan real-time PCR assay based on DNA polymerase gene for rapid detection of Orf infection.

Both conventional and real time PCR (rt-PCR) assays based on the amplification of a 103bp fragment from the DNA polymerase (DNA pol) gene (conserved, non-structural) of Orf virus (ORFV) were developed for detection and semi-quantitation of ORFV DNA from infected cell culture and clinical samples. The latter technique was based on TaqMan chemistry. The rt-PCR assay was specific and sensitive as it could detect as low as 3.5fg or 15 copies of ORFV genomic DNA. Both intra- (0.38-1.0%) and inter-assay (0.53-2.87%) variabilities of rt-PCR were within the acceptable range meaning the high efficiency and reproducibility of the assay. The rt-PCR was applied successfully to detect ORFV DNA from suspected clinical samples. Further, the assay has shown a relative diagnostic sensitivity and specificity of 100% and 93.5%, respectively, when compared to B2L gene based semi-nested PCR implying a wide potential of this rt-PCR for rapid field diagnosis of Orf in sheep and goats.

Immunogenicity of highly conserved recombinant VacJ outer membrane lipoprotein of Pasteurella multocida.

Bacterial lipoproteins are emerging targets for inducing protective immunity against many infectious diseases. VacJ is a highly conserved and widely distributed outer membrane lipoprotein of Pasteurella multocida strains, which are known to affect a wide range of domestic as well as wild animals and birds. In the present study, the gene encoding for mature VacJ outer membrane lipoprotein of P. multocida serogroup B:2 strain P52 was cloned and over-expressed in Escherichia coli as a fusion protein. The purified recombinant VacJ protein (∼44kDa) was used for immunizing mice (6/group) along with adjuvants (FCA and alum) in two experiments. Immunization of mice with rVacJ (30μg and 75μg/mice) elicited humoral immune response with significant (P<0.01) rise in antigen-specific titers of IgG and its subtypes (IgG1 and IgG2a). No protection was noticed in mice immunized with rVacJ (30μg) along with FCA followed by challenge with 100 LD50 of the homologous strain. On the contrary, higher rVacJ dose (75μg) along with FCA and alum provided 66.7% and 50% protection respectively, at reduced challenge dose (8 LD50). The study indicated that a lipidated recombinant VacJ lipoprotein with suitable adjuvants could potentially act as candidate antigen for vaccine development against pasteurellosis in livestock.

Expression and purification of recombinant type IV fimbrial subunit protein of Pasteurella multocida serogroup B:2 in Escherichia coli

Pasteurella multocida serogroup B:2, a causative agent of haemorrhagic secpticaemia (HS) in cattle and buffalo especially in tropical regions of Asia and African countries, is known to possess a type IV fimbriae (pili) as one of the virulent factors. In the present study, ptfA gene encoding for type IV fimbrial subunit of P. multocida serogroup B:2 (strain p52), an Indian HS vaccine strain, has been cloned and over-expressed in recombinant Escherichia coli. The recombinant type IV fimbrial subunit protein (∼31 kDa) including N-terminus histidine tag was purified under denaturing condition and confirmed by western blotting. A homology model of HS causing P. multocida serogroup B:2 fimbrial subunit has also been discussed. The study indicated the potential possibilities to use the recombinant fimbrial protein in developing HS subunit vaccine along with suitable adjuvant.

Expression of P32 protein of goatpox virus in Pichia pastoris and its potential use as a diagnostic antigen in ELISA.

The present study was undertaken to express goatpox virus (GTPV) P32 protein in Pichia pastoris and evaluate its potential use as a diagnostic antigen in ELISA. The amplified P32 gene of GTPV was cloned into pPICZalphaA vector and characterized by PCR, restriction enzyme digestion and sequencing. The characterized linear recombinant plasmids were transformed in Pichia host GSII5 strain by electroporation and the zeocin resistant Pichia transformant containing P32 gene was selected and confirmed by PCR. The expression of P32 protein in Pichia was induced with 0.5% methanol at 30 degrees C. The optimum expression was observed at 72 h post-induction and the yield was 100 mg/L of culture. The expressed protein was precipitated with polyethylene glycol and analyzed by SDS-PAGE and Western blot using GTPV specific serum and GTPV-P32 protein specific monoclonal antibody. Further, the protein precipitated with acetone was evaluated as diagnostic antigen in indirect ELISA in order to replace the whole GTPV. The standardized P32 protein based indirect ELISA had relative specificity and sensitivity of 84.2% and 94.2-100%, respectively when compared with serum neutralization test and whole virus based indirect ELISA. This study showed a potential of the yeast expressed GTPV-P32 protein as safe antigen in ELISA for seroepidemiological study of the capripox infection in sheep and goats, in India as well as capripox enzootic countries.

Comparative sequence analysis of poxvirus A32 gene encoded ATPase protein and carboxyl terminal heterogeneity of Indian orf viruses

Thirteen orf virus (ORFV) isolates from natural outbreaks in sheep and goats belonging to different geographical regions of India were analysed on the basis of ORF108 (a homologue of poxviral A32 gene), which is known to encode for ATPase and involved in virion DNA packaging. Comparative sequence analysis of ATPase proteins revealed highly conserved N-terminal region with five different motifs [Walker A, Walker B, A32L specific motifs (III and IV) and a novel AYDG (motif-V)] among all poxviruses and divergent carboxyl terminus with either single or double RGD sequences among all Indian ORFV isolates. A homology model and secondary structure predictions of N-terminal region of ORFV A32 revealed that most of the poxviruses including ORFV ATPase protein belong to a distinct clade of the HerA/FtsK super family of DNA packaging proteins. Despite differences in host cell specificity and poxvirus infections among animals, DNA packaging motor domain of poxviruses presumed to share remarkable similarities as indicated by the presence of conserved ATPase motifs in the present investigation. The study also indicated the circulation of heterogeneous strains of ORFV in India and possibilities of differentiation of ORFV strains based on C-terminal heterogeneity.

Animal poxvirus vaccines: a comprehensive review

The family Poxviridae includes several viruses of medical and veterinary importance. Global concerted efforts combined with an intensive mass-vaccination campaign with highly efficaceious live vaccine of vaccinia virus have led to eradication of smallpox. However, orthopoxviruses affecting domestic animals continue to cause outbreaks in several endemic countries. Different kinds of vaccines starting from conventional inactivated/attenuated to recombinant protein-based vaccines have been used for control of poxvirus infections. Live virus homologous vaccines are currently in use for diseases including capripox, parapox, camelpox and fowlpox, and these vaccines are highly effective in eliciting (with the exception of parapoxviruses) long-lasting immunity. Attenuated strains of poxviruses have been exploited as vectored vaccines to deliver heterologous immunogens, many of them being licensed for use in animals. Worthy of note are vaccinia virus, fowlpox virus, capripoxvirus, parapoxvirus and canary pox, which have been successfully used for developing new-generation vaccines targeting many important pathogens. Remarkable features of these vaccines are thermostability and their ability to engender both cellular and humoral immune responses to the target pathogens. This article updates the important vaccines available for poxviruses of livestock and identifies some of the research gaps in the present context of poxvirus research.

Molecular characterization of Indian sheeppox and goatpox viruses based on RPO30 and GPCR genes

Sheeppox and goatpox are economically important diseases of small ruminants caused by sheeppox virus (SPPV) and goatpox virus (GTPV), respectively. Although SPPV and GTPV have host preference, some strains may infect both sheep and goats. As capripox viruses (SPPV, GTPV and LSDV) are antigenically related but genetically distinct, their differentiation requires analysis at molecular level. In the present study, RPO30 and GPCR genes of eight Indian SPPV and GTPV isolates were PCR amplified, cloned and sequences are genetically and phylogenetically analyzed. The RPO30 gene of SPPV and GTPV had lineage-specific signatures, and deletion of 21-nucleotide exclusively present in SPPV. Similarly, GPCR gene also had lineage-specific signatures for SPPV and GTPV. Phylogenetic analysis of capripox viruses based on RPO30 and GPCR genes revealed three distinct lineage-specific clusters as per their host origin. Our study supports that both RPO30 and GPCR genes could be used for differentiation of SPPV and GTPV as well as for molecular epidemiological studies. The study also highlights the distinct lineage specificities of the Indian SPPV and GTPV isolates including vaccine strains.