Gnanavel Venkatesan

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.

Zoonotic cases of camelpox infection in India

This study reports the first conclusive evidence of zoonotic camelpox virus (CMLV) infection in humans associated with outbreaks in dromedarian camels (Camelus dromedaries) in northwest region of India during 2009. CMLV infection is usually restricted to camels and causes localised skin lesions but occasionally leads to generalised form of disease. However, the present outbreak involved camel handlers and attendants with clinical manifestations such as papules, vesicles, ulceration and finally scabs over fingers and hands. In camels, the pock-like lesions were distributed over the hairless parts of the body. On the basis of clinical and epidemiological features coupled with serological tests and molecular characterization of the causative agent, CMLV zoonosis was confirmed in three human cases. Clinical samples such as skin scabs/swabs and blood collected from affected animals and humans were analysed initially, for the presence of CMLV-specific antigen and antibodies by counter immunoelectrophoresis (CIE); serum neutralization test (SNT); plaque-reduction neutralization test (PRNT) and indirect immunoperoxidase test which was later confirmed by amplification of CMLV-specific ankyrin repeat protein (C18L) gene. Virus isolation was successful only from samples collected from camels. Further, sequence analyses based on three full-length envelope protein genes (A27L, H3L and D8L) revealed 95.2-99.8% and 93.1-99.3% homology with other Orthopoxviruses at nucleotide and amino acid levels, respectively. Phylogram of the three genes revealed a close relationship of CMLV with Variola virus (VARV). Considering the emerging and re-emerging nature of the virus, its genetic relatedness to VARV, zoonotic potential and productivity losses in camels; the control measures are imperative in curtailing economic and public health impact of the disease. This is the first instance of laboratory confirmed camelpox zoonosis in India.

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.

Prevalence of peste des petits ruminants among sheep and goats in India

This study measured the clinical prevalence of peste des petits ruminants (PPR) among sheep and goats in India between 2003 and 2009 by analyzing clinical samples from suspected cases of PPR that were submitted to the Rinderpest and Allied Disease Laboratory, Division of Virology, IVRI, Mukteswar for PPR diagnosis. PPR outbreaks were confirmed by detecting PPR virus (PPRV)-specific antigen in the clinical samples. Clinical samples (blood, nasal swabs, spleen, lymph node, kidney, liver, intestine, and pooled tissue materials) were taken from a total of 592 sheep and 912 goats in different states of India and screened for the presence of PPRV antigen using a monoclonal antibody-based sandwich ELISA kit. A total of 20, 38, and 11 laboratory-confirmed PPR outbreaks occurred among sheep, goat, and combined sheep and goat populations, respectively. Our findings provide evidence of widespread PPR endemicity in India. The underlying reasons could be variations in husbandry practices in different geographical regions, agro-climatic conditions, and livestock migration. Furthermore, decrease in the number of PPR outbreaks over time might be due to the effectiveness of current live PPR vaccines and timely vaccination of target species. Vaccination against PPR has been practiced in India since 2002 to control this disease.

Sequence and phylogenetic analyses of the structural genes of virulent isolates and vaccine strains of peste des petits ruminants virus from India.

Peste des petits ruminants (PPR) is an acute, highly contagious, notifiable and economically important transboundary viral disease of sheep and goats. In this study, sequence and phylogenetic analyses of structural protein genes, namely the nucleocapsid (N), the matrix (M), the fusion (F) and the haemagglutinin (H) coding sequences of virulent and vaccine strains of PPR virus (PPRV), were undertaken to determine the genetic variations between field isolates and vaccine strains. The open reading frame (ORF) of these genes of the isolates/strains was amplified by RT-PCR, cloned and sequenced. The ORF of N, M, F and H genes was 1578, 1008, 1641 and 1830 nucleotides (nt) in length and encodes polypeptides of 525, 335, 546 and 609 amino acids (aa), respectively, as reported earlier. Comparative sequence analyses of these four genes of isolates/strains were carried out with published sequences. It revealed an identity of 97.7-100% and 97.7-99.8% among the Asian lineage IV and 89.6-98.7% and 89.8-98.9% with other lineages of PPRV at nt and aa levels, respectively. The phylogenetic analyses of these isolates based on the aa sequences showed that all the viruses belonged to lineage IV along with other Asian isolates. This is in agreement with earlier observations that only PPRV lineage IV is in circulation in India since the disease was first reported. Further, sequence analysis of the thermostable/thermo-adapted vaccine strains showed no significant changes in the functional or structural surface protein-coding gene sequences. It is important to monitor the circulation of the PPRV in susceptible animals by H gene-based sequence comparisons in addition to the F gene- and N gene-based approaches to identify the distribution and spread of virus in the regular outbreaks that occur in endemic countries like India.

Comparative Efficacy of Conventional and TaqMan Polymerase Chain Reaction Assays in the Detection of Capripoxviruses from Clinical Samples

Sheeppox and goatpox are economically important viral diseases of sheep and goats, respectively. Both diseases are reportable to the World Organization for Animal Health. To implement a control and eradication program for these diseases, a rapid and user-friendly diagnostic tool is imperative for screening. Therefore, in the present study, TaqMan quantitative polymerase chain reaction (qPCR) and conventional PCR assays targeting the DNA polymerase (DNA pol) gene were developed for the detection of Capripoxvirus DNA from clinical specimens of sheep and goats. The 2 assays used different primer sets. Conventional PCR yielded a specific product of 134 bp, whereas qPCR yielded a 180-bp product. The specificity of amplified DNA pol gene products was confirmed by their size and by sequence analysis. The 2 assays were specific for Sheeppox virus and Goatpox virus. However, in comparison to conventional PCR, the qPCR was more rapid, specific, and 100 times more sensitive, with a detection limit as low as 0.042 pg of purified DNA. The qPCR assay was more sensitive (84.05%) than conventional PCR (76.06%) when used on clinical samples (n = 71) from sheep and goats.

Isolation and identification of virulent peste des petits ruminants viruses from PPR outbreaks in India

In this study, three outbreaks of peste des petits ruminants (PPR) in goats and sheep flocks with high morbidity and considerable mortality were recorded at Jhansi and Revati in Uttar Pradesh and Bhopal in Madhya Pradesh, India during 2003–2006. Clinical samples were collected from the affected flocks for laboratory investigation. The PPR virus (PPRV) antigen/nucleic acid in the infected tissues/swab materials was demonstrated by using sandwich enzyme-linked immunosorbent assay (ELISA) and reverse transcription–polymerase chain reaction techniques, and the antibody to PPRV in serum samples was detected by competitive ELISA. The causative agent of the outbreaks, PPRV, was successfully isolated in Vero cells at first passage itself, and its identity was confirmed. The isolated PPR viruses belong to lineage IV based on phylogenetic analysis of partial fusion gene sequences and are closely related to other Asian or Indian PPRV isolates/strains.

Evaluation of efficacy of stabilizers on the thermostability of live attenuated thermo-adapted Peste des petits ruminants vaccines

In this study, thermo-adapted (Ta) PPR vaccines were assessed for their stability at 25, 37, 40, 42 and 45°C in lyophilized form using two extrinsic stabilizers {lactalbumin hydrolysate-sucrose (LS) and stabilizer E} and in reconstituted form with the diluents (1 mol/L MgSO4 or 0.85% NaCl). The lyophilized vaccines showed an expiry period of 24–26 days at 25°C, 7–8 days at 37°C and 3–4 days at 40°C. LS stabilizer was superior at 42°C with a shelf-life of 44 h, whereas in stabilizer E, a 40 h shelf-life with a comparable half-life was observed. At 45°C, the half-life in stabilizer E was better than LS and lasted for 1 day. Furthermore, the reconstituted vaccine maintained the titre for 48 h both at 4°C and 25°C and for 24–30 h at 37°C. As both the stabilizers performed equally well with regard to shelf-life and half-life, the present study suggests LS as stabilizer as a choice for lyophilization with 0.85% NaCl diluent, because it has better performance at higher temperature. These Ta vaccines can be used as alternatives to existing vaccines for the control of the disease in tropical countries as they are effective in avoiding vaccination failure due to the breakdown in cold-chain maintenance, as this vaccine is considerably more stable at ambient temperatures.

Expression of Peste des petits ruminants virus nucleocapsid protein in prokaryotic system and its potential use as a diagnostic antigen or immunogen.

In this study, both partial and full-length nucleocapsid (N) gene of Peste des petits ruminants virus (PPRV) were cloned into pET33b vector and expressed in Escherichia coli (BL21) with the objective of replacing live PPRV antigen with recombinant protein in ELISA. The expressed proteins were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot by using a PPRV N protein specific monoclonal antibody. The expressed histidine-tagged fusion proteins were purified using affinity Ni-NTA column and were assessed for their conformation in terms of reactivity by ELISA. The immunogenicity of recombinant proteins was also assessed in rabbits and anti-N antibody response against PPRV was observed in all the immunized rabbits, when tested by competitive and indirect ELISAs. In sandwich ELISA, a mean OD(492 nm) of 1.4 and 0.90 was obtained for crude lysate having expressed the N protein and the PPRV antigen, respectively. Further, the N protein was tested as a coating antigen in competitive ELISA instead of PPRV antigen for serological diagnosis of PPR infection. This indicates the diagnostic potential of the PPRV recombinant N proteins, which are safe and better alternatives to live PPRV antigen in ELISA for clinical or sero-surveillance of PPR in enzootic or non-enzootic countries.

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.