Veerakyathappa Bhanuprakash

Buffalopox: an emerging and re-emerging zoonosis.

Abstract Outbreaks of buffalopox or pox-like infections affecting buffaloes, cows and humans have been recorded in many parts of the world. Since the first outbreak in India, a large number of epidemics have occurred. Unlike in the previous years, generalized forms of the disease are now rare; however, there are severe local forms of the disease affecting the udder and teats, leading to mastitis thereby undermining the productivity of milk animals. The causative agent buffalopox virus (BPXV) is a member of the Orthopoxvirus, and is closely related to Vaccinia virus (VACV), the type-species of the genus. Earlier studies with restriction fragment length polymorphism and recent investigations involving sequencing of the genes that are essential in viral pathogenesis have shown that BPXV is phylogenetically very closely related to VACV and may be considered as a clade of the latter. The review discusses the epidemiology, novel diagnostic methods for the disease, and molecular biology of the virus, and infers genetic relationships of BPXV with other members of the genus.

Orf: an update on current research and future perspectives

Orf is one of the most widespread viral diseases worldwide, affecting mostly small ruminants and, sometimes, other species, including wild animals. Of late, there have been an increasing number of reports of new species being affected by the disease, implying a dynamic host–pathogen interaction. The causative agent, orf virus, has been extensively investigated over recent years, owing to its zoonotic importance and ability to cross-infect other species sporadically. The evasive mechanisms that the virus has developed to adapt and grow in the presence of an active immune response helps to explain the ability of the virus to repeatedly reinfect the same host. The apparent diversity in the antigenic/immune targets of different orf virus strains involved in such repeat infections may also be contributing factors. Exposure of animals to stress or immunosupression as a result of therapy or primary viral infection can accentuate the severity of disease. Genes homologous to host cytokines or their antagonists, and which contribute to viral virulence, have been found in the viral genome. A combination of electron microscopy, histology and PCR is the most accurate laboratory approach for confirmation of the disease, although clinical signs are often typical. However, some infections may be confounded by similar clinical manifestations caused by other infections. This review presents, in brief, a recent understanding of the virus at the host–pathogen level, molecular biology of the virus, disease epidemiology, clinical manifestations in man and animals, diagnostic procedures, and the economic and environmental impact of the disease.

Comparative efficacy of peste des petits ruminants (PPR) vaccines

Peste des petits ruminants (PPR) is a highly contagious, economically important viral disease of sheep and goats with high morbidity and mortality rates. In order to control the disease effectively, highly sensitive diagnostic tests coupled with potent vaccines are important pre-requisites. At present, there are three live attenuated PPR vaccines available in India including Sungri 96, Arasur 87 and Coimbatore 97. Indian Veterinary Research Institute (IVRI) Mukteswar developed the PPR Sungri 96 (isolate of goat origin) vaccine; while Tamil Nadu Veterinary and Animal Sciences University (TANUVAS) developed the Arasur 87 (isolate of sheep origin) and Coimbatore 97 (isolate of goat origin). In this study, the potency of these vaccines including a fourth vaccine from Institute of Animal Health and Veterinary Biologicals, Bangalore (IAH&VB) were tested as per the office International des Epizooties (OIE) guidelines by challenge studies in sheep and goats and their efficacies were evaluated using PPR C-ELISA. Potency tests of these vaccines in sheep and goats revealed that three of the vaccines were potent; however, the IAH &VB vaccine was comparatively less potent. The three vaccines could presumably be used for mass vaccination of both sheep and goats while contemplating PPR control program.

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.

Bluetongue vaccines: the past, present and future

Bluetongue (BT) is a noncontagious and arboviral disease of both domestic and wild ruminants. The disease is enzootic in areas where reservoirs (cattle and wild ruminants) and vectors exist for the BT virus (BTV). A total of 24 BTV serotypes have been recognized worldwide. The major control measures include restriction of animal movement, vector control applying insecticides, slaughter of infected animals and vaccination. Prophylactic immunization of sheep against BT is the most practical and effective control measure to combat BT infection. At present, attenuated vaccines are used in the Republic of South Africa, the USA and other countries. However, EU countries were using attenuated vaccines, only recently shifting to inactivated vaccines owing to their safety and efficacy. In India, inactivated vaccines are in experimental stages and are expected to be on the market shortly. Inactivated vaccines generate serotype-specific long-lasting protective immunity after two injections, and may help in controlling epidemics. Differentiating infected from vaccinated animals (DIVA) is theoretically possible with inactivated vaccines but has not yet been developed, whereas the attenuated live vaccines are not candidates for DIVA. Attenuated live vaccines are efficacious but safety issues are of great concern. New-generation vaccines (subunit, virus-like particles, core-like particles and vectored) can be employed for DIVA. Recombinant vaccines, which generate cross-protection against multiple BTV serotypes, have great potential in BT vaccine regimens. Furthermore, new-generation vaccines are safe and efficacious experimentally, but large-scale field trials are warranted. Alternative areas, such as antivirals, siRNA, interferon and nanotechnology, may be of future use in the control of BT. We give an overview of BT vaccines, starting from conventional to recent developments, and their feasibility in controlling BT infection.

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.

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.