M. Hosamani

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.

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.

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.

Sequence analysis of C18L gene of buffalopox virus: PCR strategy for specific detection and differentiation of buffalopox from orthopoxviruses

The C18L gene of buffalopox virus (BPXV), a homologue of Vaccinia virus (VACV), which encodes the ankyrin repeat protein was sequenced and analyzed to elucidate its genetic relationship with VACVs and also to devise a PCR strategy for the diagnosis of buffalopox. PCR amplification and sequencing of the C18L gene of BPXV-BP4 revealed the truncated ankyrin protein with a coding region consisting of only 50 amino acids (aa) as against a 150-aa-long peptide expressed by VACV (Copenhagen strain). BPXV-specific primers were designed and employed for sequence determination of six Indian BPXV isolates. Comparative sequence analyses of the C18L gene of BPXV isolates with that of published data of the genus orthopox viruses (OPXVs) revealed 71.2-77.3% homology at the nucleotide (nt) and 35.5-67.1% at the aa levels with VACVs. Phylogenetic analyses based on deduced aa sequences of all BPXVs showed clustering in a single group which is distinct from VACVs. Furthermore, PCR performed on the C18L gene (conventional and TaqMan) and duplex PCR based on C18L and DNA polymerase genes were developed using purified viral DNA for the specific detection and differentiation of BPXV from other OPXVs. This resulted in a specific amplicon of 368 bp from the C18L gene of BPXV. Duplex PCR resulted in 96 and 368 bp products from DNA Pol and C18L genes of BPXV and only a 96-bp amplicon of the DNA pol gene in other OPXVs. These assays were employed successfully for the differentiation of BPXV from Orthopox, Capripox and Parapox viruses as it was found to be specific only for BPXV. The authenticity of the amplicons was confirmed based on their size in agarose gel electrophoresis and sequence analysis. In contrast to the conventional PCR, the TaqMan assay was found to be rapid, specific and 100 times more sensitive with a detection limit as low as 5 pg of viral DNA. In addition, the assays were evaluated with DNA extracted from suspected clinical scab materials obtained from buffaloes, cows and human beings.

Prospects of control and eradication of capripox from the Indian subcontinent: A perspective

Sheeppox and goatpox, two endemic capripox infections in India, pose a significant economic threat to small ruminant productivity in the subcontinent. Vaccination of all susceptible sheep and goats is the feasible and sustainable means of control. Availability of effective live attenuated vaccines that are inherently thermostable and development of improved diagnostics provide the opportunities to initiate effective control measures for capripox. All animals older than 4 months can be vaccinated with the current homologous vaccines using a single vaccination by intradermal or subcutaneous routes. The success of the control program needs to be monitored by active surveillance particularly for the presence of virus, as sero-monitoring does not enable the differentiation of infection and vaccination. And also the sero-conversion following capripox vaccination is not detectable enough by the available tools. Sustained control efforts call for socio-economic and political stability, adequate infrastructure and logistic support to store and transport vaccines for reaching out vaccines to the remote end users. Availability of veterinary services, improved extension services for increased awareness among farmers, contribute significantly to the control campaigns. Poor vaccination coverage and in-adequate infrastructure in major parts of the country are some of the major elements that come in the way of effective implementation of building herd immunity through immunization.

Isolation and characterization of Indian isolates of camel pox virus

In this study, we isolated and identified three camel pox viruses (CMLV) from two outbreaks of camel pox infection in camels associated with eruptions on cheeks, nostrils, limbs, scrotum, and sheath that occurred at different places of Bikaner district, Rajasthan (India). The scab specimens collected were subjected for virus isolation in Vero cell culture, and the isolated viruses were characterized by employing polymerase chain reaction (PCR) and sequencing. The causative agent was identified as CMLV, based on A-type inclusion, B5R and C18L genes-specific PCRs and partial sequencing of these genes, which clearly confirmed that the outbreaks were caused by CMLV and identity of CMLV isolates. Further, phylogenetic analysis of partial C18L gene sequences have showed that Indian CMLV are clustered together with other reported isolates/strains.

Partial genetic characterization of viruses isolated from pox-like infection in cattle and buffaloes: evidence of buffalo pox virus circulation in Indian cows

Characterization of field isolates of viruses associated with pox-like outbreaks involving both cows (cattle) and buffaloes was carried out. PCR and electron microcopy of representative virus isolates from these animals, initially identified them as orthopoxviruses (OPXVs). Sequence and phylogenetic analyses of A-type inclusion and haemagglutinin (HA) genes of these isolates revealed a closer relationship with other OPXVs. Sequencing of the HA gene of these isolates revealed sequence identity of 96.2–99.8 and 94.6–98.7% at the nucleotide and deduced amino acid level, respectively, with VACVs, particularly with VACV-MVA, a vaccine strain. Further, C18L (ankyrin repeat protein)-gene-based BPXV-specific PCR confirmed them as BPXV. It is apparent from this study that pox-like outbreaks in cows and buffaloes in India are caused, in most cases, by BPXV. Considering the zoonotic implications of buffalopox, such outbreaks involving both buffaloes and cows in a mixed flock may pose a significant public health threat. Transmissibility of BPXV between different species including cows, buffaloes and human beings implies the potential reemergence of the virus in the subcontinent, similar to vaccinia-like outbreaks witnessed recently in other countries.