Madhusudan Hosamani

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.

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.

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.

Mixed infection of peste des petits ruminants and orf on a goat farm in Shahjahanpur, India

191 bp 200 bp N M P 1 2 3 4 5 FIG 1: Agarose gel electrophoresis of peste des petits ruminants virus (PPRV) DNA amplified by M genereverse transcriptasePCR. Lane N Healthy goat splenic tissue (negative control), Lane M 100 base pair DNA ladder marker, Lane P PRRV Sungri/ 96-vaccine virusinfected Vero (positive control), Lane 1 Blood sample from a goat with clinical signs characteristic of PPRV infection, Lane 2 Lung tissue, Lane 3 Caecum tissue, Lane 4 Splenic tissue, Lane 5 Nasal swab from a goat with nasal discharge

Novel immunogenic baculovirus expressed virus-like particles of foot-and-mouth disease (FMD) virus protect guinea pigs against challenge

Vaccination is a well accepted strategy for control of foot-and-mouth disease (FMD) in endemic countries. Currently, chemically inactivated virus antigens are used for preparation of FMD vaccine. To develop a non-infectious and safe recombinant vaccine, we expressed structural polypeptide of FMDV (O/IND/R2/75) using baculovirus expression system. We show that inclusion of mutated viral 3C protease in frame with the polypeptide (P1-2A), enhanced the yield of structural proteins. The structural proteins retained antigenicity and assembled into empty virus-like particles (VLPs). Immunization of guinea pigs with purified fractions of the VLPs resulted in humoral and cell mediated immune response by 4 weeks. The VLPs elicited comparable humoral immune response and relatively higher cell mediated immune response, when compared to conventional vaccine in guinea pigs. Further, up to 70% of the VLP immunized guinea pigs were protected against challenge with homologous guinea pig adapted virus. Our results highlight the application of recombinant FMDV VLPs in FMD vaccination.

Camelpox: epidemiology, diagnosis and control measures

Camelpox is an economically important contagious skin disease of camelids caused by camelpox virus (CMLV) and is characterized by mild local skin infection and less common severe systemic infections. The disease is confined to camel-rearing belts particularly in developing countries and causes economic impact due to considerable loss in terms of morbidity, mortality, loss of weight and reduction in milk yield. The virus has gained attention from researchers due to its recent emergence with close genetic relatedness to variola virus, the causative agent of smallpox, and carrying genes responsible for host immune evasion mechanisms. CMLV was earlier thought to be a zoonotic agent but so far little evidence has been documented from Somalia. Although the disease can be diagnosed based on clinical signs, the similar confounding skin lesions necessitate identification of infection by molecular biology based diagnostic techniques, namely restriction enzyme analysis of the virus genome and specific genes, genus- and species-specific diagnostic PCRs including real-time quantitative PCR, and sequence and phylogenetic analysis for diagnosis and differentiation of CMLV. The entire genome sequence of CMLV is known and it contains more than 211 putative genes, which code for different proteins with host range, immunomodulation, virulence and other functions. Both inactivated and live-attenuated vaccines are available in some countries. However, live vaccines are preferred as they provide long-lasting immunity. Considering the virus spreads through contaminated environments, an improved diagnostic and control method would be of immense value to curtail the infection in the field. Alternative therapeutics such as antiviral agents is an area that needs to be explored. This article discusses the epidemiology and biology of the disease, novel diagnostic approaches and control measures.

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.

Development of a liquid-phase blocking ELISA based on foot-and-mouth disease virus empty capsid antigen for seromonitoring vaccinated animals

In foot-and-mouth disease (FMD) control programme, liquid-phase blocking ELISA (LPBE) is widely used to assay vaccine-induced seroconversion. Currently, the assay utilizes inactivated FMD virus antigen for the detection of antibodies in serum samples. To develop a non-infectious substitute for the antigen in LPBE, we expressed the structural polypeptide of FMDV (serotype A) using a baculovirus expression system, and show that inclusion of viral 3C with reduced protease activity resulted in a higher yield of structural proteins. Structural proteins expressed in insect cells assembled into empty virus-like particles (VLPs) and showed antigenicity comparable to chemically inactivated FMDV. Screening of serum samples from FMD-vaccinated cattle showed that the test performance of VLP-LPBE had a correlation of 0.89 with conventional inactivated virus antigen LPBE. The VLP-LPBE developed here demonstrates the diagnostic application of recombinant FMDV VLPs in monitoring seroconversion following FMD vaccination.

Sequence analysis of capsid coding region of foot-and-mouth disease virus type A vaccine strain during serial passages in BHK-21 adherent and suspension cells.

Sequence variability within the capsid coding region of the foot-and-mouth disease virus type A vaccine strain during serial in vitro passage was investigated. Specifically, two methods of virus propagation were utilized, a monolayer and suspension culture of BHK-21 cells. At three positions (VP2(131) E-K in both monolayer and suspension passages, VP3(85) H-R in late monolayer passages and VP3(139) K-E in only suspension passages), all mapped to surface exposed loops, amino acid substitutions were apparently fixed without reversion till the end of the passage regime. Interestingly, VP2(131, 121) and VP3(85) which form part of the heparan sulphate binding pocket, showed a tendency to acquire positively charged amino acids in either monolayer or suspension environment probably to better interact with the negatively charged cell surface glycosaminoglycans. At three identified antigenically critical positions (VP2(79), VP3(139) and VP1(154)), amino acids substitutions even in the absence of immune pressure were noticed. Hence both random drift and adaptive mutations attributable to the strong selective pressure exerted by the proposed cell surface alternate receptors could play a role in modifying the capsid sequence of cell culture propagated FMDV vaccine virus, which in turn may alter the desired potency of the vaccine formulations.