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Sheeppox and goatpox are diseases of sheep and goats caused by sheeppox virus (SPPV) and goatpox virus (GTPV), respectively. These two viruses are the members of the Capripoxvirus (CaPV) genus of the Poxviridae family and closely related to lumpy skin disease virus (LSDV) of cattle, the other member of the genus [22]. The genome of CaPV consists of linear dsDNA, is of 150–160 kbp nucleotides long and has termini with cross-linked hairpins. The genome has terminally redundant sequences at both the ends and these sequences have reiterated inverted terminal sequences which are repeated in tandem. Double-stranded DNA is covalently linked at both ends [6]. The genome codes for more than 200 proteins ranging from host range to immunodominant P32, cytokines and complement analogues.
Clinically, both the diseases exhibit fever and generalised pock lesions. They cause high morbidity, mortality and trade restriction of sheep and goats and their by-products and hence, are considered as economically important [1]. Capripox viruses (CaPVs) usually have no host preference as they infect both sheep and goats [19]. However, sheeppox and goatpox are considered as different entities in India [13] and recently, it was confirmed, in other countries as well [2].
Capripox are enzootic in Africa, particularly to the north and west of the Sahara, in the Middle East and Far East, and the Indian subcontinent. In India, outbreaks of the disease occur frequently incurring economic losses to the sheep and goat industries [6, 7, 17, 20, 23, 24]. Both the diseases have major impact on small ruminant productivity in countries where disease are enzootic, due to reduced milk yield, damage to hides and mortalities [26]. Mortality in young animals can exceed 50% and occasionally up to 100% in naive animals [6]. As the losses due to capripox in Maharashtra (India) state alone, with an average morbidity and mortality, are estimated over INR 105 million (US$2.3 million) and annual loss at the national level extrapolates to INR 1250 million (US$ 27.47 million) [8].
Control and eradication of any infectious disease rely on an effective vaccine and a suitable diagnostic tool for early detection of the causative agent. For capripox control in India, live attenuated vaccines are available. Despite the availability of several diagnostic tools for capripoxviruses [19], a simpler and more effective tool is always desirable. It is difficult to differentiate SPPV and GTPV based on the clinical picture as infection and pathogenesis of the diseases are alike. They are closely related to each other antigenically, which makes them indistinguishable serologically. It is now proved that cross infections of sheeppox and goatpox can occur [7, 25]. The species of the virus is normally assumed to be defined by the host, but this is not always so and this approach needs to be replaced with molecular techniques for unequivocal differentiation of the species. It is also needed when low virulence strains cause similar infections in both sheep and goats, or some strains circulate in conflicting host species as reported earlier [13]. Recently, infection of goats by sheeppox virus has been confirmed using sequencing of the full length P32 gene in an experimental animal study [7] in India and also in Vietnam where some CaPV isolates shown distinct host preferences for goats rather than sheep [1]. So, it is essential that both species of capripoxvirus should be distinguished when both viruses affect both target species.
P32, one of the structural proteins conserved among all the capripoxviruses, contains major immunogenic determinants and has been targeted by many researchers to develop several molecular diagnostic tools for unequivocal detection and differentiation of CaPV [7, 9, 10, 12, 13, 18, 23]. It is analogous to P35 protein of vaccinia virus expressed on the envelope of the mature intracellular virion and may play a role in virus attachment, virulence and virus assembly.
Earlier, in our laboratory, duplex PCR targeting two different genes of CaPV genome for detection and differentiation of SPPV and GTPV isolates have been attempted [10]. However, differentiation of two viruses based on two different genes is not always reliable and is expensive. The final confirmation of either species from cell culture isolates or suspected clinical sample is done by commercial sequencing of the full length P32 gene of CaPV genome. As it involves more time and cost, it is not always employed. Therefore, differentiation by a simple PCR-RFLP targeting a conserved gene could be preferred to tedious cloning and sequencing. In this study, a restriction enzyme specific differentiation technique, in the form of PCR-RFLP on the attachment gene (partial gene of P32 protein) of CaPV using two different enzymes, is developed and evaluated using known CaPV isolates and clinical specimens from sheep and goats.
Differentiation of Sheeppox and Goatpox Viruses by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism
- Received Date: 28 July 2012
- Accepted Date: 29 October 2012
Abstract: In the present study, the partial gene sequences of P32 protein, an immunogenic envelope protein of Capripoxviruses (CaPV), were analyzed to assess the genetic relationship among sheeppox and goatpox virus isolates, and restriction enzyme specific PCR-RFLP was developed to differentiate CaPV strains. A total of six goatpox virus (GTPV) and nine sheeppox virus (SPPV) isolates of Indian origin were included in the sequence analysis of the attachment gene. The sequence analysis revealed a high degree of sequence identity among all the Indian SPPV and GTPV isolates at both nucleotide and amino acid levels. Phylogenetic analysis showed three distinct clusters of SPPV, GTPV and Lumpy skin disease virus (LSDV) isolates. Further, multiple sequence alignment revealed a unique change at G120A in all GTPV isolates resulting in the formation of Dra I restriction site in lieu of EcoR I, which is present in SPPV isolates studied. This change was unique and exploited to develop restriction enzyme specific PCR-RFLP for detection and differentiation of SPPV and GTPV strains. The optimized PCR-RFLP was validated using a total of fourteen (n=14) cell culture isolates and twenty two (n=22) known clinical samples of CaPV. The Restriction Enzyme specific PCR-RFLP to differentiate both species will allow a rapid differential diagnosis during CaPV outbreaks particularly in mixed flocks of sheep and goats and could be an adjunct/supportive tool for complete gene or virus genome sequencing methods.