Nucleic acid based detection of canine parvovirus and cloning and sequencing of VP2 gene
Abstract
Canine parvovirus (CPV) is the major aetiologic agent associated with diarrhoeal disease of dogs throughout the world. CPV infection causes serious dog health problems and economic losses to the owners due to mortality and high cost of treatment. Parvovirus belongs to the family parvoviridae subfamily parvovirinae and genus parvovirus. It is small, non-enveloped virus comprised of linear, negative sense, single standard DNA of about 5.2 kb. Due to mutation in critical regions virus shows genetic and antigenic diversity amongst different isolates of canine parvovirus. This sometimes leads to vaccination failures in dogs. Keeping, this point in view, the present study was undertaken to differentiate field virus from the vaccine strain of canine parvovirus. In India, an imported vaccine Nobivac DHPPi is most frequently used for vaccination of dogs. However, there are reports suggesting failure of the vaccine possibly because of emergence of new viral strains due to mutations in the VP2 gene of the virus. The VP2 gene based semi-nested PCR assay was developed and validated for direct detection of CPV in vaccine as well as faecal samples of diarrhoeic dogs. The assay was applied for amplification of viral DNA in the faecal samples of dogs to yield 747 bp products. Of 100 feacal samples (37 from diarrhoeic and 67 from non diarrhoeic) collected from various private clinics and veterinary hospitals located in Hisar district of Haryana. Twelve (32.4%) samples from diarrhoeic and 2 (3.03%) from non-diarrhoeic dogs were positive. The amplified products of both vaccine and field strains were digested with restriction enzyme (R.E) Rsa1. The analyses pattern of digested products revealed that all field virus strains of CPV differed from vaccine (CPV-2) strain. PCR based genotyping of these CPV positive field samples further suggested the predominance of CPV-2b (87%) followed by CPV-2a (7.7%). One CPV positive sample could not be typed either CPV-2a or CPV-2b suggestive of emergence of third variant. Although all these genetic variations differ only by a few aminoacids but they have greater implications for designing more efficacious vaccine strategies for better protection against variety of newly emerging variants of the virus. The PCR amplicons of 747bp of canine parvovirus were successfully cloned into pPCR-Script-Amp SK (+) plasmid vector. The clones were sequenced and the processing of the data generated by DNA sequencing confirmed the predominance of CPV2b in Haryana isolates of CPV. The CPV isolates of the present study differed with Hydrabad isolates where as these were found close to Barely isolates in sequence based phylogenetic analysis. They segregated into different clades and did not display an unequivocal geographic, temporal relationship.
This study indicated that the vaccine currently being used may not be effective in producing immune respone due to difference of the strains prevalent in Haryana.