Molecular epidemiology of canine parvovirus in southern India

Aim: The present study was conducted to isolate and characterize canine parvovirus circulating in Southern India by genetic analysis of VP2 capsid protein gene. Materials and Methods: In this study, 128 samples were collected from nine different locations covering five Southern Indian states (Pondicherry, Tamil Nadu, Kerala, Andhra Pradesh and Karnataka) . Out of 128 samples, 69 samples were found to be positive by PCR assay. Out of 69 positive samples, 36 were randomly selected and processed for virus isolation. Twenty viruses could be isolated successfully and 18 randomly selected isolate were subjected to VP2 gene sequence analysis along with 6 random clinical samples. Result: Seventeen isolates and 5 clinical samples were characterized as New CPV-2a (CPV2a with 297-Ser→Ala). But one isolate and one clinical sample had amino acids variations which were characteristics of New CPV-2b. The phylogenetic analysis revealed that one of the field isolates was found to be phylogenetically closely related to New CPV-2b strains of India; rest other sequences was found to share ancestral origins with New CPV-2a reference strains of Japan, China, Thailand and India. Conclusion: The present study revealed that the predominant CPV strain circulating in Southern India is New CPV-2a. There is also enough indication of New CPV-2b strain from different states of Southern India.


Introduction
emerged in 1978 as a highly contagious and very serious disease in dogs [1].But CPV-2 underwent Canine parvovirus (CPV) is the most significant evolutionary changes after its emergence in the late viral cause of acute haemorrhagic enteritis and 1970s and within few years CPV-2 has been globally myocarditis in puppies over the age of 3-4 months [1].
replaced by the newer antigenic variants viz., CPV-2a CPV presumably originated as a host range variant and 4,8].At present, the variants of CPV from feline panleukopenia virus (FLV) that adapted to are distributed worldwide and the original prototype 2 new canine host via wild carnivores like minks and no longer circulates in the dog population [9,10,11].foxes [2].A small, round non-enveloped virus was In 2000, a new variant of CPV which was named as observed by electron microscopy in stool specimens CPV-2c was reported in Italy [12].This new variant and tissues in affected animals.Subsequently, a novel was distinguishable from CPV 2a/2b by the parvovirus was isolated in both canine and feline cell substitution of Glu in lieu of Asn/Asp at 426 residue of cultures [3].The Canine parvovirus 2 (CPV-2) belongs the VP2 capsid protein; therefore it was also referred to to the family Parvoviridae with the genome of linear as Glu-426 mutant [12].Additional amino acid single stranded DNA of 5.2 kb in size.The CPV difference was observed in both CPV-2a and CPV-2b at genome has two open reading frames (ORF) of which position 297 (Ser to Ala) [10].This mutation was first the first ORF encodes one non structural protein (NS1) appeared in 1993 in German CPV isolates and was and the second ORF encodes two capsid proteins (VP1 designated as New CPV-2a/2b [10].and VP2) which are translated from alternatively Molecular diagnostic techniques like PCR based spliced mRNA [4].VP2 gene mainly comprises the methods had been the most reliable techniques having icosahedral capsid of CPV, and only a few amino acid high degree of sensitivity and specificity in detecting substitutions in its sequence can alter relevant CPV from faecal samples [12].Amplification of VP2 immunological characteristics of the virus [5,6,7].
gene fragment (capsid protein) using primers like Canine parvovirus (CPV) caused by CPV-2 H /H and subsequent sequencing of the PCR for rev products covering the informative amino acids would definitely help in detecting genetic variation which exists between CPV-2 and its variants [12].
Most commercial vaccines available in India contain cell line monolayer.Virus isolation was carried out as CPV-2 strain whereas; only one CPV-2b based vaccine per the procedure recommended by Hirayama [16]. is available [13].Therefore, further epidemiological The infected monolayers were harvested at the 4th day survey and molecular characterization of CPV of post infection (with or without CPE) by three cycles involving samples from larger geographical area of alternative freezing and thawing.The virus would be extremely helpful in formulating a suitable supernatants were screened for the presence of virus by vaccination strategy.
PCR using the same primer pair H and H .
for rev Hence, a study was planned on molecular survey Sequencing and phylogenetic analysis: The amplified and characterization of canine parvovirus in Southern PCR products of 18 randomly selected cell culture India using PCR and subsequent sequencing of the isolates and six randomly selected clinical samples PCR products covering a portion of VP2 gene (capsid were gel extracted and custom sequenced with primer protein).The genetic analysis of canine parvovirus pair Hfor/Hrev using the automated sequencer, strains in dog population of various states in Southern Applied Biosystem 3100.The specificity of the India will reveal the predominant CPV strain/strains sequences obtained, the nucleotide variations and responsible for causing outbreaks in a larger amino acid variations with respect to the VP2 gene geographical area.
sequence of canine parvovirus were determined using  297, 300, 305, 375 and 426, respectively [13].2011 to February 2012 (Table -1).The collected For phylogenetic analysis, 30 canine parvovirus samples were emulsified in 1 ml of 0.1 M PBS of pH o sequences from various parts of the world were 7.4 and centrifuged at 6000 rpm for 15 min at 4 C.The retrieved from the GenBank and used.The sequences supernatant was collected and used for PCR were aligned using ClustalW 1.8 program and .alnand amplification and for virus isolation [13].
.nxs files were generated.The .aln file was converted to Template DNA preparation: Hundred microlitres of the .megfile using Mega4.1 [17] and Neighbor Joining processed supernatant was used for template DNA tree (NJ tree) was constructed (bootstrap replicates = o preparation by boiling at 96 C for 10 min and chilling 1000; seed = 64,248) using Kimura 2 parameter immediately in crushed ice [14].The supernatants were method for pairwise deletion at uniform rates [13].diluted 1:10 in distilled water to reduce residual The CPV sequences of 18 cell culture isolates and inhibitors of DNA polymerase activity [15].
[12] and the products were analyzed by electrophoresis using 1.5% agarose gel in Tris acetate EDTA (TAE)

buffer (1X).
Out of 128 samples screened, 69 (53.90%)Buonavoglia designed the primer pair H /H and for rev samples were found to be positive by PCR assay using utilized them for sequencing studies of canine H primer.The disease was predominantly noticed in parvovirus since this primer pair amplifies a portion of dogs between 0-6 months (73.91%) as also reported by VP2 gene containing the critical amino acids such as other authors [13,18,19].It was well known that 297, 300, 305, 375 and 426, which helps in increased intestinal epithelial turnover caused by characterization of canine parvovirus [12].changes in the microflora, diet (weaning) and diminishing maternal antibody level were the Virus isolation: Thirty six processed clinical samples, predisposing factors to CPV infection in pups [20].The representing all the nine different locations which were occurrence of CPV enteritis, in this study, was found to positive by PCR are randomly selected were filtered be more in males (62.31%) in comparison to females using 0.22 mm membrane filter (Millipore) and the (37.68%).High percentage of occurrence of CPV filtrates were used for virus isolation in 70-75% CRFK infection in non-vaccinated animals (84.05%) the maximum identity (95-99%) obtained with VP2 indicated that current vaccines confer reasonably good gene sequence of other canine parvovirus strains protection [2, 21], though there are few reports of available in the GenBank.vaccinated animals coming down with CPV infection The partial VP2 gene of 24 sequences was aligned indicating vaccine failure [13,19,20] with the reference strains for sequence analysis.In Isolation of virus was attempted in CRFK cell line comparison to prototype CPV-2 (CPV b, M38245), the from 36 samples found positive by PCR assay cell culture isolates and the clinical samples under representing the diverse locations of collection.
study had nucleotide variations at position 3675 (T→ Twenty samples showed mild cytopathic effects in the G); 3685 (C→ G); 3698 (G→ T); 3908 (A→ G) and form of rounding, increased granularity and detached identical nucleotide (A) at position 4062 (Table 2).cells, from the third passage level onwards.Moderate Seventeen cell culture isolates and five clinical isolation rate (55.55%) in this study may be attributable samples had amino acids Ala, Gly, Tyr, Asp and Asn at to the presence of antibodies in the intestinal lumen of residues 297, 300, 305, 375 and 426 respectively.the infected dogs, which may bind virions and prevent These nucleotide variations were characteristics of viral attachment to cell receptors.Similar observations New CPV-2a (CPV-2a with nucleotide variation T→G were also made by Decaro and Mohanraj who observed at position 3675 or CPV-2a with amino acid variation that the isolation of CPV could be done only for few 297-Ser→ Ala).In one cell culture isolate JN008393 days of post infection [13,14].Cryolysates of all the (Hyderabad) and one clinical sample JF900762 twenty samples were confirmed positive by PCR assay (Palakkad) had amino acids Ala, Gly, Tyr, Asp and Asp using H /H primer pair at the third passage level.
for rev These nucleotide variations were characteristics of In this study, the amplified PCR products of New CPV-2b (CPV-2b with nucleotide variation T→G eighteen cell culture isolates and six clinical samples at position 3675 or CPV-2b with amino acid variation were gel extracted and custom sequenced using 297-Ser→Ala).Automated Sequencer, Applied Biosciences 3100.The Twenty two out of 24 sequences had a nucleotide sequences obtained were subjected to 'BLAST' to study variations which were characteristics of New CPV-2a, specificity and strain identity.Sequences obtained was also reported by other authors [10,13,15,22].This were found to be highly specific to CPV as indicated by  isolated in Northern America by Kapil [26].Therefore In addition to the nucleotide variations at positions these two non-synonymous mutations indicated that 3675, 3685, 3698, 3908 and 4062, two additional non-the CPV strains were under constant selection pressure synonymous mutations were observed in the canine and were constantly evoluting which might lead to parvovirus sequences under study.At nucleotide evolution of newer CPV strains/variants in the future.position 3756-3758 (amino acid residue 324), In addition to non-synonymous mutations, three variations at 3756 (T→ A) and 3757 (A→ T) were synonymous mutations were also observed in the observed in sequences JN008387, JF900758, sequences under study.One was at nucleotide position JN008397, JN008383, JN008384, JN008396, 3824 (amino acid residue 346), where variation (G→ JN008386, JF900762, JN008385 and JN008392.This A) was observed in sequences JN008388 and type of mutation was also reported in the CPV isolated JN008389.This mutation resulted in the codon change in China by Zhang [14].Horiuchi revealed that the from GAG→ GAA, both of them coded for Glutamic residue 324 was prone to strong positive selection in all acid (Glu).Similar codon (GAA) change at nucleotide Wang ,S. and Xia, X. ( 2010 Occurrence of canine parvovirus type 2c in the dogs with 10.Ohshima, T., Hisaka, M., Kawakami, K., Kishi, M., Tohya, haemorrhagic enteritis in India, Res Vet Sci, 88: 169-171. Y. and Mochizuki, M. (2008) Chronological analysis of canine 25.Horiuchi, M., Goto, H., Ishiguro, N. and Shinagawa, M. (1994) parvovirus type 2 isolates in Japan, J Vet Med Sci, 70: 769-775.