Identification Of Yak Respiratory Virus And Molecular Characteristics Of Yak CPV-2

Canine parvovirus(CPV-2)is a worldwide prevalence that can cause haemorrhagic enteritis and myocarditis in dogs,which causing h?ge losses to the dog industry.Being the most rapidly mutating DNA virus,there have been many epidemic strains such as CPV-2a,CPV-2b,and CPV-2c.In addition to infecting dogs,it has been confirmed that CPV-2 can infect canine animals,cats,raccoonidae and many other animals,its spectrum of infection has a growing trend.In this experiment,the molecular characteristics and genome study of the unexpectedly discovered Canine parvovirus(CPV-2)were studied on the basis of the identification of yak respiratory virus by next generation sequencing;1.Identification of Virus Species in Yak Respiratory SamplesTo investigate the diversity of viral species from nasal,30 nasal cotton swabs with obvious lung lesions from Northwest Sichuan Yak were collected..After doing viral nucleic acid extraction and reverse transcription.we used the double-Stranded cDNA to construct a library and rloaded on a HiSeq 2000 for Illumina sequencing.There were 7 virus that detected : Infectious bovine rhinotracheitis virus(IBRV),bovine viral diarrhea virus(BVDV),bovine coronavirus(BoCV),canine parvovirus(CPV-2),canine minute virus(MVC),astrovirus(Ast V)and herpesvirus-4 virus(BHV-4).Among them,BVDV,BoCV and IBRV are known as respiratory diseases syndrome viruses.CPV-2,BoCV,MVC,and BHV-4 were first discovered in yaks.This experiment provides a theoretical reference for the diagnosis and control of yak respiratory diseases.The unexpectedly detection of CPV-2 has a high abundance and its host range and cross-host transmission has received extensive attention in recent years.The genomic characteristics and variability of CPV-2 infected yaks are worthy of further study.2.Investigation of Infection and Molecular Classification of Yak CPV-2In order to identify the infection of CPV-2 in yaks,A total of 57 yak nasal swabs(containing 30 for virome)and 52 yak serums were collected.Detection of canine parvovirus in the sample using a PCR method targeting the VP2 gene with a target fragment of 567 bp containing the key amino acid sites which can be typed.the result shows that:12 samples of CPV-2 positive samples were detected in 57 yak nasal swabs,the positive rate was 21.00%;2 samples of CPV-2 positive samples were detected in 52 yak serums,and the positive rate was 3.98%.Sequencing results show that : amino acids at the position 426 of 14 CPV-2 VP2 are all Asn and at position 297 are Ala,all of the 14 are typed new 2a,the nucleotide homology between the 12 sequences of VP2 gene was 99.7%~100%.Comparative analysis of its phylogenetic trees revealed that 14 VP2 sequences were all clustered in a large 2a branch.VP2 and NS1 genes that amplified from yak serum(X1,X2)showed that at VP2,the two clustered on the same branch of 2a.There were only two common mutations at 267,440,and the 324 amino acid mutations of the X2 sample may be the reason for the genetic distance from other strains.At NS1,X1 and X2 were clustered together and there were 6 amino acid co-variation sites.Among them,175 and 402 were unique to yak serum CPV-2,and the remaining 4 amino acid mutation sites had common mutations with the 6 Chinese strains with the closest genetic relationship.In this study,CPV-2 were both detected from nasals and serums,which proved that CPV-2 can infect yaks.Their is amino acids has common variations,its biological significance needs further study.3.Genome amplification of Yak CPV-2To further understand the genomic characteristics of yak CPV-2,Based on the data obtained by the Virome and the CPV-2 genome sequence registered in GenBank,six pairs of overlapped primers were designed to amplify an approximate entire length CPV-2a genomic sequence from yak(CPV CN/ya1/2017).The near-full genome of yak CPV-2a was 4666 bp in length,contains two ORFs,with 104 bp of 5' non-coding region and 294 bp of 3' non-coding region.the ORF near the 3' end(104 to 2110bp)encodes 668 aa of non-structural protein(NS1 and NS2);the other ORF(2204 to 4372 bp)encodes a 722 aa structural protein(VP1 and VP2)near the 5' end.The homology was 99.0%~99.9% with canine gene sequence in GenBank,and the genus of CPV-2a from yak had the highest homology with the Chinese strains JL6,HB1,JL3,SD19,and SH1(99.9%).The 440 and 267 amino acid sites of the VP2 gene are mutated in this strain,which is on the loop 3 as other host range CPV-2 strains whose amino acid mutated at the same region.Its biological significance needs further study.Analysis showed that CPV CN/ya1/2017 showed a unique evolutionary relationship that clustered into an independent branch with 3 CPV-2a(SD19?SD10?SH1)and 3 CPV-2b(JL6?HB1?JL3)strainscall of these sequences were Chinese isolates since 2013.This unique evolutionary relationship of CPV-2's genome is inconsistent with VP2 but tends to be consistent with NS1.The analysis revealed that these strains have a common evolutionary feature in that their amino acids 544(Tyr?Phe)and 545(Glu?Val)sites of NS1 have been mutated which resulted in this unique clustering of the 2a and 2b strains into a new branch on the genome.The results of this experiment provide a reference for the further study of the genetic evolution of CPV-2.