Molecular Characterization And Pathogenicity Of Carnivore Parvovirus Epidemic Isolates

Carnivore parvovirus belongs to the genus Protoparvovirus within the family Parvoviridae, and including canine parvovirus(CPV), feline parvovirus(FPV), and mink enteritis virus(MEV). It caused a global pandemic disease in both wild and domestic animals in families Felidae, Canidae, Mustelidae, Procyonidae, Viverridae within the order Carnivora. The disease is the most important infectious disease to order Carnivora animals, with huge economic loss every year. The present study describes our research on the molecular diagnostics, epidemiology, immunogenicity, genetic evolution and transcriptomics of carnivore parvovirus.Here we described a novel nanoPCR assay for the clinical detection and epidemiological characterization of carnivore parvovirus. It was used for detection of CPV, MEV and FPV. This assay is based upon primers specific for the conserved region of the carnivore parvovirus NS1 gene, which encodes nonstructural protein 1. Under optimized conditions, the carnivore parvovirus nanoPCR assay had a detection limit of 8.75 × 101 copies recombinant plasmids per reaction, compared with 8.75 × 103 copies for conventional PCR analysis. Furthermore no cross reactivity was observed for the nanoPCR assay with respect to related viruses, including canine distemper virus, canine adenovirus and Aleutian mink disease parvovirus. The nanoPCR assay we have described here will be useful for the detection and epidemiological and pathological characterization of carnivore parvovirus.In 2013 and 2016, 221 fecal samples were collected from dogs in North of China with suspected CPV infection. 180 samples were found positive for CPV according to PCR results. Both the VP2 gene and NS1 gene were sequenced and amino acid mutation was analyzed, then the homologous comparison and the phylogenetic tree were analyzed. The results indicated that all CPV-2, CPV-2a, CPV-2b and CPV-2c antigenic types were prevalent in China and the CPV-2a was the major epidemic type. The CPV-2c type appeared in Beijing and Hebei provinces after 2014. We foud some new mutations both in VP2 and NS1 proteins, i.e. Ala5 Gly in VP2 and Val160 Gly, Ala586 Thr, Ala587 Thr and Leu600 Arg in NS1. One hundred and fourteen complete sequences of VP2 gene and 49 that of NS1 were obtained in the study.Isolation and identification of prevalent CPV isolates were done in the study. Of the 20 CPV isolates, we got 2, 4, 5, and 9 isolates respectively belonged to antigenic types CPV-2, new CPV-2a, new CPV-2b, and CPV-2c. All those isolates were used to infect experimentally beagle dogs, which subsequently developed the typical clinical symptoms of CPV infection. Immunogenicity test showed that CPV-2c type isolates can induce the beagle produce neutralizing antibody. In addition to synonymous mutations, the CPV-2c strains also contain a unique coding mutation in VP2 that leads to glycine at residue 5, instead of the highly conserved alanine at this position in all other CPV-2c strains sequenced to date. It was the first time that CPV-2c virus was isolated and identified in China, and the isolates of CPV-2a/b were new CPV-2a/b with Ser297 Ala mutation in the VP2 protein.To investigate the prevalence of mink enteritis virus in China, we isolated two isolates from mink with clinical signs of enteritis. These viruses were identified as MEV by electron microscope examination, serologic test, artificial infection in minks and the VP2 gene sequencing, designated MEV/LN-10 and SD12/01, respectively. Furthermore, the genetic evolution analysis of the VP2 gene indicated that the isolates shared higher homology with other MEV strains available in GenBank. Phylogenetic and recombination analyses on the complete MEV/LN-10 genome showed evidence of recombination between MEV and CPV. The genome was composed of the NS1 gene originating from CPV while the VP1 gene was of MEV origin. This is the first demonstration of recombination between a CPV and MEV in nature.Here, we apply RNA-seq technology to investigate host and virus gene transcriptome-wide before and after F81 cell with MEV infection. For studies described here, uninfected F81 cell were collected before infection(0h), and at 3h, 6h, 12 h, and 24 h after infection. Genome-wide transcriptome analysis showed that total 614 differentially expressed genes(DEGs) were identified between MEV infected F81 cell and a mock control. One hundred and twenty-three DEGs were found to be significantly upregulated and 491 downregulated DEGs were identified with at least a two-fold change. To validate these results, 14 DEGs(11 upregulated and 3 downregulated) were subjected to qRT-PCR analysis. Overall, qRT-PCR data seemed largely similar to those obtained with RNA-seq analysis. Function analysis showed that the DEGs were connected with cell proliferation, immune response, tumorigenesis, apoptosis, signaling, receptor activity, and molecular transducer activity. This study constructed the mRNA database of F81 cell infection before and after MEV.