Isolation Of A New Virulent Strain Of Mink Enteritis Parvovirus And Functional Significance Of Some Particular Amino Acid Residues In VP2 Protein

Mink enteritis parvovirus (MEV) is the pathogen of mink enteritis, an acute highly contagious enteric disease causing severe diarrhea. Immature minks (especially newly-weaned kits) are more susceptible to MEV infection and the mortality rate can reach high up to 80%, resulting in huge economic losses in the worldwide. MEV, a subspecies of the feline parvovirus (FPV), is an autonomous parvovirus of the genus Parvovirus within the family Parvoviridae. The genome is a linear negative single-stranded DNA containing two major open reading frames, ORF-L and ORF-R, respectively coding for nonstructural (NS) proteins NS1 and NS2, which are required for transcription and DNA replication, and capsid proteins VP1 and VP2 which determine virual replication, hemagglutination, antigenicity, host range and pathogenicity. VP2 protein is able to self-assemble to form a virus-like particle (VLP). In this study, we focused on functional amino acids of VP2 protein of MEV, which might be involved in viral replication, host range and pathogenicity.A virus isolated from mink showing clinical signs of enteritis was identified as a high virulent mink enteritis parvovirus (MEV) based on its biological characteristics, identified by PCR, CPE of F81 cells, electron microscopy observation, hemagglutination and hemagglutination inhibition test (HA-HI), indirect immunofluorescence assay (IFA) in vitro and animal infection experiment in vivo. The results of EFA and multiple-step growth curves showed MEV-LHV had higher infection and replication efficiencies than attenuated strain MEV-L. Sequence of the complete genome of MEV-LHV containing the palindromic termini was determined by PCR. The secondary structures and cis-acting elements of the palindromic termini were predicted. The results of comparative sequence analysis and phylogenetic analysis of the complete genomic sequences of MEV-LHV and MEV-L and full-length MEV genomic sequences in GenBank provide a better understanding of the genomic and phylogenetic characteristics of MEV and identify certain regions and sites that may be involved in viral replication and pathogenicity.Virulent strain MEV-LHV replicated to higher titers in feline F81 cells and showed stronger pathogenicity than attenuated strain MEV-L. Sequence analysis of the VP2 gene of MEV-LHV, MEV-L and other strains in GenBank revealed that 3 residues,101,232 and 411, differed between virulent and attenuated strains but were conserved within the 2 branches of phylogenetic tree. To determine the roles of these residues, site-directed mutagenesis of the VP2 gene of attenuated strain MEV-L was carried out on an infectious plasmid (pMEV-L) to replace VP2 residues 101 Ile,232 Ile and 411 Ala with Thr, Val and Glu (found in virulent strains) either separately or together. Mutant viruses were prepared by transfection of F81 cells and were identified by CPE and IFA. Results of binding and entry into F81 cells showed mutant viruses showed binding affinities similar to that of MEV-L and MEV-LHV, indicated that mutation of these residues did not affect the interaction of virus with cell receptor. The mRNA and protein expression levels of VP2, multiple-step growth curves, plague forming assay and single-round infection assay showed that introducing mutations of 101 and 411 respectively increased replication efficiency, but still to a lesser extent than that of MEV-LHV, while both MEV-L I232V and MEV-L I101T/I232V/A411E substitutions decreased transcription and replication efficiency. Mutations of 101 and 411 did not affect infectious virus production. Viruses with mutation of residue 232 (MEV-L I232V and MEV-L I101T/I232V/A411E) showed decreased infectious virus production. MEV-LHV showed higher VP2 protein expression level, replication and infectious virus production than that of MEV-L. Animals infected with these mutant viruses did not develop the clinical disease resulting from MEV-LHV infection, indicating that the 3 residues were not determinants of pathogenicity.Identification and binding of virus to its host cells are critical for virus infection. Resistance of canine cells of MEV could be overcome by expression of the feline transferring receptor (TfR) in those cells, which indicated that MEV infection of feline cells was associated with its specific ability to bind the feline TfR. MEV and CPV are subspecies of FPV. Comparative analysis of amino acid sequences of VP2 of MEV and CPV showed 6 residues,80,93,103,323,564 and 568, differed between MEV and CPV. To determine the roles of these residues, site-directed mutagenesis of the VP2 gene of pMEV-L were carried out to replace MEV VP2 residues with the CPV residues. Mutant viruses were prepared by transfection of F81 cells and were identified by CPE and IFA. The changes in MEV of VP2 residue Lys 93 to Asn or Asp 323 to Asn allowed the virus to infect dog MDCK cells and the infection efficiency was increased by changing the 2 residues together. Mutations of 93 or/and 323 residues did not affect the binding and replication efficiency. The changes in MEV of VP2 residue Lys 80, Asn 564 and Ala 568 to the CPV residue separately did not affect the binding efficiency, but decreased replication efficiency. Mutations of residues 80,564 and 568 together could decrease the binding efficiency of F81 cells and viral DNA replication, but could not inhibit the infection of F81 cells. The mutation of VP2 residue 103 did not affect host range and replication efficiency.At present, with the discovery of many new parvoviruses, its host range has been developing. The results in this thesis will provide a better understanding of the biological and genomic characteristics of MEV, and will promote a further study of the mechanism of viral replication, host range and pathogenicity.