Study On The Effects Of The Key Amino Acid Residue Mutations Of VP2 Protein On The Pathogenicity Of Novel Canine Parvovirus Isolated From Raccoon Dogs

In recent years,a novel canine parvovirus（CPV）from raccoon dogs has been widespread in China,which has seriously hindered the healthy development of raccoon dogs.The novel raccoon dog parvovirus is similar to the original CPV-2,but the VP2 protein has mutations at three amino acid sites（S27T,S297A and V562L）.These three amino acid sites are located in important regions on the capsid protein,among which the amino acid 27 is located on the VP2-N terminal connected to the outer capsid by a 5-fold axis.Between the5-fold axis and the 3-fold axis there are attachment sites that act as viral receptors;The amino acid 297 is located in the 300-loop,which plays an important role in the host range of the virus.Amino acid 562 is located near amino acids 564 and 568,and the C-terminal amino acid composition of VP2 protein seriously affects viral replication in the host.However,the effect of VP2 proteins S27T,S297A and V562L on the pathogenicity of novel raccoon dog parvovirus remains unclear.In this study,a novel raccoon dog parvovirus strain RDPV-DP1was isolated in our laboratory.Reverse genetics was used to mutate VP2 proteins S27T,S297A and V562L to investigate the effect of mutations at amino acid sites 27,297 and 562on the pathogenicity of RDPV.1.Construction of RDPV-DP1 Infectious cloneRDPV-DP1 strain was infected with CRFK cells after 32 h,viral replicative DNA（RF-DNA）was extracted,and the RF-DNA was divided into 5’ends and 3’ends by enzyme digestion,and successively connected to p UC18 vector.The infectious clone of RDPV-DP1was constructed,named p-RDPV-DP1,and analyzed by whole genome sequencing.As a result,p-RDPV-DP1 contains the complete viral genome sequence.After transfection of p-RDPV-DP1 with liposome into CRFK cells for 3 generations,the cells showed obvious cytopathy.The expression of VP2 protein was detected by indirect immunofluorescence and western blotting,indicating successful rescue of the virus,which was named rRDPV-DP1.The proliferation characteristics and induction of apoptosis of rRDPV-DP1 were further detected.The results showed that the biological characteristics of rRDPV-DP1 were consistent with RDPV-DP1,indicating that p-RDPV-DP1 could be used as a technical platform to study on the effects of key amino acid mutation of RDPV-DP1 VP2 protein on pathogenicity.2.Construction of infectious clone with key amino acid reverse-mutation of RDPV-DP1VP2 proteinIn order to investigate the effect of mutations of VP2 protein S27T,S297A and V562L on the pathogenicity of the virus,PCR and reverse genetic techniques were used to reverse mutate amino acids 27,297 and 562 of VP2 protein of RDPV-DP1.Seven infectious cloned plasmids with single amino acid mutation,double amino acid mutation and triple amino acid mutation were constructed.They are named p-RDPV-DP1^27S,p-RDPV-DP1^297S,p-RDPV-DP1^562V,p-RDPV-DP1^27S/297S,p-RDPV-DP1^27S/562V,p-RDPV-DP1^297S/562V,p-RDPV-DP1^{27S/297S/562V}.Seven infectious clones were transfected into CRFK cells by liposome transfection method,and after 3 generations of blind transmission,obvious cytopathic changes were observed in all of them.Indirect immunofluorescence detected the expression of 7 VP2 proteins of reverse-mutated F3 generation virus in cells,indicating that 7reverse-mutated viruses were successfully rescued.They are named rRDPV-DP1^27S,rRDPV-DP1^297S,rRDPV-DP1^562V,rRDPV-DP1-DP1^27S/297S,rRDPV-DP1-DP1^27S/562V,rRDPV-DP1^297S/562V,rRDPV-DP1^{27S/297S/562V}.The reverse-mutated virus will provide material data for studying the effect of three sites（S27T,S297A and V562L）on pathogenicity of RDPV-DP1.3.The biological characteristics of RDPV-DP1 VP2 protein reverse-mutated virus in vitroThe titers of seven reverse-mutated viruses at different time points were detected,the growth curves were plotted,and the DNA copies of the viruses were detected quantitatively by fluorescence.The results showed that compared with parental rRDPV-DP1,the proliferation characteristics of rRDPV-DP1^27S,rRDPV-DP1^297S,rRDPV-DP1^562V,rRDPV-DP1^27S/562Vand rRDPV-DP1^27S/297Sin CRFK cells had no significant changes（P>0.05）.However,the proliferation efficiency of rRDPV-DP1^297S/562Vand rRDPV-DP1^{27S/297S/562V}in infected CRFK cells was significantly lower than rRDPV-DP1（P<0.05）.The apoptotic levels of CRFK cells infected with rRDPV-DP1^297S/562Vand rRDPV-DP1^{27S/297S/562V}for 48 h were measured by cell loss assay.The results showed that the early and late apoptotic rates were lower than those of rRDPV-DP1（P<0.05）.The differential proteomic analysis of CRFK cells infected with rRDPV-DP1 and rRDPV-DP1^{27S/297S/562V}for 12h was performed by i TRAQ labeling combined with chromatography/mass spectrometry.Four differential proteins were detected between the two treatment groups,namely,up-regulated proteins（PPME1）;Down-regulated protein:PDZK1interacting protein 1（PDZK1IP1）;Beta-enolase 3（ENO3）;Hemoglobin alpha subunit（HBA）.By KEGG annotation,differentially expressed proteins were grouped into four signaling pathways or biological pathways,including HIF-1 signaling pathway,RNA degradation,glycolysis/gluconeogenesis,methane metabolism.The analysis of differential proteomics provides a theoretical basis for studying the response of different virulence strains infected with host cells.4.The biological characteristics of RDPV-DP1 VP2 protein reverse-mutated viruses in raccoon dogsForty-five 8-week-old raccoon dogs were tested negative for parvovirus antigen and antibody.Raccoon dogs were randomly divided into 9 groups with 5 raccoon dogs in each group.The in vivo pathogenicity of 7 reverse-mutated viruses was studied.Negative control and rRDPV-DP1 challenge group were set as positive control.Results:All raccoon dogs in rRDPV-DP1 challenge group showed symptoms such as depression and anorexia,and the morbidity rate reached 100%,and the mortality rate reached 60%in each group.Pathological changes such as intestinal congestion,swelling and intestinal wall thinning were found in autopsy,and histopathological changes such as severe intestinal villi damage and intestinal villi contraction and disintegration were found in histopathological examination.rRDPV-DP1^27S,rRDPV-DP1^297Sand rRDPV-DP1^562Vcaused water-like diarrhea,depression,anorexia and dehydration,and the incidence rate was 60%.One young raccoon dog died in each challenge group,and the tolerance raccoon dogs was good.The results of histopathological examination showed a large number of epithelial cell necrosis and intestinal villi injury and shedding in the mucosal layer of intestinal tissue.The symptoms of rRDPV-DP1^27S/297Sand rRDPV-DP1^27S/562Vafter infection were mild,including mucous diarrhea and good appetite,with an incidence of 40%and no death of raccoon dogs.The resistant animals were in good condition,and the intestinal histophiological examination revealed intestinal villus abruption and other tissue lesions of individual raccoon dog.rRDPV-DP1^297S/562V,rRDPV-DP1^{27S/297S/562V}and control group showed no symptoms after infection,with good mental state,normal appetite,normal stool,and no significant changes in intestinal tissue by pathological examination.Scoring criteria were established according to clinical symptoms and pathological changes,and each experimental animal was scored.Results:compared with rRDPV-DP1,rRDPV-DP1^27S,rRDPV-DP1^297Sand rRDPV-DP1^562V,single-amino acid reverse-mutations significantly reduced the pathogenicity of raccoon dogs.However,it still had strong pathogenicity in raccoon dogs,and the clinical symptoms and histopathological scores were significantly decreased（P<0.05）.Compared with the single amino acid site reverse-mutation,the virulence of raccoon dogs was further weakened in the challenge group of two amino acid sites reverse-mutation rRDPV-DP1^27S/297Sand rRDPV-DP1^27S/562V（P<0.01）.Compared with control group,rRDPV-DP1^297S/562Vand rRDPV-DP1^{27S/297S/562V}with the reverse-mutation of 297S and 562V showed no significant difference in virulence against raccoon dogs（P>0.05）.The results of genetic animal experiments showed that rRDPV-DP1^297S/562Vand rRDPV-DP1^{27S/297S/562V}could be used as candidates for attenuated live vaccine for further study.In summary,in this study,a new CPV strain of RDPV-DP1 derived from raccoon dogs was used as the research object,and its infectious clone p-RDPV-DP1 was constructed,and the rescue virus rRDPV-DP1 was obtained.The biological characteristics of rRDPV-DP1were consistent with those of the parent virus RDPV-DP1.Using p-RDPV-DP1 as a technical platform,reverse genetics technology was used to reverse-mutate amino acids 27,297 and562 of RDPV-DP1 VP2 protein,and 7 infectious clones with single amino acid mutation,double amino acid mutation and triple amino acid mutation were constructed,and rescue viruses were obtained.The biological characteristics of 7 reverse-mutated viruses were examined in vitro and in vivo.Results:Single and double amino acid mutations at positions27,297 and 562 of VP2 protein reduced the pathogenicity of the virus against raccoon dogs.In particular,the pathogenicity of viruses carrying A297S and L562V(rRDPV-DP1^297S/562V,rRDPV-DP1^{27S/297S/562V})decreased significantly,indicating a synergistic effect at positions 27,297,and 562 of VP2 protein.