Comparative Study On Molecular Biological Characteristics Of Two Mink Enteritis Parvoviruses

As the scale of the mink farming industry continues to increase,the epidemic diseases have seriously hampered the mink farming industry and caused significant economic losses.Mink enteritis parvoviruses(MEV)is one of the main pathogens that seriously harm mink,with high morbidity and mortality.Inoculation of mink enteritis parvovirus vaccine is an important measure to prevent and control mink viral enteritis.However,in recent years,there have been many examples of vaccine immunization failures.It is of great significance to understand the molecular evolution characteristics of MEV for the prevention and control of mink enteritis.From 2015 to 2016,our laboratory isolated 8 parvoviruses from mink in major mink breeding areas of Shandong province,China,named MEV-SD1~MEV-SD8.VP2 protein of MEV-SD7 and MEV-SD8 at position 300 is Val,however,position 300 in MEV-SD1~MEV-SD6 is Ala,which is consistent with FPV VP2 protein at position 300.This project takes MEV-SD1,MEV-SD7 as research objects to detect cross-immune reaction,infection efficiency,proliferation and cell damage by HA/HI,IFA,growth curve measurement,flow cytometry and other methods.The results show that both MEV-SD1 and MEV-SD7 can have hemagglutination inhibition reaction with anti-MEV-SD1 and anti-MEV-SD7 multi-antiserum,respectively,and HI titers are higher than 1:128.Green fluorescence intensity in CRFK cells infected with MEV-SD7 are stronger than that in MEV-SD1.Apoptosis induction and cell cycle arrest are not obvious when MEV-SD1 and MEV-SD7 infect CRFK cells for 24 h.The proportion of inducing apoptosis and cell cycle arresting in G1 phase were significantly higher in CRFK that infected with MEV-SD7 than MEV-SD1.It indicates that MEV-SD1 and MEV-SD7 have cross immune protection,but MEV-SD7 has higher infection efficiency and replication efficiency,and has a stronger ability to induce apoptosis and block the cell cycle.In order to explore the reasons for differences from the molecular level,infectious clones of MEV-SD1 and MEV-SD7 were constructed using molecular virology technology,named pM-MEV-SD1 and pM-MEV-SD7,and the complete genomes were compared and analyzed.The results showed that homology of the complete genome nucleotide sequences between MEV-SD1 and MEV-SD7 was 99.0%,and the nucleotide homology with the reference strains in GenBank was 96.2-99.9%.Phylogenetic analysis of the complete genome demonstrated that MEV-SD7 was in the same branch as MEV-LHV and MEV-L isolated from mink in China,however,MEV-SD1 was in the same branch as FPV-G isolated from the Bengal tiger(Panthera tigris)in China and FPV-HRB-CS1 isolated from the cat in China,which confirmed that MEV-SD1 belonged to FPV.The analysis of NS1 gene showed that the homology of MEV-SD1 and MEV-SD7 was 99.4%,and the amino acid homology of MEV-SD1,MEV-SD7 and the reference strains was 98.5-100%.The analysis of VP2 gene showed that the homology of MEV-SD1 and MEV-SD7 was 99.5%,and the amino acid homology of MEV-SD1,MEV-SD7 and the reference strains was 97.8-100%.Analysis of hairpin structures at both ends of the genome showed that the palindrome sequence of MEV-SD1 and MEV-SD7 at the 5' end is identical,but the terminal "U" palindrome structure and the "bubble" region of the reference strain MEV-LHV are different,however,the 3' end sequence of MEV-SD1 and MEV-SD7 "bubble" structure is completely different.Meanwhile,the positions of "long ear" and "short ear" of MEV-SD1 are reversed and the sequences are quite different compared with MEV-SD7.There is also a base pair substitution(T/A-C/G)near the nick site of NS1 protease in the 3' end of MEV-SD1.VP2 300 th amino acid is an important site of parvovirus.This study explored the effect of the 300 th amino acid mutation of VP2 on the biological characteristics of MEV-SD1 and MEV-SD7.Based on pM-MEV-SD1 and pM-MEV-SD7,A300 V and V300 A site-directed mutations were performed on the VP2 300 th amino acids of MEV-SD1 and MEV-SD7 by enzyme digestion and ligation substitution,respectively.A300 V mutation of MEV-SD1 was obtained and named MEV-SD1 VP2 300 V,as well as V300 A mutant of MEV-SD7 is named MEV-SD7 VP2 300 A.Cell activity,growth curve determination,fluorescence quantitative PCR,flow cytometry cytology and other methods were used to detect the biological characteristics of mutant viruses.IFA,CCK8 detection,growth curve measurement,qPCR,flow cytometry and other methods were used to detect and compare infection efficiency,effect on the activity of CRFK cells,proliferation,virus titer in the cell supernatant,apoptosis induction and cell cycle arrest of the parental virus and the mutant virus.The results show that A300 V can increase the replication and proliferation efficiency of MEV-SD1 in CRFK cells,while V300 A can reduce the replication and proliferation efficiency of MEV-SD7 in cells,indicating that VP2 300 V is more beneficial to the proliferation of mink enteritis parvovirus in CRFK cells.In summary,there are differences in biological characteristics of the two mink enteritis parvoviruses MEV-SD1 and MEV-SD7.The difference in the 300 th amino acids of VP2 is one of the reasons that MEV-SD1 and MEV-SD7 have different molecular biological characteristics.The difference of 3' terminal hairpin structure in MEV-SD1 and MEV-SD7 may also be the reason for differences in biological characteristics of the two viruses.There is no report yet,but its exact biological significance still needs further studies.This project lays a theoretical foundation for the in-depth research of the molecular biological characteristics of MEV,and also provide material for the preparation of MEV genetic engineering seedlings.