Molecular Epidemiological Investigation Of Muscovy Duck Reovirus And Immunogenicity Of Muscovy Duck Reovirus σC Protein

Muscovy Duck Reovirus,MDRV outbreak of muscovy duck reovirus began in Zhejiang,Jiangsu,Guangdong and other places at the end of last century.The virus isolates have the same characteristics in pathology,culture and genome,and have been popular in Muscovy Duck breeding industry so far,which seriously affects the economic health and development of muscovy duck breeding industry in China.MDRV due to its characteristics of easy variation,there are great differences in appeal,transmission,pathogenicity and mortality,so it is easy to evolve into new pathogenic strains.After MDRV infection,it often stimulates other muscovy duck virus or bacterial infection,stimulates and induces many kinds of waterfowl diseases,and causes huge economic losses to muscovy duck breeding.Therefore,timely and effective prevention and control of MDRV is of great significance.Studies have shown that there is no cross-reaction between MDRV and ARV,that is,the serological diagnostic method for the detection of avian reovirus disease has no great effect on the detection of MDRV.At present,there are few commercial kits for MDRV antibodies in the market,and the prevention of the virus is mainly attenuated live vaccine,but the attenuated live vaccine itself has the safety problems of variation,virulence and so on.Based on the epidemiological investigation of muscovy duck reovirus from Guangdong,Jiangsu and other provinces in 2016-2019,this study established a fluorescence quantitative PCR detection method for clinical diagnosis.statistical results showed that the positive rate of MDRV acid was 36.5%(35/96),among which mixed infection was more.This study designed primers according to the genome sequence of muscovy duck reovirus on the Gen Bank,sequenced the σB and σC genes of the newly isolated 7 strains of muscovy duck reovirus and performed genetic evolution analysis.The results of σB gene analysis showed that except for MDRV-SHX,the other strains were in an independent branch,and the seven strains were in the MDRV branch,which was close to the MDRV815-12 strain.σC gene analysis showed that all seven strains were in the same independent branch.Evolutionary analysis shows that there are some genetic differences between the new isolates and other MDRV viruses in China,which indicates that there is great variability in domestic MDRV.Whole genome sequencing of MDRV-ZQ strain showed that the λA,μA,and μNS sequences of MDRV-ZQ strain were most similar to those of 815-12strains(nt: 96.9%-98.1%),the most similar to ZJ2000 M in λC/μB/σA/σB sequence(nt:95.4%-98.1%),and the λB of the strain is the most similar to MW9710(nt:97.9%),the most similar to YB strain in σC,while the similarity with S14 strain in σNS sequence nucleotides was 99.7%.Phylogenetic analysis showed that the σA、σB、σC of MDRV-ZQ were all in independent branches and were the only independent strains in the branches.And we have reason to suspect that the strain was reorganized by MDRV ZJ2000M、MDRV 815-12.Using a pET43.1 a carrier with a pro-soluble expression label,the recombinant MDRV-σC protein of about 91 k Da size was successfully expressed through the prokaryotic expression system.the Western Blot identification can produce specific responses mouse source His-tag monoclonal antibody and MDRV polyantibody serum,indicating that the recombinant protein has good immunogenicity.Using recombinant protein as coating antigen,MDRV indirect antibody detection method ELISA established.The method has strong specificity,no cross reaction with positive serum such as MDPV,and good repeatability,and high coincidence rate of clinical sample detection.In this study,the prokaryotic expression of MDRV-ZQ-σC protein was used to prepare a subunit vaccine for the challenge protection test.The protective effect of the MDRV-CA strain of Miao on the current popular strain MDRV-ZQ strain is compared.The results show that both have a certain protective effect and the same protective effect.Above all,this study provides reference material for MDRV molecular epidemiology,establishes sensitive and rapid antigen and antibody detection methods,and prepares subunit vaccines according to MDRV-ZQ strains of the current major epidemic strains,which is of great significance for the prevention and treatment of the disease.