Analysis Of Pathogenicity,Sequence And Prokaryotic Expression Of ?B Protein Of N-MDRV In Guangdong

In recent years,new pathotype of muscovy duck reovirus?N-MDRV?disease has been reported in Fujian,Guangdong and Zhejiang provinces and other places,commonly knowns as“New muscovy duck liver disease”.The main lesions were irregular necrosis and bleeding of liver with a high mortality in ducklings,causing serious economic losses to the poultry industry.At present,a few studies have examined thepathogenicity and genetic evolution of N-MDRV in Guangdong province.The?B protein as the main coat-shell protein of N-MDRV,has good immunogenicity and reactivity.The aim of this study was to compare and analyze the pathogenicity and whole genome sequence of the epidemic strains of N-MDRV/SH12 and N-MDRV/DH13 in Guangdong province.Meanwhile,studies on the prokaryotic expression of high efficiency of?B protein in N-MDRV/DH13 strain.In this study,1-day-old Muscovy ducks were infected with 10⁴.00.00 ELD₅₀0 of N-MDRV/SH12 and N-MDRV/DH13 strains via the eye-nose,intramuscular injection and claw pad pathways.The mortality rate of the N-MDRV/SH12-infected ducklings was between 10%and 20%.However,the mortality rate of the N-MDRV/DH13-infected ones was 30%.In our study,the pathogenicity of N-MDRV/SH12 and N-MDRV/DH13 strains were assessed employing 1-day-old Muscovy ducklings and chicks.The results showed that Muscovy ducklings appeared anorexigenic and mild diarrhea after infection with two 2strains for 3 days.The clinical symptoms were obvious 7 days after infection.The pathological changes such as hemorrhage and necrosis of liver,enlargement and necrosis of spleen,atrophy?N-MDRV/SH12 group?or necrotic hemorrhage?N-MDRV/DH13 group?of bursa were found in ducklings.The body weight of Muscovy ducklings were significantly decreased?P<0.01?at 14 days post-infection?dpi?with N-MDRV/SH12 strain.The N-MDRV/SH12 and N-MDRV/DH13 strains were not able to cause death in chickens.Some diseased chickens,appeared anorexigenic and hairs became loose after 3 dpi.A significant decrease in body weight of N-MDRV/SH12-infected chicks was observed at 14 dpi?P<0.05?.The necropsy of the diseased chicken showed that the liver and spleen lesions were similar to“New Muscovy duck liver disease”,but there was no obvious lesion in the bursa of Fabricius.Histopathological sections showed that a large number of red blood and mononuclear cell infiltration in liver,spleen,and necrosis and decrease of lymphocytes in the spleen and bursa,with the splenic granuloma structure were seen at 7 dpi in the Muscovy ducklings.The above results showed that the pathogenicity of N-MDRV/SH12 and N-MDRV/DH13 strains infecting Muscovy ducks and chicks were different,but they all had similar patterns on the liver and spleen of Muscovy ducks and chicks.In order to study the genome structure and evolution of different pathogenic strains of N-MDRV,the whole genome sequences of N-MDRV/SH12 and N-MDRV/DH13 strains were amplified,cloned and sequenced by RT-PCR.The results showed that the nucleotide and deduced amino acid sequences of each gene fragment between N-MDRV/SH12 and N-MDRV/DH13 strains are relatively similar,which were 97.1%⁹9.8%and 97.5%¹00.0%,respectively.Furthermore,the results revealed similarity of up to 86.3%⁹9.6%and 93.3%¹00%between the N-MDRV,DRV and GRV reference strains in GenBank.The sequence and phylogenetic analysis of?C gene and its deduced protein showed that the genetic type 2 strains in the adjacent regions are closer in genetic relationship.?A,?B,?NS and?A protein mutation sites have some common mutation sites,their protein and gene sequence similarity and phylogenetic tree analysis results showed that they shared a close genetic relationship.Except?B protein gene,the sequence similarity and phylogenetic analysis of the other 9 protein gene fragments showed that the relationship between waterfowl reoviruses is closer,and had a distant relationship with ARV.Further analysis of?B protein and gene fragment by SimPlot recombination software indicated that Waterfowl reovirus gene type 2 was derived from ARV and ARV-Md.The above results indicate that the N-MDRV/SH12 and N-MDRV/DH13 strains belong to waterfowl reovirus gene type 2,and the genetic evolution of the two strains is more similar to other gene type 2 strains of the same branch.Differences in the protein sites of the virus strain are likely to lead to differences in the pathogenicity of the virus.To compare the sequence variation sites and potential glycosylation sites of the N-MDRV/SH12 and N-MDRV/DH13 strains with 10 strains of gene type 2 reference strains,we found that,except?B protein,the prospective glycosylation sites of other proteins were changed due to protein sequence variation.The variation of 109 and 270 sites of?NS protein altered the chemistry of N-MDRV/SH12 and N-MDRV/DH13 proteins at pH 7.0.The changes of single amino acid sites of?C and?B proteins of the 2 strains had a little effect on the predicted epitope of the antigenic protein.Whether the mutation of its amino acid site is related to the pathogenicity of the strain or not,remains to be determined.To explore the prokaryotic gene expression of pathogenicity-related?B proteins of N-MDRV,the?B gene of highly pathogenic N-MDRV/DH13 strain was selectedfor downstream applications.Recombinant pET-32a?+?-?B-BL21?DE3?vector was constructed and induced by IPTG to express?B protein.Under the optimal conditions of induction,the expression of?B protein was 32.3%of the total bacterial protein.SDS-PAGE analysis revealed that?B recombinant proteins exist mainly in the form of inclusion bodies.After purification by Ni²⁺column,high-purity soluble?B protein was obtained.Moreover,and the recombinant protein can be detected by Western blotting.This research provides a framework for the exploration of N-MDRV?B protein for diagnostic and vaccine strategies.