Quasispecies And Evolution Of Enterovirus HY12 And The Effect Of VP1 Mutation On Viral Replication

Bovine enteroviruses(BEVs)belongs to the genus of Enterovirus within the family of Picornaviridae.These viruses are the leading etiological agents related to respiratory and digestive diseases in animals and caused a significant economic loss to the livestock industry.We reported the identification of a novel entervirus E isolates HY12 from a cattle herd with an outbreak of a severe respiratory disease and enteritis in Jilin Province in 2012.BEV infections were an emerging disease in China in recent years and the biological characteristics and pathogenic mechanism of the BEV strains were barely studied.In this study,RT-PCR were performed and the complete genome sequence of HY12 was obtained by joining the overlapped fragment sequences.After assembling the sequences,the complete genome of HY12 was made up of 7469 nucleotides and an open reading frame(ORF)encoding a large precursor polyprotein of 2176 amino acids.Sequences alignment analysis of the sequence regions of 5′UTR,VP1,VP2,VP3,VP4,3D and the complete genome sequences revealed that HY12 was a complex intraserotypic recombination of enteroviruses E in the evolution,and HY12 strain belongs to subgenotype 3 within enteroviruses E.In addition,HY12 strain has several unique mutations in the capsid proteins,especially in VP1 and VP4.Degenerated nucleotide sequences were revealed in the same position indicating an existence of quasispecies in the HY12 enteroviruses.Evolution and variation of different passages of HY12 quasispecie showed quasispecie has diversity and complexity,and the mutation rate and complexity gradually increased with the increasing passages,the proportion of the master sequence and the sequences homology gradually decreased with the increasing passages.Analysis of biological characteristics showed both of virus titer and plaque size were larger after 40 passages.Sequences alignment analysis showed that a nonsynchronous mutation at position 272(272 G>A)in VP1 gene in passage 40,leading to the arginine(R)to histidine(H)change at amino acid 91(R91H).Unique mutation R91 H altered the antigenicity and the tertiary structure of HY12-VP1.An infectious clone of HY12 strain was constructed and HY12 strain was rescued by means of molecular biology,reverse genetics and Virology,Reverse genetics was used to further dissect the effect of this amino acid mutation.The virus with R91 H mutation was rescued,termed r HY12(H).Characterization of the r HY12(H)showed the virus had a higher virus titer and plaque is significantly larger than that of parental HY12 and rescued r HY12.In addition,the mutation of R91 H enhanced translational activities and RNA replication in infected vero cells.Animal experiments showed that the pathogenicity of r HY12(H)significantly increased compared with parental HY12 and rescued r HY12 in ICR suckling mice at age of 3Days.Taken together,the results in this study demonstrated a unique mutation of R91 H in VP1 contributes to the significant increase of viral replication and its pathogenenicity,thus laying a basis for future exploration of the pathogenic mechanism underlying the HY12 virus infection,elucidating the interaction between viruses and host,and providing the fundamentals for the prevention and control of this newly emerging epidemic disease.