Amino Acid Mutations On EV71 Of C4 Subgenotype Circulating In Mainland China And Their Effects On Viral Biological Characteristics

Enterovirus 71(EV71),a member of the Enterovius genus within picornavidae family,is one of the major causative agents of hand foot mouse disease(HFMD)and herpangina,which were general mild and self-limited.However,a few cases could develop severe neurological complications which might lead to life-time sequela or death,such as aseptic meningitis,brainstem encephalitis,acute flaccid paralysis,and pulmonary edma.In the past decades,EV71 had spread across the Asian Pacific and posed great threat to children health,and has been regarded as the most significant pathogen of Genus Enterovius post poliovirus’ eradication in this region.In mainland China,EV71 has been circulating for more than twenty years since its first identification in an adult case in 1989.In 1990 s and early 2000 s,the virus was only sporadic in southern provinces until several sudden outbreaks of EV71-associated HFMD in Shandong and Anhui provinces in 2007-2008 which led to severe clinical outcome and tens of deaths among children.Considering that the impact of the disease on children’s health,HFMD was classified into notifiable C disease in China on May 2,2008 and following intensive active surveillance revealed the disease had spread nationwide and the majority of cases with severe clinical outcome were contributed to EV71 infection.Genetically,EV71 virus can be divided into genotype A,B,and C and the latter two are further classified into B1-5 and C1-5 sub-genotypes.Etiological monitoring demonstrated that the viruses circulating in mainland China were all C4 sub-genotype.During 2003-2004,a new lineage C4 a distinct from former C4 b emerged and was responsible for large-scale epidemics in mainland China in recent years.In comparison with C4 b,C4a seemed to take on stronger transmissibility because of its broadly geological distribution and persistence of the virus-associated disease transmission.Retrospective investigations revealed that the natural factors,human behaviors and genetic background of host might throw impacts on the epidemic of the disease,but the underlying mechanism for stronger transmissibility of C4 a virusremains unclear.The biological characteristic of pathogen is a key aspect as it might determine the clinical manifestation,severity as well as the epidemic intensity of disease.Investigation on discrepancy between two lineages of C4 virus circulating in mainland China and biological features changes associated with the genetic discrepancies could provide new insight into the molecular mechanism of pathogenesis and shed new lights on the prevention and control of the virus-associated disease in mainland China.In this study,22 genomes of viral isolates from Fujian province during 2003-2013 were sequenced.Together with 48 genomic sequences of viral isolates from other provinces in mainland China during 1998-2013,all of viruses could phylogenically be divided into two lineages of C4 a and C4 b with 13 notable amino acid substitutions on viral polyprotein.Two mutations,one(histidine to glutamine)at aa22 of VP1 structural protein and another(lysine to arginine)at aa140 of 3D polymerase were introduced into pcrXL149-08,an EV71 infectious clone constructed previously.From transfected cells,clone derived viruses(CDVs)harbored single or double mutations were rescued.The RNA copies of CDVs with VP1/22 H,determined by real-time RT-PCR method established in this study,were higher than that with VP1/22 Q at 72 hpi on RD cells.One step growth experiments showed that,at the early stage of viral infection,more CDVs with VP1/22 H attached to the host cells than CDVs with VP1/22 Q.The following experiments demonstrated more CDVs with VP1/22 H,regardless K or R at aa140 of 3D protein,attached to the hSCARB2 gene(human scavenger receptor class B member 2)tranfected 293 T cells.Furthermore,the results of immuno-precipitation showed that the hSCARB2 receptor molecules binded preferably CDVs with VP1/22 H to that with VP1/22 Q.The main findings in this study included as follows:1.There were 13 amino acid substitutions on viral polyprotein,which distributed differently among two lineages of C4 subgenotype EV71.2.The mutation(histidine to glutamine)on aa22 of VP1 protein could reduce the attachment of C4 genotype EV71 virus to host cell.3.The mutation(lysine to arginine)on aa140 of 3D protein could not influence viral replication of C4 genotype EV71 virus.In summary,the substitution of glutamine for histidine at aa22 of VP1 of C4subgenotype virus could reduce viral attachment to host cell.Considering that the residue at aa22 of VP1 was glutamine on C4 b virus,whereas histidine on C4 a virus,the amino acid substitution might explain to some extent why the C4 a virus had stronger transmissibility than C4 b virus.