The Mechanisms Behind The Predominance Of Recombinant GⅡ.P16-GⅡ.2 Norovirus

BackgroudNorovirus(NoV)is considered as the most common cause of the outbreaks of viral acute gastroenteritis(AGE)worldwide.Based on the amino acid sequence divergence of capsid protein(VP1),NoVs can be subdivided into 7 genogroups(GⅠ-GⅦ)and more than 30 genotypes.Since 1996,GⅡ.4 has been the predominant genotype causing NoV AGE outbreaks worldwide,and new variants emerge every 2-3 years via epochal evolution.However,during 2014 to 2015,GⅡ.17 NoV emerged and became predominant in China and some parts in Asia,suggesting that non-GⅡ.4 NoV can also contribute to the predomination in NoV AGE outbreaks.Interestingly,the outbreak Network data showed that the number of NoV AGE outbreaks in China increased sharply in the winter of 2016,which is much higher than those of the same period in previous years,suggesting that a novel NoV variant may emerge.Objective1.To investigate the main pathogen that caused the sharp increase of NoV outbreaks in China in 2016-2017,and its epidemiological characteristics.2.To reveal the origin,evolutionary dynamics and transmission rountine of 2016-2017 GⅡ.P16-GⅡ.2 NoV3.To explore the mechanism behind the predominance of 2016-2017 GⅡ.P16-GⅡ.2 NoV.Methods1.The fecal samples from patients during 2016 to 2017 NoV AGE outbreaks were collected.The one-step reverse transcription polymerase chain reaction(RT-PCR)was used to detect No Vs.The sequences were then used for genotyping,and the sample informative data were used for the statistical analysis of epidemiological characteristics.The complete genome of the virus was amplified by overlapping PCR.2.The complete genome,VP1 and RdRp gene-coding sequences of 2016-2017 GⅡ.P16-GⅡ.2 NoV were amplified by RT-PCR,respectively.The bayesian phytogeny and spatio-temporal transmission of 2016-2017 GⅡ.P16-GⅡ.2 NoVs were analyzed based on the MCMC method and BSSVS model in BEAST v.1.8.2.3.GII.2 NoV P protein was expressed in prokaryotic system and purified by affinity chromatography.Saliva-based histo-blood group antigen(HBGA)binding assays were conducted using the P particle proteins by ELISA.The crystal structure of GⅡ.2 P dimer was performed by X-ray diffraction(XRD).4.The enzyme activities of NoV RdRp and its mutants were perfomed in vitro assay.X-ray diffraction was used to analyze the crystal structure of NoV GII.P16 RdRp.The NoV RNA loads in fecal samples were quantified by Real-time PCR.Results1.(1)A total of 243 NoV AGE outbreaks were reported from January 2016 to June 2017 in China,of which the number of NoV outbreaks increased sharply from November to December 2016,accounting for 51%,and reached 39 outbreaks in December.(2)The typing data of 231 NoV outbreaks showed that 78.8%(182 to 231)were caused by GⅡ.P16-GⅡ.2 NoV,84.4%(65/77)of which were caused by GⅡ.P16-GⅡ.2 NoV during November to December 2016,suggesting that GⅡ.P16-GⅡ.2 became a predominant genotype causing NoV outbreaks in China in the winter of 2016.(3)GⅡ.P16-GⅡ.2 NoV caused outbreak mainly in winter and spring,and distributed mainly in the eastern areas of China,especially in Guangdong Province.Most of the outbreak sites are involving kindergartens,(4)The phylogenetic and recombination analysis showed that BJSMQ was recombined by GⅡ.16 and GⅡ.2 NoVs in their overlap region of ORF1 and ORF2.2.(1)Based on the VP 1-and RdRp-encoding genes,the analyses of divergence times collectively indicated that the 2016-2017 GⅡ.P16-GⅡ.2 NoV evolved from the 2011-2012 GⅡ.P16-GII.2 NoV in 2012-2013,(2)The VP1 nucleotide subsititute rate of 2016-2017 GII.P16-GIL2 NoV was accelerated,which led to the obvious genetic diversity.The viruses could be divided into three subgroups(SC1-SC3).The SCI strains are important causes of epidemics in China;whereas SC2 viruses exhibited the broadest geographic distribution,desipite only a few cases,suggesting an advantage of SC2 viruses in global spread.In addition,the strains(e.g.,BJSMQ)with Va1256Ile mutation,were rarely detected in the pre-2016 GⅡ.2 viruses,but became the dominant strain in the later stage of the outbreak.(3)Phylogenetic analysis showed that Guangdong Province located in the Pearl River Delta of China is the epicenter of 2016-2017 GⅡ.P16-GⅡ.2 NoV outbreaks,which is similar to Hong Kong in the Pearl River Delta that was an epicenter of 2014-2015 GⅡ.17 NoV outbreaks.3.(1)Sequence analysis showed that GⅡ.2 NoV VP1 nucleotide sequences have obvious time-evolution characteristics in recent 40 years,but their protein sequences remain stable.However,the codon usage bias of 2016-2017 GⅡ.P16-GⅡ.2 NoV altered,which may lead to the improvement of viral adaptability to humans.(2)Sequence analysis showed that there was no obvious antigenic change in 2016-2017 GⅡ.P16-GⅡ.2 NoV In addition,the saliva binding assay showed that the HBGA binding spectrum of 2016-2017 GⅡ.P16-GⅡ.2 NoV was similar to those of previous GⅡ.2 strain.Both of them bound type B and type AB saliva.(3)Interestingly,the novel Val256Ile mutant BJSMQ P proteins bound additional type A saliva samples compared with those of the early strain GZ20435 and pre-2016 strain SMV.The crystal structure of BJSMQ P dimer was analyzed.BJSMQ P dimer structure showed that Asp382 has restored the conventional orientation to form a functional HBS,which may explain the gain of the expanded HBGA-binding spectrum of BJSMQ.In addition,the antigen prediction showed that the residue 256 in VP1 protein may involve in the formation of antigen epitopes,and it is speculated that the antigenicity of novel Val256Ile variants may also be affected.4.(1)Sequence analysis showed that the minor amino acid substitutions were presented in the nonstructural roteins of novel GⅡ.P16,and four unique amino acid mutations on the surface of its RdRp protein.(2)The enzyme activity assay in vitro showed that the catalytic activity of BJSMQ RdRp was significantly higher than those of GⅡ.P16,GⅡ.P4 and GⅡ.P17 RdRp.BJSMQ reverse mutagenesis showed that the enzyme catalytic activity of Thr293Ser RdRp mutant was significantly weaker than that of wild type.The analysis of crystal structure showed that the 293 residue on RdRp altered the direction in the novel GⅡ.P16 NoV,which may increase the binding force of its Motif F to NTP.(3)The substitute rate of GⅡ.2 and GⅡ.4 VP1 recombinated with novel GⅡ.PP16 RdRp was significantly higher than those of GⅡ.2 and GⅡ.4 VP1 recombinated with other RdRp types.In addition,the average viral loads of 2016-2017 GⅡ.P16-GⅡ.2 NoV in the feces from children with AGE were high.Conclusion1.This study firstly reported that a recombinant norovirus GⅡ.P16-GⅡ.2 has replaced the previously prevalent GⅡ.4 and GⅡ.17 as the main cause of NoV outbreaks in China in 2016-2017;2016-2017 GⅡ.P16-GⅡ.2 NoV was most likely to evolve from the 2011-2012 GⅡ.P16-GⅡ.2 NoV in 2012-2013.Besides,the virus showed a rapid genetic diversifcation afer its emergence.3.A further variant with a single Val256Ile mutation and the conventionally orientated Asp382 in VP1 protein led to an expanded HBGA-binding spectrum,which was the dominated cause of outbreaks in the late epidemic period.4.Point amino acid substitutions were identified on the non-structural proteins of 2016-2017 GⅡ.P16-GⅡ.2 NoVs.In particular,the single amino acid mutation Ser293Thr in its polymerase that led to the increase of its activity,are most likely to cause the sudden epidemics of 2016-2017 GⅡ.PI6-GⅡ.2 No Vs.5.The virus can be traced back to Pearl River Delta,China,which highlights the necessity of enhanced global surveillance for potential epidemics of rare-genotype noroviruses,especially in the Pearl River Delta region of China.