The Study On The Genome Of CA24Variant Isolated From The Acute Hemorrhagic Conjunctivitis Outbreaks In Zhejiang Province During2002to2010

Objectives1. To analyze the variation and evolution characteristics of CA24v epidemic strainsisolated from Zhejiang province during2002to2010, based on the analyze of nucleotide, aminoacid, glycosylation sites of every gene and complete genome.2. To study the differences between the domestic and foreign epidemic strains and thevirus strains circulated from Zhejiang province.3. To detect the recombination events in each gene and the whole genome of the epidemicstrains isolated at home and abroad.4. To calculate the evolutionary rate and divergence time of VP1and3C gene, as toestimate the evolutionary characteristics of CA24v virus.MethodsThirteen Zhejiang CA24v strains were proliferated by Hep-2cells, identified by real-timefluorescent quantitative RT-PCR, amplified with RT-PCR assay. The obtained sequences wereanalysed with domestic and foreign CA24v epidemic strains isolated during1970to2011usingsome biological softwares, and detected recombination events in each gene and completesequence. Finally, the rate of nucleotide substitution and the time of most recent commonancestor(TMRCA) in CA24virus clade CA24v, based on the nucleotide sequences of VP1and3C gene, were calculated with Bayesian Markov Chain Monto Carlo(Bayesian-MCMC) andmolecular clock model.Results1. The whole sequence of three Zhejiang CA24v virus have7456-7458nucleotide.Compared with the two epidemic strains prevalence in2002, the epidemic strain isolated in2010have inserted two T bases in ribosome binding sites of the5’non coding region.2. The number of amino acid variation of VP1, VP2and VP3cell epitope zones are16,7and3respectively. Other genes also have a certain number of amino acid mutation, the entropy of some amino acids is also high, but the value of Ka/Ks is not greater than one. Thereare a total of four glycosylation sites mutation in the domestic and foreign CA24v epidemicstrains circulation during1970to2011. Recombination events were found in only individualepidemic strains.3. All the Zhejiang CA24virus epidemic strains separated during2002to2010belongedto CA24v type, the CA24v epidemic strains isolated in2002,2007-2008and2010weregathered in the C2, C3and C4small branches respectively.4. The average evolutionary rate of VP1gene was4.86×10-3substitution/site/year(95%HPD:3.522×10-3-6.167×10-3). The estimated date on TMRCA of clade CA24andCA24v of CA24virus was1909(95%HPD:1885-1930), while the divergence time of cladeCA24v was1942(95%HPD:1927-1958). The average evolutionary rate of3C gene was4.737×10-3substitution/site/year(95%HPD:3.237×10-3-6.434×10-3). The estimated date onTMRCA of clade CA24and CA24v of CA24virus was1926(95%HPD:1879-1942), while thedivergence time of clade CA24v was1961(95%HPD:1954-1969).ConclusionsThe amino acid sequence of CA24v virus epidemic strains was very stable. The CA24prototype virus was not the pathogen of acute hemorrhagic conjunctivitis, just an intestinal viruswhich had a close evolution relationship with CA24v leading to acute hemorrhagicconjunctivitis outbreaks. The most recent common ancestor of the CA24type and CA24v typeappeared during the late of19th century to the early of1940s, the CA24v began to divideabout1950. We should pay much attention to these trends, strengthen the research of themolecular epidemiology, recombination and evolution direction in CA24v virus.