| Background:Human enterovirus (EV) are members of the genus Enterovirus within the family Picornaviridae, order Picornavirales, which can be divided into four groups:EV-A, EV-B, EV-C and EV-D, and EV has been found to 119 serotypes at present, among which 64 were serotyped by early neutralization test. Because of so many serotypes of EV, newly determined EV was limited by the short supply of specific antisera, neutralization test has so many disadvantages such as time consuming, the serum supply constraints, the virus antigenic drift, recombination, specimens mixed several EV, et, al, which influence the results of neutralization test and can not quickly make the identification, especially to deal with the circulations of more and more new or variant of EV in human population. Because VP1 is an important protein coding region, containing significant antigenic determinants, and also as genotyping target region. The newly determined EV in recent years are all genotyping based on the VP1 nucleotide sequence. And the results are consistent with the serological typing, which has emerged as one of the new gold standards for EV typing.Most EV infections are asymptomatic or only cause mild symptoms, such as non-specific fever, rash, or mild upper respiratory tract symptoms (for example common cold), also can cause a diversity of clinical diseases, including acute hemorrhagic conjunctivitis, aseptic meningitis, acute flaccid paralysis, hand, foot and mouth disease, myocarditis and neonatal septicemia. Because of so many serotypes of EV, and can cause many mild or severe diseases, or the formation of asymptomatic infection, which has been received concerns of more and more national disease control and prevention department in recent years.Our country is vast in territory, where contains so many EV serotypes, and also occurs epidemic outbreaks all the year. Xinjiang Uighur Autonomous Region is located in the center of the Asia-Europe continent. It is valuable to study genetic characterizations of EV in Xinjiang. In the present study, we conducted study on serotype distribution of EV-C circulated in Xinjiang in 2011 and further molecular epidemiological study.Objective:By study the serotype distribution and genetic characterizations of EV-C circulated in Xinjiang in 2011, to preliminarily elucidate the serotype and genotype distribution in Xinjiang, to enrich our country EV-C seed bank, to further master the basic epidemic state of EV-C circulated in Xinjiang even the Mainland of China, to provide scientific material for the diagnosis, prevention and control of infectious disease caused by EV.Methods:To isolate HEV according to standard protocols, RNA extraction of positive isolations, RT-PCR of the VP1 coding region, then determine the nucleotide sequence, build the phylogenetic tree and conduct genetic characterizations. Based on the genetic distance of the full-length VP1 region, we selected 17 strains of EV-C as representative strains to conduct the complete genome sequences determination and study on genomic characteristics. As to the first discovery of two CVA1 strains which can propagate and produce CPE on RD cell, we compared the discrepant sites between the two CVA1 isolates and the prototype strain in P1 region, and compared the discrepant sites among four virus passages of continuous CVA1-8-124 passages, and conducted temperature sensitivity test of these four virus passages, and conducted the preliminary exploration of the virus receptor of two Xinjiang CVA1 isolates.Results:We isolated 32 strains belonging to 8 serotypes of EV-C from Xinjiang in 2011, they are 2 CVA1 strains,6 CVAllstrains,1 CVA13 strains,7 CVA17 strains,4 CVA20 strains,7 CVA24 strains,2 EV-C96 strains and 3 EV-C99 strains, respectively. These 8 serotypes distributed in four regions of southern Xinjiang, and Kashgar prefecture had the most serotypes of EV-C isolates. Except CVA1 can only replicate in RD cells, other 7 serotypes of EV-C can both replicate in RD and HEp-2 cells;Based on the phylogenetic tree of the full-length VP1 region, the pairwise genetic distances not only between Xinjiang EV-C isolates and the viral sequences deposited in the GenBank but also among Xinjiang EV-C isolates except CVA1 were high, which indicated that 8 serotypes of EV-C circulated in Xinjiang in 2011 were new genotypes or sub-genotypes. Not only the highest genetic divergence between Xinjiang EV-C isolates and the viral sequences deposited in the GenBank, but also the highest genetic divergence among the viral sequences deposited in the GenBank, both exceeded the generally accepted demarcation ofHEV genotyping on the nucleotide sequence level:0.25, even 0.30, however, on the amino acid level, there were still the highest genetic divergence of three serotypes, CVA13, CVA20 and CVA24 above the generally accepted demarcation of EV genotyping:0.15, so EV-C were more complex than EV-A and EV-B, and the demarcation of EV genotyping was needed adjusting and then better genetic characterizations of EV-C;The study on genomic characteristics of 17 EV-C strains as representative strains based on the genetic distances of the full-length VP1 region, was that by pairwise nucleotide sequence or amino acid sequence identities, we can not find the recombination evidence among EV-C, and by recombination analysis of the alignment of the prototype stains and Xinjiang EV-C isolates, we found that the recombination between CVA1 and CVA22 in the 5 'UTR, P2 and P3 region, the most common recombinations of Xinjiang EV-C isolates occurred among CVA11, CVA17 and CVA20, three Xinjiang CVA24 isolates were still not found specific donor sequence, and two Xinjiang EV-C99 isolates were first found intertypic recombination and the specific donor sequence, Xinjiang CVA13 isolate in the 3D region;In this study, we first isolated two CVA1 strains which can propagate and produce CPE on RD cell, and by comparing the discrepant sites between the two CVA1 isolates and the prototype strain, the discrepant sites among four virus passages of continuous passages in P1 region, we found 6 significant amino acid sites in total and these 6 sites as the priority of the site-directed mutagenesis of our next step reverse genetics research, four virus passages of CVA1-8-124 continuous passages were found to be temperature insensitive strains, and the preliminary exploration of the virus receptor of Xinjiang CVA1 isolates, was found that CVA1 can take advantage of a new and unknown virus receptor to replication on RD cell and further research were needed.Conclusions:EV-C had a wide distribution in southern Xinjiang region, and our EV isolation result was not consistent with the established research that EV-C were more sensitive to HEp-2 cell;The high genetic divergences among EV-C on the full-length VP1 region indicated that 8 serotypes of EV-C circulated in Xinjiang in 2011 were new genotypes or sub-genotypes and EV-C were more complex than EV-A and EV-B;Recombination analysis of 17 EV-C representative strains again supported the evidence that EV-C like EV-A and EV-B were multiple recombinant strains, and recombination is a very common phenomenon in EV;The preliminary exploration of the virus receptor of Xinjiang CVAl isolates, was found that CVA1 can take advantage of a new and unknown virus receptor to replication on RD cell and further research were needed... |
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