| [Background]Human enteroviruses (HEV) are members of the genus Enterovirus of the family Picornaviridae, which are common viruses replicated in human intestinal. HEV usually cause self-limited infections, and can be transmitted by the fecal-oral route, daily contact route, and respiratory route. Patients and asymptomatic infected persons are the main source of infection. Enterovirus infections are more prevalent among susceptible population, especially children less than 5 years old. HEV can replicate in human intestinal, while digestive tract symptoms are rare. Viruses cause systemic infections which are originated in the digestive tract and finally developed into viraemia, as a result of that, many kinds of diseases such as acute flaccid paralysis (AFP), meningitis or encephalitis, myocarditis, acute hemorrhagic conjunctivitis, hand-foot-and-mouth disease (HFMD) and respiratory infection can be seen on clinical. In recent years, the incidence of diseases caused by HEV infection has been showing a rising trend.Based on their pathogenesis in humans and experimental animals, differences between infectivity, and pathogenicity and antigenicity, HEVs were originally classified into four groups, poliovirus (PV,1-3), coxsackievirus A (CVA 1-22 and 24), coxsackievirus B (CVB 1-6) and echovirus (ECHO 1-7,9,11-27,29-33). With the application of molecular biology and bioinformatics technology, HEVs are classified into four species, HEV-A, HEV-B, HEV-C and HEV-D, largely on the basis of phylogenetic analysis of multiple genome regions, HEV-A includes 17 serotypes: CVA 2-8,10,12,14,16 and newer enteroviruses EV71,76, and 89-92.The genome of HEV consists of a single-stranded positive-sense RNA of approximately 7400 nucleotides. The viral genome contains a 5'- and 3'- untranslated regions (UTRs) which are essential for viral RNA replication. The genome is translated as a single large polyprotein that is composed of four capsid proteins, VP1 to VP4, and seven nonstructural proteins,2A,2B,2C,3A,3B,3C, and 3D. VP1 to VP4 capsid proteins were encoded by P1 region. The P2 and P3 regions encode nonstructural proteins and the RNA-dependent RNA polymerase. VP1 protein, the most external and immunodominant of the enterovirus capsid protein, which is the main structural of recognition and combination between viruses and cell receptor, is prone to mutation under the pressure of body's immune system. For the above reasons, nucleotide sequence analysis of VP1 protein can not only help to identify serotypes but also indicate the homologous identity and phylogenetic relationships.HEV-A can cause many kinds of diseases, such as AFP, aseptic meningitis, HFMD, herpangina and so on, of which HFMD is frequently seen, especially in China since 2007. EV71 and CVA 16 are the most common causative agents of HFMD. Shandong province is a high incidence region of HFMD and every year the epidemic of HFMD occurred since 2007. The molecular epidemiology study showed that the Shandong HFMD epidemic was mainly caused by EV71 and CVA 16, while other HEV-A played a complementary role.[Objectives]1. To describe the genotype distribution of HEV-A isolated in Shandong, and to establish a database of VP1 complete sequences of HEV-A strains in Shandong.2. To analyze the genetic characterization of main HEV-A serotypes in Shandong.3. To investigate the relationship between diseases and main types of HEV-A, and to analyze the effects of genetic evolution on the pathogenicity and epidemiology.[Methods]1. Isolation of enterovirus from stool samples of AFP cases and some of HFMD patients from 2007 was performed. All positive strains were sero-typed by neutralization test.2. Viral RNA of Unserotypable non-polio enteroviruses (NPEV) was extracted. VP1 coding region was amplified by PCR and the products were examined by agarose gel electrophoresis. Positive product was sequenced at BGI sequencing company.3. Entire VP1 coding region was obtained by sequence cut and spliced. HEV-A Shandong isolates were typed by BLAST of VP1 sequences.4. Homology analysis on VP1 sequences from Shandong strains and others submitted in GenBank were conducted by BioEdit Sequence Alignment Editor software 5.0.9.5. Phylogenetic analysis constructed by the Neighbor-Joining method based on the alignment of the complete VP1 gene sequences of Shandong HEV-A strains and the corresponding strains from GenBank were performed by Mega3.1.[Results]1. Species A HEVs of 10 genotypes were isolated and identified in Shandong Province and they were CVA4, CVA6, CVA10, CVA12, CVA14, CVA16, EV71, EV76 and EV90.2. The nucleotide and the amino acid homologies between Shandong strains and prototype of CVA4 were 83.8%-85.3% and 97%-99.6%, respectively. Mutations of nucleotide were bigger than amino acid and there were four main variation sites in amino acid sequence between Shandong strains and prototype of CVA4. CVA4 isolates segregated into five clusters and Shandong isolates were grouped to Cluster C and E.3. The nucleotide and the amino acid homologies between Shandong strains and prototype of CVA6 were 81.3%-81.5% and 94.4%-95.0%, respectively. CVA6 isolates segregated into three clusters and Shandong isolates were grouped to cluster C with a single transmission chain.4. The nucleotide and amino acid homologies between Shandong strains and prototype of CVA10 were 75.6%-76.8% and 90.2%-93.2%, respectively. CVA10 isolates segregated into seven clusters and Shandong isolates were grouped to cluster F and G.5. The nucleotide and amino acid homologies between Shandong strains and prototype of CVA16 were 75.5%-77.1% and 91.5%-92.2%. CVA16 can be divided into two genotypes, A and B. There were two lineages of B1a and B1b co-circulating in Shandong.6. The nucleotide and amino acid homologies between Shandong strains and prototype of EV71 were 82.0%-82.9% and 94.2%-95.2%. EV71 Shandong strains possessed the highest nucleotide homologies with subgenotype C4 (91.8%-93.7%). EV71 can be divided into three genotypes and Shandong isolates were grouped to subgenotype C4. A mutation at amino acid position 249 (ⅠtoⅤ) between EV71 Shandong strains and isolates belonged to subgenotype C1 to C3 was identified.7. The outbreak of HFMD in Linyi city in 2007 was mainly caused by C4a subgenotyoe of EV71. EV71 and CVA16 were dominant pathogens of HFMD epidemic in Shandong province from 2008 to 2010, other HEV-A played a subsidiary role.[Conclusions]1. EV71 and CVA16 were dominant genotypes of HEV-A in Shandong and other genotypes played a subsidiary role in outbreak of HFMD.2. HEV-A Shandong strains evaluated steadily and directionally. Compared to EV71, EV71 had a higher evolution rate. At present EV71 strains isolated in Shandong were identified to subgenotype C4a, there were two lineages of CVA16 isolates co-circulating in Shandong.3. Genetic evolution of several HEV-A genotypes showed some regional distribution characterization. Intratypic or intertypic recombination may occurred between HEV-A genotypes in the process of long-term co-evolution.4. Outbreaks and epidemics of HFMD in China mainland were mainly caused by EV71 and VP1 of EV71 evaluated steadily, no recombinant was discovered. Subgenotype C4a was the only subgenotype circulated in mainland of China since 1998, recombinant of nonstructural protein of EV71 was speculated to be one of reasons why only one single genotype of EV71 have been continuously circulating and causing epidemic in the mainland of China so far. |
PDF Full Text Request