Genotype Distribution And Molecular Epidemiology Of Human Enterovirus B Species Isolated In Shandong Province, China

[Background]Human enteroviruses, small RNA viruses in the family of Picornaviridae, based on genetic relationships between the virus strains, HEV were grouped into four species designed Human enteroviruses A, B, C and D. Aseptic Meningitis mostly associated with kinds of viruses'infection. The yearly incidences of AM are 11 to 27 per 100,000 population in the word,85% to 95% of which are associated with HEV.Eradication of wild polioviruses and the subsequent cessation of vaccination with attenuated Sabin strains may induce important changes in the transmission patterns of non polio enteroviruses. Disease caused by HEV is more and more recent years.Lots of NPEV were isolated by AFP routine surveillance of Shandong CDC, and many outbreaks of AM caused by NPEV had been reported in Shandong Province, especially AM and HFMD. Some isolates were sequenced. This study will make better knowledge of the transmission and the evolution of these viruse isolates, and will help to environmental surveillance of Shandong Province.[Objectives]1. To describe the epidemic situation of HEV-B between 1998 and 2008 in Shandong Province; research the molecular distribution of HEV-B Shandong isolates;2. To set up the data base of VP1 gene of Shandong isolates;3. To analyse the molecular characteration of the VP1 gene of Shandong isolates, compare with other isolates from other parts of the word, analyse the evolution origin.[Methods] (1)L20B, RD and HEp-2 cell lines were used for virus isolation. According to standard protocols, the microneutralization assays were carried out in 96-well tissue culture plates using an antibody pool for enterovirus.(2) Choose strains on the basis of isolation time and sites, then RNA extraction and RT-PCR were performed.(3) The combination of the two sequences yielded the entire VP1 coding region, then compared with sequences available in GenBank using BLAST. Designated the relative serotype accordance BLAST results.(4)Nucleotide sequence alignments were carried out by BioEdit 5.0.9 software.(5)Phylogenetic trees were constructed by Mega 4.1 using neighbor-joining method after estimation of genetic distance using the Kimura two-parameter method.【Results】1. Iaolation of Shandong Strains:There are 1021 NPEV strains isolated from AFP cases between 1994 and 2008; CVB3. CVB5 and E30 strains isolated from AM outbreak between 2003 to 2008.2. Molecular Distribution and Homology comparison:There are 29 serotypes, ECHO11,CVB3,ECHO6,ECHO14,ECHO25 distribute abrodly in Shandong province; There is not distinct region distribution. Homology comparison showed that: E1 isolates have the maximal nucleotide diversity (6.7%-24.6%), CVB4 have the minimum nucleotide diversity (2.9%-5.6%); CVB2 have the minimum nucleotide diversity (4.8%-7.7%), EV73 have the maximal aa diversity (24.6%), compared with relevent prototype.3. Sequence Analysis of Shandong Isolates3.1 Homologous and Phylogenetic Analysis of CVB3 Shandong IsolatesHomologous analysis showed 77.8%-80.0% nucleotide identities and 96.1%-97.5% aa identities between CVB3 Shandong isolates and relevent prototype.Nucleotide comparation showed distinct diversity between 177/2008TC/SD/ CHN and the other isolates; the numbers of aa variant are 9/10 of the four isolates.The sequences of CVB3 were grouped into 3 distinct clusters:A,B and C, Shandong CVB3 isolates segregated into clusters A. Shandong strains contained in sub-cluster A1-A3 isolated during 2004-2008.1994-2002,2004 and 2008.3.2 Homologous and Phylogenetic Analysis of CVB5 Shandong IsolatesHomologous analysis showed 81.2%-81.6% nucleotide identities and 96.8% aa identities between CVB5 Shandong isolates and relevent prototype,83.0%-94.6% nucleotide identities and 96.4%-100.0% aa identities among CVB5 Shandong isolates.There is no obvious character of the nucleotide comparation.The sequences of CVB5 were grouped into 4 distinct clusters:A,B,C and D, Shandong CVB5 isolates segregated into clusters C and D. Phylogenetic analysis showed highest nucleotides homologies between Shandong isolates and Cox B5/Zejiang/12/02 (CSF)3.3 Homologous and Phylogenetic Analysis of E6 Shandong IsolatesHomologous analysis showed 75.2%-78.8% nucleotide identities and 92.7%-95.5% aa identities between E6 Shandong isolates and relevent prototype, 77.6%-98.6% nucleotide identities and 93.7%-99.6% aa identities among E6 Shandong isolates.Nucleotide comparation showed that strains isolated from different years assembled together; aa comparation showed three intensive diversity parts.The sequences of E6 were grouped into 3 distinct clusters:A,B and C, Shandong E6 isolates segregated into clusters B and C.12 Shandong isolates divided into three different branches.3.4 Homologous and Phylogenetic Analysis of E11 Shandong IsolatesHomologous analysis showed 77.7%-80.7% nucleotide identities and 90.7%-94.8% aa identities between E11 Shandong isolates and relevent prototype, 76.4%-100.0% nucleotide identities and 91.4%-100.0% aa identities among Ell Shandong isolates.The numbers of aa variant are greater then 20; aa comparation showed assembling and intensive diversity parts.The sequences of E11 were grouped into 4 distinct clusters:A,B C and D, Shandong E11 isolates segregated into clusters A and C.12 Shandong isolates formed three new sub-clusters, A1,A2 and A3. Shandong strains contained in sub-cluster A1 isolated after 1998, contained in sub-cluster A1,A2 isolated between 1994 to 1997.3.5 Homologous and Phylogenetic Analysis of E19Shandong IsolatesHomologous analysis showed 78.6%-79.2% nucleotide identities and 97.6%-99.3% aa identities between E19 Shandong isolates and relevent prototype, 83.3%-98.6% nucleotide identities and 98.6%-99.3% aa identities among E19 Shandong isolates.Gene comparation showed similar diversity among strains isolated from the same year; 97206/SD/CHN/1997/E19 gene diversity was obvious from the others.Phylogenetic analysis showed the 9 strains isolated from China assembled together, formed a cluster.3.6 Homologous and Phylogenetic Analysis of E30 Shandong IsolatesHomologous analysis showed 81.9%-83.5% nucleotide identities and 92.4%-93.1% aa identities between E30 Shandong isolates and relevent prototype, 86.0%-98.5% nucleotide identities and 96.9%-98.9% aa identities among E30 Shandong isolates.Nucleotide comparation showed distinct diversity between 06530/SD/CHN/ 2006/E30,017/2008TC/SD/CHN and the other isolates.The sequences of E30 were grouped into 5 distinct clusters:A-E, Chinese E30 isolates segregated into clusters E, and Shandong isolates segregated into sub-clusters E1 and E2.4. Relationship between Shandong Isolates and AM Outbreak:There was an aseptic meningitis outbreak in Zhangqiu city, the age-specific peak incidence occurred in children 7 years old. Cases showed definite contact history, nervous system symptom was obvious. E30 strains isolated from this outbreak had consanguineous with strains isolated from Taishan,Zhejiang and Jiangsu.There was an aseptic meningitis outbreak in Yanzhou city, most of the cases were children 6 year old, the age-specific peak incidence occurred younger than 2 years old. Cases showed indistinct contact history, symptoms were slight. CVB5 strains isolated from this outbreak had consanguineous with strains isolated from Zhejiang 2002.There was an aseptic meningitis outbreak in Tancheng county, most of the cases were infants 1 year old. Age was older the age-specific incidence was lower. Most of the cases showed definite contact history, symptom was slight. E30 strains isolated from this outbreak belong to the same linage.[Conclusion]1. Virus of HEV-B spread abroadly in Shandong province, lots of NPEV strains isolated from AFP surveillant System every year; Different strains have different circulation mode. The change of dominant strains can result in the break out of disease.2. Homology comparison of VP1 gene showed that, the variation of Shandong isolates is distinct compared with relevent prototypes, chovirus is more active than Coxvirus.3. Shandong isolates belong to the same genotype can divided into different sub-genotype, sequence analysis showed that stains belong to the same genotype get together.4. The diseas caused by HEV-B is AM mostly. HEV-B strains isolated from HFMD some time. HEV-B stains belong to single type isolated from most diseas out breaks.5. Every starins belong to different types have different circulation mode:①The transmission of CVB3 was rapidly abroad China. Multi-transmission chains of CVB3 might have co-circulated in Shandong province the same year;②The evolution rate of CVB5 might be slower, isolates contained in sub-cluster D1 spread in Asia and European during 2002 and 2005, result in AM outbroke in Zhejiang province 2002 and Shandong province 2005.③The evolution rate of ECHO6 might be more rapidly, there were two transmission chains of ECHO6 might have co-circulated in Shandong province 2001, homologous analysis showed high nucleotide identities between strains isolated from different years, might come from different regions;④The evolution of ECHO 11 might be activity, there were two genotype of ECHO 11 might have co-circulated in Shandong province between 1994 and 1997, isolates contained in A1 sub-cluster prevalent in recent years.⑤The evolution rate of ECHO 19 might be slower, isolates caused outbreak of AM in Tai'an city 2003 prevalent abroadly in Shandong province;⑥Homologous analysis showed high nucleotide identities between ECHO30 strains isolated from outbreak of AM in Zhangqiu city and relevant prototype, viruses had strong infectious ability, transmission rate is faster, became the major strains, and there were two transmission sub-clusters of ECHO30 might have co-circulated in Shandong province between 2003 and 2008.