ObjectiveTo identify the pathogen of an aseptic meningitis outbreak which occurred in Linyi City of Shandong Province during the summer of 2009,and to analyze the genetic variations of Coxsackicvirus B5 (CVB5) isolates.MethodsForty-two cerebrospinal fluids (CSF) specimens were collected from aseptic meningitis cases and virus isolation was performed. The viral RNA was extracted and amplified from the positive specimens using reverse transcription polymerase chain reaction (RT-PCR).The partial VP1 coding region was purified and sequenced. The phylogenetic trees based on VP1 sequences were constructed among CVB5 isolates and those in GenBank.ResultsSeventeen enteroviruse strains were isolated from 42 CSF samples with 40.5％ isolation positive rate. All these strains were identified as CVB5 using both microneutralization test and molecular typing methods. Homology comparisons indicated that the nucleotide acid identities and amino acid sequence identities were 92.3 ％- 100.0％ and 98.7 ％- 100.0％,respectively among these CVB5 isolate.s,and compared with the Faulkner prototype strain,which were 81.0％-82.4％ and 96.6％97.0％,respectively.Phylogenetic analysis on VP1 sequences showed that all CVB5 could be separated into four genogroups of A,B,C and D.Isolates of this outbreak belonged to genogroup D.Interestingly,two distinct genogroups in the phylogenetic tree were observed among the 17 isolates.Conclusions CVB5 is responsible for the outbreak of aseptic meningitis in Linyi City of Shandong Province,China. The genetic diversity is high among the isolates and all belong to genogroup D.

Objective To analyze the genetic characteristics of echovirus 6 ( E-6) strains isolated from patients with acute meningitis/encephalitis syndrome ( AMES) in 2014 and sewage samples in 2013—2014 in Shandong province and to investigate their correlations.Methods Enterovirus strains were isolated from cerebrospinal fluid, stool and throat swab samples collected from 940 cases of AMES and 96 sewage samples used for environmental surveillance.The positive isolates were identified by molecular typing meth-od.Homologous and phylogenetic analyses based on the VP1 sequences of E-6 isolates were performed.Re-sults Altogether 47 E-6 strains were isolated from patients with AMES in 2014, accounting for 29.56%of all isolated enteroviruses ( EVs) strains.No E-6 strains were isolated from AMES cases in 2013.Data of the environmental surveillance showed that E-6 virus strains had been frequently detected in sewage samples since the summer of 2013 to the end of 2014.In total, 40 E-6 virus strains were isolated (7.87% of total isolated EVs strains) in 2013 and 139 E-6 virus strains (26.18%) in 2014.Phylogenetic analysis indicated that the E-6 isolates recruited in this study belonged to clusters A and C with high intracluster sequence iden-tities between AMES and environmental isolates.The nucleotide identities were 98.3%-100% among cluster A E-6 virus strains isolated from AMES cases in 2014 and 96.6%-100% among cluster A E-6 virus environ-mental isolates during the surveillance year 2013—2014.The cluster A E-6 virus strains shared 97.1%-100% nucleotide identities between the AMES and environmental isolates.For cluster C E-6 virus strains, the nucleotide identities were 100%, 98.7%-100% and 99.1%-100%, respectively.Conclusion The epidemic of viral encephalitis in Shandong province in 2014 was associated the transmission of two lineages of E-6 virus.Environmental surveillance revealed the potential epidemic of E-6 virus even before the epidemic of viral encephalitis in Shandong province, indicating the possibility of using environmental surveillance for early warning of related diseases.

Objective To analyze the expression characteristics of excision repair cross-complementing 1 (ERCC1), topoisomeraseⅡ (TOPOⅡ), ribonucleotide reductase M1 (RRM1), β3-tubulin and thymidylate synthase (TS) in lung cancer and their associations with the pathological types. Methods The immunohistochemical EnVision method was used to determine the expression of ERCC1, TOPOⅡ, RRM1,β3-tubulin and TS in 548 patients who were diagnosed as lung cancer from January 2011 to December 2014. Variance analysis was performed to analyze their expression characteristics among different pathological types and correlation. Results The expression positive rates of ERCC1, TOPOⅡ, RRM1, β3-tubulin and TS were 61.86 % (339/548), 91.06 % (499/548), 62.59 % (343/548), 73.18 % (401/548) and 70.44 % (386/548), respectively. The expression of ERCC1 was weak positive mostly (P<0.05), meanwhile the expression of TOPOⅡ was medium-strong positive mostly (P<0.05). In ERCC1 group, the positive rate of squamous cell carcinoma was higher than that of adenocarcinoma [57.39 % (167/291) vs. 42.61 % (124/291), P=0.000]. In weak positive of TOPOⅡ group, the proportion of adenocarcinoma was higher than that of squamous cell carcinoma [23.58 % (100/137) vs. 8.73 % (37/137), P=0.000]. In medium-strong positive of TOPOⅡ group, the proportion of squamous cell carcinoma was higher than that of adenocarcinoma [47.41 % (201/287/) vs. 20.28%(86/287), P=0.000]. The expressions of ERCC1, TOPOⅡ, RRM1,β3-tubulin and TS were irrelevant (r=0.4, P=0.397). Conclusions The expressions of ERCC1 and TOPOⅡ are higher in squamous cell carcinoma than those in adenocarcinoma. The expression of ERCC1 is weak positive mostly, meanwhile the expression of TOPOⅡis medium-strong positive mostly. There is no correlation between them.

Objective To analyze the genetic characterization of VP1 region of Coxsackie virus A10(CVA10)isolated from clinical specimens of hand, foot and mouth disease(HFMD) patients in Shandong Province. Methods Clinical specimens were collected from some of HFMD patients from 2008 to 2009. The virus was isolated by cell culture. Total RNA was extracted, and the VP1 genes of the isolates were amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced. The genotypes were identified by molecular typing method and bioinformatics analysis.Homologous comparison and phylogenetic analysis of representative CVA10 strains were performed.Homologous comparison between the Shandong isolates and strains obtained from GenBank were performed and phylogenetic analysis of some representative CVA10 strains were performed. Results Three hundred and thirty viruses strains were isolated from 760 clinical specimens collected from HFMD patients, and 17 of them were identified as CVA10. The homologies of nucleotide and amino acid of the 17 CVA10 strains were 82.3%-100.0% and 94.2%-100.0%, respectively. Compared with the prototype strain of CVA10 (Kowalik/USA/2003), the homologies of nucleotide and amino acid were 75.6%-76.8% and 90.2%-93.2%, respectively. Interestingly, Shandong CVA10 strains were clustered into two distinct subgroups in the phylogenetic tree. Conclusions CVA10 is one of the causative agents of HFMD. Two independently circulating subgroups of CVA10 exist in Shandong province.

To analyze the genetic characteristics of a polio-I highly variant vaccine recombinant virus in Shandong Province (China) in 2011 and to identify isolates from healthy contacts, two stool specimens from one patient with acute flaccid paralysis (AFP) and 40 stool specimens from his contacts were collected for virus isolation. The complete genome of poliovirus and VP1 coding region of the non-polio enterovirus were sequenced. Homologous comparison and phylogenetic analyses based on VP1 sequences were undertaken among coxsackievirus (CV) B1, CV-B3 isolates, and those in GenBank. One poliovirus (P1/11186), CV-A4 and CV-A8 were isolated from the AFP patient; one CV-A2, Echovirus 3 (E-3), E-12 and E-14, ten CV-B1, and five CV-B3 strains were isolated from his contacts. These results led us to believe that there may be a human enterovirus epidemic in this area, and that surveillance must be enhanced. P1/11186 was a type-1 vaccine-related poliovirus; it combined with type-2 and type-3 polioviruses in 2A and 3A regions, respectively. There were 25 nucleotide mutations with 9 amino-acid alterations in the entire genome. There were 8 nucleotide mutations with 5 amino-acid alterations in the VP1 region compared with the corresponding Sabin strains. Homology analyses suggested that the ten CV-B1 isolates had 97.0%-100% nucleotide and 98.9%-100% amino-acid identities with each other, as well as 92.6%-100% nucleotide and 99.2%-100% amino-acid identities among the five CV-B3 isolates. Phylogenetic analyses on the complete sequences of VP1 among CV-B1 and CV-B3 isolates showed that Shandong strains, together with strains from other provinces in China, had a close relationship and belonged to the same group.
Base Sequence ; Capsid Proteins ; genetics ; immunology ; Child, Preschool ; China ; Humans ; Male ; Molecular Sequence Data ; Phylogeny ; Poliomyelitis ; etiology ; prevention & control ; virology ; Poliovirus ; classification ; genetics ; immunology ; isolation & purification ; Poliovirus Vaccines ; adverse effects ; genetics ; immunology

ted in a loss of cell fusion,which suggests the 928 site on G2 is crucial for cell fusion and the fusion peptide is likely on G2.

Hand, foot and mouth disease (HFMD) is a common acute infectious disease among children. Enterovirus 71 (EV71) is one of the major pathogens of HFMD. The incidence of HFMD in our country is higher, the main target population of infection is infants and young children; EV71 is also the most important pathogen causing severe HFMD in children currently. HFMD was classified as notifiable communicable disease of Category C on May 2, 2008. Pathogenesis of HFMD is not yet clear, and there is no drug that can treat such virus effectively. The non-structural protein 3D in EV71, is one of the important targets for the development of anti-EV71 virus drugs, which catalyzes the replication and transcription of the viral genome. This review provides a summary of the structure and function of EV71 3D, which can provide reference for the development of antiviral drugs.

Objective: To characterize the etiology and epidemiological characteristics of the acute meningitis and encephalitis syndrome (AMES) in Jinan city in 2013-2016. Methods: The epidemiological data, clinical diagnosis, serum and cerebrospinal fluid (CSF) specimens were collected from 3 577 AMES cases in 6 sentinel hospitals in Jinan city in 2013-2016. Samples of all cases were made sero-diagnosis for Immunoglobulin (Ig) M antibody to Japanese encephalitis virus (JEV) and negative cases of JEV for enterovirus (EV), mumps virus (MuV) and herpes simplex virus (HSV) by enzyme-linked immunosorbent assay (ELISA). Virus isolation and molecular identification were performed. Positive rates were analyzed by Chi-square test. Results: In 2013-2016, the positive rates of JEV, EV, MuV and HSV were 9.0% (322/3 577 cases), 22.1% (643/2 916 cases), 9.9% (289/2 916 cases), 26.9% (783/2 916), respectively. Of these, the positive rates of JEV were 32.9% (261/794), 1.2% (14/1 175), 1.0% (8/807) and 4.9% (39/801 cases); EV: 19.5% (91/466), 35.1% (342/974 cases), 15.5% (115/743) and 13.0% (95/733); MuV: 9.2% (43/466), 14.4% (140/974), 9.0% (67/743) and 5.3% (39/733). HSV: 35.4% (165/466), 38.5% (375/974), 25.7% (191/743) and 7.1% (52/733). There were significant differences in positive rates of 4 kinds of viruses in 2013-2016 (P<0.001). A total of 81 EV strains belonging to 8 serotypes were isolated from 1 020 CSF specimens. The positive rates were 4.8% (6 cases), 13.1% (55 cases), 4.1% (7 cases) and 4.2% (13 cases) from 2013 to 2016. Coxsackievirus (CV) B5, echovirus (E) 6 and E30 accounted for 46% (37 isolates), 22% (18 isolates) and 21% (17 isolates) of all strains. Conclusion The AMES cases in Jinan city in 2013-2016 were mainly caused by HSV, EV, MuV, JEV. CVB5, E6 and E30 were the dominant serotypes of EV associated with AMES cases in Jinan city.

Objective: To understand the genotype distribution and molecular epidemiological characteristics of the group A rotavirus (RVA) in domestic sewage, and further explore the importance of environmental surveillance in investigating RVA regional circulation. Methods: Sewage samples were collected monthly in the city of Yantai from January 2014 to December 2016. After concentration, total RNA was extracted, and RVA VP7 and VP4 coding regions were amplified via RT-PCR. PCR products were purified, cloned and Sanger sequenced. Genotyping and phylogenetic analysis was conducted based on the sequences. Results: Thirty-six sewage samples were collected and 86.1% was positive with VP7 and VP4 sequences. A total of 205 VP7 and 239 VP4 nucleotide sequences were obtained, belonging to 4 G genotypes and 6 P genotypes. Among these, G9 (95.6%, 196/205), P[8] (58.6%, 140/239) and P[4] (28.0%, 67/239) were the most common genotypes. Phylogenetic analysis for G9, P[8] and P[4] sequences revealed co-circulation of multiple transmission chains in local population. Conclusions This study describes the genotype distribution and sequence characteristics of local RVA in Shandong province, and the result demonstrate that surveillance on environmental sewage is an effective way in investigating RVA molecular epidemiology.

Objective: To sequence the 3′UTR of enterovirus 71 strains, investigate its foundation and impact in virulence by constructing a 3′UTR-replaced recombinant cDNA infectious clone. Methods: Viral RNA of EV-A71 isolated viruses were extracted, and the nucleotide analysis was performed after sequencing. The 3′UTR of a full-length infectious clone of SDLY107 strain was replaced by its corresponding part of SDLY1 strain, and then the recombinant virus was constructed and identified. Results: The nine isolated strains were classified into sub-genotype C4a of enterovirus (EV)-A71 by analysis, and nucleotide sequence homology for 3′UTR were 94%-100%. 3′UTR of EV-A71 strains may be associated with its pathogenicity. Identification of the rescued virus by sequencing and indirect immunofluorescence confirmed the successful construction of infectious recombinant virus. Conclusions Sequence analysis indicated that the 3′UTR may be involved in the pathogenicity of EV-A71. The recombinant virus SDLY107(1-3′UTR) was rescued successfully. Our study may provide evidence for further research on the influence of 3′UTR on the virulence of enterovirus 71.