Objective To finally diagnose the outbreak of acute respiratory infection caused by Adenovirus 3 in students of the north of Jiangsu province by serologic study. Methods An enzyme-linked immunosorbent assay (ELISA) has been used to detect the adenovirus IgM in the serum of acute phase and the adenovirus IgG in the serum of recovery phase. A neutralization test has been used to detect the neutralization antibody in the serum of acute and recovery phase from cases and in the serum from the control. The SPSS11.0 has been applied to the statistical analysis of the data. Results The positive rate of IgM, IgG and recovery phase neutralization antibody of cases are 3.7%, 44.4% and 59.5% respectively, while those of control are 0%, 8.3% and 33.3% respectively, and the P values of Chi-Square are 0.510, 0.018 and 0.226 respectively. The neutralization test of paired serum of 6 in 9 cases showed that the antibody titers was obviously increased. The concordance between IgG detected by ELISA and neutralization antibody detected by neutralization test is 61.4% and the P value of Kappa is 0.070. Conclusion By the serologic study, we can finally diagnose that the outbreak of acute respiratory infection was caused by Adenovirus 3.

Objective To establish a clinical test assay for detecting common respiratory viruses and enteroviruses (EV) by using mixed cultured Madin-Darby canine kidney cells (MDCK), human epidermoid cancer cells (Hep-2) and African green monkey kidney cells (Vero) to isolate common respiratory viruses and enteroviruses. Methods Throat swabs with influenza A and B viruses,adenovirus and EV71 were incubated with mixed cultured MDCK, Hep-2 and Vero in a single vial to observe the presence of cytopathic effects. Polymerase chain reaction (PCR), reverse transcription (RT)-PCR and monoclonal antibody-based immunofluorescene assay were also used for confirmatory test. Results The sensitive cell lines developed obvious cytopathic effects to the corresponding viruses, which were confirmed by the specific green particles observed by immunofluorescence assay and specific target PCR segments. Conclusions The shell-vial of mixed cells can simultaneously isolate different common respiratory viruses and EV. The isolated pathogens can be further confirmed by antigen test and PCR. This assay may improve the diagnosis of clinical viral diseases.

Objective To evaluate shell vials of MHV,a combination of Madin-Darby canine kidney cells (MDCK),human epidermoid cancer cells (Hep-2) and African green monkey kidney cells (Vero),and conventional cell culture in detecting influenza viruses and enterovirus from fresh clinical specimens.Methods Specimens from patients with influenza-like illness and children with hand-foot-mouth disease were inoculated with both shell vials of MHV and MDCK/Vero.Then cytopathological effect (CPE) was examined daily.Influenza viruses and enteroviruses were detected by multiple reverse transcriptase-polymerase chain reaction (mRT-PCR).Results CPE of MDCK/Vero cells were stronger than the shell vials of MHV.The isolation rate of influenza virus by MHV was 24.6％ (34/138) and that by MDCK was 28.3％ (39/138),which was not significantly different (x2 =1.92,P＞0.05).That of enterovirus by MHV was 28.1％ (9/32) and that by Vero was 37.5％ (12/32),which was not significantly different (x2 =3.00.P＞0.05).Conclusions CPE in MDCK/Vero cells are easier to be observed than the shell vials of MHV.However,the shell vials of MHV are appropriate in public health emergencies,which can be used for isolation of influenza viruses and enterovirus in patients with respiratory symptoms.

Objective To evaluate the survival ability of enterovirus 71 (EV71) on different surface and under different climate.Methods Each 1 × 105 tissue culture infective dose 50 (TCID50)EV71 was added on different aseptic surface of plastic,rubber,cloth and wood,respectively.Then these materials were put into biotron (artificial climatic chamber) which could simulate different temperature and moisture.The viruses were recovered after a definite time and then inoculated into Vero cell.The cytopathic effect (CPE) was observed everyday to survey the survival ability of EV71 on different medium surface.Results The recovery rates of EV71 on medium surface ranged from 89 ％-93 ％.The survival time of EV71 on medium surface varied under different climatic conditions.The longest survival time of the virus was observed under the condition of 20 ℃ as the temperature and 80％ as the humidity.After 24 hours of incubation,the infectious titer on plastic surface reduced about 4 lg.After 72 hours of incubation,the infectious titer reduced at least 3.89 lg on cloth and wood surface.Conclusions Temperature and humidity can affect the survival time of EV71 on medium surface,which is longer in the condition of low temperature and high humidity.The survival ability of EV71 on natural cloth and wood surface is better than that on synthetic plastic surface.

Objective To analyze the VP1 and VP4 genetic region of enterovirus 71 (EV71)isolated from severe cases and mild cases with hanD-foot-mouth disease (HFMD) in Shanghai in 2011.Methods Five EV71 strains isolated from severe cases and five EV71 strains from mild cases in 2011 were included.Reverse transcription-polymerase chain reaction (RT-PCR) method was used to amplify and sequence the VP1 and VP4 genes of EV71,and then the sequencing results were compared with those of A,B,C genotype reference EV71 strains from GenBank by nucleotide alignment,amino acid alignment and phylogenetic tree analyses.Results The homogeneity between EV71 strains from severe cases and mild cases was 96.0％-98.1％ and 93.7％-99.5％ for VP1 and VP4 nucleotide sequences,respectively.The VP1 nucleotide sequences of 5 strains isolated from severe cases and 5 strains from mild cases in Shanghai shared 86.9％-98.2％ and 87.4％ 98.5％ identity with genotype C,respectively,while the homogeneity of VP4 nucleotide sequence was 85.5％-100.0％and 84.5％-99.5％,respectively.In addition,compared with the Fuyang EV71 strains (representative of C4 subtype),the strains from mild and severe cases shared homogeneity of 97.0％-98.2％ and 97.9％-98.5％ for VP1 gene,respectively,96.1％-100.0％ and 97.1％-99.5％ for VP4 gene,respectively.Among 3 strains isolated from severe cases,mutations at the residue 282 in the VP1 protein (N→S) and residue 7 in the VP4 protein (T→A) were discovered simultaneously.Conclusions The 10 EV71 strains isolated from severe and mild cases in Shanghai belong to subgenogroup C4.Among 3 strains isolated from severe cases,mutations at the residue 282 in the VP1 protein (N→S) and residue 7 in the VP4 protein(T→A) are discovered simultaneously.

Objective To study the gene characteristics of VP1 region of Enterovirus 71 (EV71) strains isolated from clinical specimens of children with hand-foot-and-mouth disease(HFMD) in Shanghai in 2010.MethodsEighteen EV71 isolates were selected from different periods of year 2010,including strains isolated from fatal cases and non-fatal cases.Complete VP1 gene (891nucleotides) of the eighteen EV71 isolates were amplified and sequenced,and then compared with that of genotype A,B,C reference EV71 strains in GeneBank by homogeneity and phylogenetic tree analyses.ResultsThe nucleotide homogeneities between these 18 Shanghai strains and the representative isolates of genotype A and B were 81.5 ％ -82.6 ％ and 83.4 ％- 84.2 ％,respectively,while the amino acid homogeneities were 94.3 ％- 95.0％ and 96.6％ -97.0％,respectively.The nucleotide and amino acid homogeneities between the 18 Shanghai strains and the representative isolates of genotype C were 87.4％- 99.2％ and 98.7％ -100.0％,respectively.Of note,the nucleotide and amino acid homogeneities between Shanghai strains and Fuyang EV71strains (representative strain of C4 subtype) appeared to be 97.8％- 99.2％ and 99.3％- 100.0％,respectively.The eighteen EV71 Shanghai strains were classified as genotype C,subgenogroup C4 in the phylogenetic tree.There was no remarkable difference in VP1 gene between the strains isolated from fatal cases and non-fatal cases.ConclusionThe EV71 strains isolated from Shanghai belong to subgenogroup C4.

Objective To evaluate the survivability of hand foot mouth disease(HFMD)virus,in tap water for daily use.Methods HFMD viruses were isolated from cases of HFMD in Shanghai and Zhejiang from in 2008.Six isolated strains (five subtype of enterovirus 71 and one coxackie virus)were selected in this study.These viruses were mixed with chloride 1.0 mg/L tap-water and then inoculated into Vero cells.The cytopathic effect (CPE)was checked everyday in order to survey the survivability of each virus strain.The decline of virus survivability was analyzed by scatter diagram.Results These six strains of HMFD virus could survive longer than one month in tap water with initial chloride concentration of 1.0 mg/L and still had celluar infectivity.The survivabilities were varied between viruses isolated from different HFMD cases.Conclusions The survivabilities of enterovirus 71 and coxackie virus stains are quite strong in water.Therefore,the transmission route of water-borne pathogens should be monitored in regions using tap water during HFMD epidemic period.

Objective To understand the genetic and antigenic variations of influenza A (H1N1) isolates in Shanghai area in winter of 2010.Methods A total of 137 throat swabs were collected from patients with influenza-like illness in the sentinel hospital in Shanghai area from December 2010 to January 2011,then inoculated into Madin-Darby canine kidney (MDCK) cells.The types of influenza were identified by direct immunofluorescence assay (DIF) and influenza A (H1N1) subtype was determined by reverse transcriptase-polymerase chain reaction (RT-PCR).The mutations of gene and amino acid locus were analyzed through the whole genome sequencing of hemagglutinin (HA),neuraminidase (NA) and polymerase (PB2) segments from some influenza A (H1N1) isolates.Results Total of 53 human influenza virus strains were isolated including 48 influenza A (H1N1) virus strains.Nineteen strains were selected for sequencing by simple random sampling.The phylogenetic tree of HA gene revealed that the latest isolates and most of influenza A (H1N1) viruses isolated before June 2010 were not in the same stem.Analysis of amino acid residues in HA protein showed that mutations were found in antigenic determinant region in some strains.Residues at the enzyme active sites of NA protein were strictly conservative,no change was observed in amino acid residues which were related to drug resistance against oseltamivir and zanamivir.The 627 and 701 residues in PB2 protein were glutamic acid and aspartic acid,respectively,which was still the feature of avian influenza virus,but E677G mutation was detected.Conclusion Compared to influenza A (H1N1) strains isolated in spring and summer,some variations have been detected in the strains isolated in Shanghai area in winter of 2010,some antigen drift and adaptive evolution in mammalian hosts have appeared.

Objective To investigate the distribution and genetic characteristic of etiological agents among children with hand,foot and mouth disease(HFMD)in Shanghai and neighbor areas in 2008.Methods Throat swabs were collected from the inpatients with HFMD from May to June 2008 in Pediatrics Hospital affiliated to Fudan University,Shanghai,and Deqing,Zhejiang Province.Cerebral spinal fluid(CSF)from some patients were collected as well.Vero,MRC-5 and RD ceils were used to isolate the possible pathogens by observing cytopathic effect(CPE).Enterovirus genus,Coxsaekie virus group A type 16(CoxA16)and enterovirus type 71(EV71)were detected by reverse transcriptase-polymerase chain reaction(RT-PCR),and finally identified by sequencing.Results A total of 107 swabs and 22 CSF samples were collected from all 100 inpatients.Swabs of 50 children caused CPE observed.Among them,enteroviruses accounted for 74.0%(37/50),which including 26 (52.0%)of EV71,10(20%)of CoxAl6 and 1(2.0%)of CoxB3,and 13(26.0%)of other pathogens.All the 26 EV71 strains were similar with the isolates from Zhejiang Province and Fuyang,Anhui Province in 2008,which belonged tO genotype Cl all the 10 CoxAl6 strains belonged to genetic lineages C.Conclusions The causative agents of HFMD are complicated.CoxA16 and EV71 are predominant among children with HFMD in Shanghai and neighbor areas in 2008,while the pathogens of some patients are still unknown.

Objective To test the levels of enterovirus 71 (EV71) antibody among children of different ages in Shanghai in 2011,and to investigate the relationship between antibody levels and virus infection.Methods EV71 antibody was detected by microneutralization assay from the serum specimens of healthy children of different ages collected during July to August,2011.The results were analyzed by t test for quantitative data with normal distribution,and by x2 test for count data.Results The positive rate of EV71 antibody among the 93 serum specimens was 58.1％ (54/93).The geometric mean titer (GMT) of EV71-specific neutralizing antibody was 1 ∶ 14.48.The positive rate of EV71 antibody in infants less than 6 months old was 87.5％ (21/24),and the GMT was 1∶29.56.In children aged 2 to 3 years,the positive rate of EV71 antibody decreased to 3.7％ (1/27),and GMT decreased to 1∶4.21,which were both statistically significantly lower than those less than 6 months old (x2 =36.37,t=7.58; both P＜0.01).The positive rate of EV71 antibody increased to 83.3％ (20/24) in children aged 5 to 6 years,with GMT reaching 1∶21.74.Whereas in children aged 7 to 8 years,the positive rate was 66.7％ (12/18) and GMT was 1∶20.76,without statistically significant difference compared with those aged 5 to 6 years (x2 =1.58,t=0.597; both P＞0.05).No statistically significant difference was found between boys and girls in positive rate of EV71 antibody [62.7 ％ (32/51) vs 52.4 ％ (22/42),x2 =1.02,P＞0.05] or GMT (1 ∶ 16.23 vs 1 ∶ 12.61,t=0.881,P＞0.05).Conclusions Children aged 2 to 3 years were at higher risk for EV71 infection,with EV71 antibody level significantly lower than other age groups.