RT-nPCR Assays for Amplification and Sequencing of VP1 Genes in Human Enterovirus A-D from Clinical Specimens / 生物医学与环境科学（英文）

Objective@#To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses (HEVs) from clinical samples and to contribute to etiological surveillance of HEV-related diseases.@*Methods@#A panel of RT-nPCR assays, consisting of published combined primer pairs for VP1 genes of HEV A-C and in-house designed primers for HEV-D, was established in this study. The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID @*Results@#The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID @*Conclusion@#This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A-D, providing rapid, sensitive, and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.

Genetic characteristics of human enterovirus A group 71 type in Xining City in 2017 / 中华实验和临床病毒学杂志

Objective@#To investigate the genetic characteristics of enterovirus A group 71 type (EV-A71) and etiological features of hand, foot and mouth disease (HFMD) in Xining city in 2017.@*Methods@#The pharyngeal swab specimens were collected from HFMD patients, and detected by real-time reverse transcription-polymerase chain reaction (RT-PCR). For EV-A71 positive samples, virus isolation was performed. Then RNA of EV-A71 strains was extracted, and then VP1 coding region was amplified by RT-PCR. The phylogenetic tree was constructed by comparing with other genotypes and sub-genotypes strains of EV-A71.@*Results@#Total 57 strains of EV-A71 were isolated in Xining city in 2017, which all belonged to genotype C4a, and could be divided into two different lineages by phylogenetic analysis. The epidemic strains of EV-A71 in Xining City was closely related to other provinces of China in 2017.@*Conclusions@#C4a was the dominant genotype of EV-A71 in Xining city in 2017, and no other genotype was detected. In addition, EV-A71 isolated from Xining city was co-evolved with EV-A71 in other provinces of China.

Post-marketing safety analysis of inactivated enterovirus A71 vaccines / 中华预防医学杂志

Objectives@#To evaluate the safety of inactivated enterovirus A71(EV-A71) vaccines after large-scale immunization in the community.@*Methods@#We selected EV-A71 susceptible people (healthy children) aged 6-59 months in vaccination clinics from 89 counties in Zhejiang Province between April 2016 and March 2018. All local and systematic adverse actions were collected by 30 min on-site inspection, within 3 days and 4-30 days follow-up. Chi-square test and Fisher′s exact test were used to compare the difference of AEs incidence in various characteristics among two groups.@*Results@#A total of 71 663 doses of vaccines were included for active safety analysis, which included 37 331 doses in boys and 34 332 doses in girls. Among all the doses, children aged 6 to 11 months, 12 to 23 months and 24 to 59 months were received 13 707, 32 639 and 25 317 doses respectively. The incidence of adverse reactions within 30 min, 3 days and 4-30 days were 0.33% (239 doses), 1.58% (1 133 doses) and 0.34% (244 doses) respectively. Adverse reactions within 3 days were 1 372 doses, with a incidence of 1.91%; among all the cases, 539 doses (0.75%) were grade 1, 677 doses (0.94%) were grade 2 and 156 doses (0.22%) were grade 3, no grade-4 adverse reaction was reported. The common local adverse reactions were redness, swelling and pruritus, with the incidence rates were 0.05% (39 doses), 0.02% (16 doses) and 0.02% (12 doses), respectively, while the most common systemic adverse reaction was pyrexia with an incidence of 1.19% (856 doses), followed by diarrhea and anorexia with the incidence rates were 0.15% (104 doses) and 0.13% (90 doses) respectively.@*Conclusion@#Most adverse actions of EV-A71 vaccines were mild and moderate and majority of them were common adverse actions. No new adverse reactions were found in the study.

Association between the single nucleotide polymorphism at locus 1165 of β1-adrenoceptor and infection by Enterovirus A71 / 中华实验和临床病毒学杂志

Objective@#To detect the single nucleotide polymorphism (SNP) at locus 1 165 of β1-adrenoceptor (β1-AR) and to investigate the association between the SNP and the infection by enterovirus A71(EV-A71).@*Methods@#Polymerase chain reaction (PCR) amplification technique was used to detect the SNP at locus 1 165 of β1-AR between hand, foot and mouth disease (HFMD) and healthy controls by sanger sequencing method .@*Results@#There was a G1165C SNP and three kinds of genotypes (GG, GC, CC) in β1-AR gene in the 77 cases of EV-A71 HFMD patients and 66 cases of healthy controls. For HFMD patients, frequencies of GG, GC and CC genotypes of the G1165C locus were 10%, 47% and 43%, respectively, and alleles frequency of G and C were 34% and 66%, respectively. But in healthy children, GG, GC, CC genotype frequencies were 7%, 41% and 52%, respectively, and G and C allele frequencies were 28% and 72% respectively. Chi-square analysis showed that there were no significant differences in distribution of genotypes (χ2=1.154, df=2, P=0.562) and alleles frequency (χ2=1.091, df=2, P=0.296) between the EV-A71-infected group and the healthy control group. Between mild and severe EV-A71-infected group, there were no significant differences in distribution of genotypes (χ2=3.945, df=2, P=0.139) and alleles frequency (χ2=3.763, df=2, P=0.052).@*Conclusions@#The 1 165 SNP in the coding region of β1-AR was not associated with EV-A71 infection and its severity.

Study on virulence related sites based on human enterovirus A71 genome / 中华实验和临床病毒学杂志

Objective@#To explore the virulence related risk factors based on the enterovirus A71 (EV-A71) genome.@*Methods@#The pairwise distance of each section of gene between mild and fatal cases was analyzed. The ⅴ domain of 5′UTR from mild and fatal cases in this study were constructed. Amino acid sequences of EV-A71 were analyzed to find the potential virulence regions which were statistically different between fatal and mild cases.@*Results@#The two EV-A71 genome sequences in this study belonged to C4a genotype with the genomic homology of 96.2%-97.5%. The nucleotides in the ⅴ domains of the 5 ′UTR of EV-A71 from mild and fatal cases were the same. Each gene of EV-A71 from 31 mild cases and 30 fatal cases shared high homology. A total of four potential virulence sites (2 A: R68 M、2C: K41R、3 A: T/V47 A and 3C: I158 V) which were significantly different between mild cases and fatal cases were obtained.@*Conclusions@#The four sites in the unstructured protein coding region might be related with the virulence of EV-A71.

Analysis of Coxsackie virus A6 and A10 detection in hand foot and mouth disease in Guiyang area during 2015 / 重庆医学

Objective To understand the enterovirus(EV) pathogenic composition of Coxsackie virus(CV)A6 and A10 causing hand foot and mouth disesse(HFMD) in Guiyang area during 2015 to provide a basis for the prevention and treatment of HFMD in local area.Methods The specific primers were respectively designed according to CVA6 and CVA10 sequence published in GenBank.The fluorescence quantitative RT-PCR method system was established.The gene sequencing method was used for conducting verification.Then this method was used to detect the clinical samples from 607 cases of HFMD.Results A total of 607 samples of suspected HFMD were detected,the overall positive rate was 59.47 ％ (361/607),in which EV71 accounted for 7.25 ％ (44/607),CVA16 for 11.37％(69/607),EV71+CVA16 double positive accounted for 0.16％ (1/607) and other EV for 40.69％ (247/607).The positive samples of CVA6,CVA10 and CVA6+CVA10 detected by the established real time fluorescence RT-PCR were 11 cases,71 cases and 1 case.Conclusion CVA6 and CVA10 are the main pathogens causing new onset HFMD in Guiyang area and the CVA10 monitoring should be strengthened.

Pattern Classification of Enterovirus 71-Associated Hand, Foot, and Mouth Disease in Chinese Medicine: A Retrospective Study in 433 Cases / 中国结合医学杂志

<p><b>OBJECTIVE</b>To determine whether patterns of enterovirus 71 (EV71)-associated hand, foot, and mouth disease (HFMD) were classified based on symptoms and signs, and explore whether individual characteristics were correlated with membership in particular pattern.</p><p><b>METHODS</b>Symptom-based latent class analysis (LCA) was used to determine whether patterns of EV71-HFMD existed in a sample of 433 cases from a clinical data warehouse system. Logistic regression was then performed to explore whether demographic, and laboratory data were associated with pattern membership.</p><p><b>RESULTS</b>LCA demonstrated a two-subgroup solution with an optimal fit, deduced according to the Bayesian Information Criterion minima. Hot pattern (59.1% of all patients) was characterized by a very high fever and high endorsement rates for classical HFMD symptoms (i.e., rash on the extremities, blisters, and oral mucosa lesions). Non-hot pattern (40.9% of all patients) was characterized by classical HFMD symptoms. The multiple logistic regression results suggest that white blood cell counts and aspartate transaminase were positively correlated with the hot pattern (adjust odds ratio=1.07, 95% confidence interval: 1.006-1.115; adjust odds ratio=1.051, 95% confidence interval: 1.019-1.084; respectively).</p><p><b>CONCLUSIONS</b>LCA on reported symptoms and signs in a retrospective study allowed different subgroups with meaningful clinical correlates to be defined. These findings provide evidence for targeted prevention and treatment interventions.</p>

ARRDC4 promotes EV71-triggered IL-6 production by enhancing the activation of NF-κB and MAPK signaling pathway / 军事医学

Objective To study the role of arrestin domain-containing protein 4( ARRDC4) in regulation of enterovirus 71(EV71) triggered innate IL-6 production and the underlying mechanism .Methods THP-1-derived macrophages (t-M?) were transfected with ARRDC4 specific siRNA and negative control siRNA .The expression and production of IL-6, replication and virus titer of EV71, and the activation of signaling pathway adaptors were analyzed with quantitative real -time PCR, ELISA and Western blot.Results Upon EV71 infection, ARRDC4 was upregulated.ARRDC4 silencing could enhance mRNA expression and production of IL-6, thus increasing the replication and virus titer of EV71.In ARRDC4 silenced t-M?, the activation of p-65,IκBα,ERK,JNK and p38 was promoted.Conclusion ARRDC4 promotes EV71-t riggered IL-6 production by enhancing the activation of NF-κB and MAPK signaling pathway to inhibit EV71 infection, contributing to positive regulation of anti-EV71 innate immune responses .

ARRDC4 promotes EV71-triggered IL-6 production by enhancing the activation of NF-κB and MAPK signaling pathway / 军事医学

Objective To study the role of arrestin domain-containing protein 4( ARRDC4) in regulation of enterovirus 71(EV71) triggered innate IL-6 production and the underlying mechanism .Methods THP-1-derived macrophages (t-M?) were transfected with ARRDC4 specific siRNA and negative control siRNA .The expression and production of IL-6, replication and virus titer of EV71, and the activation of signaling pathway adaptors were analyzed with quantitative real -time PCR, ELISA and Western blot.Results Upon EV71 infection, ARRDC4 was upregulated.ARRDC4 silencing could enhance mRNA expression and production of IL-6, thus increasing the replication and virus titer of EV71.In ARRDC4 silenced t-M?, the activation of p-65,IκBα,ERK,JNK and p38 was promoted.Conclusion ARRDC4 promotes EV71-t riggered IL-6 production by enhancing the activation of NF-κB and MAPK signaling pathway to inhibit EV71 infection, contributing to positive regulation of anti-EV71 innate immune responses .

Cell injury induced by 3D protein of different enterovirus 71 strains / 中华传染病杂志

Objective To compare the degree of cell injury induced by 3D protein (SDLY11 and SDLY107) of enterovirus 71 (EV71) strains.Methods EV71 strains SDLY11 and SDLY107 were respectively isolated from children with mild and severe hand foot mouth disease.The target genes 11-3D-Flag and 107-3D-Flag were amplified by reverse transcription-polymerase chain reation (RT-PCR) and inserted into the eukaryotic expression vector pcDNA3.1.The recombinant plasmids 11-3D-Flag-pcDNA3.1 and 107-3D-Flag-pcDNA3.1 were transformed into Escherichia.coli DH5α, respectively, and were identified by enzyme digestion and sequencing.The recombinant plasmids were transfected into rhabdomyosarcoma (RD) cells, respectively.Expression of 3D protein was detected by indirect immunofluorescence assay and western blot.Cell injury induced by 3D protein was detected with lactate dehydrogenase (LDH) test, cell proliferation was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenylthiazolium bromide (MTT) test, and cell apoptosis was detected with Annexin-V and PI.Multiple comparisons among groups were analyzed using LSD-t test if multiple sets of variables were consistent with homogeneity of variance.If not, Dunnett T3 test was used.Results The 1 400 bp fragments were amplified by reverse tramscription (RT)-polymerase chain reaction (PCR), and the recombinant plasmids were digested by enzyme and the 1 400 bp and 5 400 bp fragments were obtained and identified.Gene sequencing showed that the sequences were consistent with the target genes.The specific fluorescence was observed by indirect immunofluorescence assay, and the western blot showed that the molecular weight of the target protein was 55×103.The LDH test showed that the A490 of SDLY11 3D protein transfection group (0.790±0.048) was higher than that of SDLY107 3D protein transfection group (0.641±0.018).The difference was statistically significant (t=5.14, P<0.05).The cell membrane damage caused by SDLY11 3D protein was more severe than SDLY107 3D protein.The MTT test showed that the A570 of SDLY11 3D protein transfection group (1.028±0.020) was lower than that of SDLY107 3D protein transfection group (1.081±0.002), and the difference was statistically significant (t=3.31, P<0.05).The effect on cell proliferation activity of SDLY11 3D protein was greater than SDLY107 3D protein.The results of Annexin-V/PI showed that the percentage of apoptotic cells of SDLY11 3D protein transfection group and SDLY107 3D protein transfection group were (1.471±0.246)% and (1.465±0.237)%, respectively, and the difference was not statistically significant (t=0.04, P=0.973).Conclusions Compared with the SDLY11 3D protein, SDLY107 3D protein induces slighter cell injury, has weaker effect on cell proliferation activity, and is more favorable for virus replication in cells.

The pulmonary edema caused by enterovirus71 / 中国医师杂志

Enterovirus 71 (EV71) infection can cause hand,foot and mouth disease,due to its neurotropic nature,severe cases can affect the central nervous system and emerge neurogenic pulmonary edema (NPE),respiratory and circulatory failure,and even lead to death.The advances in the pathogenesis of severe HFMD neurogenic pulmonary edema after EV71 infection in recent years are described.

Neurologic Manifestations of Enterovirus 71 Infection in Korea

Enterovirus 71 frequently involves the central nervous system and may present with a variety of neurologic manifestations. Here, we aimed to describe the clinical features, magnetic resonance imaging (MRI) findings, and cerebrospinal fluid (CSF) profiles of patients presenting with neurologic complications of enterovirus 71 infection. We retrospectively reviewed the records of 31 pediatric patients hospitalized with acute neurologic manifestations accompanied by confirmed enterovirus 71 infection at Ulsan University Hospital between 2010 and 2014. The patients' mean age was 2.9 ± 5.5 years (range, 18 days to 12 years), and 80.6% of patients were less than 4 years old. Based on their clinical features, the patients were classified into 4 clinical groups: brainstem encephalitis (n = 21), meningitis (n = 7), encephalitis (n = 2), and acute flaccid paralysis (n = 1). The common neurologic symptoms included myoclonus (58.1%), lethargy (54.8%), irritability (54.8%), vomiting (48.4%), ataxia (38.7%), and tremor (35.5%). Twenty-five patients underwent an MRI scan; of these, 14 (56.0%) revealed the characteristic increased T2 signal intensity in the posterior region of the brainstem and bilateral cerebellar dentate nuclei. Twenty-six of 30 patients (86.7%) showed CSF pleocytosis. Thirty patients (96.8%) recovered completely without any neurologic deficits; one patient (3.2%) died due to pulmonary hemorrhage and shock. In the present study, brainstem encephalitis was the most common neurologic manifestation of enterovirus 71 infection. The characteristic clinical symptoms such as myoclonus, ataxia, and tremor in conjunction with CSF pleocytosis and brainstem lesions on MR images are pathognomonic for diagnosis of neurologic involvement by enterovirus 71 infection.

Hand, foot and mouth disease in yangpu district of shanghai: An analysis of epidemiologic characteristics in 2009-2013 / 第二军医大学学报

Objective To analyze the etiological and epidemiological characteristics of hand, foot and mouth disease (HFMD) in Yangpu District of Shanghai from 2009 to 2013, providing evidence for its prevention and control. Methods HFMD surveillance and report were done using the National Disease Supervision Information Management System. Descriptive epidemiological method was used to analyze the 2009-2013 epidemiologic characters and etiological characters (partial patients) of HFMD in Yangpu District, Shanghai. Nucleic acid of enterovirus (EV) genome was detected by real-time PCR in some patients. Results A total of 4 974 cases of HFMD were reported during 2009-2013 in Yangpu District, Shanghai, with the average annual incidence rate of 755.3 per million, with no death cases and 9 (0.18%) severe cases. The peak incidence was observed in the period of April to July. The epidemic peak had an increasing tendency and could be delayed. The incidence rates of HFMD were increased at an annual basis(Ptrend<0.05) in all communities and were significantly different between different communities (P<0.05). Most of the cases were less than 5-year-old scattered and childcare children. The main epidemic strains were EV71 and Cox A16 in 2010, and other enterovirus and Cox A16 in 2011-2013. The proportions of other enterovirus strains were increased, with overlapping phenomenon found in the pathogenic spectrum curves before and after the epidemic peak. Conclusion The 2009-2013 epidemiology of HFMD in Yangpu District of Shanghai had prominent seasonal, regional and population characteristics. Epidemic superiority strains vary annually and influence the epidemic trend and severity.

Etiology of hand foot and mouth disease in Guiyang area during 2012 / 重庆医学

Objective To understand the pathogen distribution situation among the children inpatients with hand ,foot and mouth diseases(HFMD) in Guiyang area during 2012 to provide the basis for the diagnosis ,treatment and prevention .Methods The data in 3 179 cases of HFMD were collected .The fluorescence quantitative RT‐PCR was adopted to perform the genotyping on universal enterovirus ,enterovirus 71(EV71)and Coxsackie virus A16(CA16) .Results A total 3 179 samples of HFMD were col‐lected ,among them ,151 cases (4 .75% ) were CA16 positive ,331 cases (10 .41% ) were EV71 positive ,7 cases (0 .22% ) were CA16 and EV71 co‐infection ,and 897 cases(28 .22% )were the other enterovirus .The whole year had 2 peaks of onset ,which were April to July and October to November .The onset age focused on the children aged under 5 years old (96 .16% ) ,among them ,0-3 years old had the highest onset ,moreover male children were more than female .Conclusion The etiology distribution of children HFMD in Guiyang area during 201 was dominated by the other genotypes of enterovirus and EV71 .

Application of combined detection of enterovirus nucleic acid and antibody in early etiological diagnosis for hand-foot-and-mouth disease / 中华检验医学杂志

Objective To assess the value of combined detection of enterovirus nucleic acid and antibody in early etiological diagnosis for hand-foot-and-mouth disease ( HFMD).Methods A case-control study was conducted.A total of 1 066 cases of children clinically diagnosed with HFMD from Hangzhou Children′s Hospital were involved into the research group from January to June 2014, consisting of 401 common cases and 665 severe cases; Throat swabs and serum samples from these children underwent combined detection for EV71/CA16/EV of enterovirus nucleic acid by fluorescence quantitative RT-PCR and for EV71/CA16-IgM by ELISA.All data were analyzed with SPSS 16.0.Results The total positive rate of enterovirus nucleic acid EV71/CA16/EV by fluorescence quantitative RT-PCR in the 1 066 cases of children clinically diagnosed with HFMD was 75.52%( 805/1 066 ) ( 95%CI: 72.80%-78.05%).But the total positive rate of combined detection was 91.46%( 975/1 066 ) ( 95%CI:89%.58-93.04%).The total positive rate of combined detection is higher than that of RT-PCR test(χ2 =98.338,P=0.000).The positive rate of EV71 type of combined detection was 64.63%(689/1 066)(95%CI:61.67%-67.49%),which is 15.38%higher than that of RT-PCR test 49.25%(525/1 066)(95%CI:46.21%-52.29%)(χ2 =51.453, P=0.000).In 665 severe cases of HFMD, the total positive rate of combined detection was 96.69%(643/665)(95%CI:94.95%-97.87%), which is higher than that of RT-PCR test 79.25%(527/665)(95%CI:75.92%-82.22%)(χ2 =95.607, P =0.000).In the severe cases, the positive rate of EV71 type of combined detection was 87.52%( 582/665 ) ( 95%CI:84.71%-89.89%) , which is 18.95% higher than that of RT-PCR test 68.57%(456/665) (95%CI:64.87%-72.06%) (χ2 =69.665, P=0.000).In the fatal cases, the positive rate of EV71 type of combined detection was 95.92%(94/98) (95%CI:89.28%-98.68%).Conclusions The combined detection of enterovirus nucleic acid and specific IgM antibody can significantly increase the positive rate of HFMD, especially for severe cases.The combine detection increases both the total positive rate and EV71 positive rate.Thus it has a high potential for becoming a new guidelines for laboratory diagnosis of HFMD.

Sequence analysis of VP1 and VP4 genes of enterovirus 71 strains isolated from severe and mild hanD-foot-mouth disease cases in Shanghai, China, 2011 / 中华传染病杂志

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.

Establishment and clinical application of triplex taqman probes real-time PCR for pathogenesis of hand foot mouth disease / 中华检验医学杂志

Objective To establish the triplex Taqman probes real-time RT-PCR method for simultaneously detecting of EV71,CA16 and EV.Methods Retrospective study.Specific primers and probes were designed based on conserved regions of EV71,CA16 and EV.The sensitivity,specificity and reproducibility were assessed by the optimized reaction system.A total of 176 throat swabs as the experimental group were collected from children with suspected hand foot mouth disease (HFMD),who admitted from April 2012 to July 2012 in Nanjing Children's Hospital affiliated to Nanjing Medical University.During this time,10 cases of healthy children,10 cases of outpatients with flu-like symptoms and 90 cases of inpatients in pneumology department of our hospital were recruited as control group,whose throat swabs were also collected.All of 286 samples were tested by the triplex Taqman probes real-time RT-PCR for simultaneously detecting EV71,CA16 and EV.SPSS13.0 was used to analyze the results.Results The sensitivities of the triplex Taqman probes real-time RT-PCR was 1.0 × 103 copies per milliliter for EV71,CA16 and EV.It showed 100％ specificity for 9 enterovirus and 3 non-enterovirus.Analysis with 1.0 × 103-1.0 × 105 copies per milliliter constructed plasmids demonstrated high reproducibility with coefficient of variation of 0.44％-1.04％ for EV71,0.38％-0.73％ for CA16,and 0.46％-0.90％ for EV.More over 176 samples collected from children with suspected HFMD were detected by triplex Taqman probes real-time RTPCR and real-time RT-PCR.The results showed 97.2％ (171/176)agreement and 0.94 Kappa value with high concordance.Conclusions The triplex Taqman probes real-time RT-PCR detecting EV71,CA16 and EV simultaneously has been established successfully.The assay,with high sensitivity and specificity,provide good basis for the rapid clinical diagnosis of EV71,CA16 and EV and open up broad prospects for clinical and relevant researches.

Risk Factors for Neurologic Complications of Hand, Foot and Mouth Disease in the Republic of Korea, 2009

In 2009, the first outbreak of hand, foot and mouth disease (HFMD) or herpangina (HP) caused by enterovirus 71 occurred in the Republic of Korea. This study inquired into risk factors associated with complications of HFMD or HP. A retrospective medical records review was conducted on HFMD or HP patients for whom etiologic viruses had been verified in 2009. One hundred sixty-eight patients were examined for this investigation. Eighty patients were without complications while 88 were accompanied by complications, and 2 had expired. Enterovirus 71 subgenotype C4a was the most prevalent in number with 67 cases (54.9%). In the univariate analysis, the disease patterns of HFMD rather than HP, fever longer than 4 days, peak body temperature over 39degrees C, vomiting, headache, neurologic signs, serum glucose over 100 mg/dL, and having an enterovirus 71 as a causative virus were significant risk factors of the complications. After multiple logistic analysis, headache (Odds ratio [OR], 10.75; P < 0.001) and neurologic signs (OR, 42.76; P < 0.001) were found to be the most significant factors. Early detection and proper management of patients with aforementioned risk factors would be necessary in order to attain a better clinical outcome.

Survey on the enterovirus 71 survival ability on different surfaces under different climate / 中华传染病杂志

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.

Survivability of hand foot mouth disease virus in tap water / 中华传染病杂志

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.