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@#ObjectiveTo analyze the genome-wide characteristics of 17 strains of Coxsackievirus A6(CVA6)that cause hand,foot and mouth disease(HFMD)and herpangina(HA)in Yunnan Province in 2018,and understand the genetic differences between different pathogenic CVA6.MethodsA total of 1 909 stool samples clinically diagnosed as HFMD and HA in Kunming Children′s Hospital in 2018 were randomly selected for detection using enterovirus group A universal primers and screening of CVA6 positive samples. The CVA6 whole genome sequence was amplified with CVA6 whole genome primers,spliced by BioEdit splicting software,and analyzed for the whole genome characteristics by BioEdit,MEGA 7.0,Simplot,Heml 1.0 and Phyre2softwares.ResultsA total of 929 CVA6 positive samples were screened,and 17CVA6 complete gene sequences were obtained(9 of which were clinically diagnosed as HFMD and 8 were clinically diagnosed as HA). All 17 CVA6 strains were in type IV clade on the whole phylogenetic tree. No significant recombination occurred in HA and HFMD representative strains,while mutations occurred in non-structural protein 3D region. HFMD and HA representative strains showed differences in VP1 loci S597T,Q705L and Q663L. Online predictive analysis showed that the secondary structure of VP1 was consistent with that of CVA6 with no change.ConclusionThe 17 CVA6 strains causing HFMD and HA had high genomic homology,as well as nucleotide and amino acid differences,which may affect the replication and adaptability of CVA6.
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Objective To predict and analyze the physicochemical properties, structural characteristics, and antigenic epitopes of viral protein (VP) VP1 of Coxsackievirus A10 (CV-A10) by bioinformatics methods. Methods The physicochemical properties and structural characteristics of CV-A10 VP1 were predicted by ProtParam, SOPMA, SWISS-MODEL, PDBsum, and ProSA-web. The antigenic epitopes of CV-A10 VP1 were predicted and analyzed by DNAstar, ABCpred, Bepipred 2.0, ElliPro, DiscoTope-2.0, NetMHCpan-4.1, NetMHCIIpan-4.0, Consurf, VaxiJen v.2.0, AllerTOP v.2.0, ToxinPred2, and IEDB immunogenicity. Results Bioinformatics analysis showed that CV-A10 VP1 was a basic, unstable, and hydrophilic protein, of which the secondary structure mainly consisted of random coil. The analysis revealed that CV-A10 VP1 had multiple potential B and T cell antigenic epitopes as well as a dominant antigenic epitope based on the potential epitope. Conclusion CV-A10 VP1 has multiple potential sites that induce specific humoral and cellular immunity, providing important support for its experimental identification, molecular epidemiological studies, and vaccine development.
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Objective:To analyze the etiological distribution and phylogenetic characteristics of hand, foot and mouth disease (HFMD) in Qujing city of Yunnan Province in 2020.Methods:Stool samples were collected from HFMD cases in Qujing city in 2020 and virus RNA was extracted directly from treated stool suspensions. Virus VP4 gene sequences were firstly amplified using MD91/OL68-1 primer pairs and sequenced, then the virus serotypes were determined by BLAST search on the GenBank. Virus entire VP1 gene sequences were amplified and sequenced. Virus serotypes were identified online using Enterovirus Genotyping Tool Version 0.1. Sequences of reference virus genotypes/sub-genotypes were downloaded according to references. Phylogenetic trees were constructed by MEGA5.2 software and the genetic characteristics were analyzed.Results:A total of 47 strains of enteroviruses (EVs) were detected with a detection rate of 10.22% (47/460). The detected viruses were coxsackievirus A4 (CVA4, 0.65%, 3/460), CVA6 (7.83%, 36/460), CVA10 (0.87%, 4/460) and CVA16 (0.87%, 4/460). All were enterovirus species A (EVA), while other group viruses were not detected. The predominant virus was CVA6, accounting for 7.83% (36/460). EVA71 was not detected. CVA4 strains of C2 and C4 subgenotypes were co-circulating strains in Qujing city. CVA6 subgenotype D3a and CVA16 subgenotype B1a were also circulated in Qujing city. All CVA10 strains were in a separate lineage.Conclusions:Similar to the previous situation in China, the detection rates of EVA71 and CVA16 were very low, even zero. This study showed that CVA6 was the predominant virus, indicating a HFMD outbreak caused by CVA6 in Qujing city in 2020. The phylogenetic analysis showed CVA10 isolates belonged to a separate lineage, which might be unique to Qujing city. Laboratory and molecular epidemiological surveillance of non-EVA71 and non-CVA16 viruses, especially CVA6 and CVA10 should be strengthened in the future.
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Enterovirus 71 (EV71) infection is more likely to cause hand, foot and mouth disease (HFMD) in children, which can lead to neurogenic complications and higher mortality. As a commonly used clinical medicine, Reduning injection (RDN) helps to shorten the symptoms of patients with HFMD and facilitate the early recovery of children. However, the regulatory mechanism of RDN on the HFMD immune system disorder caused by EV71 remains to be discussed. This study collected detailed treatment data of 56 children with HFMD who entered the affiliated Children's Hospital of Nanjing Medical University during 2019. Retrospective analysis of clinical data showed that the symptoms of the RDN treatment group were improved compared with the untreated group. To explore its mechanism, the relevant detection indicators were detected by flow cytometry, enzyme-linked immunosorbent assay and real-time quantitative PCR. It was found that the number and function of innate immune (ILCs) and adaptive immunity (Th1, Th2 and secreted cytokines) were reduced, suggesting that RDN plays a role by regulating cellular immunity. The in vitro differentiation inhibition test further confirmed that RDN affected Th1 differentiation by inhibiting the expression of transcription factors on the basis of Th1 cell differentiation in vitro.
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To analyze the epidemiology,genetic variation and genetic evolution of coxsackievirus A4 (CVA4) in patients with hand,foot and mouth disease in Fujian,the virus isolates were molecular typed and amplified the whole VP1 region by RT-PCR,then the genetic variation and evolution were studied.The results showed that 33 CVA4 cases (8.1％) were confirmed from the 407 non-EV71 non-CVA16 HFMD cases in Fujian Province during 2011 and 2014,accounting for 31 cases in 2012 and 2 cases in 2014.Compared with common characteristics of the HFMD epidemic,no specificity in the distribution of CVA4 cases was found between gender and age groups.Sequence analysis of VP1 nucleotide sequences of Fujian CVA4 isolates showed that the nucleotide and amino acid sequences similarity were 92.6 ％-100 ％ and 95.7 ％-100 ％ respectively,low similarity with the prototype (83.7％-85.4％,96.1％-99.0％) and abroad isolates (82.1％-89.1％,90.4％-99.6％) both in nucleotide and amino acid sequences,high similarity with domestic isolates both in nucleotide and amino acid sequences,with the similarity of 87.9％ 99.2 ％ and 96.1％-100 ％.The results from phylogenetic tree showed that the genetic distance between Fujian CVA4 isolates and the prototype and abroad strains was far,and the genetic distance was close to domestic isolates in China.The distribution of the phylogenetic trees of Fujian CVA4 strains showed multiple branches.Therefore,CVA4 is the major HFMD associated-pathogen other than EV71,CVA 16,CVA6,and CVA10 in Fujian Province from 2011 to 2014.CVA4 strains from Fujian Province is co-circulating and co-evolving with other domestic isolates.There is existence of multiple closely related CVA4 transmission chains in various regions of Fujian.
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To analyze the epidemiology,genetic variation and genetic evolution of coxsackievirus A4 (CVA4) in patients with hand,foot and mouth disease in Fujian,the virus isolates were molecular typed and amplified the whole VP1 region by RT-PCR,then the genetic variation and evolution were studied.The results showed that 33 CVA4 cases (8.1％) were confirmed from the 407 non-EV71 non-CVA16 HFMD cases in Fujian Province during 2011 and 2014,accounting for 31 cases in 2012 and 2 cases in 2014.Compared with common characteristics of the HFMD epidemic,no specificity in the distribution of CVA4 cases was found between gender and age groups.Sequence analysis of VP1 nucleotide sequences of Fujian CVA4 isolates showed that the nucleotide and amino acid sequences similarity were 92.6 ％-100 ％ and 95.7 ％-100 ％ respectively,low similarity with the prototype (83.7％-85.4％,96.1％-99.0％) and abroad isolates (82.1％-89.1％,90.4％-99.6％) both in nucleotide and amino acid sequences,high similarity with domestic isolates both in nucleotide and amino acid sequences,with the similarity of 87.9％ 99.2 ％ and 96.1％-100 ％.The results from phylogenetic tree showed that the genetic distance between Fujian CVA4 isolates and the prototype and abroad strains was far,and the genetic distance was close to domestic isolates in China.The distribution of the phylogenetic trees of Fujian CVA4 strains showed multiple branches.Therefore,CVA4 is the major HFMD associated-pathogen other than EV71,CVA 16,CVA6,and CVA10 in Fujian Province from 2011 to 2014.CVA4 strains from Fujian Province is co-circulating and co-evolving with other domestic isolates.There is existence of multiple closely related CVA4 transmission chains in various regions of Fujian.
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Objective To learn the epidemiological characteristics of severe hand,foot,mouth disease (HFMD)in Fengtai District,and to provide the theoretical basis for the prevention and intervention of severe hand,foot,mouth disease cases. Methods Descriptive epidemiological analysis was conducted on the incidence data of severe cases of HFMD in Fengtai District,2010 -2015.Results The reported number of severe cases of HFMD was highest in 2010.The cases decreased year by year,and the annual incidence peak was present during June -August.The most cases were young children aged 1 -5 years. EV71 was the major pathogen,but other intestinal virus presented a rising trend.Cases were mainly floating population and presented regional distribution of small commodity wholesale market.Clinical features were mainly fever,rash,accompanied by more nervous system symptoms.The clinical symptoms include fever and rash usually accompanied by symptoms of the nervous system.Conclusion Severe cases presented a decline trend,and the proportion of other intestinal virus pathogen was increasing.More attention should be paid to other enterovirus infection of hand,foot and mouth disease among children.
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Objective To investigate the epidemiological and clinical characteristics of hand,foot and mouth disease(HFMD) complicated with central nerve system infection,in order to improve the diagnosis and treatment.Methods Total of 569 cases of HFMD complicated with central nerve system infection were enrolled.The results of pathogen detection and CSF examination were looked into,and epidemiological and clinical characteristics were analyzed retrospectively.Results HFMD of the central nervous system infections increased year by year,endanger the children's life,pathogen detection showed positive for enterovirus type 71 (640/1077,59.4％) and coxsackie virus infection in group A (158/1077,14.7％) ; Clinical manifestation:hand,foot and mouth,hips were rash (100.0％),fever(546/569,95.9.％),temperature higher than 38.5 ℃ (74.7％,425/569),there were various signs and symptoms of nervous system damage,sleepiness (187/569,32.9％),limb jitter(347/569,60.1％),easily frightened (405/569,71.2％),irritability (130/569,22.8％),convulsions (2.4％,14/569),vomiting (67/569,11.8％),choking cough (2.8 ％,16/569),irregular breathing (0.5 ％,3/569) ; Laboratory examination:the central nervous system complications in different CFPro (P =0.992),CFWBC (P =0.994) and CRP (P =0.786),no statistical difference in such aspects of brain stem encephalitis group in CFP(P =0.001),PWBC (P =0.000) and BG (P =0.000),and the differences were statistically significant; Short-term,the combination of large dose of gamma globulin and methylprednisolone had definite efficacy.Conclusion HFMD complicated with central nervous system infections have some epidemiological and clinical characteristics,early diagnosis and treatment can reduce the incidence of severe cases and mortality.
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Hand-foot-and-mouth disease (HFMD) is becoming one of the extremely common airborne and contact transmission diseases in Guangzhou, southern China, leading public health authorities to be concerned about its increased incidence. In this study, it was used an ecological study plus the negative binomial regression to identify the epidemic status of HFMD and its relationship with meteorological variables. During 2008-2012, a total of 173,524 HFMD confirmed cases were reported, 12 cases of death, yielding a fatality rate of 0.69 per 10,000. The annual incidence rates from 2008 to 2012 were 60.56, 132.44, 311.40, 402.76, and 468.59 (per 100,000), respectively, showing a rapid increasing trend. Each 1 °C rise in temperature corresponded to an increase of 9.47% (95% CI 9.36% to 9.58%) in the weekly number of HFMD cases, while a one hPa rise in atmospheric pressure corresponded to a decrease in the number of cases by 7.53% (95% CI -7.60% to -7.45%). Similarly, each one percent rise in relative humidity corresponded to an increase of 1.48% or 3.3%, and a one meter per hour rise in wind speed corresponded to an increase of 2.18% or 4.57%, in the weekly number of HFMD cases, depending on the variables considered in the model. These findings revealed that epidemic status of HFMD in Guangzhou is characterized by high morbidity but low fatality. Weather factors had a significant influence on the incidence of HFMD.
A doença de mão-pé-e-boca (HFMD) está se tornando doença extremamente comum transmitida pelo ar e contato em Guangzhou, sul da China, levando preocupação às autoridades de saúde pública acerca da sua incidência aumentada. Neste estudo foi usada parte ecológica e regressão binomial negativa para identificar o status epidêmico da HFMD e sua relação com variáveis meteorológicas. Durante 2008-2012 um total de 173.524 casos confirmados de HFMD foram apresentados, 12 com morte, elevando o índice de fatalidade a 0,69 por 10.000. As incidências anuais de 2008 a 2010 foram 60,56, 132,44, 311,40, 402,76 e 468,59 por 100.000, respectivamente, mostrando tendência de rápido aumento. Cada 1 °C de aumento da temperatura correspondeu a aumento de 9,47% (95% CI 9,36% a 9,58%) no número semanal de casos de HFMD, enquanto a 1 hPa de aumento da pressão atmosférica correspondeu a decréscimo no número de casos de 7,53% (95% CI - 7,60% a - 7,45%). De maneira semelhante cada aumento de 1% na humidade relativa correspondeu a aumento de 1,48% ou 3,3% e a um aumento de 1 metro por hora na velocidade do vento correspondeu a um aumento de 2,18% ou 4,57%, no número de casos semanais de HFMD, dependendo das variáveis consideradas no modelo. Estes achados revelaram que o status epidêmico do HFMD em Guangzhou é caracterizado por alta morbidade, mas baixa fatalidade. Fatores referentes ao tempo tiveram influência significante na incidência do HFMD.
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Adolescent , Child , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Male , Atmospheric Pressure , Hand, Foot and Mouth Disease/epidemiology , Weather , China/epidemiology , Humidity , Hand, Foot and Mouth Disease/etiology , Incidence , Risk Factors , Seasons , TemperatureABSTRACT
ObjectiveTo study the biological characterization and the genetic background of circulating CA16 strains in mainland of China for the purpose of CA16 vaccine development in the future.MethodsCA16 strains were isolated from throat swabs of patients with hand-foot-mouth disease and identified by neutralization assay and RT-PCR.The genotype of these isolates were determined by sequence alignment and phylogenetic analysis of VP1 gene.The proliferation dynamics and the plaque morphology were observed when propagated in Vero cells.The pathogenicity of these CA16 isolates was evaluated by challenging newborn mice.ResultsIn this study,six CA16 circulating isolates,BJ-1-6 were obtained.The RT-PCR products were 150 bp amplified with the general enterovirus primers and 210 bp with CA16 primers respectively,which cannot be amplified by EV71 primers.Additionally,these isolates were identified to display some obvious proliferation dynamics and plaque morphology when propagated for 96 h in Vero cells.The diameter of plaques were about 1.5 to 2 mm for BJ-1,BJ-2,BJ-4,BJ-6,4-5 mm for BJ-3 and 3 mm for BJ5,the plaques were regular except BJ-3.All the six isolates can be neutralized by the convalescent serum of patient infected with CA16.The virus titer of different isolates propagated for five passages in Vero cells was 7.0LgCCID50/ml.The sequence alignment of VP1 gene demonstrated that the genotypes of BJ-2,BJ-4,BJ5 were C1 and BJ-1,BJ-3,B J-6 were C,3 comparatively.The genetic distance of the VPI gene from theseisolates suggested that they were highly genetic identity with the homology of 90％ in nucleotide and 99％ in dedicated amino acid respectively.However,a distinctive difference in pathogenic ability in neonatal mice was found that the suckling mice challenged with BJ-3 & BJ-5 were paralyzed 4-5 d and dead 6-7d postchallenge,compared with the control group without any abnormality in the during of 14 d.ConclusionThe circulating CA16 isolates in China have different biological characteristics,different pathogenic ability and similar genetic backgrounds,which is helpful for the development of a CA16 vaccine in the future.
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Coxsackievirns A16(CVA16),together with enterovirus type 71(EV71),is responsible for most cases of hand,foot and mouth disease(HFMD) worldwide.Recent findings suggest that the recombination between CVA16 and EV71,and the co-circulation of these two viruses may have contributed to the increase of HFMD cases in China over the past few years.It is therefore important to further understand the virology,epidemlology,virus-host interactions and host pathogeuesis of CVA16.In this study,we describe the viral kinetics of CVAI6 in human rhabdomyosarcoma(RD) cells by analyzing the cytopathic effect(CPE),viral RNA replication,viral protein expression,viral RNA package and viral particle secretion in RD cells.We show that CVA16 appears to first attach,uncoat and enter into the host cell after adsorption for 1 h.Later on,CVA16 undergoes rapid replication from 3 to 6 h at MOI 1 and until 9 h at MOI 0.1.At MOI 0.1,CVA16 initiates a secondary infection as the virions were secreted before 9 h p.i.CPE was observed after 12 h p.i.,and viral antigen was first detected at 6 h p.i.at MOI 1 and at 9 h p.i.at MOI 0.1.Thus,our study provides important information for further investigation of CVA16 in order to better understand and ultimately control infections with this virus.
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The comparative analysis of the biological characterization and the genetic background study of EV71 circulating strains is commonly recognized as basic work necessary for development of an effective EV71 vaccine. In this study, we sequenced five EV71 circulating strains, isolated from Fuyang, Hefei, Kunming and Shenzhen city of China and named them FY-23, FY-22, H44, K9 and S1 respectively. The sequence alignment demonstrated their genotypes be C4. The genetic distance of the VP1 gene from these isolates suggested that they were highly co-related with genetic identity similar to other previously reported EV71 strains in China. Additionally, these strains were identified to display some obvious proliferation dynamics and plaque morphology when propagated in Vero cells. However, a distinctive difference in pathogenic ability in neonatal mice was found. Some differences in cross neutralization test & immunogenic analysis were also found. All these results are related to the biological characterization of circulating EV71 strains in China and aid in the development of an EV71 vaccine in the future.