Objective To discuss the prevalence of non-EV71,non-CA16 virus strains of hand,foot and mouth disease(HFMD) in Guangdong province between 2008 and 2009,and analyze the genetic evolution of these non-EV71,non-CA16 virus strains.Methods Isolated viruses from stool samples collected from outpatient and in-patient cases of HFMD between 2008 and 2009 by human rhabdomyosarcoma(RD) cell and HEp-2 cell,cultures that exhibited a characteristic enterovirus cytopathic effect were evaluated by RT-PCR.Those strains which identified non-EV71,non-CA16 were analyzed by VP1 sequencing and then were identified by BLAST program.A phylogenetic tree was constructed using the Neighor-Joinning method in the MEGA 4.0 software.Results Twenty-two virus strains of non-EV71,non-CA16 were obtained,and nine of the twenty-two virus strains in 2008 were classified into CA2,CA4,and CB3 by BLAST; thirteen of the twenty-two virus strains in 2009 were classified into EV80,Echo13,Echo30,CBS,Echo24,CA10,CA6,and poliovirus 1 by BLAST.The honology of all strains was low,and all the strains belonged to CA,CB,Echoviruses,Enterovirus and poliovirus subgroup.Conclusion Except for EV71 and CA16 was a major causative agent in prevail of HFMD in Guangdong province between 2008 and 2009,there also existed other subgroup Enterovirus.The other twenty-two strains respectively belonged to CA,CB,Echoviruses,Enterovirus and poliovirus subgroup,and none of those strains was predominant.Muti-species Enterovirus occurred concomitantly.

Objective To study the epidemic and genetic characteristics of coxsackievirus A6 ( CVA6) strains isolated in Guangdong province.Methods Enterovirus strains positive for neither entero-virus A71 ( EV71) nor CVA16 were isolated from Guangdong province during 2008 to 2013 to screen CVA6 isolates by real-time PCR.The entire sequences of viral genes encoding VP1 of CVA6 positive samples were amplified and sequenced.The phylogenetic analysis was performed to analyze the full-length gene sequences encoding VP1 of CVA6 isolates and sequences downloaded from GenBank by using DNAStar6.0 and MEGA5.2 software packages.Results CVA6 strains accounted for 61.4%of the 1672 non-EV71 and non-CVA16 enterovirus strains isolated in Guangdong province during year 2008 to 2013.The positive rates were respectively 10.5%(4/38), 66.7%(34/51), 36.2% (81/224), 63.0% (182/289), 62.3% (325/522) and 73.0%(400/548) from 2008 to 2013 and the differences among different years were significant (χ2=133.79, P<0.01).The CVA6 isolates could be classified into four clusters in the phylogenetic tree, designated A, B, C and D (including D1, D2 and D3 subgenogroups) genogroups.The four clusters shared nucleotide diversity ranging from 15.5% to 23.1%.The CVA6 strains isolated in Guangdong province shared 88.7%-100.0% homologies in nucleotide and 95.7%-100.0% in amino acid.Subtype D2 strains circulated during 2008 to 2012 and subtype D3 strains circulated during 2009 to 2013.Conclusion CVA6 strains were the predominant enterovirus strains among non-EV71 and non-CVA16 enterovirus strains circula-ted in Guangdong province from year 2008 to 2013.The CVA6 isolates could be classified into A, B, C and D genogroups based on the sequence analysis of VP1 region.Subgroups D2 and D3 isolates were identified and the subgroup D3 isolates were the prevalent strains in Guangdong.

Objective To understand the enterovirus infection in family close contacts of patients with hand,foot and mouth disease (HFMD) and the contamination of environment.Methods Forty-one HFMD cases confirmed by laboratory from web-based surveillance system during July to August 2010 in Guangdong Province were selected.All members of the cases′ family were investigated by collecting their information on demography,habit of domestic hygiene and hygiene status in household.The stool samples of all members and the smear samples from the surface of family belongings from 16 families were collected and the enterovirus was detected by real time quantitative polymerase chain reaction.The data were analyzed by chi square teat and t test.ResultsForty-one HFMD cases′ families and 135 close contacts were included in this survey.The infection rate of the enterovirus was 39.2％ (53/135) in all close contacts.Of all the investigated families,the infectionrate was 58.5％ (24/41) in family with one or more close contacts and 9.8％ (4/41) in family with all close contacts.The differences of infection rates of enterovirus among the members of parents (32.5％,25/77),grandparents/aunts/ uncles (43.3％,13/30) and cousins (53.6％,15/28) didn′t show statistical significance (χ2 =4.07,P=0.131).The infection rate of enterovirus in close contacts from family with more than 5 members was higher than that from family with 4 or less members (OR=1.4,95％CI 1.1-1.9).Among 135 close contacts,27.4％ (37/135) were infected with the same types of entervirus as that of HFMD case in the family and 11.9％ (16/135) were infected with the different virus types.In 33 family belongings samples from 16 families,the positive rate of enterovirus detection was 6.1％ (2/33).Between 17 families with enterovirus testing negative and 23 families with enterovirus testing positive in close contacts,there were no statistical differences of the family hygiene status,hand-washing of babysitter,disinfection of tableware and drinking,sharing towels,airing bedding articles and toy cleaning (P＞0.05).ConclusionsThe infection rate of enterovirus in close contacts of HFMD cases is high and the enterovirus contamination exists in case family environment.Management of close contacts of HFMD cases and disinfection of the family environment are important in HFMD controls.

Objective:To analyze the evolutionary characteristics and variation of etiological agent in an acute hemorrhagic conjunctivitis (AHC) outbreak in a city of Guangdong province in May, so as to provide scientific basis for formulating a new round of measures for prevention and control of AHC epidemic.Methods:In this study, 20 conjunctival swabs were collected from AHC patients, and enterovirus, human enterovirus 70 (HEV70) and coxsackievirus A 24 variant (CVA24v) nucleic acids were detected by real-time fluorescence quantitative PCR. In addition, the VP1 and 3Cpro regions of the CVA24v positive samples were sequenced to analyze their evolutionary relationship with the CVA24v strains circulating in China and abroad.Results:All the 20 eye swab samples were EV-positive, and CVA24v-positive, with a positive rate of 100.00%, and all were HEV70-negative.The genomes of CVA24v in VP1 and 3Cpro regions of CVA24v in 5 and 7 samples were successfully sequenced. Based on molecular characterization analysis of VP1 and 3Cpro regions, it was found that the CVA24v isolated in this outbreak had the greatest nucleotide similarity with the CVA24v strains isolated in Thailand in 2014 and French Reunion Islands in 2015. The phylogenetic analysis of 3Cpro and VP1 regions showed that the CVA24v isolated in this outbreak is clustered together with the CVA24v that was prevalent in Thailand in 2014 and the French Reunion Islands in 2015, and have high affinity. Compared with CVA24v isolated in Guangdong in 2010, Thailand in 2014, and French Reunion Islands in 2015, CVA24v isolated in this outbreak was replaced at 4 amino acid sites in 3Cpro region and 1 amino acid site in VP1 region.Conclusions:The cause of this outbreak is enterovirus CVA24v, which has the highest similarity to CVA24v isolated in Thailand in 2014 and in the French Reunion Islands in 2015. There were new amino acid mutations in both 3Cpro and VP1 regions.

Objective:To analyze the laboratory test results of two outbreaks of neonatal enterovirus infections in Guangdong Province in 2019 and the genetic characteristics of Echo11, aiming to provide reference for the prevention and control of neonatal enterovirus infections.Methods:The pathogenic specimens of neonatal cases suspected of enterovirus infection were collected. Fluorescence quantitative PCR and sequencing were used for enterovirus typing and identification, and virus isolation was carried out for positive specimens.The complete sequences of VP1 of Echo11 were amplified and sequenced and the phylogenetic analysis was performed using the bioinformatics software such as Danstar6, Bioedit7.09 and MEGA6.06.Results:A total of 93 specimens from 36 neonatal cases were collected. After identification, 55 specimens from 24 cases were positive for enterovirus, of which 23 cases were positive for Echo11 and one case was positive for Coxsackievirus B4(CVB4). A total of 29 enterovirus strains were isolated from the specimens of 19 cases, of which 28 were Echo11 from 18 cases, and one was CVB4. Phylogenetic analysis revealed that the nucleotide homology between the 18 strains of Echo11 in this study was 98.2%-100.0%, and the nucleotide homology between the Echo11 strains causing the two neonatal infections was 99.7%-100.0% and 99.8%-100.0%, respectively. Echo11 could be divided into six genotypes as A, B, C, D, E and F, in which genotype A and genotype C were further divided into A1-5 and C1-4, and genotype D could be divided into D1-5. The 18 strains of Echo11 in this study were all subtype D5.Conclusions:In 2019, two outbreaks of neonatal infections in medical institutions in Guangdong Province were caused by Echo11, which belonged to the genotype D5.

Objective:To study the epidemiological and genetic characteristics of coxsackievirus B3 (CVB3) circulating in Guangdong Province from 2008 to 2021.Methods:This study collected the specimens of hand, foot, and mouth disease (HFMD) cases from 2008 to 2021 that were positive for other enteroviruses except for enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and CVA6, as well as the specimens of herpangina and neonatal infection cases from 2020 to 2021. Enteroviruses in these specimens were detected and their types were identified. CVB3 strains were isolated and the entire VP1 sequences of CVB3 strains were amplified and sequenced. The genetic features of CVB3 strains were analyzed using DNAStar 7.1 and MEGA 6.06 software packages.Results:Among 3 484 HFMD cases positive for other enteroviruses from 2008 to 2021, CVB3-positive cases accounted for 1.6% (57/3 484); among 560 cases of herpangina from 2020 to 2021, CVB3-positive cases accounted for 2.1% (12/560); one neonatal infection case in 2021 was positive for CVB3. CVB3-positive cases accounted for 67.1% (47/70) in 2021 and 18.6% (13/70) in 2020, while there were less than five cases in other years. Forty-eight CVB3 strains were isolated and the entire VP1 sequences of 26 CVB3 strains were obtained. Phylogenetic analysis indicated that the CVB3 strains could be divided into eight genotypes (A-H) and the strains of genotypes A, D and E were prevalent in the Chinese mainland. The 26 CVB3 strains isolated in Guangdong Province shared 80.2%-100.0% nucleotide homology, and belonged to two genotypes of D and E, with genotype D prevalent from 2008 to 2017 and genotype E prevalent from 2020 to 2021.Conclusions:CVB3 is prevalent sporadically in Guangdong Province from 2008 to 2017, but the epidemic intensity increased during 2020 and 2021. CVB3 strains of genotypes D and E are prevalent in Guangdong Province during 2008 to 2021, with genotype E being the prevalent genotype during 2020 and 2021.

Objective: To analyze the etiological of herpangina(HA) in Guangzhou City in 2015, and to provide laboratory data for the epidemic control. Methods: Two hundred and eleven herpangina samples (stool and throat swab) were collected.Real-time (RT)-PCR and semi-nested (Sn)-PCR assays were performed to detect human enteroviruses (HEVs)-positive samples. The human rhabdomyosarcoma (RDa) cell lines were used to inoculate virus from HEVs-positive samples. The entire sequences of viral genes encoding VP1 of CVA6 positive samples or strains were amplified and sequenced. The phylogenetic analysis was performed to analyze the full-length gene sequences encoding VP1 of CVA6 by using DNAStar6.0 and MEGA5.2 software packages. Results: According to the laboratory test results, 115 cases were HEVs-positive and positive rate was 93.50%, eight serotypes of EV including CVA6, CVA10, CVA2, EV71, CVA16, CVB2, Echo14 and Echo30 were detected.The CVA6 positive rate was the highest with a percentage of 60.98%, followed by CVA10 with a percentage of 13.01%. The enterovirus positive rate of stool samples (χ²=29.88, P<0.01) and viruses isolated positive rate (χ²=8.67, P<0.01) were higher than that in throat swab samples. Phylogenetic analysis showed that all CVA6 strains detected in this study belonged to D3 subgenotype, and shared 96.9%-99.9% homologies in nucleotide and 99.0%-100.0% in amino acid. Conclusions CVA6 of the enterovirus A group accounted for the main pathogen of herpangina in Guangzhou City in Guangdong province in 2015, which belonged to D3 subgenotype.