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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.
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Objective To analyze the epidemiological characteristics of hand foot and mouth disease (HFMD) cases caused by Coxsackie virus A16 (Cox A16) in Guangdong province from 2012 to 2016.Methods The data of mild HFMD cases caused by Cox A 16 were collected from 8 sentinel hospitals in 8 prefecture-level cities in Guangdong to estimate Cox A16 infection status and its population and time distribution characteristics.Results (1) The highest estimated incidence of Cox A16 infection was in 2014 (113.0/100 000),followed by 2016 (86.4/100 000) and 2012 (79.1/100 000),while the estimated incidence was lower in 2015 (29.0/100 000) and 2013 (28.8/100 000).(2) Cox A16 was confirmed to be the predominant pathogen causing HFMD outbreaks (54.6％,89/163).The number of outbreaks in the year with high incidence (28 outbreaks) was 11.2 times higher than that in the year with low incidence (2.5 outbreaks).(3) Across all age groups,the annual estimated incidence of Cox A16 infection decreased with age (trend x2=853 905.63,P＜0.01).The incidence was highest in age group 1 year (1 449.2/100 000),followed by that in age group 3 years (1 097.0/100 000),in age group 2 years (1 083.5/100 000),in age group 4 years (687.8/100 000) and in age group 0 year (604.9/100 000).Among the age groups ＜12 months,the estimated incidence increased with age (trend g2=5 541.77,P ＜ 0.01),which was highest in age group 11-months (2 105.1/100 000),followed by that in age groups 10-months (1 448.6/100 000),9-months (938.3/100 000),8-months (703.3/100 000) and 6-months (664.6/100 000).(4) The annual incidence peak was during May (143.9/100 000)-June (131.5/100 000).Conclusion The prevalence of Cox A16 infection differed with year in Guangdong during 2012-2016.When the incidence of Cox A16 infection was high,more outbreaks occurred.The prevalence occurred mainly in nurseries and kindergartens from May to June each year.Children aged 0-4 years were the high risk group for Cox A16 infection,children aged 6-11 months were at high risk for Cox A16 infection.
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Objective To analyze the epidemiological characteristics of hand foot and mouth disease (HFMD) cases caused by Coxsackie virus A16 (Cox A16) in Guangdong province from 2012 to 2016.Methods The data of mild HFMD cases caused by Cox A 16 were collected from 8 sentinel hospitals in 8 prefecture-level cities in Guangdong to estimate Cox A16 infection status and its population and time distribution characteristics.Results (1) The highest estimated incidence of Cox A16 infection was in 2014 (113.0/100 000),followed by 2016 (86.4/100 000) and 2012 (79.1/100 000),while the estimated incidence was lower in 2015 (29.0/100 000) and 2013 (28.8/100 000).(2) Cox A16 was confirmed to be the predominant pathogen causing HFMD outbreaks (54.6％,89/163).The number of outbreaks in the year with high incidence (28 outbreaks) was 11.2 times higher than that in the year with low incidence (2.5 outbreaks).(3) Across all age groups,the annual estimated incidence of Cox A16 infection decreased with age (trend x2=853 905.63,P＜0.01).The incidence was highest in age group 1 year (1 449.2/100 000),followed by that in age group 3 years (1 097.0/100 000),in age group 2 years (1 083.5/100 000),in age group 4 years (687.8/100 000) and in age group 0 year (604.9/100 000).Among the age groups ＜12 months,the estimated incidence increased with age (trend g2=5 541.77,P ＜ 0.01),which was highest in age group 11-months (2 105.1/100 000),followed by that in age groups 10-months (1 448.6/100 000),9-months (938.3/100 000),8-months (703.3/100 000) and 6-months (664.6/100 000).(4) The annual incidence peak was during May (143.9/100 000)-June (131.5/100 000).Conclusion The prevalence of Cox A16 infection differed with year in Guangdong during 2012-2016.When the incidence of Cox A16 infection was high,more outbreaks occurred.The prevalence occurred mainly in nurseries and kindergartens from May to June each year.Children aged 0-4 years were the high risk group for Cox A16 infection,children aged 6-11 months were at high risk for Cox A16 infection.
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Objective@#To analyze the genetic characterization of glycoprotein M(gM.),glycoprotein L(gL) of varicella zoster virus.@*Methods@#According to the program of "Ministry of Science and Technology of China" , Based on the 12 suspected VZV patients monitored in Beijing (1 case), Shanghai (5 cases), Jilin (2 cases), Qinghai (1 case), Guangdong (2 case) and Sichuan (case) in 2007-2015. A total of 12 Vesicle fluid and throat swab samples were collected. Positive samples were identified by Agarose gel electrophoresis and two glycoprotein genes were amplified by polymerase chain reaction (PCR). Nucleotide sequences were determined and analyzed by PCR amplification of VZV positive specimens V-OKA-BK of the domestic varicella attenuated live vaccine and the Varilrix-1 of the imported attenuated live vaccine. Nucleotide sequences of VZV positive specimens, vaccine strains (V-OKA-BK, varilrix-1) and GenBank foreign wild strains (41 strains), parent strains (P-oka), vaccine strains (V-oka, Varilrix, Varivax) were compared using BioEdit and MEGA 5.0.@*Results@#12 specimens were VZV positive. Compared with the vaccine strains and the parent strains, the GM gene of 1 positive specimen had radical mutation at 86686 sites, which resulted in amino acid mutation, 5 positive specimens had base mutation at 87844 sites, and 30 strains of foreign wild strains had the same variation at 87 844 sites. 1 positive specimens of gL gene in 101245 sites had base mutation, and led to amino acid mutation, 6 positive specimens at 101624, 101625, 101626 sites had base of loss and the foreign wild strains in these 3 sites had the same variation. Compared with the vaccine strains, the nucleotide and amino acid homology of gM of 12 VZV positive specimens were 99.2%-100% and 98.2%-100%, respectively, and gL of those were 99.3%-100% and 98.6%-100%, respectively. Compared with 41 strains of foreign wild strains, homology of gM's nucleotides and amino acid were 99.3%-100% and 98.5%-100%, respectively; 99.1%-100% and 98.6%-100% for gL. The results of phylogenetic analysis showed that 7 VZV positive samples were on the same branch with 4 vaccine strains and p-oka strain. Based on gL, 12 VZV positive samples were on the same branch as the vaccine strains and p-oka strain.@*Conclusion@#This study demonstrates that the genes of gM, gL are highly conserved and remain stable immunogen, which may be involved in the attenuation of VZV and need to be further researched.
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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.
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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.
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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.
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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.
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Objective To understand the genetic characteristics of Enterovirus 71 ( EVT1 ) circu-lating strains of Guangdong province in 2008. Methods We isolated an EV71 strain from the fatal case of the hand, foot and mouth disease during an epidemic of Guangdong in 2008. Its complete genome was se-quenced and analyzed comparatively. Results The results showed that the full length of EV71 GDFS-3 ge-nome( not including poly A tail ) is 7405 bp. No insertion or deletion is detected in the coding region. There are several insertions and deletions in 5'and 3'UTR. Phylogenetic analysis of GDFS-3 and reference strains showed GDFS-3 strain shares the highest nueleotide homology with TW984 strain(96.0% ) but low homology with SIN5865, MS and BrCr( about 81.0% ). GDFS-3 strain also shares the highest amino acid homology with TW984 strain(99.0% ). It clustered with reference strains of CA subgenotype in the phylogenetie tree. The nucleotide identity with CA reference strains is 91.0% -95.0%. Conclusion The phylogenetic analysis based on the entire genome demonstrates that GDFS-3 strain has the nearest genetic relationship with TW984 strains ( isolated in 2004). GDFS-3 may belong to the same subgenogroup ( CA ) with Taiwan predominant strains. Otherwise,Some mutations in 5'UTR of EV71 may play the very important role in heightened viru-lence.
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<p><b>OBJECTIVE</b>To evaluate the effectiveness of small interfering RNA (siRNA) on inhibiting severe acute respiratory syndrome (SARS)-associated coronavirus replication, and to lay bases for the future clinical application of siRNA for the treatment of viral infectious diseases.</p><p><b>METHODS</b>Vero-E6 cells was transfected with siRNA before SARS virus infection, and the effectiveness of siRNA interference was evaluated by observing the cytopathic effect (CPE) on Vero-E6 cells.</p><p><b>RESULTS</b>Five pairs of siRNA showed ability to reduce CPE dose dependently, and two of them had the best effect.</p><p><b>CONCLUSION</b>siRNA may be effective in inhibiting SARS-associated coronavirus replication.</p>
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Animals , Chlorocebus aethiops , RNA, Small Interfering , Pharmacology , Severe acute respiratory syndrome-related coronavirus , Transfection , Vero Cells , Virus ReplicationABSTRACT
Objective To provide scientific evidence to identify and confirm severe acute respiratory syndrome (SARS) by laboratory detection.Methods Multiple clinical specimens were collected serially and systematically from the 4 suspected SARS patients, which occurred between Dec.2003 to Jan.2004 in Guangdong Province. The samples were tested by serologic and molecular methods.Results IgM or IgG antibodies against SARS-CoV were detectable after 6—8 days of the onset in four patients. The four-fold or greater rising in antibodies was clearly detected in three of the four patients, while the fourth patient’s seroconversion was from negative to positive. The results analysed by enzyme-linked immunosorbent assay( ELISA), immunoflourescence assay (IFA), and neutralization test were highly correlated. SARS-CoV RNA was just detected in 3 throat swab specimens from case 1 by real-time PCR. M, N and S genes were amplified by reverse transcriptase polymerase chain reaction (RT-PCR) from the positive samples. Sequencing results showed that they were SARS-CoV gene segments, and most closely matched SARS-CoV gene sequences were isolated from civet cats in Guangdong Province. Nevertheless, SARS-CoV was not isolated from any samples of the 4 patients.Conclusion Based on these results, the 4 reported cases were laboratorily confirmed as SARS cases.
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AIM: To study the pathological change in mouse organs immunitied by inactivated SARS-CoV vaccine. METHODS: Inactivated SARS-CoV vaccine was injected into BALB/c and C57BL/6 mice. Anti-SARS antibody was analyzed by ELISA. After 8 weeks, the immunitied mice were killed and those organs were analyzed by pathological methods. RESULTS: Anti-SARS antibody in mice was positive after 8 days. Only minimal injury was observed in a few lungs and livers, but the other organs were not. CONCLUSIONS: Inactivated SARS-CoV vaccine induced mice to create antibody, whereas they did not cause severe injury. This result will be valuable for vaccine into clinical research. [
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Objective:To evaluate the immunogenicity of an experimental SARS-CoV inactivated vaccine.Methods:The virus suspension of F69 strain was inactivated with 0.4% formaldehyde and purified,then used as the immune antigen combined with Freund′s adjuvant.Eight adult New Zealand rabbits were immunized 4 times with this vaccine.12 sets of rabbit serum were sampled from the third day to 74th day after first immunization.Titers of specific IgG antibody and neutralizing antibody were determined by indirect ELISA and micro-cytopathic effect neutralizing test.Results:Rapid and potent humoral immune responses were induced by F69 inactivated vaccine in all eight immunized rabbits.Both specific IgG antibody and neutralizing antibody all peaked just with 2 vaccinations,with the maximum titer of 1∶81 920 and 1∶20 480 respectively about 6 weeks after first immunization.Across neutralizing reaction existed between F69 and Z2-Y3 strains.Conclusion:F69 inactivated vaccine owns strong immunogenicity.Similar antigenic epitopes exist between the F69 strain and Z2-Y3 strain,which ensured the cross neutralizing reaction.