Analysis of pathogen detection results of neonatal Echovirus 11 infection outbreaks in Guangdong Province in 2019 / 中华微生物学和免疫学杂志

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.

A human monoclonal antibody potently pan-neutralizes SARS-CoV-2 VOCs by targeting RBD invariant sites

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of novel corona virus disease (COVID-19). The neutralizing monoclonal antibodies (mAbs) targeting the receptor binding domain (RBD) of SARS-CoV-2 are among the most promising strategies to prevent and treat COVID-19. However, SARS-CoV-2 variants of concern (VOCs) profoundly reduced the efficacies of most of mAbs and vaccines approved for clinical use. Herein, we demonstrated mAb 35B5 efficiently neutralizes both wild-type (WT) SARS-CoV-2 and VOCs, including B.1.617.2 (delta) variant, in vitro and in vivo. Cryo-electron microscopy (cryo-EM) revealed that 35B5 neutralizes SARS-CoV-2 by targeting a unique epitope that avoids the prevailing mutation sites on RBD identified in circulating VOCs, providing the molecular basis for its pan-neutralizing efficacy. The 35B5-binding epitope could also be exploited for the rational design of a universal SARS-CoV-2 vaccine.

The adenosine analogue prodrug ATV006 is orally bioavailable and has potent preclinical efficacy against SARS-CoV-2 and its variants

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the COVID-19 pandemic, is rapidly evolving. Due to the limited efficacy of vaccination in prevention of SARS-CoV-2 transmission and continuous emergence of variants of concern (VOC), including the currently most prevalent Delta variant, orally bioavailable and broadly efficacious antiviral drugs are urgently needed. Previously we showed that adenosine analogue 69-0 (also known as GS-441524), possesses potent anti-SARS-CoV-2 activity. Herein, we report that esterification of the 5-hydroxyl moieties of 69-0 markedly improved the antiviral potency. The 5-hydroxyl-isobutyryl prodrug, ATV006, showed excellent oral bioavailability in rats and cynomolgus monkeys and potent antiviral efficacy against different VOCs of SARS-CoV-2 in cell culture and three mouse models. Oral administration of ATV006 significantly reduced viral loads, alleviated lung damage and rescued mice from death in the K18-hACE2 mouse model challenged with the Delta variant. Moreover, ATV006 showed broad antiviral efficacy against different mammal-infecting coronaviruses. These indicate that ATV006 represents a promising oral drug candidate against SARS-CoV-2 VOCs and other coronaviruses.

Viral infection and Transmission in a large well-traced outbreak caused by the Delta SARS-CoV-2 variant

We report the first local transmission of the SARS-CoV-2 Delta variant in mainland China. All 167 infections could be traced back to the first index case. Daily sequential PCR testing of the quarantined subjects indicated that the viral loads of Delta infections, when they first become PCR+, were on average [~]1000 times greater compared to A/B lineage infections during initial epidemic wave in China in early 2020, suggesting potentially faster viral replication and greater infectiousness of Delta during early infection. We performed high-quality sequencing on samples from 126 individuals. Reliable epidemiological data meant that, for 111 transmission events, the donor and recipient cases were known. The estimated transmission bottleneck size was 1-3 virions with most minor intra-host single nucleotide variants (iSNVs) failing to transmit to the recipients. However, transmission heterogeneity of SARS-CoV-2 was also observed. The transmission of minor iSNVs resulted in at least 4 of the 30 substitutions identified in the outbreak, highlighting the contribution of intra-host variants to population level viral diversity during rapid spread. Disease control activities, such as the frequency of population testing, quarantine during pre-symptomatic infection, and level of virus genomic surveillance should be adjusted in order to account for the increasing prevalence of the Delta variant worldwide.

Rapid evaluation of neutralizing antibodies in COVID-19 patients

The ongoing coronavirus disease 2019 (COVID-19) pandemic calls for a method to rapidly and conveniently evaluate neutralizing antibody (NAb) activity in patients. Here, an up-conversion phosphor technology-based point-of-care testing (UPT-POCT) and a microneutralization assay were employed to detect total antibodies against the receptor-binding domain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and NAb activity in COVID-19 patients sera, respectively, in order to determine if UPT-POCT could be used as a surrogate method for rapid evaluation of serum NAb activity in COVID-19 patients. In total, 519 serum samples from 213 recovered and 99 polymerase chain reaction re-positive (RP) COVID-19 patients were used in this report. We found that UPT-POCT reporting values correlated highly with NAb titers from 1:4 to 1:1024, with a correlation coefficient r = 0.9654 (P < 0.001), as well as protection rate against RP (r = 0.9886, P < 0.0001). As a significant point for reducing re-positive rate, UPT-POCT values of 4.380 {+/-} 2.677, corresponding to NAb titer of 1:64, may be appropriate as an indicator for evaluating high efficiency of protection. This study demonstrates that the quantitative lateral flow based UPT-POCT, could be used to rapidly evaluate NAb titer, which is of importance for assessing vaccine immunization efficacy, herd immunity, and screening patient plasma for high NAbs.

Viral RNA level, serum antibody responses, and transmission risk in discharged COVID-19 patients with recurrent positive SARS-CoV-2 RNA test results: a population-based observational cohort study

SummaryO_ST_ABSBackgroundC_ST_ABSManaging discharged COVID-19 (DC) patients with recurrent positive (RP) SARS-CoV-2 RNA test results is challenging. We aimed to comprehensively characterize the viral RNA level and serum antibody responses in RP-DC patients and evaluate their viral transmission risk. MethodsA population-based observational cohort study was performed on 479 DC patients discharged from February 1 to May 5, 2020 in Shenzhen, China. We conducted RT-qPCR, antibody assays, neutralisation assays, virus isolation, whole genome sequencing (WGS), and epidemiological investigation of close contacts. FindingsOf 479 DC patients, the 93 (19%) RP individuals, including 36 with multiple RP results, were characterised by young age (median age: 34 years, 95% confidence interval [CI]: 29-38 years). The median discharge-to-RP length was 8 days (95% CI: 7-14 days; maximum: 90 days). After readmission, RP-DC patients exhibited mild (28%) or absent (72%) symptoms, with no disease progression. The viral RNA level in RP-DC patients ranged from 1{middle dot}9-5{middle dot}7 log10 copies/mL (median: 3{middle dot}2, 95% CI: 3{middle dot}1-3{middle dot}5). At RP detection, the IgM, IgG, IgA, total antibody, and neutralising antibody (NAb) seropositivity rates in RP-DC patients were 38% (18/48), 98% (47/48), 63% (30/48), 100% (48/48), and 91% (39/43), respectively. Regarding antibody levels, there was no significant difference between RP-DC and non-RP-DC patients. The antibody level remained constant in RP-DC patients pre- and post-RP detection. Virus isolation of nine representative specimens returned negative results. WGS of six specimens yielded only genomic fragments. No clinical symptoms were exhibited by 96 close contacts of 23 RP-DC patients; their viral RNA (96/96) and antibody (20/20) test results were negative. After full recovery, 60% of patients (n=162, 78 no longer RP RP-DC and 84 non-RP-DC) had NAb titres of [≥]1:32. InterpretationRP may occur in DC patients following intermittent and non-stable excretion of low viral RNA levels. RP-DC patients pose a low risk of transmitting SARS-CoV-2. An NAb titre of [≥] 1:32 may provide a reference indicator for evaluating humoral responses in COVID-19 vaccine clinical trials. FundingSanming Project of Medicine in Shenzhen, China National Science and Technology Major Projects Foundation, Special Foundation of Science and Technology Innovation Strategy of Guangdong Province of China, and Shenzhen Committee of Scientific and Technical Innovation grants.

Genomic epidemiology of SARS-CoV-2 in Guangdong Province, China

COVID-19 is caused by the SARS-CoV-2 coronavirus and was first reported in central China in December 2019. Extensive molecular surveillance in Guangdong, Chinas most populous province, during early 2020 resulted in 1,388 reported RNA positive cases from 1.6 million tests. In order to understand the molecular epidemiology and genetic diversity of SARS-CoV-2 in China we generated 53 genomes from infected individuals in Guangdong using a combination of metagenomic sequencing and tiling amplicon approaches. Combined epidemiological and phylogenetic analyses indicate multiple independent introductions to Guangdong, although phylogenetic clustering is uncertain due to low virus genetic variation early in the pandemic. Our results illustrate how the timing, size and duration of putative local transmission chains were constrained by national travel restrictions and by the provinces large-scale intensive surveillance and intervention measures. Despite these successes, COVID-19 surveillance in Guangdong is still required as the number of cases imported from other countries is increasing. HighlightsO_LI1.6 million molecular diagnostic tests identified 1,388 SARS-CoV-2 infections in Guangdong Province, China, by 19th March 2020 C_LIO_LIVirus genomes can be recovered using a variety of sequencing approaches from a range of patient samples. C_LIO_LIGenomic analyses reveal multiple virus importations into Guangdong Province, resulting in genetically distinct clusters that require careful interpretation. C_LIO_LILarge-scale epidemiological surveillance and intervention measures were effective in interrupting community transmission in Guangdong C_LI

Comparison of SARS-CoV-2 infections among 3 species of non-human primates

COVID-19, caused by SARS-CoV-2 infection, has recently been announced as a pandemic all over the world. Plenty of diagnostic, preventive and therapeutic knowledges have been enriched from clinical studies since December 2019. However, animal models, particularly non-human primate models, are urgently needed for critical questions that could not be answered in clinical patients, evaluations of anti-viral drugs and vaccines. In this study, two families of non-human primates, Old world monkeys (12 Macaca mulatta, 6 Macaca fascicularis) and New world monkeys (6 Callithrix jacchus), were experimentally inoculated with SARS-CoV-2. Clinical signs were recorded. Samples were collected for analysis of viral shedding, viremia and histopathological examination. Increased body temperature was observed in 100% (12/12) M. mulatta, 33.3% (2/6) M. fascicularis and none (0/6) of C. jacchus post inoculation of SARS-CoV-2. All of M. mulatta and M. fascicularis showed chest radiographic abnormality. Viral genomes were detected in nasal swabs, throat swabs, anal swabs and blood from all 3 species of monkeys. Viral shedding from upper respiratory samples reached the peak between day 6 and day 8 post inoculation. From necropsied M. mulatta and M. fascicularis, the tissues showing virus positive were mainly lung, weasand, bronchus and spleen. No viral genome was seen in any of tissues from 2 necropsied C. jacchus. Severe gross lesions and histopathological changes were observed in lung, heart and stomach of SARS-CoV-2 infected animals. In summary, we have established a NHP model for COVID-19, which could be used to evaluate drugs and vaccines, and investigate viral pathogenesis. M. mulatta is the most susceptible to SARS-CoV-2 infection, followed by M. fascicularis and C. jacchus. One Sentence SummaryM. mulatta is the most susceptible to SARS-CoV-2 infection as compared to M. fascicularis and C. jacchus.

Identification of a common deletion in the spike protein of SARS-CoV-2

Two notable features have been identified in the SARS-CoV-2 genome: (1) the receptor binding domain of SARS-CoV-2; (2) a unique insertion of twelve nucleotide or four amino acids (PRRA) at the S1 and S2 boundary. For the first feature, the similar RBD identified in SARs-like virus from pangolin suggests the RBD in SARS-CoV-2 may already exist in animal host(s) before it transmitted into human. The left puzzle is the history and function of the insertion at S1/S2 boundary, which is uniquely identified in SARS-CoV-2. In this study, we identified two variants from the first Guangdong SARS-CoV-2 cell strain, with deletion mutations on polybasic cleavage site (PRRAR) and its flank sites. More extensive screening indicates the deletion at the flank sites of PRRAR could be detected in 3 of 68 clinical samples and half of 22 in vitro isolated viral strains. These data indicate (1) the deletion of QTQTN, at the flank of polybasic cleavage site, is likely benefit the SARS-CoV-2 replication or infection in vitro but under strong purification selection in vivo since it is rarely identified in clinical samples; (2) there could be a very efficient mechanism for deleting this region from viral genome as the variants losing 23585-23599 is commonly detected after two rounds of cell passage. The mechanistic explanation for this in vitro adaptation and in vivo purification processes (or reverse) that led to such genomic changes in SARS-CoV-2 requires further work. Nonetheless, this study has provided valuable clues to aid further investigation of spike protein function and virus evolution. The deletion mutation identified in vitro isolation should be also noted for current vaccine development.

Characterization analysis of gM, gL genes of varicella zoster virus in six provinces of China / 中华预防医学杂志

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.

Epidemiological characteristics of Coxsackie virus A16 caused hand foot and mouth disease cases in Guangdong province, 2012-2016 / 中华流行病学杂志

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.

Epidemiological characteristics of Coxsackie virus A16 caused hand foot and mouth disease cases in Guangdong province, 2012-2016 / 中华流行病学杂志

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.

Etiological characteristics of herpangina cases in Guangzhou city in Guangdong province, 2015 / 中华实验和临床病毒学杂志

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.

Epidemic and genetic characteristics of coxsackievirus A6 strains isolated in Guangdong province from year 2008 to 2013 / 中华微生物学和免疫学杂志

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.

Phylogenetic and molecular characterization of coxsackievirus A24 variant isolates from a 2010 acute hemorrhagic conjunctivitis outbreak in Guangdong, China.

BACKGROUND: Acute hemorrhagic conjunctivitis is a common disease in China. As a notifiable disease, cases are registered by ophthalmologists on the AHC surveillance system. An AHC outbreak caused by CA24v was observed in Guangdong Province in 2007 by the National Disease Supervision Information Management System. Three years later, a larger outbreak occurred in Guangdong during the August-October period (2010). To characterize the outbreak and compare the genetic diversity of CA24v, which was determined to be the cause of the outbreak, the epidemiology and the molecular characterization of CA24v were analyzed in this study. RESULTS: A total of 69,635 cases were reported in the outbreak. 73.5% of index cases originated from students, children in kindergarten and factory workers, with the ≦ 9 age group at the highest risk. The male to female ratio was 1.84:1 among 0-19 years. 56 conjunctival swabs were collected to identify the causative agent from five cities with the AHC outbreak. 30 virus strains were isolated, and two of the genomes had the highest identity values (95.8%) with CA24v genomes. Four CA24v genotypes were identified by phylogenetic analysis for the VP1 and 3C regions. CA24v which caused the outbreak belonged to genotype IV. Furthermore, full nucleotide sequences for four representative isolates in 2010 and 2007 were determined and compared. 20 aa mutations, two nt insertions and one nt deletion were observed in the open reading frame, with 5'- and 3'- UTR respectively between them. CONCLUSIONS: CA24v was determined to be the pathogen causing the outbreak and belongs to genotype IV. VP1 is more informative than 3C(Pro) for describing molecular epidemiology and we hypothesize that accumulative mutations may have promoted the outbreak.

Study on enterovirus infection in family close contacts of patients with hand, foot and mouth disease and the contamination of environment / 中华传染病杂志

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.

Genetic analysis of a partial VP1 region and molecular identification of non-EV71, non-CAl6 virus strains of hand, foot and mouth disease(HFMD) in Guangdong province between 2008 and 2009 / 中华微生物学和免疫学杂志

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.

The complete sequence analysis of Enterovirus 71 strain from the fatal case of the hand, foot and mouth disease during an epidemic of Guangdong province in 2008 / 中华微生物学和免疫学杂志

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.

The life cycle of SARS coronavirus in Vero E6 cells.

The aim of the study was to establish the life cycle of severe acute respiratory syndrome-associated coronavirus (SARS CoV) in host cells and determine the pathogenesis of SARS. Vero E6 cells (African green monkey kidney cells) were inoculated with SARS coronavirus for 3, 7, 24, 48, and 72 hr, respectively, and were observed under electron microscope. It was found that the SARS coronavirus entered the cells through membrane fusion instead of endocytosis, and then the nucleocapsids assembled in the RER and matured by budding into the smooth vesicles, which were derived from the Golgi apparatus. The smooth vesicles fused with the cell membrane, and the mature particles were released. A special phenomenon was that some virus-like particles appeared in the nucleus. We propose a scheme of the life cycle of SARS coronavirus and discuss the mechanism of its replication in Vero E6 cells.

Inhibiting severe acute respiratory syndrome-associated coronavirus by small interfering RNA / 中华医学杂志(英文版)

<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>