Multiple Genotypes Of Echovirus 11 Circulating In Mainland China And Research On Biosafety Of Enteroviruses Laboratory

Background:The enterovirus(EV)belongs to the genus Enterovirus in the picornavirus family of the small RNA virus.At present,there are 116 serotypes of EV,which belong to 15 groups:EV-A to EV-L,and rhinovirus A-C group.EV as non-enveloped virus,stable in acidic environment,sensitive to ultraviolet light.Currently,EV inactivation methods commonly used in laboratories include thermal inactivation,ultraviolet irradiation,chlorine disinfectant and formaldehyde fumigation.As a more frequently isolated EV,E-11 has a wide spectrum of diseases:from mild non-specific symptoms to systemic diseases such as rash,fever,hand foot and mouth disease(HFMD),uveitis and severe neurological diseases,including aseptic meningitis,encephalitis,and acute flaccid paralysis(AFP).In particular,it causes the occurrence of neonatal sepsis-like disease and multisystem hemorrhagic disease,and its high mortality and mortality cause social panic.Through literature review,research on the epidemiology and genetic characteristics of E-11 and evaluationis of EV inactivation still scarce.The only studies of E-11 are limited in geographical distribution and time span.And the study of epidemiology and genetic characteristics is still blank.The laboratory has conducted related research on the E-11 strain isolated from the Tibet Autonomous Region in 1999 and the AFP cases in Shandong Province in 1999-2003.Based on the HFMD network laboratory monitoring technology platform,this study systematically studied the epidemiology,genetic characteristics and genetic evolution of the E-11 strain isolated from mainland China;For the first time in the EV Biosafety Laboratory-2(BSL-2),a systematic evaluation of the common inactivation methods of EV was carried out.Objective:To elucidate the epidemic law of E-11 in China,establish the method for determining the full-length and whole genome sequence of E-11 VP1,update the E-11 genotyping results based on the full-length of VP1,systematically describe the distribution and epidemic patterns of E-11 strains of different genotypes globally and nationally,and improve their understanding of genetic recombination and genetic evolution,attempt to analyze the relationship between different genotypes of E-11 and disease types,and provide basic data for further research of E-11,scientific basis for early warning,prevention and control of related diseases;Systematic evaluation of the inactivation effect of EV common inactivation methods,establishment of BSL-2 laboratory surface disinfection technology to ensure the safety of experimental operators,and provide reliable data for biosafety management.Methods:Based on the China AFP and HFMD network laboratory monitoring technology platform established by the Center for Disease Control and Prevention of the Chinese Center for Disease Control and Prevention,a total of 59 cases of AFP and HFMD caused by E-11 in China from 1999 to 2017 were collected.The nucleotide sequence of the full-length VP1 coding region was determined,and the full-length sequences of 500 E-11 VP1 coding regions in the GenBank database were collected and screened,and the data set of the full-length sequence of the E-11 VP1 region was established.The E-11 strain selected by the HFMD network laboratory detection platform was selected for genome-wide sequencing,and its gene sequence,amino acid variation,recombination and evolutionary rules were systematically analyzed using Sequencher,MEGA,Bioedit,and Simplot software;Using the cell culture technique,the inactivated virus was inoculated into the cells for virus titer experiments,and the cytopathic effect(CPE)was observed,and the TCID50 of the inactivated EV was calculated by the Reed-Muench method to evaluate the heat inactivation and the inactivation effect of commonly used disinfectants.Results:1.As of December 5,2018,a total of 559 nucleotide sequences of E-11 VP1 region were collected,including 33 countries separated from six continents,with a time span of 1953-2014.The full-length sequence of E-11 VP1 region of 11 disease types was 359 strains and the full-length nucleotide sequence of 200 E-11 VP1 regions isolated from 12 provinces(autonomous regions)in China from 1994 to 2017.Among them,59 were isolated from our laboratory:41 strains were derived from the AFP network laboratory monitoring technology platform in preliminary research,and 18 strains were derived from the HFMD network laboratory monitoring platform.Four of the eighteen E-11 strains from HFMD were severe HFMD,which were isolated in 2012 in Guangdong Province,2010 in Hunan Province,Hainan Province and 2017 in Hebei Province.2.According to the full length of the nucleotide sequence of VP1 region,E-11 is divided into six genotypes of A-F,genotypes A,C and D can be further divided into A1-A5,C1-C4 and D1-D5 genotypes.Genotype F is a newly generated genotype in the evolution of E-11 that proposed in this study,the detection of D5 sub-genotype in HFMD is the first report of mainland China.3.In addition to mainland China,359 E-11 strains from 33 countries on six continents isolated during 1953-2014 were identified and distributed in A4-A5,B,C2,C4,D1-D5 and F(sub)genotypes after genotyping.The sub-genotype D5 is a dominant sub-genotype of abroad which was prevalent in 1998-2014.The sub-genotype D5 was firstly isolated in Tunsia in 1998.4.After the classification of 200 E-11 strains isolated from China in 1994-2017,they were distributed in sub-genotype A1,A2,A3,B,C1,C3,D5 and E genotypes.The sub-genotype A1 was the predominant sub-genotype in mainland China during the period of 2008-2017.The sub-genotype A1 has been isolated from AFP cases for the first time since 2006,and has been in persistent epidemics until 2017.5.Seventeen E-11 strains of HFMD-derived full-length sequences were analyzed according to different genotypes and different disease types.The recombination patterns of sub-genotype D5 were the same between mild and severe cases,which showed recombination with EV-B100 and EV-B111 in the 3C region,and recombination with EV-B86 in the 3D region.The sub-genotype A1 of mild cases showed the same recombination pattern with sub-genotype D5.The total 3 severe sub-genotype A1 have different recombination pattern,one with HaN-2010-165 and HuN-2010-112 represent the performance of 5'-UTR and EV-B98 recombination,and the other is represented by GD-2012-34,which is represented by 5'-UTR with EV-B74 and 3D with EV-B86.In addition to recombination with the EV-B prototype strain,the Chinese mainland E-11 isolate was recombined with other EV-B epidemic strains that have been popular in recent years,and recombination activities are frequent.6.The heat inactivation method showed that,the inactivation logarithm of EV-A71 virus was more than 6 after heated 56? for 30 min,and inactivation at 65? for 30 min,70? for 10 min,no CPE observed.The multiple linear regression equations of inactivation temperature and treatment time were established.The results showed that for every 10 ? increase in temperature,the EV-A71 virus titer decreased by about 1.7 TCID50,and the EV-A71 virus titer decreased about every 10 minutes of inactivation time.0.2 TCID50;The evaluation of the inactivation effect of the chemical disinfectant showed that the trichloroisocyanate effervescent tablet,the virkon disinfectant powder and the new disinfectant hypochlorous acid molecule can effectively inactivate the EV-A71 virus.Conclusion:1.re-organizing the full-length sequence of the VP1 coding region of the E-11 strain available worldwide,it can be divided into six A-F genotypes,of which the F genotype is the first reported new genotype,isolated in the landlocked countries in the midwest Africa;the E-11 strain of the sub-genotype D5 was first reported in Chinese HFMD cases.2.The 200 E-11 strains isolated from mainland China in 1994-2017 could be classified into A1-A3,B,C1,C3,D5,E genotypes,and multiple genotype of E-11 circulated in mainland China,among them,A1-A3 circulated domestically in mainland China,and has no reporte in other countries.3.The genotype distribution of 200 E-11 strains according to time in mainland China showed two distinct changes.The first time was in the year of 2000,the persistent circulated sub-genotype was changed from C1 to A2.The second time was in the year of 2008,swith sub-genotype A2 to A1;Sub-genotype A1 was the absolute dominant sub-genotype of the E-11 strain in China during 2008-2017.4.The genotype distribution of E-11 strains isolated from other countries except mainland China changed significantly around 1998:from sub-genotype D4 to D5.And D5 was the absolute dominant sub-genotype outside China during 1998-2014.5.This study suggests that the E-11 strain derived from HFMD in mainland China is recombined with other EV-B enteroviruses in the 5'-UTR and 3D regions.The sub-genotype Al1 has more recombinant methods than the sub-genotype D5,may play an important role in the process of becoming a dominant sub-genotype;the HFMD-derived E-11 strain and other popular EV-B epidemic strains in recent years have frequent recombination activities,some were isolated in outbreak event.Its occurrence has become a potential hidden danger in its future disease outbreak.6.The inactivation effect of thermal and chemical disinfectants on EV-A71 is related to inactivation temperature,disinfectant concentration and treatment time.Studying the inactivation curve of chemical disinfectant on EV-A71 is of great significance for guiding the choice of chemical disinfectant concentration and treatment time.