| Background:The novel enterovirus is a general designation of a large type of enterovirus genotype which has been genotyped since 1976,including EV-A71 which has caused a widespread epidemic of hand-foot-and-mouth disease in China and EV-D68 and EV-B7 5 which caused thousands of respiratory tract-related diseases in the United States in 2014,belonging to enterovirus Group B.The first reported strain of EV-B75 was isolated from the feces of a healthy child in Ethiopia in 1974.It was finalized and officially named by the gene in 2004.At present,there are 112 reports of EV-B75 related cases worldwide,covering a wide range of clinical manifestations,including Acute flaccid paralysis,AFP),aseptic meningitis,encephalitis,acute diarrhea,respiratory tract-related symptoms,etc.However,based on the currently reported EV-B75,EV-B75 related cases still show a sporadic distribution,most of the cases are found from AFP monitoring programs in various countries,and the case distribution does not show typical temporal and spatial clustering,nor has it seen a major outbreak of cases.The EV-B75 isolate involved in this study was feces from a child with AFP and six close contacts in 2007 in Xigaze,Tibet.All seven children were under 5 years of age,and no other on-site epidemiological information was available.Objectives:In this study,seven EVs-B75 from Tibet were subjected to molecular epidemiological study to deduce their possible sources.To investigate the seroprevalence of EV-B75 among healthy people in Tibet and understand the background value of EV-B75 infection in Tibet.To explore the temperature sensitive property of EV-B75 to understand the effect of high temperature on virus proliferation cycle;To explore the tolerance of EV-B75 genotype to the commonly used enterovirus inactivation methods in laboratories,and select the laboratory biosafety prevention and control methods targeting EV-B75.The screening of mouse strains suitable for the pathogenicity study of EV-B75 and further exploration of its pathogenicity in mice to further understand the relationship between the virus and disease will also provide a basic theoretical basis for the prevention and treatment of diseases in the future.Methods:1.In this study,the samples of seven Tibetan EV-B75 isolates were purified,and the whole genome sequences of seven Tibetan isolates were obtained using primer step-by-step method and first-generation sequencing technology.The full genome sequences of seven Tibetan EV-B75 were analyzed using Sequencher,MEGA,Bioedit,Simplot and other softwares,for molecular epidemiological study of EV-B75.2.A neutralization test was conducted on 113 serum samples of healthy children and adolescents in Tibet to calculate their serum positive rates and geometric mean titers so as to evaluate the population prevalence in Tibet.3.The EV-B75 isolate was incubated at 36℃ and 39.5℃ for 4 h,8 h,16 h,24 h,48 h and 72 h,respectively,and the viral titer was measured.The growth curve was drawn to determine the sensitivity of the isolate to temperature.4.After treating the EV-B75 strain with water bath at a certain temperature and time,the residual virus titer was measured,and the commercially available chlorine-containing disinfectant and potassium monopersulfate solution were diluted to the recommended concentration according to the instructions.After treating for a certain time,the residual virus titer was measured to determine whether the laboratory commonly used virus inactivation method could effectively inactivate EV-B75 virus.5.A certain dose of virus was injected into wild-type ICR neonatal rats,wild-type C5 7 neonatal rats and A129 immunodeficient neonatal rats with interferon a receptor gene defect by intraperitoneal injection or intracranial injection,to detect the changes of daily body weight and clinical score of the neonatal rats after virus tapping,and to collect the heart,lung,spleen,intestine,brain tissue and hindlimb muscle tissue of the neonatal rats,to analyze the replication of the virus in the tissues and to determine whether the virus was efficiently replicated in the neonatal rats.Results:1.Based on the bioinformatics analysis of the whole genome sequence of seven Tibetan EV-B75 isolates,the nucleotide and amino acid similarities of the seven Tibetan isolates were 94.4%-99.9%and 99.7%-99.9%,respectively.The phylogenetic tree analysis in VP1 region also revealed that the seven Tibetan isolates clustered into one cluster,which was close to the Indian strain(India-N-694),while the Chinese Shandong strain(102-SD-CHN-97)and the Chinese Fujian strain(CHN-FJ96-71)independently clustered and formed independent branches.Based on the analysis of recombination in the P2 and P3 regions of sequence,it was possible to perform an inter-type recombination with the EV-107 isolate(TN94-0349)at position 4620-5240 and with the CVB1(1167438)at position 5340-7425.2.A total of 113 serum samples of healthy children or adolescents from Xigaze District,Tibet and Lhasa were investigated for the positive rate of EV-B75 antibody.It was found that only 12 serum samples had neutralizing antibody titer≥1:8,of which four were from Xigaze District and eight were from Lhasa City.The serum positive rate of serological investigation was 10.62%,and the titer of neutralizing antibody was 1:4.52.3.The virus levels of the 3 and 7 EV-B75 Tibetan isolates cultured at 39.5℃ for 72 h were two logTCID50 lower than that of the virus treated at 36℃ for 72 h,and all of them showed high temperature sensitivity.4.It was sensitive to inactivation by high temperature.Treatment at 65℃ for 60 min could completely inactivate EV-B75,while treatment at 70℃ for only 10 min could completely inactivate EV-B75.Sodium hypochlorite,trichloroisocyanuric acid,and potassium monopersulfate disinfectants can completely inactivate EV-B75 at the recommended concentrations and treatment duration.5.After the wild-type ICR neonatal rats were infected with 30 μL virus stock solution with the titer of 108TCID50 by intraperitoneal injection and intracranial injection,the neonatal rats grew well,and their body weights were not different from those of the negative control group.The viral load in the tissues or organs of the neonatal rats was low.After the wild-type C57 neonatal rats were infected with 30 μL virus stock solution with the titer of 108TCID50 by intracranial injection,the neonatal rats grew well,and their body weights were not different from those of the negative control group.The viral load in the tissues or organs of the neonatal rats was low.However,when 30 μL virus stock solution with the titer of 108TCID50 was infected by intracranial injection in A129 immunodeficient neonatal rats with interferon a receptor gene deficiency,the neonatal rats’ body weight increased slowly or even decreased,and there was a significant difference compared with the negative control group.After three days of exposure,the typical clinical manifestations of hind limb stiffness or even paralysis began to appear.Conclusions:1.Molecular epidemiological analysis was performed on seven Tibetan EV-B75 isolates in this study.The results showed that the seven Tibetan isolates had extremely high similarity in nucleotide sequences and belonged to the same transmission chain;Phylogenetic analysis suggested that the Tibetan isolate did not cluster with the Chinese isolate,but was closely related to the Indian isolate.Combined with geographical location and similar case information,it was speculated that the Tibetan isolate might be an Indian imported strain.The analysis of gene sequence recombination also indicates that the epidemic of EV-B75 in China may be underestimated,and EV-B75 may have widely existed in the population,but it is still in the low epidemic period.2.This study investigated the positive rate of EV-B75 antibody in 113 serum samples of healthy children or adolescents from Xigaze region and Lhasa city,Tibet.The results showed that EV-B75 did not cause large-scale transmission in Tibet,and the population prevalence in Tibet was still low.3.The seven Tibetan EV-B75 isolates were all temperature-sensitive in this study,suggesting that high temperature could affect the proliferation cycle of the virus.4.Thermal inactivation,commercial chlorine-containing disinfectants and potassium monopersulfate solution can effectively and even completely inactivate viruses under the recommended concentration and treatment time of commodities.However,when the concentration or treatment time does not meet the standards,the viruses cannot be completely inactivated,which reminds us that we should strictly follow the operation guidelines and strictly implement the inactivation and disinfection standards of viruses to reduce the occurrence of laboratory-related infections of enteroviruses in ensuring the biological safety of laboratories.5.In this study,sensitive animal strains for the pathogenicity study of EV-B75 were explored.The results showed that A129 immunodeficient neonatal rats with interferon α receptor gene deficiency could display typical clinical symptoms and observe the progression of the disease,with the mortality rate reaching 100%.However,neither wild-type ICR neonatal rats nor C57 neonatal rats could display typical clinical symptoms and the virus could not be effectively replicated in the neonatal rats.The wild-type neonatal rats were not suitable animal models for EV-B75. |
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