[Epidemiological Characteristics of Hand, Foot, and Mouth Disease and the Effect of EV71 Vaccination in Chengdu from 2012 to 2020].

Objective: To analyze the epidemiological characteristics of hand, foot, and mouth disease (HFMD) in Chengdu from 2012 to 2020, to make comparison in order to examine the changes in incidence before and after vaccination was introduced, and to provide basis for the prevention of HFMD in the future. Methods: Descriptive epidemiological methods were adopted to analyze the incidence, mortality and rate of severe cases of HFMD cases reported in Chengdu from 2012 to 2020. Results: From 2012 to 2020, the cumulative cases of HFMD reported in Chengdu were as many as 279216, of which, there were 2201 severe cases and 16 deaths. The incidence increased every other year, reaching 326.43 per 100000 person-years, the highest ever, in 2018. The rate of severe cases and mortality had shown a decreasing trend since 2016. A total of 11892 cases of EV71, CoxA16 and other enteroviruses were detected in the laboratory, accounting for 14.8%, 18.8% and 66.3%, respectively. Since 2016, HFMD cases caused by EV71 virus infection had shown an overall decreasing trend, cases caused by Cox A16 virus infection had increased every other year, and cases caused by other enteroviral infections had shown an overall increasing trend. The reported cases were mainly concentrated in children aged 0-5 years (92.1%), with those in the age group of 1-2 years reporting the highest number of cases. For children of different ages, male patients always outnumber female patients. The geographic distribution showed that the areas with high HFMD incidence were always located in the central part of Chengdu City, and the three districts with the highest incidence growth rate were Qingbaijiang District, Shuangliu District, and Longquanyi District. Temporal distribution of HFMD cases showed an obvious bimodal distribution, with most of the cases concentrated in May through August and October through December of each year. The number of new cases reached the highest (12309 cases) in July 2018. Conclusion: While continuing to promote EV71 vaccination in the future, Chengdu also needs to pay more attention to viral infection serotypes other than EV71 and Cox A16 and conduct research on multivalent vaccines against a variety of enteroviruses. The focus of prevention and control can be placed on areas with high population density, large floating populations, large numbers of agriculture-related communities, and insufficient individual awareness of hygiene. For the second circle of Chengdu city, the disinfection of agriculture-related communities should be strengthened, and information sessions or other health education activities could be organized for individuals and daycare facilities with low awareness of the importance of hygiene. In addition, more attention should be given to the prevention and control of HFMD in the high incidence seasons.

[Investigation of Virulence Variation of Enterovirus 71 Strains during Thermal Adaptive Evolution].

Objective: To study the virulence variation of enterovirus 71 (EV71) during thermal adaptive evolution, providing references for the prevention and control of the EV71-related hand, foot and mouth disease. Methods: Parental strains and thermal-adapted strains originating from EV71 sibling strains (lineage #100 and #101) were used for plaque assay validation, CCK-8 cytotoxicity experiment, and host proteomics studies after Vero cell infection. Plaque morphology and cell inhibition rate of the viral strains were obtained. Mass spectrometry was used to examine and analyze the functions of proteins that were differential expressed in the host cells. Results: Plaque morphology variation was found only in the heat-adapted strain of lineage #101. Increase in cell inhibition rate was observed in all the thermal-adapted strains, but the amount of increase varied in different strains. According to the results of clustering analysis and principal component analysis, after infection of Vero cells, the host cell protein profile of the heat-adapted strains was similar to that of the parental strains and the host cell protein profile of cold-adapted strains was similar to that of cell-adapted strains. It showed that 500 kinds of proteins presented inter-group difference in their expression, with 239 kinds being up-regulated proteins and 261 being down-regulated. The function of the up-regulated proteins were related to post-translational protein modification, while the functions of the down-regulated proteins were related to SRP-dependent cotranslational protein translocation/targeting to membrane and retrograde protein transport. Conclusion: Virulence variations of enterovirus 71 may accompany thermal adaptive evolution, but its mechanism of action still awaits further investigation.

Molecular characterization of enterovirus 71 sibling strains for thermal adaption in Vero cells with adaptive laboratory evolution.

Enterovirus 71 is the main pathogen that causes severe and fatal hand-foot-mouth-disease (HFMD) cases. As the enterovirus virus mutation has implications for pathogenesis, vaccine development, antiviral therapy, and epidemiological disease management of the virus. In this study, we investigated the variations of enterovirus 71 in thermal adaption, using the method of adaptive laboratory evolution. The sibling virus strains were isolated from a 2-year-old severe case of HFMD (#100) and her symptomless close contact (#101). Both strains were cultured in Vero cells by serial passage of 36 generations at the temperatures of 28.0 °C, 33.0 °C and 39.5 °C to construct adaptive lineages. According to the comparative analysis of phenotypes between adapted strains and parental strains, differences in growth rate were observed in the sibling lineages and a larger plaque was found mainly in the hot adapted strains for lineage #101. Two sets of adaptive strains from six time points (parental, 12th 17th, 31st, 35th passage and endpoint) were sequenced and analyzed by both Sanger sequencing and Next Generation Sequencing. Several variations in most coding genes and one reverse mutation in 5'UTR was observed, along with the identity of 99.8% for complete genome for both lineages. Notably, thermal specific non-synonymous mutations were found in the gene of VP1\VP3\3A\2C\3C. Moreover, the concurrent mutations A292G, A434G and A355C/T of sibling lineages in VP1 showed quantificational trace with distinguishing patterns for different temperatures, which were suspected to be the thermo-sensitive mutation hotspots. These results highlight the possible rules of thermal adaption in enterovirus 71, produce a novel picture of genome evolution of the virus, and shed light on viral variation and evolution.