四川地区环境污水中诺如病毒分子流行病学研究

Molecular epidemiology of norovirus in environmental sewage in Sichuan

目的 探索四川地区环境污水中诺如病毒分子流行病学特征及环境监测的意义,同时对比2种高通量测序方法在环境监测中的应用,为病毒性病原环境监测提供技术参考。方法 2019年7—12月、2022年1—6月每月中旬在四川某污水处理厂进水口采集1 000 ml水样,经阴离子膜法浓缩富集后,提取核酸用实时荧光逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)检测诺如病毒,再用扩增子测序和宏病毒组测序方法分别对诺如病毒衣壳区基因序列进行分析。结果 2019年7—12月、2022年1—6月共采集12份环境污水标本,GⅠ、GⅡ型诺如病毒检出率均为100.0%(12/12)。水样利用扩增子测序注释到17种型别诺如病毒,包括GⅠ.1~GⅠ.7、GⅡ.1~GⅡ.4、GⅡ.6、GⅡ.8、GⅡ.12、GⅡ.13、GⅡ.15、GⅡ.17,其中GⅠ.1、GⅡ.3、GⅡ.4相对丰度较高;2019年7—12月水样利用宏病毒组测序注释到4种型别诺如病毒和13种杯状病毒科其他属的病毒。结论 诺如病毒在四川地区环境污水中有很高的检出率,表明人群中有大量隐性感染者,且环境污水中检出多种在急性胃肠炎病例监测中少见或未见的诺如病毒型别,应继续加强环境监测,警惕诺如病毒急性胃肠炎的暴发流行。同时,在诺如病毒的环境监测中,扩增子测序较宏病毒组测序更为实用。

Objective To understand the molecular epidemiological characteristics of norovirus in environmental sewage and to discuss the significance of environmental surveillance in the control and prevention of norovirus acute gastroenteritis(AGE).To compared two high-throughput sequencing methods in the application of environmental surveillance in order to provide technical references for environmental surveillance of viral pathogens. Methods Sewage samples were collected monthly from July to December 2019 and from January to June 2022 from a wastewater treatment plant in Sichuan.Nucleic acid was extracted from sewage sample after concentrated by anionic membrane method.Real-time RT-PCR was used to detect norovirus.The genotypes of norovirus were determined by both amplicon sequencing and virome sequencing. Results From July to December 2019 and January to June 2022, a total of 12 sewage samples were collected.The detection rates of GⅠ and GⅡ norovirus were both 100%(12/12).By amplicon sequencing, 17 genotypes were identified in sewage samples collected, including GⅠ.1—GⅠ.7, GⅡ.1—GⅡ.4, GⅡ.6, GⅡ.8, GⅡ.12, GⅡ.13, GⅡ.15 and GⅡ.17, in which GⅠ.1, GⅡ.3, GⅡ.4 were the dominant genotypes.The water sample is sequenced by virome sequencing, only 4 genotypes of norovirus were detected. Conclusions The detection rate of norovirus in environmental sewage in Sichuan is very high, there might be a large number of asymptomatic cases in the population.In sewage, some rare or uncommon genotypes of norovirus causing AGE are also detected.Therefore, it is necessary to improve environmental surveillance system to detect new genotypes or variants of noroviruses which may potentially trigger the epidemics.Meanwhile, amplicon sequencing is more practical than virome sequencing in environmental surveillance of norovirus.