Ji Lei, Chen Liping, Xu Deshun, Wu Xiaofang, Han Jiankang
Huzhou Center for Disease Control and Prevention, Huzhou, China.
Mol Genet Genomic Med. 2018 Sep;6(5):796-804. doi: 10.1002/mgg3.446. Epub 2018 Jul 10.
Human norovirus is the leading cause of acute gastroenteritis worldwide. However, in vitro culture system is complicated for human norovirus. Sequence analysis became more useful for norovirus research, particularly when using complete genomic sequences.
Real-time RT-PCR (qPCR) was performed for norovirus detection. Three modified paris of PCR primes were designed based on the alignment of the novel GII.17 norovirus complete sequence available in Genbank., which could amplify three overlapping fragments cover the whole genome. The PCR fragments were sequencing by Sanger sequence with Primer walking methods. Genogroup and genotype were assigned using the Norovirus Noronet typing tool and the strains were named according to the time of isolation. The phylogenetic analysis was conducted using MEGA software (ver. 6.06).
One nearly complete genome sequence were obtained from sample collected from Huzhou, China. The partial genome sequence of the HuzhouNS2014603 strain is composed of 7556 nucleotides (nt).The strain was classified as GII.17 genotype both in ORF1 and ORF2, and was most closely related to the LC037415.1/Hu/GII.17/Kawasaki308 strain. Within the GII.17 cluster, the 2013/14 season strains were grouped separately from the GII.17 strains detected in 2014/15. HuzhouNS2014603 was clustered with the 2014/15 season strains. Compared with other strains selected, there are 98 variable residues across the VP1 domain. Among the 98 variable amino acids, 13 (13.3%) were observed in the shell domain and 22 (22.4%) in the P1domain; most of the substitutions and insertions were located in the P2 domain, account for 63 (64.3%).
This is the first report of the nearly complete genome of the novel GII.17 by direct sequencing method in the Huzhou area. The results of this study could be helpful for the study of the genetic evolution of the virus, the development of rapid diagnostic reagents and the design of vaccine.
人诺如病毒是全球急性胃肠炎的主要病因。然而，人诺如病毒的体外培养系统较为复杂。序列分析对诺如病毒研究更有用，尤其是在使用完整基因组序列时。
采用实时逆转录聚合酶链反应（qPCR）检测诺如病毒。基于Genbank中新型GII.17诺如病毒完整序列的比对设计了三对修饰的PCR引物，可扩增覆盖整个基因组的三个重叠片段。PCR片段采用引物步移法通过桑格测序法进行测序。使用诺如病毒NoroNet分型工具确定基因组群和基因型，并根据分离时间对菌株进行命名。使用MEGA软件（版本6.06）进行系统发育分析。
从中国湖州采集的样本中获得了一条近乎完整的基因组序列。湖州NS2014603菌株的部分基因组序列由7556个核苷酸（nt）组成。该菌株在开放阅读框1（ORF1）和开放阅读框2（ORF2）中均被归类为GII.17基因型，与LC037415.1/Hu/GII.17/川崎308菌株关系最为密切。在GII.17簇内，2013/14季节的菌株与2014/15检测到的GII.17菌株分开聚类。湖州NS2014603与2014/15季节的菌株聚类在一起。与其他选定菌株相比，VP1结构域共有98个可变残基。在这98个可变氨基酸中，13个（13.3%）出现在壳结构域，22个（22.4%）出现在P1结构域；大多数替换和插入位于P2结构域，占63个（64.3%）。
这是首次在湖州地区通过直接测序法报道新型GII.17的近乎完整基因组。本研究结果有助于该病毒的遗传进化研究、快速诊断试剂的开发及疫苗设计。
