Prevalence and genetic diversity of noroviruses in adults with acute gastroenteritis in Huzhou, China, 2013–2014

Prevalence and genetic diversity of noroviruses in adults with acute gastroenteritis in Huzhou, China, 2013-2014 Xiaofang Wu 0 1 Jiankang Han 0 1 Liping Chen 0 1 Deshun Xu 0 1 Yuehua Shen 0 1 Yunfeng Zha 0 1 Xiaojuan Zhu 0 1 Lei Ji 0 1 0 Huzhou Center for Disease Control and Prevention , 999 Changxing Road, Huzhou 313000, Zhejiang , China 1 Noroviruses in adults in Huzhou , China Norovirus (NoV) infection is the most common cause of nonbacterial acute gastroenteritis, which affects both adults and children. However, the molecular epidemiology of NoV in adults with acute gastroenteritis in China has not been investigated extensively. In this study, we investigated the occurrence of NoV infections and analyzed the genetic diversity of NoV in adults with acute gastroenteritis in Huzhou, China. A total of 796 fecal samples were collected from outpatients (C16 years of age) between March 2013 and February 2014. Real-time RT-PCR was performed to detect NoV genogroups I (GI) and II (GII). For genotyping, the capsid and RNA-dependent RNA polymerase (RdRp) genes were partially amplified and sequenced for phylogenetic analysis. NoVs were detected in 26.51 % (211/796) of the specimens, with GII being predominant, representing 96.20 % of the NoV infections. At least nine genotypes were identified among GI and GII specimens, including GI.P2/GI.2, GI.P3/GI.3, GI.P4/GI.4, GII.Pe/GII.4 Sydney_2012, GII.P12/GII.3, GII.P7/GII.6, GII.P16/GII.13, GII.Pe, and GII.Pg (RdRp only). This is the first report of a GII.P16/GII.13 recombinant virus in adults in China. GII.Pe/GII.4 Sydney_2012 was the most prevalent genotype and the only GII.4 variant identified during the study period. Our findings suggested that NoV was a common causative agent of acute gastroenteritis in adults in Huzhou, China. During the study period, the NoVs circulating in adults in Huzhou were - Acute gastroenteritis is one of the most common illnesses and a major public health problem worldwide. Since the development and application of novel sensitive molecular assays, noroviruses (NoVs) have been recognized as the leading cause of epidemics of gastroenteritis and an important cause of sporadic gastroenteritis in individuals of all ages in both developed and developing countries [4]. It is estimated that NoVs account for 12 % of severe gastroenteritis cases (hospitalized) among children less than 5 years old and 12 % of mild and moderate diarrhea cases (outpatient) among persons of all ages [26]. NoVs belong to the genus Norovirus in the family Caliciviridae. The viral genome is a single positive-strand RNA of 7.7 kb that contains three open reading frames (ORFs) [8]. ORF1 encodes several nonstructural proteins involved in replication of the genome, including RNA-dependent RNA polymerase (RdRp), nucleoside triphosphatases (NTPases), and proteases. ORF2 and ORF3 encode the major capsid protein VP1 and minor capsid protein VP2, respectively [31]. Due to the lack of a suitable cell-culture system for human NoV, genetic analysis is the principal method used to classify NoV strains. NoVs are a group of genetically diverse viruses that can be classified into six major phylogenetic clades, referred to as genogroups (GI to GVI) [21, 25, 43]. Genogroups are further classified into several genotypes. Although more than 30 genotypes within GI, GII, and GIV can infect humans, the majority of NoVrelated outbreaks and sporadic cases of acute gastroenteritis are caused by a sub-genogroup of GII.4 strains [15, 44]. During the past decade, new variants of GII.4 strains have emerged every 23 years and have replaced the previously predominant GII.4 strains [30]. Emergence of these new NoV strains has often, but not always, been associated with increases in the number of outbreaks. RNA recombination is one of the major driving forces of virus evolution, and recombination of NoV genomes increases their genetic divergence. Analysis of these recombinants has suggested that the majority of recombination points are located near or within the ORF1/ORF2 overlap [2, 3]. Acute gastroenteritis is one of the most common public health problems in both China and other countries. During the past several years, most studies of NoV infection in China focused on the role of human NoV in acute gastroenteritis in children [9, 11, 42]. Although recent studies of NoV infection have focused on adults, no detailed examination of the genotype distribution among various age groups and according to season has been published [7, 12, 33, 38]. The present study was carried out to investigate the prevalence and genetic diversity of NoVs in adults with acute gastroenteritis in Huzhou, a medium-sized city located in eastern China. Materials and methods Study population and specimen collection This study was conducted at the First Peoples Hospital in Huzhou as part of the regional NoV gastroenteritis surveillance program. During March 2013 to February 2014, a total of 796 fecal specimens were collected from outpatients (C16 years of age) with acute gastroenteritis. Acute gastroenteritis patients were defined as patients with diarrhea (three or more loose stools within 24 hours), which may be accompanied by vomiting, abdominal pain, fever, and nausea. All stool samples were freshly collected and sent to Huzhou Center for Disease Control and Prevention for routine NoV detection. Human ethics committee approval was not requested for this study, as all investigations were carried out on NoV strains; no human experimentation was conducted. The data are associated with NoV strains. No patient demographic information other than agewas included in the analysis. Viral RNA extraction Viral RNA was extracted from 200 lL of 10 % stool supernatant in MEM medium (Sigma-Aldrich, USA) using a QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturers instructions. The extracted RNA was dissolved in 60 lL of RNase-free water and stored at -70 C until used. Genogroup-specific primers and probes described by Jothikumar et al. were used to detect NoVs by real-time RT-PCR [13]. The primer and probe sets JJV1F/JJV1R/ JJV1P and JJV2F/COG2R/RING2-TP were used to screen for GI and GII NoV strains, respectively. RealFig. 1 Monthly distribution of norovirus infections from March 2013 to February 2014. The highest detection rate (50.0 %) was observed in October 2013, and the lowest (5.1 %) in July 2013 (Mar, 35.3 %; Apr, 40.9 %; May, 30.9 %; Jun, 5.8 %; Jul, 5.1 %; Aug, 35.3 %; Sep, 33.3 %; Oct, 50.0 %; Nov, 29.8 %; Dec, 16.5 %; Jan, 15.2 %; Feb, 26.5 %) Table 1 Genotype distribution of identified NoV strains Fig. 2 Phylogenetic analysis based on partial RdRp gene (A) and capsid gene (B) sequences of GI NoVs. NoV strains identified in this study are indicated by closed circles. The phylogenetic tree was generated using the neighbor-joining method, validated by 1000 bootstrap replicates. Bootstrap values C80 % are shown on the branches time RT-PCR was carried out usin (...truncated)