Emergence of norovirus GII.P16-GII.2 strains in patients with acute gastroenteritis in Huzhou, China, 2016–2017

Noroviruses (NoVs) are the second most common cause of viral acute gastroenteritis cases worldwide, affecting people in all age groups. It is estimated that NoVs account for 18% of sporadic cases of acute gastroenteritis and approximately 50% of all-cause gastroenteritis outbreaks worldwide [1, 2].

NoV is a member of the family Caliciviridae. The viral genome is a single positive-strand RNA of 7.7 kb that contains three open reading frames (ORFs) [3]. ORF1 encodes a nonstructural polyprotein required for replication, including RNA-dependent RNA polymerase (RdRp). ORF2 encodes the major capsid protein (VP1) and ORF3 encodes a minor capsid protein (VP2). VP1 is structurally divided into a shell (S) and a protruding (P) domain [4]. The P domain is further divided into P1 and the hypervariable P2 subdomain with the major antigenic sites and histoblood group antigen (HBGA) ligands located in the P2 subdomain [5]. NoV are a highly diverse group of viruses that can be genetically grouped into 7 genogroups (GI–GVII), but only norovirus GI, GII, and GIV can infect humans, with GII being the most prevalent [6, 7]. Each genogroup can be further classified into numerous genotypes based on the sequence differences of their VP1 proteins. To date, 9, 22 and 2 capsid genotypes have been recognized in GI, GII and GIV.

Despite this diversity, the NoV GII.4 virus has been responsible for most reported norovirus-associated outbreaks and sporadic cases for over two decades globally. New variants of GII.4 strains emerge every 2–3 years [8, 9]. Since 2008, almost all outbreaks and the majority of sporadic cases in Huzhou were caused by GII.4 variants, including GII.4 2006b, New Orleans 2009, and Sydney2012 [10, 11]. However, during the winter of 2014–2015, a novel GII.17 NoV strain emerged and replaced the previously dominant GII.4 genotype Sydney2012 variant, as the major cause of acute gastroenteritis outbreaks in several countries of the Asia [12]. At the same time, GII.17 emerged and became the dominant strains during the 2014–2015 epidemic season in Huzhou [13]. This was the first time that non–GII.4 norovirus might become the predominant genotype. Moreover, in late 2016, an uncommon recombinant NoV genotype called GII.P16-GII.2 caused a sharp increase in outbreaks of acute gastroenteritis in many cities of China, most of the outbreaks (78%) associated with this recombinant variant occurred in kindergartens [14]. Increased circulation of GII.P16-GII.2 recombinant was also observed concurrently in Japan, Germany and France during the same winter season [15–17]. However, we did not observe a drastic increase in sporadic NoV cases in the winter of 2016 in Huzhou. Whether GII.P16-GII.2 recombinant has emerged and been prevalent in Huzhou is unclear. Therefore, we investigate the prevalence and genetic characteristics of NoVs in the sporadic acute gastroenteritis (AGE) cases from January 2016 to December 2017 in Huzhou City, Zhejiang, China.

In this study, we analyzed NoV-associated sporadic acute gastroenteritis in Huzhou from January 2016 to December 2017. Our data showed that NoV infection was detected throughout the year, and the circulation peak occurred during the winter–spring seasons. NoV strains circulate in Huzhou exhibits considerable genetic diversity during the study period and at least 17 genotypes were identified based on both RdRp and capsid genes, with most of them were recombinant strains. The predominant NoV genotypes have changed from GII.Pe-GII.4 Sydney2012 and GII.P17-GII.17 in 2016 to GII.P16-GII.2, GII.Pe-GII.4 Sydney2012 and GII.P17-GII.17 in 2017. NoV GII.P16-GII.2 was first detected in December 2016 and rapidly became the major genotype in the Huzhou by February 2017, result in increasing detection rates of NoV infection in spring–winter seasons of 2017. It should be noted that this time point was later than the first report of NoV GII.P16-GII.2 in China. NoV GII.P16-GII.2 emerged first in August 2016 in China, it then caused a steep rise in gastroenteritis outbreaks at the end of 2016 in many cities of China [14, 24, 25]. However, our monitoring data showed that only one GII.P2-GII.2 caused gastroenteritis outbreak was detected in December in the winter of 2016 in Huzhou (data not shown). Similar finding were reported Jiangsu provinces, in which the NoV GII.P16-GII.2 genotype firstly appeared in December of 2016, and spread extensively in February and caused rapidly increased outbreaks in the late spring of 2017 [26]. Based on the above results, we speculate that the beginning time of GII.P16-GII.2 prevalence in different areas of China is not exactly the same.

For over two decades, most NoV-associated gastroenteritis cases reported all over the world have been linked to a single genotype GII.4 [8]. However, in winter 2014–2015, the GII.4 strains were unexpectedly displaced by a new GII.17 variant (GII.P17-GII.17 Kawasaki) in some Asian countries [12]. Moreover, in late 2016, an uncommon recombinant NoV genotype called GII.P16-GII.2 caused a sharp increase in outbreaks in different countries worldwide [14–17]. This was another event that non-GII.4 NoV might become the predominant genotype. Unlike epidemic pattern observed in the past years for GII.4 in Huzhou that new GII.4 variants emerged every two or three years and presented absolutely predominant during norovirus epidemic seasons [10, 11], these non-GII.4 predominant genotypes were co-circulating with GII.4 and the predominant genotypes of NoV are diverse according to month. In the Huzhou area, GII.17 was firstly detected in October 2014 and gradually replaced GII.4 Sydeny2012 as a predominant strain [27]. However, GII.4 Sydney2012 re-emerged in October 2015 [13] and presented an alternate prevalence pattern with GII.17 until the advent of GII.P16-GIII.2 at the end of 2016. In 2017, GII.P16-GII.2 was co-circulating with GII.Pe-GII.4 Sydney2012 and GII.P17-GII.17. A recent report showing that the non–GII.4 NoV remains static may explain the epidemiological pattern of non–GII.4 NoV infection [28]. The persistence and predominance of GII.4 strains due to it is an “evolving genotype” which can continuous evolve through accumulation of mutations into antigenically distinct variant that escape from herd immunity. In contrast, non-GII.4 predominant genotypes would prevail for only a short period of time as they are highly conserved or “static genotypes”, which limits their prevalence and antigenic diversity.

Globally, GII.P16-GII.2 is an uncommon genotype with low detection rate in sporadic infections before 2016, except in Japan. GII.P16-GII.2 was firstly reported in 2008 and became the most prevalent GII.2 genotype after 2009 in Japan [29–31]. Most of the pre-2016 GII.P16-GII.2 strains were detected in Japan during 2009–2014, which could be genetically subdivided into three clusters (2009–2010 cluster, 2010–2012 cluster, and 2012–2014 cluster) [31]. Phylogenetic analyses in this study indicated that the VP1 genes of 2016–2017 GII.P16-GII.2 strains were most closely related to Japan 2010–2012 cluster, whereas the RdRp genes of these strains were genetically closely related to the GII.P16-GII.4 strain identified in 2015. This finding is consistent with a recent phylogenetic study conducted in Japan [32]. The authors suggested that the VP1 of 2016 GII.P16-GII.2 strains diverged from common ancestors of the GII.P16-GII.2 cluster detected in 2010–2012 and RdRp of the 2016 strains diverged from the common ancestors of GIIP16-GII.4.

The driving force behind the rapidly spread of GII.P16-GII.2 stains in 2016–2017 season is not clear yet. Complete VP1 amino acid sequences of 2016–2017 GII.P16-GII.2 stains were aligned with GII.2 strains from 1976 to 2015 in this study. Compared with the earliest GII.2 strain(SMV/1976/USA), no common mutations were found in the amino acids of the HBGA-binding sites and the predicted epitopes, in concordance with the relatively static nature of non-GII.4 stains [28]. Just recently, Tohma K et al. investigated the evolutionary dynamics of GII.2 strains by using bioinformatics analysis [33]. They found the reemergence of GII.P16-GII.2 strains during 2016 did not involve changes in the substitution rate or acquisition of amino acid mutations in the major capsid protein and suggested that, genetic change in the RdRp may play some role in the predominance of GII.P16-GII.2 strains. However, the above study is only about the sequence analysis of the RdRp and VP1 region, further analysis using long sequences covered other nonstructural proteins and VP2 and in vitro assays such as serum inhibition/neutralization tests are required to explore the mechanisms behind the rapidly spread of GII.P16-GII.2 stains.

Our study is limited by a small sample size, single-site setting, and especially the partial genotyping of circulating NoVs. Genotyping was only successful for 140 (68.7%) detected NoVs cases. In future studies, epidemiologic and virologic surveillance should be broadened to better clarify the epidemiological patterns and genetic characteristics of NoV strains in Huzhou.

In conclusion, we report the emergence of GII.P16-GII.2 strains and characterize the molecular epidemiological patterns NoV infection between January 2016 and December 2017 in Huzhou. We found that the predominant genotypes of norovirus during our study period in Huzhou are diverse according to month. Phylogenetic analyses showed that VP1 genes of 2016–2017 GII.P16-GII.2 strains were most closely related to Japan 2010–2012 cluster and their VP1 amino acid sequences remain relatively static after one year of circulation in the human population. However, unlike the recently emerged GII.P17-GII.17 strains that predominated only in part of Asia in 2014–2015 season, the re-emerged GII.P16-GII.2 strains were almost simultaneously reported in Asia and Europe, indicating the fast spread of this genotype across continents. Continuous surveillance and further studies are needed to understand the epidemiological characteristics of GII.P16-GII.2 strains and explore the mechanisms behind the wide spread in population of this genotype.