Clinical and molecular analyses of norovirus-associated sporadic acute gastroenteritis: the emergence of GII.17 over GII.4, Huzhou, China, 2015

Background Noroviruses (NoVs) are the most common cause of non-bacterial acute gastroenteritis (AGE) in all age groups worldwide. The NoVs circulating in Huzhou over the past 7 years were predominantly GII.4 genotypes. In the winter of 2014–2015, a novel variant of NoV GII.17 emerged and became predominant. We report the epidemiological patterns and genetic characteristics of NoV after the appearance of GII.17 in Huzhou City, Zhejiang, China. Methods Between January and December 2015, 746 stool specimens collected from patients with acute gastroenteritis were screened for NoV. Real-time RT-PCR (qPCR) was performed for NoV detection. RT-PCR was used for genomic amplification and sequencing. Genogroups and genotypes were assigned using an online NoV typing tool (http://www.rivm.nl/mpf/norovirus/typingtool). Phylogenetic analyses were conducted using MEGA (ver. 6.06). Results In total, 196 (26.3%) specimens were identified as NoV-positive. NoV infection was found in all age groups tested (≤5, 6–15, 16–40, 41–60, and ≥60 years), with the 16–40-year age group having the highest detection rate (117/196, 59.7%). Of the 196 NoV-positive specimens, 191 (97.5%) viruses belonged to GII, and 4 (2.0%) to GI; one sample showed GI and GII co-infection. Overall, 117 (59.7%) viruses were sequenced, and new GII.P17/GII.17 variants were the dominant genotype, accounting for 75.2%, followed by GII.Pe/GII.4 Sydney 2012 strains (11.11%). AGE patients infected with the GII.P17/GII.17 genotypes almost all had abdominal pain and watery stools. Conclusions We report the epidemiological patterns and genetic characteristics of the emergence GII.17 over the GII.4 in Huzhou between January and December 2015. After the emergence of GII.17 in October 2014, it steadily replaced the previously circulating GII.4 Sydney 2012 strain, and continued to be dominant in 2015. Electronic supplementary material The online version of this article (doi:10.1186/s12879-016-2033-x) contains supplementary material, which is available to authorized users.


Background
Norovirus (NoV) is the most common cause of nonbacterial acute gastroenteritis (AGE) in all age groups worldwide [1]. Transmission of NoV is mainly via fecally contaminated food or water, by direct contact with patients or vomited virus, and subsequently by contact with contaminated environmental surfaces [2]. Clinical infection with NoV generally has an incubation time of 12 to 48 h, with nausea, vomiting, watery diarrhea, and abdominal pain [3].
NoV has a diameter of~38 nm and belongs to a category of small non-enveloped icosahedral viruses in the Caliciviridae family. The genome of NoV consists of ã 7.5-kb positive sense, single-stranded RNA with three open reading frames (ORF1-ORF3) [4]. Genetically, NoV has been classified into six genogroups (I-VI) that are further subdivided into over 40 genotypes [5]. Genogroups I, II, and IV have been found to infect humans, whereas genogroup III infects bovines, and genogroup V has been isolated from mice. The sub-genogroup GII.4 virus accounts for most reported cases, and has been identified as the predominant genotype globally; new GII.4 variants emerge every 2-3 years [6,7].
Did GII.17 appear in Huzhou temporarily, or did it replace GII.4 forever? Here, we report epidemiological patterns and genetic characteristics of NoV after the appearance of GII.17 in Huzhou City, Zhejiang, China.

Specimen collection
This study was part of the regional NoV gastroenteritis surveillance program conducted at the First People's Hospital in Huzhou, and was approved by the ethics committee of Huzhou Center for Disease Control and Prevention. Informed consent for the stool samples was obtained from the patients or their guardians.
The definition of acute gastroenteritis was diarrhea (≥3 loose stools within a 24-h period), possibly accompanied by vomiting, abdominal pain, fever, and nausea. All stool samples were freshly collected in a sterile container and sent to Huzhou Center for Disease Control and Prevention for immediate storage at -70°C prior to analysis.

Viral RNA extraction and norovirus detection
Viral RNA was extracted from 140 μL of supernatant of a 10% (w/v) fecal suspension using a QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. RNA extracts were subjected to the reverse transcription polymerase chain reaction (RT-PCR) or stored at -70°C until further use. Genogroupspecific primers and probes described previously were used to detect NoVs by real-time RT-PCR (qPCR) [17]. Primer and probe sets JJV1F/JJV1R/ JJV1P and JJV2F/ COG2R/RING2-TP were used to screen for GI and GII NoV strains, respectively. RT-qPCR was carried out using a One Step PrimeScript RT-PCR Kit (DRR064; TaKaRa, Dalian, China). Amplification conditions were described previously [8].

Genomic amplification for genotyping
For genotyping, the primer set JV12Y/JV13I was used to amplify the 3'-end of the RdRp (RNA-dependent RNA polymerase) gene (region A in ORF1) [18]. Primer sets G1SKF/G1SKR and G2SKF/G2SKR were used to amplify the 5'-end of the capsid protein (VP1) gene (region C in ORF2) for GI and GII, respectively [19]. RT-PCR was carried out using a One Step RNA PCR Kit (TaKaRa) with the amplification conditions described previously [8]. After amplification, 5 μL of the PCR products was visualized by agarose gel electrophoresis. The residual PCR products were purified using a QIAquick PCR purification kit (Qiagen, Leusden, The Netherlands), and the purified products were sequenced directly at both ends with amplification primers by TaKaRa Biotechnology (Dalian, China).

Sequence analysis and phylogenetic analysis
Genotypes were determined using the online NoV Typing Tool (http://www.rivm.nl/mpf/norovirus/typingtool) [20], and the strains were named according to the isolated place, time, and sample number. A phylogenetic tree was generated using the neighbor-joining method and MEGA software (ver. 6.06) [21]. The evolutionary distance was calculated based on the maximum composite likelihood model, and the reliability of each branch was assessed with 1000 bootstrap replicates.

Nucleotide sequence accession numbers
The GenBank accession numbers for sequences obtained in this study are KU662973-KU663020.

Norovirus infections and clinical features
Between January 2015 and December 2015, 746 stool specimens collected from patients (including children and adults, outpatients and inpatients) with AGE were screened for NoV. In total, 196 (26.3%) specimens were identified as NoV-positive. Among the 196 positive samples, 191 (97.45%) viruses belonged to GII, 4 (2.04%) to GI, and 1 sample showed GI and GII co-infection. The numbers of AGE patients and the monthly detection rates of NoVs are shown in Fig. 1. The NoV detection rate increased from January, reached a peak in March, and then declined. The highest detection rate was 57% in March, at which point NoV infection accounted for nearly half of all AGE patients. In contrast, the lowest NoV infection rate was in August, when only 1 of 63 samples was positive.

Genotyping and distribution of NoVs
All NoVs from samples that tested positive by real-time PCR were classified according to genotype. Regions A and C (located at RdRp and the VP1gene, respectively) were amplified with specific primers, and the PCR products were subjected to direct sequencing [22]. In total, 117 (59.69%) viruses were sequenced, and partial nucleotide sequences from two genes (capsid and RdRp) were obtained from 46 strains, which clustered into six known genotypes and one unassigned genotype: GI.P4/GI.

Phylogenetic analyses of NoV strains
In total, 46 strains with at least partial nucleotide sequences for two genes (capsid and RdRp) were subjected to a phylogenetic analysis using the MEGA software (ver. 6.06) as mentioned previously (Figs. 1 and 2). Of the two genogroup GI strains, one (HuzhouNS2015521) belonged to the GI.4 genotype, whereas the other (HuzhouNS2015434) was apparently a recombinant, for which the RdRp and capsid genes clustered with the GI.Pb and GI.6 prototype viruses, respectively. For the remaining 44 GII isolates, most of strains grouped with the novel GII.P17/GII.17 variants, which emerged as the predominant strains in Huzhou beginning in the winter of 2014. The partial sequences of the capsid gene formed a single cluster (Cluster 3 in Fig. 3b), sharing the highest identity with reference strain Kawasaki308 (accession no. LC037415). Five strains were grouped with the formerly predominant GII.4 variants GII.Pe/GII.4_Sydney_2012. The partial RdRp gene sequence of strain HuzhouNS201557 (GII.P unassigned/ GII.13 in Table 2) clustered with neither GII.P16 nor GII.P5 strains and could not be assigned by the automated norovirus typing tool, indicating that a longer sequence was needed for further validation. Additionally, one GII.P21/GII.21 and one recombinant strain GII.P12/GII.3 were circulating in Huzhou in 2015.

Discussion
In this study, we determined the levels of NoV activity in sporadic AGE from January to December 2015 in Huzhou, China. The overall prevalence of NoV infection was 26.3% (196/746). Of the 196 NoV-positive specimens, 117 were identified as NoV GII, 4 belonged to NoV GI, and 1 was a combined NoV GI and GII infection. In total, 117 viruses were sequenced, and 88 (75.21%) belonged to a novel GII.P17/GII.17 genotype. In our previous study [10], We reported the emergence and predominance of norovirus GII.17 in the same hospital from March, 2014 to February 2015. There were overlapping of using the same set of specimens of January and February 2015 between the two studies. NoV infection was detected throughout the year. The circulation peak of NoV occurred during the 2014-2015 winter-spring period. Generally, young children (aged <5 years) and older adults (aged >60 years) are the at-risk groups for more severe NoV gastroenteritis, partly due to immature and waning immunity, respectively [23]. Our previous research found that children (≤10 years) and elderly individuals (>60 years) were more likely to be infected with GII.4 [10].
In contrast, GII.17 seemed more likely to infect people aged 16-60 years, previously considered the least vulnerable population. Chan MC [24] recently reported that the median (IQR) age of GII.17 (n =128) cases was significantly older than that of GII.4 (n =163) cases (49 (9-75) versus 1 (1-8) years, the proportion of older children and young adults (aged 5-65 years) and older adults (aged > 65 years) in GII.17 cases were higher than in GII.4 cases (47.7% vs 19.0%,36.7% vs 11.0% respectively). Adults aged 16-60 years are generally regarded as non-compromised, and thus unlikely to develop severe NoV gastroenteritis that requires medical attention. Why the adults seem more susceptible to the novel GII.P17/ GII.17 virus need to be further studied.
Generally, NoV activity peaks in the winter-spring period, but we found no significant increase in the winter of 2015 compared to the summer and autumn of 2015. This may be because, after several months of exposure to NoV GII.17, the population had acquired immunity against GII.17, and the previously circulating GII.4 Sydney 2012 strain was still at low levels of activity.
Over the past two decades, NoV GII.4 variants have been responsible for the majority of both outbreaks and sporadic cases of AGE [25]. GII.4 variants have emerged every 2-3 years, and the new variants then replaced the old ones as the predominant variant. The emergence of novel GII.4 variants has caused at least six pandemics of NoV-associated acute gastroenteritis: US 95/96  The success of GII.4 viruses is due to their evolution through the accumulation of mutations into drift variants that escape immunity from previous exposure [34], intra-genotype recombination of contemporary GII.4 noroviruses that foster the emergence of novel GII.4 variants [35], and alterations in binding properties [36].
In the Huzhou area, the GII. Some other genotypes, such as GII.3, GII.6, and GII.13, were also detected in Huzhou. However, none of these non-GII.4 genotypes ever replaced the GII.4 genotypes' dominance. The novel GII.P17/GII.17 variant was first Fig. 3 Phylogenetic analysis based on RdRp (RNA-dependent RNA polymerase) genes a and partial capsid protein (VP1) genes b of genogroup II noroviruses. Phylogenetic analysis based on RdRp genes a and partial VP1 genes b of GII NoVs. The trees were constructed by the neighborjoining method with by the neighbor-joining method with the Maximum Composite Likelihood model in MEGA (version 6.0), validated by 1000 bootstrap replicates. Bootstrap values more than 70% was shown on the branches. NoV strains identified in Huzhou are designated by location, year and sample number (indicated by black triangles). Reference sequences (variants) are indicated by their genotypes and accession numbers. The GIII.2 strain (AY126474) was used as outgroup