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

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 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. Genogroup-specific 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].

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).

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.

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

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.

We compared the clinical characteristics of AGE patients infected with NoVs versus those without NoVs (Table 1). 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%). The female-to-male ratio was 0.94 (95:101) in NoV-positive patients and 94.4% (185/196) of NoV infections were detected in outpatients. The clinical features of the NoV-associated AGE patients were fever (12/196, 6.1%), watery stool (194/196, 99%), abdominal pain (176/196, 89.8%), and vomiting (42/198, 21.4%). There was no statistically significant difference between NoV-positive and -negative AGE patients in terms of sex, age, setting, or clinical features.

The novel GII.P17/GII.17 variants accounted for most of the NoV-associated AGE cases, while the GII.4 Sydney_2012 strains were previously dominant in Huzhou. We compared the clinical futures of GII.17 and GII.4 virus in the AGE patients, None significant difference were found except for the age (Additional file 1: Table S1). The proportion of children (≤5 years), young (6–15 years), adults (16–40 years) and older adults (41–60 years) and elderly patients (≥60 years) in GII.17 cases were 1.1% (1/88), 1.1% (1/88), 62.5% (55/88), 23.9% (21/88) and 11.4% (10/88), respectively. GII.4 were detected in older adults with 63.6% (7/11)). None GII.4 virus infection were founded in young (6–15 years) group, which may be due to the limited number of cases in our study.

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.4 (1/117, 0.85%), GI.Pb/GI.6 (1/117, 0.85%), GII.Pe/GII.4 (4/117, 4.27%), GII.P17/GII.17 (36/117, 30.77%), GII.P12/GII.3 (1/117, 0.85%), GII.P21/GII.21 (1/117, 0.85%), and GII. P unassigned /GII.13 (1/117, 0.85%; Table 2). For the remaining 71 viruses, 42 RdRp sequences and 29 VP1 sequences were obtained, respectively. Each consisted of four genotypes: GII.Pe (n = 4), GII.P17 (n = 36, 30.77%), GII.P21 (n = 1), and GII.P3 (n = 1); and GII.4 (n = 4), GII.17 (n = 16, 13.68%), GII.13 (n = 5), and GII.3 (n = 4). Overall, the new GII.P17/GII.17 variants were the dominant genotype, accounting for 75.21% of the viruses, followed by the GII.Pe/GII.4 Sydney_2012 strains (11.11%).

The monthly detection rates of the GII.P17/GII.17 and GII.Pe/GII.4 viruses are shown in Fig. 1. In the winter of 2014–2015, the new GII.P17/GII.17 variant first emerged in Huzhou and began to replace GII.Pe/GII.4 Sydney_2012 as the predominant strain [10]. From January to March, the GII.P17/GII.17 variant continued to predominate and kept increasing; it then declined in April and May. From October to November, the GII.Pe/GII.4 variants (Sydney 2012) re-emerged and became dominant again, indicating that the epidemiological trend in Huzhou may have changed.

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.