Emergence and predominance of norovirus GII.17 in Huzhou, China, 2014–2015

NoVs were continuously detected throughout the year. From March 2014 to February 2015, 809 stool specimens collected from sporadic cases of acute gastroenteritis were received for NoV detection. NoV infection was found in all age groups tested (≤10y, 11–20y, 21–30y, 31–40y, 41–50y, 51–60y, and >60y). Of the 809 specimens, 193 (23.9 %) were positive for NoVs. Among these, GII NoVs comprised the greatest proportion, accounting for 93.3 % (180/193), with GII.4 and GII.17 the most commonly identified strains, GI NoVs comprised 6.2 % (12/193), and GI-GII co-infections comprised only 0.5 % (1/193). The highest NoV detection rate was during October 2014 and February 2015 (Fig. 1). However, GI NoV infection peaked from April to June 2014.

NoV-positive samples were genotyped by sequencing of regions A and C. Of the 193 norovirus positive strains, 101 were genotyped using both the capsid and polymerase genes, while 10 were genotyped using the polymerase gene alone, and 3 were genotyped using the capsid gene alone (Table 1). The most prevalent NoV genotypes were GII.Pe/GII.4 (38.6 %) and GII.P13/GII.17 (36.8 %). The GII.Pe/GII.4 genotype has been reported to predominate globally in many recent surveillance studies of NoV epidemics. The extremely high percentage of GII.17 was reported for the first time in the Huzhou area.

The incidence of sporadic acute gastroenteritis was higher during the 2014–2015 seasonthan previously (data not shown). An examination of the distribution of the detected NoV variants over time highlights that the emergent GII.P13/GII.17 variant steadily replaced GII.Pe/GII.4 (GII.4 Sydney 2012) as the predominant NoV in circulation in the Huzhou area (Fig. 1), which has not occurred previously. In February 2015, all positive isolates that could be genotyped were GII.P13/GII.17. This is the first report of (i) detection of GII.P13/GII.17 in the Huzhou area, China, and (ii) of a NoV genotype being detected in greater numbers than GII.4.

Of the114 strains genotyped using two genes, 89 strainscorresponded to suspected intergenotype recombinant strains (discordant capsid and polymerase genotypes). Notable among these were GII.Pe/GII.4 (n = 44, 49.4 %), GII.P13/GII.17 (n = 42, 47.2 %), GII.P12/GII.4, GII.P7/GII.6, and GI.Pb/GI.6 (n = 1, 1.1 %). PCR of the ORF1/ORF2 junction region confirmed the recombination of these strains. Among them, recombinants GII.Pe/GII.4, GII.P7/GII.6, and GI.Pb/GI.6 have been reported previously [8]. Recombinants GII.P13/GII.17 and GII.P12/GII.4 were newly detected in China (Table 1).

The identification of discordant genotype clustering for the partial polymerase and capsid gene sequences of NoV strains resulted in detection of two new recombinants in the Huzhou area: GII.P13/GII.17 and GII.P12/GII.4 (Fig. 2). Partial capsid gene sequence analysis of GII.17 NoVs showed that strains associated with GII.P13 polymerase segregated in a distinct cluster. This recombinant was first identified in October 2014, and thereafter rapidly became dominant type. Analysis of the GII.P12/GII.4 recombinant showed that it harbored a GII.12 polymerase gene and a GII.4 capsid gene. This strain was initially, and only, identified in January 2015. The recombinant strain GII.Pe/GII.4 is also named GII.4 Sydney 2012, which was first identified in 2012 and has been the dominant circulating strain in the Huzhou area for the past 2 years.

Institutional review board approval was not requested, as this study was part of a routine laboratory-based investigation. No human experimentation was conducted; all experiments were carried out using norovirus strains. The only human materials used were stool samples that had been sent to our laboratory for routine virological diagnosis of gastroenteritis. The results reported are associated with norovirus strains, with no patient information used.

This study was conducted at the First People’s Hospital in Huzhou, Huzhou, China as part of the regional NoV gastroenteritis surveillance program. Between March 2014 and February 2015, a total of 809 fecal samples were collected from outpatients with clinical symptoms of acute gastroenteritis. Acute gastroenteritis was defined as the presence of one or more of the following symptoms: nausea, vomiting, and/or diarrhea. Fecal samples were collected during the acute phase of infection. Acute phase means the period of time when patient have the above symptoms. All samples were obtained in a sterile container and were transported to our laboratory for immediate storage at −70 °C prior to analysis.

Fecal samples were prepared as 10 % suspensions in phosphate-buffered saline (pH 7.4). Total nucleic acid was extracted from a 200 μL aliquot of fecal suspension using a QIAamp viral RNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The RNA extracts were subjected directly to reverse transcription (RT)-PCR (Polymerase Chain Reaction) or stored at −70 °C. Real-time RT-PCR (qPCR) was performed using primers and probes described previously [8]. RT-qPCR was carried out using a One Step PrimeScript RT-PCR Kit (Perfect Real Time) (TaKaRa, Dalian, China). The reaction was conducted with an initial RT step at 42 °C for 30 min, followed by 95 °C for 5 min and 40 cycles of qPCR at 95 °C for 5 s and 55 °C for 35 s.

Samples that tested positive for NoV by RT-qPCR were analyzed using two PCRs directed at region A of ORF1 (the 3′end of the polymerase gene ) using the primers JV12/ JV13, and at region C in ORF2 ( the 5′ end of the capsid gene ) using the primers G1SKF/G1SKR for GI and G2SKF/G2SKR for GII [35, 36]. RT-PCR was carried out using a One Step RNA PCR Kit (TaKaRa) with the following amplification conditions: RT at 50 °C for 30 min and denaturation at 95 °C for 3 min, followed by 35 cycles of 30 s at 94 °C, 30 s at 48 °C, and 30 s at 72 °C. A final extension step for 10 min at 72 °C was performed. After amplification, the PCR products were visualized by agarose gel electrophoresis. To analyze recombinant strains (capsid and polymerase of different genotypes), sequences covering the overlap between ORF1 and ORF2 were amplified by RT-PCR using primers JV12 and G1SKR/G2SKR when phylogenetic analyses indicated discordant clustering for partial sequences of the polymerase and capsid genes in the same sample. RT-PCR was carried out using a One Step RNA PCR Kit (TaKaRa). RT-PCR conditions were as follows: RT at 50 °C for 30 min and denaturation at 95 °C for 2 min, followed by 35 cycles of 30 s at 94 °C, 30 s at 48 °C, and 1 min at 72 °C. A final extension step was performed for 10 min at 72 °C. The PCR products were purified using a QIAquick PCR purification kit (Qiagen, Leusden, The Netherlands) according to the manufacturer’s instructions. The amplicons were sequenced by TaKaRa Biotechnology (Dalian).

Genogroup and genotype were assigned using the NoV Noronet typing tool (http://​www.​rivm.​nl/​mpf/​norovirus/​typingtool) [6] and the strains were named according to the time of isolation. The phylogenetic analysis was conducted using MEGA 5 [37]. Phylogenetic trees with 1000 bootstrap replicates were generated using the neighbor-joining method by employing molecular evolutionary genetics analysis (MEGA), version 5.0. Reference strains were downloaded from GenBank.

The GenBank accession numbers for sequences obtained in this study are KP995319-KP995341, KR361528-KR361548.

The data sets supporting the results of this article is included within the article