Molecular Epidemiology of Genogroup II Noroviruses Infection in Outpatients with Acute Gastroenteritis in Nanjing, China (2010–2013)

Objective. Human noroviruses (NoVs) of genogroup II are the most common strains detected in sporadic cases of acute nonbacterial gastroenteritis in outpatients in Nanjing. To gain insight into the molecular epidemiology of GII strains, we analyzed 75 positive NoV cases from 2010 to 2013. Methods. The sporadic cases were detected by real-time PCR with specific primers and probes to human NoVs of genogroup I or II, human sapovirus, human rotavirus, human astrovirus, and human enteric adenovirus. Human NoVs of genogroup II were further studied by VP1 amplification (RT- PCR), cloning, sequencing, and phylogenetic analysis. Results. Rotavirus and human NoVs were more frequently detected in all the cases from 2010 to 2013. Human NoVs infection was more frequent since 2011 and more frequent than rotavirus infection after 2012. Out of the 75 NoV cases of genogroup II, there were 5 GII.6, 11 GII.3, and 59 GII.4. Of the 59 GII.4, 27 cases were previous GII.4.2006b strains that circulated between 2010 and 2012; while 32 cases were the newly emerging GII.4 strains GII.4.2012 from 2011 to 2013. Conclusion. Our data confirm other studies on the rapid emergence and displacement of highly virulent GII.4 strains.

Noroviruses (NoVs), members of the genus Norovirus of the family Caliciviridae, have a 7.5 kb to 7.7 kb singlestranded genome of positive-sense RNA which contains three open reading frames (ORFs). ORF1 encodes for the nonstructural proteins with high conservation. ORF2 codes for the major capsid protein (VP1) with the highest degree of sequence variability in the genome. ORF3 codes for the minor capsid protein (VP2) with high variability. Similar to other caliciviruses, VP1 of NoVs is most important in strain diversity. According to VP1 sequencing analysis, NoVs are classified into five distinct genogroups (genogroup I [GI] to genogroup V [GV]) with at least 32 genetic clusters [6][7][8][9]. Of these, GII.4 strains have been the predominant strains over the last decade [10]. Furthermore, the GII.4 lineage has a 1.7fold higher rate of evolution on average within the capsid sequence and a greater number of nonsynonymous changes compared to other NoVs [11]. As a result, there should be a new GII.4 strain emergence every 2 to 3 years [10]. However, only a little has been known about the dominant circulating genotype in Nanjing, China.
To understand the epidemiologic patterns of GII sporadic cases in Nanjing, we analyzed NoV sporadic specimens collected by Nanjing Municipal Center for Disease Control and Prevention from 2010 to 2013.

Real-Time RT-PCR.
Viral RNA was extracted in a 10% stool suspension by SuperPure System-32 automated nucleic acid extraction and purification system (Formosa Plastic Group, Taiwan) in accordance with the manufacturer's instructions. The stool suspension was 200 L and the nucleic acid elution volume was 100 L. The nucleic acid for detection of enteric adenovirus was treated with ribonuclease at 37 ∘ C for 30 mins by RNase cocktail (Ambion (Europe) Ltd., Cambridgeshire, United Kingdom). For detecting human calicivirus (HuCV), rotavirus, human astrovirus, and enteric adenovirus, we applied primers and TaqMan probes described in the literature (Table 1). For NoV GI and GII, these primers and probes targeted NoV sequences at the ORF1-ORF2 junction, a highly conserved region of the NoV genome [12]. For human sapovirus, the primers and probe targeted a conserved region of the RNA polymerase [13]. For rotavirus, primers and probes detected rotavirus nonstructural proteins 3 [14]. For human astrovirus, primers and probes focused on the 3 end consensus region of the astrovirus genome [15]. For enteric adenovirus, the primer pair and probes were targeting conserved segments of the hexon gene of adenovirus DNA genome [16]. Amplification was carried out in a 25 L reaction volume using the Invitrogen superscript III one-step q-RT-PCR system containing 5 l of extracted sample, 0.05 M of probe, and 0.5 M of each primer. Reverse transcription was performed for 30 mins at 50 ∘ C. Platinum Taq polymerase was activated at 95 ∘ C for 2 mins, and 40 cycles of PCR were performed at 95 ∘ C for 20 s and 60 ∘ C for 30 s using an ABI 7500 FSAT SDS. Standard mode was set to collect the fluorescence of each cycle at 60 ∘ C for 30 s. The amplification results were determined as positive when Ct values were ≤37 and there was a logarithmic growth. The experiment should be repeated when Ct values were above 37 and there was a logarithmic growth curve; the amplification was determined as positive if there was still a logarithmic growth curve and negative if there was no logarithmic growth curve.

RT-PCR and Sequencing for NoVs of Genogroup II .
A 252-nucleotide (nt) region of the 3 end of the VP1 gene of 75 strains was amplified with primer set using the Qiagen onestep RT-PCR kit (Qiagen Inc., Valencia, CA). The sequencing coverage was 1×. The reaction was conducted with an initial RT step at 50 ∘ C for 30 mins, followed by PCR activation at 95 ∘ C for 5 mins, then 35 cycles of amplification (20 s at 94 ∘ C, 30 s at 56 ∘ C, and 30 s at 72 ∘ C), and a final extension step for 7 mins at 72 ∘ C in a GeneAmp PCR system 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA). The RT-PCR products were purified in 2% agarose gels and cloned into pGEM-T Easy vector (Promega, Wisconsin, USA). The restricted plasmids of 2011, 2012, and 2013 or PCR products of 2010 were purified by PEG precipitation and washed with 70% ethanol. The restricted plasmids or PCR products were bidirectionally sequenced on an ABI 3730XL DNA sequencer (Applied Biosystems, Foster City, CA, USA) using the BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems); as a result, one direction sequencing entirely covered another direction except for samples of 2010.

Sequence Editing and Analysis.
All sequences generated in this study were edited with BioEdit 7.0.1 [17] and analyzed with MegAlign 5.1 [18]. Phylogenetic tree was draw with the MEGA 5.0 software [19]. For the MEGA analysis, the neighbor-joining method [20] was used for phylogenetic reconstruction, with bootstrap analysis of 1000 replicates. The evolutionary distances were computed using the Kimura 2parameter method [21]. The translated amino acid sequences were aligned by the Clustal W method algorithm in the MEGA 5.0. Phylogenetic trees were displayed with Tree-View software [22].

Nucleotide Sequence Accession Numbers.
The VP1 sequences identified in this study were submitted to GenBank under the following accession numbers: KJ528320-KJ528394. In these positive strains, the majority (90%) was caused by rotavirus and HuCV each year. Before 2011, there were more cases caused by rotavirus strains than that caused by HuCV strains. During the year 2012, there was almost the same number of cases caused by these two pathogens. After 2012, more cases were caused by HuCV strains than by rotavirus strains (Figure 1(a)). For cases induced by the HuCV, the majority was caused by NoVs of genogroup II throughout these four years ( Figure 1(b)).

Discussion
Our previous studies showed that the incidence rate of viral diarrhea was higher than the bacterial prevalence    [9]. And VP1 exhibits the highest degree of sequence variability in the genome [26]. Then we analyzed the sequence amplified by RT-PCR with primers targeted to VP1 epitopes of NoVs of genogroup II. The 75 strains of GII viruses showed different strain diversities and could be grouped into at least 3 distinct subclusters, namely, GII.  4.2006b (namely, group 2) between 2010 and 2012, respectively. A study in Shanghai showed that the predominant norovirus genotype was GII.4 and the GII.4-2006b variant was the predominant subtype both in inpatients and outpatients between 2006 and 2011 [27]. Another report in Nanjing also found that GII. 2006b is the dominant genotype in children from July 2010 to June 2011 [28]. The newly emerging strains norovirus Hu/GII.4/Sydney/NSW0514/2012/AU were first found in 2011 in Nanjing, which was much earlier than neighborhood regions [29,30], and no other strains but the newly emerging strains were found prevalent in Nanjing in 2013, which implied the strong invasion ability of the newly emerging strains and poor human resistance to them. These results also showed evolutionary evidence for the emergence of new GII.4 subclusters (2012 sydney/AU) that gradually displaced previous GII.4 viruses in the population (2006b).
Despite the fact that the studies on NoVs for understanding antigenic sites and immunological functions are hampered by a lack of suitable animal model, some significant advances have been achieved by using virus-like particles [31] or monoclonal antibodies [32]. Besides, a number of norovirus capsid sequences have been analyzed by the evolutionary trace method and some capsid protein