Human norovirus (HuNoV) is a major causative agent of gastroenteritis in humans (Green, 2013). The HuNoV genogroup II (GII), in particular, is frequently detected in outbreaks. The HuNoV GII strains can be classified into 22 genotypes (Kroneman et al., 2013). Moreover, the most worldwide prevalent HuNoV GII genotypes belong to GII genotype 2 (GII.2), GII.3, GII.4, GII.6, and GII.17 (Centers for Disease Control and Prevention. CaliciNet Data [cited 2016])¹. Since national surveillance began, nearly 3 million cases of NoV gastroenteritis have been recorded, and Japan was experiencing its second most serious norovirus outbreak during November 2016 to February 2017 (National Institute of Infectious Diseases. Japan. Infectious Gastroenteritis. [cited 4th April 2017, in Japanese])² Importantly, HuNoV GII.2 emerged as a major cause of this outbreak in Japan, although the GII.4 strains were the most prevalent genotype during the past 10 years (National Institute of Infectious Diseases. Japan. Flash report of norovirus in Japan [cited 4th April 2017, in Japanese])³.

Very recent studies suggested that the evolutionary patterns of human and animal NoV genotypes are distinct (Kobayashi et al., 2015, 2016). Although all viral proteins may act as antigens, the HuNoV VP1 protein is also involved in viral infection. Furthermore, HuNoV frequently experiences recombination at the ORF1/ORF2 junction, resulting in new chimera viruses with different types of the RNA-dependent RNA polymerase (RdRp) genes and capsid (VP1) genes. Most studies have focused on the molecular evolution of HuNoV GII.4. Only a few examined that gene in other HuNoV genotypes, including GII.2. To gain insight into this process, we examined the molecular evolution of the GII.2 RdRp and VP1 genes, including chimera viruses, based on the full genome analyses of those detected in Japan over a period of 10 years (2004–2015 seasons).

To investigate the molecular evolution of the HuNoV VP1 and RdRp genes, 950 stool specimens were collected from various areas (13 prefectures) of Japan during the 2004–2015 seasons. These samples were obtained from patients with acute gastroenteritis due to HuNoV infections, in compliance with the Food Sanitation Law and the Law Concerning the Prevention of Infections and Medical Care for Patients of Infections of Japan. The personal data related to these samples were anonymized. RNA was extracted from 10% PBS suspensions of the specimens, and the HuNoV genomes were comprehensively analyzed by next-generation sequencing as described (Matsushima et al., 2015). Of 950 samples, the complete genome sequences of 538 strains were obtained (a success rate of 57%). Next, HuNoV genotypes were confirmed with the Norovirus Typing Tool (Version1.0), based on the nucleotide sequences of RdRp and VP1 genes as described by Kroneman et al. (2011). GII.2 strains were selected from these all genotyped strains, and then a few of strains having the undetermined base sequences (e.g., N, Y, R, and V) were omitted. Finally, 51 GII.2 strains were used for evolutionary analyses for the present study (Supplementary Table 1). The obtained nucleotide sequences for the GII.2 strains were deposited in GenBank under the accession numbers LC209431 to LC209481.

Time-scale evolutionary analyses were performed using the Bayesian Markov Chain Monte Carlo method (MCMC) with the BEAST package v1.8.3 (Drummond and Rambaut, 2007) and Tracer⁴ as a demographic model. Substitution models were calculated with Kakusan4 (Tanabe, 2011). The substitution model for the VP1 or the RdRp gene was the GTR-Γ or GTR-Γ invariant model, respectively. Based on Akaike's Information Criterion for MCMC values, we used the random local clock as a clock model, and used the logistic growth model (VP1 gene) or the constant size model (RdRp gene) as a tree model. Convergence was evaluated with an effective sample size (acceptable more than 200). The MCMC chain length was 3 × 10⁸ steps with sampling every 1,000 steps for the MCMC tree of the VP1 gene. To exactly estimate the evolutionary rates and topologies of the MCMC tree of the RdRp gene, we bound two independent data of the MCMC chains⁵. The MCMC chain length was 2 × 10⁸ steps and 4 × 10⁸ with sampling every 5,000 steps for the MCMC tree of the RdRp gene. Statistical analyses were performed with the Welch's t-test in Excel 2013.

Here we report the molecular evolution of the VP1 and RdRp genes in HuNoV GII.2. Our main findings and hypothesis are as follows. (1) Four genotypes of GII.2 (GII.P2-GII.2, GII.P16-GII.2, GII.P12-GII.2, and GII.Pe-GII.2) were detected in Japan in 2004–2015. (2) A common ancestor of the current GII.2 virus strains circulated around 1956. (3) VP1 gene evolution seems to depend on the RdRp gene. The VP1 gene in a prevalent HuNoV genotype GII.2 might evolve uniquely by transfer of the RdRp gene.

This study protocol was approved by the National Institute of Infectious Diseases Ethics Committee (No. 532).

FM designed and performed the research, analyzed the data and wrote the manuscript. KN, YD, and KH performed the research and analyzed the data. FM, SY, YU, MS, MI, NS (Sakon), NS (Shigemoto), RO, and AO contributed samples and analyzed the data; and HK and KK designed and supervised the research, analyzed the data, and wrote the manuscript. All authors contributed, read, and approved the manuscript.

This work was partly supported by a commissioned project for Research on Emerging and Re-emerging Infectious Diseases from Japan Agency for Medical Research and Development, AMED.