Genetic Analysis of Reemerging GII.P16-GII.2 Noroviruses in 2016–2017 in China

Genetic Analysis of Reemerging GII.P16-GII.2 Noroviruses in 2016–2017 in China Abstract Background During 2016–2017, the previously rare GII.P16-GII.2 norovirus suddenly emerged as the predominant genotype causing gastroenteritis outbreaks in China and other countries. Its origin, phylodynamics, and mechanism behind the predominance remain unclear. Methods Bayesian phylogenetic analyses were performed on 180 full capsid and 150 polymerase sequences of 2016–2017 GII.P16-GII.2 noroviruses in China, and those for all publicly available GII.P16 and GII.2 sequences. Saliva-based histo-blood group antigen (HBGA) binding assays and crystal structural analysis were conducted by using the P proteins of 2016–2017 GII.P16-GII.2 noroviruses. Results The reemerging GII.P16-GII.2 norovirus showed a rapid genetic diversification after its emergence in 2012–2013. The antigenicity and HBGA binding profile of the early 2016–2017 and pre-2016 GII.2 noroviruses were similar. A further variant with a single Val256Ile mutation and the conventionally orientated Asp382 in the VP1 protein showed an expanded HBGA-binding spectrum. Mutations on the surface of polymerase that could alter its function were seen, which may help to accelerate the VP1 gene evolution to 5.5 × 10–3 substitutions per site per year. This virus can be traced back to Pearl River Delta, China. Conclusions Our findings provide new insights into GII.2 norovirus epidemics and highlight the necessity of enhanced global surveillance for potential epidemics of rare-genotype noroviruses. norovirus, reemerging GII.P16-GII.2, phylogenetic analysis, histo-blood group antigen, crystal structure Noroviruses, within the family Caliciviridae, are a major cause of acute gastroenteritis (AGE) worldwide [1, 2]. The norovirus genome is composed of 3 open reading frames (ORFs), among which ORF1 encodes several nonstructural proteins, including the viral RNA-dependent RNA polymerase (RdRp), while ORF2 and ORF3 encode the major and the minor structural proteins (VP1 and VP2, respectively). Noroviruses are divided into 7 genogroups (GI–GVII) that are further divided into >30 genotypes [3], among which GI, GII, and GIV noroviruses infect humans [4]. The VP1 protein contains 2 moieties, the N-terminal shell (S) and C-terminal protruding (P) domains. The P domain, which can be further divided into P1 and P2 subdomains, is responsible for host cell attachment and antigenicity [5]. Noroviruses recognize histo-blood group antigens (HBGAs) on the host cell surface, which are important for host susceptibility to infection [6, 7]. HBGAs are complex glycans that are divided into A/B/H and Lewis types. The complex interactions between diverse noroviruses and the polymorphic HBGAs are believed to affect the host range and prevalence of the former [6–9]. During the 2016–2017 winter season, the reemergence of GII.P16-GII.2 norovirus caused a rapid increase of norovirus outbreaks in China (mainland, Hong Kong, and Taiwan), Japan, Germany, France, and the United States [10–16], replacing the previously predominated GII.4 and GII.17 noroviruses [17–19]. The GII.P16-GII.2 norovirus outbreaks were rarely reported previously, except during the 2009–2010 season in Osaka, Japan [20]. The reason for the predominance of this virus remains unclear, although one phylogenetic study suggested that RdRp may contribute to its epidemic potential [21]. Here, comprehensive analyses of viral phylogenetics, HBGA-binding profiles, and P domain structures would provide important insights into the origin and evolutionary dynamics of the reemerging GII.P16-GII.2 norovirus and the possible mechanisms behind its predominance. METHODS Disease Definition, Sample Collection, and Ethics Statement An AGE outbreak is defined as at least 20 individuals with vomiting and/or diarrhea within 1 week, associated with a common source of infection, as described previously [10]. Feces or vomitus specimens were tested for noroviruses by the local Center for Disease Control and Prevention (CDC) from Guangdong, Guangxi, Fujian, Hunan, Chongqing, Sichuan, Jiangsu, Shandong, Beijing, Liaoning, Jilin, and Heilongjiang. At least 3 norovirus-positive samples from each outbreak were transported to China CDC on ice for further study. Informed consent was obtained from patients and the parents of all children who provided specimens. The production of New Zealand rabbit antiserum was approved by the Animal Care Welfare Committee of National Institute for Viral Control and Prevention, China CDC (20160715023). Amplification of the VP1 and RdRp Genes and Complete Genomes Norovirus-positive samples were diluted 1:10 (w/v) with phosphate-buffered saline (PBS). Total viral RNA was extracted and amplified targeting the ORF1/ORF2 genes by 1-step reverse-transcription polymerase chain reaction (RT-PCR) for norovirus genotyping [10, 22]. At least 1 sample from each GII.P16-GII.2 norovirus outbreak was subjected to amplification of the capsid and RdRp genes by nested RT-PCR, as described previously [10]. One or more complete genomes of GII.P16–GII.2 strains from every city and different months were randomly selected and sequenced, as previously described [10]. Sequencing and Phylogenetic Analysis All nucleotide (nt) and amino acid (aa) sequence alignments were performed using Bioedit and MEGA (version 7.0) software. The most recent common ancestors of GII.2 VP1 and GII.P16 RdRp gene sequences were estimated using the strict molecular clock, GTR+G substitution, and Bayesian skyline coalescent models in BEAST software (version 1.8.2). Markov chain Monte Carlo sample chains were run for 4.8 × 108 and 4 × 108 steps for the VP1 and RdRp genes, respectively. Maximum-likelihood (ML) phylogenetic trees of the VP1 gene sequences were constructed using PhyML (version 3.1) software. The convergence of parameters was evaluated by Tracer (version 1.6) software. The Markov chain Monte Carlo Bayesian phylogenetic tree was reconstructed using Tree Annotator (version 1.8.3) software. Expression and Purification of P Particles and Dimers The DNA sequences of the P domain of GZ20435 and BJSMQ were amplified, and that of the prototype GII.2 strain Snow Mountain virus (SMV) (AY134748.1) was chemically synthesized. They were cloned into the PGEX-6P-1 vector and expressed in Escherichia coli BL21(DE3). The proteins were purified by glutathione-sepharose 4B and then a Superdex 20010/300GL gel filtration column. To produce antisera, New Zealand rabbits were immunized with 3 purified P particles. Saliva-Binding Assay The saliva samples from 225 individuals involving in 3 reemerging GII.P16-GII.2 norovirus outbreaks were collected in Guangdong province. HBGA phenotypes of these saliva samples were determined by enzyme immunoassays using the monoclonal antibodies specific to A, B, H1, Lea, Leb, Lex, and Ley, respectively, as previously described [19]. A total of 214 boiled saliva samples were diluted by 1:1000 with PBS, and used to coat 96-well microtiter plates at 4°C overnight. After blocking with 5% nonfat milk, purified P proteins (10 µg/mL) of BJSMQ, GZ20435, and SMV were added to the plates. Next, the corresponding rabbit anti-P-particle antibodies (GZ20435 at 1:2000, BJSMQ at 1:2500, SMV at 1:32000) were added, and bound antibodies were detected using a horseradish peroxidase–conjugated goat anti-rabbit secondary antibody (1:20000) (MP Biomedicals). The reaction was developed using a 3,3ʹ,5,5ʹ-tetramethylbenzidine kit (Invitrogen), and was measured at 450 nm using a plate reader. Oligosaccharide-Binding Assay Synthetic oligosaccharide-based binding assays were conducted for the synthetic-oligosaccharide-polyacrylamide-biotin conjugates A, B, H1, H2, H3, Lea, Leb, Lex, and Ley, Neu5Ac, Neu5Gc, sialyl-Lex, type 1, 2 precursor (GlycoTech), as previously described [23]. Protein Crystallization and Data Collection Crystals were grown in hanging drops of 1 µL BJSMQ P dimer (~10 mg/mL) and 1 µL reservoir solution (0.2 M ammonium sulfate, 0.1 M 2-(N-morpholino)ethanesulfonic acid [pH 6.5], and 30% [wt/vol] polyethylene glycol MME 5000 at 18°C. Crystals were transferred to 20% (vol/vol) ethylene glycol in mother solution as a cryoprotectant, and flash-frozen. Data collection, structure solving, and refinement of unbound structures were conducted as described previously [23]. RESULTS Characteristics of 2016–2017 Norovirus Outbreaks From January 2016 to July 2017, 277 norovirus outbreaks confirmed (see Materials and Methods for definition) in China. At least 3 samples per outbreak were tested to confirm norovirus infection, followed by sequencing and genotyping. The norovirus sequences of each outbreak shared 99%–100% nt identities. In November 2016, the number of norovirus outbreaks per month increased sharply, which peaked at 63 outbreaks in December 2016. Genotyping showed that 181 of 208 (87%) outbreaks during November 2016 to June 2017 were caused by GII.P16-GII.2 noroviruses (Figure 1A). The first outbreak of GII.P16-GII.2 norovirus (named GZ20435) was identified in Guangdong Province on 21 September 2016. This virus subsequently caused outbreaks throughout China, the majority of which occurred in southern China in late 2016, with gradual northward expansion in 2017 (Figure 1B). Figure 1. View largeDownload slide Characteristics of the norovirus outbreak in China during the 2016–2017 winter season. A, Monthly distributions of reemerging GII.P16-GII.2, GII.P2-GII.2, and other genotypes that caused norovirus outbreaks in China from January 2016 to June 2017. B, Geographic distribution of the outbreaks caused by the reemerging GII.P16-GII.2 norovirus in China. Chronological order of the outbreaks of reemerging GII.P16-GII.2 norovirus is indicated by ● (2016) and ▲ (2017). The outbreak frequency in different provinces and regions are classified into high, medium, and low based on the incidence numbers. Figure 1. View largeDownload slide Characteristics of the norovirus outbreak in China during the 2016–2017 winter season. A, Monthly distributions of reemerging GII.P16-GII.2, GII.P2-GII.2, and other genotypes that caused norovirus outbreaks in China from January 2016 to June 2017. B, Geographic distribution of the outbreaks caused by the reemerging GII.P16-GII.2 norovirus in China. Chronological order of the outbreaks of reemerging GII.P16-GII.2 norovirus is indicated by ● (2016) and ▲ (2017). The outbreak frequency in different provinces and regions are classified into high, medium, and low based on the incidence numbers. Sequence Analysis of Reemerging GII.P16-GII.2 Noroviruses The complete genomes of 60 reemerging GII.P16-GII.2 viruses have been sequenced, but only a few complete genomes of previous GII.P16-GII.2 noroviruses within a short period of time are available in GenBank, hindering genome-wide analysis. The genome sequence of the representative reemerging GII.P16-GII.2 strain SZ123 (collected at October 2016 in Guangdong) showed the highest sequence identity (95%) to the 2011–2012 GII.P16-GII.2 strain HS255. Genome scanning of SZ123 against HS255 suggested that its ORF1, VP1, and VP2 genes have undergone coevolution. The VP1 aa sequence of the first representative reemerging GII.P16-GII.2 norovirus strain GZ20435 shared a 100% identity with the GII.2 strain Vaals8/2005/NLD (BAG68716.1). Although the HBGA binding site (HBS) of all known GII.2 viruses remained conserved, several aa mutations in GZ20435 adjacent to the site I of the HBS were found, against that of SMV (Figure 2A). In the nonstructural proteins of the reemerging GII.P16-GII.2 strain, 11 unique nonsynonymous substitutions were detected compared to those of pre-2015 GII.P16 strains (Figure 2B), while 5 amino acid mutations were located in the polymerase, with 3 near locations known to impact polymerase kinetics or fidelity that were recently explained in the GII.P16-GII.4 Sydney 2012 strains [24]. A residue mutation in 3C-like, P48 protein, and NTPase, and 2 mutations in P22 protein, as well as an insertion in P48 protein, are shown in Figure 2B. All of the sequences were deposited in GenBank under accession numbers MG745985–MG746376. Figure 2. View largeDownload slide Sequence analysis of reemerging GII.P16-GII.2 noroviruses. A, The sequence analysis of histo-blood group antigen (HBGA)–binding interface of the GII.2 major structural protein VP1 protein. The HBGA-binding interfaces are composed of 3 amino acid components that are indicated with sites I, II, and III by gray square frame. The only changed amino acid in VP1 between BJSMQ and the GZ20435 is Val256Ile (highlighted by the black frame). B, List of amino acid changes in nonstructural protein of the emerging GII.P16 norovirus compared with those of the previous GII.P16 norovirus. The black background residue mutation in the emerging GII.P16 noroviruses including reemerging GII.P16-GII.2 (2016–2017) and GII.P16-GII.4/GII.3 (2015–2017). The asterisk denotes residue deletion in the P48 protein of GII.P16 noroviruses. Figure 2. View largeDownload slide Sequence analysis of reemerging GII.P16-GII.2 noroviruses. A, The sequence analysis of histo-blood group antigen (HBGA)–binding interface of the GII.2 major structural protein VP1 protein. The HBGA-binding interfaces are composed of 3 amino acid components that are indicated with sites I, II, and III by gray square frame. The only changed amino acid in VP1 between BJSMQ and the GZ20435 is Val256Ile (highlighted by the black frame). B, List of amino acid changes in nonstructural protein of the emerging GII.P16 norovirus compared with those of the previous GII.P16 norovirus. The black background residue mutation in the emerging GII.P16 noroviruses including reemerging GII.P16-GII.2 (2016–2017) and GII.P16-GII.4/GII.3 (2015–2017). The asterisk denotes residue deletion in the P48 protein of GII.P16 noroviruses. Evolutionary Analysis of the GII.2 VP1 and GII.P16 RdRp Genes We used 180 full VP1 gene sequences of the reemerging GII.P16-GII.2 noroviruses identified in our study and 159 GII.2 VP1 sequences available in GenBank to generate a time-scale evolutionary tree. It showed that all reemerging GII.P16-GII.2 viruses clustered together (Figure 3A) but relatively close to the GII.P16-GII.2 strains HS255/USA/2011 (KJ407074.2) and Miyagi1/Japan/2012 (LC145787.1). Three major clades of GII.2 strains were observed: the reemerging GII.P16-GII.2 (2016–2017), GII.P16-GII.2 (2008–2015), and GII.P2-GII.2 (2004–2010) clades. Figure 3. View largeDownload slide Markov chain Monte Carlo Bayesian phylogenetic trees of GII.2 and GII.P16 strains. A, Trees were reconstructed using 339 major structural protein (VP1)-coding nucleotide sequences of 339 GII.2 strains. The isolated area or countries of the reemerging GII.P16-GII.2 strains in subclade SC2 are denoted by the color boxes: mainland China (no box), Taiwan (red), Australia (black), Japan (blue), and Germany (purple). B, Tree of RNA-dependent RNA polymerase (RdRp)–coding genes of GII.P16 strains reconstructed by 358 RdRp nucleotide sequences. The GII.2 and GII.P16 strains in the VP1 and RdRp trees are colored according to their polymerase genotype and capsid genotype, respectively. GII.2 strains whose polymerase genotype was not available are labeled GII.NA-GII.2 in the VP1 tree. Estimated divergence times are shown on the ancestral nodes. Phylogenetic clustering is indicated by colors and names. Scale bar is actual time (years). Figure 3. View largeDownload slide Markov chain Monte Carlo Bayesian phylogenetic trees of GII.2 and GII.P16 strains. A, Trees were reconstructed using 339 major structural protein (VP1)-coding nucleotide sequences of 339 GII.2 strains. The isolated area or countries of the reemerging GII.P16-GII.2 strains in subclade SC2 are denoted by the color boxes: mainland China (no box), Taiwan (red), Australia (black), Japan (blue), and Germany (purple). B, Tree of RNA-dependent RNA polymerase (RdRp)–coding genes of GII.P16 strains reconstructed by 358 RdRp nucleotide sequences. The GII.2 and GII.P16 strains in the VP1 and RdRp trees are colored according to their polymerase genotype and capsid genotype, respectively. GII.2 strains whose polymerase genotype was not available are labeled GII.NA-GII.2 in the VP1 tree. Estimated divergence times are shown on the ancestral nodes. Phylogenetic clustering is indicated by colors and names. Scale bar is actual time (years). We also constructed a time-scale evolutionary tree of the partial GII.P16 RdRp nt sequences, including 208 from GenBank and 150 obtained in this study (Figure 3B). The reemerging GII.P16-GII.2 strains clustered closely with GII.P16-GII.3/GII.4_Sydney2012 strains (2015–2017). Together they formed an emerging GII.P16 cluster, which was subdivided into 3 subclades (I–III). Consistent with that of the VP1 sequence phylogenetic analysis, they also displayed a relationship with the 2011–2012 GII.P16-GII.2 strains. The divergence times of the phylogenetic clusters in the VP1 and RdRp trees were comparable, and showed that the most recent common ancestor of the reemerging GII.P16-GII.2 norovirus may have appeared in 2012–2013 (Figure 3A and 3B) and then formed 3 subclades (SC1–SC3). Moreover, the strains of SC1 diverged from a common ancestor of SC2 at 2014, while SC3 diverged from the ancestor at 2012. HBGA-Binding Profile of GII.2 Norovirus Like SMV, the P proteins of GZ20435 bound to B- and AB-type saliva, but BJSMQ (collected at December 2016, Beijing) gained extra binding to A-type saliva (Figure 4). Comparing BJSMQ P domain sequences with those of the GZ20435, we found a single mutation Val256Ile in the BJSMQ P domain (Figure 2A). Synthetic oligosaccharide-based binding assays were also performed, but no binding signals to type A and B oligosaccharide were observed. Figure 4. View largeDownload slide Saliva-binding profiles of the GII.2 P domain proteins of GZ20435, BJSMQ, and Snow Mountain virus (SMV). GZ20435 (A) and BJSMQ (B) represent the reemerging GII.P16-GII.2 viruses identified in Guangdong Province and Beijing City, respectively, while SMV (C) represents GII.2 strain prototype. The 3 strains bound to A-, B-, O-, and O–-type saliva. Two hundred fourteen saliva samples with known secretor (A, B, O) and nonsecretor (O–) types were used. The cutoff value of positive samples was determined as 0.15. Abbreviations: HBGA, histo-blood group antigen; OD, optical density. Figure 4. View largeDownload slide Saliva-binding profiles of the GII.2 P domain proteins of GZ20435, BJSMQ, and Snow Mountain virus (SMV). GZ20435 (A) and BJSMQ (B) represent the reemerging GII.P16-GII.2 viruses identified in Guangdong Province and Beijing City, respectively, while SMV (C) represents GII.2 strain prototype. The 3 strains bound to A-, B-, O-, and O–-type saliva. Two hundred fourteen saliva samples with known secretor (A, B, O) and nonsecretor (O–) types were used. The cutoff value of positive samples was determined as 0.15. Abbreviations: HBGA, histo-blood group antigen; OD, optical density. Structural Analysis of the P Domain of GII.P16-GII.2 Norovirus The crystal structure of the BJSMQ P dimer was solved at 1.2 Å resolution (Protein Data Bank code: 5YSX) (Figure 5A). Superposition of the BJSMQ P domain structure with that of SMV [25] revealed a high degree of structural similarity (root mean square deviation, 0.26) (Figure 5B). The BJSMQ P domain did not bind HBGA oligosaccharides, preventing their co-crystallization with an HBGA. While the BJSMQ P domain shares conserved amino acids constituting HBS (Figure 2A), P domain structural superposition among the BJSMQ, SMV, and a closely related GII.12 [25] indicated that the orientations of Asp382, an important HBS component of BJSMQ, changes dramatically compared with that of SMV. In SMV, Asp382 points away from the modeled HBGA [25], whereas Asp382 in BJSMQ restores its traditional orientation, pointing toward the modeled HBGA (Figure 5C and 5D). Since Ile256 in the BJSMQ P1 domain is far away from the HBS, its role in affecting the HBS function might be smaller than the orientation of the Asp382. Figure 5. View largeDownload slide Structural analysis of the reemerging GII.P16-GII.2 strain BJSMQ (Protein Data Bank [PDB] code: 5YSX) P domain. A, Overall structure of BJSMQ P domain shown as cartoon representation, in which P1 and P2 subdomains are shown in green and red, respectively. B, Structural comparison between BJSMQ P domain and GII.2 Snow Mountain virus (SMV) (PDB code: 4RPB) P domain (SMV P1 subdomain, yellow; P2 subdomain, blue). C and D, Structural comparisons of the histo-blood group antigen (HBGA) binding site among BJSMQ, SMV, and a closely related GII.12 virus that binds HBGA, highlighting the differently orientated Asp382 between BJSMQ and SMV in comparison with the GII.12 norovirus. All P domains are in cartoon representations with the conserved Asp (Asp382 in BJSMQ and SMV, Asp375 in GII.12 norovirus) in stick representation. GII.12, SMV, and BJSMQ are shown in green, purple, and yellow, respectively. The modeled HBGA based on the GII.12 P domain-HBGA-trisaccharide complex (PDB code: 3R6K) are in stick representation with its 2 galactoses (GAL) in yellow and the fucose (FUC) in cyan, respectively. Figure 5. View largeDownload slide Structural analysis of the reemerging GII.P16-GII.2 strain BJSMQ (Protein Data Bank [PDB] code: 5YSX) P domain. A, Overall structure of BJSMQ P domain shown as cartoon representation, in which P1 and P2 subdomains are shown in green and red, respectively. B, Structural comparison between BJSMQ P domain and GII.2 Snow Mountain virus (SMV) (PDB code: 4RPB) P domain (SMV P1 subdomain, yellow; P2 subdomain, blue). C and D, Structural comparisons of the histo-blood group antigen (HBGA) binding site among BJSMQ, SMV, and a closely related GII.12 virus that binds HBGA, highlighting the differently orientated Asp382 between BJSMQ and SMV in comparison with the GII.12 norovirus. All P domains are in cartoon representations with the conserved Asp (Asp382 in BJSMQ and SMV, Asp375 in GII.12 norovirus) in stick representation. GII.12, SMV, and BJSMQ are shown in green, purple, and yellow, respectively. The modeled HBGA based on the GII.12 P domain-HBGA-trisaccharide complex (PDB code: 3R6K) are in stick representation with its 2 galactoses (GAL) in yellow and the fucose (FUC) in cyan, respectively. Dynamic Evolution of Reemerging GII.P16-GII.2 Noroviruses We generated an ML phylogenetic tree based on VP1 nt sequences to analyze the evolutionary dynamics of the reemerging GII.P16–GII.2 noroviruses (Figure 6A). These reemerging noroviruses formed 3 major subclades (SC1–SC3) and 5 branches (BC1–BC5). SC1, which contained the first GII.P16-GII.2 strain GZ20435, accounts for about 85% of outbreaks caused by reemerging GII.P16-GII.2 norovirus. SC2 comprised a small number of strains that occurred in southern China in the early epidemic. However, SC2 exhibited a wide global spread to many countries or areas, including Australia, Japan, Taiwan, and Germany (Figure 3A). SC3 is composed of strains identified in only an outbreak. Figure 6. View largeDownload slide Dynamic evolution of reemerging GII.P16-GII.2 noroviruses. A, Maximum-likelihood phylogenetic tree of the major structural protein (VP1)-coding gene sequences of reemerging GII.P16-GII.2 strains identified in China from September 2016 to June 2017. Branches in different colors represent the regions where the strains were isolated and the most probable ancestral locations of each branch. Others (in black) represent the reemerging GII.P16-GII.2 strains that occurred in Guangxi, Fujian, Sichuan, Shandong, and Jilin provinces. Three major subclusters and 5 branches of reemerging GII.P16-GII.2 noroviruses are denoted. B, Substitution rates of the VP1-coding gene sequences of GII.2 noroviruses grouped by their different viral RNA-dependent RNA polymerase types, as calculated by BEAST software. Figure 6. View largeDownload slide Dynamic evolution of reemerging GII.P16-GII.2 noroviruses. A, Maximum-likelihood phylogenetic tree of the major structural protein (VP1)-coding gene sequences of reemerging GII.P16-GII.2 strains identified in China from September 2016 to June 2017. Branches in different colors represent the regions where the strains were isolated and the most probable ancestral locations of each branch. Others (in black) represent the reemerging GII.P16-GII.2 strains that occurred in Guangxi, Fujian, Sichuan, Shandong, and Jilin provinces. Three major subclusters and 5 branches of reemerging GII.P16-GII.2 noroviruses are denoted. B, Substitution rates of the VP1-coding gene sequences of GII.2 noroviruses grouped by their different viral RNA-dependent RNA polymerase types, as calculated by BEAST software. We estimated the VP1 nt substitution rates for all GII.2, previous GII.P2-GII.2, GII.P16-GII.2 (2012–2014), and the reemerging GII.P16-GII.2 viruses (including strains HS255 and Miyagi1). The VP1 genes of the reemerging GII.P16-GII.2 viruses showed a mean substitution rate of 5.5 × 10–3 substitutions per site per year since 2012, which was higher than those of all GII.2, the previous GII.P16-GII.2 (2012–2014), and the GII.P2-GII.2 strains (3.56 × 10–3, 2.10 × 10–3, and 1.94 × 10–3 substitutions/site/year, respectively) (Figure 6B). During the 9-month studied period, 27 nt substitutions were observed in the VP1-encoding gene, leading to no more than 2 aa mutations. However, since November 2016, the Val256Ile mutation in VP1 protein occurred in the reemerging GII.P16-GII.2 strains of SC1, and then predominated in the latest epidemic phage in China. Transmission of Reemerging GII.2-GII.P16 Norovirus in China The ML tree of VP1 genes showed that the epidemic strains from Guangdong Province segregated into 2 subclades (SC1 and SC2) and 5 branches, and shared a common ancestral node with the strains from other regions (Figure 6A). Therefore, Guangdong may be the origin of the currently reemerging GII.P16-GII.2 noroviruses in China. Combining the epidemiological data, the virus seemed to spread from Guangdong to neighboring regions of Hong Kong, Guangxi, Fujian, Chongqing, and Sichuan in late 2016. By 2017, it had further spread toward northern China, including Shandong, Beijing, Jilin, Liaoning, and Heilongjiang provinces. DISCUSSION GII.2 noroviruses were previously rare in causing AGE outbreaks. However, during the winter of 2016–2017, a GII.P16-GII.2 norovirus suddenly emerged and rapidly became predominant throughout mainland China [10]. A similar scenario was also noted in Hong Kong, Taiwan, and Germany; this virus was also detected in norovirus outbreaks in Japan, France, the United States, and Australia [11–16], indicating that this newly emerged virus had spread across continents worldwide. In this study, we provide evidence showing that antigenicity and HBGA binding profile could not well explain this sudden predominance. This suggested that minor changes in its nonstructural protein, especially the possible acquisition of a novel polymerase, may impact its epidemics. Additionally, other factors including human host immunity may contribute to its sudden predominance. Thus, future studies to clarify the roles of these factors in the reemergence and epidemiology of the GII.P16-GII.2 norovirus is necessary. The VP1 aa sequence of GZ20435, which was the first reemerging GII.P16-GII.2 strain in China, shared the same identity to those of previous GII.P2–GII.2 viruses. A recent report showed similar immunoreactivity between the 2016–2017 GII.2 and pre-2016 GII.2 viruses [26]. Therefore, our and other data suggested that the antigenicity of 2016–2017 GII.2 noroviruses causing this epidemic is highly similar to that of the previous GII.2 strains. Previous studies indicated that HBGA phenotypes are a critical host susceptible factor impacting norovirus infection and, thus, the prevalence of GII noroviruses [18, 27]. Although GZ20435 showed several aa mutations adjacent to site I of the HBS, compared with SMV, both GZ20435 and SMV bound type B and type AB saliva, showing no HBGA binding difference toward impacting the prevalence of this GII.2 virus. An obvious difficulty in this context is that all tested GII.2 VLPs or P proteins [25] of SMV, BJSMQ, and GZ20435 did not bind HBGA oligosaccharides, preventing us from obtaining further information regarding the structural basis of these viruses interacting with HBGA through co-crystallization of GII.2 P dimers in complex with a HBGA oligosaccharide in this and previous studies [25]. RdRp plays an important role in viral fitness through improvement in viral replication and transmission [21, 28]. The RdRp of the reemerging GII.P16-GII.2 norovirus harbored 5 mutations; several are close to positions that impact polymerase function and viral transmission as reported in the novel GII.P16-GII.4 Sydney_2012 strain [24]. Whether mutations in other nonstructural proteins also influence viral replication and transmission remains unknown. For example, the impacts of 1 or 2 Glu insertions in the N-terminus of its P48 protein need further research. In our case, the faster evolutionary rate of the VP1 gene supported the notion that the reemerging GII.P16-GII.2 virus acquired a novel RNA polymerase. Furthermore, since its first identification in 2015, the emerging GII.P16 gene has replaced the pre-2015 GII.P16 gene and is widely distributed among GII.3/GII.4/GII.13 noroviruses [16, 29], suggesting that it may have an advantage of viral fitness. Thus, it is logical to speculate that the insertion and mutations in nonstructural proteins, particularly in the polymerase, may contribute to the sudden increase of epidemics of the virus. The analyses of divergence times based on a large number of VP1- and RdRp-encoding genes collectively indicated that the 2016–2017 GII.P16-GII.2 norovirus evolved from the 2011–2012 GII.P16-GII.2 strains, which also supports the claims from previous studies [21]. Additionally, phylogenetic analysis indicates that after their initial emergences in 2012–2013, the reemerging GII.P16-GII.2 noroviruses underwent rapid genetic diversification. These suggest diverse sources contributing to its epidemics in 2016–2017. The SC1 strains are important causes of epidemics throughout China; whereas SC2 viruses exhibited the broadest geographic distribution, although only a few cases were reported, suggesting an advantage of SC2 viruses in global spread. The clear separation of SC3 from SC1 and SC2 suggested that the reemerging GII.P16-GII.2 noroviruses may be highly diverse. Such genetic diversity may provide a preepidemic virus pool to enable emergences of new epidemic viruses and/or support persistent survival in human populations, as shown for GII.4-GII.17 noroviruses [30, 31]. During our surveillance for almost 1 year in China, we observed 27 nt substitutions in the VP1 sequences of the reemerging GII.P16-GII.2 viruses, supporting the rapid evolution of their VP1 genes. Although few aa mutations were detected in the VP1 protein, the Val256Ile mutation in the SC1 viruses, including the late strain BJSMQ, was rarely detected in the pre-2016 GII.2 viruses. Interestingly, BJSMQ P proteins bound additional type A saliva samples compared with those of the early strain GZ20435 and pre-2016 strain SMV. The crystal structure of the SMV P dimer revealed an unconventional orientation of Asp382, which is unfavorable to HBGA binding [25]. However, the BJSMQ P dimer structure showed that Asp382 has restored the conventional orientation to form a functional HBS, which may explain the gain of the expanded HBGA-binding spectrum of BJSMQ. More studies are necessary to clarify this puzzle and the role of the Val256Ile mutation. Phylogenetic analysis suggests that Guangdong Province in the Pearl River Delta of China is the epicenter of reemerging GII.P16-GII.2 noroviruses. During 2014–2015, Hong Kong, which is a part of the Pearl River Delta, was an epicenter of GII.17 norovirus outbreaks [31–33]. We speculated that certain geographic, climatologic, and economic features and local lifestyles may facilitate the Pearl River Delta in becoming such an important epicenter. First, seawater intrusion into fresh water aquifers often occurs during winter in this region [33], which may result in exposure of local inshore shellfish to norovirus. Second, it is an important commercial and manufacturing region. A large number of immigrants are drawn here, which may lead to importation of noroviruses and contamination of the local seawater. Third, this region has a humid subtropical winter climate; high levels of humidity reportedly facilitate transmission of noroviruses [34, 35]. Thus, the Pearl River Delta should be closely monitored for future norovirus epidemic potential, besides the GII.17 and GII.P16-GII.2 genotypes. GII.4 noroviruses have predominated for >20 years, with epochal emergence of immune-escape variants driven by the fast VP1 gene evolutionary rate (4.3 × 10–3 substitutions/site/year) [36]. During 2013–2014, the broadly recognized and immune-escape GII.17 variant emerged and became predominant through its acquisition of a novel RNA polymerase that accelerated evolution of the VP1 gene to 1.2–2.1 × 10–2 substitutions per site per year [18]. The fitness of a virus is influenced by its genetic diversity [30]. In the present study, the VP1 gene of the reemerging GII.P16-GII.2 virus showed considerable genetic diversity and evolved at a high rate of 5.5 × 10–3 substitutions per site per year due to its possible acquisition of a novel polymerase. Furthermore, the reemerging GII.P16-GII.2 variant linked to HBGA binding increase was identified. We consequently anticipate that new GII.P16-GII.2 variants may emerge to infect more vulnerable populations and circulate predominantly worldwide, through fast co-evolution of VP1 and RdRp genes, as in the case of GII.4 variants. Therefore, close monitoring of the global spread of the reemerging GII.P16-GII.2 norovirus is thus required. Notes Acknowledgments. We thank George F. Gao at the Institute of Microbiology, Chinese Academy of Sciences, for help with radiograph data collection and data processing. Financial support. This study was funded by the Special National Project on Research and Development of Key Biosafety Technologies (grant number 2016YFC1201900) and the National Natural Science Foundation of China (grant numbers 31500139 and 81702007). Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. 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Genetic Analysis of Reemerging GII.P16-GII.2 Noroviruses in 2016–2017 in China

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© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
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0022-1899
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10.1093/infdis/jiy182
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Abstract

Abstract Background During 2016–2017, the previously rare GII.P16-GII.2 norovirus suddenly emerged as the predominant genotype causing gastroenteritis outbreaks in China and other countries. Its origin, phylodynamics, and mechanism behind the predominance remain unclear. Methods Bayesian phylogenetic analyses were performed on 180 full capsid and 150 polymerase sequences of 2016–2017 GII.P16-GII.2 noroviruses in China, and those for all publicly available GII.P16 and GII.2 sequences. Saliva-based histo-blood group antigen (HBGA) binding assays and crystal structural analysis were conducted by using the P proteins of 2016–2017 GII.P16-GII.2 noroviruses. Results The reemerging GII.P16-GII.2 norovirus showed a rapid genetic diversification after its emergence in 2012–2013. The antigenicity and HBGA binding profile of the early 2016–2017 and pre-2016 GII.2 noroviruses were similar. A further variant with a single Val256Ile mutation and the conventionally orientated Asp382 in the VP1 protein showed an expanded HBGA-binding spectrum. Mutations on the surface of polymerase that could alter its function were seen, which may help to accelerate the VP1 gene evolution to 5.5 × 10–3 substitutions per site per year. This virus can be traced back to Pearl River Delta, China. Conclusions Our findings provide new insights into GII.2 norovirus epidemics and highlight the necessity of enhanced global surveillance for potential epidemics of rare-genotype noroviruses. norovirus, reemerging GII.P16-GII.2, phylogenetic analysis, histo-blood group antigen, crystal structure Noroviruses, within the family Caliciviridae, are a major cause of acute gastroenteritis (AGE) worldwide [1, 2]. The norovirus genome is composed of 3 open reading frames (ORFs), among which ORF1 encodes several nonstructural proteins, including the viral RNA-dependent RNA polymerase (RdRp), while ORF2 and ORF3 encode the major and the minor structural proteins (VP1 and VP2, respectively). Noroviruses are divided into 7 genogroups (GI–GVII) that are further divided into >30 genotypes [3], among which GI, GII, and GIV noroviruses infect humans [4]. The VP1 protein contains 2 moieties, the N-terminal shell (S) and C-terminal protruding (P) domains. The P domain, which can be further divided into P1 and P2 subdomains, is responsible for host cell attachment and antigenicity [5]. Noroviruses recognize histo-blood group antigens (HBGAs) on the host cell surface, which are important for host susceptibility to infection [6, 7]. HBGAs are complex glycans that are divided into A/B/H and Lewis types. The complex interactions between diverse noroviruses and the polymorphic HBGAs are believed to affect the host range and prevalence of the former [6–9]. During the 2016–2017 winter season, the reemergence of GII.P16-GII.2 norovirus caused a rapid increase of norovirus outbreaks in China (mainland, Hong Kong, and Taiwan), Japan, Germany, France, and the United States [10–16], replacing the previously predominated GII.4 and GII.17 noroviruses [17–19]. The GII.P16-GII.2 norovirus outbreaks were rarely reported previously, except during the 2009–2010 season in Osaka, Japan [20]. The reason for the predominance of this virus remains unclear, although one phylogenetic study suggested that RdRp may contribute to its epidemic potential [21]. Here, comprehensive analyses of viral phylogenetics, HBGA-binding profiles, and P domain structures would provide important insights into the origin and evolutionary dynamics of the reemerging GII.P16-GII.2 norovirus and the possible mechanisms behind its predominance. METHODS Disease Definition, Sample Collection, and Ethics Statement An AGE outbreak is defined as at least 20 individuals with vomiting and/or diarrhea within 1 week, associated with a common source of infection, as described previously [10]. Feces or vomitus specimens were tested for noroviruses by the local Center for Disease Control and Prevention (CDC) from Guangdong, Guangxi, Fujian, Hunan, Chongqing, Sichuan, Jiangsu, Shandong, Beijing, Liaoning, Jilin, and Heilongjiang. At least 3 norovirus-positive samples from each outbreak were transported to China CDC on ice for further study. Informed consent was obtained from patients and the parents of all children who provided specimens. The production of New Zealand rabbit antiserum was approved by the Animal Care Welfare Committee of National Institute for Viral Control and Prevention, China CDC (20160715023). Amplification of the VP1 and RdRp Genes and Complete Genomes Norovirus-positive samples were diluted 1:10 (w/v) with phosphate-buffered saline (PBS). Total viral RNA was extracted and amplified targeting the ORF1/ORF2 genes by 1-step reverse-transcription polymerase chain reaction (RT-PCR) for norovirus genotyping [10, 22]. At least 1 sample from each GII.P16-GII.2 norovirus outbreak was subjected to amplification of the capsid and RdRp genes by nested RT-PCR, as described previously [10]. One or more complete genomes of GII.P16–GII.2 strains from every city and different months were randomly selected and sequenced, as previously described [10]. Sequencing and Phylogenetic Analysis All nucleotide (nt) and amino acid (aa) sequence alignments were performed using Bioedit and MEGA (version 7.0) software. The most recent common ancestors of GII.2 VP1 and GII.P16 RdRp gene sequences were estimated using the strict molecular clock, GTR+G substitution, and Bayesian skyline coalescent models in BEAST software (version 1.8.2). Markov chain Monte Carlo sample chains were run for 4.8 × 108 and 4 × 108 steps for the VP1 and RdRp genes, respectively. Maximum-likelihood (ML) phylogenetic trees of the VP1 gene sequences were constructed using PhyML (version 3.1) software. The convergence of parameters was evaluated by Tracer (version 1.6) software. The Markov chain Monte Carlo Bayesian phylogenetic tree was reconstructed using Tree Annotator (version 1.8.3) software. Expression and Purification of P Particles and Dimers The DNA sequences of the P domain of GZ20435 and BJSMQ were amplified, and that of the prototype GII.2 strain Snow Mountain virus (SMV) (AY134748.1) was chemically synthesized. They were cloned into the PGEX-6P-1 vector and expressed in Escherichia coli BL21(DE3). The proteins were purified by glutathione-sepharose 4B and then a Superdex 20010/300GL gel filtration column. To produce antisera, New Zealand rabbits were immunized with 3 purified P particles. Saliva-Binding Assay The saliva samples from 225 individuals involving in 3 reemerging GII.P16-GII.2 norovirus outbreaks were collected in Guangdong province. HBGA phenotypes of these saliva samples were determined by enzyme immunoassays using the monoclonal antibodies specific to A, B, H1, Lea, Leb, Lex, and Ley, respectively, as previously described [19]. A total of 214 boiled saliva samples were diluted by 1:1000 with PBS, and used to coat 96-well microtiter plates at 4°C overnight. After blocking with 5% nonfat milk, purified P proteins (10 µg/mL) of BJSMQ, GZ20435, and SMV were added to the plates. Next, the corresponding rabbit anti-P-particle antibodies (GZ20435 at 1:2000, BJSMQ at 1:2500, SMV at 1:32000) were added, and bound antibodies were detected using a horseradish peroxidase–conjugated goat anti-rabbit secondary antibody (1:20000) (MP Biomedicals). The reaction was developed using a 3,3ʹ,5,5ʹ-tetramethylbenzidine kit (Invitrogen), and was measured at 450 nm using a plate reader. Oligosaccharide-Binding Assay Synthetic oligosaccharide-based binding assays were conducted for the synthetic-oligosaccharide-polyacrylamide-biotin conjugates A, B, H1, H2, H3, Lea, Leb, Lex, and Ley, Neu5Ac, Neu5Gc, sialyl-Lex, type 1, 2 precursor (GlycoTech), as previously described [23]. Protein Crystallization and Data Collection Crystals were grown in hanging drops of 1 µL BJSMQ P dimer (~10 mg/mL) and 1 µL reservoir solution (0.2 M ammonium sulfate, 0.1 M 2-(N-morpholino)ethanesulfonic acid [pH 6.5], and 30% [wt/vol] polyethylene glycol MME 5000 at 18°C. Crystals were transferred to 20% (vol/vol) ethylene glycol in mother solution as a cryoprotectant, and flash-frozen. Data collection, structure solving, and refinement of unbound structures were conducted as described previously [23]. RESULTS Characteristics of 2016–2017 Norovirus Outbreaks From January 2016 to July 2017, 277 norovirus outbreaks confirmed (see Materials and Methods for definition) in China. At least 3 samples per outbreak were tested to confirm norovirus infection, followed by sequencing and genotyping. The norovirus sequences of each outbreak shared 99%–100% nt identities. In November 2016, the number of norovirus outbreaks per month increased sharply, which peaked at 63 outbreaks in December 2016. Genotyping showed that 181 of 208 (87%) outbreaks during November 2016 to June 2017 were caused by GII.P16-GII.2 noroviruses (Figure 1A). The first outbreak of GII.P16-GII.2 norovirus (named GZ20435) was identified in Guangdong Province on 21 September 2016. This virus subsequently caused outbreaks throughout China, the majority of which occurred in southern China in late 2016, with gradual northward expansion in 2017 (Figure 1B). Figure 1. View largeDownload slide Characteristics of the norovirus outbreak in China during the 2016–2017 winter season. A, Monthly distributions of reemerging GII.P16-GII.2, GII.P2-GII.2, and other genotypes that caused norovirus outbreaks in China from January 2016 to June 2017. B, Geographic distribution of the outbreaks caused by the reemerging GII.P16-GII.2 norovirus in China. Chronological order of the outbreaks of reemerging GII.P16-GII.2 norovirus is indicated by ● (2016) and ▲ (2017). The outbreak frequency in different provinces and regions are classified into high, medium, and low based on the incidence numbers. Figure 1. View largeDownload slide Characteristics of the norovirus outbreak in China during the 2016–2017 winter season. A, Monthly distributions of reemerging GII.P16-GII.2, GII.P2-GII.2, and other genotypes that caused norovirus outbreaks in China from January 2016 to June 2017. B, Geographic distribution of the outbreaks caused by the reemerging GII.P16-GII.2 norovirus in China. Chronological order of the outbreaks of reemerging GII.P16-GII.2 norovirus is indicated by ● (2016) and ▲ (2017). The outbreak frequency in different provinces and regions are classified into high, medium, and low based on the incidence numbers. Sequence Analysis of Reemerging GII.P16-GII.2 Noroviruses The complete genomes of 60 reemerging GII.P16-GII.2 viruses have been sequenced, but only a few complete genomes of previous GII.P16-GII.2 noroviruses within a short period of time are available in GenBank, hindering genome-wide analysis. The genome sequence of the representative reemerging GII.P16-GII.2 strain SZ123 (collected at October 2016 in Guangdong) showed the highest sequence identity (95%) to the 2011–2012 GII.P16-GII.2 strain HS255. Genome scanning of SZ123 against HS255 suggested that its ORF1, VP1, and VP2 genes have undergone coevolution. The VP1 aa sequence of the first representative reemerging GII.P16-GII.2 norovirus strain GZ20435 shared a 100% identity with the GII.2 strain Vaals8/2005/NLD (BAG68716.1). Although the HBGA binding site (HBS) of all known GII.2 viruses remained conserved, several aa mutations in GZ20435 adjacent to the site I of the HBS were found, against that of SMV (Figure 2A). In the nonstructural proteins of the reemerging GII.P16-GII.2 strain, 11 unique nonsynonymous substitutions were detected compared to those of pre-2015 GII.P16 strains (Figure 2B), while 5 amino acid mutations were located in the polymerase, with 3 near locations known to impact polymerase kinetics or fidelity that were recently explained in the GII.P16-GII.4 Sydney 2012 strains [24]. A residue mutation in 3C-like, P48 protein, and NTPase, and 2 mutations in P22 protein, as well as an insertion in P48 protein, are shown in Figure 2B. All of the sequences were deposited in GenBank under accession numbers MG745985–MG746376. Figure 2. View largeDownload slide Sequence analysis of reemerging GII.P16-GII.2 noroviruses. A, The sequence analysis of histo-blood group antigen (HBGA)–binding interface of the GII.2 major structural protein VP1 protein. The HBGA-binding interfaces are composed of 3 amino acid components that are indicated with sites I, II, and III by gray square frame. The only changed amino acid in VP1 between BJSMQ and the GZ20435 is Val256Ile (highlighted by the black frame). B, List of amino acid changes in nonstructural protein of the emerging GII.P16 norovirus compared with those of the previous GII.P16 norovirus. The black background residue mutation in the emerging GII.P16 noroviruses including reemerging GII.P16-GII.2 (2016–2017) and GII.P16-GII.4/GII.3 (2015–2017). The asterisk denotes residue deletion in the P48 protein of GII.P16 noroviruses. Figure 2. View largeDownload slide Sequence analysis of reemerging GII.P16-GII.2 noroviruses. A, The sequence analysis of histo-blood group antigen (HBGA)–binding interface of the GII.2 major structural protein VP1 protein. The HBGA-binding interfaces are composed of 3 amino acid components that are indicated with sites I, II, and III by gray square frame. The only changed amino acid in VP1 between BJSMQ and the GZ20435 is Val256Ile (highlighted by the black frame). B, List of amino acid changes in nonstructural protein of the emerging GII.P16 norovirus compared with those of the previous GII.P16 norovirus. The black background residue mutation in the emerging GII.P16 noroviruses including reemerging GII.P16-GII.2 (2016–2017) and GII.P16-GII.4/GII.3 (2015–2017). The asterisk denotes residue deletion in the P48 protein of GII.P16 noroviruses. Evolutionary Analysis of the GII.2 VP1 and GII.P16 RdRp Genes We used 180 full VP1 gene sequences of the reemerging GII.P16-GII.2 noroviruses identified in our study and 159 GII.2 VP1 sequences available in GenBank to generate a time-scale evolutionary tree. It showed that all reemerging GII.P16-GII.2 viruses clustered together (Figure 3A) but relatively close to the GII.P16-GII.2 strains HS255/USA/2011 (KJ407074.2) and Miyagi1/Japan/2012 (LC145787.1). Three major clades of GII.2 strains were observed: the reemerging GII.P16-GII.2 (2016–2017), GII.P16-GII.2 (2008–2015), and GII.P2-GII.2 (2004–2010) clades. Figure 3. View largeDownload slide Markov chain Monte Carlo Bayesian phylogenetic trees of GII.2 and GII.P16 strains. A, Trees were reconstructed using 339 major structural protein (VP1)-coding nucleotide sequences of 339 GII.2 strains. The isolated area or countries of the reemerging GII.P16-GII.2 strains in subclade SC2 are denoted by the color boxes: mainland China (no box), Taiwan (red), Australia (black), Japan (blue), and Germany (purple). B, Tree of RNA-dependent RNA polymerase (RdRp)–coding genes of GII.P16 strains reconstructed by 358 RdRp nucleotide sequences. The GII.2 and GII.P16 strains in the VP1 and RdRp trees are colored according to their polymerase genotype and capsid genotype, respectively. GII.2 strains whose polymerase genotype was not available are labeled GII.NA-GII.2 in the VP1 tree. Estimated divergence times are shown on the ancestral nodes. Phylogenetic clustering is indicated by colors and names. Scale bar is actual time (years). Figure 3. View largeDownload slide Markov chain Monte Carlo Bayesian phylogenetic trees of GII.2 and GII.P16 strains. A, Trees were reconstructed using 339 major structural protein (VP1)-coding nucleotide sequences of 339 GII.2 strains. The isolated area or countries of the reemerging GII.P16-GII.2 strains in subclade SC2 are denoted by the color boxes: mainland China (no box), Taiwan (red), Australia (black), Japan (blue), and Germany (purple). B, Tree of RNA-dependent RNA polymerase (RdRp)–coding genes of GII.P16 strains reconstructed by 358 RdRp nucleotide sequences. The GII.2 and GII.P16 strains in the VP1 and RdRp trees are colored according to their polymerase genotype and capsid genotype, respectively. GII.2 strains whose polymerase genotype was not available are labeled GII.NA-GII.2 in the VP1 tree. Estimated divergence times are shown on the ancestral nodes. Phylogenetic clustering is indicated by colors and names. Scale bar is actual time (years). We also constructed a time-scale evolutionary tree of the partial GII.P16 RdRp nt sequences, including 208 from GenBank and 150 obtained in this study (Figure 3B). The reemerging GII.P16-GII.2 strains clustered closely with GII.P16-GII.3/GII.4_Sydney2012 strains (2015–2017). Together they formed an emerging GII.P16 cluster, which was subdivided into 3 subclades (I–III). Consistent with that of the VP1 sequence phylogenetic analysis, they also displayed a relationship with the 2011–2012 GII.P16-GII.2 strains. The divergence times of the phylogenetic clusters in the VP1 and RdRp trees were comparable, and showed that the most recent common ancestor of the reemerging GII.P16-GII.2 norovirus may have appeared in 2012–2013 (Figure 3A and 3B) and then formed 3 subclades (SC1–SC3). Moreover, the strains of SC1 diverged from a common ancestor of SC2 at 2014, while SC3 diverged from the ancestor at 2012. HBGA-Binding Profile of GII.2 Norovirus Like SMV, the P proteins of GZ20435 bound to B- and AB-type saliva, but BJSMQ (collected at December 2016, Beijing) gained extra binding to A-type saliva (Figure 4). Comparing BJSMQ P domain sequences with those of the GZ20435, we found a single mutation Val256Ile in the BJSMQ P domain (Figure 2A). Synthetic oligosaccharide-based binding assays were also performed, but no binding signals to type A and B oligosaccharide were observed. Figure 4. View largeDownload slide Saliva-binding profiles of the GII.2 P domain proteins of GZ20435, BJSMQ, and Snow Mountain virus (SMV). GZ20435 (A) and BJSMQ (B) represent the reemerging GII.P16-GII.2 viruses identified in Guangdong Province and Beijing City, respectively, while SMV (C) represents GII.2 strain prototype. The 3 strains bound to A-, B-, O-, and O–-type saliva. Two hundred fourteen saliva samples with known secretor (A, B, O) and nonsecretor (O–) types were used. The cutoff value of positive samples was determined as 0.15. Abbreviations: HBGA, histo-blood group antigen; OD, optical density. Figure 4. View largeDownload slide Saliva-binding profiles of the GII.2 P domain proteins of GZ20435, BJSMQ, and Snow Mountain virus (SMV). GZ20435 (A) and BJSMQ (B) represent the reemerging GII.P16-GII.2 viruses identified in Guangdong Province and Beijing City, respectively, while SMV (C) represents GII.2 strain prototype. The 3 strains bound to A-, B-, O-, and O–-type saliva. Two hundred fourteen saliva samples with known secretor (A, B, O) and nonsecretor (O–) types were used. The cutoff value of positive samples was determined as 0.15. Abbreviations: HBGA, histo-blood group antigen; OD, optical density. Structural Analysis of the P Domain of GII.P16-GII.2 Norovirus The crystal structure of the BJSMQ P dimer was solved at 1.2 Å resolution (Protein Data Bank code: 5YSX) (Figure 5A). Superposition of the BJSMQ P domain structure with that of SMV [25] revealed a high degree of structural similarity (root mean square deviation, 0.26) (Figure 5B). The BJSMQ P domain did not bind HBGA oligosaccharides, preventing their co-crystallization with an HBGA. While the BJSMQ P domain shares conserved amino acids constituting HBS (Figure 2A), P domain structural superposition among the BJSMQ, SMV, and a closely related GII.12 [25] indicated that the orientations of Asp382, an important HBS component of BJSMQ, changes dramatically compared with that of SMV. In SMV, Asp382 points away from the modeled HBGA [25], whereas Asp382 in BJSMQ restores its traditional orientation, pointing toward the modeled HBGA (Figure 5C and 5D). Since Ile256 in the BJSMQ P1 domain is far away from the HBS, its role in affecting the HBS function might be smaller than the orientation of the Asp382. Figure 5. View largeDownload slide Structural analysis of the reemerging GII.P16-GII.2 strain BJSMQ (Protein Data Bank [PDB] code: 5YSX) P domain. A, Overall structure of BJSMQ P domain shown as cartoon representation, in which P1 and P2 subdomains are shown in green and red, respectively. B, Structural comparison between BJSMQ P domain and GII.2 Snow Mountain virus (SMV) (PDB code: 4RPB) P domain (SMV P1 subdomain, yellow; P2 subdomain, blue). C and D, Structural comparisons of the histo-blood group antigen (HBGA) binding site among BJSMQ, SMV, and a closely related GII.12 virus that binds HBGA, highlighting the differently orientated Asp382 between BJSMQ and SMV in comparison with the GII.12 norovirus. All P domains are in cartoon representations with the conserved Asp (Asp382 in BJSMQ and SMV, Asp375 in GII.12 norovirus) in stick representation. GII.12, SMV, and BJSMQ are shown in green, purple, and yellow, respectively. The modeled HBGA based on the GII.12 P domain-HBGA-trisaccharide complex (PDB code: 3R6K) are in stick representation with its 2 galactoses (GAL) in yellow and the fucose (FUC) in cyan, respectively. Figure 5. View largeDownload slide Structural analysis of the reemerging GII.P16-GII.2 strain BJSMQ (Protein Data Bank [PDB] code: 5YSX) P domain. A, Overall structure of BJSMQ P domain shown as cartoon representation, in which P1 and P2 subdomains are shown in green and red, respectively. B, Structural comparison between BJSMQ P domain and GII.2 Snow Mountain virus (SMV) (PDB code: 4RPB) P domain (SMV P1 subdomain, yellow; P2 subdomain, blue). C and D, Structural comparisons of the histo-blood group antigen (HBGA) binding site among BJSMQ, SMV, and a closely related GII.12 virus that binds HBGA, highlighting the differently orientated Asp382 between BJSMQ and SMV in comparison with the GII.12 norovirus. All P domains are in cartoon representations with the conserved Asp (Asp382 in BJSMQ and SMV, Asp375 in GII.12 norovirus) in stick representation. GII.12, SMV, and BJSMQ are shown in green, purple, and yellow, respectively. The modeled HBGA based on the GII.12 P domain-HBGA-trisaccharide complex (PDB code: 3R6K) are in stick representation with its 2 galactoses (GAL) in yellow and the fucose (FUC) in cyan, respectively. Dynamic Evolution of Reemerging GII.P16-GII.2 Noroviruses We generated an ML phylogenetic tree based on VP1 nt sequences to analyze the evolutionary dynamics of the reemerging GII.P16–GII.2 noroviruses (Figure 6A). These reemerging noroviruses formed 3 major subclades (SC1–SC3) and 5 branches (BC1–BC5). SC1, which contained the first GII.P16-GII.2 strain GZ20435, accounts for about 85% of outbreaks caused by reemerging GII.P16-GII.2 norovirus. SC2 comprised a small number of strains that occurred in southern China in the early epidemic. However, SC2 exhibited a wide global spread to many countries or areas, including Australia, Japan, Taiwan, and Germany (Figure 3A). SC3 is composed of strains identified in only an outbreak. Figure 6. View largeDownload slide Dynamic evolution of reemerging GII.P16-GII.2 noroviruses. A, Maximum-likelihood phylogenetic tree of the major structural protein (VP1)-coding gene sequences of reemerging GII.P16-GII.2 strains identified in China from September 2016 to June 2017. Branches in different colors represent the regions where the strains were isolated and the most probable ancestral locations of each branch. Others (in black) represent the reemerging GII.P16-GII.2 strains that occurred in Guangxi, Fujian, Sichuan, Shandong, and Jilin provinces. Three major subclusters and 5 branches of reemerging GII.P16-GII.2 noroviruses are denoted. B, Substitution rates of the VP1-coding gene sequences of GII.2 noroviruses grouped by their different viral RNA-dependent RNA polymerase types, as calculated by BEAST software. Figure 6. View largeDownload slide Dynamic evolution of reemerging GII.P16-GII.2 noroviruses. A, Maximum-likelihood phylogenetic tree of the major structural protein (VP1)-coding gene sequences of reemerging GII.P16-GII.2 strains identified in China from September 2016 to June 2017. Branches in different colors represent the regions where the strains were isolated and the most probable ancestral locations of each branch. Others (in black) represent the reemerging GII.P16-GII.2 strains that occurred in Guangxi, Fujian, Sichuan, Shandong, and Jilin provinces. Three major subclusters and 5 branches of reemerging GII.P16-GII.2 noroviruses are denoted. B, Substitution rates of the VP1-coding gene sequences of GII.2 noroviruses grouped by their different viral RNA-dependent RNA polymerase types, as calculated by BEAST software. We estimated the VP1 nt substitution rates for all GII.2, previous GII.P2-GII.2, GII.P16-GII.2 (2012–2014), and the reemerging GII.P16-GII.2 viruses (including strains HS255 and Miyagi1). The VP1 genes of the reemerging GII.P16-GII.2 viruses showed a mean substitution rate of 5.5 × 10–3 substitutions per site per year since 2012, which was higher than those of all GII.2, the previous GII.P16-GII.2 (2012–2014), and the GII.P2-GII.2 strains (3.56 × 10–3, 2.10 × 10–3, and 1.94 × 10–3 substitutions/site/year, respectively) (Figure 6B). During the 9-month studied period, 27 nt substitutions were observed in the VP1-encoding gene, leading to no more than 2 aa mutations. However, since November 2016, the Val256Ile mutation in VP1 protein occurred in the reemerging GII.P16-GII.2 strains of SC1, and then predominated in the latest epidemic phage in China. Transmission of Reemerging GII.2-GII.P16 Norovirus in China The ML tree of VP1 genes showed that the epidemic strains from Guangdong Province segregated into 2 subclades (SC1 and SC2) and 5 branches, and shared a common ancestral node with the strains from other regions (Figure 6A). Therefore, Guangdong may be the origin of the currently reemerging GII.P16-GII.2 noroviruses in China. Combining the epidemiological data, the virus seemed to spread from Guangdong to neighboring regions of Hong Kong, Guangxi, Fujian, Chongqing, and Sichuan in late 2016. By 2017, it had further spread toward northern China, including Shandong, Beijing, Jilin, Liaoning, and Heilongjiang provinces. DISCUSSION GII.2 noroviruses were previously rare in causing AGE outbreaks. However, during the winter of 2016–2017, a GII.P16-GII.2 norovirus suddenly emerged and rapidly became predominant throughout mainland China [10]. A similar scenario was also noted in Hong Kong, Taiwan, and Germany; this virus was also detected in norovirus outbreaks in Japan, France, the United States, and Australia [11–16], indicating that this newly emerged virus had spread across continents worldwide. In this study, we provide evidence showing that antigenicity and HBGA binding profile could not well explain this sudden predominance. This suggested that minor changes in its nonstructural protein, especially the possible acquisition of a novel polymerase, may impact its epidemics. Additionally, other factors including human host immunity may contribute to its sudden predominance. Thus, future studies to clarify the roles of these factors in the reemergence and epidemiology of the GII.P16-GII.2 norovirus is necessary. The VP1 aa sequence of GZ20435, which was the first reemerging GII.P16-GII.2 strain in China, shared the same identity to those of previous GII.P2–GII.2 viruses. A recent report showed similar immunoreactivity between the 2016–2017 GII.2 and pre-2016 GII.2 viruses [26]. Therefore, our and other data suggested that the antigenicity of 2016–2017 GII.2 noroviruses causing this epidemic is highly similar to that of the previous GII.2 strains. Previous studies indicated that HBGA phenotypes are a critical host susceptible factor impacting norovirus infection and, thus, the prevalence of GII noroviruses [18, 27]. Although GZ20435 showed several aa mutations adjacent to site I of the HBS, compared with SMV, both GZ20435 and SMV bound type B and type AB saliva, showing no HBGA binding difference toward impacting the prevalence of this GII.2 virus. An obvious difficulty in this context is that all tested GII.2 VLPs or P proteins [25] of SMV, BJSMQ, and GZ20435 did not bind HBGA oligosaccharides, preventing us from obtaining further information regarding the structural basis of these viruses interacting with HBGA through co-crystallization of GII.2 P dimers in complex with a HBGA oligosaccharide in this and previous studies [25]. RdRp plays an important role in viral fitness through improvement in viral replication and transmission [21, 28]. The RdRp of the reemerging GII.P16-GII.2 norovirus harbored 5 mutations; several are close to positions that impact polymerase function and viral transmission as reported in the novel GII.P16-GII.4 Sydney_2012 strain [24]. Whether mutations in other nonstructural proteins also influence viral replication and transmission remains unknown. For example, the impacts of 1 or 2 Glu insertions in the N-terminus of its P48 protein need further research. In our case, the faster evolutionary rate of the VP1 gene supported the notion that the reemerging GII.P16-GII.2 virus acquired a novel RNA polymerase. Furthermore, since its first identification in 2015, the emerging GII.P16 gene has replaced the pre-2015 GII.P16 gene and is widely distributed among GII.3/GII.4/GII.13 noroviruses [16, 29], suggesting that it may have an advantage of viral fitness. Thus, it is logical to speculate that the insertion and mutations in nonstructural proteins, particularly in the polymerase, may contribute to the sudden increase of epidemics of the virus. The analyses of divergence times based on a large number of VP1- and RdRp-encoding genes collectively indicated that the 2016–2017 GII.P16-GII.2 norovirus evolved from the 2011–2012 GII.P16-GII.2 strains, which also supports the claims from previous studies [21]. Additionally, phylogenetic analysis indicates that after their initial emergences in 2012–2013, the reemerging GII.P16-GII.2 noroviruses underwent rapid genetic diversification. These suggest diverse sources contributing to its epidemics in 2016–2017. The SC1 strains are important causes of epidemics throughout China; whereas SC2 viruses exhibited the broadest geographic distribution, although only a few cases were reported, suggesting an advantage of SC2 viruses in global spread. The clear separation of SC3 from SC1 and SC2 suggested that the reemerging GII.P16-GII.2 noroviruses may be highly diverse. Such genetic diversity may provide a preepidemic virus pool to enable emergences of new epidemic viruses and/or support persistent survival in human populations, as shown for GII.4-GII.17 noroviruses [30, 31]. During our surveillance for almost 1 year in China, we observed 27 nt substitutions in the VP1 sequences of the reemerging GII.P16-GII.2 viruses, supporting the rapid evolution of their VP1 genes. Although few aa mutations were detected in the VP1 protein, the Val256Ile mutation in the SC1 viruses, including the late strain BJSMQ, was rarely detected in the pre-2016 GII.2 viruses. Interestingly, BJSMQ P proteins bound additional type A saliva samples compared with those of the early strain GZ20435 and pre-2016 strain SMV. The crystal structure of the SMV P dimer revealed an unconventional orientation of Asp382, which is unfavorable to HBGA binding [25]. However, the BJSMQ P dimer structure showed that Asp382 has restored the conventional orientation to form a functional HBS, which may explain the gain of the expanded HBGA-binding spectrum of BJSMQ. More studies are necessary to clarify this puzzle and the role of the Val256Ile mutation. Phylogenetic analysis suggests that Guangdong Province in the Pearl River Delta of China is the epicenter of reemerging GII.P16-GII.2 noroviruses. During 2014–2015, Hong Kong, which is a part of the Pearl River Delta, was an epicenter of GII.17 norovirus outbreaks [31–33]. We speculated that certain geographic, climatologic, and economic features and local lifestyles may facilitate the Pearl River Delta in becoming such an important epicenter. First, seawater intrusion into fresh water aquifers often occurs during winter in this region [33], which may result in exposure of local inshore shellfish to norovirus. Second, it is an important commercial and manufacturing region. A large number of immigrants are drawn here, which may lead to importation of noroviruses and contamination of the local seawater. Third, this region has a humid subtropical winter climate; high levels of humidity reportedly facilitate transmission of noroviruses [34, 35]. Thus, the Pearl River Delta should be closely monitored for future norovirus epidemic potential, besides the GII.17 and GII.P16-GII.2 genotypes. GII.4 noroviruses have predominated for >20 years, with epochal emergence of immune-escape variants driven by the fast VP1 gene evolutionary rate (4.3 × 10–3 substitutions/site/year) [36]. During 2013–2014, the broadly recognized and immune-escape GII.17 variant emerged and became predominant through its acquisition of a novel RNA polymerase that accelerated evolution of the VP1 gene to 1.2–2.1 × 10–2 substitutions per site per year [18]. The fitness of a virus is influenced by its genetic diversity [30]. In the present study, the VP1 gene of the reemerging GII.P16-GII.2 virus showed considerable genetic diversity and evolved at a high rate of 5.5 × 10–3 substitutions per site per year due to its possible acquisition of a novel polymerase. Furthermore, the reemerging GII.P16-GII.2 variant linked to HBGA binding increase was identified. We consequently anticipate that new GII.P16-GII.2 variants may emerge to infect more vulnerable populations and circulate predominantly worldwide, through fast co-evolution of VP1 and RdRp genes, as in the case of GII.4 variants. Therefore, close monitoring of the global spread of the reemerging GII.P16-GII.2 norovirus is thus required. Notes Acknowledgments. We thank George F. Gao at the Institute of Microbiology, Chinese Academy of Sciences, for help with radiograph data collection and data processing. Financial support. This study was funded by the Special National Project on Research and Development of Key Biosafety Technologies (grant number 2016YFC1201900) and the National Natural Science Foundation of China (grant numbers 31500139 and 81702007). Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. 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The Journal of Infectious DiseasesOxford University Press

Published: Mar 29, 2018

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