Detection and analysis of recombination in GII.4 norovirus strains causing gastroenteritis outbreaks in Alberta

Recombination is an important mechanism generating genetic diversity in norovirus (NoV) that occurs commonly at the NoV polymerase-capsid (ORF1/2) junction. The genotyping method based on partial ORF2 sequences currently used to characterize circulating NoV strains in gastroenteritis outbreaks in Alberta cannot detect such recombination events and provides only limited information on NoV genetic evolution. The objective of this study was to determine whether any NoV GII.4 strains causing outbreaks in Alberta are recombinants. Twenty stool samples collected during outbreaks occurring between July 2004 and January 2012 were selected to include the GII.4 variants Farmington Hills 2002, Hunter 2004, Yerseke 2006a, Den Haag 2006b, Apeldoorn 2007, New Orleans 2009, and Sydney 2012 based on previous NoV ORF2-genotyping results. Near full-length NoV genome sequences were obtained, aligned with reference sequences from GenBank and analyzed with RDPv4.13. Two sequences corresponding to Apeldoorn 2007, and Sydney 2012 were identified as recombinants with breakpoints near the ORF1/2 junction and putative parental strains as previously reported. We also identified, for the first time, a non-recombinant sequence resembling the ORF2–3 parent of the recombinant cluster Sydney 2012 responsible for the most recent pandemic. Our results confirmed the presence of recombinant NoV GII.4 strains in Alberta, and highlight the importance of including additional genomic regions in surveillance studies to trace the evolution of pandemic NoV GII.4 strains.     

Molecular epidemiology of norovirus associated with gastroenteritis and emergence of norovirus GII.4 variant 2012 in Japanese pediatric patients

In late 2012, an outbreak of acute gastroenteritis due to norovirus variant Sydney_2012 occurred and have been reported from many counties. In this study, we described surveillance study of the incidence of norovirus infections among Japanese pediatric patients in association with gastroenteritis and investigated the antigenic change of the new variant Sydney_2012 circulated in Japanese populations. A total of 2381 fecal specimens collected from children with acute gastroenteritis in Hokkaido, Tokyo, Shizuoka, Kyoto, Osaka, and Saga from 2009 to 2013 were examined for norovirus and further analyzed molecularly. A high proportion (39.3%) of norovirus positive samples and several genotypes were detected. Norovirus GII.4 dominated over other genotypes (71.4%). The Den_Haag_2006b (43.2%) was detected as the predominant variant and co-circulated with New_Orleans_2009 (17.8%) until March 2012. Subsequently, they were displaced by Sydney_2012. The Sydney_2012 variant has been responsible for the majority of norovirus infections in 2012–2013 (85.7%). Although Sydney_2012 variant has a common ancestor with New_Orleans_2009 variant, analysis of P2 sub-domain showed a high level of diversity in comparison with other variants in four amino acid changes at the antigenic sites. The change in particular residue 393 of new variant may affect HBGA recognition. Analysis of noroviruses circulating in the past 4 years revealed a change of predominant variant of norovirus GII.4 in each epidemic season. The change of amino acid in putative epitopes may have led the virus escape from the existing herd immunity and explain the increase of new variant outbreaks.     

Analysis of GII.P7 and GII.6 noroviruses circulating in Italy during 2011–2016 reveals a replacement of lineages and complex recombination history

Noroviruses are important human enteric pathogens and monitoring their genetic diversity is important for epidemiological surveillance, vaccine development, and understanding of RNA viruses evolution. Epidemiological investigations have revealed that genogroup II, genotype 6 noroviruses (GII.6) are common agents of gastroenteritis. Upon sequencing of the ORF2 (encoding the viral capsid), GII.6 viruses have been distinguished into three variants. Sentinel hospital-based surveillance in Italy revealed that GII.6 noroviruses were the second most common capsid genotype in 2015, mostly in association with a GII.P7 ORF1 (encoding the viral polymerase). Upon molecular characterization of the ORF1 and ORF2, the GII.P7_GII.6 epidemic viruses circulating in 2014–2015 (variant GII.6b) were different from those that circulated sporadically in 2011–2013 (variant GII.6a). Analysis of the ORF1 (GII.P7) and ORF2 (GII.6) sequences available in the databases unveiled marked genetic diversity and peculiarities in the phylogenetic segregation patterns, suggesting multiple recombination events. Phylogenetic analyses suggest that recent GII.P7_GII.6b viruses were circulating as early as 2008, and formed a genetically homogenous group that emerged globally.     

Characterization of GII.4 noroviruses circulating among children with acute gastroenteritis in Pune,India: 2005–2013

Genogroup II genotype 4 noroviruses (GII.4 NoVs), an important cause of sporadic childhood gastroenteritis worldwide, undergo continuous evolution leading to the periodic emergence of novel variants. The present study was undertaken for surveillance of GII.4 NoVs and identification and characterization of GII.4 variants circulating among children with sporadic gastroenteritis in Pune, India during 2005–2013. Among the 12 GII genotypes detected in the study, GII.4 was predominant. Sequencing and phylogenetic analysis of ORF2 (major capsid protein VP1 gene) of the GII.4 NoVs revealed circulation of seven GII.4 variants, Hunter_2004 (2005–2007), Yerseke_2006a (2006), DenHaag_2006b (2007), Osaka_2007 (2007–2009), Apeldoorn_2007 (2008), New Orleans_2009 (2008–2012) and Sydney_2012 (2013), with the Pune strains grouping with the contemporary global reference strains. The Hunter_2004, Osaka_2007 and New Orleans_2009 variants showed prolonged circulation, with the Hunter_2004 and New Orleans_2009 variants differentiating into temporally separated sub-clusters. Analysis of VP1 sequences and predicted structures of the GII.4 variants identified variant specific amino acid positions, particularly in and near (within 8A^°) the epitopes A-E, displaying differences in the sequence and physicochemical characteristics of the different variants. Comparison with the reference strains of each of the GII.4 variants revealed up to 11 amino acid substitutions at the variant specific positions in the GII.4 strains from Pune. Amino acid variations were also noted among the strains of the same GII.4 variant in Pune. The strains of different sub-clusters identified in the Hunter_2004 and New Orleans_2009 variants showed differences in sequence and physicochemical properties of either or all of the epitopes A, C and E. The study thus describes the temporal variations and diversity of the GII.4 strains in Pune and emphasizes continuous monitoring and analysis of the GII.4 variants.     

Identification of a novel codon in the norovirus GII.4 polymerase region which acts as a marker of major GII.4 norovirus epidemics

Noroviruses are considered the most common cause of outbreaks of non-bacterial gastroenteritis in humans and the GII.4 genotype the most common norovirus genotype. Previous studies have shown that two adjacent codons acted as markers of the severity of GII.4 norovirus outbreak epidemics. In this study, a further such codon was identified at nucleotide position 4670–4672 relative to the norovirus strain Lordsdale virus (GenBank accession no. X86557). Taken together, the data indicate these epidemic marker sites occur, on average, about once in 30 amino acids (aa) in the polymerase region. None of the variant forms associated with the three codons resulted in an aa change. The three codons were not associated with the active sites of the polymerase gene. It is possible changes in these marker sites may influence norovirus virulence by altering the timing of co-translational folding in the norovirus genome.     

Recombinant norovirus GII.g/GII.12 gastroenteritis in children

Recombinant GII.g/GII.12 norovirus (NoV) strains emerged in 2008 in Australia and subsequently have been associated with gastroenteritis outbreaks worldwide. In the winter season 2009-2010 GII.12 strains caused 16% of the NoV outbreaks in the United States. During 2009-2010 we also identified GII.g/GII.12 strains during surveillance of sporadic cases of gastroenteritis in Italian children. Severity scores were calculated for the GII.g/GII.12 NoV infections using the Vesikari scale and in two out of three paediatric cases they exceeded the median value calculated for concomitant GII.4 infections. Upon sequence analysis, the Italian strains were found to be recombinant viruses and displayed different patterns of nucleotide polymorphisms. Phylodynamic analysis with other GII.g/GII.12 recombinants showed a high rate of evolution, comparable to the rates observed for GII.4 viruses. The mechanisms leading to worldwide emergence of GII.12 NoV strains in 2008-2010 are not clear. Monitoring of GII.12 NoV circulation is necessary to understand these mechanisms of evolution.     

Occurrence of Norovirus GII.4 Sydney Variant-related Outbreaks in Korea

Human noroviruses are major causative agents of food and waterborne outbreaks of nonbacterial acute gastroenteritis. In this study, we report the epidemiological features of three outbreak cases of norovirus in Korea, and we describe the clinical symptoms and distribution of the causative genotypes. The incidence rates of the three outbreaks were 16.24% (326/2,007), 4.1% (27/656), and 16.8% (36/214), respectively. The patients in these three outbreaks were affected by acute gastroenteritis. These schools were provided unheated food from the same manufacturing company. Two genotypes (GII.3 and GII.4) of the norovirus were detected in these cases. Among them, major causative strains of GII.4 (Hu-jeju-47-2007KR-like) were identified in patients, food handlers, and groundwater from the manufacturing company of the unheated food. In the GII.4 (Hu-jeju-47-2007KR-like) strain of the norovirus, the nucleotide sequences were identical and identified as the GII.4 Sydney variant. Our data suggests that the combined epidemiological and laboratory results were closely related, and the causative pathogen was the GII.4 Sydney variant strain from contaminated groundwater.     

Phylogenetic analyses of Norovirus strains detected in Uruguay reveal the circulation of the novel GII.P7/GII.6 recombinant variant

Noroviruses (NoV) are one of the major etiological agent of acute gastroenteritis (AGE) outbreaks worldwide. Distinct NoV genotypes have been associated with different transmission patterns and disease severity in humans. Therefore, it is important to identify genetically different NoV genotypes circulating in a particular region. However, genotyping has become a challenge due to recombination events occurring mainly nearby ORF1/ORF2 junction of NoV genome, leading to distinct genotypes with polymerase and capsid regions derived from parenteral strains. Taking this into account, ORF1/ORF2 sequences were obtained from NoV strains collected from patients with AGE in Uruguay. This study reveals in silico evidences of recombination events taking place in four out of six strains analyzed for which its polymerase gene and its capsid region correspond to GII.P7 and to GII.6 genotype, respectively. These results also reveal the circulation of a GII.P7/GII.6 recombinant variant in the natural populations of NoV strains in the northwestern region of Uruguay. As far as we know this is the first report about the circulation of a NoV GII.P7/GII.6 recombinant variant in the Americas.     

Detection of novel GII.17 norovirus in Argentina, 2015

During the winter of 2014–2015 a novel GII.17 norovirus strain emerged as a cause of large gastroenteritis outbreaks in Asia; displacing the long-term predominant strain, GII.4. Although sporadically detected, the emerging GII.17 virus was described in North America and Europe. In this study, we describe the presence of this novel strain in Argentina (South America), and provide new information on the genetic diversity of GII.17 noroviruses.Ten stool samples from individuals (1–88 years old; median: 5 years old) experiencing gastroenteritis symptoms from San Martín de los Andes, Argentina were tested for Norovirus using RT-PCR. Subsequently, Norovirus positive samples were analyzed by sequencing. Norovirus was found in four out of 10 samples received. Partial sequencing of the ORF2 was available for 3/4 samples: two samples belonged to genotype GII.4 and one to genotype GII.17 (Arg13099). Sequence analyses of the VP1 encoding region revealed that the GII.17 Argentinean strain presented characteristics from both, the new (cluster C), and older (cluster A and B) GII.17 strains. Phylogenetic and sequence analyses of the RdRp region showed that this strain was closely related to strains from genotypes GII.P3, GII.P13 and GII.P17; however, did not cluster within any of them.This study represents the first report of this emergent strain in South America, and presents further evidence of the genetic plasticity of the GII.17.     

Complete nucleotide sequence analysis of the norovirus GII.17: A newly emerging and dominant variant in China, 2015

Norovirus is an important pathogen which accounts for majority of the viral related acute gastroenteritis. Recently, a variant of genotype GII.17 was reported to be predominant over GII.4 and accounted for several acute gastroenteritis outbreaks in Asia. In the current study, the full genome of a norovirus strain ZHITHC-12 isolated during this outbreak period in China was identified and characterized. The viral genome was 7557 nucleotides in length and a phylogenetic analysis based on full length genome sequences indicated that ZHITHC-12 belonged to GII.17 genotype. A further phylogenetic analysis based on all available polymerase and capsid sequences showed that ZHITHC-12 was in Cluster III on both phylogenetic trees and grouped with other strains also isolated during 2013 to 2015. Moreover, homology modeling analysis based on GII norovirus capsid 5BSX template revealed that substitutions, mutations, and more importantly, deletions and insertions, occurred at or near the putative epitopes and histo-blood group antigen (HBGA) binding sites in its protruding P2 domain, which might confer new antigenic or biological properties for this novel variant. In summary, the first full genome and capsid protein structure of a novel norovirus GII.17 variant isolated in China was extensively characterized. The data would be helpful not only for the epidemiology study, but also for the diagnostic tool development and effective vaccine design in the future.     

Comparative analysis of South African norovirus GII.4 strains identifies minor recombinant variants

Recombination within the norovirus (NoV) GII.4 genotype is well documented as a mechanism by which novel variants evolve. Norovirus GII.4 has been the predominant NoV genotype detected in South Africa (SA) in recent years and putative NoV recombinants were previously identified in SA based on partial regions of the viral genome. The objective of this study was to determine the complete genome sequence of representative NoV GII.4 variants that have circulated in SA between 2009 and 2013 and to compare major and minor GII.4 variants based on nucleotide sequence. The complete genomes of 11/27 GII.4 strains could be amplified in three or five overlapping segments, these included major variants New_Orleans_2009 and Sydney_2012 as well as three types of minor GII.4 variants. Phylogenetic and recombination analysis identified GII.4 recombinants with breakpoints located at or near the ORF1/2 junction. Apart from recombinants already recognised as major variants (GII.P4 New_Orleans_2009/GII.4 Sydney_2012 (n = 2) and GII.Pe/GII.4 Sydney_2012 (n = 2)) four further recombinant strains were detected (GII.P4 New_Orleans_2009/GII.4 Hunter_2004 (n = 1) and GII.P4 Yerseke_2006a/GII.4 Apeldoorn_2007 (n = 3)) that were attributed to three distinct minor variants. The encoded proteins with the highest diversity were p48 (Nterm), p22, VP1 and VP2. Analysis of antigenic sites in VP1 revealed mutations at epitopes A, B, C, D and E, with epitopes A and D being most variable. The high variation at epitope D was reflected in structural differences in models of GII.4 variants in the epitope D loop region (aa 393–395). Major and minor variants could not be distinguished based on specific sequence differences. HBGA-binding studies will be necessary to assess the effect of the observed amino acid differences in the P2 domain of these GII.4 strains.     

Molecular epidemiology of noroviruses detected in Nepalese children with acute diarrhea between 2005 and 2011: Increase and predominance of minor genotype GII.13

Noroviruses, an important cause of acute gastroenteritis, possess a highly divergent genome which was classified into five genogroups and dozens of genotypes. However, changes in genotype distribution over time were poorly understood, particularly in developing countries. We therefore conducted a molecular epidemiological study which characterized the norovirus strains detected in 4437 Nepalese children with acute diarrhea between November 2005 and January 2011 to gain insight into how their genotypes changed over time. Of the 356 samples positive for noroviruses, 277 (78%) were successfully genotyped into 22 capsid genotypes; GII.4 (n = 113), GII.3 (n = 38) and GII.13 (n = 37) were the majority. Interestingly, GII.13 accounted for only 1.7% (4/230) between 2005 and 2008 (period 1) but increased substantially to 26.2% (33/126) between 2009 and 2011 (period 2). Phylogenetic analysis of the VP1 nucleotide sequences of 35 GII.13 strains indicated that they clustered into two lineages named NPL2008 and NPL2009 to which two period 1 strains and 33 period 2 strains belonged, respectively. Lineage NPL2009 contained GII.13 strains that were detected in a large-scale gastroenteritis outbreak in Germany in 2012. Both Nepalese and German VP1 sequences carried two substitutions, H378N and V394Q, in the putative histo-blood group antigen (HBGA)-binding sites. As to the polymerase genotypes of Nepalese strains, the period 1 strains possessed GII.Pm, but the period 2 strains possessed GII.P13, GII.P16, and GII.P21. Together with recent reports on the predominance of GII.P13/GII.13 and GII.P16/GII.13 in India and GII.P16/GII.13 in European countries, this study predicts that genotype GII.13 which was previously regarded as a minor genotype has a potential to become an epidemiologically important genotype.     

重组GII型诺如病毒长沙株全基因组序列的测定与分析

目的 对2016年长沙地区一起由GII型诺如病毒引起的急性胃肠炎暴发疫情致病原进行全基因组序列测定,掌握其基因类型、分子进化特征和抗原重组情况。方法 提取疫情中患者粪便标本的总RNA,反转录成cDNA,PCR扩增病毒全基因组并采用Sanger法测序,比对拼接后获得病毒全基因组序列;通过BLAST比对和诺如病毒在线分型工具(typing online tool)确定其基因型别;从GenBank中下载GII型诺如病毒参考序列,采用DNA Star软件进行序列多序列比对和同源性分析,绘制系统遗传进化树,基因重组特征分析采用SimPlot软件。结果 通过一代测序获得病毒基因组序列长7491bp,有3个开放阅读框(ORF),长度分别为5100bp,1647bp,765bp。多序列比对和同源分析发现ORF1区与GII.P12型代表株同源性最高,VP1区则与GII.3型同源性最高;因此,将该毒株命名为Hu/GII.P12-GII.3/CS02/2016 /CHN。分子遗传进化分析显示其与中国其他地区如北京、上海、广东等地流行的GII.P12-GII.3重组型诺如病毒亲缘关系最为接近。抗原重组分析发现Hu/GII.P12-GII.3/CS02/2016 /CHN长沙株重组位点在5080bp,为ORF1与 ORF2重叠区的起点。结论 除了GII.P16-GII.2重组诺如病毒的广泛流行外,长沙地区仍存在GII.P12/GII.3重组诺如病毒的散发流行,需加其强监测。     

Analysis of uncommon norovirus recombinants from Manaus,Amazon region,Brazil: GII.P22/GII.5, GII.P7/GII.6 and GII.Pg/GII.1

Norovirus (NoV) is responsible for outbreaks and sporadic cases of nonbacterial acute gastroenteritis in humans worldwide. The virus consists of small round particles containing a single-stranded RNA genome that is divided into three Open Reading Frames. NoV evolves via mechanisms of antigenic drift and recombination, which lead to the emergence of new strains that are capable of causing global epidemics. Recombination usually occurs in the ORF1/ORF2 overlapping region and generates strains with different genotypes in the polymerase and capsid region. The primary objective of this study was to analyze recombination in positive-NoV samples. Specimens were collected during 2011, 2012 and 2014, from children under two years of age presenting gastrointestinal symptoms such as vomiting and diarrhea. The partial polymerase (B region), capsid (D region) genes and the ORF1-ORF2 overlap regions were sequenced in each sample. The recombinant analyses were performed in the Simplot software v.3.5.1 and RDP4 Beta v. 4.6 program. These analyses showed that GII.Pg/GII.1, GII.P7/GII.6, and GII.P22/GII.5 were recombinant strains. To our knowledge, this is the first time that the GII.P22/GII.5 and GII.Pg/GII.1 strains were described in South America and the GII.P7/GII.6 was detected in Northern of Brazil.     

Emergence of norovirus GII.2 and its novel recombination during the gastroenteritis outbreak in Japanese children in mid-2016

In mid-2016, norovirus GII.2 emerged as a major cause of gastroenteritis outbreak in Japan with overall detection rate of 56.3% of norovirus cases. The differences in ORF1 and ORF2 of some norovirus GII were observed. Inter-subtype recombinants GII.Pe/GII.2, GII.P16/GII.2 and GII.P17/GII.2 were detected. Three amino acid substitutions were noted at P2 antigenic site of GII.P16/GII.2 recombinants. Furthermore, this study revealed that the current immunochromatographic kit available in Japan could be used effectively for the detection of recent GII.2 genotype.     

Epidemic viral gastroenteritis in Queensland coincides with the emergence of a new norovirus variant

Norovirus infections cause widespread morbidity and have significant economic impact on the community. An increase in outbreaks of norovirus gastroenteritis in hospitals, nursing homes and in the community was observed in Queensland in 2004. Molecular analysis of positive samples indicated the emergence of a single strain of norovirus. A 252 nucleotide sequence from the polymerase region (POL) was compared to sequences of the new variant genotype GII.4 that has caused epidemics in the Northern Hemisphere in 2002 and 2003. Sequence analysis indicated greater than 95 per cent similarity in the POL between the Queensland strain and the Northern Hemisphere 2002/3 GII.4 variant. Phylogenetic analysis revealed that the Queensland strain forms a branch within the GII.4 genotype separate from the 2002 variant from Europe and North America. Although norovirus genotype GII.4 had circulated in Queensland in the past, the 2004 strain was characterised specifically by three nucleotides not present in any other sequences held in our database covering the years 2002-June 2004.     

Novel surveillance network for norovirus gastroenteritis outbreaks, United States

CaliciNet, the outbreak surveillance network for noroviruses in the United States, was launched in March 2009. As of January 2011, twenty state and local health laboratories had been certified to submit norovirus sequences and epidemiologic outbreak data to CaliciNet. During the network's first year, 552 outbreaks were submitted to CaliciNet, of which 78 (14%) were associated with foodborne transmission. A total of 395 (72%) outbreaks were typed as GII.4, of which 298 (75%) belonged to a new variant, GII.4 New Orleans, which first emerged in October 2009. Analysis of the complete capsid and P2 region sequences confirmed that GII.4 New Orleans is distinct from previous GII.4 variants, including GII.4 Minerva (2006b).     

Novel GII.12 norovirus strain, United States, 2009-2010

In October 2009, a novel GII.12 norovirus strain emerged in the United States and caused 16% of all reported norovirus outbreaks during the winter season. Sequence analysis demonstrated a recombinant virus with a P2 region that was largely conserved compared with previously sequenced GII.12 strains.     

Molecular characterization of new emerging GII.17 norovirus strains from South China

Noroviruses are still the primary cause of non-bacterial acute gastroenteritis worldwide. Recently, a novel GII.17 norovirus variant emerged and caused an infection peak in the cold season of 2014/2015 in some Asian countries, including China. In this study, in order to understand the evolutionary advantage of the novel variant, complete genomic sequences of GII.17 NoV strains from South China were comprehensively analyzed. Pairwise alignments of new GII.17 genomes with representative sequences of each GII genotype were performed. Inconsistent homology was observed between different protein-encoding regions, of which VPg (NS5) and P2 were found to be the most conserved and variable ones, respectively. The differences between new sequences and other reported GII.17 genomes were also compared, and 84 mismatched nucleotides were found, resulting in 15 amino acid changes. Then, all capsid sequences of different GII.17 NoV variants were collected for multiple alignments, and a total of 87 spots were identified during their evolution process. Homology modeling of capsid proteins of four GII.17 variants was carried out based on comparison with GII.4 VA387 strain, and structural differences were mainly embodied in five extended loops. Furthermore, positions of potential conformational epitope regions of new GII.17 variants were found more similar or adjacent to those of GII.4 rather than those of the former GII.17 variants. In summary, nine GII.17 strains from South China were extensively characterized based on their complete genomes, and a different distribution pattern of epitope residues was predicted on the new GII.17 variant capsid from that of the former ones.