Detection and genetic characterization of the emergent GII.17_2014 norovirus genotype among children with gastroenteritis from Northern Brazil

Norovirus is the most important cause of viral gastroenteritis outbreaks worldwide. Recently, a novel GII.17 norovirus variant emerged and caused epidemics in Asian countries, replacing the GII.4 Sydney 2012 strain in hospitalized cases. In this study we describe the emergence of this novel NoV GII.17_2014 strain in Brazil.     

北京地区诺如病毒分子生物学特点初步研究

目的了解北京地区诺如病毒的分子生物学特点。方法收集北京市2007年1—3月非细菌性胃肠炎暴发和散发病例，采集患者粪便标本，使用逆转录聚合酶链反应（RT—PCR）对粪便标本进行诺如病毒RNA检测，对RT—PCR阳性标本的PCR产物进行克隆测序。结果检测38例粪便标本，共有27例阳性。随机选择其中4例PCR产物进行克隆测序，获得4株诺如病毒序列进行比对分析，结果显示，该4株病毒序列与诺如病毒GⅡ／4型参考株同源性最高，其中与荷兰和日本提交的诺如病毒GⅡ／4型变异株最为接近，同源性分别为97．8％～98．5％和95．2％～95．9％。系统发生树分析表明，该4株诺如病毒与荷兰、日本流行的GⅡ／4型变异株处于同一分支上。结论北京地区存在诺如病毒GⅡ／4型变异株流行，其与2006年荷兰和日本的诺如病毒流行株属同一类GⅡ／4型变异株。     

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.     

Emergence of GII.e as a major ORF 1 norovirus genotype and its associated ORF 2 GII.4 variant forms

The noroviruses are a major cause of outbreaks of gastroenteritis. The norovirus genotype “GII.e”, identified by ORF (Open Reading Frame) 1 nucleotide sequencing, appears to be an obligatory recombinant, in that no unique GII.e ORF 2 genotype has been identified. In 2012 GII.e norovirus became the predominant ORF 1 genotype in norovirus outbreaks in Victoria, Australia, and the current study documents changes in the ORF 1 region of GII.e norovirus since it first emerged in 2008, as well as in the ORF 2 genotypes associated with GII.e norovirus. GII.e norovirus underwent significant genetic change in ORF 1 between 2010 and 2012 and this genetic change corresponded to a significant increase in the prevalence of the virus. Nucleotide sequencing of the ORF 2 region of GII.e specimens showed that in 2008–2009, all the ORF 2 sequences corresponded to the GII.4 (2007) variant, in 2010 all the ORF 2 sequences corresponded to the GII.4 (2012-like) variant and in 2012 all the ORF 2 sequences corresponded to the GII.4 (2012) variant, the GII.4 (2012-like) variant, or the GII.4 (2009-like) variant. The evidence indicated that the development of the 2012 GII.e epidemic strains was due to evolutionary change rather than a novel recombination event. The results also support the notion that ORF 1 is critical in determining the virulence of a norovirus strain.     

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.     

2012-2014年湖南省感染性腹泻哨点医院儿童诺如病毒感染及基因型别分析

目的 了解湖南省感染性腹泻哨点医院儿童诺如病毒的感染状况及基因型别。 方法 2012年1月-2014年12月采集湖南省哨点医院腹泻患儿的粪便标本,应用诺如病毒特异性引物进行扩增,选择扩增阳性产物进行基因测序和进化分析。 结果 936份粪便标本RT-PCR检测诺如病毒核酸,阳性100份,阳性率10.68%。对40份诺如病毒核酸阳性标本进行基因序列测定与分析,38份为GⅡ型,其中31份为GⅡ.4型。在31株GⅡ.4型中,Sydney 2012和GII.4 Den Haag 2006b各栓出14株。 结论 2012-2014年湖南省哨点医院儿童腹泻病例中诺如病毒的感染率较高,GⅡ.4是优势株,其中GⅡ.4 sydney 2012和Den Haag 2006b是主要型别。     

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.     

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.     

Rotavirus infection in Brazil: epidemiology and challenges for its control

Worldwide, rotaviruses account for 600,000 to 870,000 deaths per year among infants and young children. In Brazil, rotaviruses were first seen in 1976 by scanning electron microscopy of stool samples from diarrheic infants in Belém, Pará. Hospital-based studies have shown that rotaviruses are associated with 12-42% of cases of acute diarrhea. In addition, community-based studies yielded an average of 0.25 rotavirus-related diarrheal episodes per child per year. G types 1 to 4 account for about two-thirds of circulating strains, but the (unusual) P[8],G5 genotype has been claimed to cause over 10% of rotavirus diarrheal episodes. It has been shown that over 70% of children develop rotavirus antibodies by the age of 4-5 years. The tetravalent rhesus-human rotavirus vaccine (RRV-TV) conferred 35% protection according to a two-year follow-up study in Belém, Pará, Brazil, but reached an efficacy of 60% during the first year of life. RRV-TV was also shown to be 75% protective against very severe gastroenteritis in northern Brazil. Vaccination with RRV-TV has been suspended recently in the United States because of the detection of intussusception as a side effect. Therefore, further vaccine trials in Brazil will probably involve rotavirus candidate vaccines other than RRV-TV.     

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.     

Genome characterization and temporal evolution analysis of a non-epidemic norovirus variant GII.8

Noroviruses are the primary cause of non-bacterial acute gastroenteritis worldwide, and GII.8 belongs to a non-epidemic genotype with a limited understanding currently. In this study, we assembled the first GII.8 norovirus genome from China and clarified the temporal evolutionary process of this non-epidemic variant. Using the “4+1+1” application strategy with newly designed primer sets, the genome of one GII.8 strain GZ2017-L601 from China was firstly sequenced that comprised 7476 nucleotides. The homology of the new genome and the previous only GII.8 genome reached 93.8% identity at the nucleotide level, but only 10, 6, 7 amino acid mutations occurred in three ORFs. When compared the new strain with other GII reference strains, p22 and P2 were calculated as the variable encoding regions, and NTPase, VPg, 3CL, RdRp and S were shown as the conserved ones. We then reconstructed the evolutionary process of the GII.8 genotype using other available sequences in GenBank. Based on the partial N/C region, all GII.8 strains could be subdivided chronologically into four clusters, which spans 1967–1994, 1997–2005, 2003–2009, and 2007–2017, respectively. Moreover, differences of capsid P proteins between GII.8 strains and the epidemic GII.4 strain VA387 were also compared. There existed 147/310 distinct amino acid sites in the alignment, including two insertion and three deletion mutations. Distribution of antigen epitopes of two GII.8 variants was comparable, but the numbers of antigenic sites of GII.8 strains were less than that of VA387. In summary, the first GII.8 genome from China was assembled and extensively characterized, and a time-order evolutionary process of this genotype was identified with a static pattern of antigenic variations.     

Novel intergenotype human norovirus recombinant GII.16/GII.3 in Bangladesh

Noroviruses (NoVs) are one of the major etiological agents of acute gastroenteritis in all age groups. In this study, we identified an intergenotype NoV recombinant strain in the fecal specimens of two male infants with acute diarrhea in Bangladesh. Phylogenetic analysis showed that the identified strains were recombinant NoV strains with a GII.3 capsid and a GII.16 polymerase gene. The recombination breakpoint was located in the ORF1/ORF2 overlap region. To the best of our knowledge this is the first report of a NoV recombinant GII.16/GII.3 strain worldwide.     

2016 - 2018无锡市腹泻病疫情中诺如病毒的分子流行病学特征

目的 鉴定无锡市2016年3月 - 2018年3月急性腹泻病突发疫情中诺如病毒的感染流行情况,并对病原体进行分子特征研究。方法 对疫情上送的暴发急性病例标本,采用荧光PCR初步判定病原体,常规RT - PCR检测诺如病毒RNA聚合酶和衣壳蛋白基因片段,并进行序列测定和分子特征分析。结果 在30起暴发疫情中共有162份标本经荧光PCR检测均为诺如病毒核酸阳性,其中154份标本RT - PCR检测判为GII型诺如病毒,120份标本成功测序。测序结果显示,2016 - 2018年突发疫情中有5种基因型,分别是GII.P16 - GII.2、GII.P17 - GII.17、GII.4_Sydney2012、GII.4_NewOrleans和GI.6,2017年之前以GII.17病毒株流行为主,2017之后新的GII.P16 - GII.2重组株成为无锡市病毒性腹泻疫情中的绝对优势株。结论 诺如病毒是无锡市腹泻病疫情的最常见病原体,其最新的优势流行株是GII.P16 - GII.2重组株,和全国趋势一致。     

Gastroenteritis Outbreaks Caused by Norovirus GII.17, Guangdong Province,China, 2014–2015

In the past decade, the most prevalent norovirus genotype causing viral gastroenteritis outbreaks worldwide, including China, has been GII.4. In winter 2014–15, norovirus outbreaks in Guangdong, China, increased. Sequence analysis indicated that 82% of the outbreaks were caused by a norovirus GII.17 variant.     

北京地区诺如病毒感染的流行病学调查

目的调查北京地区诺如病毒感染的流行情况。方法对暴发疫情的急性胃肠炎患者和散发诺如病毒阳性患者全部进行现场问卷式调查，并采集患者粪便标本，应用ELISA和RT—PCR方法检测诺如病毒，选择阳性PCR产物进行克隆测序。结果2006年11月至2007年3月，北京市共计报告急性诺如病毒胃肠炎暴发疫情8起，经现场流行病学调查全部确认为院内感染暴发。在报告的409例急性散发病毒性胃肠炎病例中，共检出诺如病毒阳性患者158例，检出率38．63％。其中40～44岁年龄组检出率最高，为55．00％，55～59岁年龄组检出率最低，为21．74％。阳性患者中年龄最大者91岁，最小者仅6个月，平均年龄为40岁。男性患者84例，女性74例。序列分析结果显示，北京地区诺如病毒流行株与2006年荷兰和日本的诺如病毒流行株最为接近，为GⅡ／4型变异株。结论诺如病毒是2006—2007年北京地区冬季病毒性腹泻的一个重要病原体，北京地区诺如病毒流行株与2006年荷兰和日本的诺如病毒流行株属同一类GⅡ／4型变异株。     

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.     

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.