Detection of GII‐4/2006b variant and recombinant noroviruses in children with acute gastroenteritis,South Korea

Norovirus (NoV), a single‐stranded, positive RNA virus, is an important etiologic agent of acute gastroenteritis in children worldwide. In this study, a total of 434 fecal samples collected from 434 children with acute gastroenteritis in Seoul, between September 2007 and July 2008 were tested to determine the molecular epidemiology of NoVs and characterize recombinant strains by using RT‐PCR followed by sequencing. Of the 434 specimens, NoV, rotavirus, and adenovirus were detected in 155 (35.8%), 72 (16.6%), and 19 specimens (4.3%), respectively. NoV GI was detected in 7 specimens (1.6%) and GII in 148 (34.1%) specimens. Phylogenetic analysis of capsid sequences in the GII‐positive specimens revealed the presence of the following strains: GII‐4, 111 (75.0%); GII‐3, 35 cases (23.6%); GII‐6b, 1 case; and GII‐16, 1 case. Most of the GII‐4 strains were grouped with the GII‐4/2006b variant with 98–100% nucleotide identity. Eleven strains were identified as recombinant (GII‐4/GII‐3 in 10 cases and GII‐b polymerase/GII‐16 capsid in 1 case) by sequencing based on the RdRP and capsid genes. The putative recombination point in the recombinant strains was the ORF‐1/ORF2 overlap, located at nucleotide 5,046 with reference to Lordsdale. In conclusion, GII‐4/2006b variants were detected predominantly and a new recombinant strain (GII‐4/GII‐3) was found in the Korean children with gastroenteritis. Continuous monitoring of the genetic diversity of NoVs is important to determine the trend of the predominant genotype and new recombinant strain. J. Med. Virol. 82:146–152, 2010. © 2009 Wiley‐Liss, Inc.     

Prevalence and genetic diversity of norovirus infection in Tunisian children (2007–2010)

Viral gastroenteritis can be a life‐threatening disease in infants and young children, especially in developing countries. The aim of this study was to continue the epidemiological surveillance of norovirus (NoV) infections in Tunisian children suffering from acute gastroenteritis. Surveillance was initiated in January 2003, to monitor potential variations in strains over time, in terms of frequency and diversity of NoV genotypes, and more particularly the potential emergence of new GII.4 variants following the 2004 Hunter variant. From April 2007 to April 2010, a total of 407 stool specimens were collected from sporadic cases (238 inpatients and 169 outpatients). Furthermore, 28 stool samples were collected from children involved in 3 gastroenteritis outbreaks. Stool specimens were screened for NoV genogroup I (GI) and II (GII) by RT‐PCR. NoV strains were genotyped, and variants identified, based on sequence and phylogenetic analyses of the polymerase and capsid genes. NoVs were detected in 38 sporadic cases (9.3%) and 21 epidemic cases (75%). Great diversity was observed throughout the period, with seven distinct NoV genotypes characterized in sporadic cases, and three in outbreaks. GIIb/II.3 and GII.4 were predominant globally, with fluctuations of their prevalence over time. Interestingly, the Hunter variant, which was the unique GII.4 variant observed from 2003 to April 2007 in the region of Monastir, was replaced by the 2006b variant. NoV is an important enteropathogen responsible for viral gastroenteritis among infants and children in Tunisia, and the infecting strains between 2007 and 2010 were different from those in previous years. J. Med. Virol. 85: 1100–1110, 2013. © 2013 Wiley Periodicals, Inc.     

Epidemiological,clinical, and molecular features of norovirus infections in western India

The study was conducted to investigate the molecular epidemiology of noroviruses (NoVs) from western India. A total of 830 fecal specimens were collected during July 2005–June 2007 from children, ≤7 years of age suffering from acute gastroenteritis in Pune, Nagpur, and Aurangabad cities. All the specimens were subjected to RT‐PCR, sequencing and phylogenetic analysis for detection and characterization of Genogroup I (GI) and GII NoVs. NoV positivity varied between 6.3% and 12.6% in different cities with the predominance of GII (96.6%). NoV infections were very common in the patients ≤2 years of age. A majority (55%) of the patients suffered from severe disease, however, vomiting was not experienced in 35%. Coinfections with rotaviruses were found in 10% cases. Summer month seasonality supported NoV infections in western India. The phylogenetic analysis of partial RNA polymerase and VP1 (capsid) genes identified 2 GI (GI. 2 and GI.6) and 5 GII (GII.4, GII.6, GII.7, GII.8, and GII.14) genetic clusters with possible occurrence of “2007 new‐variant” of GII.4. Six different combinations of RdRp and capsid genes (GII.b/GII.3, GII.b/GII.4, GII.d/GII.3, GII.b/GII.18, GII.1/GII.12 and GII.3/GII.13) were also identified. GII.4 (52%) prevailed in 2005–2006 while the predominance of probable recombinant NoV strains (58%) was noted in 2006–2007 with the contribution of GII.b/GII.3 at 79% level. GII.b/GII.18 type identified in 37% infections in 2005–2006 was completely replaced by GII.b/GII.3 type in 2006–2007. This is the first report that highlights the norovirus epidemiology and strain diversity demonstrating possible circulation of new variants in patients with acute gastroenteritis from western India. J. Med. Virol. 81:922–932, 2009. © 2009 Wiley‐Liss, Inc.     

Surveillance of pathogens causing gastroenteritis and characterization of norovirus and sapovirus strains in Shenzhen,China, during 2011

Viral gastroenteritis is one of the most common diseases in humans, and it is primarily caused by rotaviruses (RVs), astroviruses (AstVs), adenoviruses (AdVs), noroviruses (NoVs), and sapoviruses (SaVs). In this study, we determined the distribution of viral gastroenteritis and human calicivirus (HuCVs) in acute gastroenteritis patients in Shenzhen, China, during 2011. Real-time RT-PCR was used to detect norovirus (NoV), group A rotavirus (RV), adenovirus (AdV), and astrovirus (AstV). From a total of 983 fecal samples, NoV was detected in 210 (21.4 %); RoV in 173 (17.6 %); AstV in 10 (1.0 %); and AdV in 15 (1.5 %). Mixed infections involving two NoVs were found in 21 of the 387 pathogen-positive stool specimens. NoV and SaV genotypes were further tested using RT-PCRs and molecular typing and phylogenetic analysis were then performed based on the ORF1-ORF2 region for NoV and a conserved nucleotide sequence in the capsid gene for SaV. Of the 68 typed strains that were sequenced and genotyped, five were NoV G1 (7.5 %) and 63 were NoV GII (96.6 %). GII strains were clustered into five genotypes, including GII.4 (65.1 %; 36 GII.4 2006b and five GII.4 New Orleans), GII.3 (28.6 %), GII.2 (3.2 %), GII.6 (1.6 %), and GII.1 (1.6 %). While all fecal specimens were tested for SaVs, 15 (1.5 %) were positive, and of these, 12 isolates belonged to G1.2, and the remaining three SaV strains belonged to the SaV GII genogroup. Although various HuCVs were detected in acute gastroenteritis patients, NoV GII.4 2006b was more prevalent than the other HuCVs.     

Recombinant norovirus implicated in gastroenteritis outbreaks in Hiroshima Prefecture, Japan

Norovirus (NoV) is a major etiological agent of acute gastroenteritis outbreaks worldwide. A total of 314 fecal specimens collected from patients of 39 NoV gastroenteritis outbreaks in Hiroshima Prefecture, Japan, between December 2001 and April 2006 were tested for the occurrence of recombinant NoVs. Sixteen genotypes (GI/1, GI/2, GI/4, GI/7, GI/8, GI/11, GI/14, GII/2, GII/3, GII/4, GII/5, GII/6, GII/8, GII/12, GII/14, and GII/untypeable) were detected in the 39 outbreaks based on capsid sequences and GII/4 was predominant recently. Twelve strains detected in 11 (28.2%) of the 39 outbreaks were suspected to be recombinants by using Simplot and Recco analyses and five recombinant genotypes, GII/4-GII/12 (five strains), GIIb-GII/3 (four strains), GII/4-GII/2 (one strain), GII/4-GII/14 (one strain), and GI/2-GI/8 (one strain), were identified based on RNA-dependent RNA polymerase and capsid sequences. None of the strains genotyped as GII/4 based on the capsid sequence was identified as a recombinant. The putative recombination points in the recombinant strains were placed either upstream or downstream of the open reading frame (ORF) 1 and ORF2 overlap. The present study indicates the following: (a) recombination among ORFs is common in nature, (b) the involvement of recombinant NoVs in gastroenteritis outbreaks is extensive even in a local area such as Hiroshima Prefecture, Japan, and (c) the conserved region (ORF1 and ORF2 overlap) has a meaningful function against the recombination event.     

Molecular epidemiology of norovirus gastroenteritis outbreaks in North Carolina, United States: 1995-2000

Noroviruses (NoVs) are the most common cause of acute non-bacterial gastroenteritis outbreaks in the US. We investigated 16 gastroenteritis outbreaks in North Carolina (NC), from 1995 to 2000, to further characterize the epidemiology of NoV using RT-PCR on stool and ELISA on sera. NoV were identified in 14 outbreaks by RT-PCR. Sequence analyses of the amplicons indicated the outbreak strains belonged to the following clusters: five GII/4, three GI/3, one GI/4, one GII/2, one GII/5, one GII/7, and one GII/13 (prototype strain). We detected NoV in stool samples from one outbreak but could not determine its specific cluster within the GII genogroup based on polymerase sequence analysis. The five GII/4 strains were classified as the "95/96 US common strain" and occurred throughout the 5-year period. In contrast to national trends, the majority (86%) of NoV outbreaks identified in North Carolina were foodborne. Of the 12 food-related NoV outbreaks, we were able to document transmission by food handlers in two outbreaks. Person-to-person transmission from primary cases was suggested in three outbreaks. Our results indicate that NoVs are important agents of viral gastroenteritis outbreaks in NC.     

Emerging GII.4 norovirus variants affect children with diarrhea in Palermo,Italy in 2006

Although the genetic/antigenic heterogeneity of human noroviruses (NoVs) is impressive, a few genogroup II strains of genotype 4 (GII.4) are dominant worldwide. GII.4 NoVs evolve rapidly and in the last 15 years six epidemic variants have been identified. In 2005–2006, surveillance of sporadic viral gastroenteritis in children in Palermo, Italy, resulted in the detection of NoV strains in 20.9% of the patients admitted to hospital. By restriction fragment length polymorphism (RFLP) and sequence analysis of region A in the RNA‐dependent RNA‐polymerase (RdRp) gene, 59 NoV strains were successfully characterized. Eighty‐one percent of the strains were characterized as GII.4, 14% as GIIb/Hilversum and 5% as GI.1. Phylogenetic analysis of region A and of the ORF1/ORF2 overlapping region of the GII.4 strains recovered in Palermo in the years 2002–2006 revealed the sequential emergence of four variants, GII.4 2002, 2004, 2006a, and 2006b. The variant GII.4 2006a was detected in June and July, 2006, while the variant 2006b first appeared in August, 2006, becoming predominant thereafter. Based on these findings, the dynamics of replacement and circulation of the GII.4 NoV variants in Italy in 2005–2006 appear to have matched the temporal pattern observed in Europe during the same period. J. Med. Virol. 81:139–145, 2009. © 2008 Wiley‐Liss, Inc.     

Clinical and molecular epidemiology of norovirus infection in childhood diarrhea in China

A prospective investigation was carried out among pediatric outpatients and inpatients with acute non‐dysenteric diarrhea between August, 2008 and July, 2009 in Shanghai, Hangzhou, Chongqing, and Tianjin, China. One step real‐time RT‐PCR was used for detection of norovirus (NoV) genogroups I and II (GI, GII). The NoV genotypes were classified based on partial capsid sequences. Rotavirus (RV) was detected in parallel. Among 4,123 fecal samples from outpatients, 1,067 (25.9%) were NoV‐positive, of which 1,051 (98.5%) belonged to GII and 1,309 (31.7%) were RV‐positive. In the inpatient group (n = 317), 25.6% were NoV‐positive and 41.6% were RV‐positive. Four hundred and fifty‐one out of 1,067 NoV‐positive strains were sequenced and genotyped and 6 typed strains were GI (3 GI.3, 2 GI.5, 1 GI.4) and 445 typed strains were GII. GII strains clustered into nine genotypes including GII.4 2006b (69.2%), the only GII.4 variant identified in this study, followed by GII.3 (23.8%), GII.6 (3.6%), GII.12 (1.3%), GII.2 (0.9%), GII.13 (0.4%), GII.14 (0.2%), GII.7 (0.2%), and GII.16 (0.2%). A peak of NoV infections was observed during the cold season in Tianjin, while NoV activity was higher between late summer and autumn and lower during winter in Shanghai, Hangzhou, and Chongqing. NoV is a common causative agent of childhood diarrhea in China and the seasons of NoV‐associated diarrhea varies between regions. The results show that NoV GII.4 2006b was the predominant strain circulating in China between 2008 and 2009. J. Med. Virol. 84:145–151, 2011. © 2011 Wiley Periodicals, Inc.     

Detection of genogroup IV noroviruses in environmental and clinical samples and partial sequencing through rapid amplification of cDNA ends

Noroviruses (NoVs) give rise to clinically relevant gastroenteritis in all age groups and are widely distributed in both clinical and environmental settings. NoVs are classified into five genogroups (GI to GV), of which GI, GII and GIV infect humans. While data on the epidemiology of human NoVs GI and GII have been steadily increasing, very little information has been published on the spread of GIV in either the health care system or the environment, resulting in a lack of information about its clinical significance and pathogenesis. In order to investigate the distribution of GIV strains in the environment, we analyzed sewage samples collected from five treatment plants, by using newly designed nested RT-PCR assays. A collection of clinical stool samples, originating from pediatric patients with symptoms of acute gastroenteritis, previously analyzed in our laboratory for the presence of NoV GI or GII, was also analyzed for the presence of GIV norovirus. Results of this work attest to the presence of GIV in both clinical and environmental contexts and underline the importance of routinely screening for this genogroup, along with GI and GII, in order to better understand its distribution, prevalence and role during epidemics, which is probably underestimated.     

Detection and molecular characterization of noroviruses and sapoviruses in children admitted to hospital with acute gastroenteritis in Vietnam

Noroviruses (NoV) and sapoviruses (SaV) are recognized as important causes of acute gastroenteritis in children worldwide. In this study, the prevalence and genetic variability of NoV and SaV were determined in hospitalized children <5 years of age with acute gastroenteritis in Hanoi, Vietnam. A total of 501 fecal specimens collected between November-2007 and October-2008, that previously had been tested for rotavirus (RV), were tested for NoV and SaV by realtime RT-PCR. Positive samples were genotyped by conventional RT-PCR followed by sequencing. GII NoV was detected in 180 (36%) and SaV in 7 (1.4%) of the samples. NoV was detected year-round ranging from 9.5% in April to 81.5% in September among RV negative samples. NoV GII.4 Minerva (2006b) was the dominant genotype (93%) with a few other genotypes detected including GII.3 (4.4%), GII.13 (1.7%), and GII.2 (0.6%) but no GI strains. Only GI and GII SaV strains were detected in this study. No difference in NoV prevalence between age groups was noted. Frequency of vomiting or fever was similar between children with NoV and RV infection, yet, NoV caused diarrhea with longer duration. In conclusion, NoV is the second most frequent cause of diarrhea in hospitalized children in North Vietnam.     

Emergence of Norovirus GII.4 variants in acute gastroenteritis outbreaks in South Korea between 2006 and 2013

BackgroundNew emerging strains of noroviruses (NoVs) often increase acute gastroenteritis (AGE) outbreaks worldwide.ObjectiveWe analyzed the epidemiological features and genotypic patterns of NoVs in AGE outbreaks.Study designTo elucidate the public health impact of NoVs during AGE outbreaks in South Korea, a molecular and epidemiological investigation was performed with 318 AGE outbreaks reported from the Gyeonggi province of South Korea during the period from 2006 to 2013.ResultsNoVs were associated with 102 (32.1%) of the AGE outbreaks. Epidemiological data revealed that the majority of NoV outbreaks were in the student group (47.1%), and the majority of AGE patients were identified in schools (68.8%). NoV genogroup (G) II strains were associated with 94 (92.2%) of the NoV outbreaks, and GII.4 strains were predominantly associated with 57.6% (n = 49) of NoV GII outbreaks. Four GII.4 variants (2006b, 2007, 2009 and 2012 variants) emerged and showed different contributions to NoV outbreak activity. The 2006b variant was predominantly associated with NoV outbreaks during the early years of the study period, and was subsequently displaced by the New Orleans 2009 variant, and most recently by the Sydney 2012 variant. In addition, the GII.2, GII.14, and GII.17 strains have recently been often associated with NoV AGE outbreaks.ConclusionsThe emergence of new NoV GII.4 variants significantly affected the NoV outbreak activity in South Korea during the period from 2006 to 2013. The surveillance for new emerging strains affecting NoV outbreak activity should be intensified to develop an adequate policy to prevent further NoV outbreaks.     

Characterisation of norovirus strains in rural Ghanaian children with acute diarrhoea

The incidence of calicivirus infection in Ghana and many other African countries is not known. Thirteen (15.9%) of the 82 diarrhoeic stool samples tested for caliciviruses were positive for noroviruses (NoVs). NoVs were present in all age groups and were detected only during the diarrhoea peak that coincided with the peak rotavirus season. Ten (76.9%) of the NoV detected were genogroup II (GII) NoVs and the remaining three (23.1%) genogroup I (GI) NoVs. The predominant GII detected was GII-4 (60%, 6/10). Three of the GII NoVs were determined to be recombinants of GII-8/GII-14 as deduced from the sequencing of the region spanning the Orf1/2 junction. The GII genotypes formed four clusters with published GII sequences. The data shown enhances understanding of NoV diversity in Ghanaian children and demonstrate the global spread of distinct common genotypes to African countries.     

Distribution of norovirus and sapovirus genotypes with emergence of NoV GII.P16/GII.2 recombinant strains in Chiang Mai,Thailand

Norovirus (NoV) and sapovirus (SaV) are recognized as the causative agents of acute gastroenteritis, and NoV is one of the leading pathogens reported worldwide. This study reports on the distribution of NoV and SaV genotypes in children hospitalized with acute gastroenteritis in Chiang Mai, Thailand, from January 2015 to February 2017. From a total of 843 stool samples, 170 (20.2%) and 16 (1.9%) were identified as having NoV and SaV infections, respectively. Two samples (0.2%) were positive for both NoV and SaV. Of these, NoV GII.4 (57.2%) was the dominant genotype, followed by GII.2, GII.3, GII.17, GII.6, GII.7, GII.13, GII.14, GII.15, GII.21, GI.6, and GI.5. Among the NoV GII.4 variants, Sydney 2012 was the dominant variant during the period 2015-2016, while the other variants detected in this study were Asia 2003 and New Orleans 2009. Interestingly, an increase of NoV GII.2 was observed in 2016 and 2017. Characterization of partial RNA-dependent RNA polymerase and VP1 nucleotide sequences of GII.2 strains revealed that more than half of the GII.2 strains circulating in 2016 and 2017 were recombinant strains of GII.P16/GII.2. For SaV, the majority of strains belonged to GI.1 (55.6%) and GI.2 (33.3%), while GII.5 accounted for 11.1%. In conclusion, this study demonstrates the diversity of NoV and SaV, and the emergence of NoV GII.P16/GII.2 recombinant strains in 2016 and 2017 in Chiang Mai, Thailand.     

Enteric viruses in healthy children in Cameroon: viral load and genotyping of norovirus strains

Data regarding prevalence of noroviruses (NoVs) among asymptomatic persons are scarce. The current study carried out on samples from Cameroon describes the asymptomatic shedding of NoVs and other enteric viruses in healthy children and in adults infected with HIV but lacking symptoms of gastroenteritis. Enteric viruses were common with a prevalence of 53.7% in the children, and 35.5% in the adult participants. Multiple enteric viruses (2–5 agents) were detected in fecal samples from 65% of the children, and co‐infection with NoV was demonstrated in almost all cases of multiple infections. NoV viral loads in the healthy children were within disease causing range and significantly higher than those observed in the adults (P < 0.01). Sequencing and genotyping of NoV strains by phylogeny showed a marked diversity within two distinct genogroups, GI and GII, and strains clustered with genotypes GI.3, GII.17, GII.8, and GII.4. Genetic similarities to recent outbreak strains from other continents suggest a rapid circulation of NoVs that includes healthy children, who may constitute a reservoir for pathogenic NoVs. J. Med. Virol. 83:2135–2142, 2011. © 2011 Wiley Periodicals, Inc.     

Emerging norovirus GII.4 2008 variant detected in hospitalised paediatric patients in South Africa

BackgroundNoroviruses (NoVs) are important enteric pathogens that cause gastroenteritis worldwide. The first documented NoV outbreaks in South Africa (SA) were described in 1993. The current NoV prevalence and circulating genotypes are unknown. SA lacks NoV outbreak reporting systems and therefore the number and impact of NoV infections is underestimated.ObjectivesThis study aimed to determine the prevalence and genetic diversity of NoV infections in hospitalised paediatric patients with gastroenteritis in SA during 2008.Study designStool specimens referred for virological analysis from hospitalised children ≤13 years, with gastroenteritis, were screened for rotavirus, human adenovirus and human astrovirus by enzyme immunoassay and for NoV genogroup I (GI), II (GII) and sapovirus by real-time RT-PCR. NoV strains were genotyped, and variants identified, based on sequence and phylogenetic analyses of the 5′ end or the full length of the capsid gene, respectively.ResultsRotavirus was the most prevalent virus detected in 24.2% (61/252) of specimens, followed by NoV in 14.3% (35/245) and adenovirus, astrovirus and sapovirus in 9.6%, 6.7% and 4% of specimens, respectively. NoVs were only detected in children ≤2 years. The GII NoVs (89%) predominated and eight types were identified with GII.4 (43%) detected most frequently. The emerging 2008 GII.4 variant represented 80% of the GII.4 strains.ConclusionsA diverse range of NoV genotypes were identified in hospitalised children with gastroenteritis. The 2008 GII.4 variant was the most frequently detected strain in the study. This is the first report of NoV GII.4 viruses in SA.     

Norovirus GII.4 variant 2006b caused epidemics of acute gastroenteritis in Australia during 2007 and 2008

BackgroundOver the last decade, four epidemics of norovirus-associated gastroenteritis have been reported in Australia. These epidemics were characterized by numerous outbreaks in institutional settings such as hospitals and nursing homes, as well as increases in requests for NoV testing in diagnostic centers. During 2007 and 2008, widespread outbreaks of acute gastroenteritis were once again seen across Australia, peaking during the winter months.ObjectivesThe primary objective of this study was to characterize two winter epidemics of NoV-associated gastroenteritis in 2007 and 2008 in Australia. Following this, we aimed to determine if these epidemics were caused by a new GII.4 variant or a previously circulating NoV strain.Study designNoV-positive fecal samples (n = 219) were collected over a 2-year period, December 2006 to December 2008, from cases of acute gastroenteritis in Australia. NoV RNA was amplified from these samples using a nested RT-PCR approach targeting the 5′ end of the capsid gene, termed region C. Further, characterization was performed by sequence analysis of the RdRp and capsid genes and recombination was identified using SimPlot.ResultsFrom 2004 to 2008, peaks in the numbers of NoV-positive EIA tests from the Prince of Wales Hospital Laboratory correlated with the overall number of gastroenteritis outbreaks reported to NSW Health, thereby supporting recent studies showing that NoV is the major cause of outbreak gastroenteritis. The predominant NoV GII variant identified during the 2007–2008 period was the GII.4 pandemic variant, 2006b (71.51%, 128/179), which replaced the 2006a variant identified in the previous Australian epidemic of 2006. Four novel GII variants were also identified including the three GII.4 variants: NoV 2008, NoV Osaka 2007 and NoV Cairo 2007, and one novel recombinant NoV designated GII.e/GII.12.ConclusionThe increase in acute gastroenteritis outbreaks in 2007 and 2008 were associated with the spread of the NoV GII.4 variant 2006b.