Norovirus Genome Research Articles

Complete genome sequence and phylogenetic analysis of a recombinant Korean norovirus, CBNU1, recovered from a 2006 outbreak

We have determined the complete nucleotide and deduced amino acid sequences of the RNA genome of CBNU1, a human norovirus (NoV) recovered from a 2006 outbreak in South Korea. The genome of 7547 nucleotides, excluding a 3′-poly(A) tail of 11–105 nucleotides, encodes three overlapping open reading frames (ORFs): ORF1 (nucleotides 5–5104), ORF2 (nucleotides 5085–6731), and ORF3 (nucleotides 6731–7495). In a comparison to 108 other currently available completely sequenced NoVs representing all five genogroups (GI–GV) except GIV, the CBNU1 strain was highly similar to GII.3 NoVs. Multiple sequence alignments of the completely sequenced NoV genomes revealed five hypervariable regions throughout their genomes: two in ORF1, one in ORF2, and two in ORF3. At both the nucleotide and amino acid levels, genome-based phylogenetic analyses invariably showed that the CBNU1 strain was most closely related to three GII.3 NoVs: the American Texas/TCH04-577 and the two Japanese Saitama U18 and Saitama U201 strains; furthermore, these genome-based phylogenetic topologies corresponded most closely to those based on the ORF2 genes, as compared to those based on the ORF1 and ORF3 genes. Subsequent ORF2-based phylogenetic analyses of a selection of 126 other NoVs representing all 19 GII genotypes, in combination with genome-based Simplot analyses, showed that the CBNU1 strain was a recombinant GII.3 NoV with a breakpoint at the ORF1/ORF2 junction between two putative parent-like strains, Guangzhou/NVgz01 and Texas/TCH04-577. Overall, the CBNU1 strain represents the first Korean human NoV whose genome has been completely sequenced and for which its relationship with a large panel of genetically diverse NoVs has been extensively characterized.

Different Genotypic Sapoviruses Detected in Two Simultaneous Outbreaks of Gastroenteritis among Schoolchildren in the Same School District in Hokkaido, Japan

Norovirus (NV) and sapovirus (SV) are members of the family Caliciviridae and are etiologic agents for acute gastroenteritis in humans (1,2). Presently, NV is the more causative of the two for the outbreak of gastroenteritis, whereas only a few outbreaks of SV have been reported. Acute gastroenteritis due to SV occurs mainly in infants and young children (2). Previously, we reported on outbreaks of SV-associated gastroenteritis in Hokkaido, Japan (3). Here, we describe two outbreaks that occurred almost simultaneously among schoolchildren in the same district in Hokkaido. The SV genomes were detected and differentiated into a genogroup I genotype 2 (GI/2) and a GI/3. The first outbreak (Outbreak 1) occurred at an elementary school between March 8 and 23 in 2009. A total of 48 schoolchildren developed gastroenteric symptoms such as vomiting and diarrhea. The second outbreak (Outbreak 2) occurred at a junior high school between March 11 and 23 in 2009. Finally, a total of 74 students suffered from gastroenteritis. These two outbreaks occurred in the same school district. Nine stool specimens from Outbreak 1 were collected from 1 to 13 days of illness, while 11 stool specimens from Outbreak 2 were collected from 1 to 8 days of illness. The epidemic curves of these two outbreaks are shown in Figs. 1 and 2. Each stool specimen was prepared as 10% emulsions with nuclease-free distilled water (Nippon Gene Co., Tokyo, Japan) and was centrifuged at 3,000 rpm for 20 min at 4°C. The supernatant was collected and transferred to microtubes, followed by centrifugation at 10,000 rpm for 10 min at 4°C. Extraction of viral RNA and reverse transcription (RT) were carried out as described previously (4). The cDNA was used for the polymerase chain reaction (PCR) and real-time PCR. Two sets of primers, P1/P3 and NV82/SM82/NV81, and another two sets of primers, COG1F/G1SKR and COG2F/ G2SKR, were used to amplify the polymerase and the capsid protein regions of the NV genome, respectively (5–8). To amplify the polymerase and capsid protein regions of the SV genome, the sets of primers SV-r-c/sapp36 and SV-F13/SVF14/SV-R13/SV-R14 were used. The conditions of PCR amplification were performed as described previously (4,9, 10). The PCR products were purified and directly sequenced using the Big Dye terminator cycle sequencing kit (Applied Biosystems, Foster City, Calif., USA). Nucleotide sequences determined in this study have been submitted to Genbank/ EMBL/DDBJ (accession no. AB518056 and AB518057). Multiple alignment and phylogenetic analyses were carried out based on partial capsid nucleotide sequences (approxiFig. 1. Number of patients and virus detection for norovirus (NV) and/ or sapovirus (SV) of gastroenteritis in Outbreak 1 from March 8 to 23, 2009. Specimens which were positive for NV and/or SV genomes were indicated. Two specimens which were collected in March 13 or 16 were positive for both NV and SV genomes.

Functional Analysis of RNA Structures Present at the 3′ Extremity of the Murine Norovirus Genome: the Variable Polypyrimidine Tract Plays a Role in Viral Virulence

Interactions of host cell factors with RNA sequences and structures in the genomes of positive-strand RNA viruses play various roles in the life cycles of these viruses. Our understanding of the functional RNA elements present in norovirus genomes to date has been limited largely to in vitro analysis. However, we recently used reverse genetics to identify evolutionarily conserved RNA structures and sequences required for norovirus replication. We have now undertaken a more detailed analysis of RNA structures present at the 3' extremity of the murine norovirus (MNV) genome. Biochemical data indicate the presence of three stable stem-loops, including two in the untranslated region, and a single-stranded polypyrimidine tract [p(Y)] of variable length between MNV isolates, within the terminal stem-loop structure. The well-characterized host cell pyrimidine binding proteins PTB and PCBP bound the 3'-untranslated region via an interaction with this variable sequence. Viruses lacking the p(Y) tract were viable both in cell culture and upon mouse infection, demonstrating that this interaction was not essential for virus replication. However, competition analysis with wild-type MNV in cell culture indicated that the loss of the p(Y) tract was associated with a fitness cost. Furthermore, a p(Y)-deleted mutant showed a reduction in virulence in the STAT1(-/-) mouse model, highlighting the role of RNA structures in norovirus pathogenesis. This work highlights how, like with other positive-strand RNA viruses, RNA structures present at the termini of the norovirus genome play important roles in virus replication and virulence.

Real-time molecular beacon NASBA for rapid and sensitive detection of norovirus GII in clinical samples

To improve the sensitivity and efficiency of the real-time nucleic acid sequence based amplification (NASBA) assay targeting the open reading frame 1-2 (ORF1-ORF2) junction of the norovirus (NoV) genome, a selection of clinical samples were analyzed. The assay results were compared with those of TaqMan and conventional reverse transcription PCR (RT-PCR) and a commercial enzyme-linked immunoassay (ELISA) for the specific detection of GII NoV in 96 fecal samples. Based on end-point dilution, the two real-time assays had similar sensitivities (0.01 particle detectable units), two log10 cycles greater than that of conventional RT-PCR. GII NoV was detected in 88.54% of the samples by real-time NASBA, in 86.46% by TaqMan RT-PCR, in 81.25% by conventional RT-PCR, and in 65.7% by ELISA. The two real-time assays were in agreement for 88.5% of the samples. These results demonstrate that real-time NASBA with a molecular beacon probe is highly sensitive, accurate, and specific for NoV detection in clinical samples. Applying this technique to samples with complex matrix and low viral loads, such as food and environmental samples, could be useful for the detection of NoVs and will improve the prevention of NoV outbreaks.

Norovirus distribution within an estuarine environment.

Human norovirus (NoV) has been studied extensively as an important cause of gastroenteritis outbreaks worldwide. While oysters are a primary vehicle for infection, few studies have examined the wider distribution of NoV in the estuarine environment. Active shellfish-harvesting areas in Georgia were examined for the prevalence, genotype diversity, and concentrations of NoV in a variety of estuarine sample types over the course of 1 year. Of the 225 samples (9 oyster, 72 water, 72 63- to 200-microm plankton, and 72 >200-microm plankton) collected from 12 stations across two estuaries, 21 samples (9.3%) tested positive for NoV. By sample type, 55.0% (5/9) of oysters, 8.3% (6/72) of water samples, 11.1% (8/72) of 63- to 200-microm plankton samples, and 2.8% (2/72) of >200-microm plankton samples were positive for human NoV. The two NoV-positive >200-microm plankton samples, which contained mainly zooplankton, had the greatest quantity of NoV genomes (3.5 x 10(13) and 1.7 x 10(15) genomes g(-1)) of any sample tested. The majority, 90.5% (19/21), of the samples tested positive for genogroup I NoV, and only 9.5% (2/21) of the samples tested positive for genogroup II. The high concentrations of NoV in plankton samples compared to water and oyster samples were unexpected and provide new insights into the presence and distribution of human NoV in the water environment.

Occurrence and persistence of enteroviruses, noroviruses and F-specific RNA phages in natural wastewater biofilms

Enteroviruses and noroviruses are pathogenic viruses excreted by infected individuals. Discharged in wastewaters, some of these viruses can be captured by biofilms. In the present study, we assessed the occurrence and persistence of these viruses in wastewaters and in corresponding biofilms. Natural wastewaters and biofilms were analyzed monthly from January to July using real-time RT-PCR. Enterovirus RNA was detected in wastewater in June while norovirus RNA was detected from January to March. In contrast, biofilm analysis revealed the presence of both enterovirus and norovirus genomes throughout the study period. For instance, enterovirus and norovirus genogroups (GG) I and II were detected in 50, 46 and 37% of the biofilm samples, respectively ( n = 24). In a laboratory experiment, persistence of norovirus GGI RNA (quantified using molecular techniques) and F-specific bacteriophages (quantified using both culture and molecular techniques) was assessed in wastewater and corresponding naturally-contaminated biofilms at both 4 and 20 °C. The concentrations of viral genomes (norovirus GGI and F-specific RNA phage) were very stable in biofilms. Indeed, no significant decrease was observed during the persistence experiment that lasted 49 days. Furthermore, regardless of our experimental conditions, viral genome and infectious F-specific bacteriophages persisted longer in biofilm than in wastewater. According to our results, wastewater biofilms may contribute to the persistence and dispersal of pathogenic viruses outside of epidemic periods.

Tracking the transmission routes of genogroup II noroviruses in suspected food‐borne or environmental outbreaks of gastroenteritis through sequence analysis of the P2 domain

The aim of this study was to apply sequence analysis of a hyper variable region of the norovirus (NoV) genome in order to identify point source outbreaks associated with suspect food or water. The hyper-variable region of the gene encoding the P2 domain was chosen as small differences in sequence are likely to indicate virus from different sources whereas identical sequence may reveal transmission routes and the source of contamination. Strains with 100% similarity were considered as originating from a common source, whereas, strains with one or more mutations in the hyper variable region sequenced were regarded as representing unrelated transmission events. This study was able to identify a point source outbreak of a dominant strain, GII-4, on a cruise ship but also of a less common strain, GII-2, between two schools. Also identical GII-3 strains were demonstrated in food handlers amongst the same outbreak; however epidemiologically related outbreaks showed different GII-3 strains indicating multiple sources of contamination.

Epidemiological study of bovine norovirus infection by RT-PCR and a VLP-based antibody ELISA

Noroviruses, belonging to the family Caliciviridae, have been identified in human beings and in several animal species including cattle. The distribution of bovine norovirus infections was investigated by both RT-PCR to detect norovirus genomes and a virus-like particles-based ELISA to detect genotype 2 bovine norovirus antibodies. During a 1-year systematic study, a virus prevalence of 7.5% (CI 95%: [3.7; 13.4%]) (10 out of 133 samples) was found in stool samples from diarrhoeic calves screened by RT-PCR. Nucleotide sequencing performed on the polymerase region classified all the norovirus amplicons in the bovine norovirus genotype 2. Rather surprisingly, some rotavirus sequences were also detected. On the basis of the polymerase region, genotype 1 bovine norovirus was not identified. Other enteropathogens were found in all samples. By ELISA, a genotype 2 seroprevalence of 93.2% (CI 95%: [90.4; 95.3%]) was found from calves and adult cattle. Antibody levels against genotype 2 bovine noroviruses rose in the first 6 months of life and were maintained in adults. Together the results of virus prevalence and seroprevalence studies suggest that bovine norovirus infection occurs early in life and that re-infection with serologically related bovine noroviruses strains could occur in adult cattle as reported for rotaviruses. The antibody rise against genotype 2 bovine noroviruses in the adult cattle also suggests a short lived and/or strain specific immunity as already shown in human noroviruses. Genotype 2 bovine noroviruses are endemic in the region investigated.

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.

Phylogenetic prediction of cis-acting elements: a cre-like sequence in Norovirus genome?

BackgroundDiscrete RNA structures such as cis-acting replication elements (cre) in the coding region of RNA virus genomes create characteristic suppression of synonymous site variability (SSSV). Different phylogenetic methods have been developed to predict secondary structures in RNA viruses, for high-resolution thermodynamic scanning and for detecting SSSV. These approaches have been successfully in predicting cis-acting signals in different members of the family Picornaviridae and Caliciviridae. In order to gain insight into the identification of cis-acting signals in viruses whose mechanisms of replication are currently unknown, we performed a phylogenetic analysis of complete genome sequences from 49 Human Norovirus (NoV) strains.FindingsThe complete coding sequences of NoV ORF1 were obtained from the DDBJ database and aligned. Shannon entropy calculations and RNAalifold consensus RNA structure prediction identified a discrete, conserved, invariant sequence region with a characteristic AAACG cre motif at positions 240 through 291 of the RNA dependant RNA polymerase (RdRp) sequence (relative to strain [EMBL:EU794713]). This sequence region has a high probability to conform a stem-loop.ConclusionA new predicted stem-loop has been identified near the 5' end of the RdRp of Human NoV genome. This is the same location recently reported for Hepatovirus cre stem-loop.

Inhibition of norovirus replication by morpholino oligomers targeting the 5′-end of the genome

Noroviruses are an important cause of non-bacterial epidemic gastroenteritis, but no specific antiviral therapies are available. We investigated the inhibitory effect of phosphorodiamidiate morpholino oligomers (PMOs) targeted against norovirus sequences. A panel of peptide-conjugated PMOs (PPMOs) specific for the murine norovirus (MNV) genome was developed, and two PPMO compounds directed against the first AUG of the ORF1 coding sequence near the 5′-end of the genome proved effective in inhibiting MNV replication in cells. A consensus PPMO (designated Noro 1.1), designed to target the corresponding region of several diverse human norovirus genotypes, decreased the efficiency of protein translation in a cell-free luciferase reporter assay and inhibited Norwalk virus protein expression in replicon-bearing cells. Our data suggest that PPMOs directed against the relatively conserved 5′-end of the norovirus genome may show broad antiviral activity against this genetically diverse group of viruses.

Single Base Substitutions in the Capsid Region of the Norovirus Genome during Viral Shedding in Cases of Infection in Areas Where Norovirus Infection Is Endemic

Norovirus (NoV) infections are the major cause of food- and waterborne nonbacterial gastroenteritis in Japan. Some individuals showed long-term excretion of the virus into feces in 29 outbreaks of acute nonbacterial gastroenteritis that occurred in Toyama Prefecture, Japan, in fiscal year 2006. In one of these cases, single base substitutions from A to G in the capsid region of the NoV genome were commonly detected in two individuals during virus shedding by direct sequencing of PCR products. The A-to-G substitution was accompanied by an N-to-S amino acid change. The population of clones that possessed A at the corresponding site was gradually replaced by those with G during the infectious course. Although other substitutions were observed in the complete open reading frame 2 sequence, they were not common in these two individuals. NoVs are capable of evolving in the gastroenteric tract.

Development of a DNA Microarray for the Simultaneous Detection and Genotyping of Noroviruses

Current methods for detecting and genotyping noroviruses focus on the use of reverse transcriptase (RT)–mediated PCR. A major drawback of this approach is that short target RT-PCR products do not always encompass sequences that can be compared among research laboratories, resulting in difficulties for molecular epidemiology. We describe the use of a microarray-based system for simultaneous detection and molecular characterization of noroviruses. The protocol generates a 917-bp RT-PCR product that encompasses two major regions currently used for detection and analysis of norovirus genomes. The PCR products are then hybridized to an oligonucleotide array (NoroChip) based on 50-mer features, which allows for both confirmation of reaction specificity and molecular characterization of the amplified genome. Parallel sequence analyses of amplicons revealed that our microarray data were robust in separating genogroups I and II, and further subtyping to the cluster level was possible. This approach, combining detection and characterization, overcomes the need for expensive and time-consuming sequence analysis of amplified genome targets for molecular epidemiology.

Translation Termination Reinitiation between Open Reading Frame 1 (ORF1) and ORF2 Enables Capsid Expression in a Bovine Norovirus without the Need for Production of Viral Subgenomic RNA

A generally accepted view of norovirus replication is that capsid expression requires production of a subgenomic transcript, the presence of capsid often being used as a surrogate marker to indicate the occurrence of viral replication. Using a polymerase II-based baculovirus delivery system, we observed capsid expression following introduction of a full-length genogroup 3 norovirus genome into HepG2 cells. However, capsid expression occurred as a result of a novel translation termination/reinitiation event between the nonstructural-protein and capsid open reading frames, a feature that may be unique to genogroup 3 noroviruses.

Rapid and Sensitive Detection of Norovirus Genomes in Oysters by a Two-Step Isothermal Amplification Assay System Combining Nucleic Acid Sequence-Based Amplification and Reverse Transcription-Loop-Mediated Isothermal Amplification Assays

We developed a two-step isothermal amplification assay system, which achieved the detection of norovirus (NoV) genomes in oysters with a sensitivity similar to that of reverse transcription-seminested PCR. The time taken for the amplification of NoV genomes from RNA extracts was shortened to about 3 h.

Amplification by long RT-PCR of near full-length norovirus genomes

A long RT-PCR method was developed to amplify the norovirus genome. Starting from RNA extracted directly from clinical samples and using broadly reactive primers, it can generate near full-length amplicons that allow for easy determination of the near complete genomic sequence. Two norovirus isolates from Toronto, Canada, in 2002 and 2005 were sequenced. This approach will facilitate molecular epidemiology studies of noroviruses.

Molecular epidemiology of norovirus gastroenteritis in Soma, Japan, 2001–2003

The aim of the present paper was to investigate the molecular epidemiology of norovirus gastroenteritis in Japan using polymerase chain reaction (PCR) and subsequent phylogenetic analysis. From September 2001 to August 2003, 515 stool samples or rectal swabs were collected from almost all children visiting the Department of Pediatrics, Public Soma General Hospital with gastroenteritis. Samples were examined on reverse transcription (RT)-PCR to detect norovirus genome. The nucleotide sequences of the PCR products were determined and phylogenetic analysis performed. The norovirus genome was detected in 66 samples. The peak season of norovirus gastroenteritis was from November 2001 to February 2002 and from September 2002 to December 2002. Norovirus gastroenteritis occurred most frequently in 1-year-old children. Norovirus strains produced four distinct clusters on phylogenetic analysis. Some strains detected in Soma were closely related to the strains detected in other regions in the world. The Mexico type and Lordsdale type were predominant in the 2001/2002 and 2002/2003 seasons, respectively, and the outbreaks continued for several months. Genetically different noroviruses might cause repeated gastroenteritis outbreaks every year in the Soma area. The long duration of the outbreak by a predominant strain in an epidemic season and the prevalence of infection mainly in the young age group suggested that norovirus epidemics were caused by person-to-person transmission rather than foodborne transmission. Based on molecular epidemiology, it is suggested that the annual prevalence of norovirus gastroenteritis in the Soma area might be caused by person-to-person transmission of genetically different norovirus strains, which might be transmitted from other region in the world.

Quantitative study of persistence of human norovirus genome in water using TaqMan real-time RT-PCR

To evaluate the persistence of human norovirus (NoV) in different types of water at various temperatures using conventional and TaqMan real-time reverse transcription-PCR (RT-PCR). Water from different sources was spiked with NoV and incubated at different temperatures over a 3-month period. NoV viral RNA was amplified by one-step TaqMan real-time RT-PCR and by conventional two-step RT-PCR. NoV persisted in mineral and tap water for over 2 months at all tested temperatures but disappeared after 100 days. At 4 and -20 degrees C, viral degradation was slower than that at 25 degrees C. In river water and effluent from primary sewage treatment, a slight reduction in viral load was observed after 1 month at 4 degrees C. This is the first demonstration of medium-to-long-term survival of human NoVs in different types of water using TaqMan real-time detection. NoV genome may persist for long periods of time in different types of water. Quantitative TaqMan real-time RT-PCR is a sensitive system that allows accurate evaluation of the persistence of human NoVs in different water samples. Our study is one of the few to demonstrate the ability of NoV to survive for a long time in water.

Characterisation of a novel recombination event in the norovirus polymerase gene

This communication describes a novel recombination event in the norovirus genome. Similarity plot analysis of a nucleotide fragment (1003 bp) amplified from a norovirus positive clinical specimen (IrlN05771) identified a previously undescribed recombination point in the 3′ region of the polymerase gene (nucleotide position 4889 bp). Nucleotide multiple alignments demonstrated that Irl05N771 shared 78.6% and 94% identity with all other Irish norovirus sequences before and after the recombination point, respectively and confirmed the sequence as a genogroup II/type 4 recombinant. Irl05N771 shared more identity with Asian norovirus sequences. This is the first description of recombination within the norovirus polymerase and highlights the continuous genetic evolution of noroviruses.

Use of modified oligonucleotides to compensate for sequence polymorphisms in the real-time detection of norovirus

Noroviruses are major causative agents of sporadic and outbreak-associated nonbacterial gastroenteritis worldwide. Real-time RT-PCR has rapidly become the principle means for norovirus detection due to its sensitivity and specificity; however sequence variations in the Norovirus genome can cause problems for real-time chemistries. Using a combination of modified bases and minor groove binder-conjugated Eclipse hybridization probes, a one step real-time RT-PCR assay was designed and optimized to detect norovirus genogroups I and II. Additionally, an RNA internal control was incorporated into the assay to monitor nucleic acid extraction and amplification inhibition. As part of this study, a combination of 36 stool and RNA samples were tested and results were compared to a previously described TaqMan assay. An overall correlation of 97% was obtained. After 7 months of clinical testing, a percent positive rate of 39% was determined, with genogroup II accounting for 98% of the positives. Overall, the Eclipse assay is a sensitive and robust method for detecting and typing norovirus genogroups I and II as well as useful for routine clinical testing. Incorporating modified bases helps overcome design limitations due to single nucleotide polymorphisms.