Complete genome sequences of novel canine noroviruses in Hong Kong

We report the complete genome sequences of two novel isolates of norovirus isolated from the fecal swab specimens of dogs in Hong Kong. The complete viral genome is approximately 7.6 kb in length and consists of 3 overlapping open reading frames encoding the ORF1 polyprotein, VP1, and VP2, respectively. Analysis of the VP1 sequence suggested that these noroviruses are divergent from known noroviruses and may represent a novel phylogenetic clade within the genus.     

Genome-wide analyses of human noroviruses provide insights on evolutionary dynamics and evidence of coexisting viral populations evolving under recombination constraints

Norovirus is a major cause of acute gastroenteritis worldwide. Over 30 different genotypes, mostly from genogroup I (GI) and II (GII), have been shown to infect humans. Despite three decades of genome sequencing, our understanding of the role of genomic diversification across continents and time is incomplete. To close the spatiotemporal gap of genomic information of human noroviruses, we conducted a large-scale genome-wide analyses that included the nearly full-length sequencing of 281 archival viruses circulating since the 1970s in over 10 countries from four continents, with a major emphasis on norovirus genotypes that are currently underrepresented in public genome databases. We provided new genome information for 24 distinct genotypes, including the oldest genome information from 12 norovirus genotypes. Analyses of this new genomic information, together with those publicly available, showed that (i) noroviruses evolve at similar rates across genomic regions and genotypes; (ii) emerging viruses evolved from transiently-circulating intermediate viruses; (iii) diversifying selection on the VP1 protein was recorded in genotypes with multiple variants; (iv) non-structural proteins showed a similar branching on their phylogenetic trees; and (v) contrary to the current understanding, there are restrictions on the ability to recombine different genomic regions, which results in co-circulating populations of viruses evolving independently in human communities. This study provides a comprehensive genetic analysis of diverse norovirus genotypes and the role of non-structural proteins on viral diversification, shedding new light on the mechanisms of norovirus evolution and transmission.     

Detection and phylogenetic analysis of norovirus in Corbicula fluminea in a freshwater river in Japan

To study the molecular epidemiology of noroviruses (NoVs) in bivalves residing in freshwater rivers, we detected, quantified and phylogenetically analyzed the NoV genome in purified concentrates obtained from the gills and digestive diverticula of Corbicula fluminea in a freshwater river in Gunma Prefecture, Japan. We detected the NoV genome in 35 of the 58 C. fluminea samples. Based on our phylogenetic analysis, the NoV genome detected in the samples was classified into 4 genotypes (GI/1, GI/2, GI/3 and GI/4) in genogroup I and 5 genotypes (GII/3, GII/4, GII/5, GII/8 and GII/12) in genogroup II. The phylogenetic tree showed wide genetic diversity among the genogroups. In addition, more than 10(4) copies of the NoV genome were detected in 2 of 35 samples. These results suggest that the freshwater bivalve C. fluminea is a reservoir for NoVs, similar to seawater bivalves such as oysters.     

Genomic surveillance of coxsackievirus A10 reveals genetic features and recent appearance of genogroup D in Shanghai,China, 2016-2020

Coxsackievirus A10 (CVA10) is one of the major causative agents of hand, foot and mouth disease (HFMD). To investigate the epidemiological characteristics as well as genetic features of CVA10 currently circulating in Shanghai, China, we collected a total of 9,952 sporadic HFMD cases from January 2016 to December 2020. In the past five years, CVA10 was the fourth prevalent causatives associated with HFMD in Shanghai and the overall positive rate was 2.78%. The annual distribution experienced significant fluctuations over the past five years. In addition to entire VP1 sequencing, complete genome sequencing and recombination analysis of CVA10 isolates in Shanghai were further performed. A total of 64 near complete genomes and 11 entire VP1 sequences in this study combined with reference sequences publicly available were integrated into phylogenetic analysis. The CVA10 sequences in this study mainly belonged to genogroup C and presented 91%-100% nucleotide identity with other Chinese isolates based on VP1 region. For the first time, our study reported the appearance of CVA10 genogroup D in Chinese mainland, which had led to large-scale outbreaks in Europe previously. The recombination analysis showed the recombination break point located between 5,100 nt and 6,700 nt, which suggesting intertypic recombination with CVA16 genogroup D. To conclusion, CVA10 genogroup C was the predominant genogroup in Shanghai during 2016-2020. CVA10 recombinant genogroup D was firstly reported in circulating in Chinese mainland. Continuous surveillance is needed to better understand the evolution relationships and transmission pathways of CVA10 to help to guide disease control and prevention.     

Analysis of amino acid variation in the P2 domain of the GII-4 norovirus VP1 protein reveals putative variant-specific epitopes

Background

Human noroviruses are a highly diverse group of viruses classified into three of the five currently recognised Norovirus genogroups, and contain numerous genotypes or genetic clusters. Noroviruses are the major aetiological agent of endemic gastroenteritis in all age groups, as well as the cause of periodic epidemic gastroenteritis. The noroviruses most commonly associated with outbreaks of gastroenteritis are genogroup II genotype 4 (GII-4) strains. The relationship between genotypes of noroviruses with their phenotypes and antigenic profile remains poorly understood through an inability to culture these viruses and the lack of a suitable animal model.

Methodology/Principal Findings

Here we describe a study of the diversity of amino acid sequences of the highly variable P2 region in the major capsid protein, VP1, of the GII-4 human noroviruses strains using sequence analysis and homology modelling techniques.

Conclusions/Significance

Our data identifies two sites in this region, which show significant amino acid substitutions associated with the appearance of variant strains responsible for epidemics with major public health impact. Homology modelling studies revealed the exposed nature of these sites on the capsid surface, providing supportive structural data that these two sites are likely to be associated with putative variant-specific epitopes. Furthermore, the patterns in the evolution of these viruses at these sites suggests that noroviruses follow a neutral network pattern of evolution.     

Acute diarrhea in west african children: diverse enteric viruses and a novel parvovirus genus

Parvoviruses cause a variety of mild to severe symptoms or asymptomatic infections in humans and animals. During a viral metagenomic analysis of feces from children with acute diarrhea in Burkina Faso, we identified in decreasing prevalence nucleic acids from anelloviruses, dependoviruses, sapoviruses, enteroviruses, bocaviruses, noroviruses, adenoviruses, parechoviruses, rotaviruses, cosavirus, astroviruses, and hepatitis B virus. Sequences from a highly divergent parvovirus, provisionally called bufavirus, were also detected whose NS1 and VP1 proteins showed <39% and <31% identities to those of previously known parvoviruses. Four percent of the fecal samples were PCR positive for this new parvovirus, including a related bufavirus species showing only 72% identity in VP1. The high degree of genetic divergence of these related genomes from those of other parvoviruses indicates the presence of a proposed new Parvoviridae genus containing at least two species. Studies of the tropism and pathogenicity of these novel parvoviruses will be facilitated by the availability of their genome sequences.     

Structural Evolution of the Emerging 2014-2015 GII.17 Noroviruses

Recent reports suggest that human genogroup II genotype 17 (GII.17) noroviruses are increasing in prevalence. We analyzed the evolutionary changes of three GII.17 capsid protruding (P) domains. We found that the GII.17 P domains had little cross-reactivity with antisera raised against the dominant GII.4 strains. X-ray structural analysis of GII.17 P domains from 2002 to 2014 and 2015 suggested that surface-exposed substitutions on the uppermost part of the P domain might have generated a novel 2014-2015 GII.17 variant.     

Rapid detection of noroviruses in fecal samples and shellfish by nucleic acid sequence-based amplification

The purpose of this study was to determine the efficacy of a nucleic acid sequence-based amplification (NASBA) method of detecting noroviruses in artificially and naturally contaminated shellfish. We used 58 fecal samples that tested positive for noroviruses with electron microscopy (EM) to develop an NASBA assay for these viruses. Oligonucleotide primers targeting the polymerase coding region were used to amplify the viral RNA in an isothermal process that resulted in the accumulation of RNA amplicons. These amplicons were detected by hybridization with digoxigenin-labeled oligonucleotide probes that were highly specific for genogroup I (GI) and genogroup II (GII) of noroviruses. The expected band of 327 bp appeared in denaturing agarose gel without any nonspecific band. The specific signal for each amplicon was obtained through Northern blotting in many repeats. All fecal samples of which 46 (79.3%) belonged to GII and 12 (20.6%) belonged to GI were positive for noroviruses by EM and by NASBA. Target RNA concentrations as low as 5 pg/ml were detected in fecal specimens using NASBA. When the assay was applied to artificially contaminated shellfish, the sensitivity to nucleic acid was 100 pg/1.5 g shellfish tissue. The potential use of this assay was also confirmed in naturally contaminated shellfish collected from different ponds in Guangzhou city of China, of which 24 (18.76%) out of 128 samples were positive for noroviruses; of these, 19 (79.6%) belonged to GII and 5 (20.4%) belonged to GI. The NASBA assay provided a more rapid and efficient way of detecting noroviruses in fecal samples and demonstrated its potential for detecting noroviruses in food and environmental samples with high specificity and sensitivity.     

Characterization of an enteropathogenic bovine calicivirus representing a potentially new calicivirus genus

Bovine enteric caliciviruses (BEC) are associated with diarrhea in young calves. The BEC strains detected in Europe form a third genogroup within the genus "Norwalk-like viruses" (NLV) of the family Caliciviridae. In this report, we present sequence, clinical, and histological data characterizing a novel enteropathogenic BEC strain, NB, detected in fecal specimens from calves in the United States. The complete RNA genome of the NB virus is 7,453 bases long and is organized into two open reading frames (ORFs). ORF-1 is 2,210 amino acids long and encodes a large nonstructural polyprotein contiguous with the major capsid protein (VP1), similar to the lagoviruses and "Sapporo-like viruses" (SLV). The conserved calicivirus motifs were identified in the nonstructural proteins. ORF-2 is located at the 3' end of the genome and encodes a small basic protein (VP2) of 225 amino acids. The 5' and 3' untranslated regions are 74 and 67 bases long, respectively. Among caliciviruses, NB virus shows amino acid identities of 14.1 to 22.6% over the entire ORF-1 nonstructural-protein sequence with NLV, SLV, vesivirus, and lagovirus strains, while the overall sequence identity of the complete NB VP-1 with other caliciviruses is low, varying between 14.6 and 26.7%. Phylogenetic analysis of the complete VP1 protein, including strains from all four calicivirus genera, showed the closest grouping of NB virus to be with viruses in the genus Lagovirus, which cause liver infections and systemic hemorrhage in rabbits. In gnotobiotic calves, however, NB virus elicited only diarrhea and intestinal lesions that were most severe in the upper small intestine (duodenum and jejunum), similar to the NLV BEC strains. The tissues of major organs, including the lung, liver, kidney, and spleen, had no visible microscopic lesions.     

Covariation of major and minor viral capsid proteins in norovirus genogroup II genotype 4 strains

We report sequence hypervariability in the viral protein 1 (VP1) interaction domain of VP2 in the norovirus (NoV) genogroup II genotype 4 (GII.4) lineage on 3 levels: (i) the global evolution of pandemic/epidemic strains from the mid-1970s through post-2006, (ii) the local emergence of an epidemic strain, and (iii) an immunocompromised patient chronically shedding NoV. When a quantitative yeast two-hybrid assay was used, VP2 was found to interact with VP1 in a time-ordered, strain-dependent manner among 3 NoV GII.4 strains. Our findings suggest that VP1 and VP2 may covary in virus evolution and that sequence hypervariability of VP2 may be functionally driven. Further investigations are warranted.     

Structural basis for norovirus inhibition and fucose mimicry by citrate

Human noroviruses bind with their capsid-protruding domains to histo-blood-group antigens (HBGAs), an interaction thought to direct their entry into cells. Although human noroviruses are the major cause of gastroenteritis outbreaks, development of antivirals has been lacking, mainly because human noroviruses cannot be cultivated. Here we use X-ray crystallography and saturation transfer difference nuclear magnetic resonance (STD NMR) to analyze the interaction of citrate with genogroup II (GII) noroviruses. Crystals of citrate in complex with the protruding domain from norovirus GII.10 Vietnam026 diffracted to 1.4 Å and showed a single citrate bound at the site of HBGA interaction. The citrate interaction was coordinated with a set of capsid interactions almost identical to that involved in recognizing the terminal HBGA fucose, the saccharide which forms the primary conserved interaction between HBGAs and GII noroviruses. Citrate and a water molecule formed a ring-like structure that mimicked the pyranoside ring of fucose. STD NMR showed the protruding domain to have weak affinity for citrate (460 μM). This affinity, however, was similar to the affinities of the protruding domain for fucose (460 μM) and H type 2 trisaccharide (390 μM), an HBGA shown previously to be specifically recognized by human noroviruses. Importantly, competition STD NMR showed that citrate could compete with HBGA for norovirus binding. Together, the results suggest that citrate and other glycomimetics have the potential to block human noroviruses from binding to HBGAs.     

Function of VP2 protein in the stability of the secondary structure of virus-like particles of genogroup II norovirus at different pH levels: Function of VP2 protein in the stability of NoV VLPs

VP2 is the minor structural protein of noroviruses (NoV) and may function in NoV particle stability. To determine the function of VP2 in the stability of the NoV particle, we constructed and purified two kinds of virus-like particles (VLPs), namely, VLPs (VP1) and VLPs (VP1+VP2), from Sf9 cells infected with recombinant baculoviruses by using a Bac-to-Bac® baculovirus expression system. The two kinds of VLPs were treated with different phosphate buffers (pH 2 to pH 8); the secondary structure was then analyzed by far UV circular dichroism (CD) spectroscopy. Results showed that significant disruptions of the secondary structure of proteins were not observed at pH 2 to pH 7. At pH 8, the percentages of a-helix, β-sheet, and β-turn in VLPs (VP1) were decreased from 11% to 8%, from 37% to 32%, and from 20% to 16%, respectively. The percentage of coil was increased from 32% to 44%. By contrast, the percentages of α-helix, β-sheet, and β-turn in VLPs (VP1+VP2) were decreased from 11% to 10%, from 37% to 35%, and from 20% to 19%, respectively. The percentage of coil was increased from 32% to 36%. VLPs (VP1+VP2) was likely more stable than VLPs (VP1), as indicated by the percentage of the secondary structures analyzed by CD. These results suggested that VP2 could stabilize the secondary structure of VLPs under alkaline pH conditions. This study provided novel insights into the molecular mechanism of the function of VP2 in the stability of NoV particles.     

Characterization of a broadly reactive monoclonal antibody against norovirus genogroups I and II: recognition of a novel conformational epitope

Norovirus, which belongs to the family Caliciviridae, is one of the major causes of nonbacterial acute gastroenteritis in the world. The main human noroviruses are of genogroup I (GI) and genogroup II (GII), which were subdivided further into at least 15 and 18 genotypes (GI/1 to GI/15 and GII/1 to GII/18), respectively. The development of immunological diagnosis for norovirus had been hindered by the antigen specificity of the polyclonal antibody. Therefore, several laboratories have produced broadly reactive monoclonal antibodies, which recognize the linear GI and GII cross-reactive epitopes or the conformational GI-specific epitope. In this study, we characterized the novel monoclonal antibody 14-1 (MAb14-1) for further development of the rapid immunochromatography test. Our results demonstrated that MAb14-1 could recognize 15 recombinant virus-like particles (GI/1, 4, 8, and 11 and GII/1 to 7 and 12 to 15) and showed weak affinity to the virus-like particle of GI/3. This recognition range is the broadest of the existing monoclonal antibodies. The epitope for MAb14-1 was identified by fragment, sequence, structural, and mutational analyses. Both terminal antigenic regions (amino acid positions 418 to 426 and 526 to 534) on the C-terminal P1 domain formed the conformational epitope and were in the proximity of the insertion region (positions 427 to 525). These regions contained six amino acids responsible for antigenicity that were conserved among genogroup(s), genus, and Caliciviridae. This epitope mapping explained the broad reactivity and different titers among GI and GII. To our knowledge, we are the first group to identify the GI and GII cross-reactive monoclonal antibody, which recognizes the novel conformational epitope. From these data, MAb14-1 could be used further to develop immunochromatography.     

High-Resolution Functional Profiling of the Norovirus Genome

Human noroviruses (HuNoV) are a major cause of nonbacterial gastroenteritis worldwide, yet details of the life cycle and replication of HuNoV are relatively unknown due to the lack of an efficient cell culture system. Studies with murine norovirus (MNV), which can be propagated in permissive cells, have begun to probe different aspects of the norovirus life cycle; however, our understanding of the specific functions of the viral proteins lags far behind that of other RNA viruses. Genome-wide functional profiling by insertional mutagenesis can reveal protein domains essential for replication and can lead to generation of tagged viruses, which has not yet been achieved for noroviruses. Here, transposon-mediated insertional mutagenesis was used to create 5 libraries of mutagenized MNV infectious clones, each containing a 15-nucleotide sequence randomly inserted within a defined region of the genome. Infectious virus was recovered from each library and was subsequently passaged in cell culture to determine the effect of each insertion by insertion-specific fluorescent PCR profiling. Genome-wide profiling of over 2,000 insertions revealed essential protein domains and confirmed known functional motifs. As validation, several insertion sites were introduced into a wild-type clone, successfully allowing the recovery of infectious virus. Screening of a number of reporter proteins and epitope tags led to the generation of the first infectious epitope-tagged noroviruses carrying the FLAG epitope tag in either NS4 or VP2. Subsequent work confirmed that epitope-tagged fully infectious noroviruses may be of use in the dissection of the molecular interactions that occur within the viral replication complex.     

Murine noroviruses comprising a single genogroup exhibit biological diversity despite limited sequence divergence

Viruses within the genus Norovirus of the family Caliciviridae are the major cause of acute, nonbacterial gastroenteritis worldwide. Human noroviruses are genetically diverse, with up to 57% divergence in capsid protein sequences, and comprise three genogroups. The significance of such genetic diversity is not yet understood. The discovery of murine norovirus (MNV) and its ability to productively infect cultured murine macrophages and dendritic cells has provided an opportunity to determine the functional consequences of norovirus diversity in vitro and in vivo. Therefore, we compared the full-length genomes of 21 new MNV isolates with five previously sequenced MNV genomes and demonstrated a conserved genomic organization consisting of four open reading frames (ORFs) and a previously unknown region of nucleotide conservation in ORF2. A phylogenetic analysis of all 26 MNV genomes revealed 15 distinct MNV strains, with up to 13% divergence at the nucleotide level, that comprise a single genotype and genogroup. Evidence for recombination within ORF2 in several MNV genomes was detected by multiple methods. Serological analyses comparing neutralizing antibody responses between highly divergent strains suggested that the MNV genogroup comprises a single serotype. Within this single genogroup, MNV strains exhibited considerable biological diversity in their ability to grow in culture and to infect and/or persist in wild-type mice. The isolation and characterization of multiple MNV strains illustrate how genetic analysis may underestimate the biological diversity of noroviruses and provide a molecular map for future studies of MNV biology.