Comparative murine norovirus studies reveal a lack of correlation between intestinal virus titers and enteric pathology

Human noroviruses are significant emerging pathogens, causing the majority of non-bacterial gastroenteritis outbreaks worldwide. The recent discovery of 30 murine norovirus strains is beginning to facilitate a detailed investigation of norovirus pathogenesis. Here, we have performed an in vivo comparative analysis of two murine norovirus strains, MNV-1 and MNV-3. In immunocompetent mice, MNV-1 caused modest intestinal pathology whereas MNV-3 was attenuated compared to MNV-1. Surprisingly though, MNV-3 reached higher titers in intestinal tissue than MNV-1. MNV-3 also displayed attenuation in mice deficient in the critical interferon signaling molecule STAT-1, demonstrating that MNV-3 attenuation is not a result of increased interferon sensitivity. Importantly, MNV-3-infected mice lost weight and developed gastric bloating and diarrhea in STAT1^−/− mice, from which all animals recovered. This disease profile recapitulates several key features of acute gastroenteritis experienced by people infected with a human norovirus.     

Investigation of the impact of the common animal facility contaminant murine norovirus on experimental murine cytomegalovirus infection

Murine norovirus (MNV) is a recently discovered pathogen that has become a common contaminant of specific pathogen-free mouse colonies. MNV-1 induces a robust interferon-β response and causes histopathology in some mouse strains, suggesting that it may impact other mouse models of infection. Despite many concerns about MNV-1 contamination, there is little information about its impact on immune responses to other infections. This study addresses whether MNV-1 infection has an effect on a model of murine cytomegalovirus (MCMV) infection. Exposure to MNV-1 resulted in a decreased CD8 T cell response to immunodominant MCMV epitopes in both BALB/c and C57BL/6 mice. However, MNV-1 did not impact MCMV titers in either mouse strain, nor did it stimulate reactivation of latent MCMV. These data suggest that while MNV-1 has a mild impact on the immune response to MCMV, it is not likely to affect most experimental outcomes in immunocompetent mice in the MCMV model.     

Subcellular localization of the MNV-1 ORF1 proteins and their potential roles in the formation of the MNV-1 replication complex

Human noroviruses are the leading cause of nonbacterial gastroenteritis worldwide and are now recognised as a significant human pathogen. Whereas human noroviruses cannot be cultivated in the laboratory, mouse norovirus 1 (MNV-1) is easily cultivated and has a defined tropism for cells of a mononuclear origin. As such, MNV-1 provides an ideal opportunity to study many aspects of norovirus biology and replication. Previously, we have shown that MNV-1 RNA replication is associated with components of the early and late secretory pathway and that all six open reading frame 1 (ORF1) proteins are associated with the viral dsRNA within the replication complex (RC) during the course of infection. In this study, we further characterise the subcellular localisation of the MNV-1 ORF1 proteins when recombinantly expressed in cells. We show that two MNV-1 proteins, NS1-2 and NS4, associate with the endoplasmic reticulum and endosomes, respectively. Whereas NS6 (the viral protease) appeared to localize within the cytoplasm and to mitochondria, NS7 (the viral polymerase) was observed to localize diffusely within the cytoplasm and within the nucleus, and NS3 localized to discrete foci within the cytoplasm which were of unknown origin. Based on the localization patterns observed we propose a model by which NS1-2 and NS4 may recruit host membranes to the MNV-1 RC during replication.     

Genetic heterogeneity, evolution, and recombination in noroviruses

Norovirus is one of the most common causes of nonbacterial gastroenteritis in humans. A total of 603 fecal specimens collected from sporadic pediatric cases of acute gastroenteritis in Japan from 2004 to 2005 were tested for the presence of norovirus by RT-PCR. It was found that 51 (8.5%) specimens were positive for norovirus. The norovirus genotypes detected in this study were GII/1, GII/2, GII/3, GII/4, GII/6, and GII/7. Of these, GII/3 was the most predominant (52.9%), followed by GII/4 (37.2%) and others. It was noticed that four distinct types of recombinant noroviruses were co-circulating and the variant norovirus GIIb suddenly emerged to be the leading strain in Japan for the first time. A novel norovirus nomenclature was proposed, in which worldwide noroviruses were classified into seven distinct genogroups (I-VII). Norovirus GI and GII consisted of 16 genotypes with 32 subgenotypes and 23 genotypes with 34 subgenotypes, respectively. Of note, human and porcine noroviruses had a close genetic relationship within GII. Interestingly, multiple short amino acid motifs located at N terminus, S domain, P1 domain, P2 domain, and C terminus of capsid gene correctly defined the phylogenetic norovirus genogroups, genotypes, and subgenotypes. Another interesting feature of the study was the identification of eight hitherto unreported recombinant noroviruses. It was noteworthy that three different types (intergenogroup, intergenotype, and intersubgenotype) of recombination in noroviruses were also found. This is the first report to demonstrate the existence of intergenogroup and intersubgenotype recombinations in noroviruses and highlights a possible route of zoonoses in humans because porcine, bovine and murine noroviruses belong to genogroups II, III, and V, respectively.     

Norovirus mixed infection in an oyster-associated outbreak: an opportunity for recombination

Summary We describe an outbreak of gastroenteritis in which the nucleic acid of three distinct noroviruses was amplified from the same fecal sample. To enable the separate amplification of each virus, an inclusion/exclusion RT-PCR primer design strategy was developed. This paired a virus-specific exclusion primer (designed with the exact sequence of one virus in a region displaying low conservation among the three viruses) with a virus-nonspecific inclusion primer (designed in a conserved region). Thus, in each reaction the exclusion primer provided specificity for a single virus, and the inclusion primer increased the sensitivity and allowed hybridization in a region of unknown sequence. Analysis of the partial genomic sequences of the three viruses (3.6–3.8 kb) indicated that each virus belonged to a separate genogroup II cluster, and each displayed evidence of a potential recombination event when the sequences were compared with other published norovirus sequences. Our results, which show a mixed norovirus infection in a single individual, confirm the need to be aware of the possibility of mixed norovirus infections, and of the possibility of genomic recombination causing anomalies in phylogenetic analyses in such instances.     

Mutation in a Lordsdale norovirus epidemic strain as a potential indicator of transmission routes

An increase in norovirus outbreaks was reported internationally during 2002 and 2003 and was also observed in Oxfordshire (United Kingdom) hospitals. To understand their epidemiological relationships, viruses from 22 outbreaks (15 from one hospital) were subjected to nucleotide sequencing. The 3'-terminal 3,255 nt or complete genomes were determined for 49 viruses. All outbreaks were caused by a genogroup II norovirus related to the Lordsdale virus (GII 4), common in healthcare settings. The norovirus mutation rate was sufficiently high that the 3,255-nucleotide sequences allowed separate and potentially connected outbreaks to be identified, since all outbreaks with identical sequences were temporally or geographically linked. The high mutation rate was further indicated by four mutations and three microheterogeneities in 3,255 nucleotides during 17 days of norovirus shedding by an immunocompromised patient. The data suggested that multiple virus introductions from the community, occasional transmission among wards, and one instance of ongoing environmental contamination had occurred. The accumulation, or lack, of mutations within an outbreak was also used to indicate the predominant transmission route. In an outbreak where person-to-person spread was thought to predominate, six mutations were detected throughout the genome, whereas one mutation was detected when point source infection was suspected. This norovirus epidemic strain differed from its closest previously described relative by 11.4 to 13.6% in the outer P2 domain of the capsid, which also had a single-amino-acid insertion. Alterations to the capsid structure compared to previous noroviruses may explain the increased number of outbreaks during 2002 and 2003.     

Quantitative detection of norovirus excretion in pediatric patients with cancer and prolonged gastroenteritis and shedding of norovirus

Although chronic courses of norovirus infection have been described in immunocompromised patients, little is known about noroviral shedding and correlation with clinical symptoms in these patients. In this report, the quantitative courses of norovirus excretion in nine pediatric patients with hematologic and oncologic disorders and prolonged gastroenteritis were investigated. In a retrospective study multiple fecal samples from nine pediatric cancer patients were examined by a one-step real-time PCR. Clinical data of the patients were reviewed and virological data were correlated with clinical symptoms. All nine patients presented with prolonged illness and prolonged noroviral shedding. Vomiting and diarrhea were associated with high norovirus concentrations and norovirus excretion declined slowly in the patients. Retrospectively, initial PCR-testing for norovirus was performed with a median of 7 days after onset of symptoms. This finding hints at the difficulty of obtaining early diagnosis of the infection in these children. The patients were shedding high norovirus concentration over a long period of time. Results of sequential quantitative PCR-testing for norovirus correlated with clinical symptoms. Both clinical symptoms and quantitative PCR-testings help to define the severity of norovirus infection and to estimate the risk for transmission. To prevent the spread of the disease, usage of virocidal disinfectants and isolation procedures should be maintained as long as patients are positive for noroviruses. Since vomiting is frequent in pediatric patients with oncological conditions, a screening program for rapid detection of norovirus infection in this group of patients should be considered.     

Complete genome analysis of a novel norovirus GII.4 variant identified in China

The complete genome sequence of a novel norovirus strain GZ2010-L87 identified in Guangzhou was analyzed phylogenetically in this study. The RNA genome of the GZ2010-L87 strain is composed of 7,559 nucleotides. The phylogenetic analysis based on open reading frame (ORF) 2 revealed that the strain belongs to the GII.4 genotype, forming the new cluster GII.4-2009 which was also identified in Asia and the USA since 2009. Furthermore, phylogenetic analyses of the full genome and the different open reading frame sequences of GZ2010-L87 and other representative strains suggested that the novel strain did not undergo recombination. Comparative analysis with the consensus sequence of 31 completely sequenced norovirus GII.4-2009 genomes showed 86 mismatched nucleotides (56 in ORF1, 16 in ORF2, and 14 in ORF3), resulting in 19 amino acid changes (9 in ORF1, 3 in ORF2, and 7 in ORF3). Furthermore, 12 variable sites were found on the capsid protein of norovirus GII.4-2009, and most were located at the P2 domain. Meanwhile, based on comparison with other GII.4 clusters, 14 sites were shown specific to the novel cluster. In summary, the genome of the new GII.4-2009 variant GZ2010-L87, which was first identified in China, was extensively characterized with a large panel of genetically diverse noroviruses. The genomic information obtained from the novel variant can be used not only as a full-length norovirus sequence standard in China but also as reference data for future evolution research.     

Genetic variants of norovirus of GII.6 genotype

Noroviruses mainly belonging to the GII.4 genotype are etiological agents of acute enteric infections. However, noroviruses belonging to other genotypes also play a role in some epidemiological periods or individual geographical regions. In recent years, GII.6 noroviruses have become the second most important etiologic agent of norovirus infection outbreaks after GII.4. The goal of the work is to characterize genetic variants of norovirus genotype GII.6 using phylogenetic analysis of noroviruses genome sequences present in the GenBank and NoroNet databases, as well as in the Nizhny Novgorod territory. Noroviruses of the GII.6 genotype circulating during sporadic morbidity, as well as causing an outbreak of acute enteric infection in Nizhny Novgorod, were identified using sequencing of the genome region coding for the N/S domain of VP1 capsid protein. Comparative phylogenetic analyses of sequences obtained in this study and sequences presented in international genetic databases was conducted using Mega 5.2 software. Existing of three genetic variants of the GII.6 genotype differing in genes coding for capsid protein (GII.6a (Seacroft_1990), GII.6b (Saitama_1997), and GII.6c (Shizuoka_2008)) in combination with two genotypes differing in gene coding for polymerase (P6 and P7) was confirmed. It has been shown that these genotypes have been co-circulating since the 1970s. This fact demonstrates that there are differences in evolution processes between minor norovirus genotypes and the dominant GII.4 genotype, new epidemic variants of which are completely replaced by new ones over several years. Noroviruses GII.6 that circulate in Nizhny Novgorod and other Russian cities belong to the GII.6a and GII.6b genovariants. Recombinant noroviruses GII.P7_GII.6c have been becoming the most widespread in Asia and Europe in recent years. Noroviruses of the GII.6c genovariant have not been detected in Russia, but the probability of its appearing as a cause of outbreaks of acute enteric infections in our country cannot be excluded.     

Characterization of a cross-reactive linear epitope in human genogroup I and bovine genogroup III norovirus capsid proteins

The Southampton norovirus (SV) capsid protein was expressed as VLPs by recombinant baculoviruses in insect cells and was used to immunize mice for the production of monoclonal antibodies (mAbs). One mAb, CM54, showed broad cross-reactivity to genogroup I (GI) noroviruses, but was not reactive to GII capsid proteins. Interestingly mAb CM54 reacted to a bovine norovirus capsid protein. Immunoblot analysis indicated the binding site for CM54 was located in the shell domain between amino acid residues 102-225 of the SV capsid protein. The epitope was mapped to high resolution using a peptide array and was located to the sequence LEDVRN at amino acid residues 162-167. Alignment of norovirus capsid protein sequences confirmed the epitope sequence was common to particular groups of human and bovine noroviruses. Modeling of the epitope onto the recombinant NV capsid protein revealed it was located to the inner surface of the shell domain.     

Clinical features and molecular epidemiology of rotavirus and norovirus infections in Libyan children

Rotaviruses and noroviruses are leading viral causes of diarrhoea in children. A cross‐sectional study was undertaken among children aged <5 years with acute gastroenteritis at Al‐Jala Children's Hospital, Tripoli, Libya, from October 2007 to September 2008. Of 1,090 fecal samples collected, 260 from inpatients and 830 from outpatients, all inpatients and approximately a third of outpatients, selected systematically, were investigated for rotavirus and norovirus infection by ELISA and real‐time RT‐PCR, respectively. Of 520 fecal samples examined (inpatients = 260, outpatients = 260), 164 (31.5%) had rotavirus and 91 (17.5%) had norovirus detected. Rotavirus was identified more often among inpatients than outpatients (35.8% vs. 27.3% respectively, P = 0.038). Norovirus was detected more commonly among outpatients than inpatients (21.2% vs. 13.8% respectively, P = 0.028). The peak incidence of infection with both viruses was among children aged between 6 and 11 months. The number of rotavirus cases was highest between November and June with a peak detection rate of 50% in January. Norovirus occurred most commonly from May through August with a peak detection rate of 47% in August. The most prevalent rotavirus genotypes were P[8], G9 (n = 116, 65.9%), followed by P[8],G1 (n = 49, 27.8%); a single P[9], G3 strain was detected. There were seven distinct electropherotypes among the G9 strains and all belonged to VP7 Lineage III. Among 91 noroviruses identified, 90 were genogroup II. Of 26 genogroup II noroviruses examined, all were genotype GII.4. Rotaviruses and noroviruses are both important causes of gastrointestinal infection among young children in Libya. J. Med. Virol. 83:1849–1856, 2011. © 2011 Wiley‐Liss, Inc.     

An RNA extraction protocol for shellfish-borne viruses

The GPTT virus RNA extraction method, originally developed for extraction of human norovirus and hepatitis A virus RNAs from contaminated shellfish, was evaluated for extraction of RNA from Aichi virus strain A846/88 (AiV), coxsackievirus strains A9 (CAV9) and B5 (CBV5), murine norovirus (strain MNV-1), and the norovirus surrogate, feline calicivirus (FCV) strain KCD, for the purpose of RT-PCR detection within seeded oyster (Crassostrea virginica) extracts. The RT-PCR equivalent sensitivities observed within seeded oysters as compared to virus stocks were 0.68, 6.8, 26, 5.6, and 14.5 RT-PCR(50) units when assaying 10% of total RNA extracted from seeded oyster extracts for CAV9, CBV5, AiV, FCV, and MNV-1, respectively. For oysters exposed to virus-contaminated seawater, the detection equivalent sensitivities observed were 680, 68, 2600, 560, and 14.5 RT-PCR(50) for CAV9, CBV5, AiV and FCV, and MNV-1, respectively. These results indicate that the GPTT method can be used as a general viral RNA extraction method for multiple picornaviruses and caliciviruses that could potentially contaminate shellfish.     

In vivo tropisms and kinetics of rat theilovirus infection in immunocompetent and immunodeficient rats

Rat theilovirus (RTV) is a cardiovirus related to Theiler's murine encephalomyelitis virus. While RTV is a prevalent viral pathogen of rats used in biomedical research, the pathogenesis and characterization of RTV infections is not well understood. In the studies reported herein, we used immunohistochemistry to identify viral antigens in enterocytes of the small intestines of Sprague-Dawley (SD) rats. Fecal viral shedding in immunocompromised and immunocompetent rats following oral gavage with RTV1 was high for the first 2 weeks of infection with persistent shedding of high viral loads being observed in immunocompromised nude rats but not in immunocompetent rats. RTV was also detected in mesenteric lymph nodes and spleen of immunocompromised rats but not immunocompetent rats. In addition, the magnitude of serum antibody responses differed between immunocompetent rat strains with Brown Norway and SD rats having a significantly higher antibody response than CD or Fischer 344 rats. These data suggest that RTV1 has a tropism for the epithelial cells of the small intestine, immunocompetent rats have differing serum antibody responses to RTV infection, and sustained fecal shedding and extraintestinal dissemination of RTV1 occurs in rats deficient in T cell-dependent adaptive immunity. RTV infection in immunocompromised and immunocompetent rats has merit as a model for further studies of theilovirus pathogenesis following oral viral exposure.     

实验小鼠感染鼠诺如病毒的检测

目的了解实验小鼠鼠诺如病毒（murine norovirus,MNV）的携带情况及其基因特征。方法在广州某实验动物中心抽取两个种系的实验小鼠,采集其粪便标本,应用逆转录-聚合酶链式反应（RT-PCR）方法检测粪便标本的MNVORF2特异性基因（547bp,MNVnt5104～5650）。对阳性PCR产物进行测序并以测序结果构建基因进化树分析。结果共采集实验小鼠粪便标本20份,粪便来源的小鼠外表健康、活泼、进食正常,没有腹泻现象。MNV的阳性率为30.0%（6/20）,阳性实验小鼠种类包括昆明小鼠（3/13）和NIH小鼠（3/7）。本研究检测的6个MNVs在进化树上独立一小分支,与参考株MNV2、3、4最为相近;核酸序列比较发现6个MNVs核酸序列的同源性最高,为98.3%～99.8%。结论该实验动物中心的小鼠存在较高的MNV携带状况,并可能在饲养设施内传播,有必要进一步探讨实验小鼠携带MNV的生物学意义。     

Molecular epidemiology of norovirus infections in sporadic cases of viral gastroenteritis among children in Northern Italy

Surveillance of norovirus infections in sporadic cases of pediatric gastroenteritis admitted to a main hospital in Northern Italy during a full-year period (2002) showed that noroviruses (10.4%) were the second most common causative viral agent, following rotaviruses (21.1%), and noroviruses (81%) were mostly implicated in mixed infections. The epidemic period of norovirus was September-December, with September and November as months of major prevalence (33.3 and 38.5%, respectively). Six distinct norovirus genotypes were detected (GI.7, GII.1, GII.2, GII.4, GII.7, GII, not assigned named GIIb), and the predominant genotype was GII.4. A "new GII.4 2002 variant" accounted for 82.9% of total strains. Since the severity of norovirus symptoms does not usually require admission to hospital, the burden of norovirus disease in the general children population may be much higher than that suggested by the present hospital-based investigation.