Molecular characterization of three novel murine noroviruses

Murine noroviruses (MNV) comprise a group of newly recognized pathogens infecting laboratory mice. The first reported murine norovirus, murine norovirus 1 (MNV-1), produces a transient infection with a short duration of fecal shedding after infection of immunocompetent laboratory mice. Our laboratory subsequently isolated three novel murine noroviruses, murine norovirus 2 (MNV-2), murine norovirus 3 (MNV-3), and murine norovirus 4 (MNV-4), that have markedly different pathogenicity from MNV-1 by producing persistent infections and prolonged fecal shedding in infected immunocompetent mice. In this study, the nucleotide sequences and the predicted amino acid sequences of the three novel murine noroviruses were determined and compared to each other, MNV-1, and other previously described human and animal noroviruses. The three novel murine norovirus strains were shown to be related to each other and MNV-1 by sequence and phylogenetic analysis even though MNV-2, MNV-3 and MNV-4 all display markedly different biologic behavior from that of MNV-1. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers DQ223041, DQ223042, and DQ223043.     

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.     

Development of a Taqman RT-PCR assay for the detection and quantification of negatively stranded RNA of human enteroviruses: evidence for false-priming and improvement by tagged RT-PCR

Human enteroviruses are among the most common viruses infecting humans. These viruses are known to be able to infect a wide range of tissues and are believed to establish persistent infections. Enteroviruses are positive-sense single-stranded RNA viruses whose replication involves the synthesis of negative strand intermediates. Therefore, the specific detection of negatively stranded viral RNA in tissues or cells is a reliable marker of active enteroviral replication. The present report presents the development of a real-time RT-PCR allowing the specific detection and quantification of negatively stranded viral RNA. Since it was known that specific amplification of single-stranded RNA can be made difficult by false-priming events leading to false-positive or overestimated results, the assay was developed by using a tagged RT primer. This tagged RT-PCR was shown to be able to amplify specifically negative RNA of enteroviruses grown in cell cultures by preventing the amplification of cDNAs generated by false-priming.     

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.     

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.     

Mosquito La protein binds to the 3' untranslated region of the positive and negative polarity dengue virus RNAs and relocates to the cytoplasm of infected cells

The untranslated regions (UTRs) of the positive and negative strand RNAs of several viruses are major binding sites for cellular and viral proteins. Human La autoantigen is one of the cellular proteins that interacts with various positive strand RNA viral genomes including that of dengue virus (DEN) within the 5'- and 3'-UTRs of positive (+) and the 3'-UTR of negative strand (-) RNA, and with the nonstructural proteins NS3 and NS5, that form DEN replicase complex. Since DEN replicates in human and mosquito cells, some functional interactions have to be conserved in both hosts. In the present report, we demonstrate that mosquito La protein interacts with the 3'-UTRs of (+) and (-) polarity viral RNAs. The localization of La protein, examined by confocal microscopy, indicates that La protein is redistributed in DEN-infected cells. Furthermore, the presence of La protein in an in vitro replication system inhibited RNA synthesis in a dose-dependent manner, suggesting that La protein plays an important role in dengue virus replicative cycle.     

The viral RNA 3′- and 5′-end structure and mRNA transcription of infectious salmon anaemia virus resemble those of influenza viruses

The nucleotide sequences of the termini of two of the genomic segments of the negative strand RNA virus infectious salmon anaemia virus (ISAV) were determined. The sequence of the terminal 9 nucleotides at both ends of the viral RNAs was identical, and showed distinctive sequence homology with the conserved terminal sequences found in the orthomyxoviruses. For both ISAV genomic segments a computer-based secondary structure modelling indicated that the terminal 21-24 nucleotides were able to form self-complementary panhandle structures. Comparison with ISAV-derived mRNA sequences showed that ISAV mRNAs have heterogeneous 5'-ends, and are polyadenylated from a signal sequence 13-14 nucleotides downstream of the 5'-end terminus of the vRNA. Furthermore, the in vitro replication of ISAV was hindered by the RNA polymerase II inhibitor alpha-amanitin. These findings indicate that the mechanisms for replication of ISAV are similar to those of the orthomyxoviruses, and add to the previously reported structural similarities between ISAV and the orthomyxoviruses.     

Experimental CVB3-induced chronic myocarditis in two murine strains: Evidence of interrelationships between virus replication and myocardial damage in persistent cardiac infection

In order to analyse the relationships between enteroviral replication and the myocardial damage at the onset of chronic cardiac infection, 2 mouse strains with different degrees of immunological competence (NMRI nu/nu, DBA/2) were infected by a myocarditic Coxsackie virus B3 (CVB3-M1) variant. At 31 days post-inoculation, plaque-forming assay, polymerase chain reaction (RT-PCR), and immunohistochemistry were carried out for detecting viruses and viral components in the myocardium. The virological findings were related to histopathological changes in the myocardium as well to the dilatation of both cardiac ventricles. Chronic myocardial lesions characterized by large fibrosis areas and interstitial inflammatory infiltrates were detected together with cardiomegalia in 52.6% (10/19) of athymic mice and in 9% (2/22) of euthymic mice. Viral replication foci were located and were found only in myocarditic cells adjacent to myocardial inflammatory lesions by immunostaining myocardial tissue sections with anti-serum to VP1 virus capsid protein. Using PCR followed by microwell capture hybridization assay, a large excess of viral positive strand RNA over negative strand was semiquantified in heart tissue from mice with chronic myocarditis, whereas approximately equal amounts of plus and minus strand RNA were detected in cases of persistent cardiac infection without chronic myocardial injuries. These findings provide evidence of the major role of viral replication in the pathogenesis of chronic murine CVB3-induced cardiomyopathy. The results indicate that the cardiac persistence of enteroviral RNAs can be observed without chronic cardiomyopathy, which could be explained by a defective viral positive RNA replication. J. Med. Virol 52:206–214, 1997. © 1997 Wiley-Liss, Inc.     

Strand specific quantitative real-time PCR to study replication of hepatitis C virus genome

Qualitative detection of negative hepatitis C virus (HCV) RNA has been used widely to demonstrate HCV replication. However, relative quantitation of both positive and negative HCV RNA strands has never been reported for studying viral genome replication. A strand specific real-time PCR carried out in the highly conserved 5'-non-coding region of HCV genome and monitored either by the DNA binding dye SYBR Green I or by molecular beacons is described. Using these techniques, it was found that negative HCV RNA strand was a 100-1000 times less abundant than the positive strand in the liver of HCV infected patients.     

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.     

Development and validation of a duplex quantitative real-time RT-PCR assay for simultaneous detection and quantitation of foot-and-mouth disease viral positive-stranded RNAs and negative-stranded RNAs

A simplified, cost-effective, two-step duplex quantitative real-time RT-PCR assay was shown to detect and quantify foot-and-mouth disease virus positive-stranded RNAs and negative-stranded RNAs simultaneously for improved investigation of the state of virus infection and replication. The primers and Taqman probes were selected from the coding regions of 2B gene and 3D gene respectively, which have the least variations among serotypes. Cells infected acutely, tissue samples and single cell samples were used for evaluation of the assay. At the early stages of virus infection in vitro, the replication level reached a peak at 9 h.p.i. and the negative strands were detectable until 3 h.p.i. The kinetics of ratios of positive strands to negative strands (+RNA/−RNA) in vivo in the liver, kidney and spleen were similar, which demonstrated that the replication dynamics were similar in the three organs. 55 single cell samples out of 187 were positive by both positive strands qPCR and negative strands qPCR, the ratios (+RNA/−RNA) ranged from 15.6 to 1463.4 which showed considerable difference among single cell samples, indicating that active viral replication differs greatly in single cells. A duplex quantitative real-time RT-PCR was validated as effective and reliable.     

Structural and inhibitor studies of norovirus 3C-like proteases

Noroviruses have a single-stranded, positive sense 7–8 kb RNA genome, which encodes a polyprotein precursor processed by a virus-encoded 3C-like cysteine protease (3CLpro) to generate mature non-structural proteins. Because processing of the polyprotein is essential for virus replication, norovirus 3CLpro has been targeted for the discovery of anti-norovirus small molecule therapeutics. Thus, we performed functional, structural and inhibition studies of norovirus 3CLpro with fluorescence resonance energy transfer (FRET) assay, X-ray crystallography, and NMR spectroscopy with a synthetic protease inhibitor. Three 3CLpro from Norwalk virus (NV, genogroup I), MD145 (genogroup II) and murine norovirus-1 (MNV-1, genogroup V) were optimized for a FRET assay, and compared for the inhibitory activities of a synthetic protease inhibitor (GC376). The apo 3D structures of NV 3CLpro determined with X-ray crystallography and NMR spectroscopy were further analyzed. In addition, the binding mode of NV 3CLpro-GC376 was compared with X-ray crystallography and NMR spectroscopy. The results of this report provide insight into the interaction of NV 3CLpro with substrate/inhibitor for better understanding of the enzyme and antiviral drug development.     

Binding of Cellular Proteins to the Leader RNA of Equine Arteritis Virus

The genome of equine arteritis virus (EAV) produces a 3 coterminal-nested set of six subgenomic (sg) viral RNAs during virus replication cycle, and each set possesses a common leader sequence of 206 nucleotides (nt) in length derived from the 5 end of the viral genome. Given the presence of the leader region within both genomic and sg mRNAs, it is likely to contain cis-acting signals that may interact with cellular or viral proteins for RNA synthesis. Gel mobility shift assays indicated that proteins in Vero cell cytoplasmic extracts formed complexes with the positive (+) and negative (-) strands of the EAV leader RNA. Several cell proteins with molecular masses ranging from 74 to 31 kDa and 58 to 32 kDa were detected in UV-induced cross-linking assays with the EAV leader RNA (+) and (-) strands, respectively. In both cases, intense bands were observed at the 58–52 kDa molecular weight markers. Results from competition gel mobility shift assays using overlapping cold RNA probes spanning the leader RNA (+) strand indicated that nt 140–206 are not necessary for binding to cell proteins.     

Identification of an Arabidopsis thaliana protein that binds to tomato mosaic virus genomic RNA and inhibits its multiplication

The genomic RNAs of positive-strand RNA viruses carry RNA elements that play positive, or in some cases, negative roles in virus multiplication by interacting with viral and cellular proteins. In this study, we purified Arabidopsis thaliana proteins that specifically bind to 5' or 3' terminal regions of tomato mosaic virus (ToMV) genomic RNA, which contain important regulatory elements for translation and RNA replication, and identified these proteins by mass spectrometry analyses. One of these host proteins, named BTR1, harbored three heterogeneous nuclear ribonucleoprotein K-homology RNA-binding domains and preferentially bound to RNA fragments that contained a sequence around the initiation codon of the 130K and 180K replication protein genes. The knockout and overexpression of BTR1 specifically enhanced and inhibited, respectively, ToMV multiplication in inoculated A. thaliana leaves, while such effect was hardly detectable in protoplasts. These results suggest that BTR1 negatively regulates the local spread of ToMV.     

Evidence that the GBV-C/hepatitis G virus is primarily a lymphotropic virus

GB virus-C and the hepatitis G virus (GBV-C/HGV) are variants of the same positive sense RNA flavivirus, initially thought to be associated with hepatitis. The tissue tropism of GBV-C/HGV in normal subjects has not been evaluated to date using an extended tissue spectrum. Therefore, the sites of GBV-C/HGV replication were investigated in serum and twenty-three tissues collected during post-mortem examination of four apparently healthy individuals who died accidental deaths, who were infected with GBV-C/HGV. All were anti-HIV and anti-HCV negative and three out of four were HBsAg negative. Tissues were collected carefully to prevent cross contamination. A highly strand-specific RT-PCR assay was employed for the detection of either GBV-C/HGV positive strand RNA (virion) or negative strand RNA (replicative intermediary). Strand specificity of the RT-PCR assay was assessed with synthetic positive-and negative strand GBV-C/HGV RNA generated from a plasmid, using T7 and T3 RNA polymerases. The spleen and bone marrow biopsies were found to be uniformly positive for both negative-and positive strand GBV-C/HGV RNA. In addition, one cadaver was positive for both RNA strands in the kidney, and another positive for both in the liver. No negative strand RNA was detected in the following: brain, muscle, heart, thyroid, salivary gland, tonsil, lung, lymph nodes, gall bladder, pancreas, oesophagus, stomach, small bowel, large bowel, adrenal gland, gonad, aorta, skin and cartilage. This preliminary study concludes that GBV-C/HGV is a lymphotropic virus that replicates primarily in the spleen and bone marrow.     

Construction of an internal RT-PCR standard control for the detection of human caliciviruses in stool

RT-PCR is the most sensitive assay for the detection of human caliciviruses (HuCV) in stool and environmental samples. However, false negative results are commonly obtained due to the presence of RT-PCR inhibitors. In order to exclude such false negative results, an internal control (IC) was developed for the assay by cloning a 319 nt sequence of the Norwalk virus (NV) polymerase containing a 156 nt cDNA insert. The RT-PCR assay was carried out using RNA derived from the constructed plasmid and a primer set previously described for calicivirus detection, resulting in a 475 nt product. Distinct bands of the internal control and the viral specific RT-PCR products (319 nt) were obtained when the internal control was added to the samples. Similar results were also obtained when both the control RNA and viral RNA were seeded into stool samples from asymptomatic volunteers, or when the internal control was included into positive samples. Since the primer set used in the assays can detect a wide range of strains in both norovirus and sapovirus genera, this internal control should have a broad application for the diagnosis of human caliciviruses diagnosis in both clinical and environmental samples.