A chimeric bovine enteric calicivirus: evidence for genomic recombination in genogroup III of the Norovirus genus of the Caliciviridae

The Norovirus genus of the Caliciviridae encompasses viruses that cause outbreaks of gastroenteritis in human and viruses that have been associated with diarrhea in cattle. The two bovine noroviruses, Bo/Newbury2/76/UK and Bo/Jena/80/DE, represent two distinct genetic clusters in the newly described genogroup III. In the present study, Jena-like polymerase sequences were identified for the first time in the UK, but one of these, Bo/Thirsk10/00/UK, was a chimeric virus. Bo/Thirsk10/00/UK had a Jena-like polymerase gene but Newbury2-like capsid and ORF3 genes by comparison of their genome organization, nucleotide, and amino acid identities and phylogenetic analyses. The present study is one of few studies to clearly demonstrate the existence of chimeric genomes in the Norovirus genus and the first, to our knowledge, to identify a chimeric genome in genogroup III. It provides additional support that genomic recombination is part of the natural evolution of noroviruses and is relevant to the diagnosis and immunological control of norovirus diarrhea outbreaks.     

Complete genomic characterization and antigenic relatedness of genogroup III,genotype 2 bovine noroviruses

Summary. Bovine enteric noroviruses form a genogroup, III, distinct from the 2 human norovirus genogroups, I and II. Two genogroup III genotypes were suggested by partial genomic analyses. In the present study, analysis of the full-length genome sequence of Bo/Newbury2/76/UK and the more contemporary Newbury2-like virus, Bo/Dumfries/1994/UK, showed that both were 7311 nucleotides in length and had three open reading frames (ORFs), amino acids motifs typical of noroviruses, and 95% or greater amino acid identities to each other in all regions of their genome. Apart from the ORF1 NTPase region, their ORF1 regions had less than 90% identity to the genogroup III genotype 1 Bo/Jena/80/DE virus, confirming two genogroup III genotypes. A close antigenic relationship was demonstrated by ELISA between the genotype 2 viruses, which will allow their serological diagnosis. Deceased [Fourth Author]     

Genome sequence and molecular characterization of Homalodisca coagulata virus-1, a novel virus discovered in the glassy-winged sharpshooter (Hemiptera: Cicadellidae)

The complete nucleotide sequence of a novel single-stranded RNA virus infecting the glassy-winged sharpshooter, Homalodisca coagulata, has been determined. In silico analysis of H. coagulata virus-1 (HoCV-1) revealed a 9321-nt polyadenylated genome encoding two large open reading frames (ORF1 and ORF2) separated by a 182-nt intergenic region (IGR). The deduced amino acid sequence of the 5'-proximal ORF (ORF1, nt 420-5807) exhibited conserved core motifs characteristic of the helicases, cysteine proteases, and RNA-dependent RNA polymerases of other insect-infecting picorna-like viruses. A structural model created using Mfold exposed a series of stem loop (SL) structures immediately preceding the second ORF which are analogous to an internal ribosome entry site (IRES), suggesting that ORF2 begins with a noncognate GCA triplet rather than the canonical AUG. This 3' ORF2 (5990-8740) showed significant similarity to the structural proteins of members of the family Dicistroviridae, particularly those belonging to the genus Cripavirus. Evidence demonstrating relatedness of these viruses regarding genome organization, amino acid sequence similarity, and putative replication strategy substantiate inclusion of HoCV-1 into this taxonomic position.     

Genotype 1 and genotype 2 bovine noroviruses are antigenically distinct but share a cross-reactive epitope with human noroviruses

The bovine enteric caliciviruses Bo/Jena/1980/DE and Bo/Newbury2/1976/UK represent two distinct genotypes within a new genogroup, genogroup III, in the genus Norovirus of the family Caliciviridae. In the present study, the antigenic relatedness of these two genotypes was determined for the first time to enable the development of tests to detect and differentiate between both genotypes. Two approaches were used. First, cross-reactivity was examined by enzyme-linked immunosorbent assay (ELISA) using recombinant virus-like particles (VLPs) and convalescent-phase sera from calves infected with either Jena (genotype 1) or Newbury2 (genotype 2). Second, cross-reactivity was examined between the two genotypes with a monoclonal antibody, CM39, derived using Jena VLPs. The two genotypes, Jena and Newbury2, were antigenically distinct with little or no cross-reactivity by ELISA to the heterologous VLPs using convalescent calf sera that had homologous immunoglobulin G titers of log10 3.1 to 3.3. CM39 reacted with both Jena and heterologous Newbury2 VLPs. The CM39 epitope was mapped to nine amino acids (31PTAGAQIAA39) in the Jena capsid protein, which was not fully conserved for Newbury2 (31PTAGAPVAA39). Molecular modeling showed that the CM39 epitope was located within the NH2-terminal arm inside the virus capsid. Surprisingly, CM39 also reacted with VLPs from two genogroup II/3 human noroviruses by ELISA and Western blotting. Thus, although the bovine noroviruses Jena and Newbury2 corresponded to two distinct antigenic types or serotypes, they shared at least one cross-reactive epitope. These findings have relevance for epidemiological studies to determine the prevalence of bovine norovirus serotypes and to develop vaccines to bovine noroviruses.     

A picorna-like virus from the red imported fire ant, Solenopsis invicta: initial discovery, genome sequence, and characterization

We report the first discovery and genome sequence of a virus infecting the red imported fire ant, Solenopsis invicta. The 8026 nucleotide, polyadenylated, RNA genome encoded two large open reading frames (ORF1 and ORF2), flanked and separated by 27, 223, and 171 nucleotide untranslated regions, respectively. The predicted amino acid sequence of the 5' proximal ORF1 (nucleotides 28 to 4218) exhibited significant identity and possessed consensus sequences characteristic of the helicase, cysteine protease, and RNA-dependent RNA polymerase sequence motifs from picornaviruses, picorna-like viruses, comoviruses, caliciviruses, and sequiviruses. The predicted amino acid sequence of the 3' proximal ORF2 (nucleotides 4390-7803) showed similarity to structural proteins in picorna-like viruses, especially the acute bee paralysis virus. Electron microscopic examination of negatively stained samples from virus-infected fire ants revealed isometric particles with a diameter of 31 nm, consistent with Picornaviridae. A survey for the fire ant virus from areas around Florida revealed a pattern of fairly widespread distribution. Among 168 nests surveyed, 22.9% were infected. The virus was found to infect all fire ant caste members and developmental stages, including eggs, early (1st-2nd) and late (3rd-4th) instars, worker pupae, workers, sexual pupae, alates ( male symbol and female symbol ), and queens. The virus, tentatively named S. invicta virus (SINV-1), appears to belong to the picorna-like viruses. We did not observe any perceptible symptoms among infected nests in the field. However, in every case where an SINV-1-infected colony was excavated from the field with an inseminated queen and held in the laboratory, all of the brood in these colonies died within 3 months.     

Genetic heterogeneity of bovine noroviruses in Italy

By screening 104 faecal samples from asymptomatic calves in Italy, bovine norovirus RNA was detected with a prevalence rate of 10.5 % (11/104). A continuous sequence spanning the RdRp region and the 5′ end of the capsid gene was generated for 7 of the 11 strains. Upon phylogenetic analysis, five strains were grouped with GIII.2 Newbury2-like viruses, and one strain was grouped with GIII.1 Jena-like noroviruses. Interestingly, one strain (80TE/IT) was genetically related to the GIII.1/Jena/80/De in the RdRp but resembled the GIII.2/Newbury2/76/UK in the capsid gene, suggesting a recombination event occurring in the ORF1/ORF2 junction region.     

Complete genome sequence of switchgrass mosaic virus, a member of a proposed new species in the genus Marafivirus

The complete genome sequence of a virus recently detected in switchgrass (Panicum virgatum) was determined and found to be closely related to that of maize rayado fino virus (MRFV), genus Marafivirus, family Tymoviridae. The genomic RNA is 6408 nucleotides long. It contains three predicted open reading frames (ORFs 1-3), encoding proteins of 227?kDa, 43.9?kDa, and 31.5?kDa, compared to two ORFs (1 and 2) for MRFV. The complete genome shares 76?% sequence identity with MRFV. The nucleotide sequence of ORF2 of this virus and the amino acid sequence of its encoded protein are 49?% and 77?% identical, respectively, to those of MRFV. The virus-encoded polyprotein and capsid protein aa sequences are 83?% and 74-80?% identical, respectively, to those of MRFV. Although closely related to MRFV, the amino acid sequence of its capsid protein (CP) forms a clade that is separate from that of MRFV. Based on the International Committee on Taxonomy of Viruses (ICTV) sequence-related criteria for delineation of species within the genus Marafivirus, the virus qualifies as a member of a new species, and the name Switchgrass mosaic virus (SwMV) is proposed.     

Genetic characterization of the Korean porcine reproductive and respiratory syndrome viruses based on the nucleocapsid protein gene (ORF7) sequences

To investigate the genetic characteristics of the Korean porcine reproductive and respiratory syndrome virus (PRRSV), we determined the complete sequence of the nucleocapsid protein gene (ORF7) from 105 PRRSV isolates from all nine Korean prefectures during the years 2003 through 2006. These sequences were then analyzed along with the published ORF7 sequences for two Korean PRRS viruses (PL97-1/1997 and LMY/2002) and 36 non-Korean viruses. The ORF7 nucleotide sequence identities among the 107 Korean PRRS viruses ranged from 86.2 to 100%, corresponding to 85.4 to 100% identity at the amino acid level. All of the Korean isolates examined belonged to the North American genotype. The ORF7 gene sequence from the North American prototype virus (VR-2332) and its derived vaccine virus (Ingelvac PRRS MLV) was 90.0–100% identical to the various ORF7 sequences of the Korean isolates, with corresponding amino acid identities from 91.0 to 100%. In the phylogenetic tree obtained by neighbor-joining analysis, all of the Korean PRRSVs were divided into four groups. Our ORF7 sequence data also revealed no correlations between the date or place of collection and the distribution of PRRSV in Korea. North American genotype PRRSVs may have been introduced into Korean swine herds some time ago; these viruses apparently radiated nationwide within a relatively short period of time. Within the North American genotype PRRSVs from around the world, the Korean PRRSVs did not emerge as a single independent clade overall, and their immediate relationships with the PRRSVs from other countries could not be determined.     

Genetic and evolutionary perspectives on genogroup III, genotype 2 bovine noroviruses

Bovine noroviruses are enteric pathogens that are detected in stool samples from cattle. Five genogroups are currently described in the genus Norovirus (family Caliciviridae), and within the genogroups, sequences are further divided into genotypes according to genetic homology and phylogenetic relationships. In this study, stool specimens from Belgian cattle were screened by RT-PCR. All of the sequences that were detected were phylogenetically related to genogroup III genotype 2 bovine noroviruses, confirming their higher prevalence in comparison with strains from genotype 1. When other sequences from around the world were introduced, phylogenetic inferences allowed neither the determination of phylogenetic lineages over time nor the deduction of topotypes for genotype 2 bovine noroviruses. Three complete genotype 2 bovine norovirus sequences were also compared genetically (Newbury2/1976 /UK, Dumfries/1994/UK and B309/2003/BE). Interestingly, the genetic divergence of the complete genomes of these three strains was relatively low, but a region of the N-terminal protein encoded by ORF1, the hypervariable region of the capsid gene encoded by ORF2, and a region of the minor structural protein encoded by ORF3 seem to be the most exposed to genetic evolution. Bayesian inference also showed that genetic evolution of genogroup III, genotype 2 bovine noroviruses over a 30-year period seemed to be lower than that already reported for noroviruses from the genotypes 3 and 4 in genogroup II.     

Epidemiology of human Sapporo-like caliciviruses in the South West of England: molecular characterisation of a genetically distinct isolate

Human enteric caliciviruses have been assigned to two distinct genera: the Norwalk-like viruses (NLVs) and the Sapporo-like viruses (SLVs). During a 3-year surveillance of gastroenteritis in the South West of England during November 1997-2000, a total of 27 clinical samples containing SLVs were collected. PCR amplicons covering a region of the RNA polymerase gene were obtained from 18 of the SLV samples. Sequence analysis of the PCR products indicated that the SLV isolates could be assigned to one of the two major genetic groups represented by Sapporo and London/92 caliciviruses. One of these isolates belonging to the London/92 group (Bristol/98) was subjected to a complete genome sequence analysis. The full genomic sequence of the Bristol/98 isolate was determined from RNA extracted from a single stool sample and consists of 7490 nucleotides, excluding the poly(A) tail. The genome is organised into two open reading frames (ORFs), similar to that of Manchester SLV although the small ORF overlapping the region encoding the capsid protein observed in Manchester SLV is absent in Bristol/98 SLV. The polyprotein (ORF1) of Bristol/98 SLV consists of 2,280 amino acids and, as observed in all SLVs, the structural protein is encoded in frame and contiguous with the 3' terminus of the ORF1. Phylogenetic studies based on complete capsid sequences and genome arrangements within the SLVs indicate that the human enteric viruses within the "Sapporo-like" virus clade should be divided into two distinct genetic groups analogous to the assignment of the Norwalk-like viruses.     

Identification of a porcine calicivirus related genetically to human sapoviruses

Whether animals may act as reservoirs for human caliciviruses is unclear. By sequence analysis of a short fragment of the RNA-dependent RNA polymerase (RdRp) region, porcine sapovirus (SaV) strains that genetically resemble human SaVs have been detected in piglets, but more-informative sequences (capsid gene) were not available for a precise characterization. In this study, the 3' terminus (the 3' end of open reading frame 1 [ORF1], including the polymerase complex and the complete capsid; ORF2; and the 3' untranslated region) of one such human SaV-like strain, 43/06-18p3/2006/It, was determined, revealing that these viruses are more related genetically to human (47.4 to 54.9% amino acid identity) than to animal (35.2 to 44.7% amino acid identity) SaVs in the capsid gene. In addition, the recombination-prone RdRp-capsid junction region was highly conserved with those of human SaVs of genogroup GI. The presence of porcine viruses similar to human SaVs is a significant finding because of the potential for zoonotic infections or generation of porcine/human recombinants.     

New Calicivirus isolated from walrus

The nucleotide sequence and genome organization of a new member of Caliciviridae was determined. Cell culture inoculated with fecal matter from walrus was used to recover fragments of a new virus by Suppression Subtractive Hybridization (SSH). The isolate was identified as a member of the Vesivirus genus of Caliciviridae and designated the name Walrus Calicivirus (WCV). Sets of PCR primers spanning the entire putative genome were designed using known sequences of other vesiviruses. The assembled genome was 8289 nucleotides (nt) long and shared no more than 87% identity with sequences of the other members of the genus Vesivirus. The largest open reading frame (ORF1) between positions 4-5646 encoded a polyprotein. ORF2, found at position 5652-7778, encoded a putative capsid protein. ORF3 overlapped ORF2 and encoded a small basic protein. Comparative analysis of multiple caliciviral capsid proteins was performed to propose a uniform capsid structural organization for this viral family.     

Novel circular single-stranded DNA virus from turkey faeces

Recently, a novel group of unclassified single-stranded (ss) circular small DNA viruses (called stool-associated circular virus; SCV) were identified in fecal samples of three mammalian species, namely, chimpanzee (ChiSCV), pig (PoSCV) and cattle (BoSCV). In this study, a novel genomic relative of stool-associated circular virus (TuSCV, KF880727) was detected in faeces of an avian species, namely, domestic turkey (Meleagris gallopavo). The complete TuSCV genome is 2479 nt long and has two open reading frames (ORF), which are bidirectionally transcribed and separated by intergenic regions. The ORF1 (replicase) and ORF2 (capsid) proteins have 77 % and 48 % aa sequence identity to different porcine-origin SCVs.     

Euprosterna elaeasa virus genome sequence and evolution of the Tetraviridae family: Emergence of bipartite genomes and conservation of the VPg signal with the dsRNA Birnaviridae family

The Tetraviridae is a family of non-enveloped positive-stranded RNA insect viruses that is defined by the T = 4 symmetry of virions. We report the complete Euprosterna elaeasa virus (EeV) genome sequence of 5698 nt with no poly(A) tail and two overlapping open reading frames, encoding the replicase and capsid precursor, with ∼67% amino acid identity to Thosea asigna virus (TaV). The N-terminally positioned 17 kDa protein is released from the capsid precursor by a NPGP motif. EeV has 40 nm non-enveloped isometric particles composed of 58 and 7 kDa proteins. The 3′-end of TaV/EeV is predicted to form a conserved pseudoknot. Replicases of TaV and EeV include a newly delineated VPg signal mediating the protein priming of RNA synthesis in dsRNA Birnaviridae. Results of rooted phylogenetic analysis of replicase and capsid proteins are presented to implicate recombination between monopartite tetraviruses, involving autonomization of a sgRNA, in the emergence of bipartite tetraviruses. They are also used to revise the Tetraviridae taxonomy.     

Identification and molecular characterization of a single-stranded circular DNA virus with similarities to Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1

A putative circular single-stranded DNA (ssDNA) virus was recovered from Hypericum japonicum collected in Vietnam. The viral isolate was tentatively named Hypericum japonicum-associated circular DNA virus (HJasCV). HJasCV shares 58.7-65.4% nucleotide sequence identity with Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) and SsHADV-1-like viruses. Like this group of viruses, the genome of HJasCV (2 200 nt) has two large ORFs, one in the virion-sense and the other in the complementary-sense DNA. The proteins encoded in the virion-sense and complementary-sense ORFs share 39-46 % and 45-67 % amino acid sequence identity with the putative capsid and replication-associated proteins (Reps), respectively, of SsHADV-1 and SsHADV-1-like viruses. The putative Rep of HJasCV contains all of the motifs related to rolling-circle replication. Its 111-bp intergenic region (IR) contains a hairpin structure with a geminivirus-like nonanucleotide sequence, TAATGTTAT, at the apex of the loop. Phylogenetic analysis revealed that HJasCV forms a monophyletic clade with SsHADV-1 and SsHADV-1-like viruses.     

Genomic characterization of swine caliciviruses representing a new genus of Caliciviridae

This study reports the molecular characterization of novel caliciviruses, the St-Valérien-like viruses, which were isolated from pig feces in the province of Quebec, Canada between 2005 and 2007. The genomes of St-Valérien-like viruses contain 6409 nucleotides and include two main open reading frames (ORFs). ORF1 encodes the non structural (NS) polyprotein and the major capsid protein (VP1) while ORF2 encodes the putative basic minor capsid protein. Typical conserved amino acid motifs predict a gene order reminiscent of calicivirus genomes. Phylogenetic, pairwise homology, and distance analyses performed on complete genomic sequences and partial amino acid sequences from the NTPase, polymerase, and major capsid protein segregated the St-Valérien-like viruses in a unique cluster sharing a common root with the Tulane virus and the noroviruses. Based on the genomic analyses presented, the St-Valérien-like viruses are members of a new genus of Caliciviridae for which we propose the name Valovirus.     

Isolation and characterization of Solenopsis invicta virus 3, a new positive-strand RNA virus infecting the red imported fire ant, Solenopsis invicta

We report the discovery of a new virus from the red imported fire ant, Solenopsis invicta. Solenopsis invicta virus 3 (SINV-3) represents the third virus discovered from this ant species using the metagenomics approach. The single (positive)-strand RNA, monopartite, bicistronic genome of SINV-3 was sequenced in entirety (GenBank accession number FJ528584), comprised of 10,386 nucleotides, and polyadenylated at the 3′ terminus. This genome size was confirmed by Northern analysis. The genome revealed 2 large open reading frames (ORFs) in the sense orientation with an untranslated region (UTR) at each end and between the two ORFs. The 5′ proximal ORF (ORF 1) encoded a predicted protein of 299.1 kDa (2580 amino acids). The 3′ proximal ORF (ORF 2) encoded a predicted protein of 73.2 kDa (651 amino acids). RNA-dependent RNA polymerase (RdRp), helicase, and protease domains were recognized in ORF 1. SDS-PAGE separation of purified SINV-3 particles yielded 2 bands (ostensibly capsid proteins) with a combined molecular mass of 77.3 kDa which was similar to the mass predicted by ORF 2 (73.2 kDa). Phylogenetic analysis of the conserved amino acid sequences containing domains I to VIII of the RdRp from dicistroviruses, iflaviruses, plant small RNA viruses, picornaviruses, and 4 unassigned positive-strand RNA viruses revealed a trichotomous phenogram with SINV-3 and Kelp fly virus comprising a unique cluster. Electron microscopic examination of negatively stained samples of SINV-3 revealed isometric particles with apparent projections and a diameter of 27.3 ± 1.3 nm. SINV-3 was successfully transmitted to uninfected workers by feeding. The minus (replicative) strand of SINV-3 was detected in worker ants indicating replication of the virus. The possibility of using SINV-3 as a microbial control agent for fire ants is discussed.     

Complete sequence of the Pepino mosaic virus RNA genome

We have determined the complete nucleotide sequence (Accession No. AF484251) of the Pepino mosaic virus (PepMV) RNA genome. PepMV is the etiological agent of a new disease which affects tomato crops in Europe and North America. The PepMV genome consists of one single stranded positive sense RNA 6410 nt long that contains five open reading frames (ORFs). ORF 1 is the putative RNA dependent RNA polymerase (RdRp), as it has the characteristic methyltransferase, NTP-binding and polymerase motifs. ORF 2 to 4 form the PepMV triple gene block. ORF 5 codes for the capsid protein. Two short untranslated regions flank the coding regions and there is a poly(A) tail at the 3'end of the genomic RNA. Thus, the genome organization of PepMV is that of a typical member of the genus Potexvirus. The nucleotide sequence obtained shares an overall 99% identity with the genomic RNA of a PepMV isolate from UK which has been partially sequenced. Protein coded by ORF4 is the least conserved between both isolates (95% amino acid identity), whereas proteins coded by ORF3 and ORF5 are identical.     

Sequence Analysis of the Gene Encoding the Capsid Protein of the Snow Mountain Human Calicivirus

Snow Mountain virus (SMV) is the reference strain for serotype 3 as determined by immune electron microscopy of the human caliciviruses that are associated with epidemic gastroenteritis. In order to establish the genetic relationship of its capsid protein with those from other human caliciviriuses, the sequence of the open reading frame 2 (ORF2) encoding the SMV capsid protein was determined. The SMV ORF2 sequence was 1626 nucleotides in length and the deduced protein of 542 amino acids had a calculated molecular weight of 59.2 kD. The SMV capsid sequence showed approximately 48 and 77% amino acid sequence identity with the capsid proteins of the Norwalk (serotype 1) and Hawaii (serotype 2) human calicivirus reference strains, respectively, a finding consistent with its serotypic distinctiveness. Furthermore, the predicted amino acid sequence of the SMV capsid was found to share highest sequence identity (98%) with the Melksham human calicivirus in database searches. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analysis of the complete genome sequence of acute bee paralysis virus shows that it belongs to the novel group of insect-infecting RNA viruses

The complete genome sequence of acute bee paralysis virus (ABPV) was determined. The 9470 nucleotide, polyadenylated RNA genome encoded two open reading frames (ORF1 and ORF2), which were separated by 184 nucleotides. The deduced amino acid sequence of the 5' ORF1 (nucleotides 605 to 6325) showed significant similarity to the RNA-dependent RNA polymerase, helicase, and protease domains of viruses from the picornavirus, comovirus, calicivirus, and sequivirus families, as well as to a novel group of insect-infecting RNA viruses. The 3' ORF2 (nucleotides 6509-9253) was proposed as encoding a capsid polyprotein with three major structural proteins (35, 33, and 24 kDa) and a minor protein (9.4 kDa). This was confirmed by N-terminal sequence analysis of two of these proteins. The overall genome structure of ABPV showed similarities to those of Drosophila C virus, Plautia stali intestine virus, Rhopalosiphum padi virus, and Himetobi P virus, which have been classified into a novel group of picorna-like insect-infecting RNA viruses called cricket paralysis-like viruses. It is suggested that ABPV belongs to the cricket paralysis-like viruses.