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PubMed2026

Detection of feline kobuvirus in filtered fecal inocula and molecular epidemiological characterization of feline kobuvirus in Japan.

Feline kobuvirus (FKoV) is an enteric virus detected in cats in several countries, but its prevalence and genomic characteristics in Japan remain unclear. Here, we report the detection of FKoV in specific-pathogen-free (SPF) cats inoculated with filtered fecal material that had tested negative for FKoV by an initial RT-PCR assay. The inoculum was derived from a shelter-housed cat positive for a feline norovirus (FNoV) strain (KU22; GVI.1; GenBank accession no. LC935948), which shared 99% VP1 nucleotide identity with the Japanese strain M49-1. FKoV RNA was undetectable in all four inoculated cats at day 0 using a degenerate universal kobuvirus primer set, but was detected in all four cats at days 5, 20, and 30 post inoculation. FKoV RNA in the inoculum was negative by the initial RT-PCR assay but positive by the degenerate universal kobuvirus primer set, indicating that FKoV was present in the inoculum but was missed by the initial assay; FKoV RNA was subsequently detected in the inoculated cats, consistent with inoculum-associated introduction of FKoV. Near-complete genome sequencing confirmed that the Japanese strain belonged to the feline kobuvirus lineage, representing the first near-complete FKoV genome sequence from Japan. In a field survey of 83 domestic cats, FKoV RNA was detected in 10 samples (12.0%), with higher prevalence in cats with gastroenteritis than in apparently healthy cats (40.0% vs. 8.2%; Bonferroni-adjusted p = 0.049), and exclusively in cats younger than 1 year. These findings provide genomic and epidemiological data on FKoV in Japan and highlight the need for broad-spectrum screening of biological inocula used in experimental infection studies.

Animals↗

PubMed2003

Isolation and characterization of a new species of kobuvirus associated with cattle.

A cytopathic agent was isolated using Vero cells from the culture medium of HeLa cells that had been used for more than 30 years in our laboratory. This agent, termed U-1 strain, was serially passed in Vero cells with distinct CPE. Particles of U-1 strain negatively stained with phosphotungstic acid exhibited a distinct surface that resembled Aichi virus. The RNA genome of U-1 strain comprises 8374 nt, with a genome organization analogous to that of picornaviruses. Possible cleavage sites of the large ORF, which encoded a leader protein prior to the capsid protein region, were assigned following amino acid alignment with Aichi virus. The virus sequence had 33 and 75 % amino acid identity with the Aichi virus VP1 and 3D regions, respectively, but no more than 23 and 36 % with those of the prototype strains of other PICORNAVIRIDAE: The dendrogram based on the P1, P2 and P3 proteins indicated that U-1 strain is genetically included in the genus Kobuvirus but is distinct from Aichi virus. Of 72 cattle sera, 43 (59.7 %) were positive for neutralizing antibody against U-1 strain at a titre of 1 : 8 or more. However, sera from 190 humans, 242 monkeys, 139 pigs, 5 horses, 22 dogs and 9 cats did not neutralize U-1 strain at a 1 : 4 dilution. RNA corresponding to U-1 strain was detected in 12 (16.7 %) of 72 faecal samples from cattle by RT-PCR. These results indicated that U-1 strain, suspected to be a contaminant from calf sera, is a new species of the genus Kobuvirus, now termed bovine kobuvirus.

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PubMed2004

Phylogeny of the Picornaviridae and differential evolutionary divergence of picornavirus proteins.

A rooted phylogenetic analysis of the viral family Picornaviridae identified several major clusters within this viral family. The genus Parechovirus, including Ljungan virus, was found to be basal to the family. Within the family, there were two major subgroups: (1) a cluster including viruses assigned to the genera Enterovirus and Rhinovirus; and (2) a cluster including Teschovirus, Cardiovirus, Erbovirus, Aphthovirus, and probably Kobuvirus. Within the former cluster, the genera Enterovirus and Rhinovirus were found not to be monophyletic. Principal components analysis was used to identify different patterns with regard to the relative sequence divergence of the 11 polyproteins encoded by the picornavirus genome. In comparisons of closely related pairs of viruses, the nonstructural proteins were generally more conserved than the capsid proteins 1B, 1C, and 1D. This pattern is most likely explained by the predominance of host antibody-driven selection on the differentiation of closely related viral genomes. However, the same pattern was not seen in more distantly related pairs of viruses, which showed a number of distinct patterns with regard to the relative conservation of the 11 proteins.

Biological Evolution↗

PubMed2003

Genomic evidence that simian virus 2 and six other simian picornaviruses represent a new genus in Picornaviridae.

Analysis of the VP1 capsid protein coding region of simian virus (SV) 2, SV16, SV18, SV42, SV44, SV45, and SV49 demonstrates that they are clearly distinct from members of the Enterovirus genus and from members of other existing picornavirus genera. To further characterize this group of viruses and to clarify their classification within the Picornaviridae, we have determined the complete genomic sequence of SV2 (8126 nucleotides). The genome was typical of members of Picornaviridae, encoding a single open reading frame. The putative polyprotein contained typical picornavirus protease cleavage sites, yielding mature proteins homologous to each of the known picornavirus proteins. SV2 contained an amino-terminal extension of the reading frame, which was analogous to the leader protein of members of the Aphthovirus, Cardiovirus, Erbovirus, Kobuvirus, and Teschovirus genera, but there was no significant amino acid homology with any of these known leader proteins. The 2A protein also aligned poorly with the 2A proteins of other picornaviruses. The deduced amino acid sequences of the SV2 structural and nonstructural proteins were related to but phylogenetically distinct from those of enteroviruses and human rhinoviruses. The major distinguishing features of SV2 were the presence of a type 2 internal ribosome entry site in the 5'-NTR, a putative leader protein encoded upstream of the structural proteins, and an unusually large 2A protein. On the basis of the molecular analysis, we propose that SV2, SV16, SV18, SV42, SV44, SV45, SV49, and porcine enterovirus 8 be classified as members of a new genus in Picornaviridae and that SV2 (strain 2383) be designated as the type strain.

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PubMed2002

Phylogenetic analysis of Ljungan virus and A-2 plaque virus, new members of the Picornaviridae.

In addition to the viruses belonging to the nine proposed genera of the Picornaviridae, Enterovirus, Rhinovirus, Cardiovirus, Aphtovirus, Hepatovirus, Parechovirus, Kobuvirus, Erbovirus and Teschovirus, two new members of this family have recently been discovered. Three strains of Ljungan virus (LV) were isolated from bank voles (Clethrionomys glareolus) and A-2 plaque virus (A-2) was isolated from human sera. To study the genetic relationship between these recently discovered viruses and the members of the family Picornaviridae, an evolutionary analysis has been carried out using the amino acid sequences of the two nonstructural proteins 2C and 3D. Phylogenetic analysis using prime members of the nine genera support the division of picornaviruses into the proposed genera. The study also supports a previous suggestion based on analysis of partial sequences of the structural proteins that LV is more related to the genus of Parechovirus than to other picornaviruses, but also shows that the three LV strains used in the comparison constitute a distinct monophyletic group, clearly separated from the parechoviruses. The analyses using the 2C and 3D sequences clearly showed that A-2 was related to the genera of Rhinovirus and Enterovirus, but it was not possible to group the A-2 with high confidence into one of the genera. Comparison using the VP1 protein sequences of Enterovirus and Rhinovirus showed that although the A-2 virus is positioned between the two genera, the virus is more related to the genus of Enterovirus than to Rhinovirus. Our analysis of the three LV strains based on the phylogenetic analysis of the 2C and 3D proteins suggests that the strains used in this study constitute a monophyletic group clearly related to Parechovirus of Picornaviridae. The taxonomic position of the A-2 virus is presently uncertain but available data indicate that this virus may be classified as a member of the genus of Enterovirus.

Animals↗

PubMed2001

Construction of an infectious cDNA clone of Aichi virus (a new member of the family Picornaviridae) and mutational analysis of a stem-loop structure at the 5' end of the genome.

Aichi virus is the type species of a new genus, Kobuvirus, of the family Picornaviridae. In this study, we constructed a full-length cDNA clone of Aichi virus whose in vitro transcripts were infectious to Vero cells. During construction of the infectious cDNA clone, a novel sequence of 32 nucleotides was identified at the 5' end of the genome. Computer-assisted prediction of the secondary structure of the 5' end of the genome, including the novel sequence, suggested the formation of a stable stem-loop structure consisting of 42 nucleotides. The function of this stem-loop in virus replication was investigated using various site-directed mutants derived from the infectious cDNA clone. Our data indicated that correct folding of the stem-loop at the 5' end of the positive strand, but not at the 3' end of the negative strand, is critical for viral RNA replication. The primary sequence in the lower part of the stem was also suggested to be crucial for RNA replication. In contrast, nucleotide changes in the loop segment did not so severely reduce the efficiency of virus replication. A double mutant, in which both nucleotide stretches of the middle part of the stem were replaced by their complementary nucleotides, had efficient RNA replication and translation abilities but was unable to produce viruses. These results indicate that the stem-loop at the 5' end of the Aichi virus genome is an element involved in both viral RNA replication and production of infectious virus particles.

Amino Acid Sequence↗

PubMed2002

Molecular analysis of three Ljungan virus isolates reveals a new, close-to-root lineage of the Picornaviridae with a cluster of two unrelated 2A proteins.

Ljungan virus (LV) is a suspected human pathogen recently isolated from bank voles (Clethrionomys glareolus). In the present study, it is revealed through comparative sequence analysis that three newly determined Swedish LV genomes are closely related and possess a deviant picornavirus-like organization: 5' untranslated region-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3' untranslated region. The LV genomes and the polyproteins encoded by them exhibit several exceptional features, such as the absence of a predicted maturation cleavage of VP0, a conserved sequence determinant in VP0 that is typically found in VP1 of other picornaviruses, and a cluster of two unrelated 2A proteins. The 2A1 protein is related to the 2A protein of cardio-, erbo-, tescho-, and aphthoviruses, and the 2A2 protein is related to the 2A protein of parechoviruses, kobuviruses, and avian encephalomyelitis virus. The unprecedented association of two structurally different 2A proteins is a feature never previously observed among picornaviruses and implies that their functions are not mutually exclusive. Secondary polyprotein processing of the LV polyprotein is mediated by proteinase 3C (3C(pro)) possessing canonical affinity to Glu and Gln at the P1 position and small amino acid residues at the P1' position. In addition, LV 3C(pro) appears to have unique substrate specificity to Asn, Gln, and Asp and to bulky hydrophobic residues at the P2 and P4 positions, respectively. Phylogenetic analysis suggests that LVs form a separate division, which, together with the Parechovirus genus, has branched off the picornavirus tree most closely to its root. The presence of two 2A proteins indicates that some contemporary picornaviruses with a single 2A may have evolved from the ancestral multi-2A picornavirus.

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PubMed2006

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.

Animals↗