ICTV Virus Taxonomy Profile: Picornaviridae.

The family Picornaviridae comprises small non-enveloped viruses with RNA genomes of 6.7 to 10.1 kb, and contains >30 genera and >75 species. Most of the known picornaviruses infect mammals and birds, but some have also been detected in reptiles, amphibians and fish. Many picornaviruses are important human and veterinary pathogens and may cause diseases of the central nervous system, heart, liver, skin, gastrointestinal tract or upper respiratory tract. Most picornaviruses are transmitted by the faecal-oral or respiratory routes. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Picornaviridae, which is available at www.ictv.global/report/picornaviridae.

Comprehensive full-length sequence analyses of human parechoviruses: diversity and recombination.

Human parechoviruses (HPeVs) are highly prevalent pathogens among very young children. Although originally classified into two serologically distinct types, HPeV1 and -2, recent analyses of variants collected worldwide have revealed the existence of 12 further types classified genetically by sequence comparisons of complete genome sequences or the capsid (VP1) gene. To investigate the nature of HPeV evolution, its population dynamics and recombination breakpoints, this study generated 18 full-length genomic sequences of the most commonly circulating genotypes, HPeV1 and -3, collected over a time span of 14 years from The Netherlands. By inclusion of previously published full-length sequences, 35 sequences were analysed in total. Analysis of contemporary strains of HPeV1 and those most similar to the prototype strain (Harris) showed that HPeV1 variants fall into two genetically distinct clusters that are much more divergent from each other than those observed within other HPeV types. Future classification criteria for HPeVs may require modification to accommodate the occurrence of variants with intermediate degrees of diversity within types. Recombination was frequently observed among HPeV1, -4, -5 and -6, but was much more restricted among HPeV3 strains. Favoured sites for recombination were found to flank the capsid region, and further sites were found within the non-structural region, P2. In contrast to other HPeV types, the majority of the HPeV3 sequences remained monophyletic across the genome, a possible reflection of its lower diversity and potentially more recent emergence than other HPeV types, or biological and/or epidemiological constraints that limit opportunities for co-infections with potential recombination partners.

Widespread recombination within human parechoviruses: analysis of temporal dynamics and constraints.

Human parechoviruses (HPeVs), members of the family Picornaviridae, are classified into six types. To investigate the dynamics and likelihood of recombination among HPeVs, we compared phylogenies of two distant regions (VP1 and 3Dpol) of 37 HPeV isolates (types 1 and 3-5) and prototype sequences (types 1-6). Evidence for frequent recombination between HPeV1, 4, 5 and 6 was found. The likelihood of recombination was correlated with the degree of VP1 divergence and differences in isolation dates, both indicative of evolutionary times of divergence. These temporal dynamics were found to be most similar to those of human enterovirus species B variants. In contrast, HPeV3 remained phylogenetically distinct from other types throughout the genome. As HPeV3 is equally divergent in nucleotide sequence from the other HPeV types, its genetic isolation may reflect different biology and changed cellular tropisms, arising from the deletion of the RGD motif, and likely use of a non-integrin receptor.

A reliable RT-PCR/RFLP assay for the molecular classification of enterovirus reference and wild type strains to either of the two genetic clusters on the basis of 5'-UTR.

The available sequence data from a large segment of the 5'-UTR of all enterovirus reference and wild type strains were analysed in an attempt to discover possible restriction sites for reliable, serotypic identification of wild type isolates. No combination of restriction endonucleases, though, was found to produce serotype-, or group-specific haplotypes. Thirteen restriction enzymes were predicted to differentiate between representatives of the two enterovirus genetic clusters on the basis of 5'-UTR. One of these enzymes, BstOI, was tested in practice for the differentiation of 61 enterovirus reference strains from 56 different serotypes and 82 wild type strains which belong to the two genetic clusters on the basis of 5'-UTR. All the representatives of the two clusters were successfully differentiated with the specific restriction enzyme. Consequently, the simple RFLP-based assay presented here could be used as a very rapid and reliable means for the initial determination of whether a clinical isolate of unknown identity belongs to either of the 'poliovirus-like', or the 'CBV-like' genetic cluster on the basis of 5'-UTR classification of human enteroviruses.

A preliminary investigation comparing pre-operative morphine and buprenorphine for postoperative analgesia and sedation in cats.

OBJECTIVE: To compare the postoperative analgesic and sedative properties of buprenorphine and morphine in cats. STUDY DESIGN: Prospective, randomized, blinded study. ANIMALS: Thirty-two domestic cats undergoing surgery. METHODS: Cats received pre-anaesthetic medication with acepromazine (0.05 mg kg-1) given intramuscularly and were randomly allocated to group M and given morphine (0.1 mg kg-1) intramuscularly (IM) or to group B and given buprenorphine (0.01 mg kg-1) IM. Anaesthesia was induced with propofol and maintained with halothane in oxygen and nitrous oxide. Pain and sedation scores using visual analogue scales, and heart and respiratory rates, were measured immediately before, and 30, 60, 120, 180, 300 and 420 minutes after anaesthesia. RESULTS: Pain scores were significantly lower at 60, 120 and 180 minutes after anaesthesia in group B. Group B also had higher heart rates at 30 minutes. There were no other statistically significant differences between the groups. CLINICAL RELEVANCE: Buprenorphine (0.01 mg kg-1) appeared to provide better postoperative analgesia than morphine (0.1 mg kg-1) and may also have a longer duration of action.

Arginine-glycine-aspartic acid motif is critical for human parechovirus 1 entry.

The human parechovirus 1 RGD motif in VP1 was studied by mutagenesis. An RGD-to-RGE change gave only revertant viruses with a restored RGD, while deletion of GD was lethal and nonrevertable. Mutations at the +1 and +2 positions had some effect on growth properties and a +1 M-to-P change was lethal. These studies indicate that the RGD motif plays a critical role in infectivity, presumably by interacting with integrins, and that downstream amino acids can have an influence on function.

Molecular detection and identification of an enterovirus during an outbreak of aseptic meningitis.

Stool samples from sixteen cases of children with meningitis originating from four different and geographically isolated parts of Greece were investigated for enteroviruses. The conventional method of cell culture in four different cell lines was initially used for the isolation of enteroviruses. The results showed a cytopathic effect (CPE) in all cases after two, or even more successive passages in only one cell line (RD), although a less-than-satisfactory CPE was obtained in many cases. Seroneutralization with RIVM mixed hyperimmune antisera followed and the isolates were typed as Coxsackie B viruses. The method of RT-PCR with enterovirus-specific primers targeted to the highly conserved 5'-UTR of the genome was initially used for the detection of enteroviruses from the inoculated cell cultures. A positive RT-PCR result was obtained for all of the clinical samples rapidly and accurately and the isolates were further characterized with the aid of Restriction Fragment Length Polymorphism (RFLP) analysis and Single Strand Conformation Polymorphism analysis (SSCP) of the amplicons. The RFLP analysis showed first of all that the isolates had an identical restriction pattern with Coxsackie B5 Faulkner reference strain with 4 out of 5 restriction enzymes and secondly, both RFLP and SSCP analysis indicated the epidemiological association of the isolates. The speed of the molecular methodology that was used in comparison with the conventional methods and its possible significance for the description of virus evolution and circulation in the populations is discussed.

In vivo and in vitro identification of structural and sequence elements of the human parechovirus 5' untranslated region required for internal initiation.

Sequence analysis of the picornavirus echovirus 22 led to its classification as the first member of a new genus, Parechovirus, and renaming as human parechovirus type 1 (HPeV1). Although distinct from other genera in most of the genome, the 5' untranslated region (5'UTR) shows similarities to that of cardio/aphthoviruses in some of its structural domains (A to L). The 5'UTR plays an important role in picornavirus translation initiation and in RNA synthesis. To investigate translation in HPeV1, we engineered an extensive range of mutations (including precise deletions and point mutations) into the 5'UTR. Their effects were studied both by in vitro transcription-translation using a bicistronic construct and by in vivo studies using an infectious, full-length HPeV1 cDNA. These approaches allowed the HPeV1 internal ribosome entry site (IRES) to be mapped. Deletions within the first 298 nucleotides had little impact in the in vitro system, while deletions of nucleotides 298 to 538 had a significant effect. Precise removal of domains H and L (nucleotides 287 to 316 and 664 to 682, respectively) did not significantly reduce translation efficiency in vitro, while domains I, J, and K (nucleotides 327 to 545, 551 to 661, and 614 to 645, respectively) appeared to have much more important roles. Mutation of a phylogenetically conserved GNRA motif (positions 421 to 424) within domain I severely reduced translation. We also confirmed the identity of the AUG (positions 710 to 712) which initiates the open reading frame, the positive identification of which has not been possible previously, as the N terminus of the polyprotein is blocked and not amenable to sequence analysis. This is therefore important in understanding parechovirus genome organization. Mutation of the AUG or an upstream polypyrimidine tract leads to aberrant translation, suggesting they both form part of the parechovirus Yn-Xm-AUG motif. In vivo experiments confirmed the importance of domains I, J, and K, the conserved GNRA motif, polypyrimidine sequences, and AUG, as mutations here were lethal. These features are also important in the IRES elements of cardio/aphthoviruses, but other features reported to be part of the IRES of some members of these genera, notably domains H and L, do not appear to be critical in HPeV1. This adds weight to the idea that there may be functional differences between the IRES elements of different picornaviruses, even when they share significant structural similarity.

Human parechoviruses--biology and clinical significance.

A new genus of the family Picornaviridae, Parechovirus, has recently been recognised on the basis of distinctive biological and molecular properties. In particular: parechoviruses exhibit characteristic effects on the host cell; cleavage of the capsid protein VP0, required for maturation of the virus particle in most other picornaviruses, does not occur; there is a unique extension, which is highly basic in character, to the N-terminus of the capsid protein VP3; and the 2A protein, in common with those of only two other known picornaviruses, is a homologue of a family of cellular proteins involved in the control of cell proliferation. The type member of the Parechovirus genus is a frequent human pathogen, formerly known as echovirus 22, which has been renamed human parechovirus 1. The genus also includes the closely related virus, human parechovirus 2 (formerly echovirus 23). Human parechoviruses generally cause mild, gastrointestinal or respiratory illness, but more serious consequences of infection, such as myocarditis and encephalitis have been reported. Most infections occur in young children. Ljungan virus, a newly identified virus of rodents, shares a number of molecular features with the human parechoviruses, raising important questions about the evolution of parechoviruses and their introduction into the human population.

The 2A proteins of three diverse picornaviruses are related to each other and to the H-rev107 family of proteins involved in the control of cell proliferation.

The 2A protein appears to be diverse among picornaviruses, in contrast to the other non-structural proteins, which have homologous structures and functions. In enteroviruses and rhinoviruses, 2A is a trypsin-like protease involved in protein processing and in shut-off of host-cell macromolecular synthesis. The aphthovirus and cardiovirus 2A is associated with an unusual processing event at the 2A/2B junction. It is shown here that the 2A protein of several diverse picornaviruses, the human parechoviruses, Aichi virus and avian encephalomyelitis virus, possess previously unrecognized conserved motifs and are likely to have a common function. Moreover, these motifs, a conserved histidine and flanking amino acids, an asparagine-cysteine dipeptide and a putative transmembrane domain, are characteristic of a family of cellular proteins, at least two of which are involved in the control of cell growth. These observations have important implications for an understanding of picornavirus genome structure and evolution, as well as pointing to possible functions of 2A in these viruses.

The crystal structure of coxsackievirus A9: new insights into the uncoating mechanisms of enteroviruses.

BACKGROUND: Coxsackievirus A9 (CAV9), a human pathogen causing symptoms ranging from common colds to fatal infections of the central nervous system, is an icosahedral single-stranded RNA virus that belongs to the genus Enterovirus of the family Picornaviridae. One of the four capsid proteins, VP1, includes the arginine-glycine-aspartate (RGD) motif within its C-terminal extension. This region binds to integrin alpha v beta 3, the only receptor for CAV9 to be conclusively identified to date. RESULTS: The crystal structure of CAV9 in complex with the antiviral compound WIN 51711 has been solved to 2.9 A resolution. The structures of the four capsid proteins, VP1 to VP4, resemble those of other picornaviruses. The antiviral compound is bound in the VP1 hydrophobic pocket, and it is possible that the pocket entrance contains a second WIN 51711 molecule. Continuous electron density for the VP1 N terminus provides a complete picture of the structure close to the fivefold axis. The VP1 C-terminal portion is on the outer surface of the virus and becomes disordered five-residues N-terminal to the RGD motif. CONCLUSIONS: The RGD motif is exposed and flexible in common with other known integrin ligands. Although CAV9 resembles coxsackie B viruses (CBVs), several substitutions in the areas implicated in CBV receptor attachment suggest it may recognise a different receptor. The structure along the fivefold axis provides new information on the uncoating mechanism of enteroviruses. CAV9 might bind a larger natural pocket factor than other picornaviruses, an observation of particular relevance to the design of new antiviral compounds.

Molecular analysis of human parechovirus type 2 (formerly echovirus 23).

Picornaviruses have been divided into five genera until recently, when a sixth genus, Parechovirus, was defined. Human parechovirus type 1 (HPeV1; formerly echovirus 22) was the first recognized member of this genus and preliminary sequence analysis of echovirus 23 [now renamed human parechovirus type 2 (HPeV2)] suggested that it is also a parechovirus. Here we describe the complete nucleotide and predicted amino acid sequences of HPeV2, which indicate a close relationship to HPeV1 throughout the genome. Sequence covariance in the 5' untranslated region allows a prediction of the secondary structure, which indicates that these parechoviruses have a type 2 internal ribosome entry site, most closely related to that of cardioviruses. Overall, HPeV2 has 87.9% amino acid identity with HPeV1, most divergence being seen in regions of the capsid proteins that probably define antigenic sites. The N-terminal sequence extension to VP3, seen only in parechoviruses, is highly basic in both viruses, but has a variable sequence, suggesting that it does not have a sequence-specific role. There is an RGD motif near the C terminus of VP1, in an analogous location to that in HPeV1 which is believed to be functionally significant. The results confirm that both viruses are parechoviruses and give insights into the molecular features of this genus.

Sequence, exon-intron organization, transcription and mutational analysis of prnA, the gene encoding the transcriptional activator of the prn gene cluster in Aspergillus nidulans.

The prnA gene codes for a transcriptional activator that mediates proline induction of four other genes involved in proline utilization as a nitrogen and/or carbon source in Aspergillus nidulans. In this paper, we present the genomic and cDNA sequence and the transcript map of prnA. The PrnA protein belongs to the Zn binuclear cluster family of transcriptional activators. The gene shows a striking intron-exon organization, with the putative nuclear localization sequence and the Zn cluster domain in discrete exons. Although the protein sequence presents some interesting similarities with the isofunctional protein of Saccharomyces cerevisiae Put3p, a higher degree of similarity is found with a functionally unrelated protein Thi1 of Schizosaccharomyces pombe. A number of mutations mapping in the prnA gene were sequenced. This comprises a deletion that results in an almost complete loss of the prnA-specific mRNA, a mutation in the putative nuclear localization signal, a proline to leucine mutation in the second loop of the zinc cluster and a cold-sensitive mutation in the so-called 'central region'. Other complete or partial loss of function mutations map in regions of unknown function. We establish that the transcription of the gene is neither self-regulated nor significantly affected by carbon and/or nitrogen metabolite repression.

A comparison of preoperative morphine and buprenorphine for postoperative analgesia for arthrotomy in dogs.

The aim of this study was to compare morphine with the partial agonist, buprenorphine, for postoperative analgesic effects, when administered preoperatively for elective arthrotomy in dogs. Fifty two dogs were anaesthetized for stifle, elbow, or hock arthrotomy. The dogs were premedicated 30 min prior to induction of anaesthesia with 0.03 mg/kg acepromazine intramuscularly, and either 0.3 mg/kg morphine or 0.01 mg/kg buprenorphine intramuscularly (allocated randomly). Anaesthesia was induced with thiopentone and maintained with halothane in an oxygen/nitrous oxide mixture. Pain and sedation were assessed preoperatively, and 0.5, 1, 2, 3, 5, and 7 h after the halothane was switched off, with a visual analogue scale scoring system. Pain scores did not differ significantly (morphine group median postoperative score from 30 to 40 mm, buprenophine group median postoperative score from 36 to 43 mm) and analgesia was considered adequate in the majority of cases (score less than 40 mm). Morphine produced significantly more sedation at 0.5 h after anaesthesia only. It was concluded that both opioids are equally suitable analgesics for postoperative analgesia for the elective arthrotomy in dogs.

An RNA tertiary structure in the 3' untranslated region of enteroviruses is necessary for efficient replication.

RNA tertiary structures, such as pseudoknots, are known to be biologically significant in a number of virus systems. The 3' untranslated regions of the RNA genomes of all members of the Enterovirus genus of Picornaviridae exhibit a potential, pseudoknot-like, tertiary structure interaction of an unusual type. This is formed by base pairing between loop regions of two secondary structure domains. It is distinct from a potential, conventional pseudoknot, studied previously in poliovirus, which is less conserved phylogenetically. We have analyzed the tertiary structure feature in one enterovirus, coxsackievirus A9, using specific mutagenesis. A double mutant in which the potential interaction was destroyed was nonviable, and viability was restored by introducing compensating mutations, predicted to allow the interaction to reform. Phenotypic pseudorevertants of virus mutants, having mutations designed to disrupt the interaction, were all found to have acquired nucleotide changes which restored the potential interaction. Analysis of one mutant containing a single-base mutation indicated a greatly increased temperature sensitivity due to a step early in replication. The results show that, in addition to secondary structures, tertiary RNA structural interactions can play an important role in the biology of picornaviruses.

Genetic and phylogenetic clustering of enteroviruses.

Genetic and phylogenetic analysis of enteroviruses showed that in the 5'NCR enteroviruses formed three clusters: polioviruses (PVs), coxsackievirus A type 21 (CAV21), CAV24 and enterovirus type 70 (ENV70) formed one cluster; coxsackievirus B isolates (CBVs), CAV9, CAV16, ENV71, echovirus type 11 (EV11), EV12 and all partially sequenced echoviruses and swine vesicular disease virus (SVDV) belonged to another cluster and bovine enteroviruses (BEVs) formed the third cluster. In the capsid coding region five clusters were seen: PVs, CAV21 and CAV24 formed one cluster (PV-like); ENV70 formed a cluster of its own; all CBVs, CAV9, EV11, EV12 and SVDV formed the third cluster (CBV-like); CAV16, CAV2 and ENV71 belonged to the fourth cluster (CAV16-like) and BEVs formed their own cluster (BEV-like). In the 3'NCR the same clusters were seen as in the coding region suggesting a close association of the 3'NCR with viral proteins while the cellular environment may be more important in the evolution of the 5'NCR. Secondary structures were predicted in the 3'NCR, which showed two different patterns among the five clusters. A potential pseudoknot region common in all five clusters was identified. Although the BEV-like viruses formed a separate cluster in all genomic regions, in the coding region they seem to be phylogenetically related to the CAV16-like viruses.

The coxsackievirus A9 RGD motif is not essential for virus viability.

An RGD (arginine-glycine-aspartic acid) motif in coxsackievirus A9 has been implicated in internalization through an interaction with the integrin alpha v beta 3. We have produced a number of virus mutants, lacking the motif, which have a small-plaque phenotype in LLC-Mk2 and A-Vero cells and are phenotypically normal in RD cells. Substitution of flanking amino acids also affected plaque size. The results suggest that interaction between the RGD motif and alpha v beta 3 is not critical for virus viability in the cell lines tested and therefore that alternative regions of the CAV-9 capsid are involved in internalization.