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.

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.

Preliminary crystallographic analysis of coxsackievirus A9.

Coxsackievirus A9 has been crystallized as small rhombic dodecahedra of maximum dimension 0.3 mm. These crystals have been shown, using synchrotron radiation, to diffract X-rays to beyond 3 A, and to have a stability in the beam comparable to that of other related virus crystals. The unit cell is tetragonal with dimensions a = b = 495 A, c = 695 A and alpha = beta = gamma = 90 degrees, with a space group of P4n22. A substantial body of diffraction data has been collected and this crystal form appears to be suitable for structure determination. Phasing of these data will be attempted using molecular replacement.

The genome of echovirus 11.

Echoviruses are the largest enterovirus subgroup consisting of 32 serotypes. They are common human pathogens causing, for example, meningitis, encephalitis and exanthema, but in spite of their clinical importance, relatively little is known about their biology. To illuminate the molecular characteristics of echoviruses, we have completed the genomic sequence of serotype 11. The RNA genome is 7438 nucleotides in length and it codes for a 2195 amino acid long polyprotein. When compared to other sequenced enteroviruses, echovirus 11 (EV11) shows remarkable similarity with coxsackie B viruses (CBVs) and coxsackievirus A9 (CAV9). On the basis of amino acid sequence homology in the capsid region, CAV9 is the virus most closely related to EV11. These two viruses have an apparent insertion sequence located at the C-terminus of the VP1 polypeptide. EV11, however, lacks the RGD motif found in the corresponding region of CAV9. The organization of the 5' end noncoding region resembles that of other enteroviruses, but contains a 12 nucleotides long poly-U stretch not seen in any other enterovirus sequenced to date.

Pathogenetic differences between coxsackie A and B virus infections in newborn mice.

Coxsackieviruses are divided into A and B subgroups on the basis of their pathogenicity in newborn mice. Although used in the classification of these viruses, our understanding of the details of the infection is incomplete due to the lack of sensitive and specific techniques to localize the viruses in affected tissue. We have used in situ hybridization to detect coxsackievirus genomes in tissues of newborn mice after infection by five serotypes (A2, A9, A21, B3 and B4) through different administration routes. Our results indicate that coxsackie A viruses are able to affect both skeletal and heart muscle while the coxsackievirus B subgroup infects a wide range of tissues. In addition to striated muscle these include central nervous system, liver, exocrine pancreas and brown fat. This model will make it possible to analyze molecular factors determining tissue tropism.

Molecular identification of viruses in sudden infant death associated with myocarditis and pericarditis.

A subset of infants dying suddenly and unexpectedly have myocarditis with or without pericarditis found at autopsy. To address whether viruses known to cause infantile myocarditis and pericarditis might be present in such infants, we examined myocardium, liver and skeletal muscle for the presence of genomic sequences from adenovirus, cytomegalovirus, enterovirus and echovirus 22/23 in infants enrolled in a comprehensive evaluation protocol. We studied eight infants who died suddenly and unexpectedly with histologic evidence of myocarditis and/or pericarditis detected at postmortem examination. One infant with myocarditis and pericarditis had adenovirus genome detected in the myocardium. In an additional infant with severe pericarditis alone, enterovirus genome was detected in the liver. Although echovirus 22/23 has been associated with myopericarditis in young infants, no previous studies have used molecular methods to search for the genomic sequences of these viruses in clinical samples. No echovirus 22/23 genome was detected in the patients reported here. The significance of enterovirus and adenovirus genome in the tissues of two patients dying suddenly and unexpectedly remains speculative but raises the possibility that pathogenic viruses may cause little or no clinical symptoms and yet be contributory to sudden death in young infants.

Detection of enteroviruses using cDNA and synthetic oligonucleotide probes.

This study compares the detection of enterovirus RNA by cDNA probes prepared from both the 5' and 3' end of the genome of coxsackie A21 and B4 with the use of synthetic oligonucleotides prepared from short but highly conserved sequences in the 5' end non-coding region of the picornavirus genome. The cDNA probes detected enteroviruses with a variable level of sensitivity which presumably depended on the degree of genomic homology with the detecting probes. Generally probes from coxsackievirus A21 detected more enteroviruses than did similar probes from coxsackievirus B4. Probes from the 5' end of the genome of both viruses were more sensitive than 3' end probes. In contrast, synthetic oligonucleotides detected all enteroviruses efficiently suggesting that these probes could be useful as 'universal' probes to detect any enterovirus. This paper discusses the application of these probes in the diagnosis and differentiation of enteroviruses.

Identification of rhinoviruses by cDNA probes.

We have used nucleic acid hybridization for the detection and grouping of human rhinoviruses (HRV) according to their genetic relationships. Fifteen rhinovirus reference strains, seventy-one clinical isolates and four enteroviruses were propagated in cell cultures, spotted onto membrane filters and hybridized with radioactively labelled cDNA probes covering different parts of the genomes of HRV-1B, HRV-2, HRV-14, HRV-85 and HRV-89. When the rhinovirus and enterovirus reference strains were tested, the 5' probe of HRV-2 hybridized with thirteen of the fifteen HRV reference strains, with poliovirus type 3 and with ECHO virus 11. The HRV-14 5' probe reacted with eleven HRV reference strains and with all the enteroviruses studied. Sixty-nine of the 71 clinical isolates were recognised by the HRV-2 5' probe, whereas the HRV-14 probe from the same part of the genome hybridized with 54 field isolates. One of the two isolates that remained negative with the HRV-2 5' probe was detected with the HRV-2 probe that derived from the P2 region of the genome, and the other isolate was not detected by any of the probes. Probes from other parts than the 5' end of the genome were generally more specific, and clusters could be formed based on the reactivity of the HRV strains with these probes.

Radioimmunolocalization of tumours by external scintigraphy after administration of 131I antibody to human chorionic gonadotrophin: preliminary communication.

(131)Iodine ((131)I) labelled antibody directed against human chorionic gonadatrophin (hCG) was given on 21 occasions to 18 patients with hCG-producing neoplasms. Tumours were localized by external scintigraphy in 13 of 21 investigations. Positive results were obtained reliably when serum hCG exceeded 500 miu/ml and in some cases sensitivity was comparable to that of computerized tomography. A positive result probably implies viability in the tumour and this was of practical value in discriminating between necrotic deposits and living tumour before surgery.

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.

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.

Genetic diversity of enterovirus subgroups.

Enterovirus serotypes were studied using nucleic acid hybridization and nucleotide sequence analysis. A great majority of enteroviruses could be roughly divided into two larger subgroups the first consisting of poliovirus and certain coxsackievirus A serotypes. The second subgroup included coxsackie B viruses, most ECHO viruses, enterovirus 71 and representatives of coxsackie A viruses. Enterovirus 70 showed low homology to the viruses in both groups. Interestingly, ECHO virus 22 failed to react with any of the hybridization probes indicating a relatively distant relationship. The close relationship between coxsackie B and ECHO viruses as well as between polio and certain coxsackie A viruses was also evident when nucleotide sequences of the 3' end noncoding parts were compared.

Sequences in the 5' non-coding region of human rhinovirus 14 RNA that affect in vitro translation.

A subgenomic cDNA clone from human rhinovirus 14 (HRV-14), comprising the 5' non-coding region and the first 1182 nucleotides of the coding sequence, has been inserted into a vector under the control of the T7 promoter, and RNA was transcribed. Deletions in the 5' non-coding sequence modulated viral polyprotein synthesis significantly in a reticulocyte lysate system. Removal of the first 491 nucleotides had little effect, but deletion of a further 55 nucleotides (491 to 546) significantly increased the efficiency of the translation process. Further deletion to nucleotide 621 almost abolished translation, suggesting an essential role for the 546 to 621 nucleotide sequence. The efficiency of the translation process can also be influenced by the addition of ribosomal salt wash prepared from uninfected HeLa cells.

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 nucleotide sequence of coxsackievirus A9; implications for receptor binding and enterovirus classification.

The complete nucleotide sequence of the genome of coxsackievirus A9 (CAV-9) has been determined from cDNA cloned in Escherichia coli. Excluding the 3' poly(A) stretch, the RNA genome is 7452 nucleotides long and encodes a single polyprotein of 2201 amino acids. Comparison of the nucleotide and predicted amino acid sequences with those of the coxsackieviruses B1, B3 and B4 reveals a surprising degree of homology, with overall amino acid homologies of 86.9%, 86.2% and 87.0%, respectively. In contrast, there is much less homology to another coxsackie A virus, CAV-21, 60.4% overall amino acid homology. This demonstrates the high degree of diversity within the CAV group and indicates that the current classification does not directly correlate with molecular genetic properties. One major feature of CAV-9 is an insertion, relative to all other enteroviruses sequenced to date, which is located at the C terminus of VP1, and includes an arginine-glycine-aspartic acid tripeptide. Such sequences in a number of other proteins are known to have activity in promoting attachment to cell receptors and the implications for CAV-9 receptor binding are discussed.

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.

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.