An insect picornavirus may have genome organization similar to that of caliciviruses.

Computer-assisted analysis of the amino acid sequence of the product encoded by the sequenced 3' portion of the cricket paralysis virus (CrPV), an insect picornavirus, genome showed that this protein is homologous not to the RNA-directed RNA polymerases, as originally suggested, but to the capsid proteins of mammalian picornaviruses. Alignment of the CrPV protein sequence with those of picornavirus and calicivirus capsid proteins demonstrated that the sequenced portion of the insect picornavirus genome encodes the C-terminal part of VP3 and the entire VP1. Thus CrPV seems to have a genome organization distinct from that of other picornaviruses but closely resembling that of caliciviruses, with the capsid proteins encoded in the 3' part of the genome. On the other hand, the tentative phylogenetic trees generated from the VP3 alignment revealed grouping of CrPV with hepatitis A virus, a true picornavirus, not with caliciviruses. Thus CrPV may be a picornavirus with a calicivirus-like genome organization. Different options for CrPV genome expression are discussed.     

The picornavirus avian encephalomyelitis virus possesses a hepatitis C virus-like internal ribosome entry site element

Avian encephalomyelitis virus (AEV) is a picornavirus that causes disease in poultry worldwide, and flocks must be vaccinated for protection. AEV is currently classified within the hepatovirus genus, since its proteins are most closely related to those of hepatitis A virus (HAV). We now provide evidence that the 494-nucleotide-long 5' untranslated region of the AEV genome contains an internal ribosome entry site (IRES) element that functions efficiently in vitro and in mammalian cells. Unlike the HAV IRES, the AEV IRES is relatively short and functions in the presence of cleaved eIF4G and it is also resistant to an inhibitor of eIF4A. These properties are reminiscent of the recently discovered class of IRES elements within certain other picornaviruses, such as porcine teschovirus 1 (PTV-1). Like the PTV-1 IRES, the AEV IRES shows significant similarity to the hepatitis C virus (HCV) IRES in sequence, function, and predicted secondary structure. Furthermore, mutational analysis of the predicted pseudoknot structure at the 3' end of the AEV IRES lends support to the secondary structure we present. AEV is therefore another example of a picornavirus harboring an HCV-like IRES element within its genome, and thus, its classification within the hepatovirus genus may need to be reassessed in light of these findings.     

Similarity in gene organization and homology between proteins of animal picornaviruses and a plant comovirus suggest common ancestry of these virus families

The amino acid sequences deduced from the nucleic acid sequences of several animal picornaviruses and cowpea mosaic virus (CPMV), a plant virus, were compared. Good homology was found between CPMV and the picornaviruses in the region of the picornavirus 2C (P2-X protein), VPg, 3C pro (proteinase) and 3D pol (RNA polymerase) regions. The CPMV B genome was found to have a similar gene organization to the picornaviruses. A comparison of the 3C pro (proteinase) regions of all of the available picornavirus sequences and CPMV allowed us to identify residues that are completely conserved; of these only two residues, Cys-147 and His-161 (poliovirus proteinase) could be the reactive residues of the active site of a proteinase with analogous mechanism to a known proteinase. We conclude that the proteinases encoded by these viruses are probably cysteine proteinases, mechanistically related, but not homologous to papain.     

Complete Nucleotide Sequence and Genetic Organization of Aichi Virus, a Distinct Member of the Picornaviridae Associated with Acute Gastroenteritis in Humans

The complete nucleotide sequence of a novel enteric virus, Aichi virus, associated with nonbacterial acute gastroenteritis in humans was determined. The Aichi virus genome proved to be a single-stranded positive-sense RNA molecule with 8,251 bases excluding a poly(A) tail; it contains a large open reading frame with 7,302 nucleotides that encodes a potential polyprotein precursor of 2,433 amino acids. The genome contains a 5′ nontranslated region (NTR) with 712 bases and a 3′ NTR with 240 bases followed by a poly(A) tail. The structure of the genome, VPg–5′ NTR–leader protein–structural proteins–nonstructural proteins–3′ NTR–poly(A), was found to be typical of a picornavirus. The VP0-VP3 and VP3-VP1 cleavage sites were determined to be Q-H and Q-T, respectively, by N-terminal amino acid sequence analyses using purified virion proteins. Possible cleavage sites, Q-G, Q-A, and Q-S, which cleave P2 and P3 polyproteins were found to be similar to those of picornaviruses. A dendrogram based on 3D^pol proteins indicated that Aichi virus is genetically distinct from the known six genera of picornaviruses including entero-, rhino-, cardio-, aphtho-, and hepatovirus and echovirus 22. Considering this together with other properties of the virus (T. Yamashita, S. Kobayashi, K. Sakae, S. Nakata, S. Chiba, Y. Ishihara, and S. Isomura, J. Infect. Dis. 164:954–957, 1991), we propose that Aichi virus be regarded as a new genus of the family Picornaviridae.     

昆虫小RNA病毒研究进展

昆虫小RNA病毒依据基因组结构特点分为家蚕软化病毒组、蟋蟀痹病毒组和豌豆蚜病毒组。昆虫小RNA病毒蛋白质翻译具有独特性：不依赖于帽子结构,不需要启始因子eIF1、eIF1A和帽子结合蛋白eIF4E;而且蟋蟀痹病毒组和豌豆蚜病毒组结构蛋白翻译从内部核糖体进入位点独立启始,启始密码子是CUU。该文还简介了昆虫小RNA病毒复制机理及与其它动、植物小RNA病毒或类小RNA病毒的亲缘关系等的研究进展。     

Rapid identification of known and new RNA viruses from animal tissues

Viral surveillance programs or diagnostic labs occasionally obtain infectious samples that fail to be typed by available cell culture, serological, or nucleic acid tests. Five such samples, originating from insect pools, skunk brain, human feces and sewer effluent, collected between 1955 and 1980, resulted in pathology when inoculated into suckling mice. In this study, sequence-independent amplification of partially purified viral nucleic acids and small scale shotgun sequencing was used on mouse brain and muscle tissues. A single viral agent was identified in each sample. For each virus, between 16% to 57% of the viral genome was acquired by sequencing only 42-108 plasmid inserts. Viruses derived from human feces or sewer effluent belonged to the Picornaviridae family and showed between 80% to 91% amino acid identities to known picornaviruses. The complete polyprotein sequence of one virus showed strong similarity to a simian picornavirus sequence in the provisional Sapelovirus genus. Insects and skunk derived viral sequences exhibited amino acid identities ranging from 25% to 98% to the segmented genomes of viruses within the Reoviridae family. Two isolates were highly divergent: one is potentially a new species within the orthoreovirus genus, and the other is a new species within the orbivirus genus. We demonstrate that a simple, inexpensive, and rapid metagenomics approach is effective for identifying known and highly divergent new viruses in homogenized tissues of acutely infected mice.     

Toward genetics-based virus taxonomy: comparative analysis of a genetics-based classification and the taxonomy of picornaviruses

Virus taxonomy has received little attention from the research community despite its broad relevance. In an accompanying paper (C. Lauber and A. E. Gorbalenya, J. Virol. 86:3890-3904, 2012), we have introduced a quantitative approach to hierarchically classify viruses of a family using pairwise evolutionary distances (PEDs) as a measure of genetic divergence. When applied to the six most conserved proteins of the Picornaviridae, it clustered 1,234 genome sequences in groups at three hierarchical levels (to which we refer as the "GENETIC classification"). In this study, we compare the GENETIC classification with the expert-based picornavirus taxonomy and outline differences in the underlying frameworks regarding the relation of virus groups and genetic diversity that represent, respectively, the structure and content of a classification. To facilitate the analysis, we introduce two novel diagrams. The first connects the genetic diversity of taxa to both the PED distribution and the phylogeny of picornaviruses. The second depicts a classification and the accommodated genetic diversity in a standardized manner. Generally, we found striking agreement between the two classifications on species and genus taxa. A few disagreements concern the species Human rhinovirus A and Human rhinovirus C and the genus Aphthovirus, which were split in the GENETIC classification. Furthermore, we propose a new supergenus level and universal, level-specific PED thresholds, not reached yet by many taxa. Since the species threshold is approached mostly by taxa with large sampling sizes and those infecting multiple hosts, it may represent an upper limit on divergence, beyond which homologous recombination in the six most conserved genes between two picornaviruses might not give viable progeny.     

Detection of a genome-linked protein (VPg) of hepatitis A virus and its comparison with other picornaviral VPgs.

The nucleotide sequence corresponding to the P3 region of the hepatitis A virus (HAV) polyprotein genome was determined from cloned cDNA and translated into an amino acid sequence. Comparison of the amino acid sequences of the genome-linked proteins (VPgs) of other picornaviruses with the predicted amino acid sequence of HAV was used to locate the primary structure of a putative VPg within the genome of HAV. The sequence of HAV VPg, like those of other picornaviral VPg molecules, contains a tyrosine residue as a potential binding site for HAV RNA in position 3 from its N terminus. The potential cleavage sites to generate VPg from a putative HAV polyprotein are between glutamic acid and glycine at the N terminus and glutamic acid and serine or glutamine and serine at the C terminus. A synthetic peptide corresponding to 10 amino acids of the predicted C terminus of HAV VPg induced anti-peptide antibodies in rabbits when it was conjugated to thyroglobulin as a carrier. These antibodies were specific for the peptide and precipitated VPg, linked to HAV RNA, from purified HAV and from lysates of HAV-infected cells. The precipitation reaction was blocked by the synthetic peptide (free in solution or coupled to carrier proteins) and prevented by pretreatment of VPg RNA with protease. Thus, our predicted amino acid sequence is colinear with the nucleotide sequence of the VPg gene in the HAV genome. From our results we concluded that HAV has the typical organization of picornavirus genes in this part of its genome. Similarity among hydrophobicity patterns of amino acid sequences of different picornaviral VPgs was revealed in hydropathy plots. Thus, the VPg of HAV appears to be closely related to VPg1 and VPg2 of foot-and-mouth disease virus. In contrast, HAV VPg has a unique isoelectric point (pI = 7.15) among the picornavirus VPgs.     

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.     

Picornavirus RNA-dependent RNA polymerase

Replication of the picornavirus genome is catalysed by a viral encoded RNA-dependent RNA polymerase, termed 3D polymerase. Together with other viral and host proteins, this enzyme performs its functions in the cytoplasm of host cells. The crystal structure of 3D polymerase from a number of picornaviruses has been determined. This review discusses the structure and function of the poliovirus 3D polymerase. The high error rates of 3D polymerase result in high sequence diversity such that virus populations exist as quasispecies. This phenomenon is thought to facilitate survival of the virus population in complex environments.     

Molecular cloning and sequence determination of the genomic regions encoding protease and genome-linked protein of three picornaviruses.

To investigate the degree of similarity between picornavirus proteases, we cloned the genomic cDNAs of an enterovirus, echovirus 9 (strain Barty), and two rhinoviruses, serotypes 1A and 14LP, and determined the nucleotide sequence of the region which, by analogy to poliovirus, encodes the protease. The nucleotide sequence of the region encoding the genome-linked protein VPg, immediately adjacent to the protease, was also determined. Comparison of nucleotide and deduced amino acid sequences with other available picornavirus sequences showed remarkable homology in proteases and among VPgs. Three highly conserved peptide regions were identified in the protease; one of these is specific for human picornaviruses and has no obvious counterpart in encephalomyocarditis virus, foot-and-mouth disease virus, or cowpea mosaic virus proteases. Within the other two peptide regions two conserved amino acids, Cys 147 and His 161, could be the reactive residues of the active site. We used a statistical method to predict certain features of the secondary structures, such as alpha helices, beta sheets, and turns, and found many of these conformations to be conserved. The hydropathy profiles of the compared proteases were also strikingly similar. Thus, the proteases of human picornaviruses very probably have a similar three-dimensional structure.     

Equine Rhinitis A Virus and Its Low pH Empty Particle: Clues Towards an Aphthovirus Entry Mechanism?

Equine rhinitis A virus (ERAV) is closely related to foot-and-mouth disease virus (FMDV), belonging to the genus Aphthovirus of the Picornaviridae. How picornaviruses introduce their RNA genome into the cytoplasm of the host cell to initiate replication is unclear since they have no lipid envelope to facilitate fusion with cellular membranes. It has been thought that the dissociation of the FMDV particle into pentameric subunits at acidic pH is the mechanism for genome release during cell entry, but this raises the problem of how transfer across the endosome membrane of the genome might be facilitated. In contrast, most other picornaviruses form ‘altered’ particle intermediates (not reported for aphthoviruses) thought to induce membrane pores through which the genome can be transferred. Here we show that ERAV, like FMDV, dissociates into pentamers at mildly acidic pH but demonstrate that dissociation is preceded by the transient formation of empty 80S particles which have released their genome and may represent novel biologically relevant intermediates in the aphthovirus cell entry process. The crystal structures of the native ERAV virus and a low pH form have been determined via highly efficient crystallization and data collection strategies, required due to low virus yields. ERAV is closely similar to FMDV for VP2, VP3 and part of VP4 but VP1 diverges, to give a particle with a pitted surface, as seen in cardioviruses. The low pH particle has internal structure consistent with it representing a pre-dissociation cell entry intermediate. These results suggest a unified mechanism of picornavirus cell entry.     

Molecular and biological characteristics of echovirus 22, a representative of a new picornavirus group.

Recent sequence analysis revealed that the human pathogen echovirus 22 (EV22) is genetically distant from all the other picornaviruses studied to date (T. Hyypiä, C. Horsnell, M. Maaronen, M. Khan, N. Kalkkinen, P. Auvinen, L. Kinnunen, and G. Stanway, Proc. Natl. Acad. Sci. USA 89:8847-8851, 1992). We have further characterized the biological properties of the virus and show here that the virion has properties similar to those of other picornaviruses. However, the protein composition is unique, in that most copies of one of the three major capsid proteins, VP0, do not undergo the further processing to VP2 and VP4 observed during the maturation of the virus in previously studied picornaviruses. Alignment of the capsid protein sequences with those of other picornaviruses revealed, furthermore, that the VP3 polypeptide contains an apparent insertion of approximately 25 amino acids at its amino terminus. An arginine-glycine-aspartic acid (RGD) motif is found in VP1, and by using synthetic peptides, it was shown that this sequence plays a role in cell surface receptor recognition. Finally, EV23 was shown to share remarkable identity with EV22 in certain parts of the genome and also belongs to this previously unrecognized picornavirus group.     

The 5' untranslated region of Rhopalosiphum padi virus contains an internal ribosome entry site which functions efficiently in mammalian, plant, and insect translation systems

Rhopalosiphum padi virus (RhPV) is one of several picorna-like viruses that infect insects; sequence analysis has revealed distinct differences between these agents and mammalian picornaviruses. RhPV has a single-stranded positive-sense RNA genome of about 10 kb; unlike the genomes of Picornaviridae, however, this genome contains two long open reading frames (ORFs). ORF1 encodes the virus nonstructural proteins, while the downstream ORF, ORF2, specifies the structural proteins. Both ORFs are preceded by long untranslated regions (UTRs). The intergenic UTR is known to contain an internal ribosome entry site (IRES) which directs non-AUG-initiated translation of ORF2. We have examined the 5' UTR of RhPV for IRES activity by translating synthetic dicistronic mRNAs containing this sequence in a variety of systems. We now report that the 5' UTR contains an element which directs internal initiation of protein synthesis from an AUG codon in mammalian, plant, and Drosophila in vitro translation systems. In contrast, the encephalomyocarditis virus IRES functions only in the mammalian system. The RhPV 5' IRES element has features in common with picornavirus IRES elements, in that no coding sequence is required for IRES function, but also with cellular IRES elements, as deletion analysis indicates that this IRES element does not have sharply defined boundaries.     

Evolution of Picornaviridae: An examination of phylogenetic relationships and cophylogeny

Picornaviruses are responsible for some of the most common and debilitating illnesses affecting humans and animals worldwide. To extend our knowledge of the evolution of picornaviruses and their molecular epidemiology, phylogenetic relationships among 11 genera and the unassigned seal picornavirus type 1 were estimated from the conserved proteins 2C, 3C^pro, and 3D^pol. Each gene was analyzed separately and as a combined dataset. Different tree topologies were recovered from each gene. However, their sequences were determined to be combinable based on our finding of no recombination among genera and failing to reject the hypothesis of homogeneity among datasets using ILD tests. The combined data tree topology was identical to the 3D^pol gene tree; a topology largely consistent with previous phylogenetic hypotheses based on 3D^pol and the coding genome. Phylogenetic trees estimated from six phenotypic characters were not congruent with those recovered from molecular datasets; further supporting the hypothesis that viral phenotypes are highly plastic. Finally, we tested the hypothesis of host–virus cophylogeny. Both global and individual tests of the relationships between host and virus trees failed to detect a significant association. These results emphasize the importance of horizontal transmission among host species for picornavirus diversification rather than vertical transmission accompanying speciation.     

Homologous sequences in non-structural proteins from cowpea mosaic virus and picornaviruses

Computer analyses have revealed sequence homology between two non-structural proteins encoded by cowpea mosaic virus (CPMV), and corresponding proteins encoded by two picornaviruses, poliovirus and foot-and-mouth disease virus. A region of 535 amino acids in the 87-K polypeptide from CPMV was found to be homologous to the RNA-dependent RNA polymerases from both picornaviruses, the best matches being found where the picornaviral proteins most resemble each other. Additionally, the 58-K polypeptide from CPMV and polypeptide P2-X from poliovirus contain a conserved region of 143 amino acids. Based on the homology observed, a genetic map of the CPMV genome has been constructed in which the 87-K polypeptide represents the core polymerase domain of the CPMV replicase. These results have implications for the evolution of RNA viruses, and mechanisms are discussed which may explain the existence of homology between picornaviruses (animal viruses with single genomic RNAs) and comoviruses (plant viruses with two genomic RNAs).     

Complete genome sequence of a novel picorna-like virus isolated from Spodoptera exigua

The complete genome sequence and the gene organization of a novel insect picorna-like virus, Spodoptera exigua virus (SeV), were determined. The genomic RNA of the SeV was 9501 nt in length excluding the poly(A) tail and contained a single, large open reading frame (nt 392–9424) encoding a 3010 aa polyprotein. Sequence comparisons with other viral polyproteins revealed that the consensus sequences for picornavirus RNA helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp) proteins are found on the genome in that order from the 5′ to the 3′ end. In terms of sequence similarity, identity, and genome organization, SeV resembled insect picorna-like viruses belonging to the genus Iflavirus. A phylogenetic analysis based on the eight conserved domains in the RdRp sequence showed that SeV was most closely related to the Perina nuda virus and Ectropis obliqua picorna-like virus, suggesting that these three insect picorna-like viruses might share a common ancestor.     

A distinct group of hepacivirus/pestivirus-like internal ribosomal entry sites in members of diverse picornavirus genera: evidence for modular exchange of functional noncoding RNA elements by recombination

The 5' untranslated regions (UTRs) of the RNA genomes of Flaviviridae of the Hepacivirus and Pestivirus genera contain internal ribosomal entry sites (IRESs) that are unrelated to the two principal classes of IRESs of Picornaviridae. The mechanism of translation initiation on hepacivirus/pestivirus (HP) IRESs, which involves factor-independent binding to ribosomal 40S subunits, also differs fundamentally from initiation on these picornavirus IRESs. Ribosomal binding to HP IRESs requires conserved sequences that form a pseudoknot and the adjacent IIId and IIIe domains; analogous elements do not occur in the two principal groups of picornavirus IRESs. Here, comparative sequence analysis was used to identify a subset of picornaviruses from multiple genera that contain 5' UTR sequences with significant similarities to HP IRESs. They are avian encephalomyelitis virus, duck hepatitis virus 1, duck picornavirus, porcine teschovirus, porcine enterovirus 8, Seneca Valley virus, and simian picornavirus. Their 5' UTRs are predicted to form several structures, in some of which the peripheral elements differ from the corresponding HP IRES elements but in which the core pseudoknot, domain IIId, and domain IIIe elements are all closely related. These findings suggest that HP-like IRESs have been exchanged between unrelated virus families by recombination and support the hypothesis that RNA viruses consist of modular coding and noncoding elements that can exchange and evolve independently.     

Terminal RNA replication elements in human parechovirus 1

To define structural elements critical for RNA replication in human parechovirus 1 (HPeV1), a replicon with chloramphenicol acetyltransferase as a reporter gene and an infectious virus cDNA clone have been used. It was observed that there are cis-acting signals required for HPeV1 replication located within the 5'-terminal 112 nucleotides of the genome and that these include two terminal stem-loops, SL-A and SL-B, together with a pseudoknot element. Significant disruption of any of these structures impaired both RNA replication and virus growth. In view of the similarity in terminal structures to several picornaviruses, such as cardioviruses and hepatoviruses, the insights generated in this work are of wider significance for understanding picornavirus replication.