Complete genome analysis of three novel picornaviruses from diverse bat species

Although bats are important reservoirs of diverse viruses that can cause human epidemics, little is known about the presence of picornaviruses in these flying mammals. Among 1,108 bats of 18 species studied, three novel picornaviruses (groups 1, 2, and 3) were identified from alimentary specimens of 12 bats from five species and four genera. Two complete genomes, each from the three picornaviruses, were sequenced. Phylogenetic analysis showed that they fell into three distinct clusters in the Picornaviridae family, with low homologies to known picornaviruses, especially in leader and 2A proteins. Moreover, group 1 and 2 viruses are more closely related to each other than to group 3 viruses, which exhibit genome features distinct from those of the former two virus groups. In particular, the group 3 virus genome contains the shortest leader protein within Picornaviridae, a putative type I internal ribosome entry site (IRES) in the 5'-untranslated region instead of the type IV IRES found in group 1 and 2 viruses, one instead of two GXCG motifs in 2A, an L→V substitution in the DDLXQ motif in 2C helicase, and a conserved GXH motif in 3C protease. Group 1 and 2 viruses are unique among picornaviruses in having AMH instead of the GXH motif in 3C(pro). These findings suggest that the three picornaviruses belong to two novel genera in the Picornaviridae family. This report describes the discovery and complete genome analysis of three picornaviruses in bats, and their presence in diverse bat genera/species suggests the ability to cross the species barrier.     

Complete genome sequence of a novel picornavirus, canine picornavirus, discovered in dogs

We discovered a novel canine picornavirus in fecal, nasopharyngeal, and urine samples from dogs. The coding potential of its genome (5'-VP4-VP2-VP3-VP1-2A-2B-2C-3A-3B-3C(pro)-3D(pol)-3', where 3C(pro) is 3C protease and 3D(pol) is 3D polymerase) is similar to those of other picornaviruses, with putative P1, P2, and P3 sharing 54% to 58%, 60%, and 64% to 67% amino acid identities with bat picornavirus groups 1, 2, and 3.     

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.     

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.     

Naturally Acquired Picornavirus Infections in Primates at the Dhaka Zoo

The conditions in densely populated Bangladesh favor picornavirus transmission, resulting in a high rate of infection in the human population. Data suggest that nonhuman primates (NHP) may play a role in the maintenance and transmission of diverse picornaviruses in Bangladesh. At the Dhaka Zoo, multiple NHP species are caged in close proximity. Their proximity to other species and to humans, both zoo workers and visitors, provides the potential for cross-species transmission. To investigate possible interspecies and intraspecies transmission of picornaviruses among NHP, we collected fecal specimens from nine NHP taxa at the Dhaka Zoo at three time points, August 2007, January 2008, and June 2008. Specimens were screened using real-time PCR for the genera Enterovirus, Parechovirus, and Sapelovirus, and positive samples were typed by VP1 sequencing. Fifty-two picornaviruses comprising 10 distinct serotypes were detected in 83 fecal samples. Four of these serotypes, simian virus 19 (SV19), baboon enterovirus (BaEV), enterovirus 112 (EV112), and EV115, have been solely associated with infection in NHP. EV112, EV115, and SV19 accounted for 88% of all picornaviruses detected. Over 80% of samples from cages housing rhesus macaques, olive baboons, or hamadryas baboons were positive for a picornavirus, while no picornaviruses were detected in samples from capped langurs or vervet monkeys. In contrast to our findings among synanthropic NHP in Bangladesh where 100% of the picornaviruses detected were of human serotypes, in the zoo population, only 15% of picornaviruses detected in NHP were of human origin. Specific serotypes tended to persist over time, suggesting either persistent infection of individuals or cycles of reinfection.     

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.     

Two Closely Related Novel Picornaviruses in Cattle and Sheep in Hungary from 2008 to 2009, Proposed as Members of a New Genus in the Family Picornaviridae

Two novel picornaviruses were serendipitously identified in apparently healthy young domestic animals—cattle (Bos taurus) and, subsequently, sheep (Ovis aries)—in Hungary during 2008 and 2009. Complete genome sequencing and comparative analysis showed that the two viruses are related to each other and have identical genome organizations, VPg + 5′ UTR^IRES-II[L/1A-1B-1C-1D-2A^NPG↓P/2B-2C/3A-3B^VPg-3C^pro-3D^pol] 3′ UTR-poly(A). We suggest that they form two novel viral genotypes/serotypes, bovine hungarovirus 1 (BHuV-1; GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"JQ941880","term_id":"406066046","term_text":"JQ941880"}}JQ941880) and ovine hungarovirus 1 (OHuV-1; GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"HM153767","term_id":"404897591","term_text":"HM153767"}}HM153767), which may belong to a potential novel picornavirus genus in the family Picornaviridae. The genome lengths of BHuV-1 and OHuV-1 are 7,583 and 7,588 nucleotides, each comprising a single open reading frame encoding 2,243 and 2,252 amino acids, respectively. In the 5′ untranslated regions (5′ UTRs), both hungaroviruses are predicted to have a type II internal ribosome entry site (IRES). The nucleotide sequence and the secondary RNA structure of the hungarovirus IRES core domains H-I-J-K-L are highly similar to that of human parechovirus (HPeV) (genus Parechovirus), especially HPeV-3. However, in the polyprotein coding region, the amino acid sequences are more closely related to those of porcine teschoviruses (genus Teschovirus). Hungaroviruses were detected in 15% (4/26) and 25% (4/16) of the fecal samples from cattle and sheep, respectively. This report describes the discovery of two novel picornaviruses in farm animals, cattle and sheep. The mosaic genetic pattern raises the possibility that hungaroviruses, human parechoviruses, and porcine teschoviruses may be linked to each other by modular recombination of functional noncoding RNA elements.     

Duck Hepatitis A virus possesses a distinct type IV internal ribosome entry site element of picornavirus

Sequence analysis of duck hepatitis virus type 1 (DHV-1) led to its classification as the only member of a new genus, Avihepatovirus, of the family Picornaviridae, and so was renamed duck hepatitis A virus (DHAV). The 5' untranslated region (5' UTR) plays an important role in translation initiation and RNA synthesis of the picornavirus. Here, we provide evidence that the 651-nucleotide (nt)-long 5' UTR of DHAV genome contains an internal ribosome entry site (IRES) element that functions efficiently in vitro and within BHK cells. Comparative sequence analysis showed that the 3' part of the DHAV 5' UTR is similar to the porcine teschovirus 1 (PTV-1) IRES in sequence and predicted secondary structure. Further mutational analyses of the predicted domain IIId, domain IIIe, and pseudoknot structure at the 3' end of the DHAV IRES support our predicted secondary structure. However, unlike the case for the PTV-1 IRES element, analysis of various deletion mutants demonstrated that the optimally functional DHAV IRES element with a size of approximately 420 nt is larger than that of PTV-1 and contains other peripheral domains (Id and Ie) that do not exist within the type IV IRES elements. The domain Ie, however, could be removed without significant loss of activity. Surprisingly, like the hepatitis A virus (HAV) IRES element, the activity of DHAV IRES could be eliminated by expression of enterovirus 2A protease. These findings indicate that the DHAV IRES shares common features with type IV picornavirus IRES elements, whereas it exhibits significant differences from type IV IRESs. Therefore, we propose that DHAV possesses a distinct type IV IRES element of picornavirus.     

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.     

Characterization of a canine homolog of human Aichivirus

Many of our fatal "civilization" infectious diseases have arisen from domesticated animals. Although picornaviruses infect most mammals, infection of a companion animal is not known. Here we describe the identification and genomic characterization of the first canine picornavirus. Canine kobuvirus (CKoV), identified in stool samples from dogs with diarrhea, has a genomic organization typical of a picornavirus and encodes a 2,469-amino-acid polyprotein flanked by 5' and 3' untranslated regions. Comparative phylogenetic analysis using various structural and nonstructural proteins of CKoV confirmed it as the animal virus homolog most closely related to human Aichivirus (AiV). Bayesian Markov chain Monte Carlo analysis suggests a mean recent divergence time of CKoV and AiV within the past 20 to 50 years, well after the domestication of canines. The discovery of CKoV provides new insights into the origin and evolution of AiV and the species specificity and pathogenesis of kobuviruses.     

Unique characteristics of a picornavirus internal ribosome entry site from the porcine teschovirus-1 talfan

The teschoviruses constitute a recently defined picornavirus genus. Most of the genome sequence of the porcine teschovirus-1 (PTV) Talfan and several other strains is known. We now demonstrate that initiation of protein synthesis occurs at nucleotide (nt) 412 on the PTV Talfan RNA and that nt 1 to 405 contains an internal ribosome entry site (IRES) that functions efficiently in vitro and within mammalian cells. In comparison with other picornavirus IRES elements, the PTV IRES is relatively short and lacks a significant polypyrimidine tract near the 3' end. Expression of an enterovirus 2A protease, which induces cleavage of eIF4G within the translation initiation complex eIF4F, has little effect on the PTV IRES activity within BHK cells. The PTV IRES has a unique set of properties and represents a new class of picornavirus IRES element.     

A comparative analysis of parechovirus protein structures with other picornaviruses

Parechoviruses belong to the genus Parechovirus within the family Picornaviridae and are non-enveloped icosahedral viruses with a single-stranded RNA genome. Parechoviruses include human and animal pathogens classified into six species. Those that infect humans belong to the Parechovirus A species and can cause infections ranging from mild gastrointestinal or respiratory illness to severe neonatal sepsis. There are no approved antivirals available to treat parechovirus (nor any other picornavirus) infections. In this parechovirus review, we focus on the cleaved protein products resulting from the polyprotein processing after translation comparing and contrasting their known or predicted structures and functions to those of other picornaviruses. The review also includes our original analysis from sequence and structure prediction. This review highlights significant structural differences between parechoviral and other picornaviral proteins, suggesting that parechovirus drug development should specifically be directed to parechoviral targets.     

Screening of feral and wood pigeons for viruses harbouring a conserved mobile viral element: characterization of novel Astroviruses and Picornaviruses

A highly conserved RNA-motif of yet unknown function, called stem-loop-2-like motif (s2m), has been identified in the 3' end of the genomes of viruses belonging to different RNA virus families which infect a broad range of mammal and bird species, including Astroviridae, Picornaviridae, Coronaviridae and Caliciviridae. Since s2m is such an extremely conserved motif, it is an ideal target for screening for viruses harbouring it. In this study, we have detected and characterized novel viruses harbouring this motif in pigeons by using a s2m-specific amplification. 84% and 67% of the samples from feral pigeons and wood pigeons, respectively, were found to contain a virus harbouring s2m. Four novel viruses were identified and characterized. Two of the new viruses belong to the genus Avastrovirus in the Astroviridae family. We propose two novel species to be included in this genus, Feral pigeon astrovirus and Wood pigeon astrovirus. Two other novel viruses, Pigeon picornavirus A and Pigeon picornavirus B, belong to the Picornaviridae family, presumably to the genus Sapelovirus. Both of the novel picornaviruses harboured two adjacent s2m, called (s2m)(2), suggesting a possible increased functional effect of s2m when present in two copies.     

Eukaryotic initiation factor 4G-poly(A) binding protein interaction is required for poly(A) tail-mediated stimulation of picornavirus internal ribosome entry segment-driven translation but not for X-mediated stimulation of hepatitis C virus translation

Efficient translation of most eukaryotic mRNAs results from synergistic cooperation between the 5' m(7)GpppN cap and the 3' poly(A) tail. In contrast to such mRNAs, the polyadenylated genomic RNAs of picornaviruses are not capped, and translation is initiated internally, driven by an extensive sequence termed IRES (for internal ribosome entry segment). Here we have used our recently described poly(A)-dependent rabbit reticulocyte lysate cell-free translation system to study the role of mRNA polyadenylation in IRES-driven translation. Polyadenylation significantly stimulated translation driven by representatives of each of the three types of picornaviral IRES (poliovirus, encephalomyocarditis virus, and hepatitis A virus, respectively). This did not result from a poly(A)-dependent alteration of mRNA stability in our in vitro translation system but was very sensitive to salt concentration. Disruption of the eukaryotic initiation factor 4G-poly(A) binding protein (eIF4G-PABP) interaction or cleavage of eIF4G abolished or severely reduced poly(A) tail-mediated stimulation of picornavirus IRES-driven translation. In contrast, translation driven by the flaviviral hepatitis C virus (HCV) IRES was not stimulated by polyadenylation but rather by the authentic viral RNA 3' end: the highly structured X region. X region-mediated stimulation of HCV IRES activity was not affected by disruption of the eIF4G-PABP interaction. These data demonstrate that the protein-protein interactions required for synergistic cooperativity on capped and polyadenylated cellular mRNAs mediate 3'-end stimulation of picornaviral IRES activity but not HCV IRES activity. Their implications for the picornavirus infectious cycle and for the increasing number of identified cellular IRES-carrying mRNAs are discussed.     

The mechanism of translation initiation on Aichivirus RNA mediated by a novel type of picornavirus IRES

Picornavirus mRNAs contain IRESs that sustain their translation during infection, when host protein synthesis is shut off. The major classes of picornavirus IRESs (Types 1 and 2) have distinct structures and sequences, but initiation on both is determined by their specific interaction with eIF4G. We report here that Aichivirus (AV), a member of the Kobuvirus genus of Picornaviridae, contains an IRES that differs structurally from Type 1 and Type 2 IRESs. Its function similarly involves interaction with eIF4G, but its eIF4G-interacting domain is structurally distinct, although it contains an apical eIF4G-interacting motif similar to that in Type 2 IRESs. Like Type 1 and Type 2 IRESs, AV IRES function is enhanced by pyrimidine tract-binding protein (PTB), but the pattern of PTB's interaction with each of these IRESs is distinct. Unlike all known IRESs, the AV IRES is absolutely dependent on DHX29, a requirement imposed by sequestration of its initiation codon in a stable hairpin.     

Differential utilization of poly(rC) binding protein 2 in translation directed by picornavirus IRES elements

The translation of picornavirus genomic RNAs occurs by a cap-independent mechanism that requires the formation of specific ribonucleoprotein complexes involving host cell factors and highly structured regions of picornavirus 5' noncoding regions known as internal ribosome entry sites (IRES). Although a number of cellular proteins have been shown to be involved in picornavirus RNA translation, the precise role of these factors in picornavirus internal ribosome entry is not understood. In this report, we provide evidence for the existence of distinct mechanisms for the internal initiation of translation between type I and type II picornavirus IRES elements. In vitro translation reactions were conducted in HeLa cell cytoplasmic translation extracts that were depleted of the cellular protein, poly(rC) binding protein 2 (PCBP2). Upon depletion of PCBP2, these extracts possessed a significantly diminished capacity to translate reporter RNAs containing the type I IRES elements of poliovirus, coxsackievirus, or human rhinovirus linked to luciferase; however, the addition of recombinant PCBP2 could reconstitute translation. Furthermore, RNA electrophoretic mobility-shift analysis demonstrated specific interactions between PCBP2 and both type I and type II picornavirus IRES elements; however, the translation of reporter RNAs containing the type II IRES elements of encephalomyocarditis virus and foot-and-mouth disease virus was not PCBP2 dependent. These data demonstrate that PCBP2 is essential for the internal initiation of translation on picornavirus type I IRES elements but is dispensable for translation directed by the structurally distinct type II elements.     

Functional and structural similarities between the internal ribosome entry sites of hepatitis C virus and porcine teschovirus, a picornavirus

Initiation of protein synthesis on picornavirus RNA requires an internal ribosome entry site (IRES). Typically, picornavirus IRES elements contain about 450 nucleotides (nt) and use most of the cellular translation initiation factors. However, it is now shown that just 280 nt of the porcine teschovirus type 1 Talfan (PTV-1) 5' untranslated region direct the efficient internal initiation of translation in vitro and within cells. In toeprinting assays, assembly of 48S preinitiation complexes from purified components on the PTV-1 IRES was achieved with just 40S ribosomal subunits plus eIF2 and Met-tRNA(i)(Met). Indeed, a binary complex between 40S subunits and the PTV-1 IRES is formed. Thus, the PTV-1 IRES has properties that are entirely different from other picornavirus IRES elements but highly reminiscent of the hepatitis C virus (HCV) IRES. Comparison between the PTV-1 IRES and HCV IRES elements revealed islands of high sequence identity that occur in regions critical for the interactions of the HCV IRES with the 40S ribosomal subunit and eIF3. Thus, there is significant functional and structural similarity between the IRES elements from the picornavirus PTV-1 and HCV, a flavivirus.     

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.     

Picornavirus internal ribosome entry site elements can stimulate translation of upstream genes

Certain viral and cellular mRNAs initiate translation cap-independently at internal ribosome entry site (IRES) elements. Picornavirus IRES elements are widely used in dicistronic or multicistronic vectors in gene therapy, virus replicon systems, and analysis of IRES function. In such vectors, expression of the upstream gene often serves as internal control to standardize the readings of IRES-driven downstream reporter activity. Picornaviral IRES elements translate optimally at up to 120 mM K(+) concentration, whereas genes used as upstream reporters usually have lower salt optima when present in monocistronic mRNAs. However, here we show that such reporter genes are efficiently translated at higher K(+) concentrations when placed upstream of a functional picornavirus IRES. This translation enhancement occurs in cis, is independent of the nature of the first reporter and of second reporter translation, and is conferred by the IRESs of picornaviruses but not of hepatitis C virus. A defective picornavirus IRES with a deletion killing IRES activity but leaving the binding site for initiation factor eIF4G intact retains translation enhancement activity. Translation enhancement on a capped mRNA is disabled by m(7)GDP. In addition, the C-terminal fragment of eIF4G can confer translation enhancement also on uncapped mRNA. We conclude that whenever eIF4F has been captured to a dicistronic mRNA by binding to a picornavirus IRES via its eIF4G moiety, it can be provided in cis to the 5'-end of the RNA and there stimulate translation initiation, either by binding to the cap nucleotide using its eIF4E moiety or by binding to the RNA cap-independently using its eIF4G moiety.