Identification of amino acid residues of Ljungan virus VP0 and VP1 associated with cytolytic replication in cultured cells

Ljungan virus is a picornavirus isolated from Swedish and North American rodents. Replication of Ljungan virus in cultured cells normally induces a weak and delayed cytopathic effect compared to that of many other picornaviruses. However, efficiently replicating Ljungan virus variants may evolve during serial passages in cell culture. In this study, we evaluate the significance of three substitutions in capsid protein VP0 and VP1 of a cell-culture-adapted variant of the Swedish Ljungan virus 145SL strain. In contrast to the parental strain, this 145SLG variant grows to high titers in green monkey kidney cells and induces a distinct cytopathic effect. Reverse genetic analyses demonstrated that each one of the individual capsid substitutions contributes to lytic replication in cell culture, but also that expression of all three substitutions results in a 100- to 500-fold increase in viral titers compared to viruses encoding single capsid substitutions. In addition, as indicated by detection of activated caspase-3 and DNA fragmentation, there seems to be an association between increased replication efficiency of lytic Ljungan virus variants and induction of an apoptotic response in infected green monkey kidney cells.     

Molecular analysis of duck hepatitis virus type 1 indicates that it should be assigned to a new genus

The genome sequences of three duck hepatitis virus type 1 (DHV-1) strains were determined. Comparative sequence analyses showed that they possessed a typical picornavirus genome organization apart from the unique possession of three in-tandem 2A genes. The 2A1 protein of DHV-1 is an aphthovirus-like 2A protein; the 2A2 protein is not related to any known picornavirus protein; the 2A3 protein is a human parechovirus-like 2A protein. Several other features were found to be unique to the DHV-1 genome when compared with other picornaviruses: (i) the 3' UTR of DHV-1 was composed of 314 nt, the largest among the picornaviruses; (ii) pair-wise amino acid sequence identities between polyprotein of DHV-1 and other picornaviruses are all less than 30%. The pair-wise amino acid sequence identities in the 3D region of DHV-1 with LV and HPeV-1 is only 38.6 and 36.6%, respectively, and less than 30% with all other picornaviruses; (iii) the DHV-1 capsid polypeptide VP0 is not proteolytically cleaved into VP4 and VP2; and (iv) phylogenetic and evolutionary analysis of DHV-1 reveals a new picornavirus clade. It is therefore proposed that DHV-1 should be assigned to a new genus in the Picornaviridae.     

Replication of Ljungan virus in cell culture: the genomic 5'-end, infectious cDNA clones and host cell response to viral infections

Ljungan virus (LV) is a picornavirus recently isolated from bank voles (Clethrionomys glareolus). The previously uncharacterised 5′-end sequence of the LV genome was determined. Infectious cDNA clones were constructed of the wild type LV prototype strain 87-012 and of the cytolytically replicating cell culture adapted variant 87-012G. Virus generated from cDNA clones showed identical growth characteristics as uncloned virus stocks. Cell culture adapted LV, 87-012G, showed a clear cytopathic effect (CPE) at 3–4 days post-infection (p.i.). Virus titers, determined by plaque titration, increased however only within the first 18 h p.i. Replication of LV (+) strand RNA was determined by real-time PCR and corresponded in time with increasing titers. In contrast, the amounts of the replication intermediate, the (−) strand, continued to increase until the cells showed CPE. This indicates separate controlling mechanisms for replication of LV (+) and (−) genome strands. Replication was also monitored by immunofluorescence (IF) staining. IF staining of both prototype 87-012 and the CPE causing 87-012G showed groups of 5–25 infected cells at 48 h p.i., suggesting a, for picornaviruses, not previously described direct cell-to-cell transmission.     

Isolation and identification of an enterovirus 77 recovered from a refugee child from Kosovo, and characterization of the complete virus genome

The complete nucleotide sequence of an enterovirus 77 isolate is reported. The virus designated FR/CF496-99 (France/Clermont-Ferrand 496-1999) was recovered from the feces of a 4-year-old child hospitalized for Salmonella gastroenteritis. The virus was identified by a molecular typing assay based on the genomic sequence encoding the VP1 capsid protein. The phylogenetic analysis based on the VP1 sequence demonstrated that the enterovirus isolated in the child clustered with viruses included in the human enterovirus B species (HEV-B) and was most closely related to enterovirus 77. A sliding window analysis of the complete genome showed an overall nucleotide similarity >80% between the P3 genomic region of the FR/CF496-99 isolate and that of the echovirus 30 prototype strain. A comparative analysis based on partial 3D(pol) sequences showed that the FR/CF496-99 virus was more closely related to recent enteroviruses from different serotypes and different geographical areas than to the prototype strains collected in the 1950s. This suggests that, in this enterovirus, the 3D(pol) encoding sequence is of recent origin.     

Picornavirus proteins share antigenic determinants with heat shock proteins 60/65

Immunological cross-reactions between enteroviruses and islet cell autoantigens have been suggested to play a role in the etiopathogenesis of insulin dependent diabetes mellitus (IDDM). In the nonobese diabetic mouse, an autoimmune model of IDDM, one of the reactive beta cell autoantigens is the heat shock protein 60 (HSP60). These studies were prompted by sequence homology discovered between the immunogenic region in HSP60 and two regions in enterovirus capsid proteins, one in the VP1 protein and the other in the VP0, the precursor of VP2 and VP4 proteins. Possible immunological cross-reactions between enterovirus proteins and heat shock proteins were studied by EIA and immunoblotting by using purified virus preparations, viral expression proteins VP1 and VP0, and recombinant HSP60/65 proteins, and corresponding polyclonal antisera. The HSP60/65 family of proteins is highly conserved and there is a striking degree of homology between bacterial and human heat shock proteins. Rabbit antibodies to HSP65 of Mycobacterium bovis that reacted with human HSP60 were also found to recognise capsid protein VP1 of coxsackievirus A9, VP1, and/or VP2 of coxsackievirus B4. Both viruses were also recognised by antisera raised against HSP60 of Chlamydia pneumoniae. In addition to the capsid proteins derived from native virions, antisera to both bacterial HSP proteins recognised expression protein VP1 of coxsackievirus A9. The cross-reactivity was also demonstrated the other way around; antisera to purified virus particles reacted with the HSP 60/65 proteins to some extent. These results suggest that apart from the well-documented sequence homology between the 2C protein of coxsackieviruses and the beta-cell autoantigen glutamic acid decarboxylase, there are other motifs in picornavirus proteins homologous to islet cell autoantigens, which might induce cross-reacting immune responses during picornavirus infections.     

Genetic analysis of the VP1 region of enterovirus 71 reveals the emergence of genotype A in central China in 2008

Enterovirus 71 (EV71) strains from children were characterized by full-length VP1 nucleotide sequencing. Out of 22 clinical specimens, five isolates identified as EV71 were recovered by virus isolation. The VP1 sequences of the five isolates had more than 97.4% sequence identity with prototype virus BrCr, clustering in the genotype A lineage. This represents the first record of genotype A EV71 in China since the BrCr prototype strain was discovered in the USA in 1969.     

Molecular cloning and sequencing of coat protein-encoding cDNA of rice tungro spherical virus—A plant picornavirus

Rice tungro spherical virus (RTSV) was shown to have three coat protein (CP) species by high resolution NaDodSO₄-PAGE and Western blot analyses. The sequence of a coat protein-expressing cDNA clone that was identified and selected from a RTSV cDNA library showed that the insert was composed of 2823 bp with only one large open reading frame (ORF) coding for 941 amino acids. The positions of the three coat proteins were located in the putative polyprotein by N-terminal microsequencing and were shown to start at amino acids 287, 495, and 698 for CP-1, CP-2, and CP-3, respectively. The coat proteins are expressed as a polyprotein at the 5 region of the viral RNA genome, and all are cleaved at glutamine carboxy termini, presumably by picornavirus 3C-type of protease(s). Sequence comparisons of coat proteins revealed that there are high amino acid homologies between CP-2 of RTSV and VP3s of encephalomyocarditis virus (EMCV) and Theiler's murine encephalomyelitis virus (TMEV). These results indicate that RTSV is a plant picornavirus.     

The genomic map of hepatitis A virus: an alternate analysis

Recently Najarian et al. reported a complete cDNA sequence of the genomic RNA of hepatitis A virus (HAV) and the amino acid sequence inferred from it. As a picornavirus, HAV contains a single-stranded plus-sense RNA encoding a single 'polyprotein' which is post-translationally cleaved to yield the mature structural and non-structural proteins. In order to identify putative cleavage sites a combined function of predicted secondary structure and hydropathy was calculated by Najarian et al. for the polyproteins of HAV and poliovirus type 1 (Mahoney) (PV-1) and the two plots were aligned on the basis of a short homology in capsid protein VP3. Several of the proteins thereby predicted fail to conserve features found in all other picornaviruses that have been examined and, indeed, on the basis of these predictions HAV would hardly be a picornavirus. By an alternate analysis utilizing the computer programs FASTP and PRTALN we find that a putative protein processing map which does preserve these features can be constructed.     

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.     

Characterization of polyclonal antibodies against the capsid proteins of Ljungan virus

Ljungan virus (LV) is a suspected human pathogen isolated from voles in Sweden and North America. To enable virus detection and studies of localization and activity of virion proteins, polyclonal antibodies were produced against bacterially expressed capsid proteins of the LV strain, 87-012G. Specific detection of proteins corresponding to viral antigens in lysates of LV infected cells was demonstrated by immunoblotting using each one of the generated polyclonal antibodies. In addition, native viral antigens present in cell culture infected with LV strains 87-012G or 145SLG were detected in ELISA and by immunofluorescence using the antibodies against the VP0 and VP1 proteins. The anti-VP3 antibody did not react with native proteins of the LV virion, suggesting that the VP3 is less potent in evoking humoral response and may have a less exposed orientation in the virus capsid. No activity of the antibodies was observed against the closely related human parechovirus type 1. The polyclonal antibody against the VP1 protein was further used for detection of LV infected myocytes in a mouse model of LV-induced myocarditis. Thus, polyclonal antibodies against recombinant viral capsid proteins enabled detection of natural LV virions by several different immunological methods.     

Human rhinovirus 87 and enterovirus 68 represent a unique serotype with rhinovirus and enterovirus features

It has recently been reported that all but one of the 102 known serotypes of the genus Rhinovirus segregate into two genetic clusters (C. Savolainen, S. Blomqvist, M. N. Mulders, and T. Hovi, J. Gen. Virol. 83:333-340, 2002). The only exception is human rhinovirus 87 (HRV87). Here we demonstrate that HRV87 is genetically and antigenically highly similar to enterovirus 68 (EV68) and is related to EV70, the other member of human enterovirus group D. The partial nucleotide sequences of the 5' untranslated region, capsid regions VP4/VP2 and VP1, and the 3D RNA polymerase gene of the HRV87 prototype strain F02-3607 Corn showed 97.3, 97.8, 95.2, and 95.9% identity to the corresponding regions of EV68 prototype strain Fermon. The amino acid identities were 100 and 98.1% for the products of the two capsid regions and 97.9% for 3D RNA polymerase. Antigenic cross-reaction between HRV87 and EV68 was indicated by microneutralization with monotypic antisera. Phylogenetic analysis showed definite clustering of HRV87 and EV68 with EV70 for all sequences examined. Both HRV87 and EV68 were shown to be acid sensitive by two different assays, while EV70 was acid resistant, which is typical of enteroviruses. The cytopathic effect induced by HRV87 or EV68 was inhibited by monoclonal antibodies to the decay-accelerating factor known to be the receptor of EV70. We conclude that HRV87 and EV68 are strains of the same picornavirus serotype presenting features of both rhinoviruses and enteroviruses.     

Sequence analysis of a duck picornavirus isolate indicates that it together with porcine enterovirus type 8 and simian picornavirus type 2 should be assigned to a new picornavirus genus

In a 1990 outbreak, a virus isolated in Taiwan from the intestines of ducks showing signs of hepatitis was tentatively classified as a picornavirus on the basis of physical, chemical, and morphological characteristics. The virus was cloned and then found not to be type 1 duck hepatitis virus (DHV-1) or a new serotype of duck hepatitis virus (N-DHV) by serum neutralization. Complete genome sequencing indicated that the virus genome had 8351 nucleotides and the typical picornavirus genome organization (i.e., 5' untranslated region (UTR)-L-P1 (VP 4-2-3-1)-P2 (2A-B-C)-P3 (3A-B-C-D)-3' UTR-poly A). One open reading frame encoded 2521 amino acids, which makes this virus one of the largest picornaviruses, second only to equine rhinitis B virus of the genus Erbovirus. Its L protein was the largest within the family Picornaviridae (451 amino acids) and suspected to be a trypsin-like protease. The 235-nucleotide 3' UTR region was of intermediate size, quite long compared to other picornaviruses but shorter than other picornaviruses of duck-origin (DHV-1 and N-DHV) and had four regions of secondary structure. The 2A protein was composed of only 12 amino acids, which is the shortest of any member of the family Picornaviridae. Phylogenetic analysis of the polyprotein and 3D sequences indicated that this virus (named duck picornavirus [DPV]) together with porcine enterovirus type 8 virus and several simian picornaviruses form a distinct branch of the family Picornaviridae and should be assigned to a new picornavirus genus.     

Cell-free translation of foot-and-mouth disease virus RNA into identifiable non-capsid and capsid proteins.

Foot-and-mouth disease virus (a member of the picornavirus group) RNA could be translated effectively in an S-30 extract from Ehrlich ascites tumour cells. This translation was inhibited by aurintricarboxylic acid, cycloheximide, puromycin and RNase. Cell-free products of translation were identified by disc gel electrophoresis and immunoprecipitation with specific antisera. Gel electrophoresis of the products without prior immunoprecipitation suggested the synthesis of some of the non-capsid proteins and capsid proteins VP1, VP2 and VP3 of the virus. Immunoprecipitations with antisera against whole virus and VP3 indicated the synthesis of VP3 and of at least two additional peptides of 100 000 and 56 000 daltons containing antigenic sites of VP3. Gel electrophoresis after immunoprecipitation with antiserum against virus infection-associated antigen indicated the synthesis of a different 56 000-dalton protein appearing to resemble non-capsid protein NCVP5. The amount of foot-and-mouth disease virus and VP3-specific peptides in the virus RNA-directed products were measured by immunoprecipitation.     

Ljungan virus is endemic in rodents in the UK

Ljungan virus is a recently identified member of the family Picornaviridae that was isolated from bank voles in Sweden. It has been shown to cause type 1 diabetes-like symptoms and myocarditis in bank voles (Myodes glareolus), and it has been suggested that it has zoonotic potential. Here, we show for the first time that Ljungan virus is prevalent (20–27 % positive by PCR) in four species of UK rodent (Myodes glareolus [bank vole], Apodemus sylvaticus [wood mouse], Microtus agrestis [field vole] and Mus musculus [house mouse]). Sequence analysis showed that Ljungan virus of genotypes 1 and 2 were present, although genotype 1 was more prevalent and more frequently associated with brain tissue. This study highlights the prevalence of Ljungan virus in the UK and the need for confirmation of its zoonotic potential.     

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

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

Complete nucleotide sequence of segment 5 of epizootic haemorrhagic disease virus; the outer capsid protein VP5 is homologous to the VP5 protein of bluetongue virus

The complete nucleotide sequence of a cDNA clone representing the segment 5 RNA of epizootic haemorrhagic disease virus (EHDV) United States serotype 1 was determined. The 5' and 3' termini of the RNA are complementary and are capable of forming secondary structures. The comparison of the predicted amino acid sequence of the encoded outer capsid protein (VP5) with the sequences of VP5 from four serotypes of bluetongue virus, the prototype orbivirus, revealed that the protein shares 59% to 62% homologies with various BTV serotypes, including a single conserved glycine residue at the amino terminus. The sequence has been submitted to the Genebank databox (X55782).