Phylogenetic analysis of Ljungan virus and A-2 plaque virus, new members of the Picornaviridae

In addition to the viruses belonging to the nine proposed genera of the Picornaviridae, Enterovirus, Rhinovirus, Cardiovirus, Aphtovirus, Hepatovirus, Parechovirus, Kobuvirus, Erbovirus and Teschovirus, two new members of this family have recently been discovered. Three strains of Ljungan virus (LV) were isolated from bank voles (Clethrionomys glareolus) and A-2 plaque virus (A-2) was isolated from human sera. To study the genetic relationship between these recently discovered viruses and the members of the family Picornaviridae, an evolutionary analysis has been carried out using the amino acid sequences of the two nonstructural proteins 2C and 3D. Phylogenetic analysis using prime members of the nine genera support the division of picornaviruses into the proposed genera. The study also supports a previous suggestion based on analysis of partial sequences of the structural proteins that LV is more related to the genus of Parechovirus than to other picornaviruses, but also shows that the three LV strains used in the comparison constitute a distinct monophyletic group, clearly separated from the parechoviruses. The analyses using the 2C and 3D sequences clearly showed that A-2 was related to the genera of Rhinovirus and Enterovirus, but it was not possible to group the A-2 with high confidence into one of the genera. Comparison using the VP1 protein sequences of Enterovirus and Rhinovirus showed that although the A-2 virus is positioned between the two genera, the virus is more related to the genus of Enterovirus than to Rhinovirus. Our analysis of the three LV strains based on the phylogenetic analysis of the 2C and 3D proteins suggests that the strains used in this study constitute a monophyletic group clearly related to Parechovirus of Picornaviridae. The taxonomic position of the A-2 virus is presently uncertain but available data indicate that this virus may be classified as a member of the genus of Enterovirus.     

Longitudinal observation of parechovirus in stool samples from Norwegian infants

Parechoviruses are assumed to be common infectious agents, but their epidemiologic and pathogenic properties are not well known. The aim of the present study was to assess the prevalence and molecular epidemiology of Parechovirus in Norwegian infants, as well as to investigate whether the presence of virus correlated with symptoms of infection. A group of 102 infants was longitudinally followed: 51 infants with a high genetic risk for type 1 diabetes (aged 3-35 months), and 51 children without this genotype (aged 3-12). Stool samples were obtained each month, and symptoms of infection were recorded regularly on questionnaires. Human parechovirus was detected in 11.3% of 1,941 samples examined by real-time RT-PCR. There was a distinct seasonality, peaking from September to December. By 12 months of age, 43% of the infants had had at least one infection, while 86% of the infants had encountered the virus by the end of the second year. Based on the VP1 sequence, human parechovirus 1 was the most prevalent type (76%), followed by human parechovirus 3 (13%), human parechovirus 6 (9%), an unclassified human parechovirus (1%), and human parechovirus 2 (1%). Ljungan virus, a murine parechovirus, was examined with a separate real-time RT-PCR, but no virus was detected. There was no significant association between infections and the following symptoms: coughing, sneezing, fever, diarrhea or vomiting. In conclusion, human parechovirus infects frequently infants at an early age without causing disease.     

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.     

Cross antigenicity among enteroviruses as revealed by immunoblot technique

Antigenic relationships of various human and two animal picornaviruses were investigated by the immunoblotting ("Western blot") technique. The viruses included all coxsackievirus B types (1-6), poliovirus types 1-3, several strains of echovirus 11, EMC virus, and FMDV. Antisera included human sera and sera from rabbits hyperimmunized with either purified picornaviruses, viral structural polypeptides (VP8), boiled or "sample-boiled" virions. Group-specific reactions of various extent were observed among the human picornaviruses, but not with EMC virus. These reactions were obtained with human sera (whole serum, IgG- and IgM-fraction) as well as with "monospecific" (neutralization test) rabbit antisera. Among cross reacting polypeptides VP1 was predominant with the notable exception of coxsackie B4, where VP1 (defined according to cleavage pattern) migrates in our gel system as second largest polypeptide. Antisera prepared vs VP1 had neutralizing activity as demonstrated with five different echovirus 11 strains (titers up to 2000). Antisera vs VP1 (and other VP8) exhibited cross-reactivity in the immunoblots. Antisera to the three poliovirus types (and to certain echovirus 11 strains) showed a surprisingly narrow cross-reacting spectrum which--in the case of poliovirus--could not be broadened by additional hyperimmunization of the rabbits with heated poliovirus 2. The significance of these results for a diagnostic ELISA in patients with picornavirus infections is dealt with.     

Identification of human parechovirus genotype,HPeV-12, in a paralytic child with diarrhea

BackgroundNew genotypes of human parechoviruses have been readily identified after improvement of diverse diagnostic tools. We hereby report the detection of a new genotype, HPeV 12, from a child presented with diarrhea and paralysis.ObjectivesThe genetic variability of human parechoviruses has recently expanded defining 16 genotypes however data available covers only 11 genotypes. The present study was designed to determine the genetic characterization of human parechovirus identified in a child with gastroenteritis and acute flaccid paralysis (AFP).Study designStool samples are referred to Virology Department, NIH-Pakistan for the routine detection of enteroviruses and polioviruses through cell culture and RT-PCR. Five of isolates showing cytopathic effect on L20B cell line but negative for poliovirus were further explored for human parechovirus using multiple cell lines and RT-PCR.ResultsHuman Coxsackie A virus type 2, 3, 6 and 20 were found in four samples whereas the fifth sample contained human parechovirus genotype 12. Efficient growth of human parechovirus was found on L20B cells while Vero and LLC-MK2 cells showed no apparent cytopathic effect.ConclusionsThis study describes the detection of a new human parechovirus genotype (HPeV-12) in a paralytic child with diarrhea. Human parechoviruses are now considered as potential pathogens that may cause a number of serious clinical complications especially in infants and young children. These findings emphasize to conduct large scale epidemiological surveys in the country to understand their association with clinical diseases especially gastroenteritis, respiratory and neurological disorders.     

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.     

Biochemistry and pathogenicity of echovirus 9 I. Characterization of the virus particles of strains Barty and Hill

Strain Barty, a mouse-pathogenic echovirus 9, which is known to cause epidemic disease in man, and strain Hill, the prototype of echovirus 9, were characterized and compared. Strain Barty virion particles sediment in a low-salt-sucrose gradient with 156 S, whereas particles of the Hill strain form fast-sedimenting aggregates. Plaque formation of the Hill strain is inhibited under an overlay containing agar; virus strain Barty, however, forms plaques. Isoelectric focusing revealed that Barty virion particles exist essentially in one conformation with a characteristic isoelectric point; virions of the Hill strain were found to behave like a mixture of particles with various isoelectric points. Thus, the two echovirus 9 strains, apparently, have a different capsid surface. These divergent capsid surface structures include not only the adsorption site(s)-strain Barty adsorbs less efficiently to cells in culture than strain Hill-but also the determinants inducing neutralizing antibodies: serologically, the two strains are clearly distinguishable. The capsid protein patterns of the two strains in SDS-PAGE show a characteristic difference: VPl of strain Barty is a double band, whereas strain Hill contains a single band in a position just between the two VP1 bands of strain Barty. In addition, fingerprinting to the capsid proteins revealed that the two echovirus 19 strains differ not only in VP1 but probably also in VP3. These determinants of capsid proteins may cause the observed divergent behaviors of the two virus strains, and-possibly-influence pathogenicity.     

Human parechovirus 1 infections in young children--no association with type 1 diabetes

The epidemiology, transmission and clinical symptoms of human parechoviruses [HPeV, classified earlier as enteroviruses; echovirus 22 (HPeV1) and echovirus 23 (HPeV2)] remain poorly characterized. Enteroviruses and one parechovirus species, the Ljungan virus, have been associated with type 1 diabetes in humans and rodents. The occurrence of human parechovirus 1 (HPeV1) infections in young children and their possible association with type 1 diabetes was evaluated. The prospective birth cohort study comprised 221 Finnish children carrying genetic type 1 diabetes susceptibility and who were observed from birth. Thirty-four children developed multiple diabetes-associated autoantibodies, and 18 children progressed to clinical type 1 diabetes during the follow-up. HPeV1 infections were diagnosed by measuring neutralizing antibodies from the follow-up sera taken every 3-12 months. In addition, viral RNA was analysed by RT-PCR from stool samples taken every month from six of the participants. HPeV1 infections were found to occur early in childhood. The median age of infection was 18 months and 20% of the children had had an infection by the age of 1 year. The number of infections started to increase from the age of 6 months and most children had their first infection by 36 months. Nearly all (99%) mothers were HPeV1 antibody positive. No difference was found in infection frequency between boys and girls, nor between prediabetic, diabetic and control subjects. Most infections (87%) occurred during autumn, winter and spring.     

Molecular characterization of the newly identified human parvovirus 4 in the family Parvoviridae

Human parvovirus 4 (PARV4) is an emerging human virus, and little is known about the molecular aspects of PARV4 apart from its incomplete genome sequence, which lacks information of the termini. We analyzed the gene expression profile of PARV4 using a nearly full-length HPV4 genome in a replication competent system in 293 cells. We found that PARV4 utilizes two promoters to transcribe non-structural protein- and structural protein-encoding mRNAs, respectively, which were polyadenylated at the right end of the genome. Three major proteins, including the large non-structural protein NS1a, whose mRNA is spliced, and capsid proteins VP1 and VP2, were detected. Additional functional analysis of the NS1a revealed its capability to induce cell cycle arrest at G2/M phase in ex vivo-generated human hematopoietic stem cells. Taken together, our characterization of the molecular features of PARV4 suggests that PARV4 represents a new genus in the family Parvoviridae.     

Sequence analysis of monoclonal antibody resistant mutants of type O foot and mouth disease virus: evidence for the involvement of the three surface exposed capsid proteins in four antigenic sites.

Sequence analysis of monoclonal antibody resistant mutants of type O foot and mouth disease virus has been performed. Distinct clusters of amino acid substitutions conferring resistance to neutralization at each of the four previously defined antigenic sites (McCahon et al., 1989, J. Gen. Virol. 70, 639-645) have been identified. One site corresponds to the well-known 140-160 region of VP1, a second site is also on VP1, one site is on VP2, and the fourth site is on VP3. All of the amino acid substitutions identified are located on the surface of the virus. Despite the differences in three-dimensional structure between FMDV and other picornaviruses the neutralizing antigenic sites occur in analogous positions on the capsid surface.     

Molecular and biological analysis of echovirus 9 strain isolated from a diabetic child

The full-length infectious cDNA clone was constructed and sequenced from the strain DM of echovirus 9, which was recently isolated from a 6-week-old child at the clinical onset of type 1 diabetes. Parallel with the isolate DM, the full-length infectious cDNA clone of the prototype strain echovirus 9 Barty (Barty-INF), was constructed and sequenced. Genetic relationships of the sequenced echo 9 viruses to the other members of the human enterovirus type B species were studied by phylogenetic analyses. Comparison of capsid protein sequences showed that the isolate DM was closely related to both prototype strains: Hill and Barty-INF. The only exception was the inner capsid protein VP4 where serotype specificity was not evident and the isolate DM clustered with the strain Hill and the strain Barty-INF with echovirus 30 Bastianni. Likewise, the nonstructural protein coding region, P2P3, of isolate DM was more similar to strain Hill than to strain Barty-INF. However, like echovirus 9 Barty, the isolate DM contained the RGD-motif in the carboxy terminus of capsid protein VP1. By blocking experiments using an RGD-containing peptide and a polyclonal rabbit antiserum to the alpha(v)beta(3)-integrin, it was shown that this molecule works as a cellular receptor for isolate DM. By using primary human islets, it was shown that the isolate DM is capable of infecting insulin-producing beta-cells like the corresponding prototype strains did. However, only isolate DM was clearly cytolytic for beta-cells. The infectious clones that were made allow further investigations of the molecular features responsible for the diabetogenicity of the isolate DM.     

Molecular analysis of duck hepatitis virus type 1

The genome sequence of a duck hepatitis virus type 1 (DHV-1) strain was determined. Comparative sequence analysis showed that the genome possesses a typical picornarivus organization and also exhibits several unique features, such as the similarity of internal ribosome entry site to that of Porcine teschovirus 1 and Hepatitis C virus, the presence of a longest 3' untranslated region and a shorter leader protein in the Picornaviridae, the absence of a predicted maturation cleavage of VP0, the association of an aphthovirus-like 2A1 and parechovirus-like 2A2, and the unprecedented presence of an AIG1 domain in the N-terminus of 2A2. It is concluded that DHV-1 belongs to a new group of the family Picornaviridae that may form a separate genus most closely related to the genus Parechovirus.     

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.     

Isolation and preliminary characterization of antigenic variant of echovirus type 11

Nosocomial infection with echovirus type 11 resulting in aseptic meningitis occurred among newborn babies in a hospital neonatal room at Fukui city. The virus was identified as a variant of echovirus type 11 by cross-neutralization tests with antisera against the prototype Gregory strain and the current Fukui isolate. Fukui isolates expressed strain specific antigen(s) in addition to type specific common antigen(s), but lacked a certain antigen(s) which was present in the prototype strain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of virus polypeptides revealed that the capsid proteins VP2 and VP3 of Fukui strain migrated more rapidly than the Gregory strain, while both strains had the same migration pattern of VP1 protein on which the antigenic determinants responsible for virus neutralization were present. The current strain produced large plaques and was more thermoresistant, suggesting some alterations in the structural proteins of the virus.     

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.     

A new picornavirus isolated from bank voles (Clethrionomys glareolus)

A previously unknown picornavirus was isolated from bank voles (Clethrionomys glareolus). Electron microscopy images and sequence data of the prototype isolate, named Ljungan virus, showed that it is a picornavirus. The amino acid sequences of predicted Ljungan virus capsid proteins VP2 and VP3 were closely related to the human pathogen echovirus 22 (approximately 70% similarity). A partial 5' noncoding region sequence of Ljungan virus showed the highest degree of relatedness to cardioviruses. Two additional isolates were serologically and molecularly related to the prototype.     

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.     

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.