Genomic characterization of Sebokele virus 1 (SEBV1) reveals a new candidate species among the genus Parechovirus.

We determined the genomic features and the taxonomic classification of Sebokele virus 1 (SEBV1), a previously unclassified arbovirus isolated in 1972 from rodents collected in Botambi, Central African Republic. The complete genome sequence was obtained using a deep sequencing approach (Illumina technology) and dedicated bioinformatics workflows for data analysis. Molecular analysis identified SEBV1 as a picornavirus, most closely related to Ljungan viruses of the genus Parechovirus. The genome has a typical Ljungan virus-like organization, including the presence of two unrelated 2A protein motifs. Phylogenetic analysis confirmed that SEBV1 belongs to the parechovirus phylogroup and was most closely related to the Ljungan virus species. However, it appeared clearly distinct from all members of this phylogroup, suggesting that it represents a novel species of the genus Parechovirus.

Highly divergent neurovirulent vaccine-derived polioviruses of all three serotypes are recurrently detected in Finnish sewage.

In Finland, surveillance of potential re-emergence of poliovirus transmission is mainly based on environmental surveillance, i.e. search for infectious poliovirus in sewage samples. Since December 2008, 21 genetically highly divergent, neurovirulent vaccine-derived polioviruses (VDPV) have been isolated from sewage in Tampere, Finland. While the source of the VDPV is unknown, characteristics of the viruses resemble those of strains isolated from immunodeficient, persistently infected persons. No cases of suspected poliomyelitis have been reported in Finland since 1985.

A poliovirus neutralization epitope expressed on hybrid hepatitis B surface antigen particles.

The hepatitis B virus (HBV) envelope protein carrying the surface antigen (HBsAg) is assembled with cellular lipids in mammalian cells into empty viral envelopes. In a study to evaluate the capacity of such particles to present foreign peptide sequences in a biologically active form, in-phase insertions were created in the S gene encoding the major envelope protein. One of the sequences inserted was a synthetic DNA fragment encoding a poliovirus neutralization epitope. Mammalian cells expressing the modified gene secreted hybrid particles closely resembling authentic 22-nanometer HBsAg particles. These particles reacted with a poliovirus-specific monoclonal antibody and induced neutralizing antibodies against poliovirus. The results indicate that empty viral envelopes of HBV may provide a means for the presentation of peptide sequences and for their export from mammalian cells.

Nucleotide sequence and expression of the diphtheria tox228 gene in Escherichia coli.

The complete nucleotide sequence of the diphtheria tox228 gene encoding the nontoxic serologically related protein CRM228 has been determined. A comparison of the predicted amino acid sequence with the available amino acid sequences from the wild-type toxin made it possible to deduce essentially the entire nucleotide sequence of the wild-type tox gene. The signal peptide of pro-diphtheria toxin and the putative tox promoter have been identified, a highly symmetrical nucleotide sequence downstream of the toxin gene has been detected; this region may be the corynebacteriophage beta attachment site (attP). The cloned toxin gene was expressed at a low level in Escherichia coli.

Genetic features of polioviruses isolated in Tunisia, 1991-2006.

BACKGROUND: Genetic characterisation of polioviruses remains highly important even in countries where wild poliovirus circulation has been interrupted. Sequence data on representative wild strains from all geographical regions is required for surveillance purposes and surveillance for vaccine-related isolates with increased potential for transmissibility in humans should continue. OBJECTIVE: To report the genetic characteristics of wild and vaccine-related polioviruses isolated in Tunisia from 1991 to 2006. STUDY DESIGN: Wild isolates were sequenced in the VP1 genomic region and compared to each other. Vaccine-related isolates were assessed for genetic recombination by PCR/RFLP and sequence analysis of the 3D region. Recombinant viruses were assessed for genetic drift in the VP1 region. RESULTS: The VP1 sequences of the last wild isolates, all from serotype3, showed 97.7-98.7% nucleotide homology. Nineteen percent of vaccine-related isolates were vaccine/vaccine intertypic recombinants. No recombinant with non-poliovirus enteroviruses was identified. Mutational differences in the VP1 sequences of recombinant viruses ranged from 0.0% to 0.7% indicating a limited replication period. CONCLUSIONS: This study provides sequence data on wild polioviruses from Tunisia/North Africa and shows that in countries with continuous high vaccine coverage transmission of vaccine-related polioviruses is time-limited.

[Genetic evolution of poliovirus: success and difficulties in the eradication of paralytic poliomyelitis]. / Evolution génétique du poliovirus: succès et difficultés de l'éradication de la poliomyélite paralytique.

Poliovirus, the aetiological agent of poliomyelitis, is an enterovirus of the Picronaviridae family. Despite the success of the World Health Organisation (WHO) worldwide vaccination campaign against poliomyelitis, poliovirus remains a public health problem in several developing countries, in Africa and Asia in particular. This is partly due to the considerable capacity of poliovirus strains to circulate and spread in populations with insufficient vaccine coverage. In addition, the attenuated strains of the oral polio vaccine (OPV) may rapidly evolve a neurovirulent phenotype, causing rare cases of paralytic poliomyelitis. The recent occurrence of epidemics associated with vaccine-derived poliovirus (VDPV) has highlighted the emergence of recombinant strains with genomes constituted of sequences from OPV strains together with sequences from non-polio enteroviruses. In this review, after briefly describing the molecular biology of poliovirus and the pathogenesis of poliomyelitis, we will provide an overview of the current situation concerning poliomyelitis prophylaxis and the strategies developed to fight this disease. We will also deal with the issue of the possible re-emergence of poliovirus after declaration of the eradication of wildtype poliovirus.

The molecular characterization of poliovirus strains by the RT-PCR-RFLP assay and its use in the active surveillance for acute flaccid paralysis cases in Romania between 2001-2006.

Poliomyelitis, an acute disease of the the central nervous system can be controlled through the use of inactivated virus vaccine (IPV) or live attenuated vaccine (OPV). The goal of the Global Polio Eradication Initiative is to stop the global transmission of poliovirus. Our study is a retrospective and prospective study that was made because in 2002 was isolated from one acute flaccid paralysis AFP case and eight healthy contacts belonging to the same small socio-cultural group having a low vaccine coverage living in Babadag a town in Romania a serotype 1 vaccine-derived poliovirus (VDPV), recombinant S1/S2/S1. The 67 poliovirus strains isolated in Romania between 2001-2006 from acute flaccid paralysis cases (AFP) (n=20, age = 3 months - 10 years), facial paralysis cases (FP) (n=5, age = 3 months - 3.7 years) and healthy's AFP contacts (n=42, age = 2 months - 5.10 years) were molecular investigated to confirm the vaccine origin of these strains and for the detection of recombinant strains. The identification of these strains was achieved through reverse transcription (RT), polymerase chain reaction (PCR) and restriction fragment length polymorphisme assays (RFLP) applied to two sequences of the viral genome, which are located in VP1-2A (2870 nt - 3648 nt) and 3D (6086nt - 6516 nt) regions. For the strains investigated in VP1-2A region, the RFLP profils after digestion with 3 restriction enzymes (Rsal, Ddel, Hinf l) were Sabin-like only with one exception, VDPV strain. In 3D region after digestion with one, two or three enzymes the genomes of most poliovirus (PV) strains were found to be similar to the original vaccine Sabin strain. In 9 AFP cases the profils detected after RFLP in 3D region were S3/S1 (n=3); S3/52 (n=2); S2/S1 (n=2), S2/S1 + S2 (n=2). In 3 FP cases the profils were S3/S2 (n=2), 53/S1 (n=1). In 11 healthy contacts the RFLP profiles in 3D region were S3/S2 (n=6); S1/S1 +S3 (n=1); S3/S1 + S2 (n=1), S3/S2 + S3 (n=1), S2 + S3/S1 (n=1), S1/S2 (n=1).

Insertions in the hepatitis B surface antigen. Effect on assembly and secretion of 22-nm particles from mammalian cells.

The envelope protein of hepatitis B virus carrying the surface antigen, HBsAg, has the unique property of mobilizing cellular lipids into spherical or elongated particles, about 22 nm in diameter, which are secreted from mammalian cells. We have created mutant envelope proteins by insertion of various sequences of different lengths into two regions of the S gene encoding the major envelope protein. S genes carrying inserts in phase with HBsAg were expressed in mouse L cells from the simian virus 40 early promoter. Various single or double inserts in the two major hydrophilic domains of HBsAg were compatible with secretion of 22-nm particles. In all mutant envelope proteins studied, the HBsAg domains required for intracellular aggregation appeared to be intact. However, assembly into particles was not sufficient to assure transport into the extracellular space. The 22-nm HBsAg particle may be a useful vehicle for the export and presentation of foreign peptide sequences.

Construction and characterization of hybrid hepatitis B antigen particles carrying a poliovirus immunogen.

The hepatitis B surface antigen (HBsAg) has the unique property of assembling with cellular lipids into spherical or elongated particles of 22 nm diameter which are secreted by mammalian cells expressing HBsAg. We have studied the structural requirements for particle formation and secretion by creating in-phase insertions into different regions of the S gene of the hepatitis B virus, coding for HBsAg. Modified genes were integrated into an appropriate vector and expressed in mouse L cells. Various single and double inserts in the two major hydrophilic domains of HBsAg were compatible with particle synthesis and secretion. The level of secretion was influenced by the length of the insert, its primary structure, and the site of insertion into the HBsAg molecule. One of the inserted sequences was a synthetic DNA fragment encoding a continuous type 1 poliovirus neutralization epitope (the C3 epitope). Mammalian cells expressing the modified hepatitis B virus S gene secreted hybrid particles carrying the poliovirus antigen. The hybrid polio-HBsAg particles reacted with a monoclonal antibody specific for the C3 epitope and induced poliovirus neutralizing antibodies at low, but significant, titers in mice and at high titers in rabbits. However, the immune response to HBsAg was weaker to hybrid particles than to unmodified HBsAg particles. By cotransfection with two different plasmids carrying either modified or unmodified genes, we obtained phenotypically mixed particles containing both polio-HBsAg and HBsAg molecules. Inoculated into rabbits, the mixed particles induced high antibody titers against both poliovirus and HBsAg.

Enhancement of humoral immunity to SIVenv following simultaneous inoculation of mice by three recombinant adenoviruses encoding SIVenv/poliovirus chimeras, Tat and Rev.

A means of inducing gene expression by simultaneous infection with three recombinant adenoviruses (Ad) is described. The simian immunodeficiency virus (SIV) envelope-coding region was placed under the control of the human immunodeficiency virus type 1 (HIV-1) Tat and Rev proteins provided in trans by distinct Ad vectors (Ad-tat; Ad-rev). Coinfection of cells with the three recombinant adenoviruses led to induction of high levels of SIV env mRNA and protein synthesis, while inoculation of mice elicited anti-Env antibodies. Insertion of the poliovirus VP1 neutralization epitope (C3) in the V1 hypervariable region of SIV envelope not only proved to be highly immunogenic for the poliovirus epitope but also enhanced the kinetics of anti-SIV Env antibody production. By contrast, insertion in V4 elicited no anti-C3 response and only normal anti-Env responses.

[Intense campaign for vaccination with the oral polio vaccine: what are the repercussions on the enterovirus world?]. / Campagnes intensives de vaccination avec le vaccin polio oral: quelles répercussions sur le monde des entérovirus? Groupe d'Etude Entérovirus.

To eradicate poliomyelitis and poliovirus, intensive vaccination campaigns with oral polio-vaccine (OPV) have been organised. Eradication campaigns may well be successful because the antiviral immunity and the local intestinal immunity due to OPV in particular avoids and/or limits poliovirus circulation. These campaigns give interesting opportunities for studying the impact of viral vaccines on the viral world in terms of ecological and genetic virology. The pre-eradication phase we are now entering brings with it two kinds of problems. First, the major disadvantage of OPV is the genetic and phenotypic variability of the vaccine strains. This variability leads to the spread of potentially pathogenic strains, which can be implicated in vaccine-associated paralytic poliomyelitis (VAPP). Genetic changes are characterised by point mutations and by genetic exchanges among OPV strains, between OPV and wild strains and perhaps between poliovirus and non-polio enteroviruses (ENPV). The fact that a few OPV mutant strains have been shown to multiply and/or to circulate for long periods suggests that OPV could sustain a reservoir of pathogenic poliovirus strains. Second, there are ecological considerations. The disappearance of wild poliovirus through OPV vaccination could be due not only to antiviral local immunity but also to competition between OPV strains and wild strains for infecting the digest tract. Moreover, a competition between OPV and other enteroviruses may take place in a common ecological niche. To our knowledge, the possible impact of intensive OPV vaccination campaigns on the ENPV populations has never been considered. Because the goal of poliovirus eradication may be reached in the near future, there is worry as to the possible evolution of ENPV towards highly epidemic and pathogenic strains. This is leading those laboratories involved in poliomyelitis surveillance not only to search for remaining wild poliovirus strains but also to study the possible long-term circulation of OPV strains and to develop efficient ENPV surveillance.

Enhancement of gene expression by somatic hybridization with primary cells: high-level synthesis of the hepatitis B surface antigen in monkey Vero cells by fusion with primary hepatocytes.

Vero cells transfected with the S gene encoding the surface antigen (HBsAg) of the hepatitis B virus (HBV) synthesize HBsAg at low levels. We have obtained a large increase in S gene expression by somatic hybridization of Vero cells with primary hepatocytes, which are the natural target cells for HBV infection. Fusion with cells other than hepatocytes did not enhance expression of the S gene. The Vero/hepatocyte hybrid clones analyzed are stable and have maintained a high level of HBsAg synthesis over prolonged periods. Hybrid cell lines may be of general interest for the high-level synthesis of proteins using cloned genes.

Circulation of a type 1 recombinant vaccine-derived poliovirus strain in a limited area in Romania.

After intensive immunisation campaigns with the oral polio vaccine (OPV) as part of the Global Polio Eradication Initiative, poliomyelitis due to wild viruses has disappeared from most parts of the world, including Europe. Here, we report the characterization of a serotype 1 vaccine-derived poliovirus (VDPV) isolated from one acute flaccid paralysis (AFP) case with tetraplegia and eight healthy contacts belonging to the same small socio-cultural group having a low vaccine coverage living in a small town in Romania. The genomes of the isolated strains appeared to be tripartite type 1/type 2/type 1 vaccine intertypic recombinant genomes derived from a common ancestor strain. The presence of 1.2% nucleotide substitutions in the VP1 capsid protein coding region of most of the strains indicated a circulation time of about 14 months. These VDPVs were thermoresistant and, in transgenic mice expressing the human poliovirus receptor, appeared to have lost the attenuated phenotype. These results suggest that small populations with low vaccine coverage living in globally well-vaccinated countries can be the origin of VDPV emergence and circulation. These results reaffirm the importance of active surveillance for acute flaccid paralysis and poliovirus in both polio-free and polio-endemic countries.

Structural factors modulate the activity of antigenic poliovirus sequences expressed on hybrid hepatitis B surface antigen particles.

We have studied the functional expression of antigenic poliovirus fragments carried by various hybrid hepatitis B surface antigen (HBsAg) particles. Several constructions were made by using two different insertion sites in the HBsAg molecule (amino acid positions 50 and 113) and two different sequences, one derived from poliovirus type 1 (PV-1) and the other from PV-2. The inserted fragments each encompassed residues 93 to 103 of the capsid protein VP1, a segment which includes the linear part of the neutralization antigenic site 1 of the poliovirus. The antigenicity and immunogenicity of the hybrid particles were evaluated and compared in terms of poliovirus neutralization. A high level of antigenic and immunogenic activity of the PV-1 fragment was obtained by insertion at position 113 but not at position 50 of HBsAg. However, a cooperative effect was observed when two PV-1 fragments were inserted at both positions of the same HBsAg molecule. Antibodies elicited by the PV-2 fragment inserted at amino acid position 113 did not bind or neutralize the corresponding poliovirus strain. They did, however, bind a chimeric poliovirus in which the homologous antigenic fragment of PV-1 had been replaced by that of PV-2. The only virions that were neutralized by these antibodies were certain mutants carrying amino acid substitutions within the PV-2 fragment. These results show that position, constraints from the carrier protein, and nature of the inserted sequences are critically important in favoring or limiting the expression of antigenic fragments as viral neutralization immunogens.

Diphtheria toxin promoter function in Corynebacterium diphtheriae and Escherichia coli.

The expression of the diphtheria tox228 gene encoding the nontoxic, serologically related CRM228 mutant diphtheria toxin has been analyzed in Corynebacterium diphtheriae and Escherichia coli. The diphtheria toxin promoter has been used to direct the expression of beta-galactosidase in E.coli, and the efficiency of promotion has been compared to that obtained with the lac promoter. Expression in C.diphtheriae is known to be dependent on the absence of iron, and we present for the first time direct evidence that this regulation occurs at the level of transcription. The 5' end of toxin mRNA maps at the same position in C.diphtheriae and E.coli, suggesting identical sequences to be recognized by C.diphtheriae and E.coli RNA polymerase. The diphtheria toxin promoter carries at position -34 a TTGATT sequence closely related to the E.coli -35 consensus sequence and in the -14 to -8 region a set of overlapping sequences with complete or partial homology to the E.coli -10 consensus sequence.

Point mutations involved in the attenuation/neurovirulence alternation in type 1 and 2 oral polio vaccine strains detected by site-specific polymerase chain reaction.

One of the problems raised by the use of the attenuated oral poliovirus vaccine (OPV) Sabin strains is the genetic instability of the attenuated phenotype upon multiplication in vivo. Nucleotide sites critical for attenuation were identified for each of the three poliovirus serotypes. One important position lies in the 5' non-coding region of the genome of each of the three OPV strains, at nucleotide 480 in type 1, 481 in type 2 and 472 in type 3. Point mutations at these positions were usually selected upon multiplication in vivo as substitutions of the vaccine-type residue. The reversion was found to correlate with an increased degree of neurovirulence. To screen easily for this mutation in a great number of strains, we developed a site-specific polymerase chain reaction method based on the property of the Taq polymerase to elongate only primers with a perfect homology at the 3' extremity. We screened for this mutation in five type 1 and nine type 2 polio vaccine-derived strains isolated from vaccine-associated paralytic poliomyelitis (VAPP) cases and in 16 such strains isolated from healthy vaccinees. All 14 strains isolated from VAPP presented the reversion. Of the eight pairs of type 1 isolates from healthy vaccinees, four presented the reversion 3 days after vaccine administration and all but one at 7 days postvaccination. These results support the involvement of the 5' non-coding specific nucleotide sites in the reversion to neurovirulence of attenuated polio vaccine strains upon multiplication in the human gut.(ABSTRACT TRUNCATED AT 250 WORDS)

Tripartite genome organization of a natural type 2 vaccine/nonvaccine recombinant poliovirus.

Intertypic vaccine/vaccine recombinant polioviruses are frequently isolated from vaccine-associated paralytic poliomyelitis cases (VAPP). We identified a vaccine/nonvaccine poliovirus recombinant as the causative agent of a lethal VAPP. Partial RNA sequencing revealed a tripartite recombinant structure of the viral genome. This consisted of a central capsid core of vaccine origin flanked by two units of nonvaccine origin. The first nonvaccine genomic unit spanned the whole 5' noncoding region, and the second one almost the entire nonstructural protein-coding region and the 3' noncoding region. Amino acid and nucleotide sequence similarities in the 3' and 5' unidentified regions indicated that the viral donor(s) were poliovirus species, suggesting recombination between a vaccine-derived and a wild poliovirus. The nonvaccine donor(s) could not be identified among the investigated wild polioviruses cocirculating in the same geographical area. This is the first report of a natural recombination event occurring in the 5' genomic extremity of poliovirus. The neurovirulence for transgenic mice and the pathogenicity for humans of the recombinant suggested that the modular genomic organization of this virus might have conferred a selective advantage over its vaccine parent.

Mouse adaptation determinants of poliovirus type 1 enhance viral uncoating.

Most poliovirus (PV) strains, such as PV type 1/Mahoney, cannot infect the mouse central nervous system. We previously identified two determinants of mouse adaptation of PV type 1/Mahoney at positions 22 and 31 of the viral capsid proteins VP1 and VP2, respectively (T. Couderc, J. Hogle, H. Le Blay, F. Horaud, and B. Blondel, J. Virol. 67:3808-3817, 1993). These residues are located on the interior surface of the capsid. In an attempt to understand the molecular mechanisms of adaptation of PV to mice, we investigated the effects of these two determinants on the viral multiplication cycle in a human cell line. Both determinants enhanced receptor-mediated conformational changes leading to altered particles of 135S, one of the first steps of uncoating, and viral internalization. Furthermore, the residue at position 22 of VP1 appears to facilitate RNA release. These results strongly suggest that these determinants could also facilitate conformational changes mediated by the PV murine receptor and internalization in the mouse nerve cell, thus allowing PV to overcome its host range restriction. Moreover, both mouse adaptation determinants are responsible for defects in the assembly of virions in human cells and affect the thermostability of the viral particles. Thus, these mouse adaptation determinants appear to control the balance between the viral capsid plasticity needed for the conformational changes in the early steps of infection and the structural requirements which are involved in the assembly and the stability of virions.

Presentation and immunogenicity of the hepatitis B surface antigen and a poliovirus neutralization antigen on mixed empty envelope particles.

Insertion of a synthetic DNA fragment encoding a poliovirus neutralization epitope into the S gene encoding the major envelope protein of hepatitis B virus has yielded hybrid (HBsPolioAg) particles closely resembling authentic 22-nm antigen (HBsAg) particles by expression of the modified gene in mammalian cells. In mice, these hybrid particles induce neutralizing antibodies against poliovirus but only weak immune response to HBsAg (F. Delpeyroux, N. Chenciner, A. Lim, Y. Malpièce, B. Blondel, R. Crainic, S. Van der Werf, and R. E. Streeck, Science 233:472-474, 1986). By cotransfection with different plasmids carrying either modified or unmodified S genes, we have now obtained mixed particles presenting both HBsAg and HBsPolioAg. When such particles were inoculated into rabbits, antibodies to both poliovirus and to HBsAg were induced. Moreover, the titers of neutralizing antibodies to poliovirus induced by HBsPolioAg were much higher than those previously obtained in mice. The design of multivalent particles carrying various peptide sequences or presenting several heterologous epitopes may therefore be possible.

A mutation in the RNA polymerase of poliovirus type 1 contributes to attenuation in mice.

The attenuated Sabin strain of poliovirus type 1 (PV-1) differs from the neurovirulent PV-1 Mahoney strain by 55 nucleotide mutations. Only one of these mutations (A-480-->G, in the 5' noncoding (5' NC) region of the genome, is well characterized, and it confers a strong attenuating effect. We attempted to identify genetic attenuation determinants in the 3'-terminal part of the Sabin 1 genome including the 3D polymerase (3Dpol) gene and the 3' NC region. Previous studies suggested that some of the 11 mutations in this region of the Sabin 1 genome, and in particular a mutation in the polymerase gene (U-6203-->C, Tyr-73-->His), are involved to some extent in the attenuation of PV-1. We analyzed the attenuating effect in the mouse model by using the mouse-adapted PV-1/PV-2 chimeric strain v510 (a Mahoney strain carrying nine amino acids of the VP1 capsid protein from the Lansing strain of PV-2). Mutagenesis of locus 6203 was performed on the original v510 (U-6203-->C) and also on a hybrid v510/Sabin 1 (C-6203-->U) carrying the downstream 1,840 nucleotides of the Sabin 1 genome including the 3Dpol and 3' NC regions. Statistical analysis of disease incidence and time to disease onset in numerous mice inoculated with these strains strongly suggested that nucleotide C-6203 is involved in the attenuation of the Sabin 1 strain. Results also suggested that, among the mutations located in the 3Dpol and 3' NC regions, nucleotide C-6203 may be the principal or the only one to be involved in attenuation in this mouse model. We also found that the effect of C-6203 was weaker than that of nucleotide G-480; the two nucleotides acted independently and may have a cumulative effect on attenuation. The U-6203-->C substitution also appeared to contribute to the thermosensitivity of the Sabin 1 strain.