Highly heterogeneous temperature sensitivity of 2009 pandemic influenza A(H1N1) viral isolates, northern France.

We assayed the temperature sensitivity of 2009 pandemic influenza A(H1N1) viral isolates (n=23) and seasonal influenza A(H1N1) viruses (n=18) isolated in northern France in 2007/08 and 2008/09. All isolates replicated with a similar efficiency at 34 °C and 37 °C, and with a lower efficiency at 40 °C. The pandemic viral isolates showed a stronger heterogeneity in their ability to grow at the highest temperature, as compared with the seasonal isolates. No statistically significant difference in temperature sensitivity was observed between the pandemic viral isolates from severe and mild cases of influenza. Our data point to the impact of temperature sensitivity on the genetic evolution and diversification of the pandemic influenza A(H1N1) virus since its introduction into the human population in April 2009, and call for close surveillance of this phenotypic marker related to host and tissue tropism.

[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.

[Poliovirus and apoptosis]. / Poliovirus et apoptose.

Poliovirus is the causal agent of paralytic poliomyelitis. Flaccid paralysis characteritic of poliomyelitis result from the destruction of motor neurons, the specific target cells of poliovirus in the central nervous system (CNS). The development of new animal and cell models has allowed the key steps of the pathogenesis of poliomyelitis to be investigated at the molecular level. In particular, it has been shown that poliovirus-induced apoptosis is an important component of the tissue injury in the CNS of infected mice that leads to paralysis. In this review, the molecular biology of poliovirus and the pathogenesis of poliomyelitis will be briefly described, and then several models of poliovirus-induced apoptosis will be considered ; the role of the cellular receptor of poliovirus, CD155, in the modulation of apoptosis will also be addressed.

Cloning and developmental expression of zebrafish GTP cyclohydrolase I.

GTP cyclohydrolase I (GCH) catalyses the conversion of GTP to dihydroneopterin triphosphate, initiating the pteridine pathway. The final product tetrahydrobiopterin (H4biopterin) is the cofactor for neurotransmitter synthesis and for tyrosine supply during melanogenesis. Sepiapterin accumulates as a pigment. We cloned the zebrafish gch cDNA, which encodes a protein highly homologous to other vertebrate sequences and characterised the recombinant enzyme. By in situ hybridisation, we found that gch is expressed in both the melanophore and xanthophore lineages during early development. gch-expression almost disappears after 3 days post-fertilisation (dpf), despite further sepiapterin synthesis. gch-transcripts are also located in catecholaminergic neurons, within the central nervous system and in the arch-associated neurons.

Inhibition of poliovirus RNA synthesis as a molecular mechanism contributing to viral persistence in the mouse central nervous system.

Many survivors of poliomyelitis, several decades after the acute phase of the disease, develop a set of new muscle symptoms called post-polio syndrome. The persistence of poliovirus (PV) in the central nervous system (CNS) may be involved in the aetiology of this syndrome. By using a mouse model, we have shown that PV persists in the CNS of paralysed mice for over a year after the acute disease. Detection of PV plus- and minus-strand RNAs in the spinal cord of paralysed mice suggested continuous PV RNA replication in the CNS. However, infectious PV particles could not be recovered from homogenates of CNS from paralysed mice beyond 20 days post-paralysis, indicating that PV replication was restricted. In an attempt to identify the molecular mechanism by which PV replication was limited, PV plus- and minus-strand RNA levels were estimated in the CNS of persistently infected mice by a semi-quantitative RT-nested PCR method. Results revealed that RNA replication was inhibited at the level of plus-strand RNA synthesis during persistent infection. Similar results were obtained in neuroblastoma IMR-32 cell cultures persistently infected with PV Restriction of PV RNA synthesis could be involved in persistence by limiting PV replication.

Poliovirus persistence in human cells in vitro.

Poliovirus (PV) can persist in vivo in the intestine of immunocompromised hosts for years. Moreover, immunocompetent individuals who have survived paralytic poliomyelitis sometimes develop the post-poliomyelitis syndrome (PPS), consisting of a variety of symptoms including new muscular atrophies. PPS may be due to PV persistence. We have developed models of PV persistence in neural cells and epidermoid cells. Cell determinants are of crucial importance for the establishment of persistent infections in human neuronal cells, whereas viral determinants play the primary role in human epidermoid HEp-2 cells. The results obtained with these in vitro models show the capacity of PV to persist and reveal a virus and cell co-evolution involving PV-receptor interactions. In addition, they suggest that several mechanisms are used by PV to establish and maintain persistent infections.

Development of the pteridine pathway in the zebrafish, Danio rerio.

In the zebrafish, the peripheral neurons and the pigment cells are derived from the neural crest and share the pteridine pathway, which leads either to the cofactor tetrahydrobiopterin or to xanthophore pigments. The components of the pteridine pattern were identified as tetrahydrobiopterin, sepiapterin, 7-oxobiopterin, isoxanthopterin, and 2,4,7-trioxopteridine. The expression of GTP cyclohydrolase I activity during the first 24-h postfertilization, followed by 6-pyruvoyl-5,6,7,8-tetrahydropterin synthase and sepiapterin reductase, suggest an early supply of tetrahydrobiopterin for neurotransmitter synthesis in the neurons and for tyrosine supply in the melanophores. At 48-h postfertilization, sepiapterin formation branches off the de novo pathway of tetrahydrobiopterin synthesis. Sepiapterin, via 7,8-dihydrobiopterin and biopterin, serves as a precursor for the formation of 7-oxobiopterin, which may be further catabolized to isoxanthopterin and 2,4,7-trioxopteridine. Neither 7, 8-dihydrobiopterin nor biopterin is a substrate for xanthine oxidoreductase. In contrast, both of these compounds are oxidized at C-7 by a xanthine oxidase variant form, which is inactivated by KCN, but is insensitive to allopurinol. The oxidase and the dehydrogenase form of xanthine oxidoreductase as well as the xanthine oxidase variant have specific developmental patterns. It follows that GTP cyclohydrolase I, the formation of sepiapterin, and the xanthine oxidoreductase family control the pteridine pathway in the zebrafish.

An approach to understanding the mechanisms of poliovirus persistence in infected cells of neural or non-neural origin.

BACKGROUND: Poliovirus (PV) is the etiologic agent of paralytic poliomyelitis, which is sometimes followed, after decades of clinical stability, by new symptoms, including progressive muscular atrophy, collectively known as the post-polio syndrome. This raises the question of possible PV persistence in post polio patients. OBJECTIVE: To test the capacity of PV to establish persistent infections in human cells, three models were developed. STUDY DESIGN: This review focuses on the viral and cellular parameters involved in persistent PV infection. RESULTS: Many PV strains, which are generally lytic in primate cell lines, are able to establish persistent infections in human neuroblastoma cells. During persistent infection, PV mutants (PVpi) are consistently selected, and several of their capsid substitutions occur at positions known to be involved in PV-PV receptor interactions. PVpi have a particular property: they can establish persistent infections in non-neural HEp-2 cells. PV can also persistently infect primary cultures of human fetal brain cells and the majority of cells which survive infection belong to the neuronal lineage. CONCLUSIONS: The results obtained with the three models of persistent PV infection in human cells suggest that several mechanisms are used by PV to establish and maintain persistent infections in neural and non-neural cells. The interactions of the virus with its receptor seem to be a key-step in all cases. In the future, the elucidation of the etiology of the post-polio syndrome will require the characterization of PV sequences having persisted for decades in post-polio patients.

Structural investigation of the cofactor-free chloroperoxidases.

The structures of cofactor-free haloperoxidases from Streptomyces aureofaciens, Streptomyces lividans, and Pseudomonas fluorescens have been determined at resolutions between 1.9 A and 1.5 A. The structures of two enzymes complexed with benzoate or propionate identify the binding site for the organic acids which are required for the haloperoxidase activity. Based on these complexes and on the structure of an inactive variant, a reaction mechanism is proposed for the halogenation reaction with peroxoacid and hypohalous acid as reaction intermediates. Comparison of the structures suggests that a specific halide binding site is absent in the enzymes but that hydrophobic organic compounds may fit into the active site pocket for halogenation at preferential sites.

Characterization of two polymorphic sites in the human kinin B1 receptor gene: altered frequency of an allele in patients with a history of end-stage renal failure.

On the basis of the genomic structure of the human B1 receptor (B1R) for kinins, the presence of possible allelic polymorphisms of this gene was investigated using restriction fragment-length polymorphism and single-strand conformation polymorphism. The frequencies of the found alleles were determined in healthy volunteers and in patients with a history of end-stage renal failure, because there is evidence for a nephroprotective action of the kallikrein-kinin system. An A1098-->G polymorphism has been identified in exon 3 in a minority of volunteer blood donors, and is located 35 nucleotides downstream from the stop codon and 14 nucleotides upstream from the polyadenylation signal. The frequency of the G allele is 4.4% in the control sample and not significantly altered in patients with a history of end-stage renal failure. A second and more frequent polymorphism (18.1% of the alleles in the control group, prevalence of 33.3%) consists of a single base substitution (G-699-->C) in the putative promoter region. This polymorphism is significantly less frequent in the population of renal failure patients (prevalence of 20.6%) and determines an increased activity of the promoter function in constructions involving a reporter gene. The altered prevalence of this allele was also found in some etiologic subgroups of uremic patients. This study confirms the mapping of the B1R gene to 14q32. Other investigators have mapped the bradykinin B2 receptor (B2R) gene to a close site on human chromosome 14. A previously described B2R polymorphism (exon 2, C181-->T) had an allele frequency of 9.7% in the control sample and appears to be clinically neutral. The polymorphism of the B1R promoter may be a marker of prognostic significance for the preservation of renal function in diseased individuals.

One amino acid change on the capsid surface of poliovirus sabin 1 allows the establishment of persistent infections in HEp-2c cell cultures.

Poliovirus mutants (PVpi) selected during the persistent infection of human neuroblastoma cells can establish secondary persistent infections in nonneural HEp-2c cells (I. Pelletier, T. Couderc, S. Borzakian, E. Wyckoff, R. Crainic, E. Ehrenfeld, and F. Colbère-Garapin, 1991, Virology, 180, 729-737). Previous results from our laboratory have also shown that, in the genome of PVpi S11 derived from the Sabin 1 strain, the genomic region involved in this phenotype contains 11 missense mutations which map exclusively to the genes encoding the capsid proteins VP1 and VP2. We report here the identification of precise viral determinants able to confer the capacity to establish persistent infections in HEp-2c cell cultures to the lytic Sabin 1 strain. We used a strategy based on the observation that PVpi, after a few months of persistent infection in HEp-2c cells, tend to regain a more lytic phenotype in uninfected HEp-2c cell cultures. We constructed mutant viruses carrying only a few mutations potentially involved in the phenotype of persistence. Two mutations were identified, one corresponding to the substitution His>Tyr of amino acid 142 of VP2 and another corresponding to the substitution Val>Ile of amino acid 160 of VP1. Mutants carrying one or the other of the two determinants established persistent infections in HEp-2c cell cultures in about 20% of the infections. Higher frequencies were obtained with the mutant carrying both determinants (30%), and with PVpi S11 (63%), indicating that the effects of several determinants can be cumulative. The two determinants are localized on the capsid surface in a region known to be involved in the interactions between poliovirus and its cell receptor and in fact, we demonstrate here that in the case of the two persistent mutants, these interactions are modified.

Two amino acid substitutions in the type 3 poliovirus capsid contribute to the establishment of persistent infection in HEp-2c cells by modifying virus-receptor interactions.

After 2.5 months of persistent infection in human neuroblastoma cells by the type 3 poliovirus (PV3) wild-type Leon strain, a mutant (PVpi), L2-2, capable of establishing a persistent infection in nonneural HEp-2c cells was isolated. Sequence analysis of the viral capsid protein genes revealed the presence of seven missense mutations, three of which were also present in a second PVpi, suggesting that they could be important determinants of the persistent phenotype. When the three mutations were introduced into the lytic Leon strain separately, in pairs or all together, all but one of the viruses was capable of establishing a persistent infection. However, aside from the triple mutant, only one mutant virus, bearing a Leu at position VP213 in the capsid interior and an Asn at position VP1290 on the capsid surface, was capable of establishing persistent infections in more than 30% of the cultures. When present together, these two determinants affect the early steps of the virus cycle including cell binding and the receptor-mediated conformational changes believed to be necessary for viral penetration and uncoating. In fact, this persistent double mutant appears to undergo a novel capsid transition when in contact with the human PV receptor, altering from the native virion which sediments at 160S to a form which sediments at about 147S. We propose that this modification could be the mechanism by which PV3 is able to establish persistent infections in HEp-2c cell cultures.

Molecular mechanisms of poliovirus persistence: key role of capsid determinants during the establishment phase.

As viral persistence is of major medical importance, well-characterized, simple models are needed to improve our understanding of persistent infections. We have chosen to study the molecular mechanisms of viral persistence with the poliovirus (PV), because this picornavirus is one of the best characterized animal viruses, it infects the central nervous system which is a target organ for viral persistence, and it belongs to the Picornaviridae family of viruses, which includes several naturally persisting viruses. We have developed models of PV persistence in neuronal and epidermoid cells, and the present review will focus on the latter one because both lytic and persistent PV strains can be used to study the PV-HEp-2 cell interactions. The viral determinants of persistence have been investigated with this model, and PV determinants have proven to be of crucial importance for the establishment of persistence in HEp-2 cells. Precise determinants of PV persistence have been identified for PV serotypes 1 and 3, in capsid proteins VP1 and VP2. These determinants modify the early steps of the PV cycle, and in particular, the conformational modifications of the capsid following virus adsorption onto its receptor. These results permit us to propose several hypotheses concerning PV persistence and the early steps of the PV cycle.

Cell clones cured of persistent poliovirus infection display selective permissivity to the wild-type poliovirus strain Mahoney and partial resistance to the attenuated Sabin 1 strain and Mahoney mutants.

We report the isolation and characterization of HEp-2c cell clones obtained after two successive persistent poliovirus (PV) infections. Once cured, some of the cell clones displayed selective permissivity toward the wild-type Mahoney strain and partial resistance to particular mutants of this strain, including the Sabin 1 strain. Two cell clones, CI 4 and CI 10, were studied in greater detail. The cytopathic effects of Mahoney infection were comparable in the cell clones and in HEp-2c cells. The cytopathic effects of infection by Sabin 1 or Mahoney mutants were greatly delayed in CI 4 and CI 10. In the genomic region encoding the capsid proteins, determinants involved in the resistance of the cell clones to the Mahoney mutants were localized in the amino-terminal part of VP1 (amino acids 22 and 43), the B-C loop of VP1 (amino acids 94-102), and the loop of VP3 connecting its amino-terminal to beta strand B (amino acid 60). These genomic regions are thought to be involved in the early steps of viral infection. Virus adsorption was slower and less efficient on CI 10 cells than on parental HEp-2c cells. Virus adsorption was faster on CI 4 than on HEp-2c cells, and at least as efficient, but there was less receptor-induced structural modification of the capsid, a step that is required for decapsidation. Furthermore, infection of CI 4 by a Mahoney mutant in which the B-C loop of VP1 has been deleted was affected in the later steps of infection. These results indicate that, in cells cured of persistent PV infection, poliovirus multiplication was restricted at several stages and particularly at two steps of virus entry: adsorption and/or the uncoating transitions following adsorption onto the receptor.

A catalytic triad is required by the non-heme haloperoxidases to perform halogenation.

The bacterial non-heme haloperoxidases are highly related to an esterase from Pseudomonas fluorescens, at structural and functional levels. Both types of enzymes displayed brominating activity and esterase activity. The presence of the serine-hydrolase motif Gly-X-Ser-X-Gly, in the esterase as well as in all aligned haloperoxidase sequences, strongly suggested that they belong to the serine-hydrolase family. Sequence alignment with several serine-hydrolases and secondary structure superimposition revealed the striking conservation of structural features characterising the alpha/beta-hydrolase fold structure in all haloperoxidases. These structural predictions allowed us to identify a potential catalytic triad in haloperoxidases, perfectly matching the triad of all aligned serine-hydrolases. The structurally equivalent triad in the chloroperoxidase CPO-P comprised the amino acids Serine 97, Aspartic acid 229 and Histidine 258. The involvement of this catalytic triad in halogenation was further assessed by inhibition studies and site-directed mutagenesis. Inactivation of CPO-P by PMSF and DEPC strongly suggested that the serine residue from the serine-hydrolase motif and an histidine residue are essential for halogenation, similar to that demonstrated for typical serine-hydrolases. By site-directed mutagenesis of CPO-P, Ser-97 was exchanged against alanine or cysteine, Asp-229 against alanine and His-258 against glutamine. Western blot analysis indicated that each mutant gene was efficiently expressed. Whereas the mutant S97C conserved a very low residual activity, each other mutant S97A, D229A or H258Q was totally inactive. This study gives the direct demonstration of the requirement of a catalytic triad in the halogenation mechanism.

A bacterial esterase is homologous with non-haem haloperoxidases and displays brominating activity.

Screening GenBank indicated that an esterase from Pseudomonas fluorescens had high sequence similarity with bacterial non-haem haloperoxides. However, this homology was limited to two distinct domains of the published esterase sequence. As errors in the published sequence were suspected, the esterase gene was sequenced again. The revised sequence displayed between 40 and 50% identical amino acids with the haloperoxidases, but distributed along the whole sequence. In addition to the structural homologies with haloperoxidases, the esterase also displayed functional homology. The recombinant esterase, purified from Escherichia coli cells, was capable of both ester hydrolysis and halogenation, as detected in situ by the formation of bromophenol blue or spectrophotometrically by the bromination of monochlorodimedon. The esterase is thus a bifunctional enzyme. The sequence analysis and the biochemical investigations show that the esterase belongs to the haloperoxidase family. It also possessed, however, a typical feature of serine-hydrolases, namely the consensus motif Gly-X-Ser-X-Gly around the active serine of the catalytic triad. By alignment of the esterase with different serine-hydrolase sequences, it was possible to identify the other two residues of the triad. The triad comprised the residues Ser95, Asp223 and His252. Interestingly, a structurally equivalent catalytic triad was also identified in the sequences of all bacterial non-haem haloperoxidases, in highly conserved domains. The presence of a catalytic triad in haloperoxidases is expected to be important in the mechanism of halogenation.

Substitutions in the capsids of poliovirus mutants selected in human neuroblastoma cells confer on the Mahoney type 1 strain a phenotype neurovirulent in mice.

Poliovirus (PV) type 1 mutants selected in human neuroblastoma cells persistently infected (PVpi) with the wild-type Mahoney strain exhibited a mouse-neurovirulent phenotype. Four of the five substitutions present in the capsid proteins of a PVpi were demonstrated to extend the host range of the Mahoney strain to mice. These new mouse-neurovirulent determinants were located in the three-dimensional structure of the viral capsid; two of them (residues 142 of VP2 and 60 of VP3) were located in loops exposed at the surface of the protein shell, whereas the other two (residues 43 of VP1 and 62 of VP4) were located on the inside of the capsid. VP1 residue 43 and VP2 residue 142 substitutions were also selected in a PVpi derived from the attenuated Sabin strain. We suggest that the selective pressure of human neuroblastoma cell factor(s) involved in early steps of PV multiplication could be responsible for the arising of amino acid substitutions which confer adaptation to the mouse central nervous system to PV.

Cloning of a second non-haem bromoperoxidase gene from Streptomyces aureofaciens ATCC 10762: sequence analysis, expression in Streptomyces lividans and enzyme purification.

The gene for BPO-A1, one of two non-haem bromoperoxidases in the tetracycline and 7-chlorotetracycline producer Streptomyces aureofaciens ATCC 10762, was cloned in the positive selection vector pIJ699 and expressed in Streptomyces lividans TK64. The cloned bromoperoxidase was over-produced up to 2800-fold by the S. lividans TK64 transformant. By taking advantage of the over-production of BPO-A1 and the heat stability of the enzyme, a new and simple purification procedure was developed. Subcloning into the vector pIJ487 and screening of recombinants by a newly developed histochemical assay located the bpoA1 gene on a 2.1 kb BamHI-HindIII fragment. The nucleotide sequence of the 2.1 kb fragment was determined; the bpoA1 gene was identified within the sequence on the basis of the biased codon usage of Streptomyces genes and the presence of a nucleotide sequence encoding the N-terminal amino acid sequence obtained from the purified BPO-A1. Comparison of the deduced primary structure of BPO-A1 with those deduced for the non-haem chloroperoxidase CPO-P from Pseudomonas pyrrocinia and the bromoperoxidase BPO-A2 from S. aureofaciens ATCC 10762 gave amino acid sequence identities of 49% and 40%, respectively.