Bruno Blondel

Co-circulation and evolution of polioviruses and species C enteroviruses in a district of Madagascar.

Between October 2001 and April 2002, five cases of acute flaccid paralysis (AFP) associated with type 2 vaccine-derived polioviruses (VDPVs) were reported in the southern province of the Republic of Madagascar. To determine viral factors that favor the emergence of these pathogenic VDPVs, we analyzed in detail their genomic and phenotypic characteristics and compared them with co-circulating enteroviruses. These VDPVs appeared to belong to two independent recombinant lineages with sequences from the type 2 strain of the oral poliovaccine (OPV) in the 5′-half of the genome and sequences derived from unidentified species C enteroviruses (HEV-C) in the 3′-half. VDPV strains showed characteristics similar to those of wild neurovirulent viruses including neurovirulence in poliovirus-receptor transgenic mice. We looked for other VDPVs and for circulating enteroviruses in 316 stools collected from healthy children living in the small area where most of the AFP cases occurred. We found vaccine PVs, two VDPVs similar to those found in AFP cases, some echoviruses, and above all, many serotypes of coxsackie A viruses belonging to HEV-C, with substantial genetic diversity. Several coxsackie viruses A17 and A13 carried nucleotide sequences closely related to the 2C and the 3Dpol coding regions of the VDPVs, respectively. There was also evidence of multiple genetic recombination events among the HEV-C resulting in numerous recombinant genotypes. This indicates that co-circulation of HEV-C and OPV strains is associated with evolution by recombination, resulting in unexpectedly extensive viral diversity in small human populations in some tropical regions. This probably contributed to the emergence of recombinant VDPVs. These findings give further insight into viral ecosystems and the evolutionary processes that shape viral biodiversity.

Detection by monoclonal antibodies of an antigenic determinant critical for poliovirus neutralization present on VP1 and on heat-inactivated virions.

Hybridoma cell lines were established against poliovirus type 1 (Mahoney) heat-denatured virions (C particles). Each anti-C monoclonal antibody (McAb) immunoprecipitated specifically one of the individualized poliovirus capsid polypeptides VP1, VP2, or VP3. One of the anti-C McAb (C-3), reacting with VP1, neutralized homologous virus and immunoprecipitated infectious D particles. Its properties have been compared to those of a neutralizing anti-D McAb (D-Ic). In contrast with the C-3 antigenic site, the D-Ic epitope was not present on C particles nor on individualized structural polypeptide. This demonstrates that C-3 and D-Ic epitopes represent two independent antigenic determinants, both critical for poliovirus neutralization.

Early Phosphatidylinositol 3-Kinase/Akt Pathway Activation Limits Poliovirus-Induced JNK-Mediated Cell Death

ABSTRACT Poliovirus (PV)-induced apoptosis seems to play a major role in tissue injury in the central nervous system (CNS). We have previously shown that this process involves PV-induced Bax-dependent mitochondrial dysfunction mediated by early JNK activation in IMR5 neuroblastoma cells. We showed here that PV simultaneously activates the phosphatidylinositol 3-kinase (PI3K)/Akt survival signaling pathway in these cells, limiting the extent of JNK activation and thereby cell death. JNK inhibition is associated with PI3K-dependent negative regulation of the apoptosis signal-regulating kinase 1, which acts upstream from JNK in PV-infected IMR5 cells. In poliomyelitis, this survival pathway may limit the spread of PV-induced damage in the CNS.

Poliovirus induces Bax-dependent cell death mediated by c-Jun NH2-terminal kinase.

ABSTRACT Poliovirus (PV) is the causal agent of paralytic poliomyelitis, a disease that involves the destruction of motor neurons associated with PV replication. In PV-infected mice, motor neurons die through an apoptotic process. However, mechanisms by which PV induces cell death in neuronal cells remain unclear. Here, we demonstrate that PV infection of neuronal IMR5 cells induces cytochrome c release from mitochondria and loss of mitochondrial transmembrane potential, both of which are evidence of mitochondrial outer membrane permeabilization. PV infection also activates Bax, a proapoptotic member of the Bcl-2 family; this activation involves its conformational change and its redistribution from the cytosol to mitochondria. Neutralization of Bax by vMIA protein expression prevents cytochrome c release, consistent with a contribution of PV-induced Bax activation to mitochondrial outer membrane permeabilization. Interestingly, we also found that c-Jun NH2-terminal kinase (JNK) is activated soon after PV infection and that the PV-cell receptor interaction alone is sufficient to induce JNK activation. Moreover, the pharmacological inhibition of JNK by SP600125 inhibits Bax activation and cytochrome c release. This is, to our knowledge, the first demonstration of JNK-mediated Bax-dependent apoptosis in PV-infected cells. Our findings contribute to our understanding of poliomyelitis pathogenesis at the cellular level.

Molecular aspects of poliovirus biology with a special focus on the interactions with nerve cells

Poliovirus (PV), the pathogenic agent of paralytic poliomyelitis, is the prototype of the picornavirus family. Although paralytic poliomyelitis has been nearly totally eradicated in most industrialized countries, PV continues to be an important public health problem in many developing countries. Moreover, in industrialized countries, two current concerns are the occurrence, albeit at a very low frequency, of vaccine-associated paralytic poliomyelitis, due to the genetic instability of the attenuated oral PV strains in vaccines, and the emergence of a neuro-muscular pathology in many survivors of the acute disease, called the post-polio syndrome. PV has been targeted by the World Health Organization for world-wide eradication in the coming decade and continues to be the subject of intensive research. The advances made in the molecular biology of PV, taken together with the development of new animal and cell models, have permitted a new look at a key step in the pathogenesis of poliomyelitis, i.e. the interactions between PV and nerve cells. These aspects of PV biology are developed in this review according to three themes: (i) the PV host range; (ii) the molecular determinants of PV neurovirulence and attenuation; and (iii) the persistence of PV in nerve cells, which has proven to be an interesting new domain in the field of PV research.

Poliovirus-Induced Apoptosis Is Reduced in Cells Expressing a Mutant CD155 Selected during Persistent Poliovirus Infection in Neuroblastoma Cells

ABSTRACT Poliovirus (PV) can establish persistent infections in human neuroblastoma IMR-32 cells. We previously showed that during persistent infection, specific mutations were selected in the first extracellular domain of the PV receptor (CD155) of these cells (N. Pavio, T. Couderc, S. Girard, J. Y. Sgro, B. Blondel, and F. Colbère-Garapin, Virology 274:331-342, 2000). These mutations included the Ala 67 → Thr substitution, corresponding to a previously described allelic form of the PV receptor. The mutated CD155Thr67 and the nonmutated IMR-32 CD155 (CD155IMR) were expressed independently in murine LM cells lacking the CD155 gene. Following infection of the cells with PV, we analyzed the death of cells expressing these two forms of CD155. Levels of DNA fragmentation, caspase activity, and cytochrome c release were lower in LM-CD155Thr67 cells than in LM-CD155IMR cells. Thus, the level of apoptosis was lower in cells expressing mutated CD155 selected during persistent PV infection in IMR-32 than in cells expressing the wild-type receptor.

Silencing viruses by RNA interference

Abstract Post-transcriptional gene silencing (PTGS) makes possible new approaches for studying the various steps of the viral cycle. Plus-strand RNA viruses appear to be attractive targets for small interfering RNAs (siRNAs), as their genome functions as both mRNA and replication template. PTGS creates an alternative to classic reverse genetics for viruses with either negative-strand or double-stranded RNA genomes and for those with a large genome. PTGS allows modification of the expression of a given cellular gene as a means to elucidate its role in the viral cycle and in virus–host cell interactions, and to investigate cellular pathways involved in viral pathogenesis. It also allows the creation of new animal models of human diseases. In addition, PTGS already appears to be a promising new therapeutic tool to fight viral multiplication and dissemination through the host and to prevent inflammation and virus-induced pathogenesis, including virus-induced tumorigenesis.

Bax Is Activated during Rotavirus-Induced Apoptosis through the Mitochondrial Pathway

ABSTRACT Rotaviruses are the leading cause of infantile viral gastroenteritis worldwide. Mature enterocytes of the small intestine infected by rotavirus undergo apoptosis, and their replacement by less differentiated dividing cells probably leads to defective absorptive function of the intestinal epithelium, which, in turn, contributes to osmotic diarrhea and rotavirus pathogenesis. Here we show that infection of MA104 cells by the simian rhesus rotavirus strain RRV induced caspase-3 activation, DNA fragmentation, and cleavage of poly(ADP-ribose) polymerase; all three phenomena are features of apoptosis. RRV induced the release of cytochrome c from mitochondria to the cytosol, indicating that the mitochondrial apoptotic pathway was activated. RRV infection of MA104 cells activated Bax, a proapoptotic member of the Bcl-2 family, as revealed by its conformational change. Most importantly, Bax-specific small interfering RNAs partially inhibited cytochrome c release in RRV-infected cells. Thus, mitochondrial dysfunction induced by rotavirus is Bax dependent. Apoptosis presumably leads to impaired intestinal functions, so our findings contribute to improving our understanding of rotavirus pathogenesis at the cellular level.

Calcium Flux between the Endoplasmic Reticulum and Mitochondrion Contributes to Poliovirus-Induced Apoptosis

ABSTRACT We show that poliovirus (PV) infection induces an increase in cytosolic calcium (Ca2+) concentration in neuroblastoma IMR5 cells, at least partly through Ca2+ release from the endoplasmic reticulum lumen via the inositol 1,4,5-triphosphate receptor (IP3R) and ryanodine receptor (RyR) channels. This leads to Ca2+ accumulation in mitochondria through the mitochondrial Ca2+ uniporter and the voltage-dependent anion channel (VDAC). This increase in mitochondrial Ca2+ concentration in PV-infected cells leads to mitochondrial dysfunction and apoptosis.

An ex vivo murine model to study poliovirus-induced apoptosis in nerve cells.

Paralytic poliomyelitis results from destruction of motor neurons owing to poliovirus (PV) replication. Using a mouse model, we have previously shown that PV kills neurons of the central nervous system (CNS) as a result of apoptosis (Girard et al., Journal of Virology 73, 6066-6072, 1999). We report the development of mixed mouse primary nerve cell cultures from the cerebral cortex of neonatal mice transgenic for the human PV receptor. These cultures contained all three main cell types of the CNS, i.e. neurons, astrocytes and oligodendrocytes. All three cell types were susceptible to PV infection and virus replication in the cultures led to DNA fragmentation characteristic of apoptosis. PV-induced apoptosis was inhibited by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD.FMK), indicating that this process involved caspases. Thus, these mixed mouse primary nerve cell cultures are a new in vitro model for studying the molecular mechanisms of PV-induced apoptosis in nerve cells.