Florian Horaud

Molecular Pathogenesis of Neural Lesions Induced by Poliovirus Type 1

Using in situ hybridization techniques for viral RNA and employing a specific riboprobe, we have detected virus in neural cells of monkeys infected with poliovirus type 1 (PV-1) by the intraspinal route. In monkeys paralysed after inoculation of a neurovirulent revertant of PV-1/Sabin strain, viral RNA was detected in motor neurons and their processes, and in polymorphonuclear and small neural cells. Quantitative in situ hybridization provided evidence of viral replication in individual cells suggesting that the death of motor neurons was due to the direct effect of poliovirus replication in these cells. The histological study of neural lesions of monkeys paralysed after infection with the attenuated Sabin strain of PV-1 revealed two major differences compared to monkeys infected with a virulent strain: (i) the number of destroyed motor neurons was reduced and limited to the site of inoculation and (ii) the inflammatory reaction was localized but more intense. An account is given of the difference in histopathology induced by virulent and attenuated strains of PV-1 in the central nervous system.

Poliovirus permissivity and specific receptor expression on human endothelial cells

To test the role of the endothelial cells (EC) in the poliomyelitis pathogenesis, their sensitivity to poliovirus infection was determined at different times after isolation from the human umbilical vein. While 80% of EC were permissive for poliovirus after 4 days of in vitro primary cultures, only 6% of freshly isolated EC were susceptible to poliovirus infection. A progressive development of this susceptibility was observed during the first 3 days of culture. In an attempt to explain the mechanism of the appearance of cell permissivity for poliovirus, the expression of the poliovirus receptor on EC was studied by cytofluorometric analysis using an anti-receptor monoclonal antibody. The number of poliovirus receptor molecules in the EC population was found to increase with time. This paralleled the increase of the poliovirus-binding capacity of EC cultures. In contrast, the efficiency of viral internalization did not appear to be dependent on the age of culture. These results indicate that the development of EC permissivity for poliovirus in vitro is mainly dependent on the expression of poliovirus receptor on the cell surface.

Analysis of neutralization-escape mutants selected from a mouse virulent type 1/type 2 chimeric poliovirus : identification of a type 1 poliovirus with antigenic site 1 deleted

A chimeric type 1/type 2 poliovirus (v510), in which the antigenic site 1 (Ag1) of poliovirus type 1 (PV-1) Mahoney was replaced by the corresponding site of poliovirus type 2 (PV-2) Lansing, is known to be neurovirulent for mice and neutralized by both type 1 and type 2 monoclonal antibodies. Neutralization-escape mutants to monoclonal antibodies specifically recognizing the PV-2 sequence were obtained from v510. The nucleotide sequence and the mouse neurovirulence of mutants were determined. Amino acid substitutions obtained inside the replaced sequence, at positions 95 and 99, and outside this site, at positions 93 or 104, rendered the virus attenuated for mice. One of the escape mutants harboured a deletion of the entire substituted nonapeptide sequence in v510. This particular virus, which is a PV-1 Mahoney lacking the natural Ag1 loop, does not react with PV-2-specific monoclonal antibodies, has a ts phenotype, is heat-labile and is devoid of neurovirulence for mice.

Molecular pathogenesis of type 2 poliovirus in mice

Lansing strain of poliovirus type 2 (PV-2) produces a fatal paralytic disease in mice after intracerebral inoculation. To identify virus-containing nerve cells in poliovirus-infected mice, we developed a technique of in situ hybridization for viral RNA with a poliovirus-specific riboprobe. Large numbers of genomes were found in motoneurons and their processes, as well as in ghosts of neurons with or without inflammatory cells. This indicates that the death of motoneurons is due to a direct effect of viral replication. The presence of viral RNA in neuronal processes suggests an axonal dissemination of poliovirus within the central nervous system (CNS). Detection of viral genomes in small cells located in anterior and in posterior horns shows that motoneurons are not the only cells susceptible to poliovirus in the CNS. To see if PV-2/Lansing still exits in nature, PV-2 isolated from the CNS and from the gut of two human cases of paralytic poliomyelitis were characterized by determining epitope maps and phenotypic markers and by sequencing regions of the genome coding for antigenic sites and the 5′ non-coding region. Comparison of virus isolated from the intestine and from the CNS of the same subject revealed a high degree of homology, suggesting that virus isolated from the gut is representative of virus in the neuronal lesions. The isolate from a case had more homology to PV-2/Lansing, whereas the other isolate showed more homology to PV-2/Sabin. These results show that, despite intensive vaccination with oral polio vaccine for 25 years, wild PV-2/Lansing-like viruses, pathogenic for mice, still circulate in nature ad induce human paralytic cases.

Comparative expression of the hepatitis B surface antigen gene in biochemically transformed human, Simian and murine cells

In this paper, we show that the pattern of expression of the human hepatitis B surface antigen (HBs Ag) gene, transfected along with a dominant selectable marker into mammalian cells, is complex. In human hepatoma (HepG2) cells, late transient expression occurs and permanent expression takes place at high frequencies in the selected clones. In HeLa and human xeroderma pigmentosum (GM4312A)-derived cells, the late transient expression is barely seen or absent and permanent expression is only seen in a few selected clones. In monkey kidney Vero cells, late transient expression has been described and we show in this report that only 5% of the selected clones are capable of expressing HBs Ag in a permanent manner. In most of the Vero clones, the absence of HBs Ag expression is mainly due to HBs Ag gene rearrangements. We have selected and amplified more than 500 transfected Vero clones and have characterized in detail one clone (GAR1412) which is a permanent high-level HBs Ag expressor.

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.

Viral safety of blood derivatives: an overview

The causes of past accidental viral transmissions associated with blood derivatives are reviewed briefly, as are the safety procedures instituted in Europe on the basis of viral validation studies. A comprehensive five-class classification system for delineating viral risk according to source material and comparative viral safety is presented. Newly emerging viruses are briefly discussed in view of the viral safety of blood products.