Live rubella virus vaccine long-term persistence as an antigenic trigger of cutaneous granulomas in patients with primary immunodeficiency.

Granulomas may develop as a response to a local antigenic trigger, leading to the activation of macrophages and T-lymphocytes. Primary immunodeficiency (PID) is associated with the development of extensive cutaneous granulomas, whose aetiology remains unknown. We performed high-throughput sequencing of the transcriptome of cutaneous granuloma lesions on two consecutive index cases, and RT-PCR in a third consecutive patient. The RA27/3 vaccine strain of rubella virus-the core component of a universally used paediatric vaccine-was present in the cutaneous granuloma of these three consecutive PID patients. Controls included the healthy skin of two patients, non-granulomatous cutaneous lesions of patients with immunodeficiency, and skin biopsy samples of healthy individuals, and were negative. Expression of viral antigens was confirmed by immunofluorescence. Persistence of the rubella vaccine virus was also demonstrated in granuloma lesions sampled 4-5 years earlier. The persistence of the rubella virus vaccine strain in all three consecutive cutaneous granuloma patients with PID strongly suggests a causal relationship between rubella virus and granuloma in this setting.

[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.

Poliovirus, pathogenesis of poliomyelitis, and apoptosis.

Poliovirus (PV) is the causal agent of paralytic poliomyelitis, an acute disease of the central nervous system (CNS) resulting in flaccid paralysis. 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 PV-induced apoptosis is an important component of the tissue injury in the CNS of infected mice, which leads to paralysis. In this review the molecular biology of PV and the pathogenesis of poliomyelitis are briefly described, and then several models of PV-induced apoptosis are considered; the role of the cellular receptor of PV, CD155, in the modulation of apoptosis is also addressed.

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.

Expression of mutated poliovirus receptors in human neuroblastoma cells persistently infected with poliovirus.

Poliovirus (PV) is able to establish persistent infections in human neuroblastoma IMR-32 cells [Colbère-Garapin et al. (1989) Proc. Natl. Acad. Sci. USA 86, 7590]. During persistent infection, PV mutants are selected that display substitutions of residues in regions of the capsid known to interact with the PV receptor (PVR), a glycoprotein of the immunoglobulin superfamily. The mechanism of persistent infection in IMR-32 cells may therefore involve the selection of mutant PVRs. To test this hypothesis, the sequences of the PVR mRNAs in uninfected IMR-32 cells and in two independent IMR-32 cell cultures persistently infected with the Mahoney strain of PV type 1 (PV1/Mahoney) were determined. The PVR mRNA population of uninfected cells was homogeneous, and no mutation was repeatedly found, whereas that of persistently infected cells displayed missense mutations. Particular mutations were repeatedly detected, and all of them mapped to the N-terminal domain of PVR (domain 1), which interacts directly with PV. These mutations generated several types of PVR variants with the following substitutions: Ala67-->Thr alone, Ala67-->Thr associated with Gly39-->Ser, and Arg104-->Gln. Functional analysis of PVR in murine LM cells, stably expressing each of the PVR forms, showed that the PVR forms selected during persistent infection conferred on LM cells partial resistance to PV1/Mahoney-induced lysis. Although adsorption onto PVR seemed to be independent of the PVR form, an analysis of the conformational changes of the capsid during the early steps of the PV cycle provided evidence that the Ser39/Thr67 and Gln104 substitutions almost halved the conversion of 160S infectious particles into 135S A particles associated with the PV-PVR interaction. Altogether, these findings indicate that during persistent infection, specific mutations were selected in the domain 1 of PVR and that these mutations increased the resistance of cells to PV-induced lysis. These results are discussed in view of the position of the mutations on PVR.

Poliovirus induces apoptosis in the mouse central nervous system.

Poliovirus (PV) is the etiological agent of human paralytic poliomyelitis. Paralysis results from the destruction of motoneurons, a consequence of PV replication. However, the PV-induced process leading to the death of motoneurons is not well known. We investigated whether PV-induced central nervous system (CNS) injury is associated with apoptosis by using mice as animal models. Transgenic mice expressing the human PV receptor were infected intracerebrally with either the neurovirulent PV-1 Mahoney strain or a paralytogenic dose of the attenuated PV-1 Sabin strain. Nontransgenic mice were infected with a mouse-adapted PV-1 Mahoney mutant. DNA fragmentation was demonstrated in CNS tissue from paralyzed mice by visualization of DNA oligonucleosomal laddering and by enzyme-linked immunosorbent assay. Viral antigens and DNA fragmentation detected by the in situ terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling technique were colocalized in neurons of spinal cords from paralyzed mice. In addition, morphological changes characteristic of cells undergoing apoptosis were observed in motoneurons by electron microscopy. Thus, we show that PV multiplication and CNS injury during paralytic poliomyelitis are associated with apoptosis.

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.

Persistent poliovirus infection in mouse motoneurons.

Poliovirus (PV) is the causal agent of paralytic poliomyelitis. Many survivors of the acute disease, after decades of clinical stability, develop new muscular symptoms called postpolio syndrome. It has been hypothesized that the persistence of PV in the spinal cord is involved in the etiology of this syndrome. To investigate the ability of PV to persist in the spinal cord after the onset of paralysis, we exploited a mouse model in which most animals inoculated with a mouse-adapted mutant survived after the onset of paralysis. Light microscopy and ultrastructural immunohistochemical studies and reverse transcription followed by nested PCR performed on spinal cord from paralyzed mice demonstrated that PV persisted in the mouse spinal cord for at least 12 months after the onset of paralysis. This mouse model provides a new tool for studying poliomyelitis evolution after the onset of paralysis.

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.

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.

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.

Molecular characterization of mouse-virulent poliovirus type 1 Mahoney mutants: involvement of residues of polypeptides VP1 and VP2 located on the inner surface of the capsid protein shell.

Most poliovirus (PV) strains, including PV PV-1/Mahoney, are unable to cause paralysis in mice. Determinants for restriction of PV-1/Mahoney in mice have been identified by manipulating PV-1 cDNA and located on the viral capsid protein VP1. These determinants consist of a highly exposed amino acid sequence on the capsid surface corresponding to the B-C loop (M. Murray, J. Bradley, X. Yang, E. Wimmer, E. Moss, and V. Racaniello, Science 241:213-215, 1988; A. Martin, C. Wychowski, T. Couderc, R. Crainic, J. Hogle, and M. Girard, EMBO J. 7:2839-2847, 1988) and of residues belonging to the N-terminal sequence located on the inner surface of the protein shell (E. Moss and V. Racaniello, EMBO J. 10:1067-1074, 1991). Using an in vivo approach, we isolated two mouse-neurovirulent PV-1 mutants in the mouse central nervous system after a single passage of PV-1/Mahoney inoculated by the intracerebral route. Both mutants were subjected to two additional passages in mice, plaque purified, and subsequently characterized. The two cloned mutants, Mah-NK13 and Mah-NL32, retained phenotypic characteristics of the parental PV-1/Mahoney, including epitope map, heat lability, and temperature sensitivity. Mah-NK13 exhibited slightly smaller plaques than did the parental virus. The nucleotide sequences of the mutant genomes were determined, and mutations were identified. Mutations were independently introduced into the parental PV-1/Mahoney genome by single-site mutagenesis. Mutated PV-1/Mahoney viruses were then tested for their neurovirulence in mice. A single amino acid substitution in the capsid proteins VP1 (Thr-22-->Ile) and VP2 (Ser-31-->Thr) identified in the Mah-NK13 and Mah-NL32 genomes, respectively, conferred the mouse-virulent phenotype to the mouse-avirulent PV-1/Mahoney. Ile-22 in VP1 was responsible for the small-plaque phenotype of Mah-NK13. Both mutations arose during the first passage in the mouse central nervous system. We thus identified a new mouse adaptation determinant on capsid protein VP1, and we showed that at least one other capsid protein, VP2, could also express a mouse adaptation determinant. Both determinants are located in the inside of the three-dimensional structure of the viral capsid. They may be involved in the early steps of mouse nerve cell infection subsequent to receptor attachment.

[Palliative treatment by endoprosthesis of icterus caused by distal biliary tumoral obstruction]. / Traitement palliatif par endoprothèse des ictères par obstacle tumoral biliaire sous-hilaire.

Between March 1982 and December 1987, 466 patients (256 women, 210 men, mean age 73 years) with tumor obstruction of the common bile duct were referred to our department. The causes of obstruction were carcinoma of the pancreas (298 patients), carcinoma of the ampulla of Vater (32 patients) and carcinoma of the common bile duct (136 patients). Endoscopical insertion of a biliary prosthesis was initially possible in 377 patients (81%). In case of failure, patients were referred to the radiologist for percutaneous drainage. Successful drainage was obtained in 58 patients with an overall success rate of 93% (435 patients). Endoscopic replacement was necessary in 170 cases for 114 patients and was successful in 155 (91%). Pruritus was relieved in 89% of the patients. Serum bilirubin levels decreased more than 75% after initial endoscopic endoprosthesis, repeated endoscopic endoprosthesis and percutaneous prosthesis insertion in 80%, 79%, and 62% of the patients, respectively. Short term complications of endoscopic endoprosthesis occurred in 28% of patients with a mortality rate of 8%. Percutaneous prosthesis complications occurred in 33% of patients with a mortality rate of 11%. In the long term, cholangitis was the main complication and occurred in 27% of patients with a delay of 103 +/- 105 days. Intestinal obstruction was observed in 7% of patients. The average life expectancy of endoscopic endoprosthesis and percutaneous prosthesis was 109 +/- 157 and 92 +/- 101 days, respectively. The average life expectancy of patients was 163 +/- 224 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Persistent poliovirus infection: establishment and maintenance involve distinct mechanisms.

Mutants of poliovirus (PV) with highly modified biological properties can be selected in vitro in cells of neural origin. Mutations accumulate in the genome of type 1 PV strains selected in human neuroblastoma cells, modifying cell specificity and conferring to the virus the ability to persist in such nonneural cells as HEp-2c (Pelletier et al., Virology 180, 729 1991). With this cell system, we have both parent lytic strains and persistent PV mutants; these were used to study the mechanisms of the establishment and maintenance of the persistent infection. We found that a persistent infection was established when the lytic potential of the virus was reduced; this involved both an early and a late event of the virus cycle for the type 1 mutants. In contrast, maintenance of the infection did not correlate with the reduced lytic potential of the viruses, but rather with the selection of mutant cell populations of various phenotypes. Two cell lines, representative of two phenotypes, were studied in greater detail. In the first one, HEp-S32 (cl7), the PV receptor was not detected by cytofluorometry and viral genomes were detected by in situ hybridization in 2% of the cells. In the second cell line, HEp-S31 (cl18), 97% of the cells expressed the PV receptor, viral genomes were detected in 9-10% of the cells, and viral antigens in 5-10% of the cells. With this cell line, the cure of the culture or, alternatively, the lysis of the majority of cells, could be induced under specific culture conditions. We propose a model involving an equilibrium between an abortive and a lytic infection to explain the properties of cells persistently infected with PV.

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.

Use of type 1/type 2 chimeric polioviruses to study determinants of poliovirus type 1 neurovirulence in a mouse model.

We previously described the characteristics of a type 1/type 2 (PV-1/PV-2) chimeric poliovirus, v510, which contains the six amino acids specific for PV-2 in the B-C loop of VP1. This virus was found to be mouse-adapted, as PV-2 and in contrast with PV-1. Determinants of host range were studied in detail and are reported here. PV-1/PV-2 chimeras containing partial PV-1----PV-2 substitutions in the B-C loop of VP1 were obtained by making use of a mutagenesis cartridge on PV-1 cDNA. Analysis of mouse neurovirulence of these chimeras, when correlated with the three-dimensional structure of the v510 capsid, revealed that PV-2 residues important for mouse tropism are those which determine the particular conformation of the B-C loop of VP1 in v510. The mutation of the adenine residue at position 480 of the 5' noncoding region into a guanine residue has been shown to be an important determinant of PV-1 attenuation in monkeys. We show that introduction of this mutation in the v510 genome results in a virus which is partially attenuated for mice. This suggests that analysis of genomic determinants important for PV-1 neurovirulence could be carried out in a mouse model by making use of a mouse-adapted PV-1/PV-2 chimera.

Characterization of persistent poliovirus mutants selected in human neuroblastoma cells.

Six Sabin-derived persistent poliovirus mutants were selected in human neuroblastoma IMR-32 cells. The mutants had a titer 30 to 10(5) times lower in nonneural HEp-2c cells than in IMR-32 cells. When the growth cycles of persistent viruses in the two cell lines were compared, the most striking feature was a delay of 2 to 4 hr in virus release from HEp-2c cells. In Hep-2c cells, type 1 mutants could spontaneously establish a persistent infection in the absence of any exogenous viral inhibitor. Mutations at a rate of 1 every 210 nucleotides had accumulated in the genome of the type 1 mutants selected in neuroblastoma cells, modifying cell specificity and conferring the ability to persist in some nonneural cells. These results indicate that mutants of poliovirus with highly modified biological properties can be selected in vitro in cells of neural origin.