Enhancement of antigen-specific interleukin 2 production by adding liposomes to rabies antigens for priming.

Antigen-specific IL-2 production was assessed, using splenocytes from rabies immune mice incubated for 24 h with rabies virus antigen. The antigenic material used for in vivo priming was either purified glycoprotein from rabies virus, or the inactivated virus. The time between priming, harvesting and restimulation of the splenocytes was 7 days. It was found that when antigenically inert liposomes were injected, together with antigenic material, to the prospective splenocyte donor mice, IL-2 production was enhanced. This augmentation was observed particularly when priming was performed with the inactivated rabies virus.

Interleukin 2 increases protection against experimental rabies.

Vaccination with either whole inactivated rabies virus or immunosome (rabies glycoprotein anchored on liposomes) induces a high level of interleukin 2 (IL 2) production after in vitro specific stimulation of splenocytes from primed mice (9). On the contrary, infection with a live rabies virus does not specifically induce the production of IL 2: splenocytes from ill mice previously infected with wild rabies virus cannot be specifically stimulated by rabies antigens, whereas they can be non-specifically stimulated by a mitogen (Concanavalin A (Con A]. When injected in mice, exogenous IL 2 (purified rat IL 2 or human recombinant IL 2) exhibits an adjuvant effect on rabies virus vaccine or subunit vaccine tested in a pre-exposure potency test (NIH test). When injected in hamsters, according to a post-exposure potency test (infection with a wild rabies virus followed by vaccination), IL 2 has no adjuvant effect on the rabies vaccine. Nevertheless, when injected alone, IL 2 protects thirty to fifty percent of the infected animals treated (1 hour, 3 and 7 days post-infection) with 10 international units of human recombinant IL 2.

Short report: isolation and partial characterization of a Puumala virus from a human case of nephropathia epidemica in France.

Puumala (PUU) virus (Bunyaviridae: Hantavirus), the etiologic agent of nephropathia epidemica (NE), the mid form of hemorrhagic fever with renal syndrome, is enzootic in Europe and has been known to occur in France since 1983. We report the first isolation of PUU virus in France and western Europe from a case of NE acquired in France. The virus was isolated from a serum collected in the acute phase of the clinical course by successive blind passages in Vero E6 cells. Serologic typing using monoclonal antibodies confirmed the identity of the virus as PUU. The sequence of an 832-nucleotide fragment of the virus medium RNA segment obtained by the polymerase chain reaction (PCR) also classified it as a PUU virus. The sequence of this isolate from a human case in France is closely related to the sequence of a PUU virus obtained by the PCR from a German patient.

From rabies to rabies-related viruses.

Antigenic differences between rabies virus strains characterized with monoclonal antibodies presently define at least four serotypes within the Lyssavirus genus of the Rhabdoviridae family: classical rabies virus strains (serotype 1), Lagos bat virus (serotype 2), Mokola virus (serotype 3) and Duvenhage virus (serotype 4). The wide distribution of rabies-related virus strains (serotypes 2, 3 and 4) and above all, the weak protection conferred by rabies vaccines against some of them (principally Mokola virus) necessitates the development of new specific vaccines. We first determined the complete nucleotide sequence of a rabies virus strain of serotype 1 (Pasteur virus) and characterized the structure of the viral genes and their regulatory sequences. We then extended this study to the Mokola virus genome. Five non-overlapping open reading frames were found in both viruses and had similar sizes and positions in both. Similarities were also found in the mRNA start and stop sequences and at the genomic extremities. Comparison of both genomes helps to analyze the basis of the particular antigenicity of these two serotypes. The sequence homology in the region coding for the viral glycoprotein was only 58% between the two viruses, compared with 94% between different rabies virus strains within serotype 1. This comparison, extended to other unsegmented negative strand RNA viruses, gives new insight into the understanding of rhabdoviruses and paramyxoviruses. Furthermore, molecular cloning provides a rationale for the genetic engineering of a future vaccine.

Recent data on the epidemiology and prophylaxis of human rabies in France.

Rabies is an animal disease which is transmitted to man only by accident, most often through the bite (more rarely after scratches or licks of mucosa) of a rabid animal, domestic or wild. A good knowledge of the epizootiology of animal rabies is therefore necessary to establish, on solid grounds, the prophylaxis of human rabies. Inter-human transmission of rabies being an exceptional event which will be considered separately, the epidemiology of human rabies mainly studies the sources and circumstances of human exposure to rabid animals, which differ according to the epizootiology of animal rabies in a given country: either enzootic (or hyperenzootic) canine rabies, or enzootic selvatic rabies. It appears that the risk of human rabies is higher in the first situation for two reasons: (i) rabies viruses show an increased virulence due to numerous serial passages in dogs (viruses with short incubation period) and (ii) high frequency of dog-man contacts due to the high density of both populations.

[Antigenic variants among street rabies virus isolates from France, Africa, Madagascar and Asia. Preliminary results with antinucleocapsid monoclonal antibodies]. / Variantes antigéniques du virus rabique: souches des rues de France, d'Afrique, de Madagascar et d'Asie. Résultats preliminaires obtenus avec des anticorps monoclonaux antinucléocapside.

A panel of monoclonal antibodies directed against the nucleocapsid antigens of rabies virus allowed a rapid characterization of isolates by an indirect immunofluorescence test on brain smears of infected animals. Analysis of 77 isolates (71 from animals and 6 from humans) showed a great diversity. Isolates from different areas showed different patterns of reactivity. No relation was found between animal species and antigenic patterns specificity.

[Correlations between immunoenzymatic test, neutralization test and fluorescent focus reduction test in rabies antibody titration]. / Corrélations entre l'epreuve immunoenzymatique, la séroneutralisation et la réduction de foyers fluorescents pour le titrage des anticorps rabiques.

Three methods have been used and compared for the detection and titration of antibodies in the serum of humans receiving post-exposure treatment with tissue culture inactivated rabies vaccines: mouse seroneutralization test, fluorescent focus reduction test and immunoenzymatic test. Results have shown that the fluorescent focus reduction test has a sensitivity and specificity which are equivalent to the mouse seroneutralization test with the advantage of giving results in 30 hr instead of 21 days, and that the immunoenzymatic test which avoids the use of laboratory animals and/or cell cultures, and which gives results in 5 hr, may advantageously replace the neutralization methods, particularly for the testing of large numbers of sera.

New vaccines for immunization of man: new approaches towards the prevention of rabies in man.

Cell culture rabies vaccines for human use, highly immunogenic and well tolerated, are now used for pre-exposure immunization as well as for post-exposure treatment. Presently available cell culture rabies vaccines induce immunity against the SAD modified live rabies virus used for oral immunization of foxes. They also induce immunity against the newly identified European bat rabies virus (Duvenhage).

[Ecology of indigenious arbovirus in Alsace. Tick Central European Encephalitis. I.--Complex Ixodes ricinus--bank voles. II.--Study of bank voles population immunity. III.--Virologic results in bank voles population (author's transl)]. / Ecologie d'un arbovirus indigène en Alsace. L'encéphalite à tiques. I.--Complexe Ixodes ricinus--campagnol roussâtre. II.--Etude de l'immunité d'une population de campagnols roussâtres. III.--Résultats virologiques observés dans une population de campagnols roussâtres.

I.--After showing that bank voles are parasited only by Ixodes ricinus larvae, the authors attempt to found different factors (demographic, biometric, and sexual) who favor individual parasitism. The authors conclude to absent of anti tick immunity for this rodent specie. II.--The search for anti-central european encephalitis antibodies (I.H.A.) are shown that 2 p. cent animals were immuns. Yearly and monthly chronologies of antibodies apparition are shown, factors favoring the growth of specific Central european encephalitis antibodies are discussed. III.--The Central european encephalitis tick viral infection of bank vole is studied according to the number of viral strains isolated from different viscera. The monthly chronology of this infection is shown.

Treatment of rabies in mice and foxes with antiviral compounds.

Thirty four chemical compounds were injected into rabies infected mice by intramuscular (i.m.) route. Twenty four compounds such as well known therapeutic agents: amantadine, lipacids, phenol compounds, didemnin-B, procaine, nucleosides analogues (ribavirin, tiazofurin, pyrazofurin) had no effect. Two compounds had a slight effect not justifying to consider them as possible therapeutic agents: selenazofurin and an analogue of ribavirin (RTA). Eight heteropolyanions (HPA), which have a related chemical structure, were efficient providing 100% protection. Nineteen compounds were injected into rabies infected mice by the intracerebral (i.c.) route. Fourteen compounds such as ribavirin, RTA, selenazofurin, tiazofurin and 9 HPA compounds had no effect. Five other HPA compounds (HPA 23-39-46-51-56) were efficient preventing the development of clinical infection in some mice. Whatever was the treatment route, treated surviving mice developed rabies neutralizing antibodies. No proof of viral multiplication was found in their brains. As some HPA compounds did produce a therapeutic effect in mice, two of them HPA 23 and HPA 39 were administered to rabies-infected foxes. In foxes the compounds prolonged the mean survival time and increased the number of survivors. These data suggest that chemotherapy might be worthwhile when vaccination was impossible.

Lack of interferon induction in man by two rabies tissue culture vaccines.

Both neutralising antibody and interferon play a part in protection of animals against death from rabies virus infection. Interferon induction was therefore sought in 53 volunteers within 24 hours of receiving human diploid cell strain vaccine or fetal bovine kidney cell vaccine given either intramuscularly or intradermally. Repeat observations were made in 18 subjects following a second dose of vaccine seven days later. No interferon was detected in any sample tested although no subject had any detectable rabies neutralising antibody on day 0. The sensitivity of the interferon assay, and comparison with other studies are discussed. An interferon inducer suitable for human use should be sought as an alternative to, or a replacement for, passive rabies immunization.

[The efficacy of the ERA rabies vaccine, in mice against 4 antigenic variants of rabies virus]. / Eficácia da vacina anti-rábica ERA em camundongos, frente a quatro diferentes variantes antigênicas do vírus da raiva.

ERA anti-rabies vaccine prepared in kidney tissue culture was evaluated against four different antigenic strains of rabies virus in mice: two of them dog strains, C/SP and C/NG, another a bat vampire strain, DR-19, and the CVS strain. The CVS antigenic characteristics were determined by means the antinucleocapsid monoclonal antibodies technique. Twenty one days old mice were vaccinated, intramuscularly, in the inner side of the thigh, with 0.05 ml of vaccine and challenged at 42 days old, together with those of the control group, intramuscularly, in the inner side of the thigh, with 0.05 ml of the corresponding viral strain dilution. The ERA anti-rabies vaccine protected 100% of all the mice challenged with C/SP, C/NG and DR-19 strains and 83% of those challenged with CVS. The control groups mortality rate varied between 70 and 90%.

[Rabies encephalomyelitis with myocarditis and pancreatitis. Report on a case recently imported into France]. / Encéphalomyélite rabique avec myocardite et pancréatite. Observation d'un cas récent importé en France.

We report the case of a 29 year-old man who died from rabies in France, following a dog-bite during a trip in Mexico. Although it was clinically suspected, the diagnosis was uncertain until he died because of digestive, cardiac and psychiatric misleading symptoms associated to the neurologic disorders. Post mortem diagnosis was based upon virological study in immunofluorescence on cerebral smears, viral isolation on cell-culture, and ELISA. It was confirmed by light microscopy examination which showed numerous Negri bodies, and ultrastructural study of the rhabdovirus in the central nervous system. Extranervous lesions, especially myocarditis and pancreatitis, were observed and their meaning is discussed. The physician is exceptionally confronted to the diagnosis of human rabies in France. Nevertheless, the lack of compulsory antirabic vaccination and the increase of touring in enzootic countries increase the risk of infection. As an intra vitam diagnosis in frequently lacking, the diagnosis of rabies infection needs a complete post mortem virological study as well as an histological and ultrastructural examination of the central nervous system.

[A simplified method of vaccination against rabies after exposure]. / Schéma simplifié pour la vaccination antirabique après exposition.

Vaccination against rabies after exposure to the risk of contamination is currently performed according to the WHO recommendations: 6 injections in 6 visits. We have studied the serological effectiveness of a simplified method using the human diploid-cell rabies vaccine: the vaccine is injected subcutaneously at the rate of 2 doses on day 0, then one dose on day 7 and one dose on day 21. Fifty subjects were vaccinated, and antiglycoprotein antibodies were assayed in serum by the immunoenzymatic technique (EU/ml). Seroconversion with titres above 0.5 EU/ml was observed in all subjects. The antibody titres were 0.127 +/- 1.57, 11.31 +/- 8.87, 10.2 +/- 7.55, and the numbers of subjects with protective titres were 0/50, 11/50, 46/47 and 36/37 on days 0, 7, 21 and 90 respectively. No undesirable side-effect was recorded. The good results obtained with this 2+1+1 vaccination schedule (4 injections in 3 visits) suggest that the recommended method of post-exposure vaccination could be replaced by this simplified method.

[Post-exposure antirabies vaccination. Early serological response to vaccine cultivated on VERO cells using a reduced 2-1-1 schedule]. / Vaccination antirabique après exposition. Réponse sérologique précoce au vaccin cultivé sur cellules VERO utilisé en schéma réduit 2-1-1.

OBJECTIVES: An abbreviated 2-1-1 schedule for post-exposure rabies vaccination would theoretically lead to more rapid production of specific antibodies than the classical schedule. We measured early serological response to the 2-1-1 schedule. METHODS: Patients consulting the antirabies centre of the Epinal hospital from June 1992 to June 1993 who had never been vaccinated and whose exposure history justified antirabies vaccination were included in this study. Fifty subjects were vaccinated with PVRV (purified vero rabies vaccine, Pasteur Institute) cultured on VERO (vervet monkey origin) cells using the abbreviated 2-1-1 schedule of 2 doses (0.5 ml = 2.5 IU/dose) on day 0 and 1 dose on days 7 and 21. Antirabies antibodies were assayed using the Platelia Rage immunoenzyme method (Diagnostic Pasteur) on day 21. Titres above 0.5 IU were considered to give protection and non-protected subjects were seen again on day 28 for a supplementary dose. RESULTS: Only 34 subjects (68%) had protective antibody titres on day 21, but by day 28, 48 (96%) had acquired immunity. In this study population, the age range was from 1 to 83 years and age over 30 years appeared to delay antibody formation. CONCLUSION: These findings emphasize the importance of initial antirabies immunoglobulins if short incubation in suspected and the need for serological follow-up if delayed antibody formation is suspected (subjects over 30).

[Hemorrhagic fever with renal syndrome due to Hantaan virus or a serologically related virus]. / Fièvre hémorragique avec syndrome rénal due au virus Hantaan ou à un virus sérologiquement apparenté.

Seven cases of acute renal failure consecutive to haemorrhagic fever with renal syndrome (HFRS) due to the Hantaan virus or to a serologically related virus are reported. These cases were observed in north-eastern France between March, 1983 and January, 1984. All patients were of rural origin and had been in contact with field mice. The predominant initial clinical symptoms were signs of infection and diffuse muscle pain, without evidence of haemorrhage. However, massive proteinuria was noted, and acute anuric renal failure unaccompanied by oedema or arterial hypertension developed. Renal biopsy performed in 2 patients showed tubular and interstitial nephritis but no glomerular or vascular lesions. Two patients only required haemodialysis. All patients recovered within 2 to 8 weeks without sequelae. Antibodies directed against the Hantaan virus were detected by indirect immunofluorescence tests, and seroconversion could be demonstrated in 2 patients seen at a sufficiently early stage. The risk of epidemics suggested by this small outbreak of HFRS can only be evaluated after an exhaustive epidemiological study.

[Arboviruses in Iran (author's transl)]. / Arbovirus en Iran.

During an entomological survey conducted in april-june 1978 in the province of Khorassan in the eastern part of Iran, ixodid and argasid ticks were collected for arbovirus research. Five serotypes of arboviruses were obtained: Quaranfil, Thogoto, Wad Medani, Wanowrie and Crimean Haemorrhagic Fever-Congo. The isolation of all these arboviruses is reported for the first time in Iran.