Paul-Pierre Pastoret

An approach to the control of disease transmission in pig-to-human xenotransplantation

Abstract: Although several major immunologic hurdles need to be overcome, the pig is currently considered the most likely source animal of cells, tissues and organs for transplantation into humans. Concerns have been raised with regard to the potential for the transfer of infectious agents with the transplanted organ to the human recipient. This risk is perceived to be increased as it is likely that the patient will be iatrogenically immunocompromised and the organ‐source pig may be genetically engineered in such a way to render its organs particularly susceptible to infection with human viruses. Furthermore, the risk may not be restricted to the recipient, but may have consequences for the health of others in the community. The identification of porcine endogenous retroviruses and of hitherto unknown viruses have given rise to the most concern. We document here the agents we believe should be excluded from the organ‐source pigs. We discuss the likelihood of achieving this aim and outline the potential means by which it may best be achieved.

Virulence, immunogenicity and reactivation of bovine herpesvirus 1 mutants with a deletion in the gC, gG, gI, gE, or in both the gI and gE gene

Within the framework of developing a marker vaccine against bovine herpesvirus 1 (BHV1), several mutants with deletions in non-essential glycoprotein genes were constructed. Glycoprotein gC, gG, gI and gE single deletion mutants, a gI/gE double deletion mutant and a gE frame-shift mutant were made. The virulence and immunogenicity of these mutants were evaluated in specific-pathogen-free calves. Except for the gC deletion mutant, all mutants were significantly less virulent than the parental wild-type (wt) BHV1 strain Lam. The virulence of the gI and the gI-/gE- mutants was almost completely reduced. Upon challenge infection, the calves of the control group became severely ill, whereas all other calves remained healthy. The reduction of the virus shedding after challenge infection was related to the virulence of the strain of primary inoculation. Virus shedding was almost completely reduced in calves first inoculated with Lam-wt or with gC- and the least reduced in calves inoculated with gI- or gI-/gE-. Six weeks after challenge, all calves were treated with dexamethasone to study whether mutant or challenge virus or both could be reactivated. The gC- and the gG- mutants were reactivated, whereas none of the other mutants were reisolated. Reactivation of challenge virus was reduced in all calves inoculated with mutant viruses. The gC deletion mutant was too virulent and the gI and the gI/gE deletion mutants were the least immunogenic, but based on residual virulence and immunogenicity, both the gG and the gE deletion mutants are candidates for incorporation in live BHV1 vaccines. However, it also depends on the kinetics of the anti-gG and anti-gE antibody response after wild-type virus infection, whether these deletion mutants are really suitable to be incorporated in a marker vaccine.

Spatial and temporal dynamics of Puumala hantavirus infection in red bank vole (Clethrionomys glareolus) populations in Belgium.

Dynamics of hantavirus infection and population densities in rodents were investigated from 1996 to 1999 in southern Belgium. Evidence of Puumala infection was restricted to Clethrionomys glareolus. Although the serotype was not determined, antibodies against hantavirus were also found in eight Apodemus sylvaticus. In fall 1996, the seroprevalence in C. glareolus was high (20.1%, 37 of 184) and the infection was widely distributed in the area studied whereas a focal occurrence of positive rodents and lower seroprevalence rates were recorded in spring 1997 (14.3%, six of 42), fall 1997 (6. 6%, 11 of 166), spring 1998 (6.4%, three of 47) and fall 1998 (6.7%, 11 of 165). A pullulation of rodents was observed in spring 1999 and was associated with a markedly higher seroprevalence in C. glareolus (47.7%, 189 of 396). In all seasons, infection rates in adults were higher than in juveniles and subadults. No significant difference of prevalence was recorded between males and females. In two trapping sites, the temporary disappearance of positive animals after a crash in rodent populations suggests that a threshold in density is necessary for the maintenance of the enzootic cycle.

Behavioral, Physiologic, and Habitat Influences on the Dynamics of Puumala virus Infection in Bank Voles (Clethrionomys glareolus)

Populations of bank voles (Clethrionomys glareolus) were monitored during a 4-year study in southern Belgium to assess the influence of agonistic behavior, reproductive status, mobility, and distribution of the rodents on the dynamics of Puumala virus (abbreviation: PUUV; genus: Hantavirus) infection. Concordance was high between data from serologic testing and results of viral RNA detection. Wounds resulting from biting or scratching were observed mainly in adult rodents. Hantavirus infection in adults was associated with wounds in the fall, i.e., at the end of the breeding season, but not in spring. In addition, sexually active animals were significantly more often wounded and positive for infection. Hantavirus infection was associated with higher mobility in juvenile and subadult males. Seroconversions observed 6 months apart also occurred more frequently in animals that had moved longer distances from their original capture point. During nonepidemic years, the distribution of infection was patchy, and positive foci were mainly located in dense ground vegetation.

Ixodes ticks belonging to the Ixodes ricinus complex encode a family of anticomplement proteins.

The alternative pathway of complement is an important innate defence against pathogens including ticks. This component of the immune system has selected for pathogens that have evolved countermeasures. Recently, a salivary protein able to inhibit the alternative pathway was cloned from the American tick Ixodes scapularis (Valenzuela et al., 2000; J. Biol. Chem. 275, 18717–18723). Here, we isolated two different sequences, similar to Isac, from the transcriptome of I. ricinus salivary glands. Expression of these sequences revealed that they both encode secreted proteins able to inhibit the complement alternative pathway. These proteins, called I. ricinus anticomplement (IRAC) protein I and II, are coexpressed constitutively in I. ricinus salivary glands and are upregulated during blood feeding. Also, we demonstrated that they are the products of different genes and not of alleles of the same locus. Finally, phylogenetic analyses demonstrate that ticks belonging to the Ixodes ricinus complex encode a family of relatively small anticomplement molecules undergoing diversification by positive Darwinian selection.

USE OF RECOMBINANT VACCINIA-RABIES GLYCOPROTEIN VIRUS FOR ORAL VACCINATION OF WILDLIFE AGAINST RABIES: INNOCUITY TO SEVERAL NON-TARGET BAIT CONSUMING SPECIES

The pathogenicity of a vaccinia recombinant virus expressing the rabies glycoprotein (VVTGgRAB) was tested in several wild animal species which could compete with the natural rabies host, the red fox (Vulpes vulpes) in consuming vaccine baits in Europe. The following species were included in this study, wild boar (Sus scrofa), Eurasian badger (Meles meles), wood mouse (Apodemus sylvaticus), yellow-necked mouse (Apodemus flavicollis), bank vole (Clethrionomys glareolus), common vole (Microtus arvalis), field vole (Microtus agrestis), water vole (Arvicola terrestris), common buzzard (Buteo buteo), kestrel (Falco tinnunculus), carrion crow (Corvus corone), magpie (Pica pica) and jay (Garrulus glandarius). During the observation period, the 107 animals given the VVTGgRAB vaccine orally did not show any clinical signs. Daily monitoring for 28 days and postmortem examination did not result in the detection of pox lesions in the oral mucosa or the skin in mammals or the unfeathered portions of birds. VVTGgRAB seems to multiply in the mammalian species tested, since rabies seroconversion was observed in all of them. Birds failed to develop demonstrable rabies virus-neutralizing antibody. A seroconversion against vaccinia virus was observed in two of four wild boars. Serological results obtained in badgers and wild boars also demonstrates the absence of direct or indirect horizontal transmission oi the recombinant virus. The potential of the recombinant virus for the immunization of badgers against rabies also was investigated. Only 50% of the badgers orally administered with 1 × 1083 TCID50 of this vaccine were protected against rabies.

Molecular biology of bovine herpesvirus type 4

Bovine herpesvirus type 4 (BHV-4) is a ubiquitous virus of cattle. Its genome is a 144 +/- 6 kb double-stranded DNA consisting of a unique central part (L-DNA) flanked at both ends by tandem repeats called polyrepetitive DNA (prDNA or H-DNA). The overall arrangement of genes has been obtained by the analysis of homologies between short BHV-4 DNA sequences and corresponding genes of Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS). The gene expression is temporally regulated. Glycoprotein precursor p (gp10/gp17) is expressed as gamma 1 polypeptide. Glycoproteins gp1, gp8, gp11 and their precursors are gamma 2 proteins. The analysis of strain variations allows the definition of two types of strains, based on the DNA patterns: the Movar 33/63-like and the DN 599-like strains. Only the M40 strain, isolated in India, fails to fit this classification. The genomic variations have been compiled to build a dendrogram showing three levels of divergence between BHV-4 strains or isolates. The available molecular data indicate that the BHV-4 genome shares much similarity with the DNA of EBV and HVS, two representative members of the gammaherpesvirinae. BHV-4 may therefore be classified in the subfamily gammaherpesvirinae.

The development and use of a vaccinia-rabies recombinant oral vaccine for the control of wildlife rabies; a link between Jenner and Pasteur

To improve both safety and stability of the oral vaccines used in the field to vaccinate foxes against rabies, a recombinant vaccinia virus, which expresses the immunizing G protein of rabies virus has been developed by inserting the cDNA which codes for the immunogenic glycoprotein of rabies virus into the thymidine kinase (TK) gene of the Copenhagen strain of vaccinia virus. The efficacy of this vaccine was tested by the oral route, primarily in foxes. The immunity conferred, a minimum of 12 months in cubs and 18 months in adult animals, corresponds to the duration of the protection required for vaccination of foxes in the field. Innocuity was tested in foxes, domestic animals, and in numerous European wild animal species that could compete with the red fox for the vaccine bait. No clinical signs or lesions were observed in any of the vaccinated animals during a minimum of 28 days post vaccination. Moreover, no transmission of immunizing doses of the recombinant occurred between foxes or other species tested. To study the stability of the vaccine strain, baits containing the vaccine were placed in the field. Despite considerable variations of environmental temperatures, the vaccine remained stable for at least one month. Because bait is taken within one month, it can be assumed that most animals taking the baits are effectively vaccinated. To test the field efficacy of the recombinant vaccine, large-scale campaigns of fox vaccination were set up in a 2200 km2 region of southern Belgium, were rabies was prevalent. A dramatic decrease in the incidence of rabies was noted after the campaigns. The recombinant is presently used to control wildlife rabies in the field both in several European countries and in the United States.

Experimental rabies infection and oral vaccination in vampire bats (Desmodus rotundus)

A rabies virus variant isolated from a vampire bat (Desmodus rotundus) and characterized by genome sequencing was used for the standardization of an experimental infection in this species. The parenteral administration of 10(6) MICLD50 of this variant was capable of inducing death from rabies in 89% of animals. The mean duration of post-challenge survival was 12 days. None of the experimental rabid vampire bats showed aggressive behaviour. A vaccinia-rabies glycoprotein recombinant virus vaccine was administered orally to vampire bats on days -120, -90, -30 or -18 pre-challenge, on the same day of challenge, or on day +5 post-challenge. A significant protection was noticed only in animals vaccinated on days -18 or -30 pre-challenge. A longer period of incubation was observed in animals vaccinated 5 days post-challenge.

Experimental infection of bulls with a genital isolate of bovine herpesvirus-4 and reactivation of latent virus with dexamethasone

Five 13- to 18-month old Belgian Blue bulls were used in this experiment. Four bulls (Nos. 2, 3, 4 and 5) were inoculated intratesticularly with 10(5) plaque-forming units of bovine herpesvirus-4 (BHV-4) in each testicle (Day 0). The challenge BHV-4 strain was previously isolated from testicle cells of a bull exhibiting orchitis and azoospermia. The fifth bull (No. 1) was used as a control and received the same volume of uninfected cell culture supernatant. For 5 days, beginning on Day 51 post-infection, two bulls (Nos. 4 and 5) and the control bull (No. 1) received 0.1 mg kg-1 of dexamethasone. Unilateral castrations were then performed at regular intervals for viral examination. Treatment with dexamethasone reactivated latent BHV-4, but no clinical signs were observed in treated bulls until the end of the experiment (Day 93). Only Bull 3 showed conjunctivitis and temporary azoospermia. The virus was recovered from various samples showing that: (i) BHV-4 can be present in a latent state in the testicles and mononuclear blood cells; (ii) dexamethasone reactivates the virus; (iii) the virus is excreted by nasal and ocular routes. Each infected bull seroconverted and a booster antibody response appeared after dexamethasone treatment as shown by immunofluorescence. Neutralizing antibodies were detected in each bull by complement-dependent neutralization test with titres higher than those obtained by a classical neutralization test. No booster response of neutralizing antibodies was observed after dexamethasone treatment. The antigenically relevant envelope BHV-4 proteins were identified by Western blotting using sera samples from the animals. DNA restriction endonuclease profiles of viruses reisolated after primary infection and reactivation showed only small differences.