Colette Grillet

Development of a dual recombinant vaccine to protect small ruminants against peste-des-petits-ruminants virus and capripoxvirus infections.

ABSTRACT A recombinant capripoxvirus vaccine containing a cDNA of the peste-des-petits-ruminants virus (PPRV) fusion protein gene was constructed. A quick and efficient method was used to select a highly purified recombinant virus clone. A trial showed that a dose of this recombinant as low as 0.1 PFU protected goats against challenge with a virulent PPRV strain.

The emergence of Schmallenberg virus across Culicoides communities and ecosystems in Europe

Schmallenberg virus (SBV), a novel arboviral pathogen, has emerged and spread across Europe since 2011 inflicting congenital deformities in the offspring of infected adult ruminants. Several species of Culicoides biting midges (Diptera: Ceratopogonidae) have been implicated in the transmission of SBV through studies conducted in northern Europe. In this study Culicoides from SBV outbreak areas of mainland France and Italy (Sardinia) were screened for viral RNA. The role of both C. obsoletus and the Obsoletus complex (C. obsoletus and C. scoticus) in transmission of SBV were confirmed in France and SBV was also discovered in a pool of C. nubeculosus for the first time, implicating this species as a potential vector. While collections in Sardinia were dominated by C. imicola, only relatively small quantities of SBV RNA were detected in pools of this species and conclusive evidence of its potential role in transmission is required. In addition to these field-based studies, infection rates in colony-derived individuals of C. nubeculosus and field-collected C. scoticus are also examined in the laboratory. Rates of infection in C. nubeculosus were low, confirming previous studies, while preliminary examination of C. scoticus demonstrated that while this species can replicate SBV to a potentially transmissible level, further work is required to fully define comparative competence between species in the region. Finally, the oral competence for SBV of two abundant and widespread mosquito vector species in the laboratory is assessed. Neither Aedes albopictus nor Culex pipiens were demonstrated to replicate SBV to transmissible levels and appear unlikely to play a major role in transmission. Other vector competence data produced from studies across Europe to date is then comprehensively reviewed and compared with that generated previously for bluetongue virus.

New avian paramyxoviruses Type I strains identified in Africa provide new outcomes for phylogeny reconstruction and genotype classification

Newcastle disease (ND) is one of the most lethal diseases of poultry worldwide. It is caused by an avian paramyxovirus 1 that has high genomic diversity. In the framework of an international surveillance program launched in 2007, several thousand samples from domestic and wild birds in Africa were collected and analyzed. ND viruses (NDV) were detected and isolated in apparently healthy fowls and wild birds. However, two thirds of the isolates collected in this study were classified as virulent strains of NDV based on the molecular analysis of the fusion protein and experimental in vivo challenges with two representative isolates. Phylogenetic analysis based on the F and HN genes showed that isolates recovered from poultry in Mali and Ethiopia form new groups, herein proposed as genotypes XIV and sub-genotype VIf with reference to the new nomenclature described by Diel’s group. In Madagascar, the circulation of NDV strains of genotype XI, originally reported elsewhere, is also confirmed. Full genome sequencing of five African isolates was generated and an extensive phylogeny reconstruction was carried out based on the nucleotide sequences. The evolutionary distances between groups and the specific amino acid signatures of each cluster allowed us to refine the genotype nomenclature.

Development and evaluation of a real-time quantitative PCR assay for Culicoides imicola, one of the main vectors of Bluetongue (BT) and African Horse Sickness (AHS) in Africa and Europe

The current microscopy method for identifying the Culicoides imicola Kieffer, 1913 species can be time and labour intensive. There is a need for the development of a rapid and quantitative tool to quantify the biting midges C. imicola ss in light trap catches. A reproducible and sensitive real-time polymerase chain reaction method that targets the internal transcribed spacer (ITS-1) of ribosomal DNA of C. imicola ss species was developed. This real-time PCR assay was first performed on 10-fold serial dilutions of purified plasmid DNA containing specific C. imicola ss ITS-1. It was then possible to construct standard curves with a high correlation coefficient (r2=0.99) in the range of 10(-2)-10(-8) ng of purified DNA. The performances of this PCR were evaluated in comparison with morphological determination on Culicoides trapped along the Mediterranean coastal mainland France. ROC statistical analysis was carried out using morphology as gold standard and the area under the ROC curve had a satisfactory value of 0.9752. The results indicated that this real-time PCR assay holds promise for monitoring C. imicola ss population in both surveillance and research programmes because of its good specificity (92%) and sensitivity (95%).

Description of the outbreak of bluetongue in Corsica in 2003, and lessons for surveillance

Since 1999, several serotypes of bluetongue virus (btv) have been isolated in the western part of the Mediterranean basin, and since 2000, Corsica has been exposed to three different serotypes: btv serotype 2 in 2000, btv serotype 4 (btv-4) in 2003 and btv serotype 16 in 2004. In 2000 there were no surveillance systems for bluetongue, but in 2003, active surveillance of the circulation of btv and its vector Culicoides species, aided by a raised level of awareness in farmers and veterinarians, made it possible to study the introduction of btv-4. The monitoring and analysis of the seroconversions of sentinel herds of goats, clinical signs and meteorological variables showed that the serotype had been present in the island since May that year, but clinical signs were first observed only in October. Moreover, the weather conditions and wind patterns were suitable for the transport of Culicoides species from Sardinia in May. These observations suggest that btv had been transported on air currents from a southern infected area, and that it could have spread without causing clinical signs of disease for a few months.