Sylvie Brunet

Comparison between acute oral/respiratory and chronic stomatitis/gingivitis isolates of feline calicivirus: pathogenicity, antigenic profile and cross-neutralisation studies

Summary. Feline calicivirus (FCV) is a major oral and respiratory pathogen of cats, able to induce subclinical infection as well as acute disease. It is also characterized by a high degree of antigenic variation.This work sought to address the question of the existence of distinct biotypes of FCV. Eight French, 6 British and 9 American FCV isolates, responsible for acute oral/respiratory disease or chronic gingivitis/stomatitis, were compared for their pathogenicity, antigenic profiles and serological relationships. Antigenic profiles were assessed by an indirect immunofluorescence assay with a large panel of characterized monoclonal antibodies. Cross-neutralisation assays were performed with specific cat antisera collected at 30 days p.i., then analysed by calculation of antigenic bilateral relatedness and dominance.Whatever their pathogenic origin, all the isolates induced an acute upper-respiratory tract infection in oronasally infected SPF kittens. Their antigenic profiles were different and did not correlate with their geographical or pathological origin. Cross-neutralisation studies and calculation of the mean bilateral relatedness allowed us to distinguish chronic original isolates from acute original ones. This study did not confirm the existence of FCV biotypes but showed that the chronic carrier state is related to the emergence of antigenically distant viruses.

A Synthetic Biology Approach for a Vaccine Platform against Known and Newly Emerging Serotypes of Bluetongue Virus

ABSTRACT Bluetongue is one of the major infectious diseases of ruminants and is caused by bluetongue virus (BTV), an arbovirus existing in nature in at least 26 distinct serotypes. Here, we describe the development of a vaccine platform for BTV. The advent of synthetic biology approaches and the development of reverse genetics systems has allowed the rapid and reliable design and production of pathogen genomes which can be subsequently manipulated for vaccine production. We describe BTV vaccines based on “synthetic” viruses in which the outer core proteins of different BTV serotypes are incorporated into a common tissue-culture-adapted backbone. As a means of validation for this approach, we selected two BTV-8 synthetic reassortants and demonstrated their ability to protect sheep against virulent BTV-8 challenge. In addition to further highlight the possibilities of genome manipulation for vaccine production, we also designed and rescued a synthetic BTV chimera containing a VP2 protein, including regions derived from both BTV-1 and BTV-8. Interestingly, while the parental viruses were neutralized only by homologous antisera, the chimeric proteins could be neutralized by both BTV-1 and BTV-8 antisera. These data suggest that neutralizing epitopes are present in different areas of the BTV VP2 and likely “bivalent” strains eliciting neutralizing antibodies for multiple strains can be obtained. IMPORTANCE Overall, this vaccine platform can significantly reduce the time taken from the identification of new BTV strains to the development and production of new vaccines, since the viral genomes of these viruses can be entirely synthesized in vitro. In addition, these vaccines can be brought quickly into the market because they alter the approach, but not the final product, of existing commercial products.

Efficacy of a bivalent inactivated non-adjuvanted feline calicivirus vaccine : Relation between in vitro cross-neutralization and heterologous protection in vivo

Feline calicivirus (FCV) is a major pathogen of the cat characterized by a strong genomic, antigenic and clinical diversity. Despite vaccination, FCV infection is highly prevalent, and for a few years, outbreaks of virulent systemic disease (VSD) have been reported in North America and Europe. An inactivated non-adjuvanted bivalent vaccine was recently developed by combining antigens derived from two broadly cross-reactive FCV strains. The antigenic relatedness between the vaccine strains and other antigenic variants was demonstrated by cross-neutralization studies in vitro. This study showed that vaccine-induced protection against heterologous challenges was correlated to in vitro cross-neutralization, and it validated the use of cross-neutralization tests to select vaccine FCV strains. This correlation applies also for the highly virulent strains causing VSD (VS-FCV).

Rdl Gene Polymorphism and Sequence Analysis and Relation to In Vivo Fipronil Susceptibility in Strains of the Cat Flea

ABSTRACT The &ggr;-amino butyric acid (GABA)-gated chloride ion channel in the insect central nervous system is the target of cyclodiene and phenylpyrazole insecticides. Resistance to dieldrin has been reported in several insect species and was associated with a point mutation (Ala285 to Ser substitution) in the M2 transmembrane domain of the GABA-gated chloride ion channel (the resistant to dieldrin [Rdl] gene). A partial Rdl gene sequence was reported previously in specimens of the cat flea, Ctenocephalides felis (Bouché). Because the presence of the Rdl gene mutation coincided with a reduction in susceptibility to fipronil in some insect species, it has been inferred that a similar association may exist in cat fleas. The Rdl gene sequence was evaluated in 20–50 fleas each from six cat flea strains shown previously to be fully susceptible to fipronil. Total DNA or RNA from fleas was extracted using a commercial kit, and the sequence encompassing the single nucleotide polymorphism (SNP) position Rdl was amplified by polymerase chain reaction (PCR) or reverse transcription-PCR. Amplification products were sequenced on both strands. All tested strains were homozygous for the mutant allele (T nucleotide at SNP position); amino acid sequencing demonstrated the Ala285 to Ser substitution. The results of this study indicated that the Rdl gene mutation was uniformly present as homozygous alleles in strains of fleas that have been shown to be fully susceptible to topically applied fipronil and that the efficacy of fipronil against cat fleas was not impacted by the Rdl gene mutation.

Time-Course Analysis of Main Markers of Primary Infection in Cats with the Feline Immunodeficiency Virus

Studies of the response of the immune system to feline immunodeficiency virus (FIV) during primary infection have shown that a subpopulation of CD8+ T-cells with an activated phenotype and reduced expression of the CD8β chain (denoted CD8β low T cells) expands to reach up to 80% of the total CD8+ T cell count. The expansion of this subpopulation is considered to be a signature of FIV and an indicator of immune system alteration. We use a simple mathematical formalism to study the relationships over time between the dose of infection, the size of the CD8β low population, and the circulating viral load in cats infected with FIV. Viremia profiles are described using a combination of two exponential laws, whereas the CD8β low percentage (out of the total CD8+ population) is represented by a Gompertz law including an expansion phase and a saturation phase. Model parameters are estimated with a population approach using data from 102 experimentally infected cats. We examine the dose of infection as a potential covariate of parameters. We find that the rates of increase of viral load and of CD8β low percentage are both correlated with the dose of infection. Cats that develop strong acute viremia also show the largest degree of CD8β low expansion. The two simple models are robust tools for analysing the time course of CD8β low percentage and circulating viral load in FIV-infected cats and may be useful for generating new insights on the disease and on the design of therapeutic strategies, potentially applicable to HIV infection.

Diversity of trends of viremia and T-cell markers in experimental acute feline immunodeficiency virus infection.

Objective The early events of human immunodeficiency virus infection seem critical for progression toward disease and antiretroviral therapy initiation. We wanted to clarify some still unknown prognostic relationships between inoculum size and changes in various immunological and virological markers. Feline immunodeficiency virus infection could be a helpful model. Methods Viremia and T-cell markers (number of CD4, CD8, CD8βlowCD62Lneg T-cells, CD4/CD8 ratio, and percentage of CD8βlowCD62Lneg cells among CD8 T-cells) were measured over 12 weeks in 102 cats infected with different feline immunodeficiency virus strains and doses. Viremia and T-cell markers trajectory groups were determined and the dose-response relationships between inoculum titres and trajectory groups investigated. Results Cats given the same inoculum showed different patterns of changes in viremia and T-cell markers. A statistically significant positive dose-response relationship was observed between inoculum titre and i) viremia trajectory-groups (r = 0.80, p<0.01), ii) CD8βlowCD62Lneg cell-fraction trajectory-groups (r = 0.56, p<0.01). Significant correlations were also found between viremia and the CD4/CD8 ratio and between seven out of ten T-cell markers. Conclusions In cats, the infectious dose determines early kinetics of viremia and initial CD8+ T-cell activation. An expansion of the CD8βlowCD62Lneg T-cells might be an early predictor of progression toward disease. The same might be expected in humans but needs confirmation.