Denis Soubieux

A novel chicken lung epithelial cell line: Characterization and response to low pathogenicity avian influenza virus

Avian influenza virus (AIV) infections of the chicken occur via the respiratory route. Unlike ducks which are considered as a natural AIV reservoir, chickens are highly susceptible to AIV infections and do not possess the RIG-I pattern recognition receptor involved in triggering the antiviral interferon response. To study the chicken innate immune response to AIV in the respiratory tract, we established an epithelial cell line (CLEC213) from lung explants of white leghorn chickens. CLEC213 cells exhibited a polyhedral morphology and formed cohesive clusters bound through tight junctions as assessed by electron microscopy. Expression of E-cadherin but not vimentin could be detected as expected for cells of epithelial origin. In addition, CLEC213 cells showed characteristics similar to those of mammalian type II pneumocytes, including the presence of intracytoplasmic vacuoles filled with a mucopolysaccharide material, alkaline phosphatase activity, transcription of chicken lung collectins genes (cLL and SPA), and some intracytoplasmic lamellar-like bodies. CLEC213 cells showed a constitutive expression level of TLR3 and TLR4 and were responsive to stimulation with the respective agonists, poly (I:C) and LPS: between 4h and 24h after treatment, a strong increase in the expression of IFN-α, IFN-β and IL-8 genes could be detected. Furthermore, CLEC213 cells supported efficient growth of the low pathogenicity avian influenza virus H6N2 (A/duck/France/05057a/2005) in the presence or the absence of trypsin in the culture media. At 4h post-infection, the H6N2 virus induced highly elevated levels of expression of IFN-α and IL-8, moderately elevated levels of LITAF, TGF-β4 and CCL5. However, an increase of IFN-β gene expression could not be detected in response to AIV infection. In conclusion, like mammalian type II pneumocytes, CLEC213 are able to mount a robust cytokine and chemokine immune response to microbial patterns and viral infection. We hypothesize that they could derive from lung atrial granular cells. The involvement of such type of lung epithelial cells in the respiratory tract defence of the chicken can thus be further studied.

Role of nonclassical class I genes of the chicken major histocompatibility complex Rfp-Y locus in transplantation immunity

The chicken major histocompatibility complex (MHC) genes are organized into two genetically independent clusters which both possess class I and class IIβ genes: the classical B complex and the Restriction fragment pattern-Y (Rfp-Y) complex. In this study, we have examined the role of Rfp-Y genes in transplantation immunity. For this we used three sublines, B19H1, B19H2 and B19H3, derived from a line fixed for B19. Southern blots, PCR-SSCP assays using primers specific for Rfp-Y genes, and Rfp-Y class I allele-specific sequencing show that the polymorphisms observed in B19H1, B19H2 and B19H3 are due to the presence of three different Rfp-Y haplotypes. The Rfp-Y class I (YF) alleles in these three haplotypes are highly polymorphic, and RT-PCR shows that at least two YF loci are expressed in each subline. The three sublines show Rfp-Y-directed alloreactivity in that Rfp-Y-incompatible skin grafts are rejected within 15 days, a rate intermediate between that seen in B-incompatible rejection (7 days) and that observed for grafts within the sublines (20 days). We conclude that Rfp-Y has an intermediate role in allograft rejection, likely to be attributable to polymorphism at the class I loci within this region.

In vivo metabolism of aminopyrine by the larvae of the helminthHeligmosomoides polygyrus

The in vivo N-dealkylation of [13C-2]-labeled aminopyrine by the L1–L2 larvae ofHeligmosomoides polygyrus was demonstrated by the use of a sensitive gas chromatography-mass spectrometry method. This is the first evidence for the possible existence of a cytochrome P-450-dependent activity in helminths.

The RNA-binding domain of influenzavirus non-structural protein-1 cooperatively binds to virus-specific RNA sequences in a structure-dependent manner

Influenzavirus non-structural protein NS1 is involved in several steps of the virus replication cycle. It counteracts the interferon response, and also exhibits other activities towards viral and cellular RNAs. NS1 is known to bind non-specifically to double-stranded RNA (dsRNA) as well as to viral and cellular RNAs. We set out to search whether NS1 could preferentially bind sequence-specific RNA patterns, and performed an in vitro selection (SELEX) to isolate NS1-specific aptamers from a pool of 80-nucleotide(nt)-long RNAs. Among the 63 aptamers characterized, two families were found to harbour a sequence that is strictly conserved at the 5′ terminus of all positive-strand RNAs of influenzaviruses A. We found a second virus-specific motif, a 9 nucleotide sequence located 15 nucleotides downstream from NS1’s stop codon. In addition, a majority of aptamers had one or two symmetrically positioned copies of the 5′-GUAAC / 3′-CUUAG double-stranded motif, which closely resembles the canonical 5′-splice site. Through an in-depth analysis of the interaction combining fluorimetry and gel-shift assays, we showed that NS1’s RNA-binding domain (RBD) specifically recognizes sequence patterns in a structure-dependent manner, resulting in an intimate interaction with high affinity (low nanomolar to subnanomolar KD values) that leads to oligomerization of the RBD on its RNA ligands.

Vaccination against Marek's disease reduces telomerase activity and viral gene transcription in peripheral blood leukocytes from challenged chickens.

We investigated whether telomerase activity and viral gene transcription were associated with protection against the RB-1B strain of Mareks disease virus (MDV) in chickens vaccinated with Rispens CVI988 or the herpes virus of turkey (HVT). Telomerase activity in peripheral blood leukocytes (PBLs) seemed to be an appropriate marker of lymphoma and levels of viral transcription were correlated with the virulence of MDV strains. Vaccinated protected birds had lower levels of telomerase activity and RB-1B viral gene transcription than unvaccinated chickens infected with RB-1B. The decrease in RB-1B viral transcription was more marked in chickens vaccinated with CVI988 than in those vaccinated with HVT. Indeed, RB-1B viral transcription was not detectable after 14 days post-challenge. In conclusion, telomerase activity and gene transcription in challenge MDV strains are potential new reliable criteria of protection in vaccinated chickens.

Flow-cytometry analysis of sheep-nematode egg populations.

Abstract Flow cytometry was applied to the analysis of nematode populations. Three strains of Haemonchus contortus susceptible or resistant to anthelmintics were studied. Eggs were chosen for these analyses. Data on light-scatter emissions and native green fluorescence were collected. In addition, the size of the eggs (image analysis), the hatching rate, and the susceptibility to benzimidazoles were measured. The results showed that nematode eggs are a suitable material for multiparametric flow-cytometry analyses. Forward-scatter emission is a discriminating parameter for the egg size. The hatching rate and side-scatter emission have a significantly positive relationship. For resistant strains, the rate of resistance shows a significant regression on the native green-fluorescence pulses that might reflect the state of oxidation of associated flavin molecules.