Host gene expression signatures discriminate between ferrets infected with genetically similar H1N1 strains.

Different respiratory viruses induce virus-specific gene expression in the host. Recent evidence, including those presented here, suggests that genetically related isolates of influenza virus induce strain-specific host gene regulation in several animal models. Here, we identified systemic strain-specific gene expression signatures in ferrets infected with pandemic influenza A/California/07/2009, A/Mexico/4482/2009 or seasonal influenza A/Brisbane/59/2007. Using uncorrelated shrunken centroid classification, we were able to accurately identify the infecting influenza strain with a combined gene expression profile of 10 selected genes, independent of the severity of disease. Another gene signature, consisting of 7 genes, could classify samples based on lung pathology. Furthermore, we identified a gene expression profile consisting of 31 probes that could classify samples based on both strain and severity of disease. Thus, we show that expression-based analysis of non-infected tissue enables distinction between genetically related influenza viruses as well as lung pathology. These results open for development of alternative tools for influenza diagnostics.

Transcriptome sequencing and development of an expression microarray platform for the domestic ferret.

BACKGROUND: The ferret (Mustela putorius furo) represents an attractive animal model for the study of respiratory diseases, including influenza. Despite its importance for biomedical research, the number of reagents for molecular and immunological analysis is restricted. We present here a parallel sequencing effort to produce an extensive EST (expressed sequence tags) dataset derived from a normalized ferret cDNA library made from mRNA from ferret blood, liver, lung, spleen and brain. RESULTS: We produced more than 500000 sequence reads that were assembled into 16000 partial ferret genes. These genes were combined with the available ferret sequences in the GenBank to develop a ferret specific microarray platform. Using this array, we detected tissue specific expression patterns which were confirmed by quantitative real time PCR assays. We also present a set of 41 ferret genes with even transcription profiles across the tested tissues, indicating their usefulness as housekeeping genes. CONCLUSION: The tools developed in this study allow for functional genomic analysis and make further development of reagents for the ferret model possible.

Single base instability is promoted in vulvar lichen sclerosus.

Single base substitution mutations in codons 248 and 273 of TP53 and codon 12 Kirsten-ras (KRAS) are commonly found in human carcinomas. To determine whether these mutations also occur in normal and inflamed tissues from which carcinomas arise, we utilized the ultra-sensitive polymerase chain reaction/restriction endonuclease/ligase chain reaction mutation assay. Ninety samples of genital skin, including lichen sclerosus (LS) affected skin, adjacent normal and non-adjacent normal, were assayed. Mutations were detected in 103 of 349 assays and consisted of KRAS G34A, G34T, G35A, and TP53 C742T, G818C, C817T, and G818A mutations. Mutant prevalence varied from 1 to 20 per 10(6) wild-type cells. Mutations occurred significantly more frequently in LS (78/224 (35%)) than adjacent normal (20/88 (23%)) and non-adjacent normal genital skin (5/38 (13%)). KRAS G34A mutation was relatively common to all classes of specimen, whereas TP53 gene C742T and G818C mutations were significantly more frequent in LS than normal genital skin. In matched samples, immunohistochemistry evaluation of p53 protein expression revealed the presence of epidermal p53 clones in LS whose presence and number significantly correlated with the presence of TP53 C742T and G818C mutations. Based on these results, it appears oncogenic point mutations occur in normal genital skin, and are selected for in LS.