Course of seasonal influenza A/Brisbane/59/07 H1N1 infection in the ferret

Every year, influenza viruses infect approximately 5-20% of the population in the United States leading to over 200,000 hospitalizations and 36,000 deaths from flu-related complications. In this study, we characterized the immune and pathological progression of a seasonal strain of H1N1 influenza virus, A/Brisbane/59/2007 in a ferret model. The immune response of the animals showed a dose-dependent increase with increased virus challenge, as indicated by the presence of virus specific IgG, IgM, and neutralizing antibodies. Animals infected with higher doses of virus also experienced increasing severity of clinical symptoms and fever at 2 days post-infection (DPI). Interestingly, weight loss was more pronounced in animals infected with lower doses of virus compared to those infected with a higher dose; these results were consistent with viral titers of swabs collected from the nares, but not the throat. Analyzed specimens included nasal and throat swabs from 1, 3, 5, and 7 DPI as well as tissue samples from caudal lung and nasal turbinates. Viral titers of the swab samples in all groups were higher on 1 and 3 DPI and returned to baseline levels by 7 DPI. Analysis of nasal turbinates indicated presence of virus at 3 DPI in all infected groups, whereas virus was only detected in the lungs of animals in the two highest dose groups. Histological analysis of the lungs showed a range of pathology, such as chronic inflammation and bronchial epithelial hypertrophy. The results provided here offer important endpoints for preclinical testing of the efficacy of new antiviral compounds and experimental vaccines

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

<p>Abstract</p> <p>Background</p> <p>The ferret (<it>Mustela putorius furo</it>) 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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>The tools developed in this study allow for functional genomic analysis and make further development of reagents for the ferret model possible.</p

De-Novo Transcriptome Sequencing of a Normalized cDNA Pool from Influenza Infected Ferrets

The ferret is commonly used as a model for studies of infectious diseases. The genomic sequence of this animal model is not yet characterized, and only a limited number of fully annotated cDNAs are currently available in GenBank. The majority of genes involved in innate or adaptive immune response are still lacking, restricting molecular genetic analysis of host response in the ferret model. To enable de novo identification of transcriptionally active ferret genes in response to infection, we performed de-novo transcriptome sequencing of animals infected with H1N1 A/California/07/2009. We also included splenocytes induced with bacterial lipopolysaccharide to allow for identification of transcripts specifically induced by Gram-negative bacteria. We pooled and normalized the cDNA library in order to delimit the risk of sequencing only highly expressed genes. While normalization of the cDNA library removes the possibility of assessing expression changes between individual animals, it has been shown to increase identification of low abundant transcripts. In this study, we identified more than 19000 partial ferret transcripts, including more than 1000 gene orthologs known to be involved in the innate and the adaptive immune response

Genetic analysis of neurofibromatosis type 2 (NF2) patients and NF2-associated tumors with emphasis on chromosome 22 deletions

Neurofibromatosis type 2 (NF2) is an autosomal dominant disease with the hallmark of bilateral vestibular schwannomas. NF2 patients may also develop schwannomas at other locations as well as meningiomas, neurofibromas and ependymomas. NF2 shows a distinct clinical variability ranging from very mild to severe forms. Since identification of the NF2 gene in 1993, mutation-screening analyses have been performed. Mutations were, however, not found in numerous cases and the mechanism behind tumor development in NF2 is not fully understood. The correlation between type of NF2 gene mutation and clinical phenotype of patients does not provide a clear-cut explanation of the clinical variability. Also, absence of NF2 gene mutations in constitutional tissue from a related disorder, such as schwannomatosis, points towards a possible existence of an additional NF2-related locus from chromosome 22, which may modify the disease phenotype. This implies the necessity of further clarification of the genetic factors involved in NF2. Deletions on chromosome 22 and the inactivation of NF2 tumor suppressor gene are critical steps for meningioma formation. However, 40% of the tumors do not show aberrations of chromosome 22 or mutations in the NF2 gene. This suggest that alternative mechanisms are responsible for the development of a large fraction of meningiomas. We analyzed 25 meningiomas, which do not display chromosome 22 deletions, for genetic abnormalities by CGH (comparative genomic hybridization) to metaphase chromosomes. Two tumors showed l oss of chromosome 1p and 3p which suggest that deletions of both lp and 3p may contribute to meningioma turnorigenesis (paper I). In schwannomas, detailed mutation analyses of the NF2 gene showed that 60% of the tumors carry inactivating mutations. Thus, the mechanism behind the development of 40% of schwannomas is unknown. We studied 50 sporadic and NF2-associated schwannomas by high resolution LOH (loss-of-heterozygosity) on chromosome 22 and other chromosomes. Chromosome 22 deletions were detected in over 80% of the cases. Four tumors showed LOH not involving the NF2 locus. All exons of the NF2 gene were sequenced in these tumors and mutations were detected only in one case. Thus, additional regions chromosome 22 may harbor mutations possibly involved in schwannoma turnorigenesis (paper II). We also identified an early-onset NF2 patient with a large constitutional deletion on chromosome 22. Constitutional deletions of the entire NF2 gene were previously described in NF2 patients; two large deletions encompassing 700-800 kb have been reported in mildly affected subjects. Our severely affected case showed a much larger deletion stretching approximately 5 Mb towards the telomere (paper III). It may therefore be hypothesized that the severe phenotype in this patient is a result of a combined mutation in NF2 gene and in a putative modifier gene. Further mapping of deletions in this candidate region was performed by analysis of 116 NF2 patients for deletions on 22q. Analysis was carried out using high-resolution microarray-CGH on a genomic array covering at least 90% of a 7.4 Mb interval of 22q, around and distal to the NF2 locus. This is a novel approach for high-resolution detection of chromosomal abnormalities, both in constitutional and tumor-derived DNA. Deletions were detected in nine severe, nine moderate and six mild patients. Deletions in severely and moderately affected patients varied in size, whereas cases with mild NF2 displayed deletions affecting the NF2 locus only (paper IV). This result indicates that the geneotype/phenotype correlation is unlikely to exist and supports the notion that the NF2 modifier gene may exist in the vicinity of the NF2 locus. Finally, the tumor suppressor gene SMARCB1 was tested for mutations in meningioma and schwannoma. SMARCB1 is mutated in malignant rhabdoid tumors and is located on 22q11.2, a region frequently deleted in meningiomas. Fourty-three meningiomas and twenty-one schwannomas were tested for mutations in exons 2 through 8. However, no mutations were detected, suggesting that the SMARCB1 gene is not frequently involved in the pathogenesis of these tumors. We also identified the mouse ortholog and characterized different splice forms of this gene, both in human and in mouse (paper V)

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

Growth hormone overexpression in the central nervous system results in hyperphagia-induced obesity associated with insulin resistance and dyslipidemia

It is well known that peripherally administered growth hormone (GH) results in decreased body fat mass. However, GH-deficient patients increase their food intake when substituted with GH, suggesting that GH also has an appetite stimulating effect. Transgenic mice with an overexpression of bovine GH in the central nervous system (CNS) were created to investigate the role of GH in CNS. This study shows that overexpression of GH in the CNS differentiates the effect of GH on body fat mass from that on appetite. The transgenic mice were not GH-deficient but were obese and showed increased food intake as well as increased hypothalamic expression of agouti-related protein and neuropeptide Y. GH also had an acute effect on food intake following intra-cerebroventricular injection of C57BL/6 mice. The transgenic mice were severely hyperinsulinemic and showed a marked hyperplasia of the islets of Langerhans. In addition, the transgenic mice displayed alterations in serum lipid and lipoprotein levels and hepatic gene expression. In conclusion, GH overexpression in the CNS results in hyperphagia-induced obesity indicating a dual effect of GH with a central stimulation of appetite and a peripheral lipolytic effect

Classification analysis.

<p>Panel A shows the heat map of the 10 genes isolated by the USC algorithm to classify the samples with regards to infectious strain. The samples are denoted by their ID number and the euthanasia day, and are sorted according to strain (and dose for A/Cal/07 infected animals). The asterisks denote gene expression verified by qRT-PCR. Panel B shows the 7 genes that were used to classify the samples based on the cumulative histopathology score. The samples are denoted with ID number and euthanasia day, and are sorted according to cumulative histopathology score (given as numbers under the sample names). Panel C displays the 31 genes required to classify the samples based on infectious strain and histopathology score. The samples are denoted with ID number and euthanasia day, and are sorted according to strain and cumulative histopathology score (given as numbers under the heat map). All samples were correctly classified with regards to strain. The samples used to train the classification algorithm is denoted in black, correctly classified samples in the test set in green and incorrectly classified samples in red.</p

Global gene expression changes.

<p>Panel A shows a heat map of the 25% (7685 probes) most variable genes in the dataset for all samples. Each sample day represents three individual animals, except the control group (CTRL, n = 6). HD and LD indicate high dose and low dose, respectively. D1 through D7 designates the day of euthanasia. Panel B illustrates gene expression profiles of 1997 significantly changed probes with a fold change larger than +/−2 in at least one group when compared to the control group. The average fold change from the three animals within each group is shown. Red designate up-regulated genes, blue down regulated genes, where a more intense color illustrates a more pronounced fold change. The Venn diagram in panel C shows the number of probes up or down regulated after infection by any of the three strains (177 genes), by two of the three strains (228, 131 and 29 genes) and the number of probes aberrantly expressed in a strain specific pattern.</p