Increased Inducible Nitric Oxide Synthase Expression in Organs Is Associated with a Higher Severity of H5N1 Influenza Virus Infection

BACKGROUND: The mechanisms of disease severity caused by H5N1 influenza virus infection remain somewhat unclear. Studies have indicated that a high viral load and an associated hyper inflammatory immune response are influential during the onset of infection. This dysregulated inflammatory response with increased levels of free radicals, such as nitric oxide (NO), appears likely to contribute to disease severity. However, enzymes of the nitric oxide synthase (NOS) family such as the inducible form of NOS (iNOS) generate NO, which serves as a potent anti-viral molecule to combat infection in combination with acute phase proteins and cytokines. Nevertheless, excessive production of iNOS and subsequent high levels of NO during H5N1 infection may have negative effects, acting with other damaging oxidants to promote excessive inflammation or induce apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: There are dramatic differences in the severity of disease between chickens and ducks following H5N1 influenza infection. Chickens show a high level of mortality and associated pathology, whilst ducks show relatively minor symptoms. It is not clear how this varying pathogenicty comes about, although it has been suggested that an overactive inflammatory immune response to infection in the chicken, compared to the duck response, may be to blame for the disparity in observed pathology. In this study, we identify and investigate iNOS gene expression in ducks and chickens during H5N1 influenza infection. Infected chickens show a marked increase in iNOS expression in a wide range of organs. Contrastingly, infected duck tissues have lower levels of tissue related iNOS expression. CONCLUSIONS/SIGNIFICANCE: The differences in iNOS expression levels observed between chickens and ducks during H5N1 avian influenza infection may be important in the inflammatory response that contributes to the pathology. Understanding the regulation of iNOS expression and its role during H5N1 influenza infection may provide insights for the development of new therapeutic strategies in the treatment of avian influenza infection

Who wants a slimmer body? The relationship between body weight status, education level and body shape dissatisfaction among young adults in Hong Kong

Background: Body shape dissatisfaction has been thought to have an indispensable impact on weight control behaviors. We investigated the prevalence of body shape dissatisfaction (BSD) and explored its association with weight status, education level and other determinants among young adults in Hong Kong. Methods. Information on anthropometry, BSD, and socio-demographics was collected from a random sample of 1205 young adults (611 men and 594 women) aged 18-27 in a community-based household survey. BSD was defined as a discrepancy between current and ideal body shape based on a figure rating scale. Cross-tabulations, homogeneity tests and logistic regression models were applied. Results: The percentages of underweight men and women were 16.5% and 34.9% respectively, and the corresponding percentages of being overweight or obese were 26.7% and 13.2% for men and women respectively. Three-quarters of young adults had BSD. Among women, 30.9% of those underweight and 75.5% of those with normal weight desired a slimmer body shape. Overweight men and underweight women with lower education level were more likely to have a mismatch between weight status and BSD than those with higher education level. After controlling for other determinants, underweight women were found to have a higher likelihood to maintain their current body shapes than other women. Men were found to be less likely to have a mismatch between weight status and BSD than women. Conclusions: Overweight and obesity in men and underweight in women were prevalent among Hong Kong young adults. Inappropriate body shape desire might predispose individuals to unhealthy weight loss or gain behaviors. Careful consideration of actual weight status in body shape desire is needed in health promotion and education, especially for underweight and normal weight women and those with a low education level. © 2011 Cheung et al; licensee BioMed Central Ltd.published_or_final_versio

Identification of hepatotropic viruses from plasma using deep sequencing: a next generation diagnostic tool.

We conducted an unbiased metagenomics survey using plasma from patients with chronic hepatitis B, chronic hepatitis C, autoimmune hepatitis (AIH), non-alcoholic steatohepatitis (NASH), and patients without liver disease (control). RNA and DNA libraries were sequenced from plasma filtrates enriched in viral particles to catalog virus populations. Hepatitis viruses were readily detected at high coverage in patients with chronic viral hepatitis B and C, but only a limited number of sequences resembling other viruses were found. The exception was a library from a patient diagnosed with hepatitis C virus (HCV) infection that contained multiple sequences matching GB virus C (GBV-C). Abundant GBV-C reads were also found in plasma from patients with AIH, whereas Torque teno virus (TTV) was found at high frequency in samples from patients with AIH and NASH. After taxonomic classification of sequences by BLASTn, a substantial fraction in each library, ranging from 35% to 76%, remained unclassified. These unknown sequences were assembled into scaffolds along with virus, phage and endogenous retrovirus sequences and then analyzed by BLASTx against the non-redundant protein database. Nearly the full genome of a heretofore-unknown circovirus was assembled and many scaffolds that encoded proteins with similarity to plant, insect and mammalian viruses. The presence of this novel circovirus was confirmed by PCR. BLASTx also identified many polypeptides resembling nucleo-cytoplasmic large DNA viruses (NCLDV) proteins. We re-evaluated these alignments with a profile hidden Markov method, HHblits, and observed inconsistencies in the target proteins reported by the different algorithms. This suggests that sequence alignments are insufficient to identify NCLDV proteins, especially when these alignments are only to small portions of the target protein. Nevertheless, we have now established a reliable protocol for the identification of viruses in plasma that can also be adapted to other patient samples such as urine, bile, saliva and other body fluids

Representative viral proteins identified as high scoring hits during BLASTx alignments of assembled scaffolds.

<p><b>Library</b>: id of library. <b>Length (nt)</b>: Length of scaffold in nucleotides. <b>Average read ends depth</b>: (number of read ends × length of end)/length of scaffold. <b>Unknown read ends</b>: number of read ends previously binned as unknown included in the assembly of scaffold. <b>Target</b>: target sequence ID. <b>Coverage (%)</b>: percentage of target protein included in the alignment. <b>Identities (%)</b>: percentage of amino acids identical in the query and target sequences. <b>E-value</b>: E value.</p

A novel circovirus genome was assembled from sequences in sample AIH.

<p>(<b>A</b>) The schematic of Scaffold2603 shows the 580 reads used for assembly and the location of the two predicted open reading frames. The few reads matched by BLASTn to the YN-BtCV-1 (AEL87784.1) and to the RW-E (NC_013023.1) circovirus reference sequences show red segments on aligned reads that represent mismatches. (<b>B</b>) Phylogenetic tree derived from the alignment of the replicase amino acid sequences from the circoviruses named on the tree branches (the GenBank identifier is also indicated). The dendrogram was calculated using the neighbor joining method and the bootstraps option (1000 replications) of the Mega 4 software (<a href="http://www.megasoftware.net/mega4/mega.html" target="_blank">http://www.megasoftware.net/mega4/mega.html</a>). Vertical branches are arbitrary, while the length of horizontal branches is proportional to calculated mutational distances. Numbers at nodes indicate percentage bootstrap scores.</p

Schematic of bioinformatics pipeline used for processing of NGS libraries.

<p>High quality reads, excluding ribosomal and mitochondrial sequences, were aligned against the taxonomy databases of NCBI using BLASTn (taxonomic classification). Unclassified or ambiguously classified reads, together with virus, phage, and HERV sequences were assembled into scaffolds. Scaffolds were used to query the non-redundant protein database of NCBI using BLASTx to identify viral proteins with similarity to predicted polypeptides in our scaffolds (finding novel viruses). Given the large genomes of NCLDVs, hits to this class of viruses were reanalyzed with the profile hidden Markov model-based algorithm HHblits. PCR and Sanger sequencing were used to confirm the presence of novel viral-like sequences in our samples.</p

Classification of viral reads into families reveals high diversity of viruses in plasma samples.

<p>Reads were classified into families according to the taxonomy databases of NCBI. Solid bars represent DNA libraries and open bars represent RNA libraries. Data is presented in log10 scale. The names of the DNA viral families are shadowed (bottom).</p

NGS identified several classes of viruses that would otherwise remain undetected.

<p>Panels A to C show the mapping of reads along the indicated viral genome, (<b>A</b>) <i>Expected abundant viruses</i> were recovered at high and uniform coverage as illustrated for HBV (NC_003977.1) in library hbvP02D and HCV genotype 2 (NC_009823.1) in library hcvP05. (<b>B</b>) <i>Unexpected abundant viruses</i> were also found at high and uniform coverage, as illustrated for GBV-C/Hepatitis G virus (NC_001710.1) in library aihP01 and TTV 5 isolate TCHN-C1 (AF345523.1) in library nshP01D. (<b>C</b>) A few read ends mapping to HCV genotype 1 (NC_004102.1) were found in library nshP01, which initially tested negative for HCV in serological tests. (<b>D</b>) HCV was PCR amplified in one of the three samples pooled for construction of library nshP01 (upper panel). Additionally, TTV was found in samples NSH and AIH, but not in samples from patients affected by chronic hepatitis C (HCVP03 and HCVP05).</p

Viral read ends represent only a small fraction of libraries from plasma.

<p>Pie charts: Classification of reads from each library into human, bacteria, virus, and unknown categories (HERV and phage sequences are not included as they were found at low frequency, not visible at the scale of the pie charts). The number of clean reads (high-quality sequences excluding ribosomal, mitochondrial, and low complexity sequences; in black), ambiguous reads (in brackets), viral reads (in red), and the density of viral reads per million (in blue) are indicated. Bar graphs: Red bars depict total number of viral reads (normalized to 100%) and green bars represent the percentage thereof that corresponds to the most abundant virus in each library (N.I. not identified; indicates that no predominant virus was identified during BLASTn alignments).</p