Mammalian innate resistance to highly pathogenic avian influenza H5N1 virus infection is mediated through reduced proinflammation and infectious virus release.

Respiratory epithelial cells and macrophages are the key innate immune cells that play an important role in the pathogenesis of influenza A virus infection. We found that these two cell types from both human and pig showed comparable susceptibilities to initial infection with a highly pathogenic avian influenza (HPAI) H5N1 virus (A/turkey/Turkey/1/05) and a moderately pathogenic human influenza H1N1 virus (A/USSR/77), but there were contrasting differences in host innate immune responses. Human cells mounted vigorous cytokine (tumor necrosis factor alpha [TNF-α] and interleukin-6 [IL-6]) and chemokine (CXCL9, CXCL10, and CXCL11) responses to H5N1 virus infection. However, pig epithelial cells and macrophages showed weak or no TNF-α and chemokine induction with the same infections. The apparent lack of a strong proinflammatory response, corroborated by the absence of TNF-α induction in H5N1 virus-challenged pigs, coincided with greater cell death and the reduced release of infectious virus from infected pig epithelial cells. Suppressor of cytokine signaling 3 (SOCS3), a protein suppressor of the JAK-STAT pathway, was constitutively highly expressed and transcriptionally upregulated in H5N1 virus-infected pig epithelial cells and macrophages, in contrast to the corresponding human cells. The overexpression of SOCS3 in infected human macrophages dampened TNF-α induction. In summary, we found that the reported low susceptibility of pigs to contemporary Eurasian HPAI H5N1 virus infections coincides at the level of innate immunity of respiratory epithelial cells and macrophages with a reduced output of viable virus and an attenuated proinflammatory response, possibly mediated in part by SOCS3, which could serve as a target in the treatment or prevention of virus-induced hypercytokinemia, as observed for humans.

Rapid death of duck cells infected with influenza: a potential mechanism for host resistance to H5N1.

Aquatic birds are the natural reservoir for most subtypes of influenza A, and a source of novel viruses with the potential to cause human pandemics, fatal zoonotic disease or devastating epizootics in poultry. It is well recognised that waterfowl typically show few clinical signs following influenza A infection, in contrast, terrestrial poultry such as chickens may develop severe disease with rapid death following infection with highly pathogenic avian influenza. This study examined the cellular response to influenza infection in primary cells derived from resistant (duck) and susceptible (chicken) avian hosts. Paradoxically, we observed that duck cells underwent rapid cell death following infection with low pathogenic avian H2N3, classical swine H1N1 and 'classical' highly pathogenic H5N1 viruses. Dying cells showed morphological features of apoptosis, increased DNA fragmentation and activation of caspase 3/7. Following infection of chicken cells, cell death occurred less rapidly, accompanied by reduced DNA fragmentation and caspase activation. Duck cells produced similar levels of viral RNA but less infectious virus, in comparison with chicken cells. Such rapid cell death was not observed in duck cells infected with a contemporary Eurasian lineage H5N1 fatal to ducks. The induction of rapid death in duck cells may be part of a mechanism of host resistance to influenza A, with the loss of this response leading to increased susceptibility to emergent strains of H5N1. These studies provide novel insights that should help resolve the long-standing enigma of host-pathogen relationships for highly pathogenic and zoonotic avian influenza.

18S rRNA is a reliable normalisation gene for real time PCR based on influenza virus infected cells.

BACKGROUND: One requisite of quantitative reverse transcription PCR (qRT-PCR) is to normalise the data with an internal reference gene that is invariant regardless of treatment, such as virus infection. Several studies have found variability in the expression of commonly used housekeeping genes, such as beta-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), under different experimental settings. However, ACTB and GAPDH remain widely used in the studies of host gene response to virus infections, including influenza viruses. To date no detailed study has been described that compares the suitability of commonly used housekeeping genes in influenza virus infections. The present study evaluated several commonly used housekeeping genes [ACTB, GAPDH, 18S ribosomal RNA (18S rRNA), ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide (ATP5B) and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C1 (subunit 9) (ATP5G1)] to identify the most stably expressed gene in human, pig, chicken and duck cells infected with a range of influenza A virus subtypes. RESULTS: The relative expression stability of commonly used housekeeping genes were determined in primary human bronchial epithelial cells (HBECs), pig tracheal epithelial cells (PTECs), and chicken and duck primary lung-derived cells infected with five influenza A virus subtypes. Analysis of qRT-PCR data from virus and mock infected cells using NormFinder and BestKeeper software programmes found that 18S rRNA was the most stable gene in HBECs, PTECs and avian lung cells. CONCLUSIONS: Based on the presented data from cell culture models (HBECs, PTECs, chicken and duck lung cells) infected with a range of influenza viruses, we found that 18S rRNA is the most stable reference gene for normalising qRT-PCR data. Expression levels of the other housekeeping genes evaluated in this study (including ACTB and GPADH) were highly affected by influenza virus infection and hence are not reliable as reference genes for RNA normalisation.

Predicting in vivo starch digestibility coefficients in newly weaned piglets from in vitro assessment of diets using multivariate analysis.

The study was based on correlating a dataset of in vivo mean starch digestibility coefficients obtained in the immediate post-weaning phase of piglets with a range of dietary in vitro variables. The paper presents a model that predicts (R2 0.71) in vivo average starch digestibility coefficients in the 0.5 small-intestinal region of newly weaned piglets fed cereal-based diets using seven in vitro variables describing starch properties that are fundamentally associated with the quality of feed materials, i.e. hydration, structure and amylolytic digestion. The variables were: Rapid Visco Analyser (RVA; measures the viscosity of materials when sheared under defined hydration and temperature regimens); RVA end viscosity; RVA (gelatinisation) peak viscosity; DeltaH (gelatinisation enthalpy that provides an estimate of helical order or degree of crystallinity in starch); water solubility index (WSI; that denotes the amount of soluble polysaccharides released from starch granules to the aqueous phase); grain endogenous amylase (concentration of endogenous alpha-amylase in cereals, assessed by pasting cereal flours in 25 g of AgNO3, an amylase inhibitor v. water using RVA).

Comparative distribution of human and avian type sialic acid influenza receptors in the pig.

BACKGROUND: A major determinant of influenza infection is the presence of virus receptors on susceptible host cells to which the viral haemagglutinin is able to bind. Avian viruses preferentially bind to sialic acid alpha2,3-galactose (SAalpha2,3-Gal) linked receptors, whereas human strains bind to sialic acid alpha2,6-galactose (SAalpha2,6-Gal) linked receptors. To date, there has been no detailed account published on the distribution of SA receptors in the pig, a model host that is susceptible to avian and human influenza subtypes, thus with potential for virus reassortment. We examined the relative expression and spatial distribution of SAalpha2,3-GalG(1-3)GalNAc and SAalpha2,6-Gal receptors in the major organs from normal post-weaned pigs by binding with lectins Maackia amurensis agglutinins (MAA II) and Sambucus nigra agglutinin (SNA) respectively. RESULTS: Both SAalpha2,3-Gal and SAalpha2,6-Gal receptors were extensively detected in the major porcine organs examined (trachea, lung, liver, kidney, spleen, heart, skeletal muscle, cerebrum, small intestine and colon). Furthermore, distribution of both SA receptors in the pig respiratory tract closely resembled the published data of the human tract. Similar expression patterns of SA receptors between pig and human in other major organs were found, with exception of the intestinal tract. Unlike the limited reports on the scarcity of influenza receptors in human intestines, we found increasing presence of SAalpha2,3-Gal and SAalpha2,6-Gal receptors from duodenum to colon in the pig. CONCLUSIONS: The extensive presence of SAalpha2,3-Gal and SAalpha2,6-Gal receptors in the major organs examined suggests that each major organ may be permissive to influenza virus entry or infection. The high similarity of SA expression patterns between pig and human, in particular in the respiratory tract, suggests that pigs are not more likely to be potential hosts for virus reassortment than humans. Our finding of relative abundance of SA receptors in the pig intestines highlights a need for clarification on the presence of SA receptors in the human intestinal tract.

Understanding obesity among companion dogs: New measures of owner's beliefs and behaviour and associations with body condition scores.

This research aimed to improve our understanding of how owners' beliefs and behaviour are associated with obesity in companion dogs. To do this, we employed new theoretical frameworks and integrated previously reported measures to curate a collection of brief, user-friendly self-report measures to assess owner factors. The reliability and validity of these was examined in two phases of empirical research, each with a cross-sectional questionnaire design that also examined the validity of assessing body condition score (BCS) from photographs submitted by owners. Phase 1 (n = 47 dog owners from France) found that the brief owner-report measures correlated with the long-form measures (all correlations except one exceeded r = 0.70). BCS as coded from photographs were highly correlated with a vet's assessment of the same dogs (r = 0.67). Phase 2 (n = 3339 dog owners from France, Germany, the UK, Italy, and Russia) investigated which measures are associated with obesity among companion dogs. Perceptions of the dog's vulnerability to the threat of obesity, perceived weight status, perceived costs associated with ownership, normative beliefs about feeding, social support from friends, and being in the precontemplation stage of change predicted BCS alongside demographic factors (e.g., dog's age, neutered status). Taken together, the findings provide a method for assessing a wide range of factors that may be associated with obesity among companion dogs and point to potential targets for interventions designed to reduce obesity.

Differences in influenza virus receptors in chickens and ducks: Implications for interspecies transmission.

Avian influenza viruses are considered to be key contributors to the emergence of human influenza pandemics. A major determinant of infection is the presence of virus receptors on susceptible cells to which the viral haemagglutinin is able to bind. Avian viruses preferentially bind to sialic acid alpha2,3-galactose (SAalpha2,3-Gal) linked receptors, whereas human strains bind to sialic acid alpha2,6-galactose (SAalpha2,6-Gal) linked receptors. While ducks are the major reservoir for influenza viruses, they are typically resistant to the effects of viral infection, in contrast to the frequently severe disease observed in chickens. In order to understand whether differences in receptors might contribute to this observation, we studied the distribution of influenza receptors in organs of ducks and chickens using lectin histochemistry with linkage specific lectins and receptor binding assays with swine and avian influenza viruses. Although the intestinal epithelial cells of both species expressed only SAalpha2,3-Gal receptors, we found widespread presence of both SAalpha2,6-Gal and SAalpha2,3-Gal receptors in many organs of both chickens and ducks. Co-expression of both receptors may allow infection of cells with both avian and human viruses and so present a route to genetic reassortment. There was a marked difference in the primary receptor type in the trachea of chickens and ducks. In chicken trachea, SAalpha2,6-Gal was the dominant receptor type whereas in ducks SAalpha2,3-Gal receptors were most abundant. This suggests that chickens could be more important as an intermediate host for the generation of influenza viruses with increased ability to bind to SAalpha2,6-Gal receptors and thus greater potential for infection of humans. Chicken tracheal and intestinal epithelial cells also expressed a broader range of SAalpha2,3-Gal receptors (both beta(1-4)GlcNAc and beta(1-3)GalNAc subtypes) in contrast to ducks, which suggests that they may be able to support infection with a broader range of avian influenza viruses.