Highly pathogenic avian influenza virus infection in chickens but not ducks is associated with elevated host immune and pro-inflammatory responses.

Highly pathogenic avian influenza (HPAI) H5N1 viruses cause severe infection in chickens at near complete mortality, but corresponding infection in ducks is typically mild or asymptomatic. To understand the underlying molecular differences in host response, primary chicken and duck lung cells, infected with two HPAI H5N1 viruses and a low pathogenicity avian influenza (LPAI) H2N3 virus, were subjected to RNA expression profiling. Chicken cells but not duck cells showed highly elevated immune and pro-inflammatory responses following HPAI virus infection. HPAI H5N1 virus challenge studies in chickens and ducks corroborated the in vitro findings. To try to determine the underlying mechanisms, we investigated the role of signal transducer and activator of transcription-3 (STAT-3) in mediating pro-inflammatory response to HPAIV infection in chicken and duck cells. We found that STAT-3 expression was down-regulated in chickens but was up-regulated or unaffected in ducks in vitro and in vivo following H5N1 virus infection. Low basal STAT-3 expression in chicken cells was completely inhibited by H5N1 virus infection. By contrast, constitutively active STAT-3 detected in duck cells was unaffected by H5N1 virus infection. Transient constitutively-active STAT-3 transfection in chicken cells significantly reduced pro-inflammatory response to H5N1 virus infection; on the other hand, chemical inhibition of STAT-3 activation in duck cells increased pro-inflammatory gene expression following H5N1 virus infection. Collectively, we propose that elevated pro-inflammatory response in chickens is a major pathogenicity factor of HPAI H5N1 virus infection, mediated in part by the inhibition of STAT-3.

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.