Novel Reassortant H5N6 Influenza A Virus from the Lao People's Democratic Republic Is Highly Pathogenic in Chickens.

Avian influenza viruses of H5 subtype can cause highly pathogenic disease in poultry. In March 2014, a new reassortant H5N6 subtype highly pathogenic avian influenza virus emerged in Lao People's Democratic Republic. We have assessed the pathogenicity, pathobiology and immunological responses associated with this virus in chickens. Infection caused moderate to advanced disease in 6 of 6 chickens within 48 h of mucosal inoculation. High virus titers were observed in blood and tissues (kidney, spleen, liver, duodenum, heart, brain and lung) taken at euthanasia. Viral antigen was detected in endothelium, neurons, myocardium, lymphoid tissues and other cell types. Pro-inflammatory cytokines were elevated compared to non-infected birds. Our study confirmed that this new H5N6 reassortant is highly pathogenic, causing disease in chickens similar to that of Asian H5N1 viruses, and demonstrated the ability of such clade 2.3.4-origin H5 viruses to reassort with non-N1 subtype viruses while maintaining a fit and infectious phenotype. Recent detection of influenza H5N6 poultry infections in Lao PDR, China and Viet Nam, as well as six fatal human infections in China, demonstrate that these emergent highly pathogenic H5N6 viruses may be widely established in several countries and represent an emerging threat to poultry and human populations

Tropism of influenza H5N6 virus in experimentally infected chickens.

<p>Immunohistochemistry for influenza A nucleoprotein antigen (brown staining) in representative tissues infected with H5N6 virus. (A) Brain, chicken 1. Viral antigen can be seen in neurons (corners of image) and capillary endothelium (arrows). (B) Heart, chicken 4. Viral antigen is predominantly within cardiomyocytes with some antigen present in capillary endothelium (arrow). (C) Liver, chicken 4. Most of the viral antigen in liver is within sinusoidal endothelium. (D) Lung, chicken 5. Viral antigen is abundant in the respiratory parenchyma of lung and also found in the endothelium of large blood vessels (arrow). (E) Spleen, chicken 6. Viral antigen is found in histiocytic cells, but not lymphocytes. (F) Skin, with feather root and follicle, chicken 5. Viral antigen is present in feather pulp and in capillaries of the dermis (arrow). All scale bars are 100 μm.</p

Cytokine mRNA levels at necropsy in spleen (upper panel) and lung (lower panel) as measured by quantitative RT-PCR.

<p>Data represents the mean fold expression of chicken mRNA relative to each uninfected tissue type after normalizing data to the housekeeping gene GAPDH. Error bars are standard error. * = p<0.05, ** = p<0.01, compared to mRNA levels in control birds; n.s. = not significant.</p

Immune cell proportions in spleens of control (white bars) and infected (black bars) chickens.

<p>Birds were sacrificed and splenocytes stained for the expression of CD3, CD4, and CD8 cell surface antigens. Bars show the mean percentage of T cell sub-populations detected in the spleens of uninfected (control) chickens and those from infected (H5N6) chickens, harvested at the time of euthanasia. Error bars are standard error. ** = p<0.01, compared to T cell proportions in control birds; n.s. = not significant.</p

Analysis of select characteristic amino acids of emergent H5N6 avian influenza viruses.

<p>Analysis of select characteristic amino acids of emergent H5N6 avian influenza viruses.</p

Relative abundance of H5N6 virus in different tissues at post-mortem, as determined by virus titration in cell culture.

<p>Data represents the mean titre (TCID₅₀/mL) of virus from all birds using three replicate titrations of each, but excluding negative values (single colon and muscle tissues samples from two different birds). Error bars are standard error of the mean.</p

Detection of H5N6 virus shedding in infected chickens.

<p>Detection of H5N6 virus shedding in infected chickens.</p