MOESM1 of Pathobiology and innate immune responses of gallinaceous poultry to clade 2.3.4.4A H5Nx highly pathogenic avian influenza virus infection

Additional file 1. Primers used for the qRRT-PCR of innate immune response genes

DataSheet_1_Dual Host and Pathogen RNA-Seq Analysis Unravels Chicken Genes Potentially Involved in Resistance to Highly Pathogenic Avian Influenza Virus Infection.docx

Highly pathogenic avian influenza viruses (HPAIVs) cause severe systemic disease and high mortality rates in chickens, leading to a huge economic impact in the poultry sector. However, some chickens are resistant to the disease. This study aimed at evaluating the mechanisms behind HPAIV disease resistance. Chickens of different breeds were challenged with H7N1 HPAIV or clade 2.3.4.4b H5N8 HPAIV, euthanized at 3 days post-inoculation (dpi), and classified as resistant or susceptible depending on the following criteria: chickens that presented i) clinical signs, ii) histopathological lesions, and iii) presence of HPAIV antigen in tissues were classified as susceptible, while chickens lacking all these criteria were classified as resistant. Once classified, we performed RNA-Seq from lung and spleen samples in order to compare the transcriptomic signatures between resistant and susceptible chickens. We identified minor transcriptomic changes in resistant chickens in contrast with huge alterations observed in susceptible chickens. Interestingly, six differentially expressed genes were downregulated in resistant birds and upregulated in susceptible birds. Some of these genes belong to the NF-kappa B and/or mitogen-activated protein kinase signaling pathways. Among these six genes, the serine protease-encoding gene PLAU was of particular interest, being the most significantly downregulated gene in resistant chickens. Expression levels of this protease were further validated by RT-qPCR in a larger number of experimentally infected chickens. Furthermore, HPAIV quasi-species populations were constructed using 3 dpi oral swabs. No substantial changes were found in the viral segments that interact with the innate immune response and with the host cell receptors, reinforcing the role of the immune system of the host in the clinical outcome. Altogether, our results suggest that an early inactivation of important host genes could prevent an exaggerated immune response and/or viral replication, conferring resistance to HPAIV in chickens.</p

Diverse infectivity, transmissibility, and pathobiology of clade 2.3.4.4 H5Nx highly pathogenic avian influenza viruses in chickens

Clade 2.3.4.4 Eurasian lineage H5Nx highly pathogenic avian influenza virus (HPAIV) has become the globally dominant clade and caused global outbreaks since 2014. The clade 2.3.4.4 viruses have evolved into eight hemagglutinin subgroups (2.3.4.4a-h). In this study, we evaluated the infectivity, pathobiology, and transmissibility of seven clade 2.3.4.4 viruses (two 2.3.4.4a, two 2.3.4.4b, one 2.3.4.4c and two 2.3.4.4e) in chickens. The two clade 2.3.4.4e viruses caused 100% mortality and transmissibility in chickens. However, clade 2.3.4.4a and c viruses showed 80–90% mortality and 67% transmissibility. Clade 2.3.4.4b viruses showed 100% mortality, but no transmission to co-housed chickens was observed based on lack of seroconversion. All the infected chickens died showing systemic infection, irrespective of subgroup. The results highlight that all the clade 2.3.4.4 HPAIVs used in this study caused high mortality in infected chickens, but the transmissibility of the viruses in chickens was variable in contrast to that of previous Eurasian-lineage H5N1 HPAIVs. Changes in the pathogenicity and transmissibility of clade 2.3.4.4 HPAIVs warrant careful monitoring of the viruses to establish effective control strategies.</p

Oral shedding from experimentally infected falcons with avian influenza virus.

<p>Viral RNA shedding detected by RRT-PCR in oropharyngeal swabs of falcons infected via the feeding route or via the nasochoanal route. Ct, cycle of threshold. <b>A.</b> Falcons infected with A/<i>Anas plathyrhynchos</i>/Spain/1877/2009 (H7N2) LPAI virus and euthanized at 11 dpi. <b>B.</b> Falcons infected with A/Great crested grebe/Basque Country/06.03249/2006 (H5N1) HPAI virus.</p

Distribution of α-2,3 and α-2,6 receptors in the digestive tract of falcons demonstrated by means of MAAII and SNA lectin histochemistry.

<p><b>A.</b> Proventriculus stained by MAAII lectin. <b>B.</b> Duodenum stained by MAAII lectin. <b>C.</b> Rectum stained by MAAII lectin. <b>D.</b> Proventriculus stained by SNA lectin. <b>E.</b> Duodenum stained by SNA lectin. <b>F.</b> Rectum stained by SNA lectin.</p

Spatiotemporal Phylogenetic Analysis and Molecular Characterisation of Infectious Bursal Disease Viruses Based on the VP2 Hyper-Variable Region

<div><p>Background</p><p>Infectious bursal disease is a highly contagious and acute viral disease caused by the infectious bursal disease virus (IBDV); it affects all major poultry producing areas of the world. The current study was designed to rigorously measure the global phylogeographic dynamics of IBDV strains to gain insight into viral population expansion as well as the emergence, spread and pattern of the geographical structure of very virulent IBDV (vvIBDV) strains.</p><p>Methodology/Principal Findings</p><p>Sequences of the hyper-variable region of the VP2 (HVR-VP2) gene from IBDV strains isolated from diverse geographic locations were obtained from the GenBank database; Cuban sequences were obtained in the current work. All sequences were analysed by Bayesian phylogeographic analysis, implemented in the Bayesian Evolutionary Analysis Sampling Trees (BEAST), Bayesian Tip-association Significance testing (BaTS) and Spatial Phylogenetic Reconstruction of Evolutionary Dynamics (SPREAD) software packages. Selection pressure on the HVR-VP2 was also assessed. The phylogeographic association-trait analysis showed that viruses sampled from individual countries tend to cluster together, suggesting a geographic pattern for IBDV strains. Spatial analysis from this study revealed that strains carrying sequences that were linked to increased virulence of IBDV appeared in Iran in 1981 and spread to Western Europe (Belgium) in 1987, Africa (Egypt) around 1990, East Asia (China and Japan) in 1993, the Caribbean Region (Cuba) by 1995 and South America (Brazil) around 2000. Selection pressure analysis showed that several codons in the HVR-VP2 region were under purifying selection.</p><p>Conclusions/Significance</p><p>To our knowledge, this work is the first study applying the Bayesian phylogeographic reconstruction approach to analyse the emergence and spread of vvIBDV strains worldwide.</p></div

Average distribution of nucleoprotein antigen, as determined by immunohistochemistry, in tissues sampled from falcons infected via the nasochoanal route with A/Great crested grebe/Basque Country/06.03249/2006 (H5N1) HPAI virus.

<p>− = no positive cells; + = single positive cells; ++ = scattered groups of positive cells; +++ = widespread positivity.</p><p>NSL, no significant lesions; nd, not determined.</p

Distribution of α-2,3 and α-2,6 receptors in the respiratory tract of falcons demonstrated by means of MAAII and SNA lectin histochemistry.

<p><b>A.</b> Nasal turbinates stained by MAAII lectin. <b>B.</b> Trachea stained by MAAII lectin. <b>C.</b> Lung stained by MAAII lectin. <b>D.</b> Nasal turbinates stained by SNA lectin. <b>E.</b> Trachea stained by SNA lectin. <b>F.</b> Lung stained by SNA lectin.</p

Brain tissue from an experimentally H5N1 HPAI virus-inoculated falcon dead at 5 dpi.

<p><b>A.</b> Focal area of malacia in the cortex (HE stain). <b>B.</b> Immunohistochemical staining of influenza A virus antigen in brain tissue.</p

Pancreatic macroscopic lesions from a falcon experimentally infected with highly pathogenic avian influenza virus H5N1.

<p>Multifocal hemorrhagic necrosis in the pancreas of a falcon infected via the nasochoanal route with A/Great crested grebe/Basque Country/06.03249/2006 H5N1 HPAI virus (5 dpi).</p