H5N1 Highly Pathogenic Avian Influenza Virus Research Articles

Pathology and virology of natural highly pathogenic avian influenza H5N8 infection in wild Common buzzards (Buteo buteo)

Highly pathogenic avian influenza (HPAI) in wild birds is a major emerging disease, and a cause of increased mortality during outbreaks. The Common buzzard (Buteo buteo) has a considerable chance of acquiring the infection and therefore may function as bio-sentinel for the presence of virus in wildlife. This study aimed to determine the virus distribution and associated pathological changes in the tissues of Common buzzards that died with HPAI H5 virus infection during the 2020–2021 epizootic. Eleven freshly dead, HPAI H5 virus-positive Common buzzards were necropsied. Based on RT-PCR, all birds were systemically infected with HPAI H5N8 virus, as viral RNA was detected in cloacal and pharyngeal swabs and in all 10 selected tissues of the birds, with mean Ct values per tissue ranging from 22 for heart to 32 for jejunum. Based on histology and immunohistochemistry, the most common virus-associated pathological changes were necrotizing encephalitis (9/11 birds) and necrotizing myocarditis (7/11 birds). The proventriculus of two birds showed virus-associated necrosis, indicating tropism of this virus for the digestive tract. Our advice is to collect at least a miniset of samples including brain, heart, liver, and spleen, as these tissues were positive both by RT-PCR and for virus-antigen-associated lesions.

The highly pathogenic H5N1 avian influenza virus induces the mitogen-activated protein kinase signaling pathway in the trachea of two Ri chicken lines.

ObjectiveThe highly pathogenic avian influenza virus (HPAIV) is a threat to the poultry industry and economy and remains a potential source of pandemic infection in humans. Antiviral genes are considered a potential factor for studies on HPAIV resistance. Therefore, in this study, we investigated gene expression related to the mitogen-activated protein kinase (MAPK) signaling pathway by comparing non-infected, HPAI-infected resistant, and susceptible Ri chicken lines.MethodsResistant (Mx/A; BF2/B21) and susceptible Ri chickens (Mx/G; BF2/B13) were selected by genotyping the Mx and BF2 genes. Then, the tracheal tissues of non-infected and HPAIV H5N1 infected chickens were collected for RNA sequencing.ResultsA gene set overlapping test between the analyzed differentially expressed genes (DEGs) and functionally categorized genes was performed, including biological processes of the gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. A total of 1,794 DEGs were observed between control and H5N1-infected resistant Ri chickens, 432 DEGs between control and infected susceptible Ri chickens, and 1,202 DEGs between infected susceptible and infected resistant Ri chickens. The expression levels of MAPK signaling pathway-related genes (including MyD88, NF-κB, AP-1, c-fos, Jun, JunD, MAX, c-Myc), cytokines (IL-1β, IL-6, IL-8), type I interferons (IFN-α, IFN-β), and IFN-stimulated genes (Mx1, CCL19, OASL, and PRK) were higher in H5N1-infected than in non-infected resistant Ri chickens. MyD88, Jun, JunD, MAX, cytokines, chemokines, IFNs, and IFN-stimulated expressed genes were higher in resistant-infected than in susceptible-infected Ri chickens.ConclusionResistant Ri chickens showed higher antiviral activity compared to susceptible Ri chickens, and H5N1-infected resistant Ri chickens had immune responses and antiviral activity (cytokines, chemokines, interferons, and IFN-stimulated genes), which may have been induced through the MAPK signaling pathway in response to H5N1 infection.

Susceptibility of herons (family: Ardeidae) to clade 2.3.2.1 H5N1 subtype high pathogenicity avian influenza virus

ABSTRACT The pathogenicity of the H5 subtype high pathogenicity avian influenza viruses (HPAIVs) in Ardeidae bird species has not been investigated yet, despite the increasing infections reported. Therefore, the present study aimed to examine the susceptibility of the Ardeidae species, which had already been reported to be susceptible to HPAIVs, to a clade 2.3.2.1 H5N1 HPAIV. Juvenile herons (four grey herons, one intermediate egret, two little egrets, and three black-crowned night herons) were intranasally inoculated with 106 50% egg infectious dose of the virus and observed for 10 days. Two of the four grey herons showed lethargy and conjunctivitis; among them, one died at 6 days post-inoculation (dpi). The viruses were transmitted to the other two cohoused naïve grey herons. Some little egrets and black-crowned night herons showing neurological disorders died at 4–5 dpi; these birds mainly shed the virus via the oral route. The viruses predominantly replicated in the brains of birds that died of infection. Seroconversion was observed in most surviving birds, except some black-crowned night herons. These results demonstrate that most Ardeidae species are susceptible to H5 HPAIVs, sometimes with lethal effects. Herons are mostly colonial and often share habitats with Anseriformes, natural hosts of influenza A viruses; therefore, the risks of cluster infection and contribution to viral dissemination should be continuously evaluated. RESEARCH HIGHLIGHTS Clade 2.3.2.1 H5N1 HPAIV causes lethal infections in Ardeidae sp. Viruses are transmitted among grey herons. Some herons with HPAIV showed conjunctivitis or neurological symptoms. HPAIV systemically replicated in herons tissues.

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

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.

Emergence of novel avian origin H7N9 viruses after introduction of H7‐Re3 and rLN79 vaccine strains to China

In early 2021, roughly 6 months after the H7N9 H7-Re3 and H7N9 rLN79 vaccine strains were introduced into China, we monitored a number of H7N9 subtype avian influenza viruses, which could have escaped vaccine-induced immunity in live poultry markets (LPMs) in Yunnan, Hebei, Shanxi and Guangdong provinces, China. To investigate whether these viruses were a novel H7N9 variant of highly pathogenic avian influenza (HPAI) virus and whether they had the potential for further spread, we characterized the genetic evolution, antigenic divergence and pathogenicity of the viruses in the context of vaccine immunity. The results show further diversification in the HA gene of newly isolated HPAI H7N9 viruses compared with antigenic variants that emerged after the period of 2017-2019. There were clear antigenic differences between current vaccines and these viruses, and SPF broilers under vaccine protection could not resist virus challenges. Our study demonstrates that the current vaccine has insufficient protective capacity against the novel H7N9 variants under experimental conditions. A novel H7N9 immune escape virus has emerged. Faced with potential outbreaks, we should strengthen surveillance and update vaccine strains.

Baculovirus-derived influenza virus-like particle confers complete protection against lethal H7N9 avian influenza virus challenge in chickens and mice

Currently, highly pathogenic avian influenza (HPAI) H7N9 viruses still pose a potential pandemic threat. Influenza virus-like particle (VLP) is one of the most promising vaccine strategies to complement traditional egg-dependent vaccines. Here, we generated a H7N9 VLP vaccine candidate by baculovirus expression system and evaluated its efficacy in chickens and mice. The H7N9 VLP was produced through co-infection of Sf9 insect cells with three recombinant baculoviruses expressing individual HA, NA and M1 gene of the HPAI H7N9 virus A/chicken/Guangdong/GD15/2016. Intramuscular immunization of the H7N9 VLP elicited robust antibody immune responses and conferred complete clinical protection against lethal H7N9 virus challenge both in chickens and mice. Meanwhile, H7N9 VLP significantly restrained virus shedding and dramatically alleviated pulmonary lesions caused by H7N9 virus infection in birds and mice. Interestingly, chicken antibodies induced by the H7N9 VLP also had a good cross-reactivity with H7N9 field strains isolated in different years. In addition, vaccination with the H7N9 VLP elicited high T cell immunity in mouse lung, evidenced by significantly upregulated expression of IL-2, IL-4 and IFN-γ. Furthermore, the H7N9 VLP significantly decreased the expression of some key inflammatory cytokines, such as IL6, RANTES and TNF-α in mouse lung, which may partially account for its contribution to alleviate lung pathology. Therefore, our study describes the good efficacy of the HA + NA + M1-containing H7N9 VLP both in chicken and mice models, highlighting the potential of VLP-based vaccine as a critical alternative of traditional egg-based vaccine for control of H7N9 influenza virus in both humans and poultry.

Pathogenicity of three genetically distinct and highly pathogenic Egyptian H5N8 avian influenza viruses in chickens

In late 2016, Egypt encountered multiple cases of the highly pathogenic avian influenza (HPAI) virus of the H5N8 subtype. In a previous study, three distinct genotypes, including A/common-coot/Egypt/CA285/2016 (H5N8) (CA285), A/duck/Egypt/SS19/2017 (H5N8) (SS19), and A/duck/Egypt/F446/2017 (H5N8) (F446), were isolated from wild birds, a backyard, and a commercial farm, respectively, during the first wave of infection. In this current study, we investigated the differences in the pathogenicity, replication and transmissibility of the three genotypes and A/chicken/Egypt/15S75/2015 (H5N1) (S75) was used as the control. The intravenous pathogenicity index was between 2.68 and 2.9. The chicken lethal dose 50 values of F446, SS19 and CA285 were 103.7, 103.7, an 104 with a natural route of infection, respectively. These strains took longer than S75 to cause death when infection was carried out through the natural route (HPAI H5N1). After inoculation with the original concentration of 105 and 106 egg infective dose 50 (EID50), F446 had a higher mortality rate with short mean death times of 4, and 7 days, respectively compared with the other H5N8 viruses. Chickens inoculated with F446 and contacted exposed chickens infected with F446 showed the highest viral titer with remarkable differences in all H5N8 tested swabs at 2-4 days postinfection (dpi) compared to S75 at 2 dpi. This indicates that F446 had a more efficient transmission and spread from contact exposed birds to other birds. All H5N8 viruses were able to replicate systematically in all organs (trachea, brain, lung, and spleen) of the chicken with high viral titer with significantly different and more pathological changes observed in F446 than in other H5N8 viruses at 2 and 4 dpi. Compared with H5N1, we recorded a significantly high viral titer in the samples obtained from the lung, brain and both cloacal and tracheal swabs at 2 and 4 dpi, respectively and in the samples obtained from the spleen at 2 and 4 dpi among the experimental chicken. The comparative pathogenesis study revealed that in comparison with the other HPAI H5N8 viruses, the genotype F446 was more pathogenic, and showed more efficient viral replication and transmissibility in chickens in Egypt. The genotype F446 also showed a high viral titer than HPAI H5N1 and short mean death time at the third day after inoculation with 106 and 105 EID50, which revealed a conservation of certain H5N8 genotypes and a decrease in the incidence of H5N1.

Rapid Emergence of the Reassortant 2.3.4.4b H5N2 Highly Pathogenic Avian Influenza Viruses in a Live Poultry Market in Xinjiang, Northwest China

Live poultry markets (LPMs) play a key role in reassorting and spreading avian influenza viruses (AIVs). In 2018, four strains of H5N2 AIVs were isolated from domestic ducks (Anas platyrhynchos) during AIV surveillance from the LPM in Urumqi, Xinjiang, China. All gene segments of the isolates were amplified by reverse transcription-PCR and sequenced; then, the viral genetic mutations, reassortant, and origin were analyzed. Higher nucleotide identities were observed among each gene of the isolates, indicating a common ancestor. The hemagglutinin (HA) genes of the isolates all classified into the clade 2.3.4.4b; the HA, matrix protein (MP), and nonstructural protein (NS) genes were all clustered together with the local H5N6 highly pathogenic AIVs (HPAIVs) identified in the same LPM of Urumqi in July 2017; the neuraminidase albumen, polymerase basic proteins 1 and 2, polymerase acidic protein, and nucleocapsid protein genes (NA, PB1, PB2, PA, and NP) all had close phylogenetic relationships with the local H9N2 AIVs identified in the same LPM from September to October 2018. Multiple basic amino acids were present at the cleavage site of the HA protein, which was associated with HPAIVs. These results indicated that the reassortant clade 2.3.4.4b H5N2 HPAIVs were rapidly generated from reassortment between the H5N6 and H9N2 AIVs in the local LPM of Urumqi in 2018.

H9N2 virus-derived M1 protein promotes H5N6 virus release in mammalian cells: Mechanism of avian influenza virus inter-species infection in humans.

H5N6 highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4 not only exhibits unprecedented intercontinental spread in poultry, but can also cause serious infection in humans, posing a public health threat. Phylogenetic analyses show that 40% (8/20) of H5N6 viruses that infected humans carried H9N2 virus-derived internal genes. However, the precise contribution of H9N2 virus-derived internal genes to H5N6 virus infection in humans is unclear. Here, we report on the functional contribution of the H9N2 virus-derived matrix protein 1 (M1) to enhanced H5N6 virus replication capacity in mammalian cells. Unlike H5N1 virus-derived M1 protein, H9N2 virus-derived M1 protein showed high binding affinity for H5N6 hemagglutinin (HA) protein and increased viral progeny particle release in different mammalian cell lines. Human host factor, G protein subunit beta 1 (GNB1), exhibited strong binding to H9N2 virus-derived M1 protein to facilitate M1 transport to budding sites at the cell membrane. GNB1 knockdown inhibited the interaction between H9N2 virus-derived M1 and HA protein, and reduced influenza virus-like particles (VLPs) release. Our findings indicate that H9N2 virus-derived M1 protein promotes avian H5N6 influenza virus release from mammalian, in particular human cells, which could be a major viral factor for H5N6 virus cross-species infection.

Phylogenetic Analysis of H5N8 Highly Pathogenic Avian Influenza Viruses in Ukraine, 2016-2017.

Highly pathogenic avian influenza viruses (HPAIV) can be carried long distances by migratory wild birds and by poultry trade. Highly pathogenic avian influenza (HPAI) is often lethal in domestic poultry and can sporadically infect and cause severe respiratory or systemic disease in other species including humans. Since 2003, the H5 subtype of HPAIV have spread from epicenters in China to neighboring regions in East and Southeast Asia, and across Central Asia to the Indian subcontinent, Europe, Africa, and North America. Outbreaks of H5N1 HPAIV struck poultry in Ukraine in 2005. In 2016, A H5N8 clade 2.3.4.4b HPAIV outbreaks occurred in wild and domestic birds in Ukraine concurrently with outbreaks in Central Europe, Russia, and the Middle East. We report outbreaks of HPAI in domestic backyard poultry in (2016-2017) in the southern region of Ukraine, in proximity to mass gathering sites for migratory waterfowl including mute swans (Cygnus olor). All eight genome segments of three novel H5N8 HPAIV isolated in November 2016 from two domestic backyard chickens (Gallus gallus) and one backyard mallard duck (Anas platyrhynchos) found dead of HPAI in Azov-Black Sea region of Ukraine were cladistically related to H5N8 2.3.4.4b HPAI viruses isolated from wild shelduck (Tadorna tadorna) and white-fronted goose (Anser albifrons) in Askania Nova Biopreserve (Kherson district, Ukraine) in 2016-2017 and to other contemporary H5N8 HPAIV strains sequenced from wild birds and poultry in Eurasia. Amino acid variations in hemagglutinin were outside of the polybasic cleavage site (PLREKRRKR/GLF), and D224G suggested avian-like receptor binding specificity; neuraminidase did not have mutations characteristic of oseltamivir drug resistance. Outbreaks of HPAI in Ukraine highlight the continual need for biosurveillance and genomic sequencing of avian influenza viruses along wild bird flyways and interfaces with domestic poultry in Eurasia.

Proteomic analysis of differential expression of lung proteins in response to highly pathogenic avian influenza virus infection in chickens

Elucidation of the molecular pathogenesis underlying virus-host interactions is important for the development of new diagnostic and therapeutic strategies against highly pathogenic avian influenza (HPAI) virus infection in chickens. However, the pathogenesis of HPAI virus in chickens is not completely understood. To identify the intracellular signaling pathways and critical host proteins associated with influenza pathogenesis, we analyzed the lung proteome of a chicken infected with HPAI H5N1 virus (A/duck/India/02CA10/2011/Agartala). Mass spectrometry data sets were searched against the chicken UniProt reference database. At the local false discovery rate level of 5%, a total of 3313 proteins with the presence of at least one unique peptide were identified in the chicken lung proteome datasets. Differential expression analysis of these proteins showed that 247 and 1754 proteins were downregulated at 12 h and 48 h postinfection, respectively. We observed expression of proteins of the predominant signaling pathways, including Toll-like receptors (TLRs), retinoic acid-inducible gene I-like receptors (RLRs), NOD-like receptors (NLRs), and JAK-STAT signaling. Activation of these pathways is associated with the cytokine storm effect and thus may be the cause of the severity of HPAI H5N1 infection in chickens. We also observed the expression of myeloid differentiation primary response protein (MyD88), inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB), interleukin 1 receptor associated kinase 4 (IRAK4), RELA proto-oncogene NF-κB subunit (RELA), and mitochondrial antiviral signaling protein (MAVS), which are involved in critical signaling pathways, as well as other, less-commonly identified proteins such as hepatocyte nuclear factor 4 alpha (HNF4A), ELAV-like RNA binding protein 1 (ELAVL1), fibronectin 1 (FN1), COP9 signalosome subunit 5 (COPS5), cullin 1 (CUL1), breast cancer type 1 susceptibility protein (BRCA1), and the FYN proto-oncogene Src family tyrosine kinase (FYN) as main hub proteins that might play important roles in influenza pathogenesis in chickens. In summary, we identified the signaling pathways and the proteomic determinants associated with disease pathogenesis in chickens infected with HPAI H5N1 virus.

Examination of the protective efficacy of two avian influenza H5 vaccines against clade 2.3.4.4b H5N8 highly pathogenic avian influenza virus in commercial broilers

The highly pathogenic avian influenza (HPAI) H5N8 virus of clade 2.3.4.4 was detected in 2017 in Egypt, which is one of the few countries using vaccination as a control strategy in poultry farms. This study was conducted to evaluate the efficacy of the commercial recombinant turkey herpes virus-H5 (rHVT-H5) vaccine (clade 2.2), alone or in combination with commercial inactivated reverse genetically engineered H5N1 vaccine (rgH5N1) (clade 2.2), in preventing the genetically distinct HPAI H5N8 virus of clade 2.3.4.4b in commercial broiler chickens. Four experimental groups of chickens were used as follows: G1, non-vaccinated and non-challenged; G2, non-vaccinated and challenged; G3, vaccinated with rHVT-H5; and G4, prime–boost vaccinated with rHVT-H5/rgH5N1. For challenge with the Egyptian HPAI H5N8 (2.3.4.4b) virus, the groups were divided into two subgroups (A and B); chickens in subgroups A were challenged at the age of 28 days, whereas those in subgroups B were challenged at the age of 35 days. Results showed that a protective efficacy (survival rate) of 40%–50% was obtained in the vaccinated subgroups A. By delaying challenge for 1 week (subgroups B), a single rHVT-H5 vaccination provided 80% protection, whereas prime–boost vaccination induced full protection and reduced viral shedding very efficiently (1/10 birds and only detected on the 3rd day post challenge) against HPAI H5N8 virus (2.3.4.4b). Moreover, body weight loss improved from 31.39% and 43.65% in G3A and G4A, respectively, to 16.34% and 7.7% in G3B and G4B, respectively. The HI titers obtained in G3A and G4A on the challenge day (28th d) using H5N8 antigen were 3 and 3.75 log2 (p > 0.05), respectively, whereas those in G3B and G4B on the challenge day (35th d) were 6.25 and 6 log2 (p > 0.05), respectively, which increased post-challenge in all vaccinated subgroups. Therefore, the dual use of vectored rHVT-H5 and inactivated rgH5N1 vaccines in the vaccination schedule in poultry farms is the most efficient tool for preventing the disease (mortality and viral shedding) caused by the genetically distinct virus (clade 2.3.3.4b HPAI H5N8) in combination with strict biosecurity and sanitary measures.

Transgenic Chicks Expressing Interferon-Inducible Transmembrane Protein 1 (IFITM1) Restrict Highly Pathogenic H5N1 Influenza Viruses

Mammalian cells utilize a wide spectrum of pathways to antagonize the viral replication. These pathways are typically regulated by antiviral proteins and can be constitutively expressed but also exacerbated by interferon induction. A myriad of interferon-stimulated genes (ISGs) have been identified in mounting broad-spectrum antiviral responses. Members of the interferon-induced transmembrane (IFITM) family of proteins are unique among these ISGs due to their ability to prevent virus entry through the lipid bilayer into the cell. In the current study, we generated transgenic chickens that constitutively and stably expressed chicken IFITM1 (chIFITM1) using the avian sarcoma-leukosis virus (RCAS)-based gene transfer system. The challenged transgenic chicks with clinical dose 104 egg infective dose 50 (EID50) of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 (clade 2.2.1.2) showed 100% protection and significant infection tolerance. Although challenged transgenic chicks displayed 60% protection against challenge with the sub-lethal dose (EID50 105), the transgenic chicks showed delayed clinical symptoms, reduced virus shedding, and reduced histopathologic alterations compared to non-transgenic challenged control chickens. These finding indicate that the sterile defense against H5N1 HPAIV offered by the stable expression of chIFITM1 is inadequate; however, the clinical outcome can be substantially ameliorated. In conclusion, chIFITM proteins can inhibit influenza virus replication that can infect various host species and could be a crucial barrier against zoonotic infections.

Epidemiology, Genetic Characterization, and Pathogenesis of Avian Influenza H5N8 Viruses Circulating in Northern and Southern Parts of Egypt, 2017-2019.

Simple SummaryDuring 2020–2021, highly pathogenic avian influenza (HPAI) viruses of subtype H5N8 were spreading rapidly, and two genetically distinct lineages were detected in Europe, the Middle East, and Southeast Asia. HPAI H5N8 viruses have been circulating in Egyptian poultry flocks since 2016. In this study, 74 commercial chicken farms tested positive for HPAI H5N8 virus. Genetic characterization of the hemagglutinin (HA) and the neuraminidase (NA) of Egyptian HPAI H5N8 viruses showed a relationship with those recently isolated in Europe.Highly pathogenic avian influenza (HPAI) viruses of subtype H5N8 continue to circulate, causing huge economic losses and serious impact on poultry production worldwide. Recently, HPAIV H5N8 has been spreading rapidly, and a large number of HPAI H5N8 outbreaks have been reported in Eurasia 2020–2021. In this study, we conducted an epidemiological survey of HPAI H5N8 virus at different geographical locations in Egypt from 2017 to 2019. This was followed by genetic and pathogenic studies. Our findings highlight the wide spread of HPAI H5N8 viruses in Egypt, including in 22 governorates. The genetic analyses of the hemagglutinin (HA) and neuraminidase (NA) gene segments emphasized a phylogenetic relatedness between the Egyptian HPAI H5N8 viruses and viruses of clade 2.3.4.4b recently isolated in Europe. These findings suggest that a potential back transmission of Egyptian HPAI H5N8 virus has occurred from domestic poultry in Egypt to migratory wild birds, followed by further spread to different countries. This highlights the importance of continuous epidemiological and genetic studies of AIVs at the domestic–wild bird interface.

Gross pathology associated with highly pathogenic avian influenza H5N8 and H5N1 in naturally infected birds in the UK (2020-2021).

Multiple outbreaks with highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4b viruses, including H5N8 and H5N1, have occurred in the United Kingdom, as well as in other European countries, since late 2020. This report describes the pathology among poultry species (chickens, turkeys, ducks, and pheasants) and captive birds (Black Swans, a whistling duck and peregrine falcons) naturally infected with HPAIV from 22 cases of HPAIV H5N8 and two cases of HPAIV H5N1 outbreaks investigated between October 2020 and April 2021. On gross examination, pancreatic necrosis was easily identified and most commonly observed in galliformes infected with both subtypes of HPAIV but rarely in anseriformes. In addition, splenic necrosis was also frequently observed in chickens and turkeys infected with HPAIV H5N8. Other less common lesions included cardiac petechiae, serosal haemorrhages and ascites in a variety of species. Given the widespread dissemination of HPAIV infection in susceptible avian species during autumn/winter 2020-2021, these data, when evaluated along with clinical information, is a valuable first step for both veterinarians and field services to evaluate gross pathology at post-mortem to support the diagnosis of HPAIV infection.

Site-Specific Glycan-Masking/Unmasking Hemagglutinin Antigen Design to Elicit Broadly Neutralizing and Stem-Binding Antibodies Against Highly Pathogenic Avian Influenza H5N1 Virus Infections.

The highly pathogenic avian influenza (HPAI) H5N1 viruses with the capability of transmission from birds to humans have a serious impact on public health. To date, HPAI H5N1 viruses have evolved into ten antigenically distinct clades that could cause a mismatch of vaccine strains and reduce vaccine efficacy. In this study, the glycan masking and unmasking strategies on hemagglutinin antigen were used for designing two antigens: H5-dm/st2 and H5-tm/st2, and investigated for their elicited immunity using two-dose recombinant H5 (rH5) immunization and a first-dose adenovirus vector prime, followed by a second-dose rH5 protein booster immunization. The H5-dm/st2 antigen was found to elicit broadly neutralizing antibodies against different H5N1 clade/subclade viruses, as well as more stem-binding antibodies to inhibit HA-facilitated membrane fusion activity. Mice immunized with the H5-dm/st2 antigen had a higher survival rate when challenged with homologous and heterologous clades of H5N1 viruses. Mutant influenza virus replaced with the H5-dm/st2 gene generated by reverse genetics (RG) technology amplified well in MDCK cells and embryonated chicken eggs. Again, the inactivated H5N1-dm/st2 RG virus elicited more potent cross-clade neutralizing and anti-fusion antibodies in sera. Therefore, the H5N1-dm/st2 RG virus with the site-specific glycan-masking on the globular head and the glycan-unmasking on the stem region of H5 antigen can be used for further development of cross-protective H5N1 vaccines.

RNA-dependent assembly of chimeric antigen nanoparticles as an efficient H5N1 pre-pandemic vaccine platform

Highly pathogenic avian influenza viruses (HPAIVs) pose a significant threat to human health, with high mortality rates, and require effective vaccines. We showed that, harnessed with novel RNA-mediated chaperone function, hemagglutinin (HA) of H5N1 HPAIV could be displayed as an immunologically relevant conformation on self-assembled chimeric nanoparticles (cNP). A tri-partite monomeric antigen was designed including: i) an RNA-interaction domain (RID) as a docking tag for RNA to enable chaperna function (chaperna: chaperone + RNA), ii) globular head domain (gd) of HA as a target antigen, and iii) ferritin as a scaffold for 24 mer-assembly. The immunization of mice with the nanoparticles (~46 nm) induced a 25–30 fold higher neutralizing capacity of the antibody and provided cross-protection from homologous and heterologous lethal challenges. This study suggests that cNP assembly is conducive to eliciting antibodies against the conserved region in HA, providing potent and broad protective efficacy.

Evaluation of Protective Efficacy of Influenza Virus Like Particles Prepared from H5N1 Virus of Clade 2.2.1.2 in Chickens.

Highly pathogenic Avian Influenza (HPAI) viruses continue to cause severe economic losses in poultry species worldwide. HPAI virus of subtype H5N1 was reported in Egypt in 2006, and despite vaccination efforts, the virus has become endemic. The current study aims to evaluate the efficacy of a virus-like particle (VLP) based vaccine in vivo using specific pathogen-free (SPF) chickens. The vaccine was prepared from the HPAI H5N1 virus of clade 2.2.1.2 using the baculovirus expression system. The VLPs were quantitated and characterized, including electron microscopy. In addition, the protection level of the VLPs was evaluated by using two different regimens, including one dose and two-dose vaccinated groups, which gave up to 70% and 100% protection level, respectively. The results of this study emphasize the potential usefulness of the VLPs-based vaccine as an alternative vaccine candidate for the control of AIV infection in poultry.

Primary Chicken and Duck Endothelial Cells Display a Differential Response to Infection with Highly Pathogenic Avian Influenza Virus.

Highly pathogenic avian influenza viruses (HPAIVs) in gallinaceous poultry are associated with viral infection of the endothelium, the induction of a ‘cytokine storm, and severe disease. In contrast, in Pekin ducks, HPAIVs are rarely endothelial tropic, and a cytokine storm is not observed. To date, understanding these species-dependent differences in pathogenesis has been hampered by the absence of a pure culture of duck and chicken endothelial cells. Here, we use our recently established in vitro cultures of duck and chicken aortic endothelial cells to investigate species-dependent differences in the response of endothelial cells to HPAIV H5N1 infection. We demonstrate that chicken and duck endothelial cells display a different transcriptional response to HPAI H5N1 infection in vitro—with chickens displaying a more pro-inflammatory response to infection. As similar observations were recorded following in vitro stimulation with the viral mimetic polyI:C, these findings were not specific to an HPAIV H5N1 infection. However, similar species-dependent differences in the transcriptional response to polyI:C were not observed in avian fibroblasts. Taken together, these data demonstrate that chicken and duck endothelial cells display a different response to HPAIV H5N1 infection, and this may help account for the species-dependent differences observed in inflammation in vivo.

A quantitative risk assessment to evaluate the efficacy of mitigation strategies to reduce highly pathogenic avian influenza virus, subtype H5N1 (HPAI H5N1) in the Menoufia governorate, Egypt

BackgroundThe poultry industry in Egypt has been suffering from endemic highly pathogenic avian influenza (HPAI) virus, subtype H5N1 since 2006. However, the emergence of H9N2, H5N8, and H5N2 in 2011, 2016, and 2019 respectively, has aggravated the situation. Our objective was to evaluate how effective are the mitigation strategies by a Quantitative Risk Assessment (QRA) model which used daily outbreak data of HPAI-H5N1 subtype in Egypt, stratified by different successive epidemic waves from 2006 to 2016.ResultsBy applying the epidemiologic problem-oriented approach methodology, a conceptual scenario tree was drawn based on the knowledgebase. Monte Carlo simulations of QRA parameters based on outbreak data were performed using @Risk software based on a scenario-driven decision tree. In poultry farms, the expected probability of HPAI H5N1 prevalence is 48% due to failure of mitigation strategies in 90% of the time during Monte Carlo simulations. Failure of efficacy of these mitigations will raise prevalence to 70% with missed vaccination, while failure in detection by surveillance activities will raise it to 99%. In backyard poultry farms, the likelihood of still having a high HPAI-H5N1 prevalence in different poultry types due to failure of passive and active surveillance varies between domestic, mixed and reservoir. In mixed poultry, the probability of HPAI-H5N1 not detected by surveillance was the highest with a mean and a SD of 16.8 × 10–3 and 3.26 × 10–01 respectively. The sensitivity analysis ranking for the likelihood of HPAI-H5N1 in poultry farms due to missed vaccination, failure to be detected by passive and active surveillance was examined. Among poultry farms, increasing vaccination by 1 SD will decrease the prevalence by 14%, while active and passive surveillance decreases prevalence by 12, and 6%, respectively. In backyard, the active surveillance had high impact in decreasing the prevalence by 16% in domestic chicken. Whereas the passive surveillance had less impact in decreasing prevalence by 14% in mixed poultry and 3% in domestic chicken.ConclusionIt could be concluded that the applied strategies were not effective in controlling the spread of the HPAI-H5N1 virus. Public health officials should take into consideration the evaluation of their control strategies in their response.