Development of Virus-like Particle Plant-Based Vaccines against Avian H5 and H9 Influenza A Viruses.

Avian influenza A virus (AIV) is a significant cause of mortality in poultry, causing substantial economic loss, particularly in developing countries, and has zoonotic potential. For example, highly pathogenic avian influenza (HPAI) viruses of the H5 subtype have been circulating in Egypt for around two decades. In the last decade, H5N1 viruses of clade 2.2.1 have been succeeded by the antigenically distinct H5N8 clade 2.3.4.4b viruses. Furthermore, H9N2 viruses co-circulate with the H5N8 viruses in Egyptian poultry. It is widely recognised that effective vaccination against IAV requires a close antigenic match between the vaccine and viruses circulating in the field. Therefore, approaches to develop cost-effective vaccines that can be rapidly adapted to local virus strains are required for developing countries such as Egypt. In this project, the haemagglutinin (HA) proteins of Egyptian H5 and H9 viruses were expressed by transient transfection of plants (Nicotiana benthamiana). The formation of virus-like particles (VLPs) was confirmed by transmission electron microscopy. Mice were immunised with four doses of either H5 or H9 VLPs with adjuvant. Antibody and cellular immune responses were measured against the corresponding recombinant protein using ELISA and enzyme-linked immunosorbent assay (ELISpot), respectively. Chickens were immunised with one dose of H5 VLPs, eliciting HA-specific antibodies measured by ELISA and a pseudotyped virus neutralisation test using a heterologous H5 HA. In conclusion, plant-based VLP vaccines have potential for producing an effective vaccine candidate within a short time at a relatively low cost.

UK flockdown: A survey of smallscale poultry keepers and their understanding of governmental guidance on highly pathogenic avian influenza (HPAI).

The scale of the current outbreak of highly pathogenic avian influenza (HPAI) due to the A/H5N1 virus in the United Kingdom is unprecedented. In addition to its economic impact on the commercial poultry sector, the disease has devastated wild bird colonies and represents a potential public health concern on account of its zoonotic potential. Although the implementation of biosecurity measures is paramount to reducing the spread of HPAI in domestic and commercial settings, little is known about the attitudes and perspectives of backyard poultry keepers, who often keep their flocks in close proximity to the public. A large nationwide survey of backyard poultry keepers was undertaken in December 2021-March 2022, contemporaneous with the enforcement of an Avian Influenza Prevention Zone (AIPZ) and additional housing measures in England, Scotland and Wales. The survey explored keepers' understanding of the clinical manifestations of HPAI, compliance with housing and biosecurity measures, attitudes towards obligatory culling on confirmation of HPAI in their flocks, and the potential use of vaccination to control HPAI. Summary statistical analysis of the closed question responses was supplemented with qualitative data analysis and corpus linguistic approaches to draw out key themes and salient patterns in responses to open text questions. Survey responses were received from 1559 small-scale poultry keepers across the United Kingdom. Awareness of the HPAI outbreak was very high (99.0%). The majority of respondents learned of it via social media (53%), with Defra (49.7%), British Hen Welfare Trust (33.8%) and the APHA (22.0%) identified as the principal sources of information. Analysis revealed that backyard keepers lacked knowledge of the clinical signs of avian influenza and legal requirements relating to compliance with biosecurity measures. Some respondents dismissed the seriousness of HPAI and were unwilling to comply with the measures in force. The issue of obligatory culling proved highly emotive, and some expressed a lack of trust in authorities. Most respondents (93.1%) indicated a willingness to pay for vaccination if the option was available. Communications on biosecurity measures that are relevant to large-scale industrial setups are inappropriate for backyard contexts. Understanding the barriers that backyard keepers face is essential if official agencies are to communicate biosecurity information effectively to such groups. Lack of trust in authorities is likely to make elimination of the virus in the UK difficult. We make recommendations for tailoring HPAI-related information for backyard contexts, to aid future HPAI control measures in the UK.

Veterinarians' knowledge and experience of avian influenza and perspectives on control measures in the UK.

BACKGROUND: The scale of the outbreak of highly pathogenic avian influenza (HPAI) in 2021-23 due to the influenza A/H5N1 virus is unprecedented. METHODS: An online survey was designed to explore veterinarians' experiences of and confidence in treating avian species, experiences of dealing with suspected HPAI and perspectives on control measures in the UK. The survey ran between December 2021 and March 2022. RESULTS: Survey responses were received from 26 veterinarians. Although veterinarians are well placed to communicate HPAI-related information and guidance, a lack of confidence around treating birds and dealing with suspected cases of HPAI represent key barriers for non-specialist practices, and this limits opportunities to educate clients. LIMITATIONS: This study presents the views of a small group of self-selected respondents and may over-represent veterinarians with existing interests in avian species and/or avian influenza and who engage with online fora. CONCLUSIONS: Improved training and resources designed to increase confidence with avian species, along with guidance on diagnosing and reporting notifiable diseases, are needed for first opinion practices. Governing bodies should clarify regulations on treating birds in veterinary practices when HPAI outbreak numbers are high.

Exploring the responses of smallscale poultry keepers to avian influenza regulations and guidance in the United Kingdom, with recommendations for improved biosecurity messaging.

Understanding how smallscale ('backyard') poultry keepers interpret and respond to governmental directives designed to reduce the transmission of highly pathogenic avian influenza (HPAI) is of paramount importance in preparing for future HPAI outbreaks. Qualitative insights from open questions in an online survey conducted during the 2021-22 HPAI season (1,559 responses) shed light on smallscale poultry keepers' understanding of, and responses to, governmental directives to control HPAI exposure and onwards transmission. A follow-up participatory workshop (21 participants) explored the HPAI-related information sources used by smallscale poultry keepers, their trust in these sources, perceptions of HPAI-related risk, and interpretation of, opinions on and adherence to government regulations and communications regarding biosecurity and housing measures. This paper draws on a multi-scale behaviour change model to explore barriers to compliance with HPAI-related regulations. Insights from behaviour settings theory reveal how poultry-keeping settings and routines might be 'disrupted' and 're-configured' to improve long-term biosecurity and reduce the risk of HPAI exposure. The findings highlight the need for HPAI-related guidance that is tailored to smallscale poultry keepers. This guidance should include clear action points and simple, practical, affordable and sustainable suggestions for improving compliance with biosecurity measures.

Associations between small ruminant lentivirus infection and total milk yield and somatic cell count in a dairy sheep flock.

BACKGROUND: Small ruminant lentiviruses (SRLVs) are lentiviruses of sheep and goats, formerly known as maedi-visna (MV) in sheep and caprine encephalitis and arthritis in goats. In sheep, SRLVs commonly cause progressive pneumonia, wasting and indurative mastitis. SRLVs have a long latent period, and chronic production losses are often not recognised until very late. Few studies quantifying the production losses in ewes have been published, and none have been published under UK flock husbandry conditions. METHODS: Production records of milk yield and somatic cell count (SCC) from a dairy flock of 319 milking East Friesian × Lacaune ewes identified as MV infected via routine serological screening for SRLV antibodies were used in multivariable linear regression modelling to estimate the impact of SRLV status on total milk yield and SCC. RESULTS: Milk yield was reduced in seropositive ewes by 8.1%-9.2% over an entire lactation. SCC counts were not significantly different in SRLV-infected and unifected animals. LIMITATIONS: Further parameters, such as body condition score or clinical mastitis, that were not available may have clarified the underlying cause of milk yield drop. CONCLUSIONS: The study demonstrates substantial production losses in an SRLV-affected flock and highlights the impact of the virus on a farm's economic viability.

Failure to detect Schmallenberg virus RNA in ram semen in the UK (2016-2018).

Background: Schmallenberg virus (SBV) is a midge-borne arbovirus that first emerged in the European ruminant population in 2011 and has since settled to an endemic pattern of disease outbreaks on an approximately 4-year cycle when herd immunity from the previous circulation drops to a point allowing renewed widescale virus circulation. The impacts of trade restrictions on genetic products (semen, embryos) from affected areas were severe, particularly after the discovery that the virus is intermittently shed in the semen of a small number of bulls. The trade in small ruminant (ram and goat) semen is less than that of bulls; nonetheless, there has been no study into the shedding rate of SBV in ram semen. Methods: Semen samples (n = 65) were collected as part of UK ram trials and artificial insemination studies around the period of the 2016-2018 SBV recirculation. Semen was preserved in RNAlater for shipping, and RNA extraction with RNeasy and S gene RT-quantitative PCR performed for SBV nucleic acid detection. Results: No SBV RNA was detected in any samples. Conclusions: While larger numbers of animals would be needed to completely exclude the possibility of SBV shedding in ram semen, this trial nonetheless highlights that this is likely a rare event if it occurs at all and is unlikely to play a role in disease transmission.

Lipid biophysics and/or soft matter-inspired approach for controlling enveloped virus infectivity.

Proven as a natural barrier against viral infection, pulmonary surfactant phospholipids have a biophysical and immunological role within the respiratory system, acting against microorganisms including viruses. Enveloped viruses have, in common, an outer bilayer membrane that forms the underlying structure for viral membrane proteins to function in an optimal way to ensure infectivity. Perturbating the membrane of viruses using exogenous lipids can be envisioned as a generic way to reduce their infectivity. In this context, the potential of exogenous lipids to be used against enveloped virus infectivity would be indicated by the resulting physical stress imposed to the viral membrane, and conical lipids, i.e. lyso-lipids, would be expected to generate stronger biophysical disturbances. We confirm that when treated with lyso-lipids the infectivity three strains of influenza virus (avian H2N3, equine H3N8 or pandemic human influenza H1N1) is reduced by up to 99% in a cell-based model. By contrast, lipids with a similar head group but two aliphatic chains were less effective (reducing infection by only 40-50%). This work opens a new path to merge concepts from different research fields, i.e. 'soft matter physics' and virology.

Development of Plant-Based Vaccines for Prevention of Avian Influenza and Newcastle Disease in Poultry.

Viral diseases, including avian influenza (AI) and Newcastle disease (ND), are an important cause of morbidity and mortality in poultry, resulting in significant economic losses. Despite the availability of commercial vaccines for the major viral diseases of poultry, these diseases continue to pose a significant risk to global food security. There are multiple factors for this: vaccine costs may be prohibitive, cold chain storage for attenuated live-virus vaccines may not be achievable, and commercial vaccines may protect poorly against local emerging strains. The development of transient gene expression systems in plants provides a versatile and robust tool to generate a high yield of recombinant proteins with superior speed while managing to achieve cost-efficient production. Plant-derived vaccines offer good stability and safety these include both subunit and virus-like particle (VLP) vaccines. VLPs offer potential benefits compared to currently available traditional vaccines, including significant reductions in virus shedding and the ability to differentiate between infected and vaccinated birds (DIVA). This review discusses the current state of plant-based vaccines for prevention of the AI and ND in poultry, challenges in their development, and potential for expanding their use in low- and middle-income countries.

Clearance of Maedi-visna infection in a longitudinal study of naturally infected rams is associated with homozygosity for the TMEM154 resistance allele.

Maedi-visna (MV) is a lentiviral disease of sheep responsible for severe production losses in affected flocks. There are no vaccination or treatment options with control reliant on test and cull strategies. The most common diagnostic methods used at present are combination ELISAs for Gag and Env proteins with virus variability making PCR diagnostics still largely an experimental tool. To assess variability in viral loads and diagnostic tests results, serology, DNA and RNA viral loads were measured in the blood of 12 naturally infected rams repeatedly blood sampled over 16 months. Six animals tested negative in one or more tests at one or more time points and would have been missed on screening programmes reliant on one test method or a single time point. In addition the one animal homozygous for the 'K' allele of the TMEM154 E35K SNP maintained very low viral loads in all assays and apparently cleared infection to below detectable limits at the final time point it was sampled. This adds crucial data to the strong epidemiological evidence that this locus represents a genuine resistance marker for MV infection and is a strong candidate for selective breeding of sheep for resistance to disease.

Schmallenberg virus neutralising antibody responses in sheep.

BACKGROUND: Schmallenberg virus (SBV) is a midge borne virus of cattle and sheep. Infection is typically asymptomatic in adult sheep but fetal infection during pregnancy can result in abortion, stillbirth, neurological disorders and malformations of variable severity in newborn animals. It was first identified in Germany and the Netherlands in 2011 and then circulated throughout Europe in 2012 and 2013. Circulation in subsequent years was low or non-existent until summer and autumn 2016, leading to an increased incidence of deformed newborn lambs and calves in 2016-17. This study reports SBV circulation in October 2016 within a group of 24 ewes and 13 rams. The ewes were monitored at 3 times points over an 11 week period (September to December 2016). RESULTS: Most ewes displayed an increase in SBV VNT with antibody titre increases greater in older, previously exposed ewes. Two ewes had SBV RNA detectable by RT-qPCR, one on 30/09/16 and one on 04/11/16. Of these ewes, one had detectable serum SBV RNA (indicating viraemia) despite pre-existing antibody. The rams had been previously vaccinated with a commercial inactivated SBV vaccine, they showed minimal neutralising antibody titres against SBV 8 months post-vaccination and all displayed increased titre in October 2016. CONCLUSION: This data suggests that SBV circulated for a minimum period of 5 weeks in September to October 2016 in central England. Ewes previously exposed to virus showed an enhanced antibody response compared to naïve animals. Pre-existing antibody titre did not prevent re-infection in at least one animal, implying immunity to SBV upon natural exposure may not be life-long. In addition, data suggests that immunity provided by killed adjuvanted SBV vaccines only provides short term protection (< 8 months) from virus.

Recombinant Viruses for Cancer Therapy.

Recombinant viruses are novel therapeutic agents that can be utilized for treatment of various diseases, including cancers. Recombinant viruses can be engineered to express foreign transgenes and have a broad tropism allowing gene expression in a wide range of host cells. They can be selected or designed for specific therapeutic goals; for example, recombinant viruses could be used to stimulate host immune response against tumor-specific antigens and therefore overcome the ability of the tumor to evade the host's immune surveillance. Alternatively, recombinant viruses could express immunomodulatory genes which stimulate an anti-cancer immune response. Oncolytic viruses can replicate specifically in tumor cells and induce toxic effects leading to cell lysis and apoptosis. However, each of these approaches face certain difficulties that must be resolved to achieve maximum therapeutic efficacy. In this review we discuss actively developing approaches for cancer therapy based on recombinant viruses, problems that need to be overcome, and possible prospects for further development of recombinant virus based therapy.

DNA microarray global gene expression analysis of influenza virus-infected chicken and duck cells.

The data described in this article pertain to the article by Kuchipudi et al. (2014) titled "Highly Pathogenic Avian Influenza Virus Infection in Chickens But Not Ducks Is Associated with Elevated Host Immune and Pro-inflammatory Responses" [1]. While infection of chickens with highly pathogenic avian influenza (HPAI) H5N1 virus subtypes often leads to 100% mortality within 1 to 2 days, infection of ducks in contrast causes mild or no clinical signs. The rapid onset of fatal disease in chickens, but with no evidence of severe clinical symptoms in ducks, suggests underlying differences in their innate immune mechanisms. We used Chicken Genechip microarrays (Affymetrix) to analyse the gene expression profiles of primary chicken and duck lung cells infected with a low pathogenic avian influenza (LPAI) H2N3 virus and two HPAI H5N1 virus subtypes to understand the molecular basis of host susceptibility and resistance in chickens and ducks. Here, we described the experimental design, quality control and analysis that were performed on the data set. The data are publicly available through the Gene Expression Omnibus (GEO)database with accession number GSE33389, and the analysis and interpretation of these data are included in Kuchipudi et al. (2014) [1].

Longitudinal study of Asian elephants, Elephas maximus, indicates intermittent shedding of elephant endotheliotropic herpesvirus 1 during pregnancy.

INTRODUCTION: EEHV-1 is a viral infection of elephants that has been associated with a fatal haemorrhagic syndrome in Asian elephants. Previous studies have suggested that pregnant animals may shed more virus than non-pregnant animals. METHODS: This study examined whether pregnancy affected the frequency or magnitude of shedding of elephant endotheliotropic herpesvirus 1 (EEHV1) using Taq man real-time PCR on trunk washes from four female elephants from a UK collection over three time periods between 2011 and 2014. These periods included pregnancies in two animals (period 1 and period 3). Behavioural observations made by keepers were also assessed. RESULTS: During period 1 there was a high degree of social hierarchical instability which led to a hierarchy change, and was associated with aggressive behaviour. Also during period 1 EEHV-1 shedding was of a higher magnitude and frequency than in the latter two time periods. CONCLUSIONS: These results suggest that there is no clear relationship between shedding and pregnancy, and that behavioural stressors may be related to an increase in EEHV-1 shedding.

Identification of morphological differences between avian influenza A viruses grown in chicken and duck cells.

Although wild ducks are considered to be the major reservoirs for most influenza A virus subtypes, they are typically resistant to the effects of the infection. In contrast, certain influenza viruses may be highly pathogenic in other avian hosts such as chickens and turkeys, causing severe illness and death. Following in vitro infection of chicken and duck embryo fibroblasts (CEF and DEF) with low pathogenic avian influenza (LPAI) viruses, duck cells die more rapidly and produce fewer infectious virions than chicken cells. In the current study, the morphology of viruses produced from CEF and DEF cells infected with low pathogenic avian H2N3 was examined. Transmission electron microscopy showed that viruses budding from duck cells were elongated, while chicken cells produced mostly spherical virions; similar differences were observed in viral supernatants. Sequencing of the influenza genome of chicken- and duck-derived H2N3 LPAI revealed no differences, implicating host cell determinants as responsible for differences in virus morphology. Both DEF and CEF cells produced filamentous virions of equine H3N8 (where virus morphology is determined by the matrix gene). DEF cells produced filamentous or short filament virions of equine H3N8 and avian H2N3, respectively, even after actin disruption with cytochalasin D. These findings suggest that cellular factors other than actin are responsible for the formation of filamentous virions in DEF cells. The formation of elongated virions in duck cells may account for the reduced number of infectious virions produced and could have implications for virus transmission or maintenance in the reservoir host.

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.

A consensus approach to vertebrate de novo transcriptome assembly from RNA-seq data: assembly of the duck (Anas platyrhynchos) transcriptome.

For vertebrate organisms where a reference genome is not available, de novo transcriptome assembly enables a cost effective insight into the identification of tissue specific or differentially expressed genes and variation of the coding part of the genome. However, since there are a number of different tools and parameters that can be used to reconstruct transcripts, it is difficult to determine an optimal method. Here we suggest a pipeline based on (1) assessing the performance of three different assembly tools (2) using both single and multiple k-mer (MK) approaches (3) examining the influence of the number of reads used in the assembly (4) merging assemblies from different tools. We use an example dataset from the vertebrate Anas platyrhynchos domestica (Pekin duck). We find that taking a subset of data enables a robust assembly to be produced by multiple methods without the need for very high memory capacity. The use of reads mapped back to transcripts (RMBT) and CEGMA (Core Eukaryotic Genes Mapping Approach) provides useful metrics to determine the completeness of assembly obtained. For this dataset the use of MK in the assembly generated a more complete assembly as measured by greater number of RMBT and CEGMA score. Merged single k-mer assemblies are generally smaller but consist of longer transcripts, suggesting an assembly consisting of fewer fragmented transcripts. We suggest that the use of a subset of reads during assembly allows the relatively rapid investigation of assembly characteristics and can guide the user to the most appropriate transcriptome for particular downstream use. Transcriptomes generated by the compared assembly methods and the final merged assembly are freely available for download at http://dx.doi.org/10.6084/m9.figshare.1032613.

The challenge of Schmallenberg virus emergence in Europe.

The large-scale outbreak of disease across Northern Europe caused by a new orthobunyavirus known as Schmallenberg virus has caused considerable disruption to lambing and calving. Although advances in technology and collaboration between veterinary diagnostic and research institutes have enabled rapid identification of the causative agent and the development and deployment of tests, much remains unknown about this virus and its epidemiology that make predictions of its future impact difficult to assess. This review outlines current knowledge of the virus, drawing comparisons with related viruses, then explores possible scenarios of its impact in the near future, and highlights some of the urgent research questions that need to be addressed to allow the development of appropriate control strategies.

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.

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.