Summary Existing molecular diagnostic approaches for notifiable avian diseases (NADs) involve a suite of PCR assays that enable both generic detection, and where positive, subtyping of both avian influenza virus (AIV) and Newcastle disease virus (NDV). Novel rapid and direct diagnostic assays for the detection of AIV and NDV were developed and evaluated using unprocessed cloacal (C) and oropharyngeal (OP) poultry swab material. Both assays employ a closed tube direct real-time reverse transcription polymerase chain reaction (RRT-PCR) approach in which viral lysis is achieved by heat treatment and a dedicated PCR compatible buffer, followed by detection using a RRT-PCR approach. Primer and probe sets were designed using globally circulating AIV and NDV sequences collected over the preceding five years, rather than region-specific sequence datasets, so that the assays detect all circulating genotypes. Analytical performance assessment demonstrated that both assays were highly sensitive and specific, successfully detecting all unextracted target antigens without cross reactivity to a panel of other common poultry pathogens. For each assay, viral lysis and amplification were achieved directly from samples at single digit genome copy numbers. Furthermore, low levels of viral RNA could be reliably detected in the presence of C and OP matrix material, providing proof-of-concept for direct detection of these economically significant avian pathogens in a field setting. Additional use case scenarios, including pooled sample screening and combined C/OP testing from individual birds, were also explored. These findings establish a foundation for ongoing studies incorporating paired-sample testing against validated laboratory reference assays.

Abstract Background Infectious diseases including Newcastle disease (ND) impair poultry productivity and represent a significant burden to sustainable agriculture in Nigeria. This study aimed to investigate the active circulation and seroprevalence of Newcastle disease virus (NDV) caused by virulent forms of avian paramyxovirus type-1 (APMV-1) in poultry at live bird markets (LBMs) across Nigeria. Methods A cross-sectional study of 18 LBMs was conducted within three states in Nigeria (Kano, Oyo, and Abuja). Paired swab and tissue samples (n = 413) were collected from birds on FTA cards and tested for APMV-1 using real-time reverse transcription polymerase chain reaction (rRT-PCR). A subset of rRT-PCR-positive samples were selected for whole genome sequencing based on the originating species (chicken, duck, geese), date, and market location to provide a broad range of isolates for characterisation. Blood samples (n = 405) were also collected from birds and the seroprevalence of APMV-1 antibodies was measured using an enzyme-linked immunosorbent assay (ELISA). Results APMV-1 RNA was detected in 21.5% (89/413) of samples from LBMs by rRT-PCR. At least one APMV-1 positive sample was detected in 55.6% (10/18) of LBMs. The largest proportion of APMV-1-positive markets was in Kano (83.3%, 5/6), whereas the lowest was in Oyo (16.7%, 1/6). Assessment of genetic data demonstrated that genotype XIV.2 APMV-1 was circulating within Nigeria with the viruses detected clustering closely with other Nigerian isolates described previously. The seroprevalence of APMV-1 in birds was 45.9% (186/405) and 94.4% (17/18) of LBMs had at least one APMV-1 seropositive sample (i.e., with at least one APMV-1-antibody-positive bird). The LBMs in Kano had the lowest seroprevalence (88.3%, 5/6). Conclusions This study demonstrated that APMV-1 continues to circulate in LBMs in Nigeria. LBM traders, poultry producers, and related industry and policy stakeholders should be aware of the occurrence of APMV-1 and how ND may negatively impact upon poultry production and the livelihoods of poultry farmers and LBM traders. Training initiatives aimed at improving the knowledge of APMV-1 infection and improvements in biosecurity practises and the role of disease mitigation through vaccination are required to reduce the impact of this threat to food security.

A free-range organic broiler (Gallus gallus domesticus) premises in Staffordshire was infected by high pathogenicity avian influenza virus (HPAIV) H5N8 during the 2020-2021 epizootic in the United Kingdom (UK). Following initial confirmation of the infection in poultry, multiple wild bird species were seen scavenging on chicken carcasses. Detected dead wild birds were subsequently demonstrated to have been infected and succumbed to HPAIV H5N8. Initially, scavenging species, magpie (Pica pica) and raven (Corvus corax) were found dead on the premises but over the following days, buzzards (Buteo buteo) were also found dead within the local area with positive detection of HPAIV in submitted carcasses. The subacute nature of microscopic lesions within a buzzard was consistent with the timeframe of infection. Finally, a considerable number of free-living pheasants (Phasianus colchicus) were also found dead in the surrounding area, with carcasses having higher viral antigen loads compared to infected chickens. Limited virus dissemination was observed in the carcasses of the magpie, raven, and buzzard. Further, an avirulent avian paramyxovirus type 1 (APMV-1) was detected within poultry samples as well as in the viscera of a magpie infected with HPAIV. Immunohistochemistry did not reveal colocalization of avian paramyxovirus antigens with lesions, supporting an avirulent APMV-1 infection. Overall, this case highlights scenarios in which bi-directional transmission of avian viral diseases between commercial and wild bird species may occur. It also underlines the importance of bio separation and reduced access when infection pressure from HPAIV is high.

The virulent form of Avian paramyxovirus-1 (APMV-1), commonly known as Newcastle Disease Virus (NDV), is a pathogen with global implications for avian health, affecting both wild and domestic bird populations. In Pakistan, recurrent Newcastle Disease (caused by NDV) outbreaks have posed significant challenges to the poultry industry. Extensive surveillance in Pakistan over 20 years has demonstrated a dynamic genetic diversity among circulating APMV-1 strains, emphasizing the potential necessity for customized vaccination strategies and continuous surveillance. In this study, 13 APMV-1-positive isolates harboring four different APMV-1 genotypes circulating throughout Pakistan were identified. These included the highly virulent genotypes VII and XIII, genotype XXI, commonly associated with Columbiformes, and genotype II, hypothesized to have been detected following vaccination. These findings underscore the intricate interplay of mutational events and host-immune interactions shaping the evolving NDV landscape. This study advances our understanding of the evolutionary dynamics of APMV-1 in Pakistan, highlighting the need for tailored vaccination strategies and continuous surveillance to enable effective APMV-1 management in avian populations, further emphasizing the importance of globally coordinated strategies to tackle APMV-1, given its profound impact on wild and domestic birds.

The United Kingdom (UK) and Europe have seen successive outbreaks of H5N1 clade 2.3.4.4b high-pathogenicity avian influenza virus (HPAIV) since 2020 peaking in the autumn/winter periods. During the 2021/22 season, a mass die-off event of Svalbard Barnacle Geese (Branta leucopsis) was observed on the Solway Firth, a body of water on the west coast border between England and Scotland. This area is used annually by Barnacle Geese to over-winter, before returning to Svalbard to breed. Following initial identification of HPAIV in a Barnacle Goose on 8 November 2021, up to 32% of the total Barnacle Goose population may have succumbed to disease by the end of March 2022, along with other wild bird species in the area. Potential adaptation of the HPAIV to the Barnacle Goose population within this event was evaluated. Whole-genome sequencing of thirty-three HPAIV isolates from wild bird species demonstrated that there had been two distinct incursions of the virus, but the two viruses had remained genetically stable within the population, whilst viruses from infected wild birds were closely related to those from poultry cases occurring in the same region. Analysis of sera from the following year demonstrated that a high percentage (76%) of returning birds had developed antibodies to H5 AIV. This study demonstrates genetic stability of this strain of HPAIV in wild Anseriformes, and that, at the population scale, whilst there is a significant impact on survival, a high proportion of birds recover following infection.

Although commercial vaccines against Newcastle Disease have been available for decades, outbreaks still occur in the face of vaccination Further vaccination may accelerate viral evolution resulting in a further reduction in vaccine efficacy. A key question is whether genotype-matched vaccines can confer better protection against contemporary type 1 Avian Paramyxoviruses. To assess this, an in vivo vaccine-challenge study was undertaken to assess protection afforded by 'genotype-matched' and commercial vaccine formulations. Groups of chickens were vaccinated twice (prime-boost) with an inactivated preparation of either La Sota Clone 30, AV632-chicken-Cyprus-13 (genotype VII.2), or mock vaccine, and later challenged with virulent AV632-chicken-Cyprus-13. Post vaccinal serological responses differed, although both vaccination/challenge groups showed similar levels of clinical protection compared to the unvaccinated group, where 100 % mortality was observed. Shedding was significantly reduced in the vaccinated groups compared to the unvaccinated group. Virus dissemination in the tissues of vaccinated birds was comparable, but onset of infection was delayed. Two mutations were observed in the HN gene of the heterologous vaccine group; H199N and I192M, the latter thought to be associated with increased fusogenic potential. These data demonstrate that existing vaccine formulations confer similar levels of clinical protection to contemporary strains and that the antigenic heterogeneity of circulating strains does not impact upon shedding profiles in immunised birds. In conclusion, the ability of virulent APMV-1 to cause disease in vaccinated flocks is unlikely to be the result of antigenic mismatch alone, and other factors likely contribute to vaccination failure and breakthrough.

High-pathogenicity avian influenza virus (HPAIV) outbreaks devastate avian species across all sectors, having both economic and ecological impacts through mortalities in poultry and wild birds, respectively. These viruses can also represent a significant zoonotic risk.

Newcastle disease (ND) is a notifiable disease affecting chickens and other avian species caused by virulent strains of Avian paramyxovirus type 1 (APMV-1). While outbreaks of ND can have devastating consequences, avirulent strains of APMV-1 generally cause subclinical infections or mild disease. However, viruses can cause different levels of disease in different species and virulence can evolve following cross-species transmission events. This report describes the detection of three cases of avirulent APMV-1 infection in Great Britain (GB). Case 1 emerged from the 'testing to exclude' scheme in chickens in Shropshire while cases 2 and 3 were made directly from notifiable avian disease investigations in chicken broilers in Herefordshire and on premises in Wiltshire containing ducks and mixed species, respectively). Class II/genotype I.1.1 APMV-1 from case 1 shared 99.94% identity to the Queensland V4 strain of APMV-1. Class II/genotype II APMV-1 was detected from case 2 while the class II/genotype I.2 virus from case 3 aligned closely with strains isolated from Anseriformes. Exclusion of ND through rapid detection of avirulent APMV-1 is important where clinical signs caused by avirulent or virulent APMV-1s could be ambiguous. Understanding the diversity of APMV-1s circulating in GB is critical to understanding disease threat from these adaptable viruses.

Newcastle disease (ND) is a highly contagious viral respiratory and neurological disease that has a severe impact on poultry production worldwide. In the present study, an expression platform was established for the transient production in N.bethamiana of ND virus-like particles (VLPs) for use as vaccines against ND. The expression of the ND Fusion (F) and/or Hemagglutinin-neuraminidase (HN) proteins of a genotype VII.2 strain formed ND VLPs in planta as visualized under the transmission electron microscope, and HN-containing VLPs agglutinated chicken erythrocytes with hemagglutination (HA) titres of up to 13 log2.The immunogenicity of the partially-purified ND VLPs was confirmed in specific-pathogen-free White leghorn chickens. Birds receiving a single intramuscular immunization with 1024 HA units (10 log2) of the F/HN ND VLPs administered with 20% [v/v] Emulsigen®-P adjuvant, seroconverted after 14 days with F- and HN-specific antibodies at ELISA titres of 5705.17 and HI geometric mean titres (GMTs) of 6.2 log2, respectively. Furthermore, these ND-specific antibodies successfully inhibited viral replication in vitro of two antigenically closely-related ND virus isolates, with virus-neutralization test GMTs of 3.47 and 3.4, respectively. Plant-produced ND VLPs have great potential as antigen-matched vaccines for poultry and other avian species that are highly immunogenic, cost-effective, and facilitate prompt updating to ensure improved protection against emerging ND field viruses.

Newcastle disease (ND) is caused by virulent forms of avian paramyxovirus-1 (APMV-1) and is an economically important disease of poultry world-wide. Pigeon paramyxovirus 1 (PPMV-1), a sub-group of APMV-1 is endemic in Columbiformes and can cause infections of poultry. An outbreak of ND in partridges in Scotland, UK, in 2006 (APMV-1/partridge/UK(Scotland)/7575/06) was identified as a class II, genotype VI.2.1.1.2.1, more commonly associated with PPMV-1. It has been hypothesized that game birds may be a route of transmission into commercial poultry settings due to the semi-feral rearing system, which potentially brings them into contact with both wild-birds and poultry species. Therefore, the pathogenesis and transmission of APMV-1/partridge/UK(Scotland)/7575/06 in game birds and chickens was investigated, and compared to a contemporary PPMV-1 isolate, PPMV-1/pigeon/UK/015874/15. Viral shedding and seroconversion profiles demonstrated that pheasants were susceptible to infection with APMV-1/partridge/UK(Scotland)/7575/06 with limited clinical signs observed although they were able to excrete and transmit virus. In contrast, partridges and pheasants showed limited infection with PPMV-1/pigeon/UK/015874/15, causing mild clinical disease. Chickens, however, were productively infected and were able to transmit virus in the absence of clinical signs. From the data, it can be deduced that whilst game birds may play a role in the transmission and epidemiology of genotype VI.2 APMV-1 viruses, the asymptomatic nature of circulation within these species precludes evaluation of natural infection by clinical surveillance. It therefore remains a possibility that genotype VI.2 APMV-1 infection in game birds has the potential for asymptomatic circulation and remains a potential threat to avian production systems.RESEARCH HIGHLIGHTS Demonstration of infection of game birds with Pigeon paramyxovirus-1 (PPMV-1).There are differing dynamics of infection between different game bird species.Differing dynamics of infection between different PPMV-1 isolates and genotypes in game birds and chickens.

Newcastle Disease (ND), caused by virulent forms of Avian orthoavulavirus serotype-1 (AOAV-1) is an economically important avian disease worldwide. The past two incursions of ND into the United Kingdom occurred in game bird populations during 2005 and 2006. The nature of the game bird semi-feral rearing system, which can bring these birds into close contact with both wild birds and commercial or backyard poultry, has been hypothesized to act as a bridge between these two environments. As such, the risk that AOAV-1-infected game birds may pose to the UK poultry industry was investigated. Pheasants, partridges and chickens were experimentally infected with the virulent strain APMV-1/Chicken/Bulgaria/112/13, a genotype VII.2 virus associated with ND outbreaks in Eastern Europe. The study demonstrated that both chickens and pheasants are susceptible to infection with APMV-1/Chicken/Bulgaria/112/13, which results in high mortality and onward transmission. Partridges by contrast are susceptible to infection, but mortality was reduced, as was onward transmission. However, the data indicated that both pheasants and partridges may serve as intermediate hosts of AOAV-1 and may bridge the wild bird–domestic poultry interface enabling transmission into an economically damaging environment where morbidity and mortality may be high.

This focus article has been prepared by Marco Falchieri, Scott M. Reid, Craig S. Ross, Joe James, Alexander M. P. Byrne, Madalina Zamfir, Ian H. Brown and Ashley C. Banyard of the APHA; Glen Tyler and Emma Philip of NatureScot; and Will Miles of Scottish Oceans Institute, School of Biology, University of St Andrews.

Newcastle disease (ND) is a highly contagious disease of poultry caused by virulent avian paramyxovirus-1 (APMV-1) (previously termed avian avulavirus-1 and avian orthoavulavirus-1). APMV-1 is endemic in poultry in many developing countries, whilst outbreaks still occur in developed countries, affecting both commercial and backyard flocks. ND outbreaks can have substantial economic consequences due to high mortality rates and the imposition of trade restrictions. APMV-1 nucleic acid can be detected from swabs or tissues of suspected cases by PCR. Evidence of infection or vaccination may be demonstrated by the presence of specific antibodies against HN in serum samples. No anti-viral treatments exist, but vaccines are available, although there are currently concerns over their efficacy.

Veterinary RecordVolume 191, Issue 2 p. 80-81 Avian Disease Molecular detection of pigeon paramyxovirus type 1 Scott M Reid, Scott M Reid molecular resource management team leaderSearch for more papers by this authorSharon M Brookes, Sharon M Brookes lead scientist in virologySearch for more papers by this authorVivien J Coward, Vivien J Coward manager of avian virology investigation unitSearch for more papers by this authorJune K Mynn, June K Mynn avian virology investigation unit scientistSearch for more papers by this authorJayne L Cooper, Jayne L CooperSearch for more papers by this authorEric Agyeman-Dua, Eric Agyeman-Dua molecular resource management team scientistsSearch for more papers by this authorPaul Skinner, Paul Skinner research scientistSearch for more papers by this authorCraig Ross, Craig Ross Newcastle disease team leaderSearch for more papers by this authorRowena DE Hansen, Rowena DE Hansen veterinary head of exotic diseaseSearch for more papers by this authorIan H Brown, Corresponding Author Ian H Brown head of virology [email protected] Department of Virology, APHA-Weybridge, Woodham Lane, New Haw, Addlestone, Surrey, KT15 3NBSearch for more papers by this authorJo Mayers, Jo Mayers laboratory manager APHA Veterinary Investigation Centre, Staplake Mount, Starcross, Exeter, Devon, EX6 8PESearch for more papers by this authorDavid A Sutton, David A Sutton scientist Qiagen, Skelton House, Lloyd St N, Manchester, M15 6SHSearch for more papers by this author Scott M Reid, Scott M Reid molecular resource management team leaderSearch for more papers by this authorSharon M Brookes, Sharon M Brookes lead scientist in virologySearch for more papers by this authorVivien J Coward, Vivien J Coward manager of avian virology investigation unitSearch for more papers by this authorJune K Mynn, June K Mynn avian virology investigation unit scientistSearch for more papers by this authorJayne L Cooper, Jayne L CooperSearch for more papers by this authorEric Agyeman-Dua, Eric Agyeman-Dua molecular resource management team scientistsSearch for more papers by this authorPaul Skinner, Paul Skinner research scientistSearch for more papers by this authorCraig Ross, Craig Ross Newcastle disease team leaderSearch for more papers by this authorRowena DE Hansen, Rowena DE Hansen veterinary head of exotic diseaseSearch for more papers by this authorIan H Brown, Corresponding Author Ian H Brown head of virology [email protected] Department of Virology, APHA-Weybridge, Woodham Lane, New Haw, Addlestone, Surrey, KT15 3NBSearch for more papers by this authorJo Mayers, Jo Mayers laboratory manager APHA Veterinary Investigation Centre, Staplake Mount, Starcross, Exeter, Devon, EX6 8PESearch for more papers by this authorDavid A Sutton, David A Sutton scientist Qiagen, Skelton House, Lloyd St N, Manchester, M15 6SHSearch for more papers by this author First published: 22 July 2022 https://doi.org/10.1002/vetr.2057Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume191, Issue223/30 July 2022Pages 80-81 RelatedInformation

AbstractSince 2020, the UK and Europe, have experienced annual epizootics of high pathogenicity avian influenza virus (HPAIV). The first during autumn/winter 2020/21 involved the detected with six H5Nx subtypes although H5N8 HPAIV dominated in the UK. Whilst genetic assessment of the H5N8 HPAIVs within the UK demonstrated relative homogeneity, there was a background of other genotypes circulating at a lower degree with different neuraminidase and internal genes. Following a small number of summer detections of H5N1 in wild birds over the summer of 2021, autumn/winter 2021/22 saw another European H5 HPAIV epizootic, that has dwarfed the prior epizootic. This second epizootic was dominated almost exclusively by H5N1 HPAIV, although six distinct genotypes were defined. We have used genetic analysis to evaluate the emergence of different genotypes and proposed reassortment events that have been observed. The existing data suggests that the H5N1 circulating in Europe during late 2020, continued to circulate in wild birds throughout 2021, with minimal adaptation, but has then gone on to reassort with AIVs in the wild bird population. We have undertaken an in-depth genetic assessment of H5 HPAIVs detected in the UK, over the last two winter seasons and demonstrate the utility of in-depth genetic analyses in defining the diversity of H5 HPAIVs circulating in avian species, the potential for zoonotic risk and whether incidents of lateral spread can be defined over independent incursion of infection from wild birds. Key supporting data for mitigation activities.ImportanceHigh pathogenicity avian influenza virus (HPAIV) outbreaks devastate avian species across all sectors having both economic and ecological impacts through mortalities in poultry and wild birds, respectively. These viruses can also represent a significant zoonotic risk. Since 2020, the UK has experienced two successive outbreaks of H5 HPAIV. Whilst H5N8 HPAIV was predominant during the 2020/21 outbreak, other H5 subtypes were also detected. The following year there was a shift in subtype dominance to H5N1 HPAIV, but multiple H5N1 genotypes were detected. Through thorough utilisation of whole-genome sequencing, it was possible to track and characterise the genetic evolution of these H5 HPAIVs in UK poultry and wild birds. This has enabled us to assess the risk posed by these viruses at the poultry:wild bird and the avian:human interface and to investigate potential lateral spread between infected premises, a key factor in understanding threat to the commercial sector.

SARS-CoV-2 virus was first detected in late 2019 and circulated globally, causing COVID-19, which is characterised by sub-clinical to severe disease in humans. Here, we investigate the serological antibody responses to SARS-CoV-2 infection during acute and convalescent infection using a cohort of (i) COVID-19 patients admitted to hospital, (ii) healthy individuals who had experienced ‘COVID-19 like-illness’, and (iii) a cohort of healthy individuals prior to the emergence of SARS-CoV-2. We compare SARS-CoV-2 specific antibody detection rates from four different serological methods, virus neutralisation test (VNT), ID Screen® SARS-CoV-2-N IgG ELISA, Whole Antigen ELISA, and lentivirus-based SARS-CoV-2 pseudotype virus neutralisation tests (pVNT). All methods were able to detect prior infection with COVID-19, albeit with different relative sensitivities. The VNT and SARS-CoV-2-N ELISA methods showed a strong correlation yet provided increased detection rates when used in combination. A pVNT correlated strongly with SARS-CoV-2 VNT and was able to effectively discriminate SARS-CoV-2 antibody positive and negative serum with the same efficiency as the VNT. Moreover, the pVNT was performed with the same level of discrimination across multiple separate institutions. Therefore, the pVNT is a sensitive, specific, and reproducible lower biosafety level alternative to VNT for detecting SARS-CoV-2 antibodies for diagnostic and research applications. Our data illustrate the potential utility of applying VNT or pVNT and ELISA antibody tests in parallel to enhance the sensitivity of exposure to infection.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Influenza A viruses (IAV) are subject to species barriers that prevent frequent zoonotic transmission and pandemics. One of these barriers is the poor activity of avian IAV polymerases in human cells. Differences between avian and mammalian ANP32 proteins underlie this host range barrier. Human ANP32A and ANP32B homologues both support function of human-adapted influenza polymerase but do not support efficient activity of avian IAV polymerase which requires avian ANP32A. We show here that the gene currently designated as avian ANP32B is evolutionarily distinct from mammalian ANP32B, and that chicken ANP32B does not support IAV polymerase activity even of human-adapted viruses. Consequently, IAV relies solely on chicken ANP32A to support its replication in chicken cells. Amino acids 129I and 130N, accounted for the inactivity of chicken ANP32B. Transfer of these residues to chicken ANP32A abolished support of IAV polymerase. Understanding ANP32 function will help develop antiviral strategies and aid the design of influenza virus resilient genome edited chickens. https://doi.org/10.7554/eLife.45066.001 eLife digest The influenza A virus pandemic of 1918 killed more people than the armed conflicts of World War 1. Like all other pandemic and seasonal influenza, this virus originated from bird viruses. In fact, avian influenza viruses continually threaten to spark new outbreaks in humans, but pandemics do not occur often. This is because these viruses must undergo several adaptations before they can replicate in and spread between people. Viruses make new copies of themselves using the molecular machinery of the cells that they invade. The proteins that make up this machinery are often slightly different in different species, and so a virus that can replicate in cells of one species might not be able to do so when it invades a cell from another species. In 2016, researchers discovered that species differences in a cell protein called ANP32A pose a key barrier that avian influenza viruses have to overcome. Now, Long et al. – including some of the researchers involved in the 2016 study – show that the avian influenza virus cannot replicate in chicken cells that lack ANP32A. Exploring closely related versions of the genes that produce ANP32A and its relative ANP32B in different species revealed the region of the protein that the virus relies on to support its replication. Long et al. speculate that by making a few small changes to the ANP32A gene in chickens, it might be possible to generate a gene-edited chicken that is resilient to influenza. Close contact with poultry has led to hundreds of cases of ‘bird ‘flu’ in South East Asia, many of which have been fatal. Moreover, if avian influenza viruses mutate further in an infected person, a new pandemic could begin. Stopping influenza viruses from replicating in chickens would prevent people from being exposed to these dangerous viruses, whilst also improving the welfare of the chickens. https://doi.org/10.7554/eLife.45066.002 Introduction Influenza A viruses (IAV) infect a wide range of host species but originate from wild birds. Zoonotic transmission from the avian reservoir is initially restricted by host specific species barriers. Infection of new host species requires the virus to bind to cell surface receptors, utilise foreign host cellular proteins whilst evading host restriction factors in order to replicate its genome, and finally transmit between individuals of the new host. The negative sense RNA genome of influenza A virus (IAV) is replicated in the cell nucleus using a virally encoded RNA-dependent RNA polymerase, a heterotrimer composed of the polymerase basic 1 (PB1), polymerase basic 2 (PB2) and polymerase acidic (PA) proteins together with nucleoprotein (NP) that surrounds the viral RNA, forming the viral ribonucleoprotein complex (vRNP) (Te Velthuis and Fodor, 2016). Crucially, the viral polymerase must co-opt host factors to carry out transcription and replication (Te Velthuis and Fodor, 2016). The PB2 subunit is a major determinant of the host restriction of the viral polymerase (Almond, 1977). Avian IAV polymerases typically contain a glutamic acid at position 627 of PB2, and mutation to a lysine, the typical residue at this position in mammalian-adapted PB2 (Subbarao et al., 1993), can adapt the avian polymerase to function efficiently in mammalian cells. We have suggested that the restriction of avian IAV polymerase is due to a species specific difference in host protein ANP32A (Long et al., 2016). Avian ANP32A proteins have a 33 amino acid insertion, lacking in mammals, and overexpression of chicken ANP32A (chANP32A) in human cells rescues efficient function of avian origin IAV polymerases (Long et al., 2016). Removal of the 33 amino acids from chANP32A prevents polymerase rescue, whilst conversely artificial insertion of the 33 amino acids into either huANP32A or B overcomes host restriction (Long et al., 2016). A naturally occurring splice variant of avian ANP32A lacks the first four amino acids of the 33 amino acid insertion, reducing the rescue efficiency of avian IAV polymerase in human cells (Baker et al., 2018). This may be due to the disruption of a SUMOylation interaction motif, shown to enhance chANP32A’s interaction with IAV polymerase (Domingues and Hale, 2017). In human cells, both family members ANP32A and ANP32B (huANP32A/B) are utilised by human adapted IAV polymerases, and are thought to stimulate genome replication from the viral cRNA template, although the exact mechanism remains unclear (Sugiyama et al., 2015). Here we demonstrate that the avian ANP32B clade is evolutionarily distinct from mammalian and other ANP32Bs. We demonstrate that two amino acids differences, N129 and D130, in the LRR5 domain of chANP32B render it unable to interact with and support IAV polymerase function. We used CRISPR/Cas9 to remove the exon encoding the 33 amino acid insertion from chANP32A or to knockout the entire protein in chicken cells. Edited cells that expressed the short chANP32A isoform lacking the additional 33 amino acids supported mammalian-adapted but not avian IAV polymerase activity. Cells completely lacking chANP32A did not support either mammalian or avian IAV polymerase activity and were resilient to IAV infection. These results suggest a strategy to engineer IAV resilience in poultry through genetic deletion or amino acid changes of the LRR domain of ANP32A protein. Results Phylogenetic analysis identifies that avian ANP32B is a paralog of mammalian ANP32B To examine the relatedness of ANP32 proteins from different species, we constructed a phylogenetic tree using vertebrate ANP32 protein sequences using Drosophila mapmodulin protein as an outgroup. ANP32A and E homologues formed well-supported monophyletic clades which included multiple avian and mammalian species (Figure 1, Figure 1—figure supplement 1). Most vertebrate ANP32B proteins formed a monophyletic clade but this clade did not include avian ANP32B proteins. Rather, avian ANP32B proteins were strongly supported as members of a distinct clade with ANP32C from Xenopus and unnamed predicted proteins from non-placental mammals. This suggests that avian ANP32B and mammalian ANP32B are paralogues: birds have lost the protein orthologous to human ANP32B and eutherian mammals have lost the protein orthologous to avian ANP32B. Synteny provides further evidence to support the evolutionary relationship between avian ANP32B, Xenopus ANP32C, and the unnamed marsupial gene as they are all found adjacent to ZNF414 and MYO1F on their respective chromosomes (Figure 1—figure supplement 2). In humans, we found a short stretch of sequence between ZNF414 and MY01F which appears homologous to avian ANP32B (Figure 1—figure supplement 2). This provides further evidence that a functional gene orthologous to avian ANP32B has been lost in placental mammals. Figure 1 with 2 supplements see all Download asset Open asset Phylogenetic and sequence analysis reveals avian ANP32B to be a paralog of mammalian ANP32B. The best maximum-likelihood tree was calculated from a set of ANP32 proteins with mapmodulin from Drosophila melanogaster as an outgroup using RAxML with 100 bootstraps. This figure is a cladogram showing the relationships between mammalian ANP32s, avian ANP32s and ANP32s from Xenopus tropicalis. Selected bootstrap values show the relationship between different ANP32 protein clades. Avian ANP32B clade is shown in green. The full tree is shown in Figure 1—figure supplement 1. https://doi.org/10.7554/eLife.45066.003 Chicken ANP32B does not support IAV polymerase activity We and others have previously shown that both human ANP32A and B proteins support activity of a human-adapted IAV polymerase in human cells (Long et al., 2016; Sugiyama et al., 2015; Watanabe et al., 2014). Using CRISPR/Cas9, we generated human eHAP1 cells that lacked expression of both human ANP32A and ANP32B protein (Staller et al. in review). In WT eHAP1 cells, human-adapted IAV polymerase (PB2 627K), derived from an H5N1 virus A/turkey/England/50-92/1991 (50-92), was active, whereas the WT avian polymerase (PB2 627E) was not. Exogenous expression of C-terminally FLAG-tagged chANP32A could rescue the activity of avian IAV polymerase whereas expression of chANP32B-FLAG, which naturally lacks the 33 amino acid insertion, did not (Figure 2a). In double knockout cells, neither human-adapted nor avian-origin polymerase were active. Expression of chANP32A-FLAG rescued activity of both polymerases but expression of chicken ANP32B-FLAG rescued neither, despite confirmation of robust expression by western blot (Figure 2b and c). This suggests that chicken ANP32B is not functional for IAV polymerase and that the IAV polymerase activity relies on ANP32A in chicken cells. To confirm this in chicken cells, we used CRISPR/Cas9 gene editing to generate chicken DF-1 cells which lacked chANP32B but retained chANP32A expression (Figure 2—figure supplement 1). Wild type DF-1 cells had mRNAs for chANP32A, B and E (Figure 2—figure supplement 1) and supported activity of avian IAV polymerase bearing either PB2 627E or 627K. Overexpression of chANP32B-FLAG did not affect activity (Figure 2d). DF-1 bKO cells also supported activity of both polymerases and again, exogenous expression of chANP32B had no effect. Since chicken cells lacking expression of chANP32B did not demonstrate any loss of IAV polymerase activity compared to WT, this implied that chANP32B is not functional for IAV polymerase and that IAV polymerase uses solely ANP32 family member A in chicken cells. Figure 2 with 1 supplement see all Download asset Open asset Chicken ANP32B is not functional for IAV polymerase. Cells were transfected with avian H5N1 50–92 polymerase (PB2 627E or 627K) together with NP, firefly minigenome reporter, Renilla expression control, either Empty vector (control) or ANP32 expression plasmid and incubated at 37°C for 24 hr. (a) Minigenome assay in human eHAP1 cells with co-expressed Empty vector, FLAG-tagged chANP32A or chANP32B. (b) Minigenome assay in double knockout (dKO) eHAP1 cells. (c) Western blot analysis of dKO eHAP1 cell minigenome assay confirming expression of PB2 and FLAG-tagged chANP32A and B. (d) Minigenome assay in WT DF-1 cells with either co-expressed Empty vector or chANP32B. (e) Minigenome assay in DF-1 ANP32B knockout (bKO) cells with either co-expressed Empty vector or chANP32B. Data shown are firefly activity normalised to Renilla, plotted as mean ± SEM (n = 3 biological replicates). Two-way ANOVA with Dunnet’s multiple comparisons to Empty vector. ns = not significant, ****p<0.0001. https://doi.org/10.7554/eLife.45066.006 Chicken cells lacking intact ANP32A do not support avian IAV polymerase activity To investigate the function of ANP32A in chicken cells we utilised a cell type that is more amenable to genome editing and clonal growth. Primordial germ cells (PGCs) are the lineage restricted stem cells which form the gametes in the adult animal. PGCs from the chicken embryo can be easily isolated and cultured indefinitely in defined medium conditions (van de Lavoir et al., 2006; Whyte et al., 2015). Chicken PGCs can be edited using artificial sequence-specific nucleases and subsequently used to generate genome edited offspring (Park et al., 2014; Oishi et al., 2016). Under appropriate in vitro conditions PGCs can acquire pluripotency and be subsequently differentiated into multiple cell types (Matsui et al., 1992; Shim et al., 1997; Shamblott et al., 1998; Park and Han, 2000). Chicken PGC cells were genome edited using CRISPR/Cas9 and a single guide RNA which generated chANP32A knock-out cells (aKO) containing a biallelic deletion of 8 nucleotides in exon 1. PGCs lacking the 33 amino acid insertion in chANP32A were generated using a pair of guide RNAs to remove exon five resulting in chicken cells with a mammalian-like ANP32A (Δ33) (Figure 3a). The precise deletions were confirmed by Sanger sequence analysis of subcloned PCR products from genomic DNA, and both found to be homozygous at both alleles (Figure 3—figure supplement 1). We differentiated the edited chicken PGCs into fibroblast-like cells using serum induction with the aim of generating cell lines to test avian IAV polymerase activity (Figure 3—figure supplement 2). The predicted alterations of ANP32A protein in these cells were confirmed by western blot analysis of the PGC-derived fibroblast cells (Figure 3b). WT, Δ33, and aKO and PGC-derived cell lines were tested for the functional effects of alteration or loss of chANP32A expression on IAV polymerase activity measured by reconstituted minigenome assay. Both avian (PB2 627E) and human-adapted polymerase (PB2 627K) were active in WT fibroblast cells (Figure 3c). Removal of the 33 amino acids from ANP32A resulted in restriction of the 627E polymerase but not the 627K polymerase, mirroring the avian IAV polymerase phenotype observed in mammalian cells (Long et al., 2016). Both polymerases were restricted in cells lacking chANP32A (aKO). Expression of exogenous chANP32A in Δ33 and aKO cells rescued avian IAV polymerase activity (Figure 3d & e) demonstrating the specificity of the genetic alterations. The lack of polymerase activity in the aKO PGC cell line supports the hypothesis that, in the absence of chANP32A, the remaining ANP family members including chANP32B or chANP32E could not support IAV polymerase activity in chicken cells, even though ANP32B and E mRNAs were readily detected in both DF-1 and PGC cells (Figure 2—figure supplement 1 and Figure 3—figure supplement 1). Figure 3 with 2 supplements see all Download asset Open asset Chicken PGC derived fibroblast cells lacking ANP32A or the 33 amino acid insertion do not support avian IAV polymerase activity. (a) Schematic of CRISPR/Cas9 RNA guide targets used to generate aKO (exon1) and Δ33 (exon 5) PGC cell lines. (b) Western blot analysis of ANP32A and β-actin expression in WT, KO and Δ33 PGC-derived fibroblast cells. (c) Minigenome assay in WT, Δ33 or aKO PGC derived fibroblast cells with either PB2627E (black) or 627K (grey) polymerase derived from avian H5N1 50–92 virus. (d) Minigenome assay in WT, Δ33 or aKO cells with avian H5N1 50–92 PB2 627E polymerase co-transfected with Empty vector (black) or FLAG-tagged chANP32A (grey). (e) Western blot analysis of PB2, FLAG and Histone 3. Data shown are firefly activity normalised to Renilla, plotted as mean ± SEM (n = 3 biological replicates). Two-way ANOVA with Dunnet’s multiple comparisons to WT. ns = not significant, **p<0.01, ***p<0.001, ****p<0.0001. https://doi.org/10.7554/eLife.45066.008 Functional differences between chicken ANP32A and ANP32B map to the LRR domain sequence ANP32 proteins share a common domain organisation in which an N terminal domain consisting of 5 consecutive leucine rich repeats (LRR 1–5) is followed by a cap and central domain and a C terminal low complexity acidic region (LCAR). In avian ANP32A proteins (except some flightless birds) a sequence duplication, derived in part from nucleotides that encode 27 amino acids (149-175), has resulted in an additional exon and an insertion of up to 33 amino acids between the central domain and the LCAR (Figure 4a). We previously showed that insertion of the 33 amino acids from the central domain of chANP32A into the equivalent region of the human ANP32A or huANP32B proteins conferred the ability to rescue the activity of a restricted avian IAV polymerase in human cells. The equivalent 33 amino acid insertion into chANP32B (chANP32B33) did not support avian IAV polymerase activity (Figure 4b). In order to ascertain the domains of chANP32B that rendered it non-functional for IAV polymerase activity, we generated chimeric constructs between human and chicken ANP32B. To measure the rescue of avian IAV polymerase in human 293 T cells, all chimeric constructs had the 33 amino acid sequence derived from chANP32A inserted between the LRR and LCAR domains. Western blot analysis and immunofluorescence confirmed that all chimeric constructs were expressed and localised to the cell nucleus as for the wild type ANP32 proteins. (Figure 4b and Figure 4—figure supplement 1). Swapping the LCAR domain of chANP32B into huANP32B33 did not prevent the rescue of avian IAV polymerase (huANP32B33LCAR). Introduction of the central domain of chANP32B into huANP32B (huANP32B33CENT) significantly reduced rescue efficiency and swapping the LRR domain of chANP32B (huANP32B33LRR) rendered the protein non-functional to avian IAV polymerase (Figure 4b). By sequential swapping of each LRR repeat, the 5th LRR of chANP32B was found to be the domain that prevented rescue of avian IAV polymerase (Figure 4b). The fifth LRR contains five amino acid differences between human and chicken ANP32B, highlighted on the crystal structure of huANP32A, plus an additional one difference to chANP32A (Figure 4d and Figure 4—figure supplement 2). Swapping chANP32B’s fifth LRR into chANP32A also prevented rescue of avian IAV polymerase activity in human cells (chANP32ALRR5) (Figure 4c). Introduction of the single amino acid changes derived from the chANP32B LRR5 sequence into chANP32A revealed that mutations N129I and D130N significantly reduced the ability of chANP32A to rescue avian IAV polymerase activity in human cells (Figure 4c). Minigenome assays with co-expressed chANP32A or chANP32AN129I in aKO chicken fibroblast cells confirmed that the 129I mutation significantly reduced the ability of chANP32A to support avian-origin (PB2 627E) or human-adapted (PB2 627K) IAV polymerase activity (Figure 4e). Figure 4 with 2 supplements see all Download asset Open asset Lack of functional support for IAV polymerase by chicken ANP32B maps to differences in LRR5 domain. (a) Schematic of chicken ANP32A protein highlighting the different domains and LRR sequences (LRR 1–5). (b) Human 293 T cells were transfected with avian H5N1 50–92 polymerase (PB2 627E) together with NP, pHOM1-firefly minigenome reporter, Renilla expression control, either Empty vector or FLAG-tagged ANP32 expression plasmid and incubated at 37°C for 24 hr. Western blot analysis shown below (FLAG and Vinculin). (c) Minigenome assay in 293 T cells (PB2 627E) with FLAG-tagged WT or mutant chANP32A expression plasmids with associated western blot (FLAG and PCNA). (d) huANP32A crystal structure (PDB 4 × 05) with residues K116, N127, N129, D130 and K137 highlighted using UCSF Chimaera (Pettersen et al., 2004). (e) Minigenome assay of avian H5N1 50–92 polymerase with either PB2 627E or 627K in PGC-derived fibroblast aKO cells, together with co-expressed Empty vector, chANP32A or chANP32AN129I. Data shown are firefly activity normalised to Renilla, plotted as mean ± SEM (n = 3 biological replicates). One-way ANOVA with Tukey’s comparison to chANP32A (b and c) or two-way ANOVA with Dunnet’s multiple comparisons to chANP32A (e). ns = not significant, *p<0.05, **p<0.01, ****p<0.0001. https://doi.org/10.7554/eLife.45066.011 Sequence of amino acids 149–175 of the central domain of chANP32A are required to support activity of both avian and human-adapted IAV polymerase As chANP32A KO PGC-derived fibroblast cells did not support of IAV polymerase despite expressing chANP32B, we were able to use these cells to understand in more detail the sequences in chANP32A required for IAV polymerase activity. The results above showed that the 33 amino acid insertion, fifth LRR and central domain are important for the ability of chANP32A to support function of avian IAV polymerase. We performed the minigenome assay in aKO cells with polymerases containing either PB2 627E and 627K with co-expression of further chANP32 mutants including: chANP32A in which the 27 amino acids in the central domain preceding the 33 amino acid insertion were scrambled (chANP32Ascr149-175) or chANP32A with the 33 amino acid insertion scrambled (chANP32Ascr176-208) (Figure 5a). Both mutants were expressed and localised to the nucleus (Figure 5c and Figure 4—figure supplement 1). The first mutant, chANP32Ascr149-175, did not support either PB2 627E or 627K polymerase, suggesting the sequence of the central domain is important for function of IAV polymerase. The second mutant, chANP32A scr176-208, only supported PB2 627K function, confirming that the sequence of the 33 amino acid insertion, not just the extended length is required for avian IAV polymerase (PB2 627E) (Figure 5b). Figure 5 Download asset Open asset Sequence of amino acids 149–175 of the central domain of chANP32A are required to support activity of both avian and human-adapted IAV polymerase. (a) Schematic of chANP32A showing the sequence of amino acids in the central domain (149–175 or 33 amino acid insertion (176-208) and the randomly scrambled sequence in red. (b) Minigenome assay of avian H5N1 50–92 polymerase with either PB2 627E or 627K in PGC-derived fibroblast aKO cells with co-expressed Empty plasmid or FLAG-tagged WT chANP32A, chANP32Ascr149-175 or chANP32Ascr176-208 expression plasmids. (c) Western blot analysis of PB2 (627E), lamin B1 and FLAG. Data shown are firefly activity normalised to Renilla, plotted as mean ± SEM (n = 3 biological replicates). Two-way ANOVA with Dunnet’s multiple comparisons to chANP32A. ns = not significant, ****p<0.0001. https://doi.org/10.7554/eLife.45066.014 A single amino acid difference between chANP32B and chANP32A abrogates binding of chANP32A to IAV polymerase An interaction between ANP32A and IAV polymerase was demonstrated previously that is dependent on the presence of all three polymerase subunits (Mistry et al. in preparation & Baker et al., 2018; Domingues and Hale, 2017). To examine the interaction between IAV polymerase and chANP32 proteins we employed a split luciferase complementation assay as a quantitative measure of binding (Munier et al., 2013; Cassonnet et al., 2011). The C-terminus of the PB1 subunit of avian origin IAV polymerase was fused with one half of gaussia luciferase (PB1luc1) and the C-terminus of chicken ANP32A or B with the second half (chANP32Aluc2 and chANP32Bluc2) (Figure 6a). Reconstitution of PB1luc1, PB2 and PA together with chANP32Aluc2 in human 293 T cells gave a strong Normalised Luciferase Ratio (NLR) (Figure 6—figure supplement 1) with polymerases containing either PB2 627E or 627K (Figure 6b). Luciferase complementation was significantly less between polymerase and chANP32Bluc2, and even insertion of the 33 amino acids from chANP32A did not restore the signal (chANP32B33luc2) (Figure 6b). When chANP32A carried the single N129I mutation (chANP32AN129Iluc2), luciferase complementation was reduced 22-fold for PB2 627E polymerase and 52-fold forPB2 627K polymerase (Figure 6c and Figure 6—figure supplement 1). These results suggest that the loss of support of polymerase function by chANP32AN129I was due to a disruption of binding to IAV polymerase. Figure 6 with 1 supplement see all Download asset Open asset A single amino acid change (N129I) derived from chANP32B disrupts chANP32A support of influenza polymerase activity by abrogating binding to IAV polymerase. (a) Diagram of the split Gaussia luciferase system, demonstrating how ANP32 fused to luciferase fragment luc2 may bind to polymerase containing PB1 fused to luciferase fragment luc1 and complement full luciferase, which then reacts with substrate to generate a measurable bioluminescent signal. (b) Human 293 T cells were transfected with PB1 fused to luc1 (PB1luc1), PB2 (627E or K), PA and either chANP32A, chANP32B or chANP32B33 fused to luc2 (control wells were transfected with all components but with unfused PB1 and luc1 or chANP32 and luc2). (c) As (b) but with either chANP32Aluc2 or chANP32AN129Iluc2. (d) 293 T cells transfected with either chANP32Aluc2 or chANP32AN129Iluc2 and histone four fused to luc1 (or with unfused controls) or with H4luc1 and histone three fused to luc2. All data are Normalised Luciferase Ratio (n = 3 biological replicates) (Figure 6—figure supplement 1). One-way ANOVA (d) or two-way ANOVA with Dunnet’s multiple comparisons to chANP32A (b and c). ns = not significant, ****p<0.0001. https://doi.org/10.7554/eLife.45066.015 ANP32A proteins bind to histones as part of their role in chromatin regulation (Reilly et al., 2014). To measure if the mutation N129I had any effect on this cellular interaction, we generated expression plasmids that encoded human histone four with luc1 fused to the C-terminus (H4luc1) and histone 3.1 with luc2 fused to the C terminus (H3luc2). As expected, H4luc1 and H3luc2 generated a strong NLR, reflecting their interaction in the nucleosome (Luger et al., 1997). The ability of chANP32A to bind histone four was not impaired by mutation N129I, suggesting chANP32N129I was not altered in this cellular role, despite abrogation of its support of IAV polymerase (Figure 6d). Viral replication is abrogated in chicken cells lacking ANP32A The data above suggest that chANP32B cannot substitute for chANP32A in support of IAV polymerase in chicken cells. Since chicken cells that completely lack expression of chANP32A show no polymerase activity in the minigenome assay, they might be refractory to IAV infection. Multi-cycle growth kinetics of recombinant influenza A viruses were measured in WT and aKO PGC-derived fibroblast cells (Figure 7). To ensure robust infection, recombinant viruses were generated carrying H1N1 vaccine strain PR8 haemagglutinin (HA), neuraminidase (NA) and M genes; this also mitigated the risks of working with avian influenza viruses with novel antigenicity. Infectious titres of recombinant virus with internal genes of avian H5N1 virus 50–92 were not detected in the chicken cells lacking ANP32A infected at low MOI (Figure 7a). At higher MOI virus titres were significantly reduced compared to WT chicken cells, almost 325-fold less at 8 hr post infection and 16-fold less by 24 hr (Figure 7c). Similarly, at low MOI, a recombinant virus with internal genes from the H7N9 virus A/Anhui/1/2013, had limited virus growth in aKO cells but replicated efficiently in WT fibroblasts (Figure 7b). At the higher MOI, peak viral titres were 1365-fold less than in WT cells at 8 hr post infection and 100-fold less by 24 hr (Figure 7d). Since virus growth in aKO cells was observed at the higher MOI, we sequenced the PB2 gene of virus progeny to determine if this replication was due to adaptation in PB2. At 24 hr post infection the sequence of the PB2 gene from virus in supernatants of cells infected at high MOI was determined. Virus recovered from aKO and WT cells was found to be identical and contained no sequence changes compared with the inoculum, suggesting adaptation in PB2 was not required for the low level of replication seen in the aKO cells. In conclusion, PGC derived fibroblast cells lacking chANP32A were resilient to IAV replication, particularly at lower multiplicities of infection. Figure 7 Download asset Open asset Viral replication is abrogated in chicken PGC fibroblast cells lacking ANP32A. WT (black lines) or aKO (red lines) PGC-derived fibroblast cells were infected with recombinant viruses (containing PR8 HA, NA and M genes and internal genes from either H5N1 50–92 or H7N9 Anhui), at an MOI of either 0.0001 (a,b) or 1.0 (c,d), incubated at 37°C in the presence of trypsin, cell supernatants harvested at described time-points and PFU ml−1 measured by plaque assay on MDCK cells. (a) H5N1 50–92 (MOI 0.0001). b. H7N9 Anhui (MOI 0.0001). c. H5N1 50-−92 (MOI 1.0). d. H7N9 Anhui (MOI 1.0). vRNAs from supernatants 24 hr post infection MOI 1.0 (c and d) were extracted, PCR amplified and sequenced by Sanger sequencing. Limit of detection by plaque assay shown by dotted line (10PFU ml−1). n = 3 biological replicates. Multiple t-tests with Holm-Sidak comparison. ns = not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. https://doi.org/10.7554/eLife.45066.017 Discussion We show that avian origin IAV polymerases rely exclusively on chicken ANP32A family member for their replication, because they are unable to co-opt chicken ANP32B. We found avian ANP32B p

Influenza A viruses (IAV) are subject to species barriers that prevent frequent zoonotic transmission and pandemics. One of these barriers is the poor activity of avian IAV polymerases in human cells. Differences between avian and mammalian ANP32 proteins underlie this host range barrier. Human ANP32A and ANP32B homologues both support function of human-adapted influenza polymerase but do not support efficient activity of avian IAV polymerase which requires avian ANP32A. We show here that the gene currently designated as avian ANP32B is evolutionarily distinct from mammalian ANP32B, and that chicken ANP32B does not support IAV polymerase activity even of human-adapted viruses. Consequently, IAV relies solely on chicken ANP32A to support its replication in chicken cells. Amino acids 129I and 130N, accounted for the inactivity of chicken ANP32B. Transfer of these residues to chicken ANP32A abolished support of IAV polymerase. Understanding ANP32 function will help develop antiviral strategies and aid the design of influenza virus resilient genome edited chickens.

Influenza A viruses (IAV) are subject to species barriers that prevent frequent zoonotic transmission and pandemics. One of these barriers is the poor activity of avian IAV polymerases in human cells. Differences between avian and mammalian ANP32 proteins underlie this host range barrier. Human ANP32A and ANP32B homologues both support function of human-adapted influenza polymerase but do not support efficient activity of avian IAV polymerase which requires avian ANP32A. We show here that avian ANP32B is evolutionarily distinct from mammalian ANP32B, and that chicken ANP32B does not support IAV polymerase activity even of human-adapted viruses. Consequently, IAV does not replicate in chicken cells that lack ANP32A. Amino acid differences in LRR5 domain accounted for the inactivity of chicken ANP32B. Transfer of these residues to chicken ANP32A abolished support of IAV polymerase. Understanding ANP32 function will help develop antiviral strategies and aid the design of influenza virus resistant genome edited chickens.