Iceland: an underestimated hub for the spread of high-pathogenicity avian influenza viruses in the North Atlantic.

High-pathogenicity avian influenza viruses (HPAIVs) of the goose/Guangdong lineage are enzootically circulating in wild bird populations worldwide. This increases the risk of entry into poultry production and spill-over to mammalian species, including humans. Better understanding of the ecological and epizootiological networks of these viruses is essential to optimize mitigation measures. Based on full genome sequences of 26 HPAIV samples from Iceland, which were collected between spring and autumn 2022, as well as 1 sample from the 2023 summer period, we show that 3 different genotypes of HPAIV H5N1 clade 2.3.4.4b were circulating within the wild bird population in Iceland in 2022. Furthermore, in 2023 we observed a novel introduction of HPAIV H5N5 of the same clade to Iceland. The data support the role of Iceland as an utmost northwestern distribution area in Europe that might act also as a potential bridging point for intercontinental spread of HPAIV across the North Atlantic.

Experimental Pathogenicity of H9N2 Avian Influenza Viruses Harboring a Tri-Basic Hemagglutinin Cleavage Site in Sonali and Broiler Chickens.

Low-pathogenic avian influenza (LPAI) H9N2 virus is endemic in Bangladesh, causing huge economic losses in the poultry industry. Although a considerable number of Bangladeshi LPAI H9N2 viruses have been molecularly characterized, there is inadequate information on the pathogenicity of H9N2 viruses in commercial poultry. In this study, circulating LPAI H9N2 viruses from recent field outbreaks were characterized, and their pathogenicity in commercial Sonali (crossbred) and broiler chickens was assessed. Phylogenetic analysis of currently circulating field viruses based on the hemagglutinin (HA) and neuraminidase (NA) gene sequences revealed continuous circulation of G1 lineages containing the tri-basic hemagglutinin cleavage site (HACS) motif (PAKSKR*GLF) at the HA protein. Both the LPAI susceptible Sonali and broiler chickens were infected with selected H9N2 isolates A/chicken/Bangladesh/2458-LT2/2020 or A/chicken/Bangladesh/2465-LT56/2021 using intranasal (100 µL) and intraocular (100 µL) routes with a dose of 106 EID50/mL. Infected groups (LT_2-So1 and LT_56-So2; LT_2-Br1 and LT_56-Br2) revealed no mortality or clinical signs. However, at gross and histopathological investigation, the trachea, lungs, and intestine of the LT_2-So1 and LT_56-So2 groups displayed mild to moderate hemorrhages, congestion, and inflammation at different dpi. The LT 2-Br1 and LT 56-Br2 broiler groups showed nearly identical changes in the trachea, lungs, and intestine at various dpi, indicating no influence on pathogenicity in the two commercial bird species under study. Overall, the prominent lesions were observed up to 7 dpi and started to disappear at 10 dpi. The H9N2 viruses predominantly replicated in the respiratory tract, and higher titers of virus were shed through the oropharyngeal route than the cloacal route. Finally, this study demonstrated the continuous evolution of tri-basic HACS containing H9N2 viruses in Bangladesh with a low-pathogenic phenotype causing mild to moderate tracheitis, pneumonia, and enteritis in Sonali and commercial broiler chickens.

Investigating Environmental Matrices for Use in Avian Influenza Virus Surveillance-Surface Water, Sediments, and Avian Fecal Samples.

Surveillance of avian influenza viruses (AIV) in wild water bird populations is important for early warning to protect poultry from incursions of high-pathogenicity (HP) AIV. Access to individual water birds is difficult and restricted and limits sampling depth. Here, we focused on environmental samples such as surface water, sediments, and environmentally deposited fresh avian feces as matrices for AIV detection. Enrichment of viral particles by ultrafiltration of 10-L surface water samples using Rexeed-25-A devices was validated using a bacteriophage ϕ6 internal control system, and AIV detection was attempted using real-time RT-PCR and virus isolation. While validation runs suggested an average enrichment of about 60-fold, lower values of 10 to 15 were observed for field water samples. In total 25/36 (60%) of water samples and 18/36 (50%) of corresponding sediment samples tested AIV positive. Samples were obtained from shallow water bodies in habitats with large numbers of waterfowl during an HPAIV epizootic. Although AIV RNA was detected in a substantial percentage of samples virus isolation failed. Virus loads in samples often were too low to allow further sub- and pathotyping. Similar results were obtained with environmentally deposited avian feces. Moreover, the spectrum of viruses detected by these active surveillance methods did not fully mirror an ongoing HPAIV epizootic among waterfowl as detected by passive surveillance, which, in terms of sensitivity, remains unsurpassed. IMPORTANCE Avian influenza viruses (AIV) have a wide host range in the avian metapopulation and, occasionally, transmission to humans also occurs. Surface water plays a particularly important role in the epidemiology of AIV, as the natural virus reservoir is found in aquatic wild birds. Environmental matrices comprising surface water, sediments, and avian fecal matter deposited in the environment were examined for their usefulness in AIV surveillance. Despite virus enrichment efforts, environmental samples regularly revealed very low virus loads, which hampered further sub- and pathotyping. Passive surveillance based on oral and cloacal swabs of diseased and dead wild birds remained unsurpassed with respect to sensitivity.

Tetraplex Fluorescent Microbead-Based Immunoassay for the Serodiagnosis of Newcastle Disease Virus and Avian Influenza Viruses in Poultry Sera.

Virulent Newcastle disease virus (NDV) as well as highly pathogenic avian influenza (HPAIV) subtypes H5 and H7 induce contagious and lethal systemic disease in poultry. In contrast, low pathogenic AIV H5 and H7 may circulate clinically unnoticed in poultry but eventually generate HPAIV. Low pathogenic NDV strains are widely used as live-attenuated vaccines against ND. Serological tools are essential to conduct active surveillance for infections with notifiable AIV-H5, -H7 and to control vaccination against NDV and HPAIV in poultry populations. Here, recombinant nucleocapsid proteins (NP) of AIV and NDV, and haemagglutinin protein fragment-1 (HA1) of AIV subtypes H5 and H7 were expressed in E. coli. Purification and refolding were required before coating fluorescent microspheres via streptavidin-biotin linkage. The tetraplexed inhibition fluorescent microsphere immunoassay (iFMIA) was then assembled for analysis on a Luminex®-like platform (Bioplex®) using murine monoclonal antibodies specific for each of the four targets. The assay was evaluated by testing galliform poultry sera derived from experimental infections (n = 257) and from farms (n = 250), respectively. The tetraplex iFMIA compared favorably with commercially available ELISAs and the "gold standard" hemagglutination inhibition assay. Tetraplexed iFMIA provided a specific and sensitive tool to detect and discriminate AIV- and NDV-specific antibodies in the sera of galliform poultry.

Exploring surface water as a transmission medium of avian influenza viruses - systematic infection studies in mallards.

Mallards (Anas platyrhynchos) are an abundant anseriform migratory wild bird species worldwide and an important reservoir for the maintenance of low pathogenicity (LP) avian influenza viruses (AIV). They have also been implicated in the spread of high pathogenicity (HP) AIV after spill-over events from HPAIV-infected poultry. The spread of HPAIV within wild water bird populations may lead to viral contamination of natural habitats. The role of small shallow water bodies as a transmission medium of AIV among mallards is investigated here in three experimental settings. (i) Delayed onset but rapid progression of infection seeded by two mallards inoculated with either LP or HP AIV to each eight sentinel mallards was observed in groups with access to a small 100 L water pool. In contrast, groups with a bell drinker as the sole source of drinking water showed a rapid onset but lengthened course of infection. (ii) HPAIV infection also set off when virus was dispersed in the water pool; titres as low as 102 TCID50 L-1 (translating to 0.1 TCID50 mL-1) proved to be sufficient. (iii) Substantial loads of viral RNA (and infectivity) were also found on the surface of the birds' breast plumage. "Unloading" of virus infectivity from contaminated plumage into water bodies may be an efficient mechanism of virus spread by infected mallards. However, transposure of HPAIV via the plumage of an uninfected mallard failed. We conclude, surface water in small shallow water bodies may play an important role as a mediator of AIV infection of aquatic wild birds.

Comparison of genomic and antigenic properties of Newcastle Disease virus genotypes II, XXI and VII from Egypt do not point to antigenic drift as selection marker.

Newcastle disease (ND), caused by avian orthoavulavirus type-1 (NDV), is endemic in poultry in many regions of the world and causes continuing outbreaks in poultry populations. In the Middle East, genotype XXI, used to be present in poultry in Egypt but has been replaced by genotype VII. We investigated whether virus evolution contributed to superseding and focussed on the antigenic sites within the hemagglutinin-neuraminidase (HN) spike protein. Full-length sequences of an NDV genotype VII isolate currently circulating in Egypt was compared to a genotype XXI isolate that was present as co-infection with vaccine-type viruses (II) in a historical virus isolated in 2011. Amino acid differences in the HN glycoprotein for both XXI and VII viruses amounted to 11.7% and 11.9%, respectively, compared to the La Sota vaccine type. However, mutations within the globular head (aa 126-570), bearing relevant antigenic sites, were underrepresented (a divergence of 8.8% and 8.1% compared to 22.4% and 25.6% within the protein domains encompassing cytoplasmic tail, transmembrane part and stalk regions (aa 1-125) for genotypes XXI and VII, respectively). Nevertheless, reaction patterns of HN-specific monoclonal antibodies inhibiting receptor binding revealed differences between vaccine-type viruses and genotype XXI and VII viruses for epitopes located in the head domain. Accordingly, compared to Egyptian vaccine-type isolates and the La Sota vaccine reference strain, single aa substitutions in 6 of 10 described neutralizing epitopes of HN were found. However, the same alterations in neutralization sensitive epitopes were present in old genotype XXI as well as in newly emerged genotype VII isolates. In addition, isolates were indistinguishable by polyclonal chicken sera raised against different genotypes including vaccine viruses. These findings suggest that factors other than antigenic differences within the HN protein account for facilitating the spread of genotype VII versus genotype XXI viruses in Egypt.

Optimizing Release of Nucleic Acids of African Swine Fever Virus and Influenza A Virus from FTA Cards.

FTA cards and related products simplify the collection, transport, and transient storage of biological sample fluids. Here, we have compared the yield and quality of DNA and RNA released from seven different FTA cards using seven releasing/extraction methods with eleven experimental eluates. For the validation, dilution series of African swine fever virus (ASFV) positive EDTA blood and Influenza A virus (IAV) positive allantoic fluid were used. Based on our data, we conclude that direct PCR amplification without the need for additional nucleic acid extraction and purification could be suitable and more convenient for ASFV DNA release from FTA cards. In contrast, IAV RNA loads can be amplified from FTA card punches if a standard extraction procedure including a lysis step is applied. These differences between the amplifiable viral DNA and RNA after releasing and extraction are not influenced by the type of commercial FTA card or the eleven different nucleic acid releasing procedures used for the comparative analyses. In general, different commercial FTA cards were successfully used for the storage and recovery of the ASFV and IAV genetic material suitable for PCR. Nevertheless, the usage of optimized nucleic acid releasing protocols could improve the recovery of the viral genome of both viruses. Here, the application of Chelex® Resin 100 buffer mixed with 1 × Tris EDTA buffer (TE, pH 8.0) or with TED 10 (TE buffer and Dimethylsulfoxid) delivered the best results and can be used as a universal method for releasing viral DNA and RNA from FTA cards.

Influenza A Viruses and Zoonotic Events-Are We Creating Our Own Reservoirs?

Zoonotic infections of humans with influenza A viruses (IAVs) from animal reservoirs can result in severe disease in individuals and, in rare cases, lead to pandemic outbreaks; this is exemplified by numerous cases of human infection with avian IAVs (AIVs) and the 2009 swine influenza pandemic. In fact, zoonotic transmissions are strongly facilitated by manmade reservoirs that were created through the intensification and industrialization of livestock farming. This can be witnessed by the repeated introduction of IAVs from natural reservoirs of aquatic wild bird metapopulations into swine and poultry, and the accompanied emergence of partially- or fully-adapted human pathogenic viruses. On the other side, human adapted IAV have been (and still are) introduced into livestock by reverse zoonotic transmission. This link to manmade reservoirs was also observed before the 20th century, when horses seemed to have been an important reservoir for IAVs but lost relevance when the populations declined due to increasing industrialization. Therefore, to reduce zoonotic events, it is important to control the spread of IAV within these animal reservoirs, for example with efficient vaccination strategies, but also to critically surveil the different manmade reservoirs to evaluate the emergence of new IAV strains with pandemic potential.

Modulation of lethal HPAIV H5N8 clade 2.3.4.4B infection in AIV pre-exposed mallards.

In 2016/2017, a severe epidemic of HPAIV H5N8 clade 2.3.4.4 group B (H5N8B) affected Europe. To analyse the role of mallards in the spatiotemporal dynamics of global HPAIV H5N8B dispersal, mallards (Anas platyrhynchos), naturally exposed to various AIV and therefore seropositive, were challenged with H5N8B. All experiments were controlled by infection and co-housing of seronegative juvenile Pekin ducklings. All ducks that survived the first infection were re-challenged 21 dpi with the homologous H5N8B strain. After the first H5N8B infection, seropositive mallards showed only mild clinical symptoms. Moderate to low viral shedding, occurring particularly from the oropharynx and lasting for 7 days maximum, led to severe clinical disease of all contact ducklings. All challenged seronegative Pekin ducks and contact ducklings died or had to be euthanized. H5-specific antibodies were detected in surviving birds within 2 weeks. Virus and viral RNA could be isolated from several water samples until 6 and 9 dpi, respectively. Conversely, upon re-infection with homologous H5N8B neither inoculated nor contact ducklings showed any clinical symptoms, nor was an antibody titer increase of seropositive mallards or any seroconversion of contact ducklings observed. Mallard ducks naturally pre-exposed to LPAIV can play a role as a clinically unsuspicious virus reservoir for H5N8B effective in virus transmission. Mallards with homologous immunity did not contribute to virus transmission.

Variable impact of the hemagglutinin polybasic cleavage site on virulence and pathogenesis of avian influenza H7N7 virus in chickens, turkeys and ducks.

Avian influenza viruses (AIV) are classified into 16 hemagglutinin (HA; H1-H16) and 9 neuraminidase (NA; N1-N9) subtypes. All AIV are low pathogenic (LP) in birds, but subtypes H5 and H7 AIV can evolve into highly pathogenic (HP) forms. In the last two decades evolution of HPAIV H7 from LPAIV has been frequently reported. However, little is known about the pathogenesis and evolution of HP H7 from LP ancestors particularly, in non-chicken hosts. In 2015, both LP and HP H7N7 AIV were isolated from chickens in two neighbouring farms in Germany. Here, the virulence of these isogenic H7N7 LP, HP and LP virus carrying a polybasic HA cleavage site (HACS) from HP (designated LP-Poly) was studied in chickens, turkeys and different duck breeds. The LP precursor was avirulent in all birds. In contrast, all inoculated and contact chickens and turkeys died after infection with HP. HP infected Pekin and Mallard ducks remained clinically healthy, while Muscovy ducks exhibited moderate depression and excreted viruses at significantly higher amounts. The polybasic HACS increased virulence in a species-specific manner with intravenous pathogenicity indices of 3.0, 1.9 and 0.2 in chickens, turkeys and Muscovy ducks, respectively. Infection of endothelial cells was only observed in chickens. In summary, Pekin and Mallard were more resistant to HPAIV H7N7 than chickens, turkeys and Muscovy ducks. The polybasic HACS was the main determinant for virulence and endotheliotropism of HPAIV H7N7 in chickens, whereas other viral and/or host factors play an essential role in virulence and pathogenesis in turkeys and ducks.

A newly developed tetraplex real-time RT-PCR for simultaneous screening of influenza virus types A, B, C and D.

BACKGROUND: Human- or avian-to-swine transmissions have founded several autonomously circulating influenza A virus (IAV) lineages in swine populations that cause economically important respiratory disease. Little is known on other human influenza virus types, like B (IBV) and C (ICV) in European swine, and of the recently detected novel animal influenza virus type D (IDV). OBJECTIVES: Development of a cost-effective diagnostic tool for large-scale surveillance programmes targeting all four influenza virus types. METHODS: An influenza ABCD tetraplex real-time RT-PCR (RT-qPCR) was developed in the frame of this study. A selection of reference virus strains and more than 4000 porcine samples from a passive IAV surveillance programme in European swine with acute respiratory disease were examined. RESULTS: Two IBV, a single IDV but no ICV infections were identified by tetraplex RT-qPCR. IBV and IDV results were confirmed by conventional RT-PCR and partial sequence analysis. CONCLUSIONS: The tetraplex RT-qPCR proved fit for purpose as a sensitive, specific and high-throughput tool to study influenza virus transmission at the human-animal interface. Complementing close-meshed active virological and serological surveillance is required to better understand the true incidence and prevalence of influenza virus type B, C and D infections in swine.

Subtyping of Swine Influenza Viruses Using a High-Throughput Real-Time PCR Platform.

Influenza A viruses (IAVs) are important human and animal pathogens with high impact on human and animal health. In Denmark, a passive surveillance program for IAV in pigs has been performed since 2011, where screening tests and subsequent subtyping are performed by reverse transcription quantitative real-time PCR (RT-qPCR). A disadvantage of the current subtyping system is that several assays are needed to cover the wide range of circulating subtypes, which makes the system expensive and time-consuming. Therefore, the aim of the present study was to develop a high-throughput method, which could improve surveillance of swine influenza viruses (swIAVs) and lower the costs of virus subtyping. Twelve qPCR assays specific for various hemagglutinin and neuraminidase gene lineages relevant for swIAV and six assays specific for the internal genes of IAV were developed and optimized for the high-throughput qPCR platform BioMark (Fluidigm). The qPCR assays were validated and optimized to run under the same reaction conditions using a 48.48 dynamic array (48.48DA). The sensitivity and specificity was assessed by testing virus isolates and field samples with known subtypes. The results revealed a performance of the swIAV 48.48DA similar to conventional real-time analysis, and furthermore, the specificity of swIAV 48.48DA was very high and without cross reactions between the assays. This high-throughput system provides a cost-effective alternative for subtyping of swIAVs.

Engineered recombinant protein products of the avian paramyxovirus type-1 nucleocapsid and phosphoprotein genes for serological diagnosis.

BACKGROUND: Virulent Newcastle disease virus (NDV, avian Avulavirus-1, APMV-1) induces a highly contagious and lethal systemic disease in gallinaceous poultry. APMV-1 antibody detection is used for surveillance and to control vaccination, but is hampered by cross-reactivity to other subtypes of avian Avulaviruses. Data are lacking concerning the applicability of NDV V proteins as differential diagnostic marker to distinguish vaccinated from virus-infected birds (DIVA strategy). METHODS: Full length and C-terminally truncated nucleocapsid (NP) protein, and the unique C-terminal regions of the phospho- (P) and V proteins of the NDV LaSota strain were bacterially expressed as fusion proteins with the multimerization domain of the human C4 binding protein, and used as diagnostic antigens in indirect ELISA. RESULTS: When used as diagnostic antigen in indirect ELISAs, recombinant full-length proved to be a sensitive target to detect seroconversion in chickens after APMV-1 vaccination and infection, but revealed some degree of cross reactivity with sera raised against other APMV subtypes. Cross reactivity was abolished but also sensitivity decreased when employing a C-terminal fragment of the NP of NDV as diagnostic antigen. Antibodies to the NDV V protein were mounted in poultry following NDV infection but also, albeit at lower rates and titers, after vaccination with attenuated NDV vaccines. V-specific seroconversion within the flock was incomplete and titers in individual bird transient. CONCLUSIONS: Indirect ELISA based on bacterially expressed recombinant full-length NP compared favorably with a commercial NDV ELISA based on whole virus antigen, but cross reactivity between the NP proteins of different APMV subtypes could compromise specificity. However, specificity increased when using a less conserved C-terminal fragment of NP instead. Moreover, a serological DIVA strategy built on the NDV V protein was not feasible due to reduced immunogenicity of the V protein and frequent use of live-attenuated NDV vaccines.

Molecular subtyping of European swine influenza viruses and scaling to high-throughput analysis.

BACKGROUND: Swine influenza is a respiratory infection of pigs that may have a significant economic impact in affected herds and pose a threat to the human population since swine influenza A viruses (swIAVs) are zoonotic pathogens. Due to the increasing genetic diversity of swIAVs and because novel reassortants or variants may become enzootic or have zoonotic implications, surveillance is strongly encouraged. Therefore, diagnostic tests and advanced technologies able to identify the circulating strains rapidly are critically important. RESULTS: Several reverse transcription real-time PCR assays (RT-qPCRs) were developed to subtype European swIAVs in clinical samples previously identified as containing IAV genome. The RT-qPCRs aimed to discriminate HA genes of four H1 genetic lineages (H1av, H1hu, H1huΔ146-147, H1pdm) and one H3 lineage, and NA genes of two N1 lineages (N1, N1pdm) and one N2 lineage. After individual validation, each RT-qPCR was adapted to high-throughput analyses in parallel to the amplification of the IAV M gene (target for IAV detection) and the ß-actin gene (as an internal control), in order to test the ten target genes simultaneously on a large number of clinical samples, using low volumes of reagents and RNA extracts. CONCLUSION: The RT-qPCRs dedicated to IAV molecular subtyping enabled the identification of swIAVs from the four viral subtypes that are known to be enzootic in European pigs, i.e. H1avN1, H1huN2, H3N2 and H1N1pdm. They also made it possible to discriminate a new antigenic variant (H1huN2Δ146-147) among H1huN2 viruses, as well as reassortant viruses, such as H1huN1 or H1avN2 for example, and virus mixtures. These PCR techniques exhibited a gain in sensitivity as compared to end-point RT-PCRs, enabling the characterization of biological samples with low genetic loads, with considerable time saving. Adaptation to high-throughput analyses appeared effective, both in terms of specificity and sensitivity. This new development opens novel perspectives in diagnostic capacities that could be very useful for swIAV surveillance and large-scale epidemiological studies.

Natural Reassortants of Potentially Zoonotic Avian Influenza Viruses H5N1 and H9N2 from Egypt Display Distinct Pathogenic Phenotypes in Experimentally Infected Chickens and Ferrets.

The cocirculation of zoonotic highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 and avian influenza virus (AIV) of subtype H9N2 among poultry in Egypt for at least 6 years should render that country a hypothetical hot spot for the emergence of reassortant, phenotypically altered viruses, yet no reassortants have been detected in Egypt. The present investigations proved that reassortants of the Egyptian H5N1 clade 2.2.1.2 virus and H9N2 virus of the G1-B lineage can be generated by coamplification in embryonated chicken eggs. Reassortants were restricted to the H5N1 subtype and acquired between two and all six of the internal segments of the H9N2 virus. Five selected plaque-purified reassortant clones expressed a broad phenotypic spectrum both in vitro and in vivo Two groups of reassortants were characterized to have retarded growth characteristics in vitro compared to the H5N1 parent virus. One clone provoked reduced mortality in inoculated chickens, although the characteristics of a highly pathogenic phenotype were retained. Enhanced zoonotic properties were not predicted for any of these clones, and this prediction was confirmed by ferret inoculation experiments: neither the H5N1 parent virus nor two selected clones induced severe clinical symptoms or were transmitted to sentinel ferrets by contact. While the emergence of reassortants of Egyptian HPAIV of subtype H5N1 with internal gene segments of cocirculating H9N2 viruses is possible in principle, the spread of such viruses is expected to be governed by their fitness to outcompete the parental viruses in the field. The eventual spread of attenuated phenotypes, however, would negatively impact syndrome surveillance on poultry farms and might foster enzootic virus circulation.IMPORTANCE Despite almost 6 years of the continuous cocirculation of highly pathogenic avian influenza virus H5N1 and avian influenza virus H9N2 in poultry in Egypt, no reassortants of the two subtypes have been reported. Here, the principal compatibility of the two subtypes is shown by forcing the reassortment between copassaged H5N1 und H9N2 viruses in embryonated chicken eggs. The resulting reassortant viruses displayed a wide range of pathogenicity including attenuated phenotypes in chickens, but did not show enhanced zoonotic propensities in the ferret model.

Insights into genetic diversity and biological propensities of potentially zoonotic avian influenza H9N2 viruses circulating in Egypt.

Low pathogenic avian influenza (LPAI) H9N2 viruses have established endemic status in Egyptian poultry populations since 2012. Recently, four cases of human H9N2 virus infections in Egypt demonstrated the zoonotic potential of these viruses. Egyptian H9N2 viruses obtained from 2011 to 2014 phylogenetically grouped into three clusters (1-3) within subclade B of the G1 lineage. Antigenically, a close clustering of the Egyptian H9N2 viruses with other recent G1-B like H9N2 strains and a significant antigenic distance from viruses outside the G1-B lineage was evident. Recent Egyptian LPAIV H9N2 showed a tendency to increased binding with erythrocytes expressing α 2,6-linked sialic acid which correlated with the Q226L amino acid substitution at the receptor binding unit of the hemagglutinin (Q234L, H9 numbering). Sequence analyses of the N2 neuraminidase (NA) revealed substitutions in the NA hemadsorption site similar to the N2 of prepandemic H3N2/1968, but no distinct antigenic or functional characteristics of the H9N2 NA associated with increased zoonotic potential could be identified.

Heterologous post-infection immunity against Egyptian avian influenza virus (AIV) H9N2 modulates the course of subsequent infection by highly pathogenic AIV H5N1, but vaccination immunity does not.

In Egypt, zoonotic A/goose/Guangdong/1/96 (gs/GD-like) highly pathogenic avian influenza virus (HPAIV) H5N1 of clade 2.2.1.2 is entrenched in poultry populations and has co-circulated with low-pathogenic avian influenza virus H9N2 of the G1 lineage since 2010. Here, the impact of H9N2 infection or vaccination on the course of consecutive infection with a lethal Egyptian HPAIV H5N1 is studied. Three-week-old chickens were infected with H9N2 or vaccinated with inactivated H9N2 or H5N1 antigens and challenged three weeks later by an HPAIV H5N1. Interestingly, pre-infection of chickens with H9N2 decreased the oral excretion of H5N1 to levels that were comparable to those of H5N1-immunized chickens, but vaccination with inactivated H9N2 did not. H9N2 pre-infection modulated but did not conceal clinical disease by HPAIV H5N1. By contrast, homologous H5 vaccination abolished clinical syndromic surveillance, although vaccinated clinical healthy birds were capable of spreading the virus.

Isolation and genetic characterization of a novel 2.2.1.2a H5N1 virus from a vaccinated meat-turkeys flock in Egypt.

BACKGROUND: Vaccination of poultry to control highly pathogenic avian influenza virus (HPAIV) H5N1 is used in several countries. HPAIV H5N1 of clade 2.2.1 which is endemic in Egypt has diversified into two genetic clades. Clade 2.2.1.1 represents antigenic drift variants in vaccinated commercial poultry while clade 2.2.1.2 variants are detected in humans and backyard poultry. Little is known about H5N1 infection in vaccinated turkeys under field conditions. CASE PRESENTATION: Here, we describe an HPAI H5N1 outbreak in a vaccinated meat-turkey flock in Egypt. Birds were vaccinated with inactivated H5N2 and H5N1 vaccines at 8 and 34 days of age, respectively. At 72nd day of age (38 days post last vaccination), turkeys exhibited mild respiratory signs, cyanosis of snood and severe congestion of the internal organs. Survivors had a reduction in feed consumption and body gain. A mortality of ~29% cumulated within 10 days after the onset of clinical signs. Laboratory diagnosis using RT-qPCRs revealed presence of H5N1 but was negative for H7 and H9 subtypes. A substantial antigenic drift against different serum samples from clade 2.2.1.1 and clade 2.3.4.4 was observed. Based on full genome sequence analysis the virus belonged to clade 2.2.1.2 but clustered with recent H5N1 viruses from 2015 in poultry in Israel, Gaza and Egypt in a novel subclade designated here 2.2.1.2a which is distinct from 2014/2015 2.2.1.2 viruses. These viruses possess 2.2.1.2 clade-specific genetic signatures and also mutations in the HA similar to those in clade 2.2.1.1 that enabled evasion from humoral immune response. Taken together, this manuscript describes a recent HPAI H5N1 outbreak in vaccinated meat-turkeys in Egypt after infection with a virus representing novel distinct 2.2.1.2a subclade. CONCLUSIONS: Infection with HPAIV H5N1 in commercial turkeys resulted in significant morbidity and mortality despite of vaccination using H5 vaccines. The isolated virus showed antigenic drift and clustered in a novel cluster designated here 2.2.1.2a related to viruses in poultry in Israel, Gaza and Egypt. Enforcement of biosecurity and constant update of vaccine virus strains may be helpful to protect vaccinated birds and prevent spillover infection to neighbouring countries.

New real time and conventional RT-PCRs for updated molecular diagnosis of infectious bronchitis virus infection (IBV) in chickens in Egypt associated with frequent co-infections with avian influenza and Newcastle Disease viruses.

In Egypt, currently two geographically restricted genotypes of the infectious bronchitis coronavirus (IBV) are circulating with detrimental effects for poultry industry. A sensitive real-time RT-PCR assay targeting the IBV nucleocapsid gene (N) was developed to screen clinical samples for presence of IBV. Conventional RT-PCRs amplifying hypervariable regions (HVRs 1-2 and 3) of the IBV S1 gene were developed and amplificates used for nucleotide sequence-based typing of IBV field strains in Egyptian chickens directly from clinical samples.