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Cross-species transmission of influenza A virus (IAV) from wild waterfowl to poultry is the first step in a chain of events that can ultimately lead to exposure and infection of humans. Herein, we study the outcome of infection with eight different mallard-origin IAV subtypes in two different avian hosts: tufted ducks and chickens. We found that infection and shedding patterns as well as innate immune responses were highly dependent on viral subtypes, host species, and inoculation routes. For example, intraoesophageal inoculation, commonly used in mallard infection experiments, resulted in no infections in contrast to oculonasal inoculation, suggesting a difference in transmission routes. Despite H9N2 being endemic in chickens, inoculation of mallard-origin H9N2 failed to cause viable infection beyond 1 day postinfection in our study design. The innate immune responses were markedly different in chickens and tufted ducks, and despite the presence of retinoic acid-inducible gene-I (RIG-I) in tufted duck transcriptomes, it was neither up nor downregulated in response to infection. Overall, we have revealed the heterogeneity of infection patterns and responses in two markedly different avian hosts following a challenge with mallard-origin IAV. These virus-host interactions provide new insights into important aspects of interspecies transmission of IAV.IMPORTANCE Our current findings highlight important aspects of IAV infection in birds that have implications for our understanding of its zoonotic ecology. In contrast to mallards where the intestinal tract is the main site of IAV replication, chickens and tufted ducks show limited or no signs of intestinal infection suggesting that the fecal-oral transmission route might not apply to all bird IAV host species. Our results indicate that mallard-origin IAVs undergo genetic changes upon introduction into new hosts, suggesting rapid adaptation to a new environment. However, similar to the mallard, chickens and tufted ducks s

Controlling avian influenza viruses (AIVs) is mainly based on culling of the infected bird flocks or via the implementation of inactivated vaccines in countries where AIVs are considered to be endemic. Over the last decade, several avian influenza virus subtypes, including highly pathogenic avian influenza (HPAI) H5N1 clade 2.2.1.2, H5N8 clade 2.3.4.4b and the recent H5N1 clade 2.3.4.4b, have been reported among poultry populations in Egypt. This demanded the utilization of a nationwide routine vaccination program in the poultry sector. Antigenic differences between available avian influenza vaccines and the currently circulating H5Nx strains were reported, calling for an updated vaccine for homogenous strains. In this study, three H5Nx vaccines were generated by utilizing the reverse genetic system: rgH5N1_2.3.4.4, rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2. Further, the immunogenicity and the cross-reactivity of the generated inactivated vaccines were assessed in the chicken model against a panel of homologous and heterologous H5Nx HPAIVs. Interestingly, the rgH5N1_2.3.4.4 induced high immunogenicity in specific-pathogen-free (SPF) chicken and could efficiently protect immunized chickens against challenge infection with HPAIV H5N1_2.3.4.4, H5N8_2.3.4.4 and H5N1_2.2.1.2. In parallel, the rgH5N1_2.2.1.2 could partially protect SPF chickens against infection with HPAIV H5N1_2.3.4.4 and H5N8_2.3.4.4. Conversely, the raised antibodies to rgH5N1_2.3.4.4 could provide full protection against HPAIV H5N1_2.3.4.4 and HPAIV H5N8_2.3.4.4, and partial protection (60%) against HPAIV H5N1_2.2.1.2. Compared to rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2 vaccines, chickens vaccinated with rgH5N1_2.3.4.4 showed lower viral shedding following challenge infection with the predefined HPAIVs. These data emphasize the superior immunogenicity and cross-protective efficacy of the rgH5N1_2.3.4.4 in comparison to rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2.

Cross-species transmission of influenza A virus (IAV) from wild waterfowl to poultry is the first step in a chain of events that can ultimately lead to exposure and infection of humans. Herein, we study the outcome of infection with eight different mallard-origin IAV subtypes in two different avian hosts: tufted ducks and chickens. We found that infection and shedding patterns as well as innate immune responses were highly dependent on viral subtypes, host species, and inoculation routes. For example, intraoesophageal inoculation, commonly used in mallard infection experiments, resulted in no infections in contrast to oculonasal inoculation, suggesting a difference in transmission routes. Despite H9N2 being endemic in chickens, inoculation of mallard-origin H9N2 failed to cause viable infection beyond 1 day postinfection in our study design. The innate immune responses were markedly different in chickens and tufted ducks, and despite the presence of retinoic acid-inducible gene-I (RIG-I) in tufted duck transcriptomes, it was neither up nor downregulated in response to infection. Overall, we have revealed the heterogeneity of infection patterns and responses in two markedly different avian hosts following a challenge with mallard-origin IAV. These virus-host interactions provide new insights into important aspects of interspecies transmission of IAV.IMPORTANCE Our current findings highlight important aspects of IAV infection in birds that have implications for our understanding of its zoonotic ecology. In contrast to mallards where the intestinal tract is the main site of IAV replication, chickens and tufted ducks show limited or no signs of intestinal infection suggesting that the fecal-oral transmission route might not apply to all bird IAV host species. Our results indicate that mallard-origin IAVs undergo genetic changes upon introduction into new hosts, suggesting rapid adaptation to a new environment. However, similar to the mallard, chickens and tufted ducks s

Controlling avian influenza viruses (AIVs) is mainly based on culling of the infected bird flocks or via the implementation of inactivated vaccines in countries where AIVs are considered to be endemic. Over the last decade, several avian influenza virus subtypes, including highly pathogenic avian influenza (HPAI) H5N1 clade 2.2.1.2, H5N8 clade 2.3.4.4b and the recent H5N1 clade 2.3.4.4b, have been reported among poultry populations in Egypt. This demanded the utilization of a nationwide routine vaccination program in the poultry sector. Antigenic differences between available avian influenza vaccines and the currently circulating H5Nx strains were reported, calling for an updated vaccine for homogenous strains. In this study, three H5Nx vaccines were generated by utilizing the reverse genetic system: rgH5N1_2.3.4.4, rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2. Further, the immunogenicity and the cross-reactivity of the generated inactivated vaccines were assessed in the chicken model against a panel of homologous and heterologous H5Nx HPAIVs. Interestingly, the rgH5N1_2.3.4.4 induced high immunogenicity in specific-pathogen-free (SPF) chicken and could efficiently protect immunized chickens against challenge infection with HPAIV H5N1_2.3.4.4, H5N8_2.3.4.4 and H5N1_2.2.1.2. In parallel, the rgH5N1_2.2.1.2 could partially protect SPF chickens against infection with HPAIV H5N1_2.3.4.4 and H5N8_2.3.4.4. Conversely, the raised antibodies to rgH5N1_2.3.4.4 could provide full protection against HPAIV H5N1_2.3.4.4 and HPAIV H5N8_2.3.4.4, and partial protection (60%) against HPAIV H5N1_2.2.1.2. Compared to rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2 vaccines, chickens vaccinated with rgH5N1_2.3.4.4 showed lower viral shedding following challenge infection with the predefined HPAIVs. These data emphasize the superior immunogenicity and cross-protective efficacy of the rgH5N1_2.3.4.4 in comparison to rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2.

Cross-species transmission of influenza A virus (IAV) from wild waterfowl to poultry is the first step in a chain of events that can ultimately lead to exposure and infection of humans. Herein, we study the outcome of infection with eight different mallard-origin IAV subtypes in two different avian hosts: tufted ducks and chickens. We found that infection and shedding patterns as well as innate immune responses were highly dependent on viral subtypes, host species, and inoculation routes. For example, intraoesophageal inoculation, commonly used in mallard infection experiments, resulted in no infections in contrast to oculonasal inoculation, suggesting a difference in transmission routes. Despite H9N2 being endemic in chickens, inoculation of mallard-origin H9N2 failed to cause viable infection beyond 1 day postinfection in our study design. The innate immune responses were markedly different in chickens and tufted ducks, and despite the presence of retinoic acid-inducible gene-I (RIG-I) in tufted duck transcriptomes, it was neither up nor downregulated in response to infection. Overall, we have revealed the heterogeneity of infection patterns and responses in two markedly different avian hosts following a challenge with mallard-origin IAV. These virus-host interactions provide new insights into important aspects of interspecies transmission of IAV.IMPORTANCE Our current findings highlight important aspects of IAV infection in birds that have implications for our understanding of its zoonotic ecology. In contrast to mallards where the intestinal tract is the main site of IAV replication, chickens and tufted ducks show limited or no signs of intestinal infection suggesting that the fecal-oral transmission route might not apply to all bird IAV host species. Our results indicate that mallard-origin IAVs undergo genetic changes upon introduction into new hosts, suggesting rapid adaptation to a new environment. However, similar to the mallard, chickens and tufted ducks s

Cross-species transmission of influenza A virus (IAV) from wild waterfowl to poultry is the first step in a chain of events that can ultimately lead to exposure and infection of humans. Herein, we study the outcome of infection with eight different mallard-origin IAV subtypes in two different avian hosts: tufted ducks and chickens. We found that infection and shedding patterns as well as innate immune responses were highly dependent on viral subtypes, host species, and inoculation routes. For example, intraoesophageal inoculation, commonly used in mallard infection experiments, resulted in no infections in contrast to oculonasal inoculation, suggesting a difference in transmission routes. Despite H9N2 being endemic in chickens, inoculation of mallard-origin H9N2 failed to cause viable infection beyond 1 day postinfection in our study design. The innate immune responses were markedly different in chickens and tufted ducks, and despite the presence of retinoic acid-inducible gene-I (RIG-I) in tufted duck transcriptomes, it was neither up nor downregulated in response to infection. Overall, we have revealed the heterogeneity of infection patterns and responses in two markedly different avian hosts following a challenge with mallard-origin IAV. These virus-host interactions provide new insights into important aspects of interspecies transmission of IAV.IMPORTANCE Our current findings highlight important aspects of IAV infection in birds that have implications for our understanding of its zoonotic ecology. In contrast to mallards where the intestinal tract is the main site of IAV replication, chickens and tufted ducks show limited or no signs of intestinal infection suggesting that the fecal-oral transmission route might not apply to all bird IAV host species. Our results indicate that mallard-origin IAVs undergo genetic changes upon introduction into new hosts, suggesting rapid adaptation to a new environment. However, similar to the mallard, chickens and tufted ducks s

Controlling avian influenza viruses (AIVs) is mainly based on culling of the infected bird flocks or via the implementation of inactivated vaccines in countries where AIVs are considered to be endemic. Over the last decade, several avian influenza virus subtypes, including highly pathogenic avian influenza (HPAI) H5N1 clade 2.2.1.2, H5N8 clade 2.3.4.4b and the recent H5N1 clade 2.3.4.4b, have been reported among poultry populations in Egypt. This demanded the utilization of a nationwide routine vaccination program in the poultry sector. Antigenic differences between available avian influenza vaccines and the currently circulating H5Nx strains were reported, calling for an updated vaccine for homogenous strains. In this study, three H5Nx vaccines were generated by utilizing the reverse genetic system: rgH5N1_2.3.4.4, rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2. Further, the immunogenicity and the cross-reactivity of the generated inactivated vaccines were assessed in the chicken model against a panel of homologous and heterologous H5Nx HPAIVs. Interestingly, the rgH5N1_2.3.4.4 induced high immunogenicity in specific-pathogen-free (SPF) chicken and could efficiently protect immunized chickens against challenge infection with HPAIV H5N1_2.3.4.4, H5N8_2.3.4.4 and H5N1_2.2.1.2. In parallel, the rgH5N1_2.2.1.2 could partially protect SPF chickens against infection with HPAIV H5N1_2.3.4.4 and H5N8_2.3.4.4. Conversely, the raised antibodies to rgH5N1_2.3.4.4 could provide full protection against HPAIV H5N1_2.3.4.4 and HPAIV H5N8_2.3.4.4, and partial protection (60%) against HPAIV H5N1_2.2.1.2. Compared to rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2 vaccines, chickens vaccinated with rgH5N1_2.3.4.4 showed lower viral shedding following challenge infection with the predefined HPAIVs. These data emphasize the superior immunogenicity and cross-protective efficacy of the rgH5N1_2.3.4.4 in comparison to rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2.

The neuraminidase inhibitor (NAI) oseltamivir is stockpiled globally as part of influenza pandemic preparedness. However, oseltamivir carboxylate (OC) resistance develops in avian influenza virus (AIV) infecting mallards exposed to environmental-like OC concentrations, suggesting that environmental resistance is a real concern. Herein we used an in vivo model to investigate if avian influenza H1N1 with the OC-resistant mutation NA-H274Y (51833/H274Y) as compared to the wild-type (wt) strain (51833 /wt) could transmit from mallards, which would potentially be exposed to environmentally contaminated environments, to and between chickens, thus posing a potential zoonotic risk of antiviral-resistant AIV. Regardless of whether the virus had the OC-resistant mutation or not, chickens became infected both through experimental infection, and following exposure to infected mallards. We found similar infection patterns between 51833/wt and 51833/H274Y such that, one chicken inoculated with 51833/wt and three chickens inoculated with 51833/H274Y were AIV positive in oropharyngeal samples more than 2 days consecutively, indicating true infection, and one contact chicken exposed to infected mallards was AIV positive in faecal samples for 3 consecutive days (51833/wt) and another contact chicken for 4 consecutive days (51833/H274Y). Importantly, all positive samples from chickens infected with 51833/H274Y retained the NA-H274Y mutation. However, none of the virus strains established sustained transmission in chickens, likely due to insufficient adaptation to the chicken host. Our results demonstrate that an OC-resistant avian influenza virus can transmit from mallards and replicate in chickens. NA-H274Y does not constitute a barrier to interspecies transmission per se, as the resistant virus did not show reduced replicative capacity compared to the wild-type counterpart. Thus, responsible use of oseltamivir and surveillance for resistance development is warranted to limit the ris

Exchange of viral segments between one or more influenza virus subtypes can contribute to a shift in virulence and adaptation to new hosts. Among several influenza subtypes, H9N2 is widely circulating in poultry populations worldwide and has the ability to infect humans. Here, we studied the reassortant compatibility between chicken H9N2 with N1-N9 gene segments of wild bird origin, either with an intact or truncated stalk. Naturally occurring amino acid deletions in the NA stalk of the influenza virus can lead to increased virulence in both mallard ducks and chickens. Our findings show extended genetic compatibility between chicken H9Nx gene segments and the wild-bird NA with and without 20 amino acid stalk deletion. Replication kinetics in avian, mammalian and human cell lines revealed that parental chH9N2 and rH9N6 viruses with intact NA-stalk replicated significantly better in avian DF1 cells compared to human A549 cells. After introducing a stalk deletion, an enhanced preference for replication in mammalian and human cell lines could be observed for rH9N2(Delta)(H6), rH9N6(Delta) and rH9N9(Delta) compared to the parental chH9N2 virus. This highlights the potential emergence of novel viruses with variable phenotypic traits, warranting the continuous monitoring of H9N2 and co-circulating subtypes in avian hosts.

Wild migratory birds have the capability to spread avian influenza virus (AIV) over long distances as well as transmit the virus to domestic birds. In this study, swab and tissue samples were obtained from 190 migratory birds during close surveillance in Egypt in response to the recent outbreaks of the highly pathogenic avian influenza (HPAI) H5N1 virus. The collected samples were tested for a variety of AIV subtypes (H5N1, H9N2, H5N8, and H6N2) as well as other pathogens such as NDV, IBV, ILT, IBDV, and WNV. Among all of the tested samples, the HPAI H5N1 virus was found in six samples; the other samples were found to be negative for all of the tested pathogens. The Egyptian HPAI H5N1 strains shared genetic traits with the HPAI H5N1 strains that are currently being reported in Europe, North America, Asia, and Africa in 2021-2022. Whole genome sequencing revealed markers associated with mammalian adaption and virulence traits among different gene segments, similar to those found in HPAI H5N1 strains detected in Europe and Africa. The detection of the HPAI H5N1 strain of clade 2.3.4.4b in wild birds in Egypt underlines the risk of the introduction of this strain into the local poultry population. Hence, there is reason to be vigilant and continue epidemiological and molecular monitoring of the AIV in close proximity to the domestic-wild bird interface.

Wild migratory birds have the capability to spread avian influenza virus (AIV) over long distances as well as transmit the virus to domestic birds. In this study, swab and tissue samples were obtained from 190 migratory birds during close surveillance in Egypt in response to the recent outbreaks of the highly pathogenic avian influenza (HPAI) H5N1 virus. The collected samples were tested for a variety of AIV subtypes (H5N1, H9N2, H5N8, and H6N2) as well as other pathogens such as NDV, IBV, ILT, IBDV, and WNV. Among all of the tested samples, the HPAI H5N1 virus was found in six samples; the other samples were found to be negative for all of the tested pathogens. The Egyptian HPAI H5N1 strains shared genetic traits with the HPAI H5N1 strains that are currently being reported in Europe, North America, Asia, and Africa in 2021-2022. Whole genome sequencing revealed markers associated with mammalian adaption and virulence traits among different gene segments, similar to those found in HPAI H5N1 strains detected in Europe and Africa. The detection of the HPAI H5N1 strain of clade 2.3.4.4b in wild birds in Egypt underlines the risk of the introduction of this strain into the local poultry population. Hence, there is reason to be vigilant and continue epidemiological and molecular monitoring of the AIV in close proximity to the domestic-wild bird interface.

Wild migratory birds have the capability to spread avian influenza virus (AIV) over long distances as well as transmit the virus to domestic birds. In this study, swab and tissue samples were obtained from 190 migratory birds during close surveillance in Egypt in response to the recent outbreaks of the highly pathogenic avian influenza (HPAI) H5N1 virus. The collected samples were tested for a variety of AIV subtypes (H5N1, H9N2, H5N8, and H6N2) as well as other pathogens such as NDV, IBV, ILT, IBDV, and WNV. Among all of the tested samples, the HPAI H5N1 virus was found in six samples; the other samples were found to be negative for all of the tested pathogens. The Egyptian HPAI H5N1 strains shared genetic traits with the HPAI H5N1 strains that are currently being reported in Europe, North America, Asia, and Africa in 2021-2022. Whole genome sequencing revealed markers associated with mammalian adaption and virulence traits among different gene segments, similar to those found in HPAI H5N1 strains detected in Europe and Africa. The detection of the HPAI H5N1 strain of clade 2.3.4.4b in wild birds in Egypt underlines the risk of the introduction of this strain into the local poultry population. Hence, there is reason to be vigilant and continue epidemiological and molecular monitoring of the AIV in close proximity to the domestic-wild bird interface.

Wild migratory birds have the capability to spread avian influenza virus (AIV) over long distances as well as transmit the virus to domestic birds. In this study, swab and tissue samples were obtained from 190 migratory birds during close surveillance in Egypt in response to the recent outbreaks of the highly pathogenic avian influenza (HPAI) H5N1 virus. The collected samples were tested for a variety of AIV subtypes (H5N1, H9N2, H5N8, and H6N2) as well as other pathogens such as NDV, IBV, ILT, IBDV, and WNV. Among all of the tested samples, the HPAI H5N1 virus was found in six samples; the other samples were found to be negative for all of the tested pathogens. The Egyptian HPAI H5N1 strains shared genetic traits with the HPAI H5N1 strains that are currently being reported in Europe, North America, Asia, and Africa in 2021-2022. Whole genome sequencing revealed markers associated with mammalian adaption and virulence traits among different gene segments, similar to those found in HPAI H5N1 strains detected in Europe and Africa. The detection of the HPAI H5N1 strain of clade 2.3.4.4b in wild birds in Egypt underlines the risk of the introduction of this strain into the local poultry population. Hence, there is reason to be vigilant and continue epidemiological and molecular monitoring of the AIV in close proximity to the domestic-wild bird interface.

The neuraminidase inhibitor (NAI) oseltamivir is stockpiled globally as part of influenza pandemic preparedness. However, oseltamivir carboxylate (OC) resistance develops in avian influenza virus (AIV) infecting mallards exposed to environmental-like OC concentrations, suggesting that environmental resistance is a real concern. Herein we used an in vivo model to investigate if avian influenza H1N1 with the OC-resistant mutation NA-H274Y (51833/H274Y) as compared to the wild-type (wt) strain (51833 /wt) could transmit from mallards, which would potentially be exposed to environmentally contaminated environments, to and between chickens, thus posing a potential zoonotic risk of antiviral-resistant AIV. Regardless of whether the virus had the OC-resistant mutation or not, chickens became infected both through experimental infection, and following exposure to infected mallards. We found similar infection patterns between 51833/wt and 51833/H274Y such that, one chicken inoculated with 51833/wt and three chickens inoculated with 51833/H274Y were AIV positive in oropharyngeal samples more than 2 days consecutively, indicating true infection, and one contact chicken exposed to infected mallards was AIV positive in faecal samples for 3 consecutive days (51833/wt) and another contact chicken for 4 consecutive days (51833/H274Y). Importantly, all positive samples from chickens infected with 51833/H274Y retained the NA-H274Y mutation. However, none of the virus strains established sustained transmission in chickens, likely due to insufficient adaptation to the chicken host. Our results demonstrate that an OC-resistant avian influenza virus can transmit from mallards and replicate in chickens. NA-H274Y does not constitute a barrier to interspecies transmission per se, as the resistant virus did not show reduced replicative capacity compared to the wild-type counterpart. Thus, responsible use of oseltamivir and surveillance for resistance development is warranted to limit the ris

Exchange of viral segments between one or more influenza virus subtypes can contribute to a shift in virulence and adaptation to new hosts. Among several influenza subtypes, H9N2 is widely circulating in poultry populations worldwide and has the ability to infect humans. Here, we studied the reassortant compatibility between chicken H9N2 with N1-N9 gene segments of wild bird origin, either with an intact or truncated stalk. Naturally occurring amino acid deletions in the NA stalk of the influenza virus can lead to increased virulence in both mallard ducks and chickens. Our findings show extended genetic compatibility between chicken H9Nx gene segments and the wild-bird NA with and without 20 amino acid stalk deletion. Replication kinetics in avian, mammalian and human cell lines revealed that parental chH9N2 and rH9N6 viruses with intact NA-stalk replicated significantly better in avian DF1 cells compared to human A549 cells. After introducing a stalk deletion, an enhanced preference for replication in mammalian and human cell lines could be observed for rH9N2(Delta)(H6), rH9N6(Delta) and rH9N9(Delta) compared to the parental chH9N2 virus. This highlights the potential emergence of novel viruses with variable phenotypic traits, warranting the continuous monitoring of H9N2 and co-circulating subtypes in avian hosts.

The neuraminidase inhibitor (NAI) oseltamivir is stockpiled globally as part of influenza pandemic preparedness. However, oseltamivir carboxylate (OC) resistance develops in avian influenza virus (AIV) infecting mallards exposed to environmental-like OC concentrations, suggesting that environmental resistance is a real concern. Herein we used an in vivo model to investigate if avian influenza H1N1 with the OC-resistant mutation NA-H274Y (51833/H274Y) as compared to the wild-type (wt) strain (51833 /wt) could transmit from mallards, which would potentially be exposed to environmentally contaminated environments, to and between chickens, thus posing a potential zoonotic risk of antiviral-resistant AIV. Regardless of whether the virus had the OC-resistant mutation or not, chickens became infected both through experimental infection, and following exposure to infected mallards. We found similar infection patterns between 51833/wt and 51833/H274Y such that, one chicken inoculated with 51833/wt and three chickens inoculated with 51833/H274Y were AIV positive in oropharyngeal samples more than 2 days consecutively, indicating true infection, and one contact chicken exposed to infected mallards was AIV positive in faecal samples for 3 consecutive days (51833/wt) and another contact chicken for 4 consecutive days (51833/H274Y). Importantly, all positive samples from chickens infected with 51833/H274Y retained the NA-H274Y mutation. However, none of the virus strains established sustained transmission in chickens, likely due to insufficient adaptation to the chicken host. Our results demonstrate that an OC-resistant avian influenza virus can transmit from mallards and replicate in chickens. NA-H274Y does not constitute a barrier to interspecies transmission per se, as the resistant virus did not show reduced replicative capacity compared to the wild-type counterpart. Thus, responsible use of oseltamivir and surveillance for resistance development is warranted to limit the ris

Exchange of viral segments between one or more influenza virus subtypes can contribute to a shift in virulence and adaptation to new hosts. Among several influenza subtypes, H9N2 is widely circulating in poultry populations worldwide and has the ability to infect humans. Here, we studied the reassortant compatibility between chicken H9N2 with N1-N9 gene segments of wild bird origin, either with an intact or truncated stalk. Naturally occurring amino acid deletions in the NA stalk of the influenza virus can lead to increased virulence in both mallard ducks and chickens. Our findings show extended genetic compatibility between chicken H9Nx gene segments and the wild-bird NA with and without 20 amino acid stalk deletion. Replication kinetics in avian, mammalian and human cell lines revealed that parental chH9N2 and rH9N6 viruses with intact NA-stalk replicated significantly better in avian DF1 cells compared to human A549 cells. After introducing a stalk deletion, an enhanced preference for replication in mammalian and human cell lines could be observed for rH9N2(Delta)(H6), rH9N6(Delta) and rH9N9(Delta) compared to the parental chH9N2 virus. This highlights the potential emergence of novel viruses with variable phenotypic traits, warranting the continuous monitoring of H9N2 and co-circulating subtypes in avian hosts.

Exchange of viral segments between one or more influenza virus subtypes can contribute to a shift in virulence and adaptation to new hosts. Among several influenza subtypes, H9N2 is widely circulating in poultry populations worldwide and has the ability to infect humans. Here, we studied the reassortant compatibility between chicken H9N2 with N1-N9 gene segments of wild bird origin, either with an intact or truncated stalk. Naturally occurring amino acid deletions in the NA stalk of the influenza virus can lead to increased virulence in both mallard ducks and chickens. Our findings show extended genetic compatibility between chicken H9Nx gene segments and the wild-bird NA with and without 20 amino acid stalk deletion. Replication kinetics in avian, mammalian and human cell lines revealed that parental chH9N2 and rH9N6 viruses with intact NA-stalk replicated significantly better in avian DF1 cells compared to human A549 cells. After introducing a stalk deletion, an enhanced preference for replication in mammalian and human cell lines could be observed for rH9N2(Delta)(H6), rH9N6(Delta) and rH9N9(Delta) compared to the parental chH9N2 virus. This highlights the potential emergence of novel viruses with variable phenotypic traits, warranting the continuous monitoring of H9N2 and co-circulating subtypes in avian hosts.

The neuraminidase inhibitor (NAI) oseltamivir is stockpiled globally as part of influenza pandemic preparedness. However, oseltamivir carboxylate (OC) resistance develops in avian influenza virus (AIV) infecting mallards exposed to environmental-like OC concentrations, suggesting that environmental resistance is a real concern. Herein we used an in vivo model to investigate if avian influenza H1N1 with the OC-resistant mutation NA-H274Y (51833/H274Y) as compared to the wild-type (wt) strain (51833 /wt) could transmit from mallards, which would potentially be exposed to environmentally contaminated environments, to and between chickens, thus posing a potential zoonotic risk of antiviral-resistant AIV. Regardless of whether the virus had the OC-resistant mutation or not, chickens became infected both through experimental infection, and following exposure to infected mallards. We found similar infection patterns between 51833/wt and 51833/H274Y such that, one chicken inoculated with 51833/wt and three chickens inoculated with 51833/H274Y were AIV positive in oropharyngeal samples more than 2 days consecutively, indicating true infection, and one contact chicken exposed to infected mallards was AIV positive in faecal samples for 3 consecutive days (51833/wt) and another contact chicken for 4 consecutive days (51833/H274Y). Importantly, all positive samples from chickens infected with 51833/H274Y retained the NA-H274Y mutation. However, none of the virus strains established sustained transmission in chickens, likely due to insufficient adaptation to the chicken host. Our results demonstrate that an OC-resistant avian influenza virus can transmit from mallards and replicate in chickens. NA-H274Y does not constitute a barrier to interspecies transmission per se, as the resistant virus did not show reduced replicative capacity compared to the wild-type counterpart. Thus, responsible use of oseltamivir and surveillance for resistance development is warranted to limit the ris

Controlling avian influenza viruses (AIVs) is mainly based on culling of the infected bird flocks or via the implementation of inactivated vaccines in countries where AIVs are considered to be endemic. Over the last decade, several avian influenza virus subtypes, including highly pathogenic avian influenza (HPAI) H5N1 clade 2.2.1.2, H5N8 clade 2.3.4.4b and the recent H5N1 clade 2.3.4.4b, have been reported among poultry populations in Egypt. This demanded the utilization of a nationwide routine vaccination program in the poultry sector. Antigenic differences between available avian influenza vaccines and the currently circulating H5Nx strains were reported, calling for an updated vaccine for homogenous strains. In this study, three H5Nx vaccines were generated by utilizing the reverse genetic system: rgH5N1_2.3.4.4, rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2. Further, the immunogenicity and the cross-reactivity of the generated inactivated vaccines were assessed in the chicken model against a panel of homologous and heterologous H5Nx HPAIVs. Interestingly, the rgH5N1_2.3.4.4 induced high immunogenicity in specific-pathogen-free (SPF) chicken and could efficiently protect immunized chickens against challenge infection with HPAIV H5N1_2.3.4.4, H5N8_2.3.4.4 and H5N1_2.2.1.2. In parallel, the rgH5N1_2.2.1.2 could partially protect SPF chickens against infection with HPAIV H5N1_2.3.4.4 and H5N8_2.3.4.4. Conversely, the raised antibodies to rgH5N1_2.3.4.4 could provide full protection against HPAIV H5N1_2.3.4.4 and HPAIV H5N8_2.3.4.4, and partial protection (60%) against HPAIV H5N1_2.2.1.2. Compared to rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2 vaccines, chickens vaccinated with rgH5N1_2.3.4.4 showed lower viral shedding following challenge infection with the predefined HPAIVs. These data emphasize the superior immunogenicity and cross-protective efficacy of the rgH5N1_2.3.4.4 in comparison to rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2.

Cross-species transmission of influenza A virus (IAV) from wild waterfowl to poultry is the first step in a chain of events that can ultimately lead to exposure and infection of humans. Herein, we study the outcome of infection with eight different mallard-origin IAV subtypes in two different avian hosts: tufted ducks and chickens. We found that infection and shedding patterns as well as innate immune responses were highly dependent on viral subtypes, host species, and inoculation routes. For example, intraoesophageal inoculation, commonly used in mallard infection experiments, resulted in no infections in contrast to oculonasal inoculation, suggesting a difference in transmission routes. Despite H9N2 being endemic in chickens, inoculation of mallard-origin H9N2 failed to cause viable infection beyond 1 day postinfection in our study design. The innate immune responses were markedly different in chickens and tufted ducks, and despite the presence of retinoic acid-inducible gene-I (RIG-I) in tufted duck transcriptomes, it was neither up nor downregulated in response to infection. Overall, we have revealed the heterogeneity of infection patterns and responses in two markedly different avian hosts following a challenge with mallard-origin IAV. These virus-host interactions provide new insights into important aspects of interspecies transmission of IAV.IMPORTANCE Our current findings highlight important aspects of IAV infection in birds that have implications for our understanding of its zoonotic ecology. In contrast to mallards where the intestinal tract is the main site of IAV replication, chickens and tufted ducks show limited or no signs of intestinal infection suggesting that the fecal-oral transmission route might not apply to all bird IAV host species. Our results indicate that mallard-origin IAVs undergo genetic changes upon introduction into new hosts, suggesting rapid adaptation to a new environment. However, similar to the mallard, chickens and tufted ducks s

Controlling avian influenza viruses (AIVs) is mainly based on culling of the infected bird flocks or via the implementation of inactivated vaccines in countries where AIVs are considered to be endemic. Over the last decade, several avian influenza virus subtypes, including highly pathogenic avian influenza (HPAI) H5N1 clade 2.2.1.2, H5N8 clade 2.3.4.4b and the recent H5N1 clade 2.3.4.4b, have been reported among poultry populations in Egypt. This demanded the utilization of a nationwide routine vaccination program in the poultry sector. Antigenic differences between available avian influenza vaccines and the currently circulating H5Nx strains were reported, calling for an updated vaccine for homogenous strains. In this study, three H5Nx vaccines were generated by utilizing the reverse genetic system: rgH5N1_2.3.4.4, rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2. Further, the immunogenicity and the cross-reactivity of the generated inactivated vaccines were assessed in the chicken model against a panel of homologous and heterologous H5Nx HPAIVs. Interestingly, the rgH5N1_2.3.4.4 induced high immunogenicity in specific-pathogen-free (SPF) chicken and could efficiently protect immunized chickens against challenge infection with HPAIV H5N1_2.3.4.4, H5N8_2.3.4.4 and H5N1_2.2.1.2. In parallel, the rgH5N1_2.2.1.2 could partially protect SPF chickens against infection with HPAIV H5N1_2.3.4.4 and H5N8_2.3.4.4. Conversely, the raised antibodies to rgH5N1_2.3.4.4 could provide full protection against HPAIV H5N1_2.3.4.4 and HPAIV H5N8_2.3.4.4, and partial protection (60%) against HPAIV H5N1_2.2.1.2. Compared to rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2 vaccines, chickens vaccinated with rgH5N1_2.3.4.4 showed lower viral shedding following challenge infection with the predefined HPAIVs. These data emphasize the superior immunogenicity and cross-protective efficacy of the rgH5N1_2.3.4.4 in comparison to rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2.

Cross-species transmission of influenza A virus (IAV) from wild waterfowl to poultry is the first step in a chain of events that can ultimately lead to exposure and infection of humans. Herein, we study the outcome of infection with eight different mallard-origin IAV subtypes in two different avian hosts: tufted ducks and chickens. We found that infection and shedding patterns as well as innate immune responses were highly dependent on viral subtypes, host species, and inoculation routes. For example, intraoesophageal inoculation, commonly used in mallard infection experiments, resulted in no infections in contrast to oculonasal inoculation, suggesting a difference in transmission routes. Despite H9N2 being endemic in chickens, inoculation of mallard-origin H9N2 failed to cause viable infection beyond 1 day postinfection in our study design. The innate immune responses were markedly different in chickens and tufted ducks, and despite the presence of retinoic acid-inducible gene-I (RIG-I) in tufted duck transcriptomes, it was neither up nor downregulated in response to infection. Overall, we have revealed the heterogeneity of infection patterns and responses in two markedly different avian hosts following a challenge with mallard-origin IAV. These virus-host interactions provide new insights into important aspects of interspecies transmission of IAV.IMPORTANCE Our current findings highlight important aspects of IAV infection in birds that have implications for our understanding of its zoonotic ecology. In contrast to mallards where the intestinal tract is the main site of IAV replication, chickens and tufted ducks show limited or no signs of intestinal infection suggesting that the fecal-oral transmission route might not apply to all bird IAV host species. Our results indicate that mallard-origin IAVs undergo genetic changes upon introduction into new hosts, suggesting rapid adaptation to a new environment. However, similar to the mallard, chickens and tufted ducks s

The neuraminidase inhibitor (NAI) oseltamivir is stockpiled globally as part of influenza pandemic preparedness. However, oseltamivir carboxylate (OC) resistance develops in avian influenza virus (AIV) infecting mallards exposed to environmental-like OC concentrations, suggesting that environmental resistance is a real concern. Herein we used an in vivo model to investigate if avian influenza H1N1 with the OC-resistant mutation NA-H274Y (51833/H274Y) as compared to the wild-type (wt) strain (51833 /wt) could transmit from mallards, which would potentially be exposed to environmentally contaminated environments, to and between chickens, thus posing a potential zoonotic risk of antiviral-resistant AIV. Regardless of whether the virus had the OC-resistant mutation or not, chickens became infected both through experimental infection, and following exposure to infected mallards. We found similar infection patterns between 51833/wt and 51833/H274Y such that, one chicken inoculated with 51833/wt and three chickens inoculated with 51833/H274Y were AIV positive in oropharyngeal samples more than 2 days consecutively, indicating true infection, and one contact chicken exposed to infected mallards was AIV positive in faecal samples for 3 consecutive days (51833/wt) and another contact chicken for 4 consecutive days (51833/H274Y). Importantly, all positive samples from chickens infected with 51833/H274Y retained the NA-H274Y mutation. However, none of the virus strains established sustained transmission in chickens, likely due to insufficient adaptation to the chicken host. Our results demonstrate that an OC-resistant avian influenza virus can transmit from mallards and replicate in chickens. NA-H274Y does not constitute a barrier to interspecies transmission per se, as the resistant virus did not show reduced replicative capacity compared to the wild-type counterpart. Thus, responsible use of oseltamivir and surveillance for resistance development is warranted to limit the ris

Exchange of viral segments between one or more influenza virus subtypes can contribute to a shift in virulence and adaptation to new hosts. Among several influenza subtypes, H9N2 is widely circulating in poultry populations worldwide and has the ability to infect humans. Here, we studied the reassortant compatibility between chicken H9N2 with N1-N9 gene segments of wild bird origin, either with an intact or truncated stalk. Naturally occurring amino acid deletions in the NA stalk of the influenza virus can lead to increased virulence in both mallard ducks and chickens. Our findings show extended genetic compatibility between chicken H9Nx gene segments and the wild-bird NA with and without 20 amino acid stalk deletion. Replication kinetics in avian, mammalian and human cell lines revealed that parental chH9N2 and rH9N6 viruses with intact NA-stalk replicated significantly better in avian DF1 cells compared to human A549 cells. After introducing a stalk deletion, an enhanced preference for replication in mammalian and human cell lines could be observed for rH9N2(Delta)(H6), rH9N6(Delta) and rH9N9(Delta) compared to the parental chH9N2 virus. This highlights the potential emergence of novel viruses with variable phenotypic traits, warranting the continuous monitoring of H9N2 and co-circulating subtypes in avian hosts.

Wild migratory birds have the capability to spread avian influenza virus (AIV) over long distances as well as transmit the virus to domestic birds. In this study, swab and tissue samples were obtained from 190 migratory birds during close surveillance in Egypt in response to the recent outbreaks of the highly pathogenic avian influenza (HPAI) H5N1 virus. The collected samples were tested for a variety of AIV subtypes (H5N1, H9N2, H5N8, and H6N2) as well as other pathogens such as NDV, IBV, ILT, IBDV, and WNV. Among all of the tested samples, the HPAI H5N1 virus was found in six samples; the other samples were found to be negative for all of the tested pathogens. The Egyptian HPAI H5N1 strains shared genetic traits with the HPAI H5N1 strains that are currently being reported in Europe, North America, Asia, and Africa in 2021-2022. Whole genome sequencing revealed markers associated with mammalian adaption and virulence traits among different gene segments, similar to those found in HPAI H5N1 strains detected in Europe and Africa. The detection of the HPAI H5N1 strain of clade 2.3.4.4b in wild birds in Egypt underlines the risk of the introduction of this strain into the local poultry population. Hence, there is reason to be vigilant and continue epidemiological and molecular monitoring of the AIV in close proximity to the domestic-wild bird interface.

Since it was first discovered, the low pathogenic avian influenza (LPAI) H9N2 subtype has established linages infecting the poultry population globally and has become one of the most prevalent influenza subtypes in domestic poultry. Several different variants and genotypes of LPAI H9N2 viruses have been reported in Egypt, but little is known about their pathogenicity and how they have evolved. In this study, four different Egyptian LPAI H9N2 viruses were genetically and antigenically characterized and compared to representative H9N2 viruses from G1 lineage. Furthermore, the pathogenicity of three genetically distinct Egyptian LPAI H9N2 viruses was assessed by experimental infection in chickens. Whole-genome sequencing revealed that the H9N2 virus of the Egy-2 G1-B lineage (pigeon-like) has become the dominant circulating H9N2 genotype in Egypt since 2016. Considerable variation in virus shedding at day 7 post-infections was detected in infected chickens, but no significant difference in pathogenicity was found between the infected groups. The rapid spread and emergence of new genotypes of the influenza viruses pinpoint the importance of continuous surveillance for the detection of novel reassortant viruses, as well as monitoring the viral evolution.

Since it was first discovered, the low pathogenic avian influenza (LPAI) H9N2 subtype has established linages infecting the poultry population globally and has become one of the most prevalent influenza subtypes in domestic poultry. Several different variants and genotypes of LPAI H9N2 viruses have been reported in Egypt, but little is known about their pathogenicity and how they have evolved. In this study, four different Egyptian LPAI H9N2 viruses were genetically and antigenically characterized and compared to representative H9N2 viruses from G1 lineage. Furthermore, the pathogenicity of three genetically distinct Egyptian LPAI H9N2 viruses was assessed by experimental infection in chickens. Whole-genome sequencing revealed that the H9N2 virus of the Egy-2 G1-B lineage (pigeon-like) has become the dominant circulating H9N2 genotype in Egypt since 2016. Considerable variation in virus shedding at day 7 post-infections was detected in infected chickens, but no significant difference in pathogenicity was found between the infected groups. The rapid spread and emergence of new genotypes of the influenza viruses pinpoint the importance of continuous surveillance for the detection of novel reassortant viruses, as well as monitoring the viral evolution.

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 genoty

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 genoty

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 genoty

Since it was first discovered, the low pathogenic avian influenza (LPAI) H9N2 subtype has established linages infecting the poultry population globally and has become one of the most prevalent influenza subtypes in domestic poultry. Several different variants and genotypes of LPAI H9N2 viruses have been reported in Egypt, but little is known about their pathogenicity and how they have evolved. In this study, four different Egyptian LPAI H9N2 viruses were genetically and antigenically characterized and compared to representative H9N2 viruses from G1 lineage. Furthermore, the pathogenicity of three genetically distinct Egyptian LPAI H9N2 viruses was assessed by experimental infection in chickens. Whole-genome sequencing revealed that the H9N2 virus of the Egy-2 G1-B lineage (pigeon-like) has become the dominant circulating H9N2 genotype in Egypt since 2016. Considerable variation in virus shedding at day 7 post-infections was detected in infected chickens, but no significant difference in pathogenicity was found between the infected groups. The rapid spread and emergence of new genotypes of the influenza viruses pinpoint the importance of continuous surveillance for the detection of novel reassortant viruses, as well as monitoring the viral evolution.

The highly pathogenic avian influenza (HPAI) H5N8 virus was first detected in Egypt in late 2016. Since then, the virus has spread rapidly among different poultry sectors, becoming the dominant HPAI H5 subtype reported in Egypt. Different genotypes of the HPAI H5N8 virus were reported in Egypt; however, the geographic patterns and molecular evolution of the Egyptian HPAI H5N8 viruses are still unclear. Here, extensive epidemiological surveillance was conducted, including more than half a million samples collected from different poultry sectors (farms/backyards/live bird markets) from all governorates in Egypt during 2019-2021. In addition, genetic characterization and evolutionary analyses were performed using 47 selected positive H5N8 isolates obtained during the same period. The result of the conducted surveillance showed that HPAI H5N8 viruses of clade 2.3.4.4b continue to circulate in different locations in Egypt, with an obvious seasonal pattern, and no further detection of the HPAI H5N1 virus of clade 2.2.1.2 was observed in the poultry population during 2019-2021. In addition, phylogenetic and Bayesian analyses revealed that two major genotypes (G5 and G6) of HPAI H5N8 viruses were continually expanding among the poultry sectors in Egypt. Notably, molecular dating analysis suggested that the Egyptian HPAI H5N8 virus is the potential ancestral viruses of the European H5N8 viruses of 2020-2021. In summary, the data of this study highlight the current epidemiology, diversity, and evolution of HPAI H5N8 viruses in Egypt and call for continuous monitoring of the genetic features of the avian influenza viruses in Egypt.

The highly pathogenic avian influenza (HPAI) H5N8 virus was first detected in Egypt in late 2016. Since then, the virus has spread rapidly among different poultry sectors, becoming the dominant HPAI H5 subtype reported in Egypt. Different genotypes of the HPAI H5N8 virus were reported in Egypt; however, the geographic patterns and molecular evolution of the Egyptian HPAI H5N8 viruses are still unclear. Here, extensive epidemiological surveillance was conducted, including more than half a million samples collected from different poultry sectors (farms/backyards/live bird markets) from all governorates in Egypt during 2019-2021. In addition, genetic characterization and evolutionary analyses were performed using 47 selected positive H5N8 isolates obtained during the same period. The result of the conducted surveillance showed that HPAI H5N8 viruses of clade 2.3.4.4b continue to circulate in different locations in Egypt, with an obvious seasonal pattern, and no further detection of the HPAI H5N1 virus of clade 2.2.1.2 was observed in the poultry population during 2019-2021. In addition, phylogenetic and Bayesian analyses revealed that two major genotypes (G5 and G6) of HPAI H5N8 viruses were continually expanding among the poultry sectors in Egypt. Notably, molecular dating analysis suggested that the Egyptian HPAI H5N8 virus is the potential ancestral viruses of the European H5N8 viruses of 2020-2021. In summary, the data of this study highlight the current epidemiology, diversity, and evolution of HPAI H5N8 viruses in Egypt and call for continuous monitoring of the genetic features of the avian influenza viruses in Egypt.

The highly pathogenic avian influenza (HPAI) H5N8 virus was first detected in Egypt in late 2016. Since then, the virus has spread rapidly among different poultry sectors, becoming the dominant HPAI H5 subtype reported in Egypt. Different genotypes of the HPAI H5N8 virus were reported in Egypt; however, the geographic patterns and molecular evolution of the Egyptian HPAI H5N8 viruses are still unclear. Here, extensive epidemiological surveillance was conducted, including more than half a million samples collected from different poultry sectors (farms/backyards/live bird markets) from all governorates in Egypt during 2019-2021. In addition, genetic characterization and evolutionary analyses were performed using 47 selected positive H5N8 isolates obtained during the same period. The result of the conducted surveillance showed that HPAI H5N8 viruses of clade 2.3.4.4b continue to circulate in different locations in Egypt, with an obvious seasonal pattern, and no further detection of the HPAI H5N1 virus of clade 2.2.1.2 was observed in the poultry population during 2019-2021. In addition, phylogenetic and Bayesian analyses revealed that two major genotypes (G5 and G6) of HPAI H5N8 viruses were continually expanding among the poultry sectors in Egypt. Notably, molecular dating analysis suggested that the Egyptian HPAI H5N8 virus is the potential ancestral viruses of the European H5N8 viruses of 2020-2021. In summary, the data of this study highlight the current epidemiology, diversity, and evolution of HPAI H5N8 viruses in Egypt and call for continuous monitoring of the genetic features of the avian influenza viruses in Egypt.

The highly pathogenic avian influenza (HPAI) H5N8 virus was first detected in Egypt in late 2016. Since then, the virus has spread rapidly among different poultry sectors, becoming the dominant HPAI H5 subtype reported in Egypt. Different genotypes of the HPAI H5N8 virus were reported in Egypt; however, the geographic patterns and molecular evolution of the Egyptian HPAI H5N8 viruses are still unclear. Here, extensive epidemiological surveillance was conducted, including more than half a million samples collected from different poultry sectors (farms/backyards/live bird markets) from all governorates in Egypt during 2019-2021. In addition, genetic characterization and evolutionary analyses were performed using 47 selected positive H5N8 isolates obtained during the same period. The result of the conducted surveillance showed that HPAI H5N8 viruses of clade 2.3.4.4b continue to circulate in different locations in Egypt, with an obvious seasonal pattern, and no further detection of the HPAI H5N1 virus of clade 2.2.1.2 was observed in the poultry population during 2019-2021. In addition, phylogenetic and Bayesian analyses revealed that two major genotypes (G5 and G6) of HPAI H5N8 viruses were continually expanding among the poultry sectors in Egypt. Notably, molecular dating analysis suggested that the Egyptian HPAI H5N8 virus is the potential ancestral viruses of the European H5N8 viruses of 2020-2021. In summary, the data of this study highlight the current epidemiology, diversity, and evolution of HPAI H5N8 viruses in Egypt and call for continuous monitoring of the genetic features of the avian influenza viruses in Egypt.

Since it was first discovered, the low pathogenic avian influenza (LPAI) H9N2 subtype has established linages infecting the poultry population globally and has become one of the most prevalent influenza subtypes in domestic poultry. Several different variants and genotypes of LPAI H9N2 viruses have been reported in Egypt, but little is known about their pathogenicity and how they have evolved. In this study, four different Egyptian LPAI H9N2 viruses were genetically and antigenically characterized and compared to representative H9N2 viruses from G1 lineage. Furthermore, the pathogenicity of three genetically distinct Egyptian LPAI H9N2 viruses was assessed by experimental infection in chickens. Whole-genome sequencing revealed that the H9N2 virus of the Egy-2 G1-B lineage (pigeon-like) has become the dominant circulating H9N2 genotype in Egypt since 2016. Considerable variation in virus shedding at day 7 post-infections was detected in infected chickens, but no significant difference in pathogenicity was found between the infected groups. The rapid spread and emergence of new genotypes of the influenza viruses pinpoint the importance of continuous surveillance for the detection of novel reassortant viruses, as well as monitoring the viral evolution.

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 genoty

The highly pathogenic avian influenza (HPAI) H5N8 virus was first detected in Egypt in late 2016. Since then, the virus has spread rapidly among different poultry sectors, becoming the dominant HPAI H5 subtype reported in Egypt. Different genotypes of the HPAI H5N8 virus were reported in Egypt; however, the geographic patterns and molecular evolution of the Egyptian HPAI H5N8 viruses are still unclear. Here, extensive epidemiological surveillance was conducted, including more than half a million samples collected from different poultry sectors (farms/backyards/live bird markets) from all governorates in Egypt during 2019-2021. In addition, genetic characterization and evolutionary analyses were performed using 47 selected positive H5N8 isolates obtained during the same period. The result of the conducted surveillance showed that HPAI H5N8 viruses of clade 2.3.4.4b continue to circulate in different locations in Egypt, with an obvious seasonal pattern, and no further detection of the HPAI H5N1 virus of clade 2.2.1.2 was observed in the poultry population during 2019-2021. In addition, phylogenetic and Bayesian analyses revealed that two major genotypes (G5 and G6) of HPAI H5N8 viruses were continually expanding among the poultry sectors in Egypt. Notably, molecular dating analysis suggested that the Egyptian HPAI H5N8 virus is the potential ancestral viruses of the European H5N8 viruses of 2020-2021. In summary, the data of this study highlight the current epidemiology, diversity, and evolution of HPAI H5N8 viruses in Egypt and call for continuous monitoring of the genetic features of the avian influenza viruses in Egypt.

Since it was first discovered, the low pathogenic avian influenza (LPAI) H9N2 subtype has established linages infecting the poultry population globally and has become one of the most prevalent influenza subtypes in domestic poultry. Several different variants and genotypes of LPAI H9N2 viruses have been reported in Egypt, but little is known about their pathogenicity and how they have evolved. In this study, four different Egyptian LPAI H9N2 viruses were genetically and antigenically characterized and compared to representative H9N2 viruses from G1 lineage. Furthermore, the pathogenicity of three genetically distinct Egyptian LPAI H9N2 viruses was assessed by experimental infection in chickens. Whole-genome sequencing revealed that the H9N2 virus of the Egy-2 G1-B lineage (pigeon-like) has become the dominant circulating H9N2 genotype in Egypt since 2016. Considerable variation in virus shedding at day 7 post-infections was detected in infected chickens, but no significant difference in pathogenicity was found between the infected groups. The rapid spread and emergence of new genotypes of the influenza viruses pinpoint the importance of continuous surveillance for the detection of novel reassortant viruses, as well as monitoring the viral evolution.

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 genoty

Highly pathogenic avian influenza (HPAI) viruses continue to circulate worldwide, causing numerous outbreaks among bird species and severe public health concerns. H5N1 and H5N8 are the two most fundamental HPAI subtypes detected in birds in the last two decades. The two viruses may compete with each other while sharing the same host population and, thus, suppress the spread of one of the viruses. In this study, we performed a statistical analysis to investigate the temporal correlation of the HPAI H5N1 and HPAI H5N8 subtypes using globally reported data in 2015-2020. This was joined with an in-depth analysis using data generated via our national surveillance program in Egypt. A total of 6412 outbreaks were reported worldwide during this period, with 39% (2529) as H5N1 and 61% (3883) as H5N8. In Egypt, 65% of positive cases were found in backyards, while only 12% were found in farms and 23% in live bird markets. Overall, our findings depict a trade-off between the number of positive H5N1 and H5N8 samples around early 2017, which is suggestive of the potential replacement between the two subtypes. Further research is still required to elucidate the underpinning mechanisms of this competitive dynamic. This, in turn, will implicate the design of effective strategies for disease control.

Highly pathogenic avian influenza (HPAI) viruses continue to circulate worldwide, causing numerous outbreaks among bird species and severe public health concerns. H5N1 and H5N8 are the two most fundamental HPAI subtypes detected in birds in the last two decades. The two viruses may compete with each other while sharing the same host population and, thus, suppress the spread of one of the viruses. In this study, we performed a statistical analysis to investigate the temporal correlation of the HPAI H5N1 and HPAI H5N8 subtypes using globally reported data in 2015-2020. This was joined with an in-depth analysis using data generated via our national surveillance program in Egypt. A total of 6412 outbreaks were reported worldwide during this period, with 39% (2529) as H5N1 and 61% (3883) as H5N8. In Egypt, 65% of positive cases were found in backyards, while only 12% were found in farms and 23% in live bird markets. Overall, our findings depict a trade-off between the number of positive H5N1 and H5N8 samples around early 2017, which is suggestive of the potential replacement between the two subtypes. Further research is still required to elucidate the underpinning mechanisms of this competitive dynamic. This, in turn, will implicate the design of effective strategies for disease control.

Highly pathogenic avian influenza (HPAI) viruses continue to circulate worldwide, causing numerous outbreaks among bird species and severe public health concerns. H5N1 and H5N8 are the two most fundamental HPAI subtypes detected in birds in the last two decades. The two viruses may compete with each other while sharing the same host population and, thus, suppress the spread of one of the viruses. In this study, we performed a statistical analysis to investigate the temporal correlation of the HPAI H5N1 and HPAI H5N8 subtypes using globally reported data in 2015-2020. This was joined with an in-depth analysis using data generated via our national surveillance program in Egypt. A total of 6412 outbreaks were reported worldwide during this period, with 39% (2529) as H5N1 and 61% (3883) as H5N8. In Egypt, 65% of positive cases were found in backyards, while only 12% were found in farms and 23% in live bird markets. Overall, our findings depict a trade-off between the number of positive H5N1 and H5N8 samples around early 2017, which is suggestive of the potential replacement between the two subtypes. Further research is still required to elucidate the underpinning mechanisms of this competitive dynamic. This, in turn, will implicate the design of effective strategies for disease control.

Background: The tufted duck is a non-model organism that experiences high mortality in highly pathogenic avian influenza outbreaks. It belongs to the same bird family (Anatidae) as the mallard, one of the best-studied natural hosts of low-pathogenic avian influenza viruses. Studies in non-model bird species are crucial to disentangle the role of the host response in avian influenza virus infection in the natural reservoir. Such endeavour requires a high-quality genome assembly and transcriptome. Findings: This study presents the first high-quality, chromosome-level reference genome assembly of the tufted duck using the Vertebrate Genomes Project pipeline. We sequenced RNA (complementary DNA) from brain, ileum, lung, ovary, spleen, and testis using Illumina short-read and Pacific Biosciences long-read sequencing platforms, which were used for annotation. We found 34 autosomes plus Z and W sex chromosomes in the curated genome assembly, with 99.6% of the sequence assigned to chromosomes. Functional annotation revealed 14,099 protein-coding genes that generate 111,934 transcripts, which implies a mean of 7.9 isoforms per gene. We also identified 246 small RNA families. Conclusions: This annotated genome contributes to continuing research into the host response in avian influenza virus infections in a natural reservoir. Our findings from a comparison between short-read and long -read reference transcriptomics contribute to a deeper understanding of these competing options. In this study, both technologies complemented each other. We expect this annotation to be a foundation for further comparative and evolutionary genomic studies, including many waterfowl relatives with differing susceptibilities to avian influenza viruses.

Background: The tufted duck is a non-model organism that experiences high mortality in highly pathogenic avian influenza outbreaks. It belongs to the same bird family (Anatidae) as the mallard, one of the best-studied natural hosts of low-pathogenic avian influenza viruses. Studies in non-model bird species are crucial to disentangle the role of the host response in avian influenza virus infection in the natural reservoir. Such endeavour requires a high-quality genome assembly and transcriptome. Findings: This study presents the first high-quality, chromosome-level reference genome assembly of the tufted duck using the Vertebrate Genomes Project pipeline. We sequenced RNA (complementary DNA) from brain, ileum, lung, ovary, spleen, and testis using Illumina short-read and Pacific Biosciences long-read sequencing platforms, which were used for annotation. We found 34 autosomes plus Z and W sex chromosomes in the curated genome assembly, with 99.6% of the sequence assigned to chromosomes. Functional annotation revealed 14,099 protein-coding genes that generate 111,934 transcripts, which implies a mean of 7.9 isoforms per gene. We also identified 246 small RNA families. Conclusions: This annotated genome contributes to continuing research into the host response in avian influenza virus infections in a natural reservoir. Our findings from a comparison between short-read and long -read reference transcriptomics contribute to a deeper understanding of these competing options. In this study, both technologies complemented each other. We expect this annotation to be a foundation for further comparative and evolutionary genomic studies, including many waterfowl relatives with differing susceptibilities to avian influenza viruses.

Background: The tufted duck is a non-model organism that experiences high mortality in highly pathogenic avian influenza outbreaks. It belongs to the same bird family (Anatidae) as the mallard, one of the best-studied natural hosts of low-pathogenic avian influenza viruses. Studies in non-model bird species are crucial to disentangle the role of the host response in avian influenza virus infection in the natural reservoir. Such endeavour requires a high-quality genome assembly and transcriptome. Findings: This study presents the first high-quality, chromosome-level reference genome assembly of the tufted duck using the Vertebrate Genomes Project pipeline. We sequenced RNA (complementary DNA) from brain, ileum, lung, ovary, spleen, and testis using Illumina short-read and Pacific Biosciences long-read sequencing platforms, which were used for annotation. We found 34 autosomes plus Z and W sex chromosomes in the curated genome assembly, with 99.6% of the sequence assigned to chromosomes. Functional annotation revealed 14,099 protein-coding genes that generate 111,934 transcripts, which implies a mean of 7.9 isoforms per gene. We also identified 246 small RNA families. Conclusions: This annotated genome contributes to continuing research into the host response in avian influenza virus infections in a natural reservoir. Our findings from a comparison between short-read and long -read reference transcriptomics contribute to a deeper understanding of these competing options. In this study, both technologies complemented each other. We expect this annotation to be a foundation for further comparative and evolutionary genomic studies, including many waterfowl relatives with differing susceptibilities to avian influenza viruses.

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

Emerging zoonotic diseases exert a significant burden on human health and have considerable socioeconomic impact worldwide. In Asia, live animals as well as animal products are commonly sold in informal markets. The interaction of humans, live domestic animals for sale, food products, and wild and scavenging animals, creates a risk for emerging infectious diseases. Such markets have been in the spotlight as sources of zoonotic viruses, for example, avian influenza viruses and coronaviruses, Here, we bring data together on the global impact of live and wet markets on the emergence of zoonotic diseases. We discuss how benefits can be maximized and risks minimized and conclude that current regulations should be implemented or revised, to mitigate the risk of new diseases emerging in the future.

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