Differential replication characteristic of reassortant avian influenza A viruses H5N8 clade 2.3.4.4b in Madin-Darby canine kidney cell.

Late in 2016, multiple reassortant highly pathogenic (HP) avian influenza virus (AIVs) H5N8 was detected. AIVs infect different isolated hosts with a specific viral tropism. In the current study, the whole genome of the Egyptian A/chicken/NZ/2022 was genetically characterized. The H5N8-A/Common-coot/Egypt/CA285/2016, A/duck/Egypt/SS19/2017 previously isolated in Egypt, and the recently circulating A/chicken/Egypt/NZ/2022 reassortant viruses' replication, pathogenicity, and viral load in comparison to the H5N1-Clade 2.2.1.2 were investigated on Madin-Darby canine kidney cell (MDCK), by using the cytopathic effect (CPE) percent and matrix-gene reverse transcription quantitative real-time polymerase chain reaction to compute the virus titer at various points in time. The A/chicken/Egypt/NZ/2022 virus was similar to the reassortant strain clade 2.3.4.4b discovered in farms in 2016. The 2 sub-groupings of hemagglutinin (HA) and neuraminidase (NA) genes were identified (I and II); the A/chicken/Egypt/NZ/2022 HA and NA genes were associated with subgroup II. The subgroup II of the HA gene was further divided into A and B owing to acquired specific mutations. The A/chicken/Egypt/NZ/2022 in our study was associated with subgroup B. The M, NS, PB1, and PB2 genes were shown to be clustered into clade 2.3.4.4b by full genome sequence analysis; however, the PA and NP genes were found to be associated with H6N2 viruses, which had particular mutations that improved viral virulence and mammalian transmission. The current results showed that the circulating H5N8 viruses were more variable than previous viruses analyzed in 2016 and 2017. Compared to other reassortant HPAI H5N8, and HPAI H5N1, the growth kinetics of A/chicken/Egypt/NZ/2022 had a high CPE without the addition of trypsin and the most viral copies with a significant difference (P < 0.01) in comparison to HPAI H5N8 and HPAI H5N1 reassortant viruses. Accordingly, the effective viral replication of A/chicken/Egypt/NZ/2022 in the MDCK than other viruses may play a factor in the spread and maintenance of specific reassortant H5N8 influenza virus in the field.

Emergence of Highly Pathogenic Avian Influenza A Virus (H5N1) of Clade 2.3.4.4b in Egypt, 2021-2022.

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.

Molecular Epidemiology and Evolutionary Analysis of Avian Influenza A(H5) Viruses Circulating in Egypt, 2019-2021.

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.

Temporal Dynamics of Influenza A(H5N1) Subtype before and after the Emergence of H5N8.

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.