The nature of influenza virus to randomly mutate and evolve into new types is an important challenge in the control of influenza infection. It is necessary to monitor virus evolution for a better understanding of the pandemic risk posed by certain variants as evidenced by the highly pathogenic avian influenza (HPAI) viruses. This has been clearly recognized in Egypt following the notification of the first HPAI H5N1 outbreak. The continuous circulation of the virus and the mass vaccination programme undertaken in poultry have resulted in a progressive genetic evolution and a significant antigenic drift near the major antigenic sites. In order to establish if vaccination is sufficient to provide significant intra- and interclade cross-protection, lentiviral pseudotypes derived from H5N1 HPAI viruses (A/Vietnam/1194/04, A/chicken/Egypt-1709-01/2007) and an antigenic drift variant (A/chicken/Egypt-1709-06-2008) were constructed and used in pseudotype-based neutralization assays (pp-NT). pp-NT data obtained was confirmed and correlated with HI and MN assays. A panel of pseudotypes belonging to influenza Groups 1 and 2, with a combination of reporter systems, was also employed for testing avian sera in order to support further application of pp-NT as an alternative valid assay that can improve avian vaccination efficacy testing, vaccine virus selection, and the reliability of reference sera. 1. Background Egypt faced its first H5N1 outbreak in 2006 where a highly pathogenic avian influenza (HPAI) virus was detected in poultry [1]. The strategy used by the Egyptian authorities to mitigate this relied on vaccinating poultry, depopulating infected areas, and increasing awareness and biosecurity levels. Despite these efforts, by 2008, the H5N1 virus had become endemic, and vaccine-escape variants have emerged despite commercial poultry vaccines exhibiting protection in laboratory settings [2]. For each year, from 2009 through 2012, Egypt has had more laboratory-confirmed human cases reported to the WHO than any other country, and global concern regarding Egyptian H5N1 influenza viruses is currently high, as some isolates have been reported to possess at least two mutations, of the 4 (or 5) needed to confer ferret-to-ferret airborne transmissibility [3]. Despite the mass vaccination program undertaken in poultry, the continuous circulation of the virus has resulted in a progressive genetic evolution and a significant antigenic drift with multiple mutations near the major antigenic sites [4]. To date, the WHO has identified 12 new H5N1 clades, and the Egyptian
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