The NA truncation-specific reverse transcription real-time quantitative PCR (RT-qPCR) assay was optimized to work with a probe complementary to an ancestor sequence maintained within the EscEgg50A segment 6 sequence and EscEgg50A virus segment 6-specific primers (sequences are available upon request). shuffling and insertion of a short fragment originating from the PA segment. Characterization of that novel variant revealed a loss of the neuraminidase protein and enzymatic activity, but its replication efficiency remained comparable to that of the wild type. Using reverse genetics, a recombinant virus consisting of the wild-type backbone and the shortened NA segment could Vinorelbine (Navelbine) be generated; however, generation of this recombinant virus required the polybasic hemagglutinin cleavage site. Two independent repetitions starting with egg passage 30 in the presence of alternative chicken-derived immune sera selected mutants with similar but different large deletions within the NA segment without any neuraminidase activity, indicating a general mechanism. In chicken, these virus variants were avirulent, even though the HPAIV polybasic hemagglutinin cleavage site was still present. Overall, the variants reported here are the first HPAIV H5N1 strains without a functional neuraminidase shown to grow efficiently without any helper factor. These novel HPAIV variants may facilitate future studies shedding light on the role of neuraminidase in virus replication and pathogenicity. INTRODUCTION Highly pathogenic avian influenza viruses (HPAIVs) of subtype H5N1 have been circulating in many regions in Asia and Africa for up to 10 years (1), raising concerns of an influenza pandemic. While wild waterfowl serves as a virus reservoir, poultryprimarily chickensinfected with HPAIV H5N1 succumb to death due to a devastating disease. In addition, the currently used control measures (2), like culling of infected birds, restriction of movement, enforcement of biosecurity, and surveillance, lead to severe economic losses in the poultry industry worldwide. Vaccination against HPAIV H5N1 using inactivated virus preparations was implemented, particularly in developing countries, to combat the disease. However, as influenza Vinorelbine (Navelbine) A viruses continue to change their antigenicity by antigenic drift, due to base exchanges introduced during the error-prone process of genome replication by the viral polymerase complex, and by antigenic shift, which results from reassortment of genome segments from two viruses (3), vaccines have to be adapted regularly. For application in humans, the World Health Organization (WHO) predetermines the vaccine composition each season. In the veterinary field, nonhomologous vaccines are used, often resulting in nonsterile immunity in the vaccinated poultry flocks and thus a lack of disruption of infection chains. As a consequence, infection of those partially protected birds by circulating recent HPAIV H5N1 leads to the Vinorelbine (Navelbine) continuous emergence of escape variants (4C6) with Rabbit polyclonal to Ezrin an altered antigenic repertoire (6). These viruses are not neutralized by the antibodies present in the vaccinated flocks; hence, the animals are not fully protected, as demonstrated by the reoccurrence of morbidity and mortality (4). The phenomenon of antigenic escape was classically investigated by the characterization of escape variants generated by virus passaging in the presence of monoclonal antibodies (7, 8). While antigenic sites were thereby successfully identified, such a rather artificial selection is limited to epitope-specific variation only. However, analysis of the evolution of both viral surface proteins, i.e., the hemagglutinin (HA) and neuraminidase (NA), revealed several epistatic mutations, highlighting that immunoescape is a polygenic trait (9). In addition, we recently showed that cell culture passaging of HPAIV H5N1 under the selection pressure of a polyclonal chicken-derived serum resulted in attenuated viruses with numerous point mutations in several segments (10). To assess the immunoescape enabled by the considerable genetic plasticity of influenza A viruses under strong, more authentic selection pressure closer to conditions study (10), this experimental approach resulted in replication-competent and stable neuraminidase-negative attenuated H5N1 viruses with large intrasegmental deletions in segment 6 causing a complete loss of neuraminidase activity. Their generation, along with the and features, is the subject of this study. MATERIALS AND METHODS All experiments using HPAIV H5N1 were conducted in biosafety level 3+ containment facilities at the Friedrich-Loeffler-Institut (FLI), Greifswald-Insel Riems, Germany. Viruses and sera. Ancestor virus for passaging was from the 3rd (egg culture) passage of the reference strain A/Cygnus cygnus/Germany/R65/2006 (H5N1) (11)..