Construction and Immunological Activity of Recombinant Newcastle Disease Virus Expressing H5 Hemagglutinin Gene of Avian Influenza Virus

Newcastle disease virus(NDV)-expressing avian influenza virus(AIV)hemagglutinin(HA)of subtype H5 was constructed by reverse genetics.A cloned full-length copy of the genome of the lentogenic NDV strain Clone 30 was used for insertion of the ORF encoding the HA of the highly pathogenic AIV isolate A/chicken/Italy/8/98(H5N2)in the intergenic region between the NDV fusion and hemagglufinin-neuraminidase(HN)genes.Remarkably,two species of HA transcripts were detected in cells infected with the resultant NDVH5.In a second recombinant(NDVH5m),a NDV transcription termination signal-like se- quence located within the HA ORF was eliminated by silent mutations.Consequently,NDVHSm produced 2.7-fold more full- length HA transcripts,expressed higher levels of HA,and also incorporated more HA protein into its envelope than NDVH5.ND- VH5m stably expressed the modified HA gene for 10 egg passages and both recombinants were found innocuous after intracerebral inoculation of 1-day-old chickens.Immunization of chickens with NDVH5m induced NDV and AIVH5-specific antibodies and protected chickens against clinical disease after challenge with a lethal dose of velogenic NDV or highly pathogenic AIV,respec- tively.Remarkably,shedding of influenza virus was not observed.Furthermore,immunization with NDVH5m permitted serological discrimination of vaccinated and AIV field virus-infected animals based on antibodies against the nucleoprotein of AIV.Therefore, recombinant NDVH5m is suitable as a bivalent vaccine against NDV and AIV and may be used as marker vaccine for the control of avian influenza.