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In this study, we introduced by reverse genetics temperature-sensitive mutations in the PB1 and PB2 genes of an avian influenza virus, A\/Guinea Fowl\/Hong Kong\/WF10\/99 (H9N2) (WF10). Further genetic modifications were introduced into the PB1 gene to enhance the attenuated (<jats:italic>att<\/jats:italic>) phenotype of the virus in vivo. Using the<jats:italic>att<\/jats:italic>WF10 as a backbone, we substituted neuraminidase (NA) for hemagglutinin (HA) for vaccine purposes. In chickens, a vaccination scheme consisting of a single dose of an<jats:italic>att<\/jats:italic>H7N2 vaccine virus at 2 weeks of age and subsequent challenge with the wild-type H7N2 LPAI virus resulted in complete protection. We further extended our vaccination strategy against the HPAI H5N1. In this case, we reconstituted an<jats:italic>att<\/jats:italic>H5N1 vaccine virus, whose HA and NA genes were derived from an Asian H5N1 virus. A single-dose immunization in ovo with the<jats:italic>att<\/jats:italic>H5N1 vaccine virus in 18-day-old chicken embryos resulted in more than 60% protection for 4-week-old chickens and 100% protection for 9- to 12-week-old chickens. Boosting at 2 weeks posthatching provided 100% protection against challenge with the HPAI H5N1 virus for chickens as young as 4 weeks old, with undetectable virus shedding postchallenge. Our results highlight the potential of live<jats:italic>att<\/jats:italic>avian influenza vaccines for mass vaccination in poultry.<\/jats:p>","DOI":"10.1128\/jvi.00893-07","type":"journal-article","created":{"date-parts":[[2007,6,28]],"date-time":"2007-06-28T04:43:45Z","timestamp":1183005825000},"page":"9238-9248","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":80,"title":["A New Generation of Modified Live-Attenuated Avian Influenza Viruses Using a Two-Strategy Combination as Potential Vaccine Candidates"],"prefix":"10.1128","volume":"81","author":[{"given":"Haichen","family":"Song","sequence":"first","affiliation":[{"name":"Department of Veterinary Medicine, University of Maryland, College Park, and Virginia-Maryland Regional College of Veterinary Medicine, 8075 Greenmead Drive, College Park, Maryland 20742-3711"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gloria Ramirez","family":"Nieto","sequence":"additional","affiliation":[{"name":"Department of Veterinary Medicine, University of Maryland, College Park, and Virginia-Maryland Regional College of Veterinary Medicine, 8075 Greenmead Drive, College Park, Maryland 20742-3711"},{"name":"Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional de Colombia, Bogota\u0301, Colombia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel R.","family":"Perez","sequence":"additional","affiliation":[{"name":"Department of Veterinary Medicine, University of Maryland, College Park, and Virginia-Maryland Regional College of Veterinary Medicine, 8075 Greenmead Drive, College Park, Maryland 20742-3711"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.2307\/1591676"},{"key":"e_1_3_2_3_2","doi-asserted-by":"publisher","DOI":"10.1056\/NEJM199805143382002"},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.68.3.1819-1826.1994"},{"key":"e_1_3_2_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2004.04.017"},{"key":"e_1_3_2_6_2","first-page":"705","volume":"1","year":"1984","unstructured":"Clements, M. 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