Abstract: NIBRG-14 is one of the H5N1 candidate vaccine viruses developed using “6+2” approach with the hemagglutinin (HA) and neuraminidase (NA) genes derived from A/Vietnam/1194/2004(H5N1, VN1194) and the remaining six internal segments from A/Puerto Rico/8/34(H1N1, PR8). However, NIBRG-14 was reported to yield low amounts of HA antigen. This study found that the NA vRNA of VN1194 is poorly packaged (38%~68%) into the recombinant viruses with a backbone of PR8 genes, causing the formation of defective virions without the NA vRNA in viral genome. Using recombinant DNA techniques, we constructed a chimeric NA gene with the coding region of VN1194 NA flanked by the packaging signal sequence (vRNA 3′end 41bp, 5′end 67bp) of PR8 NA. The packaging of NA vRNA is completely restored in the recombinant viruses with the chimeric NA gene. Moreover, the recombinant viruses contained the chimeric NA gene replicate better in chicken embroynated eggs than recombinant viruses with wild type NA gene of VN1194, as indicated by a 10-fold increase in virus titer and 27-fold increase in HA antigen content. These findings suggest a novel strategy to improve the in ovo growth and increase the available dose of NIBRG-14 in vaccine manufacture.