title: Increase in viral yield in eggs and MDCK cells of reassortant H5N1 vaccine candidate viruses caused by insertion of 38 amino acids into the NA stalk.
abstract: BACKGROUND
  The H5N1 subtype of highly pathogenic avian influenza viruses has spread to over 63 countries in Asia, Europe, and Africa and has become endemic in poultry. Since 2004, vaccination against H5N1 influenza has become common in domestic poultry operations in China. Most influenza vaccines have been produced in embryonated chicken eggs. High yield is the essential feature of a good vaccine candidate virus.
  
  
  OBJECTIVE
  Therefore, the large-scale manufacture of such a vaccine requires that the viral yield of H5N1 reassortant vaccine viruses in eggs and MDCK cells be increased.
  
  
  METHODS
  We generated two sets of reassortant H5N1 viruses based on backbone viruses A/Chicken/F/98 (H9N2) and A/Puerto Rico/8/34 (H1N1) using reverse genetics. The HAs and NAs of the reassortants were derived from the three epidemic H5N1 strains found in China. We compared the replication properties of these recombinant H5N1 viruses in embryonated chicken eggs and MDCK cells after inserting either 20 or 38 amino acids into their NA stalks.
  
  
  RESULTS
  In this study, we demonstrated that inserting 38 amino acids into the NA stalks can significantly increase the viral yield of H5N1 reassortant viruses in both embryonated chicken eggs and MDCK cells, while inserting only 20 amino acids into the same NA stalks does not. Hemagglutinin inhibition testing and protection assays indicated that recombinant H5N1 viruses with 38 aa inserted into their NA stalks had the same antigenicity as the viruses with wt-NA.
  
  
  CONCLUSIONS
  These results suggest that the generation of an H5N1 recombinant vaccine seed by the insertion of 38 aa into the NA stalk may be a suitable and more economical strategy for the increase in viral yield in both eggs and MDCK cells for the purposes of vaccine production.
authors.affiliation: Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, 12 East Wenhui Road, Yangzhou, Jiangsu 225009, China.
authors.forename: Wenjun
authors.initials: W
authors.lastname: Zhang
authors.forename: Tao
authors.initials: T
authors.lastname: Xue
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Wu
authors.forename: Pinghu
authors.initials: P
authors.lastname: Zhang
authors.forename: Guo
authors.initials: G
authors.lastname: Zhao
authors.forename: Daxing
authors.initials: D
authors.lastname: Peng
authors.forename: Shunlin
authors.initials: S
authors.lastname: Hu
authors.forename: Xiaoquan
authors.initials: X
authors.lastname: Wang
authors.forename: Xiaowen
authors.initials: X
authors.lastname: Liu
authors.forename: Wenbo
authors.initials: W
authors.lastname: Liu
authors.forename: Xiufan
authors.initials: X
authors.lastname: Liu
mesh_terms: D000818:Animals; D002642:Chick Embryo; D002681:China; D004285:Dogs; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification; D007252:Influenza Vaccines / Q000191:economics / Q000235:genetics*; D016254:Mutagenesis, Insertional; D009439:Neuraminidase / Q000235:genetics*; D016865:Reassortant Viruses / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification; D013678:Technology, Pharmaceutical / Q000191:economics / Q000379:methods*; D014612:Vaccines; D014764:Viral Proteins / Q000235:genetics*; D014779:Virus Replication
chemical_list: D007252:Influenza Vaccines; D014612:Vaccines; D014764:Viral Proteins; C487630:NA protein, influenza A virus; D009439:Neuraminidase