1. Generation of an attenuated H5N1 avian influenza virus vaccine with all eight genes from avian virus Vaccine 2007 Huoying Shi, Xiu Fan Liu, Xiaorong Zhang, Sujuan Chen, Lei Sun and Jianhong Lu

2. Influenza virus http://www.bch.cuhk.edu.hk/influenza/virology.html (hemagglutinin) (neuraminidase) a globally important respiratory pathogen causes a high degree of morbidity and mortality in humans and animals annually

3. Avian influenza virus 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes since the late 1990s, some of avian influenza A viruses have transmitted directly from birds to humans H5 subtype highly pathogenic avian influenza (HPAI) and H5N1 (HPAIV) is continuously undergoing antigenic change to escape the host's acquired immunity

4. Avian influenza pathogenesis and epidemiology http://www.influenzareport.com/ir/ai.htm

5. 2003–2006, a highly pathogenic avian influenza A (H5N1) virus caused poultry disease in Asian countries and infected many people  most of these individuals had close contact with poultry  to development of an H5N1 AIV vaccine not all viruses that are closely related are suitable for vaccine production, some grow poorly in eggs  plasmid-based system

6. http://www.futurasciences.com/fr/print /comprendre/dossiers/doc/t/medecine -1/d/grippe-aviaire-et-prevention- chez-lhomme-33_682/c3/221/p1/ 1.live attenuated vaccine 2.cold-adaptive strain vaccine 3.capable of inducing broad mucosal and systemic responses

7. Generation of the C4/F reassortant virus and confirmed it in vitro growth properties ↓ Pathogenicity and replication in chickens ↓ Pathogenicity and replication in BALB/c mice ↓ Immunogenicity and protective efficacy of a formalin- inactivated C4/F virus vaccine

8. Receptor Basic patches Cleavage site HA2 HA1

11. C4/F 8-plasmid transfection system HA NA PB2PB1PA NP MNS HA and NA from C4/H5N1 Internal gene from F strain HA titers: 2048 ~ 20 passages in embryonated chicken eggs

12. Pathogenicity and replication in chickens Eight chickens (4-week-old SPF white leghorn chickens) Intravenously (i.v.) with 0.2 ml 1:10 dilution of stock virus 1. H5N1 2. C4/F 3. F

13. Pathogenicity and replication in BALB/c mice Eight 6–8-week-old female BALB/c mice Intranasally (i.n.) with 50×10 6  l 50% eggs infectious dose (EID 50 ) 1. H5N1 2. C4/F 3. F

14. Immunogenicity and protective efficacy of a formalin-inactivated C4/F virus vaccine White Leghorn chicken 7-day-old wt. C4/H5N1 and C4/F formalin-inactivated vaccine Single i.m. dose (3  g HA) Three weeks after immunization Immunogenicity  challenge with 100 ELD 50 of virus Protective efficacy Survival and disease sign

15. Immunogenicity and protective efficacy of a formalin-inactivated C4/F virus vaccine 3 Weeks Postvaccination Hemagglutination Inhibition 3 Weeks Postvaccination Challenge with wt. C4/H5N1 Intranasally 3 days2 weeks

16. Discussion Measures for the control of emerging and reemerging H5N1 influenza include the use of inactivated vaccines and improvement in biosecurity. 1.HPAI viruses are lethal to embryonated eggs, which limits growth in a high titer. 2.The multibasic amino acid motif at the HA cleavage site is believed to contribute to the virulence of these viruses in humans as well as in domestic poultry. 郭懿瑩

17. In this study Developed an attenuated H5N1 avian influenza virus vaccine with all eight genes from avian viruses, a reassortant C4/F converted the basic amino acids of HA gene seen in the C4/H5N1 viruses to the sequence motif seen in avirulent avian influenza viruses, was generated by reverse genetics with two surface genes from C4/H5N1 and the remaining six genes from F, which is not pathogenic and replicate for mice and low pathogenic for chickens. the C4/F reassortant showed good growth characteristic with the HA titer as high as 1:2048.

18. This data indicated that although the C4/F transfectant virus replicated in the trachea and lungs of mice, the attenuation phenotypes in the HA and the accompanying genotype are associated with a loss of virulence for chicken and mice.