Evolution Of H9N2 Influenza Viruses In China And Study On H5N1 Influenza Marker Vaccine

H9N2 avian influenza virus was firstly isolated in Guangdong province in 1994, and then the viruses were widely spread in Mainland China In this study, 27 representative strains of H9N2 viruses that isolated from chickens and ducks from different provinces in China from 1996 to 2002 are intensively analyzed, and the questions that we are trying to answer are: 1) What are the genetic relationship of these viruses? 2) Are these avian influenza viruses able to replicate in mammalian hosts? And 3) What are the antigenic relationship of these viruses? Are they antigenic drifted?To understand the pathogenicity of H9N2 viruses in chickens, 6-week-old SPF chickens were i.n. inoculated with l0^6.0EID₅₀ of virus. The results demonstrated that all of the 27 viruses are low pathogenic to SPF chickens. The antigenic analysis indicated that the H9N2 viruses circulated in China are quite different and some viruses are antigenic drifted. In the immunization protection experiment, only the chickens immunized with CK/SH/10/01 vaccine were essentially protected from homologous virus challenge, and the chickens immunized with the inactivated vaccines of CK/GX/10/99, CK/HLJ/35/00 or CK/SH/10/01 could not be fully protected against the heterologous challenge with CK/GX/10/99 or CK/SH/10/01 virus.We use BALB/c mice as a model to evaluate the ability of infection in the mammalian hosts of these H9N2 viruses. On the base of their ability to replicate and cause disease in mice, these viruses could be divided into 3 groups. The group I consists of 5 viruses, which could not be recovered from lungss of the inoculated mice and the mice stay healthy and keep getting weight during the observation period. The group II includes 14 viruses. Eleven viruses in this group could replicate well in the lungs with titers of 2.8- 5.81ogl0EID₅₀/ml on day 3 after inoculation, while 3 other viruses replicate slowly and could only be detected in the mice lungs with low titers on day 5 after inoculation. The weight losses of the mice induced by this group of viruses are less than 5%. The group III contains 8 viruses that replicate very well in the lungs on day 3 after inoculation, and the titers reach to 6.0-7.3 log10EID₅₀/ml. The mice showed disease signs and 10-20% weight loss.Phylogenic analysis revealed that all of the 27 viruses originated from CK/BJ/1/94-like virus and formed multiple genotypes through complicated reassortment with QA/HK/G1/97-, CK/HK/G9/97-, CK/SH/F/98- or TY/WI/66-like viruses. On the basis of genomic diversity, the viruses were divided into 9 genotypes. Detailed analysis indicated that 3 viruses have an S to N substitution at residue 31 in the M2, which is related with the resistance phenotype to adamantanes, and the resistance to adamantanes of these three viruses was experimentally confirmed.The present study systemically analyzed 27 previously uncharacterized H9N2 representativeviruses isolated from poultry in China, and revealed that these viruses posed multiple genotypes and big diversity of the biological properties. The low pathogenic properties to poultries make these viruses easily to be widespread; their gradually acquired ability to infect and replicate in mammalian ranks H9N2 viruses on the top of the list to cause humans influenza pandemics in the future; the antigenic diversity and the resistance to the adamantanes of these viruses will pose great difficulties for the future human influenza control caused by H9N2 viruses. Therefore, urgent attentions should be paid to the control of H9N2 influenza viruses in animals and to the human's influenza pandemic preparedness.H5N1 highly pathogenic avian influenza is responsible for one of the most devastating diseases in domestic poultries. Vaccination has been proved to be a useful tool for the avian influenza control, and a technique for differentiating infected from vaccinated animals (DIVA) is highly recommended for the field application of influenza vaccines. This study is to construct a influenza marker vaccine by using plasmid-based reverse genetics and develop a DIVA system for the vaccination. We generated a low-pathogenicity/high growth H5N1 virus, H5N1/PR8-5B19, the HA and NA gene of which were derived from GS/GD/1/96, the first H5N1 avian influenza isolates in China, and the 6 internal genes were derived from a high growth virus PR8. The HA cleavage site was modified to resemble that of low pathogenic avian influenza virus strains and 20 amino acids between 49 and 67 in the NA stalk region was replaced by 16 amino acids of an immunodominant 5B19 epitope of S2 glycoprotein of murine hepatitis virus (MHV).The H5N1/PR8-5B19 virus was low pathogenic in chickens when administered iv. or in., showing high biosafety. The H5N1/PR8-5B19 virus was not lethal to embryonated chicken eggs and replicated to very high titer, achieving to 10.51og2 after 72 hours incubation. The 5B19 epitope was stably expressed in the H5N1/PR8-5B19 virus when the recombinant virus was passaged 14 times in embryonated SPF eggs, demonstrating the stability of the introduced 5B19 epitope.The H5N1/PR8-5819 inactivated vaccine is higly immunogenic and can induce high titers of HI antibodies in the immunized chickens. In the challenge test, no signs of diseases were observed in any of the H5N1/PR8-5B19 vaccine immunized chickens. In addition, no virus was recovered from the tracheal and cloacal swabs. These results showed that the H5N1/PR8-5B19 marker vaccine was efficient to provide complete protection from lethal infection with a highly pathogenic H5N1 influenza virus.We established the indirect ELISA procedure based on the synthetic 16-mer 5B19 peptide to detect the antibody response against the 5B19 epitope in the immunized chicken serum. 2 of 10 chickens were positive for 5B19 ELISA antibody detection in 3 weeks after immunization with H5N1/PR85B19 vaccine, and 7 of 10 chickens developed detectable antibodies on 6 and 12 weeks post immunization. The ELISA antibody response could be detected in all chickens after a second dose.