| The highly pathogenic avian influenza A virus(HPAIV) H5N1 can transmit from poultry to human occasionally and cause devastating effects to agricultural poultry flocks and humans. H5N1 virus was first isolated in the domestic goose in Hong Kong 1996. Hong Kong reported the first fatal case of human in August 1997. H5N1 virus tends to spread more and more widely worldwide in recent years. WHO has documented more than 300 confirmed human fatal cases with a mortality rate about 58%. The study about the determinants of H5N1 pathogenicity mainly focused on the PB2, PB1, PA, and NS1 gene. The function of receptor binding domain(RBD) of hemagglutinin(HA) on H5N1 viral pathogenicity still unclear.HA is the most abundant antigen protein on the surface of influenza virus. HA is one of the primary glycoprotein of H5N1 viral membrane. The main function of HA is recognizing and binding to the sialic acid(SA)- linked receptors on the surface of host cells and mediating the fusion between the viral membrane and host cell membrane. HA is necessary for influenza virus to achieve viral invasion of host cells. The present study analyze the H5N1 virus mutant sites in the RBD segment after passages in vitro and in vivo. We isolated and recognized the RBD substitutions, K193 E, G225 E, and E190 G. In order to identify the specific influence of the mutation on H5N1 influenza virus, we use clinical H5N1 isolates, A/Vietnam/1194/2004(VN1194) virus as model and viruses carrying these mutations in RBD were rescued by reverse genetics techniques(RG). 1. Isolation and recognizing of RBD mutations from VN1194, and the rescuingof RG viurses containing RBD mutations.The present study inducing mutations into influenza genome by changing environment and host of viruses. We set A/Vietnam/1194/2004 virus as model strain and serial passaged viruses in MDCK cells at low temperature(33 °C) and in murine lungs. The isolates of these passaged viruses were sequenced and several mutations in RBD were recognized together with substitutions in PB2, PB1, and NA genes. Among them, the reports about RBD mutations K193 E, G225 E and E190 G were sporadic, and the functions of these sites were still unclear. According to the prediction of HA structure by mutant amino acid sequence, all of 193, 225, and 190 sites located on the key elements of RBD, 190 helix and 220 loop.In order to clear the influence of RBD substitutions on viral receptor binding, replication in vitro and in vivo, we obtained the HAs segment carrying all combinations of RBD mutations by PC R. RG viruses containing these mutatio ns were rescued by reverse genetic techniques and based on the ge nome backbone of VN1194 virus. RG viruses, r VN1194, r VN-K193 E, r VN-G225 E, r VN-K193E/G225 E, and r VN-E190 G were rescued successfully. The infective virions of containing E190 G together with other mutations were not obtained which meant the E190 G was disable to coexist with other mutations. So the r VN-E190 G was set into another group to compare with VN1194 virus. RG virus were serial passaged in MDCK cells and sequencing, all of the substitutions are genetic stable without recovery of RBD mutations. 2. Influence of RBD mutations K193 E, G225 E, and E190 G on virus receptor binding specificity.To investigate the effects of RBD mutation on H5N1 viral binding specificity, RG viruses were concentrated and purified by PEG-6000. Then the HA titers of purified viruses were tested by hemagglutination assay. The receptor binding avidity of RG viruses were detected by solid phase binding assay. The result of binding assay indicated the low temperature adapted BRD mutation K193 E have little influence on receptor binding to both type SA. The mutation G225 E of RBD significantly enhanced the viral binding avidity to α-2,3 SA. The double mutations K193E/G225 E caused little change on receptor binding avidity. The mouse lung-adapted RBD mutation E190 G slightly reduce the viral binding avidity to α-2,3 SA and enhanced the binding avidity to α-2,6 SA. However, the preference of all kinds of RG viruses were not shifted from α-2,3 SA to α-2,6 SA. 3. Influence of RBD mutations K193 E, G225 E, and E190 G on virus replication in vitro.In order to investigate the influence of RBD mutations on viral replication in vitro, different cell lines were used to test the viral growth property at different temperatures. RG viruses infected MDCK and A549 cells, respectively, and replicated under different temperatures(33°C, 37°C, and 39°C). The viral titers of supernatant were tested by plaque forming assay and the growth curves of viruses were sketched. The result of growth kinetics indicate that the low temperature adapted RBD mutation K193 E and G225 E affect little on viral replication at different temperatures. However, the K193E/G225 E mutation significantly improve the replication efficiency of VN1194 virus. The mouse lung-adapted RBD mutation relatively reduced viral growth in both cell lines, but the decrease was less than r VN-K193E/G225 E. 4. Influence of RBD mutations K193 E, G225 E, and E190 G on virulence in mice.The current study used BALB/c mouse as mammalian model to test virulence of RG viruses. The mice were intranasally infected with r VN1194, r VN-K193 E, r VN-G225 E, r VN-K193E/G225 E and r VN-E190 G viruses. The clinical symptoms, MLD50, weight change and viral load of lungs were assayed. The result of animal experiment showed that r VN-K193 and r VN-G225 E were slightly attenuated in mice. The r VN-K193E/G225 E virus was dramatically attenuated in mice and the MLD50 of it was higher than 1000 PFU considering the MLD50 of r VN1194 was only 2.65 PFU. The r VN-E190 G virus was slightly attenuated in mice.To value the protection of attenuated RG virus to wild-type H5N1 virus, the r VN-K193E/G225 E virus were inactivated by formalin and combined with Freund’s adjuvant, then subcutaneous injected BALB/c mice with combination. The mice were immunized with r VN-K193E/G225 E virus at first and third week. After immunization, the serum of above mice were collected and the microneutralization assay was practiced. The result of neutralization assay showed that the r VN-K193E/G225 E virus can induce mice to produce abundant effective antibodies to neutralizing the wild-type VN1194 virus. The mutation K193E/G225 E did not mortify the specific type of neutralizing antibodies. Furthermore, the murine lymphocytes cell line, RAW 264.7 was used to test the cytokine levels change a fter infection of RG viruses. The levels of IL-6, IFNγ, TNFα, and IP10 were tested by ELISA. The result indicated that the RG viruses can induce RAW264.7 cells produce same level of TNFα and IP10 as that of wild-type virus.The present study first reported the relationship between the RBD mutations(193, 225, and 190) and the pathogenicity of H5N1 virus. This study reveal the influence of RBD mutations K193 E, G225 E, and E190 G on viral receptor binding specificity, viral replication in vitro, and virulence in mice. The current study further our understanding of RBD’s function and the determinants of H5N1 virus.The properties of RG virus r VN-K193E/G225 E indicated that double mutant virus was significantly attenuated in mice while the in vitro replication efficiency of it was maintained. This phenotype is very valuable and it might be a useful strategy for new type of live attenuated vaccines development. This mutation could help vaccine strains replicate more efficiency in vitro to improve the viral production for industrial. On the other hand, r VN-K193E/G225 E virus can be used as a safer strain for experimental works in laboratory to investigate the sections except the RBD. |
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