Mutation Of H5Avian Influenza Virus HA Gene Affects Its Trypsin-dependence For Cell Fusion Activity And Generation And Evaluation Of Newcastle Disease Virus-vectored Nipah Encephalitis Vaccines

Part I.Mutation of H5avian influenza virus HA gene affects its trypsin-dependenence for cell fusion activityH5N1highly pathogenic avian influenza virus (HPAIV) to infect humans directly with a high fatality rate makes it a great threat to the world public health. Evolution and variation of HPAIV genome in avian species has resulted in emerging of H5N1HPAIV variants with diverse HA and change its host range,pathogenic and the ability of transmission HPAIV have been grouped into10differen clades based on their antigenic properties.The present studies of two H5N1HPAIV isolates performed extensively characterizations, results indicated that the A/BHG/Qinghai/3/2005(clade2.3)(QH) and A/CK/Shanxi/2/2006(clade7)(SX) virus respectively, are both highly pathogenic to chickens but exhibit a great difference in pathogenicity to mice. The QH virus is highly pathogenic to mice whereas the SX virus only causes mild infection. The entire HA genes of this two isolates was sequenced. Compared them with recent collect isolates H5HPAIV HA genes which have different pathogenicity,replicate ability and host range.we found a highly conserved amino acid site that a proline residue (P90), this residue in this segment of the QH HA is highly conserved among H5N1influenza viruses but it is mutated into a serine residue in the SX HA. Mutation of this conserved residue P90in QH HA into a serine residue QHP90S eliminated its fusion activity in human cells. Interestingly, cell fusion activity can be effectively restored by treatment with trypsin. Characterization of protein showed that the mutant QH-P90S HA is erpressed and transported to the cell surface similarly as the wild type QAHA. Further, with the presence of the polybasic sequence at the cleavage site, both mutant QHP90S and wild type QHHA proteins are cleaved into HA1and HA2subunits when expressed in cells in the absence of trypsin treatment. These results show that the conserved proline residue (P90) in the QHHA is critical for its trypsin independent cell fusion activity in human cells in addition to the presence of a polybasic cleavage site. Mutation of the SXHAS90P is not effect the fusion activity.After capture the90amino acid, we try to focuse on the polybasic sequence at the cleavage site, we firstly mutated both of the QH (GERRRKKR) and SX (REGGRRKR) to RETR, and construct the sequence in pCAGGS plasmide, then transfect the three kind of cells, the results show that both of the cleavage site mutant plasmids cannot cleavage into HA1and HA2, and also no fusion activity in absence of trypsin treatment. The same mutations were done to the90site mutant HA of QH and SX HA and transfect the same cells, results show that QH-HA90PS with cleavage site mutation cannot induce cell fusion in all cells without trypsin treatment, but SX-HAc90SP with cleavage site mutation plasmid remained fusion activity without trypsin treatment.To investigate the pathogenity and replication of two virus and mutant virus, we use a8-plasmid reverse genetics system, we are successfully to rescue6mutant virus QH-HA90PS,QH90PA,QH88NQ and SX-HA-90SP,SX-HAc,SX-HAc90SP which basis on the A/BGH/Qinghai/3/2005(QH-HA) and A/CK/Shanxi/2/2006(SX-HA) background. The ability to infect mammalians of these viruses was evalutated in a mouse model. The results show that QH point mutation reassortant virus were letha in mice, only intranasally with102EID50virus mices can live. SX-HA point mutation reassortant virus cannot cause death in mice model. Use QH,SX point mutation reassortant virus infect avian cell-DF-1,mammalian cell-MDCK and A549, the results show that, in the QH and SX group QH-HA, QH90PA. QH88NQ and SX-HA,SX-HA-90SP,SXc90SP reassortant virus can replicate and passage in absence of trypsin treatment, but QH-HA90PS and SX-HAc reassortant virus can replicate and passage only in the presence of trypsin treatment. This demonstrate that the cleavage site and proline90amino acid residue in the HA gene is a capital factor in virus replicate and pssage in avian and mammalian cells.In summary, the mutation of the highly conserved HA position90amino acid significantly affects its trypsin dependence of fusion activity in avian and mammalian cells. This discovery may provide a new clue to explain the infection and pathogenic mechanism of H5HPAIV.Part II.Gneration and evaluation of Newcastle disease virus-vectored Nipah encephalitis vaccinesNipah encephalitis is a deadly emerging infectious zoonosis which can cause a mortality rate of more than40%in human infection. Fruit bat is the natural host of NiV and widely distributed in Southeast Asia, South Asia and southern China.In some areas in China fruit bat serological results show that our country also faces the reality of NiV threat. NiV is very sensitive to pig, after infection symptoms despite lighter and the mortality rate is low, but large discharge virus through the natural holes, pigs areimportant intermediary amplifiying the host of NiV. Considering the huge pig-producing quantity in our country, once the pig bite by the poisonous fruit bat it, may be change into the potential source of infection to human. Therefore, the development of safe and effective NiV vaccine for pig is very important.Nipah virus (NiV), a member of the Paramyxoviridae family, causes deadly encephalitis in humans and huge economic losses to the pig industry. Most human cases result from close contact with infected pigs. Here, we generated recombinant avirulent Newcastle disease virus (NDV) LaSota strains expressing the NiV G and F proteins respectively (designated as rLa-NiVG and rLa-NiVF), and evaluated their immunogenicity in mice and pigs. Both rLa-NiVG and rLa-NiVF displayed growth properties similar to those of LaSota virus in chicken eggs. Co-infection of rLa-NiVG and rLa-NiVF caused marked syncytia formation, while intracerebral co-inoculation of these viruses in mice showed them to be highly safe in mammals. Animal immunization studies showed rLa-NiVG and rLa-NiVF induced profound Nipah neutralizing antibody responses and F protein-specific CD8+T cell responses in mice. Most importantly, rLa-NiVG and rLa-NiVF administrated alone or together, induced long-lasting neutralizing antibodies in pigs. Recombinant rLa-NiVG/F thus appear to be promising Nipah vaccine candidates for pigs and potentially humans.