Influenza A Virus Subtype Of Each Fake Anti-virus System Building And H1 Subtype Specificity Of Conservative Epitope Identification

Influenza A viruses (IAVs) cause seasonal influenza epidemics annually and recurring pandemics with severe consequences for public health. Recently, the increasing reports of H5N1subtype avian influenza A virus infections in human and the outbreak of H1N1pandemic influenza further underlie the importance of reinforcing the study and control of IAVs. Hemagglutinin (HA) and neuraminidase (NA) are two surface glycoproteins of IAVs. Based on the antigenic properties of HA and NA, IAVs have been classified into16HA subtypes and9NA subtypes.Theoretically144subtype IAVs can be formed by the free combinations of16HA subtypes and9NA subtypes. In fact, most influenza subtypes can be found in nature. Due to the high mutation and reassortation rate of influenza viruses, any influenza strain may acquire increased pathogenicity and cause pandemic irrespective of its subtype. It is very hard to know which influenza strain can cause pandemic. For the possible influenza pandemic preparedness, this study aims to establish a pseudotyping assay system combined with different HA and NA to evaluate neutralizing antibodies or inhibitors for any potential flu strain and address HA/NA coordination in influenza entry and egress. On the other hand, IAV subtype-specific diagnosis techni based on epitope provide the basis for the development of novel influenza virus surveillance technology.Eukaryotic expression plasmids separately encoding16HA subtypes and9NA subtypes were constructed. Expression of HA was verified by western blot using subtype-specific antibodies. To increase the infectivity of pseudovirus particles (PPs), the cleavage sites of14HA subtypes except H5and H7were changed from monobasic amino acids to multibasic amino acids. The expression and cleavage were verified by western blot using corresponding subtype-specific antibodies. Expression of NA was confirmed by western blot using subtype-specific antibodies and by an NA activity assay using the NA-Star Influenza Neuraminidase Inhibitor Resistance Detection Kit.144pseudovirus particles combined with16HA and9NA were packaged and their infectivities on293T cells were determined by luciferase assay. According to the HA cleavability and the infectivity of pseudovirus particles, the results can be divided into four categories:H5, H6, H7and H9HA are cleavable and PPs combined with either NA have high infectivity; H10-H16and H1HA are cleavable and PPs combined with partial NA have high infectivity; H2and H8HA are not cleavable but PPs combined with partial NA have infectivity; H3and H4HA are not cleavable and PPs combined with either NA have little infectivity. All the above results suggest that the great difference on the cleavability and infectivity of different subtype HAs and the existence of HA/NA interactions.Next, the produced PPs were characterized. The morphology of PPs was observed by the electronic microscope. HA incorporations were detected by Western blot using the corresponding subtype-specific antibodies. The activities of incorporated HAs were confirmed by a hemagglutination assay. H1, H3, H5and H7PPs can be inhibited (90%inhibition) by the corresponding antisera in a pseudotype neutralization test, suggesting that the constructed PPs can be used to evaluate neutralizing antibodies.Several studies have identified conserved epitopes within specific HA subtypes that can be used for diagnostics. However, immune specific epitopes in H1N1IAV have not been completely assessed. In this study, linear epitopes on the H1N1pdm viral HA protein were identified by peptide scanning using libraries of overlapping peptides against convalescent sera from HlNlpdm patients. One epitope, P5(aa58-72) was found to be immunodominant in patients and to evoke high titer antibodies in mice. Multiple sequence alignments and in silico coverage analysis showed that this epitope is highly conserved in influenza H1HA [with a coverage of91.6%(9860/10767)] and almost completely absent in other subtypes [with a coverage of3.3%(792/23895)]. This is a previously unidentified linear epitope, which is located outside the five well-recognized antigenic sites in HA. A peptide ELISA method based on this epitope was developed and showed high correlation (X2=58.01, P<0.01) with hemagglutination inhibition test. The highly conserved H1subtype-specific immunodominant epitope may form the basis for developing novel assays for sero-diagnosis and active surveillance against H1N1IAVs.Taken together, this study establish influenza pseudotyping systems encompassing all the16HA and9NA subtypes of IAVs for the first time, suggesting the existence of HA and NA interactiond at the pseudotype packaging and entry level. A H1-subtype specific and highly conserved linear epitope was also first identified using a pepscan approach. An epitope-ELISA method for the H1antibody detection was established. All the results provide basis for the effective survelliance of influenza epidemic.