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published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Because diagnostic tests for highly pathogenic avian influenza (HPAI) viruses require the use of replication-competent viruses in a biosafety level 3 containment, numerous studies have looked at ways to develop alternative tests. Lentiviral particles pseudotyped with H5 hemagglutinin (HA), the surface glycoprotein of influenza virus, have been described as useful and safe tools for research and serological surveillance on the HPAI viruses. However, not all H5 HA give rise to efficient H5 pseudotyped lentiviral particles (H5pp) production. HA from A/Cambodia/408008/05 H5N1 (H5Cam) and HA from A/Anhui/1/05 H5N1 (H5Anh) exhibit a dramatic difference in their ability to pseudotype lentiviral particles. H5Cam gives the highest H5pp production among all HAs tested, whereas the lowest has been observed with H5Anh. The objective of this study was to investigate the molecular determinants that govern efficient H5pp production. Based on the amino acid differences between H5Cam and H5Anh, H5Anh mutants were generated by site-directed mutagenesis. Strikingly, a single amino acid change, A134V, in the 130-loop receptor-binding domain of HA, significantly increased H5pp production with H5Anh. The finding that valine 134 is crucial for H5pp production was confirmed by reciprocal H5Cam and H5Anh mutants, which displayed either a dramatic decrease or increase in H5pp production, respectively. Influenza virus and H5pp bud at the plasma membrane, therefore changes in HA cell surface expression could affect the production of H5pp. Thus, cell surface expressions of H5Cam and H5Anh were compared by flow cytometry. Intriguingly, H5Cam displayed a higher plasma membrane expression than H5Anh, suggesting that transport is important for H5pp production. Introduction of V134A mutation in H5Cam reduced its surface expression to that of H5Anh; by contrast, H5Anh mutant harboring A134V mutation largely restored its expression. Next the effect of A134V mutation on the binding of HA to sialic acid receptors was investigated. A cell-based Enzyme-linked Immunosorbent Assay was developed to measure binding of wild-type and mutated HA. Soluble recombinant proteins were produced by mammalian cells stably transfected with HA gene ectodomain and were mostly trimeric as indicated by discontinuous native gel electrophoresis. Interestingly, H5Anh proteins exhibited a stronger binding to MDCK cells than H5Cam proteins, and introduction of A134V mutation in H5Anh proteins reduced the binding. By contrast, as predicted, the reciprocal V134A mutation induced a major increase in binding to cellular receptors. It is likely that stronger binding of H5Anh to sialic acids could hinder the release of H5pp. Consistent with this notion, the ability of H5Anh to generate H5pp was significantly increased in a sialylation deficient Lec2 cell, a CHO mutant cell line. In conclusion, H5Cam allows efficient H5pp production whereas H5Anh does not. With several lines of evidence, it is likely that the behavior of H5Anh can be explained by a stronger binding to sialic acid receptors that is dependent on a single amino acid residue at position 134. Since A134V is a naturally occurring mutation observed occasionally in human host, these results may have implications for the understanding of human host adaptations of H5N1 viruses. / published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Our systematic influenza surveillance in southern China revealed that two lineages of H9N2 influenza viruses, represented by Chicken/Beijing/1/94 and Quail/Hong Kong/G1/97, became endemic in the poultry in southern China since 1990’s. These established H9N2 lineages continually evolved to generate many different reassortants (or genotypes) and caused sporadic human infection cases. As co-circulating with H5N1 influenza viruses, the increasing genetic diversity and the capability to cause sporadic human infection make the H9N2 viruses become one of the major candidates with pandemic potential. Even though highly pathogenic H5N1 influenza viruses were seldom detected at the live-poultry markets of Hong Kong since 2002, H9N2 viruses were still commonly isolated in our surveillance program. The accumulated H9N2 isolates provided an opportunity to get insights into the continual evolution of this subtype virus in the region. In present study, we have systematically analyzed the H9N2 influenza viruses isolated from 2005 to 2010. Antigenic and phylogenetic analyses of 60 representative H9N2 viruses showed that the Ck/Bei-like H9N2 virus lineage continued endemic in the terrestrial poultry during the survey period in southern China. Genotyping analyses revealed four prevalent genotypes or reassortant variants in the field. Fifty-three of the viruses analyzed belonged to genotype B14 and B15, which were also the major reassortant variants prevailing in southern China from 2000 to 2005. The remaining seven viruses belonged to novel genotypes that have not been identified before. Our findings suggested that the Ck/Bei-like lineage continually maintained high genetic diversity in this region. The epidemiological findings showed that the isolation rate of H9N2 virus at the marketing poultry in Hong Kong was dramatically dropped down since 2009, which was different from what have observed in other provinces in southern China, but was closely correlated with the hygiene measures implemented in live-poultry markets in Hong Kong, e.g. not keeping live chicken overnight. These findings suggest the proper market policy would directly impact the prevalence of influenza virus in the field. / published_or_final_version / Microbiology / Master / Master of Philosophy

During the 1997 Hong Kong ‘bird flu’ incident, three subtypes of influenza viruses, including H5N1, H9N2 and H6N1, were co-circulated at the live-poultry markets. Genetic analyses revealed that all these viruses shared the same internal gene complex and might have been all involved in generation of the HK/97-like H5N1 virus. Subsequent epidemiological and genetic studies found that both H6N1 and H9N2 viruses became established and prevalent in minor poultry in the region. However, the genesis pathway for each of these viruses has not been defined. It is also unclear about these three subtypes further interact with each other and evolve in the field, along with the emerging reassortant variants. To address these questions, H6 subtype of avian influenza viruses isolated from terrestrial minor poultry from 2000 to 2005, and from 2006 to 2007 in our influenza surveillance in southern china has been genetically and antigenically analyzed in this study. Genetic and phylogenetic analyses of representative strains indicated that all H6N1 isolates from 2000 to 2007 had W312-like hemagglutinin and neuraminidase genes. These H6N1 viruses have become established in the minor poultry, mainly in quail and chukar, in this region. However, phylogenetic analyses revealed that the internal genes of the H6N1 virus lineage were derived from multiple origins with different evolutionary pathways. Evolution analyses of different gene segments of H6N1 viruses revealed imbalance dynamic evolutionary rates between surface genes and internal genes, which suggests that this virus lineage was more likely a descendant of the HK/97-like H5N1, rather than its precursor virus. Similar to what have been observed in the H5N1 and H9N2 virus lineages, the internal gene complex of the H6N1 viruses was found to undergo extensive reassortment. Many novel internal gene segments of H6N1 viruses were first recognized in the reassortant H9N2 virus particles, suggesting that the gene flow is likely from H9N2 to H6N1. The co-circulation of different virus lineages in southern China has greatly increased the genetic diversity of influenza viruses in this region. Analyses of the dynamics of different H6N1 reassortant variants also showed that some of them became persistent, but others were transient in the field. The increasingly diversified H6N1 and other subtypes of viruses will naturally increase the opportunity of interspecies transmission and dissemination, and may pose renewal threat for public health. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

The outbreaks of human influenza caused by highly pathogenic avian influenza H5N1 virus have attracted a lot of attention and public concern. Effective and universal vaccines may be the best means for prevention and control of the influenza. Taking into account that viral clearance and recovery from influenza A virus infection have been demonstrated to be correlated to specific cytoxic T lymphocyte (CTL) instead of neutralizing antibodies, it is important to develop effective vaccines which are capable of inducing not only neutralizing antibody but also CTL responses. Furthermore, T cell epitopes are usually more conserved than neutralizing epitopes. However, rare information concerning human T cell epitopes specific to H5N1 virus has been reported so far. This study was designed to test our hypothesis that novel and potent human CTL and Th epitopes specific to NP protein of H5N1 virus may be identified in vaccinated and/or infected HLA-A2/DR1 transgenic mice (SURE/L1), while protective epitopes may be further defined from the identified T cell epitopes in the mice challenged with lethal dose of the virus. We used SURE/L1 mouse model because it contains HLA-A2 (*0201) and -DR1 (*0701), both are the second highest frequency of HLA class I and II in Chinese. Since the NP gene is relatively conserved among different clades or strains of H5N1 virus, we selected viral protein NP as the target. Furthermore, we screened the T cell epitopes in splenocytes not only from vaccinated mice but also from survived mice infected with gradually increased dose of H5N1 virus, because the T cell epitopes identified in both vaccinated and infected mice or in infected mice alone might have higher potential to be protective epitopes. In this study, a novel HLA-DR1 (class II) restricted T cell epitope NP368-382, NPII-7, was identified in both vaccinated and infected mice. Two doses of NPII-7 peptide boosting in the mice induced very strong Th1 and CTL responses but no NP specific antibody responses. The vaccination of additional 2 doses of NPII-7 also provided partial protection against lethal challenge of H5N1 virus in the mice, whereas NP DNA vaccination alone did not show any protective effect. The protective effect may be attributed to the strong Th1 and CTL responses induced by the NPII-7 vaccination, because both NP DNA and NPII-7 vaccinations could not induce neutralizing antibody response. Notably, a HLA class II restricted peptide, NPII-7, may induce not only Th1 responses but also more strong HLA class I restricted T cell (CTL) responses. It may probably due to that the HLA-DR1 restricted T cell epitope (NENMEAMDSNTLELR) contained the full sequence of a reported HLA-A2 restricted CTL epitope (AMDSNTLEL), named NP-17 in this study. Although it needs to be further defined whether this novel epitope is really a HLA-DR1 restricted T cell epitope, or it shares the activity of HLA-A2 restricted T cell epitope, or it is just an alternate HLA-A2 restricted T cell epitope, this study has identified a novel T cell epitope and proved that it is a protective T cell epitope. / published_or_final_version / Microbiology / Master / Master of Philosophy

Avian H5N1 viruses have perpetuated in poultry and caused sporadic human transmission since 1997. Vaccine candidates for the potential pandemic caused by H5N1 viruses have been continuously updated by World Health Organization. Multiple genetic lineages of H5N1 viruses which co-circulate and rapidly evolve in different regions, together with periodic population replacement of newly emerged genetic and antigenic variants in the field, pose great challenge for H5N1 vaccine candidate selection. The complexity of avian H5N1 viruses evolution raises an important issue for studying antigenic properties and also for projecting antigenic trend of this virus since the model established for the seasonal influenza viruses may not apply to H5N1 viruses which they are still in the animal phase. In contrast, the 2009 pandemic H1N1 viruses have established as another seasonal influenza viruses in humans. How will this swine originated viruses evolve genetically and antigenically in humans? For the first in human history, we are able to track the changes of pandemic viruses from the very beginning when they transmitted to human. This study focuses on antigenic and receptor binding properties of avian H5N1 viruses from 1997 to 2010 and 2009 pandemic H1N1 viruses from 2009 to 2011. It is found that avian H5N1 viruses continue to display highly diverse antigenic profile. The newly emerged H5N1 virus variants of clade 2.3.4 in 2008 and clade 2.3.2 in 2010 exhibit distinct antigenic properties as compared to the genetically similar viruses that were characterized previously. Receptor binding analysis showed H5N1 viruses still exhibit binding preference for avian type receptor. However, analysis of escape mutants selected from H5N1 viruses exposed to H5 monoclonal antibodies in cell based assay indicates that mutations in the conserved sites may cause switch of receptor binding specificity to human type or dual specificity for both human and avian. Based on antigenic and receptor binding analyses, it is found that the 2009 pandemic H1N1 viruses isolated from 2009 to 2011 are relatively stable. Most of the antigenic variants to monoclonal antibodies are transient and not able to become prevalent. It remains to be investigated if more significant antigenic variants may emerge in the coming seasons when population immunity prevails this virus. In conclusion, this study showed that clade 2.3 avian H5N1 viruses become increasingly antigenic distinct as compared to clade 2.1 and 2.2 viruses. Antigenic variation in antigenic sites may change receptor binding specificity in avian H5N1 viruses. The 2009 pandemic H1N1 viruses remain stable up to date but continue monitoring in coming seasons is necessary. / published_or_final_version / Microbiology / Master / Master of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.