Theses and Dissertations from UMD
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Item LIVE-ATTENUATED VACCINES AGAINST INFLUENZA VIRUSES AND ROLE OF NS1 IN HOST RESPONSE(2013) Cai, Yibin; Perez, Daniel R.; Zhu, Xiaoping; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The research interests in this dissertation thesis are to develop a better live attenuated influenza virus A (LAIV) vaccine which allows mass vaccination and further understand the phenotype changes of a quail-adapted influenza virus. The recurrent outbreaks of low pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI) highlight the pressing need for the development of mass in-ovo vaccination strategies using LAIV. However, this method often results in a poor hatchability (unpublished data). Our study shows that after vaccination with a LAIV carrying the modified H7 and H9 hemagglutinin (HA) proteins by substituting the amino acids at the cleavage site for those found in the H6 HA subtype, the hatchability of egg embryos was significantly improved (≥90%). Furthermore, a single dose in-ovo vaccination in 19-day-old chicken embryo provided complete protection against homologous challenge with low pathogenic virus in ≥ 70% of chickens at 2 or 6 weeks post-hatching. Our findings indicate that the strategy of modifying the HA cleavage site in a LAIV backbone could be used for improving in-ovo vaccination. A good vaccine candidate should grow to high titer in eggs or cell culture. In this study, through egg adaptation, a LAIV (2 mouse-adapted(ma) Ca/04 H1N1:6WF10att) mounted a higher HA titre in eggs compare to its parental virus. Sequence comparison of the egg-adapted virus with its parental virus revealed only one amino acid substitution in PA protein (E59V). Using reverse genetics, E59V was introduced in PA protein of the parental virus, which renders the virus similar growth characteristics as the egg-adapted virus. Importantly, this mutation did not change the temperature sensitive (TS) phenotype of the attenuated virus. Furthermore, the E59A in PA also moderately enhanced both HA and viral titers in wild type WF10 and Turkey/04 att backbones. In contrast, E59V had no significant effect on that of PR8 or ca Ann Arbor/60 backbone viruses. Take together; these data suggest that single change in PA, E59V, is critical for some LAIV vaccines efficient growth in eggs or cell culture. A QA23 virus was previously generated through 23 serial passages of the H9N2 (A/duck/Hong Kong/702/79) virus in quail lung. Phenotypically, the QA23 viruses replicate and transmit more efficiently in both chickens and quail. Interestingly, QA23 NS1 protein was mainly distributed in the cytoplasm, which is remarkably different from the parent strain NS1 that was mainly accumulated in the nucleus. Although mutations occurred in the nuclear localization signal sequence, QA23 NS1 did not display a significant change in antagonizing IFN-β function. In addition, QA23 NS1 up-regulated the viral protein synthesis in DF1 cells and greatly reduced the apoptosis of the cell during the infection. However, there is no evidence showing that QA23 NS1 is associated the enhanced replication and transmission of the viruses in Japanese quail.Item MOLECULAR PATHOGENESIS OF INFLUENZA IN SWINE AND ENGINEERING OF NOVEL RECOMBINANT INFLUENZA VIRUSES(2011) Pena, Lindomar Jose; Perez, Daniel R; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Influenza A viruses (IAVs) belong to the family Orthomyxoviridae and represent major pathogens of both humans and animals. Swine influenza virus is an important pathogen that affects not only the swine industry, but also represents a constant threat to the turkey industry and is of particular concern to public health. In North America, H3N2 triple reassortant (TR) IAVs first emerged in 1998 and have since become endemic in swine populations. In the first part of this dissertation, we focused on the role of surface glycoproteins and PB1-F2 to unravel their roles in the virulence of TR IAVs in this important natural host. We found that surface glycoproteins are necessary and sufficient for the lung pathology, whereas the internal genes play a major role in the febrile response induced by TR H3N2 IAVs in swine. With respect to PB1-F2, we found that PB1-F2 exerts pleiotropic effects in the swine host, which are expressed in a strain-dependent manner. Pathogenicity studies in swine revealed that the presence of PB1-F2 leads the following effects in context of three TR strains tested: no effect in the context of sw/99 strain; increases the virulence of pH1N1; and decreases the virulence of ty/04. Next, we developed temperature-sensitive live attenuated influenza vaccines for use in swine and shown that these vaccines are safe and efficacious against aggressive intratracheal challenge with pH1N1. Lastly, we rearranged the genome of an avian H9N2 influenza virus to generate replication competent influenza virus vectors that provide a robust system for expression and delivery of foreign genes. As a proof-of-principle, we expressed the hemagglutinin from a prototypical highly pathogenic avian influenza virus (HPAIV) H5N1 and shown that this vectored H5 vaccine retained its safety properties in avian and mammalian species, and induced excellent protection against aggressive HPAIV H5N1 challenges in both mice and ferrets. Taken together, these studies have advanced our understanding of molecular basis of pathogenesis of influenza in the swine host and have contributed to the development of improved vaccines and influenza-based vectors with potential applications in both human and veterinary medicine.