A proper vaccination program can play a critical role in prevention and control of avian influenza (AI) in commercial poultry. Low pathogenic avian influenza viruses (LPAIV) of H5 and H7 AI subtypes cause serious economic losses to the poultry industry and have the potential to mutate to highly pathogenic AI (HPAI) strains. Due to trade implications, differentiation of infected from vaccinated animals (DIVA) is an important issue in the control of AI. Therefore, the development and characterization of vaccine candidates with DIVA properties is critical in improving vaccination programs. Keeping these aspects in mind, we investigated the role of an NS1 mutant virus as a potential live attenuated DIVA vaccine. The NS1 protein of influenza virus plays a major role in blocking the host's antiviral response. Using an eight-plasmid reverse genetics system, we recovered the low pathogenic parental (H5N3) and NS1 mutant (H5N3/NS1/144) viruses. H5N3/NS1/144 expresses only the first 144 amino acids of the NS1 protein compared to the 230 of the parental H5N3. The growth properties of H5N3 and H5N3/NS1/144 were compared in cell culture and in different age embryonated chicken eggs. Our results confirmed that NS1 is involved in down regulation of interferon as shown by IFN-beta mRNA expression analysis and by the inability of H5N3/NS1-144 to efficiently grow in older age, interferon competent, chicken embryos. However with regards to safety the virus reverted to virulence within five back passages in chickens and was therefore not a safe vaccine candidate. However the killed form of H5N3/NS1-144 was a safer alternative and it also induced antibody titers and protection not significantly different from the parental H5N3 as vaccine. To further understand the reversion of H5N3/NS1/144 to virulence, we carried out 3 independent serial passages of H5N3/NS1/144 in increasing age of embryonated chicken eggs and examined the NS1 gene for presence of mutations. RT-PCR and sequence analysis of the NS gene in all three lineages showed the presence of a 54 amino acid deletion resulting in the generation of a 87 amino acids long NS1 ORF with a point mutation (L80V) at the site of deletion. In addition, the NS1 ORF in lineages L2 and L3 presented two additional point mutations in the RNA binding domain (Q40R and T73M). To determine if these mutations played a role in increased virulence, recombinant viruses expressing these mutant NS1 proteins in the background of parental virus were generated by reverse genetics and their replication properties and pathogenicity was examined in vitro, in ovo and in vivo systems. Our results showed that the 87 amino acid long NS1 protein clearly increased virus replication and virulence specifically in interferon competent systems. In addition, the two point mutations in the RNA binding domain of NS1 ORF expressing 87 a protein slightly increased the virus virulence. Overall this study reinforces the role of NS1 in influenza virus pathogenicity and supports the use of killed inactivated NS1 mutant virus vaccines as potential DIVA vaccines.

Thesis (Ph.D.)--York University, 2008. Graduate Programme in Mathematics and Statistics. / Typescript. Includes bibliographical references (leaves 166-175). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR46017

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Influenza vaccination is the best preventive measure against influenza virus infection, and antivirals including oseltamivir are effective treatments. From a public health point of view, it is important to evaluate whether vaccination and antiviral treatment reduces transmission of the virus. I analyzed data from a community-based study of influenza virus transmission in households, and identified effectiveness of antiviral treatment in reducing duration of illness and some evidence that treatment reduced transmission to household contacts. I also analyzed data from a community-based placebo-controlled trial of influenza vaccination and confirmed efficacy of vaccination against seasonal influenza but differential efficacy against pandemic influenza possibly because of timing and mediation of seasonal influenza epidemics. In further analyses I found that antibody titers of 1:40 correlated with 50% protection against infection, and repeated vaccination with the same strains tended to be associated with reduced responses to those strains although there was no evidence of reduced efficacy. In the study, one child in each household was randomly allocated to receive vaccine or placebo and I did not identify any evidence of indirect benefits to the household members of vaccinated children. I reviewed vaccine target groups in different countries, and noted that some countries now include school-age children in their target groups based mainly on the principle of herd immunity. My findings did not support the inclusion of school-age children as a target group for vaccination in Hong Kong. Further studies should examine the indirect as well as direct benefits of vaccination in different settings in order to guide optimal influenza vaccination policies. / published_or_final_version / Community Medicine / Doctoral / Doctor of Philosophy

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Iowa, 2007. / Thesis supervisor: Gregory C. Gray. Includes bibliographical references (leaves 94-105).

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 131-151) Also available in print.

Thesis (M. P. H.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 89-94) Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 1994. / Includes bibliographical references (leaves 254-266).

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 131-151) Also available online.