Swine influenza (SI) is an acute, highly contagious, respiratory disease of swine. The causative agent of SI infections is swine influenza virus (SIV). SIV is a type A influenza virus classified into the Orthomyxoviridae family and is an enveloped particle with a genome composed of eight negative-orientated RNA segments.<p> The mortality rate of influenza disease in pigs is generally low but morbidity can reach up to 100%. SI infections considerably contribute to respiratory disease in post-weaning pigs, causing significant economic losses due to an increase in the number of days pigs need to reach market weight. In addition, SI infections possess significant human public health concerns. Vaccination is the primary method for the prevention of SI. Currently available vaccines against SI are a combination of two inactivated antigenically distinct SIVs with oil adjuvant. The application of these vaccines induce mainly humoral immune responses. In contrast, application of live attenuated influenza vaccines (LAIV) mimics natural infection and induce strong, long-lived cell-mediated and humoral immunity. Furthermore, LAIV induces cross-protective immunity against different subtypes of influenza A viruses. LAIVs are developed for human and equine influenza viruses but at present no LAIV is available for SIVs.<p> The critical step in influenza virus infection is an initial interaction between virus and cell surface carbohydrates followed by receptor-mediated endocytosis and fusion of the viral and endosomal membranes. Influenza virus entry into cells is mediated by the viral surface glycoprotein hemagglutinin (HA). HA is primary synthesized as a polypeptide in HA0 form. In order to be infectious, HA0 must be cleaved by host proteases into HA1 and HA2 subunits. Therefore, this process is crucial determinant for virus pathogenicity.<p> Our objective was to generate a live attenuated SIVs, particularly a viruses with a modified HA cleavage site resistant to activation during natural infection but which can be activated in vitro by an exogenous protease. Using the reverse genetics technique, we generated two mutant SIVs of strain A/SW/SK/18789/02 (H1N1) containing a modified cleavage site within their HA. Mutant A/SW/SK-R345V (R345V) contained a mutation within HA segment at amino acid (AA) position 345 from Arginine (Arg) to Valine (Val) while the second mutant, A/SW/SK-R345A (R345A) encoded Alanine (Ala) instead of Arginine (Arg) at position AA345 on HA. We showed that HA cleavage in both mutants was strictly dependent on the presence of human neutrophil elastase in tissue culture. These tissue-culture grown mutant SIVs showed similar growth properties in terms of plaque size and growth kinetics, compared to the wild type virus. Both mutant SIVs were able to preserve introduced mutations after multiple passages in tissue culture suggesting that AA substitution within HA cleavage site did not alter genetic stability in the presence of appropriate protease. Furthermore, these mutant SIVs were highly attenuated in pigs but capable of inducing significant cell-mediated and humoral immune responses after two vaccinations via intratracheal (IT) and intranasal (IN) routes. Immune responses induced by vaccination with elastase dependent SIV were sufficient to confer full protection against parental homologous and antigenic variant of H1N1 SIVs and partial protection from heterologous subtypic H3N2 after the challenge. Therefore, elastase-dependent mutant SIV could serve as live vaccine against antigenically distinct swine influenza viruses in pigs.

Swine influenza (SI) is an acute, highly contagious, respiratory disease of swine. The causative agent of SI infections is swine influenza virus (SIV). SIV is a type A influenza virus classified into the Orthomyxoviridae family and is an enveloped particle with a genome composed of eight negative-orientated RNA segments.<p> The mortality rate of influenza disease in pigs is generally low but morbidity can reach up to 100%. SI infections considerably contribute to respiratory disease in post-weaning pigs, causing significant economic losses due to an increase in the number of days pigs need to reach market weight. In addition, SI infections possess significant human public health concerns. Vaccination is the primary method for the prevention of SI. Currently available vaccines against SI are a combination of two inactivated antigenically distinct SIVs with oil adjuvant. The application of these vaccines induce mainly humoral immune responses. In contrast, application of live attenuated influenza vaccines (LAIV) mimics natural infection and induce strong, long-lived cell-mediated and humoral immunity. Furthermore, LAIV induces cross-protective immunity against different subtypes of influenza A viruses. LAIVs are developed for human and equine influenza viruses but at present no LAIV is available for SIVs.<p> The critical step in influenza virus infection is an initial interaction between virus and cell surface carbohydrates followed by receptor-mediated endocytosis and fusion of the viral and endosomal membranes. Influenza virus entry into cells is mediated by the viral surface glycoprotein hemagglutinin (HA). HA is primary synthesized as a polypeptide in HA0 form. In order to be infectious, HA0 must be cleaved by host proteases into HA1 and HA2 subunits. Therefore, this process is crucial determinant for virus pathogenicity.<p> Our objective was to generate a live attenuated SIVs, particularly a viruses with a modified HA cleavage site resistant to activation during natural infection but which can be activated in vitro by an exogenous protease. Using the reverse genetics technique, we generated two mutant SIVs of strain A/SW/SK/18789/02 (H1N1) containing a modified cleavage site within their HA. Mutant A/SW/SK-R345V (R345V) contained a mutation within HA segment at amino acid (AA) position 345 from Arginine (Arg) to Valine (Val) while the second mutant, A/SW/SK-R345A (R345A) encoded Alanine (Ala) instead of Arginine (Arg) at position AA345 on HA. We showed that HA cleavage in both mutants was strictly dependent on the presence of human neutrophil elastase in tissue culture. These tissue-culture grown mutant SIVs showed similar growth properties in terms of plaque size and growth kinetics, compared to the wild type virus. Both mutant SIVs were able to preserve introduced mutations after multiple passages in tissue culture suggesting that AA substitution within HA cleavage site did not alter genetic stability in the presence of appropriate protease. Furthermore, these mutant SIVs were highly attenuated in pigs but capable of inducing significant cell-mediated and humoral immune responses after two vaccinations via intratracheal (IT) and intranasal (IN) routes. Immune responses induced by vaccination with elastase dependent SIV were sufficient to confer full protection against parental homologous and antigenic variant of H1N1 SIVs and partial protection from heterologous subtypic H3N2 after the challenge. Therefore, elastase-dependent mutant SIV could serve as live vaccine against antigenically distinct swine influenza viruses in pigs.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Background Swine influenza virus (SIV) was first isolated in the United States in 1930 and was thereafter widely reported in many countries. Most SIVs that have been identified are influenza A viruses. There was no report of influenza B viruses isolated in swine. Seroepidemiological study in UK has shown a low seroprevalence of influenza B antibody in pigs. The primary serological test used to detect influenza antibody is the hemagglutination inhibition (HI)test. Enzyme-linked immunosorbent assay (ELISA) are also available commercially for detection of antibodies against influenza A viruses but not for the detection of influenza B antibodies. Objectives 1) To examine the prevalence of influenza B antibodies in pig sera sampled at the abattoir in Hong Kong. 2) To develop the cell-based ELISA assay for the detection of antibodies against influenza A and B viruses. 3) To compare the cell-based ELISA assays with three commercial ELISA kits, namely the IDVet ID Screen influenza A antibody competition ELISA, the IDEXX Influenza A Ab test and the IDEXX AI MultiS-Screen Ab test using swine sera. 4) To test swine sera using the influenza B cell-based ELISA assay to complement data on swine seroprevalence obtained with HI tests. Methods The first part of this study involved HI screening of 4643 pig sera from 2009 to 2012. These sera were tested for the presence of antibodies against B/Brisbane/60/2008 and B/Wisconsin/1/2010whichrepresent the B/Victoria and B/Yamagata lineages respectively. The second part of this study involved the development and performance evaluation of the cell-based ELISA assays. The cell-based ELISA assays were developed using influenza virus infected cells as the capture antigens and fluorescence-labelled anti-IgG antibody as the detection antibody. The viruses that were used to prepare the assays were A/California/04/2009, B/Brisbane/60/2008 and B/Wisconsin/1/2010. All three cell-based ELISA assays were tested with WHO reference sera and swine sera and the results were analyzed using paired t-test and receiver operating characteristic analysis. In addition, the results of the influenza A cell-based ELISA assay were compared with the commercial ELISA assay using Fisher’s exact two-tailed test, Pearson’s correlation analysis and Bland-Altman plot. Results A low prevalence (0.28%; 95%CI: 0.16%-0.47%) of influenza B antibody was observed inthe swine sera samples. The seroprevalence for B/Victoria was higher than that of B/Yamagatain 2010to2012. Co-existence of B/Victoria and B/Yamagata antibodies were found in the swine population during 2010 and 2011. The influenza A cell-based ELISA was found to have low sensitivity (64.1%;95%CI: 52.4%-74.4%) and high specificity (94.7%; 95%CI:80.9%-99.1%) when compared with the commercial ELISA assays. In contrast, using HI as the reference test influenza B cell-based ELISA prepared using B/Wisconsin/1/2010 infected cells were shown to have high sensitivity (92.31%; 95%CI:64.0%-99.8%) but low specificity (63.16%;95%CI:38.4%-83.7%) in detection of influenza B antibodies in swine sera. Conclusion Sporadic transmission of influenza B virus may occur in swine but there is no evidence for efficient and sustained transmission of the virus between them. Cell-based ELISA assay prepared with B/Wisconsin/1/2010 may be considered as an alternative screening testprior to HI subtyping. / published_or_final_version / Public Health / Master / Master of Public Health

Thesis (Ph. D.)--University of Wisconsin--Madison, 1967. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

Thesis (Ph. D.)--University of Hong Kong, 1998. / Includes bibliographical references (leaves 187-199).

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

Thesis (Ph. D.)--University of Wisconsin--Madison, 1961. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 54-57).

Thesis (M.S.)--University of Wisconsin--Madison, 1970. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.