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.

The 1918 “Spanish Flu” pandemic was cause by an Influenza A virus that infected 500 million people and nearly 50 million people died. Influenza viruses utilize a RNA-Dependant RNA Polymerase (RdRp) complex, composed of the PA, PB1 and PB2 proteins along with the viral nucleoprotein (NP) to mediate viral transcription and replication. The 1918 PB1 gene has been linked to increased virulence in mice and ferrets. We have investigated the role of PB1 in the transcription and replication of the 1918 virus and its relation to pathogenicity by comparing its RdRp to the RdRp of a low virulence conventional H1N1 human virus isolate, A/Canada/RV733/2007 (RV733). Contrary to previous studies, our dual-luciferase reporter assay revealed the 1918 RdRp had lower transcriptional activity than the RV733 RdRp in vitro. The 1918 NP seems to be the key determinant for the difference in transcriptional activity of the 1918 and RV733 RdRp complexes. The 1918 PB1 in the RV733 RdRp maintained high reporter expression while the RV733 PB1 in the 1918 RdRp abolished reporter expression. 1918/RV733 chimeric PB1 proteins were also generated and evaluated with the reporter assay. Recombinant RV733/1918 viruses were generated by reverse genetics and we determined that PB1 is a key determinant of the high growth phenotype of the 1918 virus, but only a minor contributor to pathogenicity. A novel role for the 1918 NP in the high growth phenotype and pathogenicity of the 1918 virus is also described.

Kyasanur Forest Disease Virus (KFDV) is a tick-borne, hemorrhagic fever-causing member of the Flaviviridae. With infections annually ranging from 50 to 1000 people in south-west India and the lack of effective treatments, a better understanding of this virus is needed. The development of a Reverse Genetics System for KFDV would provide the opportunity to address these issues in future studies. Using molecular techniques, the KFDV genome sequence was elucidated and the reverse genetics system was created. Utilizing this system live, infectious KFDV particles were produced from mammalian cell culture, thereby validating the success of the reverse genetics system. The implementation of this system will enable researchers to better study pathogenesis and disease progression, virus-host interplay, virion structure, genome replication and the emergence of effective therapeutics and vaccines.

The 1918 “Spanish Flu” pandemic was cause by an Influenza A virus that infected 500 million people and nearly 50 million people died. Influenza viruses utilize a RNA-Dependant RNA Polymerase (RdRp) complex, composed of the PA, PB1 and PB2 proteins along with the viral nucleoprotein (NP) to mediate viral transcription and replication. The 1918 PB1 gene has been linked to increased virulence in mice and ferrets. We have investigated the role of PB1 in the transcription and replication of the 1918 virus and its relation to pathogenicity by comparing its RdRp to the RdRp of a low virulence conventional H1N1 human virus isolate, A/Canada/RV733/2007 (RV733). Contrary to previous studies, our dual-luciferase reporter assay revealed the 1918 RdRp had lower transcriptional activity than the RV733 RdRp in vitro. The 1918 NP seems to be the key determinant for the difference in transcriptional activity of the 1918 and RV733 RdRp complexes. The 1918 PB1 in the RV733 RdRp maintained high reporter expression while the RV733 PB1 in the 1918 RdRp abolished reporter expression. 1918/RV733 chimeric PB1 proteins were also generated and evaluated with the reporter assay. Recombinant RV733/1918 viruses were generated by reverse genetics and we determined that PB1 is a key determinant of the high growth phenotype of the 1918 virus, but only a minor contributor to pathogenicity. A novel role for the 1918 NP in the high growth phenotype and pathogenicity of the 1918 virus is also described.

Kyasanur Forest Disease Virus (KFDV) is a tick-borne, hemorrhagic fever-causing member of the Flaviviridae. With infections annually ranging from 50 to 1000 people in south-west India and the lack of effective treatments, a better understanding of this virus is needed. The development of a Reverse Genetics System for KFDV would provide the opportunity to address these issues in future studies. Using molecular techniques, the KFDV genome sequence was elucidated and the reverse genetics system was created. Utilizing this system live, infectious KFDV particles were produced from mammalian cell culture, thereby validating the success of the reverse genetics system. The implementation of this system will enable researchers to better study pathogenesis and disease progression, virus-host interplay, virion structure, genome replication and the emergence of effective therapeutics and vaccines.

African horse sickness is an economically important equid disease caused by African horse sickness virus (AHSV). Upon infection, the virus produces seven structural proteins that constitute the virus particle, and four non-structural proteins with various supportive roles during replication. This study focused on the pleiotropic non-structural protein NS3 and its isoform, NS3A. Arthropod-borne viruses are uniquely equipped to replicate in multiple species, often with significant infection cycle differences. Notably, while AHSV infection is highly pathogenic to the mammalian host, the insect vector exhibits no detrimental effect. One potential contributing factor to these observed differences is the viral release mechanism, with NS3/A playing an essential role in mediating these final infection cycle stages. Multiple conserved protein domains have been identified for NS3/A. This study aimed to address questions regarding the way in which these domains contribute to the protein’s function, in mammalian and insect cells, respectively. The initial phase of this study involved augmenting a panel of reverse genetics-derived AHSV NS3mutants. As these viruses each express a modified version of the NS3/A protein, an in-depth investigation into the function of the different conserved domains was possible. Consequently, a comparative analysis of the wild-type and mutant virus strains was conducted in both mammalian and insect cells, using biochemical, virological and microscopy techniques. In mammalian cells, the results indicated a variable contribution of the different NS3/A domains to the cytopathic effect and in ensuring effective virus trafficking and release. The transmembrane (TM) domains were identified as essential mediators of NS3/A intracellular distribution, as the abnormal processing of the TM mutant proteins resulted in their nuclear localisation and unique interaction with another viral protein, NS1. Additionally, the TM domain disruptions resulted in cytosolic virus particle accumulation. A similar mutant phenotype was observed when the C-terminus of NS3/A was deleted, with an apparent lack of efficient virus trafficking and release. Disruption of the NS3/A late domain (LD) resulted in abnormal tethering of virions to the plasma membrane, suggesting that AHSV utilises the cellular ESCRT (Endosomal Sorting Complex Required for Transport) pathway for non-lytic release events within mammalian cells. Comparatively, while some of the NS3/A domains appear to have similar functions within insect cells, key differences were also observed. Most notably, within insect cells, NS3/A localised to the periphery of unique large cytoplasmic vesicle-like structures, likely facilitating non-lytic release. Interestingly, disruption of the TM domains of NS3/A resulted in the degradation of these mutant proteins as they could not be detected within infected cells. Correspondingly, this apparent absence of NS3/A resulted in diminished release, with most of the virus particles remaining cell-associated throughout the course of infection. Notwithstanding normal NS3/A protein localisation, deletion of the C-terminus of NS3/A also prevented proper virus egress. Disruption of the LD did not have any significant effect within insect cells, with no abnormal virus particle localisation and efficient virus release observed. Consequently, in insect cells, virus particle trafficking and release appears independent of characteristic cellular membrane trafficking systems utilised in mammalian cells. / Thesis (PhD)--University of Pretoria, 2020. / National Research Foundation (NRF) / Poliomyelitis Research Foundation / University of Pretoria Institutional Research Theme / Genetics / PhD / Unrestricted

Coronaviruses have caused high pathogenic epidemics within the human population on two occasions; in 2003 a coronavirus (HCoV-SARS) caused severe acute respiratory syndrome and in 2012 a novel coronavirus emerged named Middle East respiratory syndrome (HCoV-MERS). Four other species of coronavirus circulate endemically in the human population (HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1), which cause more benign respiratory disease than either HCoV-SARS or HCoV-MERS. The emergence of HCoV-MERS provides an additional opportunity to study the characteristics of coronaviruses. Reverse genetics can be used to study an organism’s phenotype by logical mutation of its genotype. Construction of an infectious clone construct provides a means to investigate the nature of HCoV-MERS by reverse genetics. An HCoV-MERS infectious cDNA clone system was constructed to use for reverse genetics by homologous recombination of bacterial artificial chromosomes (BACs). This system should aid in answering remaining questions of coronavirus genetics and evolution as well as expedite the development of vaccines and prophylactic treatments for HCoV-MERS. / October 2015

La génétique inverse est la manipulation génétique des virus à ARN pour créer un virus de type sauvage ou modifié. Les techniques de génétique inverse conduit à bien comprendre les fonctions des gènes viraux et leur pathogénicité dans les cellules d’hôte ou de vecteur. Les virus à ARN comme les arbovirus ceux qui ont comme vecteurs les arthropodes représente une vraie menace pour la santé humaine mondiale. Les techniques de génétique inverse sont un moyen adéquat pour protéger l’homme contre les virus par la création des vaccins at par bloquer leur transmission par les vecteurs comme les moustiques. De nos jours, la plupart des systèmes de génétique inverse se concentraient exclusivement sur l’étude d’interaction entre les virus et les cellules de mammifères. Cependant, la transmission de l'arbovirus se situe entre un hôte mammifère et un vecteur d'invertébré.Nous présentons ici ISA (Infectious-Subgenomic-Amplicons) comme une méthode universelle de génétique inverse qui a prouvé son applicabilité sur les cellules de mammifères et de moustiques pour générer des virus infections ARN simple brin de polarité positive.Ainsi, ISA est une méthode adéquate pour étudier le cycle de vie de l'arbovirus chez le moustique vecteur et l'hôte mammifère at ainsi avoir une idée générale de la circulation de l’arbovirus pour créer d’autre outils de blocage. / Reverse genetics, the genetic manipulation of RNA viruses to create a wild-type or modified virus, has led to important advances in our understanding of viral gene function and interaction with host cells. Since arboviruses the most threatening viruses to human and animal are RNA viruses, thus reverse genetics is an extremely powerful technique with important application for the protection from these viruses and to control their spread.Hitherto, most reverse genetics systems focused exclusively on mammalian cells. However, arbovirus transmission is between a mammalian host and invertebrate vector.Herein, we present ISA (Infectious-Subgenomic-Amplicons) as a universal reverse genetic method that proved it applicability to rescue infectious single stranded positive RNA viruses on mammalian and mosquito cells.Thereby, ISA is an adequate method to study the arbovirus life cycle in mosquito vector and mammalian host. Thus, providing information about the global arbovirus circulation to provide further technique that protect mammalian from their infection and inhibit vector to transmit the virus.

The thermostability of vaccines is of crucial importance in Africa, where the logistical process to get the vaccine from the manufacturer to the animal may take months, and in many remote regions transport and storage is in the absence of a cold-chain. Vaccines with improved stability and less reliance on a cold-chain are needed and could improve the longevity of immune responses elicited in vaccinated animals. In South Africa, cattle in the vaccination zone neighbouring the Kruger National Park have to be vaccinated thrice annually because of declining antibody responses at three months postvaccination. FMDV is known to be unstable, especially for O and SAT2 serotypes in mildly acidic pH conditions or at elevated temperatures, leading to dissociation of the capsid (146S particle) and loss of immunogenicity. The link between rapidly declining antibody responses and capsid stability have been reported by Doel and Baccarini, 1981. We hypothesized that more stable viruses, especially thermostability, will not only improve the protective immune response in animals but also require less frequent booster vaccinations. / The residues at the capsid inter-pentamer interfaces, and their interactions, are important for the infectivity and stability of the virion and mutations adjacent to these interfaces have an effect on the conformational stability of FMDV. However, experimental studies on the relative importance of residues and molecular interactions in viral capsid assembly, disassembly, and/or stability are still very limited, especially for the SAT serotypes of FMDV. This study investigated the effects of potential residues at the pentameric interfaces that are responsible for increased thermostability and potentially improved stability candidates were tested in small (guinea pigs) and large (cattle) animal vaccination trials to understand the role of stabilised antigens on immune responses. The biological variation in biophysical stability in SAT2 viruses in the southern Africa region was investigated to determine if any naturally occurring viruses have greater capsid thermostability. Naturally occurring stable viruses could be used as prospective candidates in vaccine production and therefore potentially result in increased duration of immune responses. / Our first aim was to investigate the role of different amino acid changes at the interface and their effect on capsid stability using models derived by Oxford University. These changes were introduced by mutating the SAT2 ZIM7/83 infectious genome-length clone (pSAT2) to derive mutated chimeric SAT2 viruses. We quantified the stabilizing effects of these mutations by using various stability assays. We established the novel thermofluor shift assay that is able to quantify the capsid stability of viruses. The growth kinetics, antigenicity, genetic stability, pH and salt sensitivity were investigated for each of the genetically engineered viruses (Chapters 2 and 3). / The second aim was to further our understanding on the correlation between improved stability and immune responses by performing small animal (Chapter 2) and large animals trials in cattle (Chapter 4) and comparing stabilised and wild-type antigens. This study for the first time for SAT vaccines, determined differences in IgG1 and IgG2 profiles, interferon gamma (IFN-γ) responses and differences in total and neutralising antibodies of stabilised and wild-type antigens over a six month period in cattle (Chapter 4). Animals were intra-dermolingually challenged with live virus to determine levels of protection the antigens have afforded. / In addition, a third aim will be to better understand the inherent thermostability variation of SAT2 viruses in the Southern African region (Chapter 5) by establishing a protocol for screening field isolates as potential vaccine strains and correlating their stability to amino acid residues at the interface of the 146S particles. / Thesis (PhD)--University of Pretoria, 2016. / Agricultural Research Council / Veterinary Tropical Diseases / PhD / Unrestricted