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Doctor of Philosophy / Department of Biology / A. Lorena Passarelli / Baculoviruses are enveloped, rod shaped viruses with circular, double-stranded DNA genomes. These viruses infect arthropods, primarily in the order Lepidoptera, although members of this virus family also infect species of Diptera, Hymenoptera, and Crustacea. The majority of these viruses undergo a bi-phasic cycle with one phase defined by the production of a budded virus (BV) form, responsible for cell to cell transmission, and the other defined by the production of an occlusion-derived virus (ODV) form, responsible for host to host transmission. The proto-typical member of the Baculoviridae family is considered to be Autographa californicaM Nucleopolyhedrovirus (AcMNPV). Its 133,894 base pair genome is predicted to encode for 156 proteins, a large number of which are essential for virus replication.. In this current work, we have further characterized two viral proteins that are highly conserved among baculoviruses. The first of these is an ortholog of the fibroblast growth factor family of proteins with sequence homology to the Drosophila Branchless protein as well as the mammalian FGF- 9, -16 and -20 subfamily. Despite its high degree of conservation among baculoviruses, the viral fibroblast growth factor (vFGF) is considered a non-essential protein, although its deletion from the genome does affect the lethality of the virus when ingested per os. In our study, we were able to localize vFGF to the membrane of BV. Its presence on the envelope affected the ability of the virus particle to bind to both heparin in vitro and to the cell surface in vivo, and may play a role in the attachment phase prior to virus entry. We also characterized AcMNPV’s open reading frame 109 (Ac-orf109). Unlike vFGF, Ac-orf109 is essential for virus replication since its deletion results in a complete lack of BV production. Transmission electron microscopy of cells transfected with an Ac-orf109 deletion virus shows the full range of virus-associated structures including mature capsid formation but there appears to be a deficiency in capsid egress out of the nucleus. Furthermore, the ODV retained in the nucleus appear to lack microvesicular membranes, an essential component for host to host transmission of infection.

O objetivo do trabalho foi estudar a eficiência, a estabilidade e a persistência de três formulações de Baculovirus anticarsia preparadas no centro piloto de formulações de defensivos agrícolas do Instituto Biológico de São Paulo, no controle da lagarta da soja Anticarsia gemmatalis, Hubner, 1818. As formulações do tipo pó molhável (PM) foram preparadas mediante impregnações diretas (pulverização) das suspensões de poliedros de vírus em inertes de origem mineral (Leucita e Talco), enquanto a preparação do patógeno em óleo emulsionável foi obtida a partir da mistura da suspensão viral com óleo de soja. Os resultados evidenciaram a superioridade da formulação PM - Leucita, quando comparada a suspensão purificada do vírus. Quanto a estabilidade dessa formulação, não foi observada queda significativa de atividade durante os 6 primeiros meses de armazenamento enquanto que, no mesmo período, o patógeno na forma de suspensões purificadas mostrou redução de 25% na eficiência de forma similar; foi também estudada, em condições de laboratório, a estabilidade das formulações de B. anticarsia (PM) - Talco e óleo emulsionavel (OE). Para a formulação PM, não foi observada queda significativa de atividade ao longo de 12 meses. No caso do material formulado em óleo vegetal, a perda de viabilidade alcançou mais de 50%. A proteção contra a radiação solar, conferida ao vírus pelas formulações PM - Leucita e OE-óleo de soja, foi avaliada após 1, 2, 7 e 14 dias de exposição das plantas de soja à radiação solar. Leucita manteve a atividade do patógeno por um período superior ao observado para as demais preparações / The purpose of the work was to study the efficiency, the stability and the persistence of three formulations of Baculovirus anticarsia, produced by Pilot Center for Formulations of Biological Institute in the Control of valvetbean caterpillar, Anticarsia gemmatalis Hübner, 1818. The results showed the superiority of the Leucite formulation, where compared to purified suspensions of the virus. Regarding the stability during the first 6 months of storing, whereas in the same period, the pathogen as purified suspension presented reduction of 25% in efficiency. In a similar way, it was also studied, in laboratory conditions, the stability of B. anticarsis (Wp) - Talcum and B. anticarsia (EO) - soybean oil. The pathogen, formulated as wettable power (Talcum), did not present significant decrease of activity throughout the 12 months, while the soybean oil lost more than 50% of viability. The protection against solar radiation, confered by formulations (Wp)-Leucite and (EO) - Soybean oil, was evaluated after the 1st, 2nd and 14th days of soybean plants exposition to solar radiation. The pathogen, with Leucite, maintained its activity in higher levels than the ones observed for the other preparations

Production of an effective vaccine and long-term treatment against human immunodeficiency virus (HIV) is elusive. In this thesis two different techniques were used in an attempt to insert HIV-neutralising monoclonal antibody (IgG1b12) sequences into a simian retroviral gene therapy agent pseudo-typed with vesicular stomatitis virus glycoprotein. Genes were encoded in either a poxvirus split-vector system or a baculovirus expression system. Both systems aim to produce replication incompetent pseudotyped virus like particles with simian origin. It is believed that the resulting non-infectious artificial lentivirus particles enter neighbouring cells, penetrate the nucleus and insert genetic material (the antibody gene) into the mammalian genome. The poxvirus split-vector system used in this project was a Vaccinia Retroviral Hybrid Vector, where recombinant modified vaccinia Ankara (MVA) is used to deliver the simian immunodeficiency virus (SIV) like particles into mammalian cells. However, the MVA system failed to express proteins of interest due to the instability of genetic insertion into the recombinant MVA genome. As an alternative strategy, two different BacMam systems were used to allow the production of VLPs, where mammalian cells are co-transduced with different recombinant baculoviruses (rBVs). VLPs were expressed either under the control of T7 RNA polymerase system or under the cytomegalovirus immediate early gene promoter. The results from the first BacMam system indicated that the T7 RNA polymerase system was not suitable to express detectable levels of proteins. The results indicated that translation of the produced mRNA by T7 promoter is inefficient, most likely because of the absence of RNA 5’ cap structure. To overcome this hybrid BV–T7 system limitation, a different system was developed. Proteins of interest from the second BacMam system were successfully expressed and detected using western blot analysis. VLPs were generated and visualised under electronic microscope. IgG1b12 was secreted in the supernatant of the transduced mammalian cells. Mammalian cells were successfully transduced with multiple different recombinant BVs simultaneously. The study establishes the feasibility of antibody gene transfer, and demonstrates the use of SIV like particles production to transduce mammalian cells using BacMam technology. The technique may have application for use as an immunotherapy of HIV infection as well providing long-acting prevention of HIV infection for those not yet infected with HIV.

Professional antigen presenting cells (pAPCs) process and present antigens on their cell surface in association with MHC class I molecules through two general pathways: direct or cross-presentation. The process of antigen presentation by pAPCs to naïve T cells resulting in their proliferation and differentiation into activated cytotoxic lymphocytes (CTLs) is called T cell priming. In these studies, we examine the cross-presentation of antigens from two non-replicating viruses: inactivated Lymphocytic Choriomeningitis virus (LCMV), and recombinant baculovirus encoding the LCMV nucleoprotein (NP). Since effective activation of pAPCs is essential for efficient priming of CD8+ T cells and CTL activation, and because infection with inactivated viruses generally induces an extremely poor level of CTL activation, we examined the activation state of pAPCs by measuring their cytokine profiles following infection to help further delineate their involvement in the CTL response to inactivated viruses. Our results indicate a pro-inflammatory cytokine mRNA upregulation in pAPCs in response to the inactivated virus, similar to the cytokine profiles subsequent to live LCMV infection, but to a lesser extent. In these studies, we also examined CTL activation following infection with inactivated LCMV and bAc-NP. We have demonstrated that the presentation of antigens from inactivated LCMV and bAC-NP results in a low level of CTL activation in vivo, though there is an undetectable level of CTL activation in vitro, in comparison to activation following infection with the live virus. Ultimately, the characterization of the cytokine profiles of pAPCs and the CD8+ T cell profiles induced in response to inactivated LCMV or the baculovirus derived NP may lead to a better understanding of how cross-presentation of these viral antigens may occur. This information may be applied to enhance the process of pAPC activation and T cell priming, for the induction of more effective cellular immune responses and the generation of stronger protective immunity. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-09-02 15:30:13.883

Baculoviruses are lytic insect viruses. Upon internalisation, the viral genome orchestrates a sequential expression process ultimately leading to lysis of the infected cell. Release of progeny capable of infecting other cells during the process completes the infection cycle. Studies of the infection cycle in cell culture are typically conducted by synchronous infection, i.e. near simultaneous infection of all cells, by means of high virus concentrations. The behaviour of the synchronously infected culture, such as the timing of onset of progeny release, is considered representative for the infection progression within individual cells. In reality, however, the synchronously infected culture only reflects the average behaviour of all infected cells. The infection progresses in individual cells display large variability; this is most obvious in the observation that within the same culture some cells undergo cell lysis at two days post infection while others remain viable up to four days post infection. Such variabilities or asynchronies observed in synchronously infected culture is the topic of this thesis. Using a simple phenomenological model, it is demonstrated that cell death and associated intracellular product release is adequately described assuming that the waiting time from infection to cell death follows a Gaussian distribution with a mean of 59 hours post infection (hpi) and a standard deviation of 15hpi. Unlike other deterministic model developed over the last decade (Licari and Bailey 1992; Nielsen 2000), this stochastic model does not make the biologically inconsistent assumption that cells continue to be metabolically active following loss of membrane integrity. While elegant in its simplicity, the model provides no explanation for the underlying stochasticity. Investigations into the cause of this dispersion of cell death highlighted further asynchronies in the specific recombinant protein yield, in viral DNA content, in virus budding rate, and in cell volume increase instead of clarifying the issue. A modelling framework developed by Licari & Bailey (1992) and later Hu & Bentley (2000) incorporates the number of infectious particles each individual cell receives as a possible source of the dispersions in the host cell responses. However, this was found NOT to be the cause of the observed asynchronies under non-substrate limiting conditions. The timing of cell death, cell volume increase, recombinant product yield, viral DNA content, and virus budding rate is identical in Sf9 cell cultures infected at multiplicities of infection of ~5, ~15, and ~45 infectious particles per cell. Cell cycle variation has previously been suggested as a possible cause for observed asynchronies in baculovirus infections (Brown and Faulkner, 1975). The cell cycle phase is indirectly linked to the cell volume, because a G2-phase cell prior to division is inherently twice the cell volume of a G1-phase cell after cell division. By the same logic, it is also apparent that a G2-phase cell possesses twice the number of ribosomes of a G1-phase cell and thus a doubled protein production capacity. The effect of the cell cycle or cell volume on the baculovirus infection was determined by splitting an exponentially growing Sf9 cell culture into 5 cell size dependent fractions by centrifugal elutriation. The subsequent infection of these fractions showed (1) no dependency of the timing of cell lysis and cell volume increase and (2) approximately twofold increase of a) recombinant protein yield, b) viral DNA concentration, and c) budded virus yield. The recombinant protein yield showed a strong proportionality to the initial cell volume and the total RNA concentration during the late phase of the infection. As argued in chapter 6, these proportionalities suggest that the observed differences in the responses of the cell fractions to the baculovirus infection are more likely caused by the difference in the protein production capacity than by cell cycle specific molecules. This investigation gave also reason to speculate that infected cells cannot progress beyond the G2/M phase, and cell cycle progression continues undisturbed until ~8hpi when all cellular DNA replication appears to cease. Resuspended, infected Sf9 cells synchronised by centrifugal elutriation showed an identical cell cycle distribution as the non-infected control cultures for the first ~8hpi; G1 and G2/M phase cell proportions remained unchanged, whereas S phase cells progress to G2/M phase. Subsequently, the non-infected control cells resumed normal cycling whereas all infected cells remained at the same cell cycle phase from 8 to 11hpi. The initial cell cycle arrests in G2/M phase in both infected and non-infected cultures were caused through medium exchange.

Master of Science / Division of Biology / Ana Lorena Passarelli / Baculoviruses are a large, diverse, and an ecologically-important group of entomopathogens. The ac78 gene of the prototype baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is one of the 38 genes conserved among all baculoviruses sequenced to date. Previous studies show that Ac78 is essential for optimal production of occlusion-derived virions (ODVs) and budded virions (BVs), which are two virion types produced during baculovirus infection. However, the biochemical mechanism by which Ac78 is involved in these processes remains unknown. The AcMNPV sulfhydryl oxidase ac92 is a conserved gene, and its product, Ac92, is ODV and BV envelope-associated. Recently, the Ac78 and Ac92 homologs in Helicoverpa armigera nucleopolyhedrovirus (HearNPV) were reported to interact and co-localize to the site of BV and ODV formation. To investigate the relationship between Ac78 and Ac92, we determined their localization in the presence and absence of AcMNPV infection, performed co-immunoprecipitations to assess interaction relationships, and provided an updated report of Ac78 and Ac92 homology with other proteins. We concluded that in the absence of viral infection, Ac78 and Ac92 localized perinuclearly in the cytoplasm and that localization of Ac92 was not affected by Ac78. During AcMNPV infection, Ac78 and Ac92 co-localized within the nucleus and surrounding virus replication and assembly sites (ring zone). Co-immunoprecipitation experiments showed that at least two differentially-tagged Ac78 proteins were part of a complex in the presence of other AcMNPV proteins. Ac78 did not associate with Ac92 during AcMNPV infection. Our characterization of the relationship between Ac78 and the AcMNPV sulfhydryl oxidase is a preliminary step in a broader effort to elucidate important biochemical pathways underlying the poorly described structural changes in capsid proteins and other proteins involved in virion stability, folding, and infectivity. In a separate project, the same approach was applied in a different virus system to determine the relationship between the small accessory protein C and the measles virus (MeV) replication complex. Co-immunoprecipitation experiments showed that during MeV infection, C associated with large protein (L) and phosphoprotein (P), which comprise the MeV replication complex, and nucleoprotein (N), which encapsidates the RNA genome. Expression constructs for full-length MeV L were generated, and L was successfully expressed following transfection. Subsequent co-immunoprecipitation experiments showed that C did not precipitate with L, P, nor N when transfected in isolation from MeV infection, indicating that another factor resulting from MeV infection is necessary for the association of C with the MeV replication complex. The results of this investigation are an important step in elucidating a biochemical mechanism underlying the function of C as a quality control factor in MeV replication. MeV has been attenuated and is a highly effective vaccine against pathogenic MeV and an active subject of clinical research as an oncolytic agent for treating a number of human cancers. Taken together, the investigations of Ac78 and C and their respective relationships with the AcMNPV sulfhydryl oxidase and the MeV replication complex adds knowledge of biochemical mechanisms underlying the important functions of small accessory proteins containing less than 200 amino acids as mediators in viral replication processes of two different viral systems.

O objetivo do trabalho foi estudar a eficiência, a estabilidade e a persistência de três formulações de Baculovirus anticarsia preparadas no centro piloto de formulações de defensivos agrícolas do Instituto Biológico de São Paulo, no controle da lagarta da soja Anticarsia gemmatalis, Hubner, 1818. As formulações do tipo pó molhável (PM) foram preparadas mediante impregnações diretas (pulverização) das suspensões de poliedros de vírus em inertes de origem mineral (Leucita e Talco), enquanto a preparação do patógeno em óleo emulsionável foi obtida a partir da mistura da suspensão viral com óleo de soja. Os resultados evidenciaram a superioridade da formulação PM - Leucita, quando comparada a suspensão purificada do vírus. Quanto a estabilidade dessa formulação, não foi observada queda significativa de atividade durante os 6 primeiros meses de armazenamento enquanto que, no mesmo período, o patógeno na forma de suspensões purificadas mostrou redução de 25% na eficiência de forma similar; foi também estudada, em condições de laboratório, a estabilidade das formulações de B. anticarsia (PM) - Talco e óleo emulsionavel (OE). Para a formulação PM, não foi observada queda significativa de atividade ao longo de 12 meses. No caso do material formulado em óleo vegetal, a perda de viabilidade alcançou mais de 50%. A proteção contra a radiação solar, conferida ao vírus pelas formulações PM - Leucita e OE-óleo de soja, foi avaliada após 1, 2, 7 e 14 dias de exposição das plantas de soja à radiação solar. Leucita manteve a atividade do patógeno por um período superior ao observado para as demais preparações / The purpose of the work was to study the efficiency, the stability and the persistence of three formulations of Baculovirus anticarsia, produced by Pilot Center for Formulations of Biological Institute in the Control of valvetbean caterpillar, Anticarsia gemmatalis Hübner, 1818. The results showed the superiority of the Leucite formulation, where compared to purified suspensions of the virus. Regarding the stability during the first 6 months of storing, whereas in the same period, the pathogen as purified suspension presented reduction of 25% in efficiency. In a similar way, it was also studied, in laboratory conditions, the stability of B. anticarsis (Wp) - Talcum and B. anticarsia (EO) - soybean oil. The pathogen, formulated as wettable power (Talcum), did not present significant decrease of activity throughout the 12 months, while the soybean oil lost more than 50% of viability. The protection against solar radiation, confered by formulations (Wp)-Leucite and (EO) - Soybean oil, was evaluated after the 1st, 2nd and 14th days of soybean plants exposition to solar radiation. The pathogen, with Leucite, maintained its activity in higher levels than the ones observed for the other preparations

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