Doctor of Philosophy / Biology / Stefan Rothenburg / Poxviruses are large double-stranded DNA viruses that collectively exhibit a broad host range. Whereas many members of the poxvirus family are capable of infecting various host species, others are restricted to only one or a very limited numbers of species, such as variola virus, which is the causative agent of smallpox and is restricted to humans. Since the entry of poxviruses is not dependent upon any specific receptors, the cell tropism is therefore fully determined by the virus’ ability to manipulate the cellular signaling networks that are responsible for antagonizing viral infections. Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is a unique antiviral protein found in most vertebrates, which serves both as a virus sensor by detecting the presence of dsRNA and an antiviral effector by suppressing cap-dependent translation during virus infection. Many viruses, including poxviruses, have therefore evolved genes that encode for PKR inhibitors, such as vaccinia virus K3L, which shows sequence homology to the N-terminal region of the eukaryotic translation initiation factor 2α (eIF2α), the substrate of PKR. K3L is able to inhibit PKR-mediated eIF2α phosphorylation in vitro and in vivo. Because K3L was shown to be indispensable for virus replication in Syrian hamster cells but not in human cells, it was categorized as a host range factor. However, the molecular basis for K3L’s host range function is not fully understood. We examined the interactions of poxvirus K3L orthologs, especially vaccinia virus K3L and M156R, the K3L ortholog in the rabbit-specific myxoma virus, and PKR from a variety of host species in multiple assays, and found that K3L and M156R inhibit PKR in a species-specific manner, which likely contributes to the cell tropism and host range for both viruses. Inactivation of M156R or K3L led to virus attenuation in cells, which could be rescued by ectopic expression of viral PKR inhibitors. We also identified the helix αG region as the main molecular determinant for PKR’s sensitivity to inhibition by K3L orthologs. In conclusion, the research summarized here indicates that the interactions of PKR and poxvirus pseudosubstrate inhibitors play important roles in virus host range and virulence.

abstract: Cell death is a powerful tool through which organisms can inhibit the spread of viruses by preventing their replication. In this work, I used viral and chemical stressors to elucidate the mechanisms by which one anti-viral system might be activated over another, focusing on the programmable death pathway necroptosis and Protein Kinase R (PKR). PKR can detect viral dsRNA and trigger antiviral effects such as cessation of translation and induction of programmed death. Necroptosis is a rapid cellular death that can be induced via sensors such as DNA-dependent activator of IFN-regulatory factors (DAI), also known as Z-DNA-binding protein 1 (ZBP1). DAI contains a Z-form nucleic acid (ZNA) binding domain. E3, the primary vaccinia virus (VACV) interferon resistance protein, contains a similar domain in its amino terminus. We have previously reported this domain to be necessary for the inhibition of both PKR activation and DAI/ZBP1-mediated necroptosis. Monkeypox virus is a reemerging human pathogen. Despite a partial amino-terminal deletion in its E3 homolog, it does not activate PKR. In chapter 2, I show that MPXV produces less dsRNA than VACV, which could explain how the virus avoids activating PKR. The amino-terminus of vaccinia is associated with ZNA binding, inhibition of PKR, and inhibition of necroptosis. To determine the roles of PKR inhibition and ZNA binding in necroptosis inhibition, I characterized the VACV mutants Za(ADAR1)-E3, which binds ZNA but does not inhibit PKR, and E3:Y48A, which cannot bind ZNA. I found that while Za(ADAR1)-E3 fails to induce necroptosis, E3:Y48A does not activate PKR but does induce necroptosis. This suggests that Z-form nucleic acid binding is not necessary for vaccinia E3-mediated inhibition of PKR, nor is the inhibition of PKR sufficient for the inhibition of necroptosis. Finally, all known ZNA-binding proteins have immune functions and home to stress granules. I asked if stress granule formation alone could lead to necroptosis. I found that in L929 cells sodium arsenite, a known inducer of stress granules, could trigger DAI-dependent necroptosis. This suggests that DAI/ZBP1 is not necessarily a sensor of viral ligands but perhaps is a sensor of stress signals brought about by infection. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2018

Several wild-type poxviruses have emerged as potential oncolytic viruses (OVs), including orf virus (OrfV), and vaccinia virus (VV). Oncolytic VVs have been modified to include attenuating mutations that enhance their tumour selective nature, but these mutations also reduce overall viral fitness in cancer cells. Previous studies have shown that a VV (Western Reserve) with its E3L gene replaced with the E3L homologue from, OrfV (designated VV-E3LOrfV), maintained its ability to infect cells in vitro, but was attenuated compared to its parental VV in vivo. Our goal was to determine the safety and oncolytic potential VV-E3LOrfV, compared to wild type VV and other attenuated recombinants. VV-E3LOrfV, was unable to replicate to the same titers and was sensitive to IFN compared to its parental virus and other attenuated VVs in normal human fibroblast cells. The virus was also less pathogenic when administered in vivo. Viral replication, spread and cell killing, as measures of oncolytic potential in vitro, along with in vivo efficacy, were also observed.. The Parapoxvirus, OrfV has been shown to have a unique immune-stimulation profile, inducing a number of pro-inflammatory cytokines, as well as potently recruiting and activating a number of immune cells. Despite this unique profile, OrfV is limited in its ability to replicate and spread in human cancer cells. Various strategies were employed to enhance the oncolytic efficacy of wild-type OrfV. A transient transfection/infection screen was created to determine if any of the VV host-range genes (C7L, K1L, E3L or K3L) would augment OrfV oncolysis. Combination therapy, including the use of microtubule targeting agents, Viral Sensitizer (VSe) compounds and the addition of soluble VV B18R gene product were employed to see if they also enhance OrfV efficacy. Unfortunately, none of the strategies mentioned were able to enhance OrfV.

Doenças infecciosas emergentes se caracterizam como enfermidades cujos patógenos evoluíram para uma nova cepa ou genótipo capaz de infectar tanto o mesmo hospedeiro quanto uma nova espécie. A ocorrência de doenças emergentes tem sido correlacionada à destruição de habitats e à perda de biodiversidade. As aves marinhas têm sido consideradas bons indicadores e sentinelas do ecossistema aquático, e uma ampla variedade de enfermidades virais têm sido investigadas e descritas em aves no mundo todo. A caracterização e o entendimento das enfermidades virais que acometem as aves marinhas que habitam a costa brasileira são de fundamental importância para a compreensão de possíveis surtos de mortalidade dentre outros fatores que interferem na conservação das aves em geral. O objetivo desta pesquisa foi investigar a ocorrência de herpesvírus, avipoxvírus e coronavírus nas espécies de aves marinhas que foram reabilitadas em três centros localizados ao longo da costa brasileira e nas colônias reprodutivas de Sula sp. e Phaeton sp. localizadas no arquipélago de Abrolhos, Bahia, Brasil, e em quatro colônias de Spheniscus magellanicus localizadas na Patagônia argentina. As análises virais foram realizadas pela técnica de PCR e RT-PCR e confirmadas pela reação de sequenciamento do amplicon identificado. Nos casos de óbito, as alterações histopatológicas foram identificadas por meio de análise microscópica. O estudo revelou a ocorrência dos três agentes virais identificados em centros de reabilitação no Brasil, além da ocorrência de herpesvírus nas populações de vida livre e ativas em seus sítios reprodutivos. Foram identificados quatro novos herpesvírus denominados: Magellanic penguin herpesvirus 1 (MagHV -1) causador de um surto de mortalidade associado a traqueíte necrótico-hemorrágica em pinguins de Magalhães em reabilitação; Magellanic penguin herpesvirus 2 (MagHV-2), identificado em pinguins de Magalhães aparentemente saudáveis nas colônias reprodutivas da Patagônia argentina; Sulid herpesvirus 1 (SuHV -1), identificado em atobás (Sula sp.) e grazinas (Phaeton sp.) nas colônias reprodutivas de Abrolhos e Thalassarchid herpesvirus 1 (ThHV -1), identificados em um albatroz de nariz amarelo (Thalassarche chlororhynchos) nas praias de Rio Grande, RS, Brasil. Também foram identificados: um novo avipox, denominado Brazilian penguinpox, causador de lesões cutâneas, esofágicas e respiratórias nos pinguins de Magalhães cutâneas, esofágicas e respiratórias nos pinguins de Magalhães em reabilitação em Santa Catarina e a identificação de dois Gammacoronavirus em três diferentes espécies assintomáticas que estavam em reabilitação em Santa Catarina e no Rio Grande do Sul. Os dados obtidos constituem uma base de informação útil para estudos futuros no campo da patologia, virologia, epidemiologia e dos impactos antrópicos na saúde das aves marinhas do cone sul. / Emerging infectious diseases are characterized as disease whose pathogens progressed to a new strain ar genotype capable of infecting the same host or a new species. The occurrence of emerging diseases has been correlated to the destruction of habitats and loss of biodiversity. Seabirds have been considered as good indicators and sentinels of aquatic ecosystem, and a wide variety of viral diseases have been investigated and described in birds worldwide. The characterization and understanding of viral diseases that affect seabirds that inhabit the Brazilian coast are of fundamental importance for the understanding of possible mortality outbreaks among other factors that influence the conservation of birds in general. The objective of this research was to investigate the occurrence of herpesvirus, coronavirus and avipoxvirus in seabird species that were rehabilitated in three centres located along the Brazilian coast and in breeding colonies of Sula sp. and Phaeton sp. located at the Abrolhos’s archipelago, Bahia, Brazil and four Spheniscus magellanicus's colonies located at Argentina's Patagonia. Viral analyzes were performed by PCR and RT-PCR and confirmed by sequencing the identified amplicon. In cases of death, the histopathological alterations were identified through optical microscopic analysis. The study revealed the occurrence of the three investigated viral agents in the Brazilian rehabilitation centres, besides the occurrence of herpesvirus in the freeliving and reproductive active seabird’s populations. Four new herpesvirus were identified and called: Magellanic penguin herpesvirus 1 (MagHV -I), causing a mortality outbreak associated with necrotic-hemorrhagic tracheitis in Magellanic penguins in rehabilitation process; Magellanic penguin herpesvirus 2 (MagHV-2), identified in apparently healthy breeding Magellanic penguins at the Argentinean Patagonia’s colonies; Sulid herpesvirus 1 (SuHV-l), identified in boobies (Sula sp.) and tropicalseabirds (Phaeton sp.) breeding colonies at Abrolhos Archipelago, and Thalassarchid herpesvirus 1 (ThHV -1), identified in a yellow nose albatross (Thalassarche chlororhynchos) that appeared on the beaches of Rio Grande, RS, Brazil. Were. also identified: a new avipox named Brazilian penguinpox causing cutaneous, esophagus and respiratory lesions in rehabilitation penguins at Santa Catarina and the identification of two Santa Catarina and the identification of two Gammacoronavirus in three different asymptomatic seabirds species that were undergoing rehabilitation in Santa Catarina and Rio Grande do Sul. The data provi de a useful information basis for further studies related to pathology, virology, epidemiology and human impacts on southern hemispheres seabirds’ health.

Cetaceans are sentinels of the marine environment, currently threatened by many factors, mainly anthropogenic. The most easily identified compromising conditions are those affecting the skin and external mucosae - good indicators of the cetaceans health status. Cutaneous and mucocutaneous lesions have been extensively reported in wild and captive cetaceans, but little is known about the involved etiological factors, evolution of the dermatological lesions and their systemic consequences. Viruses are the most commonly involved agents in cutaneous and mucocutaneous lesions, especially herpesviruses (HV) and, associated with skin and mucosal lesions with varying morphologies, and cetacean poxviruses (CePV), mainly associated with characteristic tattoo or ring skin lesions. In addition, fungal agents are also recognized as causative agents of dermatological disease in cetaceans, especially in the process known as paracoccidioidomycosis ceti, observed as raised proliferative whitish lesions, caused by non cultivable yeast of Paracoccidioides brasiliensis (order Onygenales). Despite being reported worldwide, the occurrence of these etiological agents in southern Atlantic cetaceans is still poorly understood. The goal of this study was to identify and characterize selected cutaneous and mucocutaneous pathogens (HV, CePV and P. brasiliensis) of free-ranging cetaceans from Brazil, and to design more sensitive diagnostic methods for their detection. All the studied cetaceans stranded along the coast of Brazil, between 2005 and 2015, except three wild riverine dolphins that were physically contained and released after sample collection. In order to achieve our goals, we employed molecular, histopathological, and occasionally immunohistochemical and electron microscopy techniques. The presence of HV and CePV was evaluated in cutaneous, and oral and genital mucosal samples from 115 specimens and skin samples from 113 individuals, respectively; whereas the presence of members of the genus Onygenales sp. was evaluated in four specimens presenting macroscopic compatible lesions. Skin or oral mucosal samples from four animals were HV PCR-positive: a whitish ulcerated skin lesion from a Guiana dolphin (Sotalia guianensis), a lingual sample from an Atlantic spotted dolphin (Stenella frontalis), ulcerative lesions and healthy skin samples from a dwarf sperm whale (Kogia sima), and a proliferative skin lesion from a Bolivian river dolphin (Inia boliviensis). The tree first animals were infected with alphaherpesvirus. A sequence more similar to gammaherpesvirus was obtained from the Bolivian river dolphins proliferative skin lesion. The Bolivian river dolphin sequence could possibly be a member of a new gammaherpesvirus genus. Additionally, all other available tissue samples from HV-positive specimens, aside from skin and oral mucosa, were also tested by PCR and histologically evaluated. A different alphaherpesvirus sequence was found in the stomach and in a mesenteric lymph node of the dwarf sperm whale. Microscopic findings in two HV-positive animals (chronic proliferative dermatitis in Bolivian river dolphin and Guiana dolphin) were compatible with HV. CePV was identified in tattoo skin lesions of an Atlantic bottlenose dolphin and a Guiana dolphin by established molecular methods, and poxviral particles were observed by electron microscopy. CePV-positive animals presented epidermal ballooning degeneration and occasionally small, pale eosinophilic or amphophilic intracytoplasmic inclusions, compatible with CePV. Specific amino acid motifs for all CePV were also identified, reinforcing the suggestion of the new Cetaceanpoxvirus genus. We also designed novel SYBR® Green real-time and conventional CePV PCR methods significantly more sensitive than those currently available in the literature. An additional Guiana dolphin, previously negative based in established PCR methods was diagnosed positive for CePV through these new techniques. Refractile yeasts (49 µm in diameter) were observed under light microscopy in mild granulomatous and necrotic skin lesions of four Atlantic bottlenose dolphin, and for the first time, in a skeletal muscle abscess (the former possibly indicating the invasive potential of the agent). Onygenales sp. yeasts were identified in skin lesions by immunohistochemistry and a sequence of P. brasiliensis, more similar (100% nucleotide identity) to the one described in an Atlantic bottlenose dolphin from Cuba than to human or any other terrestrial mammals cases in Brazil, was obtained from the skin lesion of one of the specimens, confirming the etiological agent of these type of lesions. Herein we report the first molecular identification of HV in South American cetaceans and in riverine dolphins worldwide. This study also describes the first amplification of CePV and P. brasiliensis in odontocetes from South America. Four of the five novel herpesvirus sequences herein identified are possibly novel species, tentatively named Delphinid HV-10, Kogiid HV-2, Kogiid HV-3 and Iniid HV-1. / Cetáceos são sentinelas do ambiente marinho, atualmente ameaçados por diversos fatores, principalmente antropogênicos. Os processos de pele e mucosas externas são os mais facilmente identificados, bons indicadores do estado de saúde em cetáceos. Lesões cutâneas e mucocutâneas já foram amplamente relatadas em cetáceos de vida livre e de cativeiro, mas pouco se sabe a respeito dos fatores etiológicos envolvidos, evolução das lesões dermatológicas e suas consequências sistêmicas. Vírus são os agentes mais comumente envolvidos em lesões cutâneas e mucocutâneas, especialmente os herpesvírus (HV), associados a lesões de morfologias variáveis em mucosas, e poxvírus dos cetáceos (Cetacean Poxvirus CePV), principalmente associados a lesões de pele tatuagem ou anel características. Agentes fúngicos também podem causar doenças dermatológicas em cetáceos, como por exemplo a paracoccidioidomicose ceti, caracterizada por lesões esbranquiçadas elevadas e proliferativas, causada por leveduras não-cultiváveis de Paracoccidioides brasiliensis (ordem Onygenales). Apesar de mundialmente reportados, a ocorrência desses agentes etiológicos em cetáceos do Atlântico Sul ainda é pouco compreendida. O objetivo desse estudo foi identificar e caracterizar patógenos cutâneos e mucocutâneos selecionados (HV, CePV e P. brasiliensis) de cetáceos brasileiros de vida livre e desenhar métodos diagnósticos mais sensíveis para sua detecção. Todos os animais estudados encalharam ao longo da costa brasileira, entre 2005 e 2015, exceto por três botos-cor-de-rosa que foram fisicamente imobilizados e liberados após a coleta de amostras. Para atingir tais objetivos, empregamos técnicas moleculares e histológicas, e ocasionalmente de imunohistoquímica e microscopia eletrônica. A presença de HV e CePV foi avaliada, respectivamente, em amostras cutâneas e de mucosa oral e genital de 115 espécimes, e amostras de pele de 113 indivíduos; enquanto a presença de membros da ordem Onygenales foi avaliada em quatro espécimes que apresentavam lesões macroscópicas compatíveis. Amostras de pele ou de mucosa oral de quatro animais foram positivas para a PCR de HV: uma lesão ulcerada de pele de coloração esbranquiçada de um boto-cinza (Sotalia guianensis), uma amostra de tecido lingual de um golfinho-pintado-do-Atlântico (Stenella frontalis), lesões ulcerativas e amostras de pele saudável de um cachalote-anão (Kogia sima), e uma lesão proliferativa de pele em boto-vermelho-boliviano (Inia boliviensis). Os primeiros três animais estavam infectados com alphaherpesvirus. Uma sequência mais similar com gammaherpesvírus foi obtida da lesão proliferativa de pele do boto-vermelho-boliviano. A sequência do boto-vermelho-boliviano possivelmente pertence a um novo gênero de gammaherpesvírus. Ademais, todas as outras amostras de tecidos disponíveis dos espécimes HV-positivos, à parte de pele e mucosa oral, também foram avaliadas por técnicas de PCR e histológicas. Uma sequência diferente de alphaherpesvírus foi encontrada no estômago e em um linfonodo mesentérico do cachalote-anão. Achados microscópicos em dois animais HV-positivos (dermatites proliferativas em boto-vermelho-boliviano e boto-cinza) eram compatíveis com HV. CePV foi identificado em lesões de pele do tipo tattoo de um golfinho-nariz-de-garrafa (Tursiops truncatus) e de um boto-cinza por meio de técnicas moleculares estabelecidas, e observação de partículas de poxvírus por microscopia eletrônica. Animais CePV-positivos apresentavam degeneração balonosa epidérmica e ocasionais inclusões intracitoplasmáticas anfofílicas ou eosinofílicas compatíveis com CePV. Motivos aminoácidos específicos para todos os CePVs também foram identificados, reforçando a sugestão de um novo gênero, chamado Cetaceanpoxvirus. Nesse estudo também foram desenvolvidas novas técnicas de PCR convencional e real-time com SYBR® Green, significativamente mais sensíveis do que os métodos atualmente disponíveis em literatura. Um boto-cinza, inicialmente negativo segundo os métodos de PCR previamente conhecidos foi diagnosticado positivo para CePV por meio das novas técnicas aqui descritas. Leveduras refratáveis (49 µm de diâmetro) foram observadas á microscopia sob a forma de lesões de pele granulomatosas moderadas e necróticas em quatro golfinhos-nariz-de-garrafa, e pela primeira vez, em um abscesso muscular (esse último um indício do potencial invasivo desse agente). Leveduras de Onygenales sp. foram identificadas em lesões de pele por meio de imunohistoquímica e uma sequência de P. brasiliensis mais semelhante (100% de identidade de nucleotídeos) áquela descrita em um gofinho-nariz-de-garrafa de Cuba do que a casos de humanos e mamíferos descritos no Brasil, foi encontrada em lesões de pele de um dos espécimes. Esse estudo relata a primeira identificação molecular de HV em cetáceos da América do Sul e em golfinhos de rio no mundo. Além disso, descrevemos a primeira amplificação de CePV e P. brasiliensis em odontocetes da América do Sul, confirmando a etiologia desse tipo de lesões. Quatro das cinco novas sequências de HV identificadas são possivelmente novas espécies, provisoriamente chamadas Delphinid HV-10, Kogiid HV-2, Kogiid HV-3 e Iniid HV-1.

Doenças infecciosas emergentes se caracterizam como enfermidades cujos patógenos evoluíram para uma nova cepa ou genótipo capaz de infectar tanto o mesmo hospedeiro quanto uma nova espécie. A ocorrência de doenças emergentes tem sido correlacionada à destruição de habitats e à perda de biodiversidade. As aves marinhas têm sido consideradas bons indicadores e sentinelas do ecossistema aquático, e uma ampla variedade de enfermidades virais têm sido investigadas e descritas em aves no mundo todo. A caracterização e o entendimento das enfermidades virais que acometem as aves marinhas que habitam a costa brasileira são de fundamental importância para a compreensão de possíveis surtos de mortalidade dentre outros fatores que interferem na conservação das aves em geral. O objetivo desta pesquisa foi investigar a ocorrência de herpesvírus, avipoxvírus e coronavírus nas espécies de aves marinhas que foram reabilitadas em três centros localizados ao longo da costa brasileira e nas colônias reprodutivas de Sula sp. e Phaeton sp. localizadas no arquipélago de Abrolhos, Bahia, Brasil, e em quatro colônias de Spheniscus magellanicus localizadas na Patagônia argentina. As análises virais foram realizadas pela técnica de PCR e RT-PCR e confirmadas pela reação de sequenciamento do amplicon identificado. Nos casos de óbito, as alterações histopatológicas foram identificadas por meio de análise microscópica. O estudo revelou a ocorrência dos três agentes virais identificados em centros de reabilitação no Brasil, além da ocorrência de herpesvírus nas populações de vida livre e ativas em seus sítios reprodutivos. Foram identificados quatro novos herpesvírus denominados: Magellanic penguin herpesvirus 1 (MagHV -1) causador de um surto de mortalidade associado a traqueíte necrótico-hemorrágica em pinguins de Magalhães em reabilitação; Magellanic penguin herpesvirus 2 (MagHV-2), identificado em pinguins de Magalhães aparentemente saudáveis nas colônias reprodutivas da Patagônia argentina; Sulid herpesvirus 1 (SuHV -1), identificado em atobás (Sula sp.) e grazinas (Phaeton sp.) nas colônias reprodutivas de Abrolhos e Thalassarchid herpesvirus 1 (ThHV -1), identificados em um albatroz de nariz amarelo (Thalassarche chlororhynchos) nas praias de Rio Grande, RS, Brasil. Também foram identificados: um novo avipox, denominado Brazilian penguinpox, causador de lesões cutâneas, esofágicas e respiratórias nos pinguins de Magalhães cutâneas, esofágicas e respiratórias nos pinguins de Magalhães em reabilitação em Santa Catarina e a identificação de dois Gammacoronavirus em três diferentes espécies assintomáticas que estavam em reabilitação em Santa Catarina e no Rio Grande do Sul. Os dados obtidos constituem uma base de informação útil para estudos futuros no campo da patologia, virologia, epidemiologia e dos impactos antrópicos na saúde das aves marinhas do cone sul. / Emerging infectious diseases are characterized as disease whose pathogens progressed to a new strain ar genotype capable of infecting the same host or a new species. The occurrence of emerging diseases has been correlated to the destruction of habitats and loss of biodiversity. Seabirds have been considered as good indicators and sentinels of aquatic ecosystem, and a wide variety of viral diseases have been investigated and described in birds worldwide. The characterization and understanding of viral diseases that affect seabirds that inhabit the Brazilian coast are of fundamental importance for the understanding of possible mortality outbreaks among other factors that influence the conservation of birds in general. The objective of this research was to investigate the occurrence of herpesvirus, coronavirus and avipoxvirus in seabird species that were rehabilitated in three centres located along the Brazilian coast and in breeding colonies of Sula sp. and Phaeton sp. located at the Abrolhos’s archipelago, Bahia, Brazil and four Spheniscus magellanicus's colonies located at Argentina's Patagonia. Viral analyzes were performed by PCR and RT-PCR and confirmed by sequencing the identified amplicon. In cases of death, the histopathological alterations were identified through optical microscopic analysis. The study revealed the occurrence of the three investigated viral agents in the Brazilian rehabilitation centres, besides the occurrence of herpesvirus in the freeliving and reproductive active seabird’s populations. Four new herpesvirus were identified and called: Magellanic penguin herpesvirus 1 (MagHV -I), causing a mortality outbreak associated with necrotic-hemorrhagic tracheitis in Magellanic penguins in rehabilitation process; Magellanic penguin herpesvirus 2 (MagHV-2), identified in apparently healthy breeding Magellanic penguins at the Argentinean Patagonia’s colonies; Sulid herpesvirus 1 (SuHV-l), identified in boobies (Sula sp.) and tropicalseabirds (Phaeton sp.) breeding colonies at Abrolhos Archipelago, and Thalassarchid herpesvirus 1 (ThHV -1), identified in a yellow nose albatross (Thalassarche chlororhynchos) that appeared on the beaches of Rio Grande, RS, Brazil. Were. also identified: a new avipox named Brazilian penguinpox causing cutaneous, esophagus and respiratory lesions in rehabilitation penguins at Santa Catarina and the identification of two Santa Catarina and the identification of two Gammacoronavirus in three different asymptomatic seabirds species that were undergoing rehabilitation in Santa Catarina and Rio Grande do Sul. The data provi de a useful information basis for further studies related to pathology, virology, epidemiology and human impacts on southern hemispheres seabirds’ health.

Several wild-type poxviruses have emerged as potential oncolytic viruses (OVs), including orf virus (OrfV), and vaccinia virus (VV). Oncolytic VVs have been modified to include attenuating mutations that enhance their tumour selective nature, but these mutations also reduce overall viral fitness in cancer cells. Previous studies have shown that a VV (Western Reserve) with its E3L gene replaced with the E3L homologue from, OrfV (designated VV-E3LOrfV), maintained its ability to infect cells in vitro, but was attenuated compared to its parental VV in vivo. Our goal was to determine the safety and oncolytic potential VV-E3LOrfV, compared to wild type VV and other attenuated recombinants. VV-E3LOrfV, was unable to replicate to the same titers and was sensitive to IFN compared to its parental virus and other attenuated VVs in normal human fibroblast cells. The virus was also less pathogenic when administered in vivo. Viral replication, spread and cell killing, as measures of oncolytic potential in vitro, along with in vivo efficacy, were also observed.. The Parapoxvirus, OrfV has been shown to have a unique immune-stimulation profile, inducing a number of pro-inflammatory cytokines, as well as potently recruiting and activating a number of immune cells. Despite this unique profile, OrfV is limited in its ability to replicate and spread in human cancer cells. Various strategies were employed to enhance the oncolytic efficacy of wild-type OrfV. A transient transfection/infection screen was created to determine if any of the VV host-range genes (C7L, K1L, E3L or K3L) would augment OrfV oncolysis. Combination therapy, including the use of microtubule targeting agents, Viral Sensitizer (VSe) compounds and the addition of soluble VV B18R gene product were employed to see if they also enhance OrfV efficacy. Unfortunately, none of the strategies mentioned were able to enhance OrfV.

<p>Orthopoxviruses encode many genes that are not essential for viral replication, which often account for differences in pathogenesis among otherwise closely related orthopoxviruses. Although dendritic cells (DCs) are essential to the generation of an effective anti-viral immune response, the effects of different orthopoxviruses on DC function is poorly understood. The objective of these studies was to determine the effect of different orthopoxviruses on DCs. Cowpox virus (CPXV) is ideally suited to this purpose because it encodes the largest and most representative set of accessory genes among orthopoxviruses, it is endemic in mouse populations, and can infect humans. </p><p>We hypothesized that CPXV would have novel mechanisms of evading the immune response that other orthopoxviruses lack, which may exert maximal effect in the context of antigen presenting cells such as DCs, allowing for discovery of novel viral strategies of immune evasion. To test this, CPXV was used to infected mouse bone marrow-derived DCs (BMDCs), and the effect of the virus on DC survival, expression of T-cell costimulatory molecules and cytokine production was determined. The effects of vaccinia virus strain Western Reserve (VV), the prototype of the species, and modified vaccinia virus strain Ankara (MVA), a promising vaccine vector, on mouse BMDCs were also determined. Confirming the hypothesis that CPXV would have different effects on mouse BMDCs from other orthopoxviruses, BMDCs infected with CPXV survived longer in culture than those infected with MVA or VV. In addition, CPXV specifically downregulated MHC I, MHC II, CD40, and CD86, and induced production of significant levels of IL-6 and IL-10.</p><p>Because IL-10 has many suppressive effects on the immune system, inducing IL-10 may provide a selective advantage to CPXV in vivo. To examine the role of IL-10 in a CPXV infection, wild type and IL-10 deficient mice were infected intranasally with CPXV. The effect of CPXV infection on disease morbidity, viral loads, inflammation and the protective immune response was determined. As expected, IL-10 was important in controlling inflammation during CPXV infection, but there was no effect on viral replication or clearance. Surprisingly, IL-10 was important in generation of a protective memory response to CPXV, which may reflect a novel role for IL-10 in the immune response.</p> / Dissertation

No description available.

The focus of this dissertation is the use of bioinformatics in the identification of highly conserved sequences among a set of poxvirus genomes and the subsequent functional analysis of the conserved functions of these sequences. A novel algorithm, Java Pattern Finder, which identifies sequences of a user-specified length that are conserved with a user-specified number of allowed differences, was used to identify near-perfectly conserved sequences among a set of poxvirus genomes. A scoring method was established to quantify the degree of conservation of these sequences and used to show that the 11 most conserved sequences were significantly more conserved than control sequences. Functional analysis showed that explanations such as low codon degeneracy or the presence of conserved promoter elements partially – but not fully – accounted for the conservation observed in these sequences, suggesting that these highly conserved regions may have novel functions in the poxvirus genome that have yet to be uncovered.