The contrasting infection strategy of herpes simplex virus (HSV) consists of an initial primary lytic infection in epithelial cells, followed by establishment of lifelong latency in sensory and autonomic neurons of the peripheral nervous system that innervate the site of infection. Any cellular stress trigger, ranging from external stimuli such as UV radiation or nerve injury to psychological and physiological stress, can reactivate HSV from latency in the neurons, resulting in recurrent disease episodes. Stress hormones and deprivation of neurotrophic factor (NTF) both have a strong correlation with HSV reactivation from neurons. However, neuronal signaling pathways cardinal to HSV latency and reactivation are still not clear. This dissertation provides new understanding of HSV latency and reactivation in response to two orthogonal stress stimuli, viz. stress hormones epinephrine (EPI) and corticosterone (CORT), as well as NTF deprivation that simulates a nerve injury in primary neuronal cultures. In this dissertation, we demonstrate that physiological stress hormones EPI and CORT differentially regulate HSV-1 and HSV-2 reactivation in adult neurons. Both EPI and CORT treatment reactivated only HSV-1 in sympathetic superior cervical ganglia (SCG) neurons, while HSV-2 was reactivated only by CORT in both sensory trigeminal ganglia (TG) neurons and sympathetic superior cervical (SCG) neurons. EPI utilized the combination of α and β adrenergic receptor complex, while CORT signaled through glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) to reactivate HSV in the neurons. NTFs are tissue-target derived growth factors required for neuronal protection and survival. Neurotrophins are also required for maintaining HSV latency, as NTF deprivation reactivates both HSV-1 and HSV-2 in adult sensory TG and sympathetic SCG neurons. In addition, assessing the temporal kinetics of HSV gene expression showed differential expression profiles of viral immediate-early (IE) genes ICP0, ICP4, ICP27 and trans-activator VP16 following treatment with stress hormones and NTF deprivation in HSV-1 and HSV-2 infected neurons. We also show that different molecular mechanisms are involved in HSV latency and reactivation, which are dependent on the stimuli and the type of neurons. Tyrosine kinase receptor-mediated PI3K-Akt-mTORC signaling cascades have been studied for their role in maintaining HSV latency. Activation of β-catenin signalosome expression has also been implicated during HSV latency and following reactivation. GSK3β is a key effector molecule that inter-connects Akt and β-catenin mediated pathways, forming an Akt-GSK3β-β-catenin signaling axis. Analyzing the Akt-GSK3β-β-catenin signaling in response to stress hormone and NTF deprivation revealed significant differences in protein expression levels between HSV-1 and HSV-2 infected sensory and sympathetic neurons. In HSV-1 infected neurons, the Akt-GSK3β-β-catenin maintains the signal transmission in order to keep the neurons alive, but HSV-2 infections obliterated the entire axis in the adult neurons, particularly in sympathetic neurons. In summary, we demonstrate that HSV-1 and HSV-2 do not have a 'one for all' infection mechanism. Establishment of latency and reactivation by HSV is virus specific, stimulus specific and neuron specific. / Doctor of Philosophy / Herpes simplex viruses (HSVs) are common global viral pathogens that are responsible for causing lifelong painful infections and debilitating disease. The two serotypes of HSV include HSV-1, which is associated with oral or ocular disease but can also cause genital disease, and HSV-2, which is predominantly associated with genital herpes. Once infected, both HSV-1 and HSV-2 are present as lifelong reservoirs in our peripheral neurons. Stress stimuli mediated by our stress hormones or external triggers, such as nerve trauma or an axonal injury, can periodically reactivate the latent virus to cause recurrent disease. Clinical manifestation of HSV recurrences range from asymptomatic viral shedding to painful blisters, cold sores, or herpetic keratitis. In some cases, the virus can spread to the central nervous system, causing encephalitis or recurrent meningitis. No vaccines have been approved yet, and the current treatment utilizes nucleoside analogs, such as acyclovir and its prodrug valacyclovir, to ameliorate the symptoms of HSV infection by halting viral replication and if taken as a daily prophylaxis, reduces the chances of clinical recurrence. Given the route and transmission efficiency of HSV, it is practically impossible to prevent herpes infection. To develop strategic therapeutic interventions to lock the virus in its latent phase in the neurons and prevent it from reactivation, a better understanding of neuronal signaling pathways cardinal to HSV latency and reactivation is necessary. However, neuronal signaling pathways cardinal to HSV latency and reactivation are still not clear. In this dissertation, we make contributions to better understand HSV latency and reactivation in response to stress stimuli. We show that different stress stimuli exert preferential reactivation between HSV-1 and HSV-2, and are further dependent upon the neurons where they establish latency. Our study specifically focuses on three neuronal stressors that have been associated with HSV recurrences: two stress hormones, epinephrine (EPI) and corticosterone (CORT), as well as deprivation of neurotrophic factors (NTF) that simulates nerve injury. We also focused on a neuronal signaling cascade involved in the response to all of these stimuli, Akt-GSK3β-β-catenin, and viral gene transcripts that respond to these stimuli during reactivation. Comprehensive understanding of the neuronal processes and viral gene transcripts involved during HSV-1 and HSV-2 reactivation in neurons will help the herpes virology field towards development of targeted therapies and vaccines to prevent reactivation and recurrent disease.

Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus, often acquired during childhood, that currently infects more than 50% of the human population. The symptoms of infection are herpetic lesions that frequently appear throughout a host's life in response to stress in the orofacial or genital region. As a pathogen, HSV-1 replicates rapidly in epithelial cells, but it is also capable of infecting neurons where it can pursue a lytic or latent infection. Latency is a state of viral quiescence where the virus can persist indefinitely yet remain poised to reactivate. Latency is unique to herpesviruses and key to HSV's success, but the molecular mechanisms that govern this state are unclear. A virus-encoded E3-ubiquitin ligase, Infected Cell protein 0 (ICP0), is often correlated with latency establishment but is detected in opposition to the state of latency. During lytic infection, ICP0 has many biological roles but primarily catalyzes the addition of ubiquitin to target substrate, marking proteins for degradation or altering their function. This ubiquitination ability allows ICP0 to alter the intracellular environment making neurons conducive to lytic or latent HSV-1 infection. ICP0's neuron-specific targets, however, are unknown, representing a significant gap in knowledge. Through the studies presented in this dissertation, we identified some of the neuron-specific ubiquitination targets of ICP0 in neurons. We utilized primary adult sensory neurons of the dorsal root ganglia and HSV-1 viral strains KOS, wild-type virus encoding a fully functional ICP0, and HSV-1 n212, encoding a truncated ICP0 protein, to illuminate the mechanisms involved in establishing and maintaining HSV latency. By using adult primary neurons and functional HSV-1 strains with and without ICP0, we were able to show that ICP0 regulates host and viral proteins during the initial onset of neuronal infection. We also show that based on neuronal conditions set forth before HSV-1 initial infection, host proteins will influence HSV-1 viral proteins to repress viral gene expression, thereby promoting the establishment of latency. / Doctor of Philosophy / Herpes simplex virus (HSV-1) is a virus, often acquired during childhood, that more than 50% of people have. Those who are infected with HSV-1 often have cold sores that appear in response to stress on the face or on the genitals. As a virus, HSV-1 replicates around the eyes, nose, and mouth but can also infect neurons where it can continue to replicate or establish latency. Latency is when the virus is inside the neurons but is unnoticeable and can reappear in response to stress. The state of latency is unique to herpesviruses and key to the success of HSV-1, but scientists are unsure of how it works. A protein made by the virus, Infected Cell Protein 0 (ICP0), is often correlated with the state of latency but is often present when the virus is not latent. ICP0 does a lot to support HSV-1, but it primarily destroys proteins that prevent the virus from replicating. By destroying proteins that prevent HSV-1 replication, ICP0 can help the virus make more viruses. The proteins that are destroyed by ICP0 are currently unknown, which represents a significant gap in knowledge. Through the research conducted in this dissertation, we identified some of the proteins that ICP0 destroys in neurons. We utilized neurons from the dorsal root ganglia and HSV-1 viral strain KOS, which encoded a functional ICP0, and n212, which encodes a nonfunctional ICP0, to study the mechanisms used by the virus to infect neurons. By using HSV-1 viruses with and without ICP0, we were able to show what proteins ICP0 destroys during infection in neurons. We were also able to show that HSV-1's ability to establish latency is dependent on how the neurons handle the initial onset of infection. Overall, a combination of host and viral proteins coordinates the virus's ability to establish latency and persist within a host.

Os vírus são responsáveis por infecções de distribuição mundial com elevadas prevalência e morbimortalidade sendo, muitas das vezes, sem tratamento efetivo disponível. Existem medicamentos fitoterápicos com atividade antibacteriana ou antiviral comprovadas, porém seu uso na prática clínica ainda é limitado. O objetivo deste trabalho foi investigar a presença ou ausência de atividade antiviral in vitro de extratos brutos e fracionados de Cocos nucifera L. em cultura de células Vero infectadas com herpes simplex vírus tipo 1 (HSV-1). Foram escolhidas doses não tóxicas dos extratos aquoso e hidroetanólico da fibra do C. nucifera, de 4 frações derivadas daquele com diferentes solventes (hexano, acetato de etila, metanol e água) e de duas substâncias isoladas a partir desta fibra (Substâncias CN1A e CN342B). O HSV-1 foi adicionado à cultura de células Vero uma hora após estas serem incubadas com os dois extratos e seus derivados. As placas foram analisadas em microscópio óptico a cada 24 horas, sendo o material colhido quando o efeito citopático viral no controle negativo atingisse mais de 80% das células e congelado para posterior titulação viral por TCID50. Utilizamos o aciclovir como droga de referência para o HSV-1. A substância CN342B foi capaz de inibir a replicação do HSV-1, com efeito antiviral comparável ao do aciclovir, enquanto que os extratos brutos, as quatro frações e a substância CN1A não foram efetivas. Em conclusão, a substância CN342B isolada das fibras do C. nucifera foi eficaz contra o HSV-1 in vitro / Viruses are responsible for infections worldwide with high prevalence and morbimortality, often with no effective treatment available. There are herbal medicines with proven antibacterial and antiviral activities; however, their use in clinical practice is still limited. The aim of this study was to investigate the presence or absence of in vitro antiviral activity of aqueous and hydroethanolic extracts from Cocos nucifera L. husk fiber, in culture of Vero cells infected with herpes simplex virus (HSV)-1. Non-toxic dosages of aqueous and hydroethanolic extracts, as well as the hexane, ethyl-acetate, methanol and end-aqueous fractions were used. In addition, two isolated substances (CN1A and CN342B) were tested. HSV-1 was added to the cell culture after one hour of incubation with the extracts and fractions. The plates were analyzed in optical microscopes each 24 hours, and cells were harvested when the cytopathic effect in negative controls were above 80% of the cells. The cells were then frozen for viral titulation (TCID50). Acyclovir was the reference drug for HSV-1. Substance CN342B, but none of the other drugs, was able to reduce HSV-1 replication, similarly to acyclovir. In conclusion, the substance CN342B isolated from C. nucifera husk fibers was effective against HSV-1 in vitro

Os vírus são responsáveis por infecções de distribuição mundial com elevadas prevalência e morbimortalidade sendo, muitas das vezes, sem tratamento efetivo disponível. Existem medicamentos fitoterápicos com atividade antibacteriana ou antiviral comprovadas, porém seu uso na prática clínica ainda é limitado. O objetivo deste trabalho foi investigar a presença ou ausência de atividade antiviral in vitro de extratos brutos e fracionados de Cocos nucifera L. em cultura de células Vero infectadas com herpes simplex vírus tipo 1 (HSV-1). Foram escolhidas doses não tóxicas dos extratos aquoso e hidroetanólico da fibra do C. nucifera, de 4 frações derivadas daquele com diferentes solventes (hexano, acetato de etila, metanol e água) e de duas substâncias isoladas a partir desta fibra (Substâncias CN1A e CN342B). O HSV-1 foi adicionado à cultura de células Vero uma hora após estas serem incubadas com os dois extratos e seus derivados. As placas foram analisadas em microscópio óptico a cada 24 horas, sendo o material colhido quando o efeito citopático viral no controle negativo atingisse mais de 80% das células e congelado para posterior titulação viral por TCID50. Utilizamos o aciclovir como droga de referência para o HSV-1. A substância CN342B foi capaz de inibir a replicação do HSV-1, com efeito antiviral comparável ao do aciclovir, enquanto que os extratos brutos, as quatro frações e a substância CN1A não foram efetivas. Em conclusão, a substância CN342B isolada das fibras do C. nucifera foi eficaz contra o HSV-1 in vitro / Viruses are responsible for infections worldwide with high prevalence and morbimortality, often with no effective treatment available. There are herbal medicines with proven antibacterial and antiviral activities; however, their use in clinical practice is still limited. The aim of this study was to investigate the presence or absence of in vitro antiviral activity of aqueous and hydroethanolic extracts from Cocos nucifera L. husk fiber, in culture of Vero cells infected with herpes simplex virus (HSV)-1. Non-toxic dosages of aqueous and hydroethanolic extracts, as well as the hexane, ethyl-acetate, methanol and end-aqueous fractions were used. In addition, two isolated substances (CN1A and CN342B) were tested. HSV-1 was added to the cell culture after one hour of incubation with the extracts and fractions. The plates were analyzed in optical microscopes each 24 hours, and cells were harvested when the cytopathic effect in negative controls were above 80% of the cells. The cells were then frozen for viral titulation (TCID50). Acyclovir was the reference drug for HSV-1. Substance CN342B, but none of the other drugs, was able to reduce HSV-1 replication, similarly to acyclovir. In conclusion, the substance CN342B isolated from C. nucifera husk fibers was effective against HSV-1 in vitro

Orientador: João Pessoa Araújo Junior / Banca: José Paes de Oliveira Filho / Banca: Clarice Weis Arns / Resumo: O herpesvírus canino (HVC-1) causa doença infecto-contagiosa que acomete cães em todo o mundo. É considerado o responsável por causar problemas reprodutivos, respiratórios, oculares, alterações neurológicas podendo levar à morte neonatos e adultos imunossuprimidos. Assim, o presente trabalho teve como objetivos isolar o HVC-1 a partir de amostras biológicas, padronizar uma técnica diagnóstica para detecção do herpesvírus canino, verificar a presença de cães infectados no estado de São Paulo e comparar 3 modificações na técnica de diluição limitante em relação à quantificação molecular pela qPCR (quantitative Polymerase Chain Reaction). Para isso, foram coletadas 139 amostras sangue, fragmentos de órgãos de 12 neonatos que foram a óbito e 5 amostras de suabes de secreção genital, de animais provenientes de canis e domicílios. Após a obtenção do DNA das amostras colhidas, foram submetidas à qPCR e snPCR (seminested Polymerase Chain Reaction) para o teste de detecção do HVC-1. Foi encontrada positividade em nove foram amostras de sangue e em fragmentos de órgãos de um animal, as amostras de secreções genitais foram todas negativas. O isolamento viral foi realizado a partir de fragmento de rim. As células infectadas apresentaram efeitos citopáticos compatíveis com as do HVC-1. As amostras positivas foram sequenciadas apresentando identidade de 100% com o HVC-1 (Genbank ™: X75765). As técnicas moleculares para a detecção do agente mostraram-se sensíveis e específicas, tanto a snPCR como a qPCR. Nessas técnicas foi possível detectar até 15,35 cópias/ μL de DNA de amostras de sangue e 1,15 cópias/μg de tecido. O uso de técnicas moleculares possibilitou a detecção de infecções ativas por HVC-1 com ótimo limiar de detecção. No entanto, mais estudos são necessários para conhecer a epidemiologia da infecção do HVC-1 na população canina no Brasil / Abstract: The canine herpesvirus (CHV-1) causes contagious disease affecting dogs worldwide. It is considered the responsible for cause reproductive problems, respiratory, eyes, neurologic changes may lead to death newborns and immunocompromised adults. Therefore, our study aimed to isolate CHV-1 from biological samples, standardize a diagnostic techniques for the detection of canine herpesvirus, verify the presence of infected dogs in São Paulo state and compare three modifications in the technique of limiting dilution in relation to molecular quantification to qPCR (by quantitative PCR to determine the viral titer). Thus, we collected 139 blood samples, organ's fragments from 12 neonates death and 5 genital secretions samples with swabs of animals from kennels and households. After obtaining the DNA from samples were accomplished the qPCR (quantitative PCR) and snPCR (seminested PCR) assay for detection of CHV-1. It was found positivity in nine blood sample and organ's fragments of an animal, the genital secretions samples were all negative. The viral isolation was performed from kidney. The infected cells showed cytopathic effects compatible with the CHV-1. Positive samples were sequenced showed 100% identity with the CHV-1 (Genbank™: X75765). The molecular techniques for detection agent were sensitive and specific, both the snPCR as the qPCR. In these techniques was enabled to detect DNA 15.35 copies/mL of blood samples and 1.15 copies/μg of tissue. The use of molecular techniques allowed the detection active infections of of HCV-1 with optimal detection limit. More studies are needed to understand the infection epidemiology of CHV-1 in the canine population in Brazil / Mestre

A fototerapia com lasers em baixa intensidade de lesões de herpes simples tem sido demonstrada clinicamente ora prevenindo a formação de vesículas, ora cicatrizando rapidamente as lesões e até aumentando o espaço de tempo entre o aparecimento dessas manifestações recorrentes. No entanto, os mecanismos básicos de ação dos lasers nessas situações são desconhecidos. Dessa forma, o objetivo do trabalho foi realizar ensaios in vitro utilizando células epiteliais em cultivo e culturas do vírus HSV-1 para estudar a interferência do laser em baixa intensidade na infecção do HSV-1. Material e Métodos: Culturas de vírus HSV-1 e de células epiteliais de macaco (linhagem Vero) infectadas ou não infectadas, crescidas em déficit nutricional (2 % de soro fetal bovino - sfb) foram utilizadas. As irradiações foram realizadas com um laser de GaAlAs (660 e 780 nm, área focal de 3,6 mm2). Uma, duas e três irradiações com intervalos de 6 h foram realizadas. Os grupos experimentais foram: Controle: não-irradiadas; 660 nm/ 3 J/cm2 (28 s); 660 nm/ 5 J/cm2 (38 s); 780 nm/ 3 J/cm2 (19 s) e, 780 nm/ 5 J/cm2 (25 s). Os efeitos citopáticos do HSV-1 e a viabilidade celular de culturas irradiadas e controles foram analisadas em 4 condições: 1) irradiação das células epiteliais não infectadas; 2) células epiteliais irradiadas antes da infecção; 3) irradiação dos vírus antes da infecção; 4) irradiação das células previamente infectadas pelo HSV-1. A viabilidade celular foi obtida pelo teste da redução do MTT e os efeitos citopáticos por observação em microscopia de luz. Resultados: A viabilidade celular de culturas irradiadas crescidas em déficit nutricional, independentemente do número de irradiações, foi sempre significantemente menor que aquela de culturas não-irradiadas e crescidas nas condições ideais de concentração de sfb (10 %). A viabilidade celular de culturas não infectadas foi similar em todos os grupos. O número de irradiações influenciou o crescimento celular positiva e proporcionalmente ao número de irradiações, exceto para o grupo 660 nm/ 3 J/cm2. Nenhuma diferença nos efeitos citopáticos foi observada entre os grupos, independentemente do número de irradiações nas 3 condições do estudo. A viabilidade celular de todos os grupos não mudou nem pela irradiação das células nem do vírus antes da inoculação nas células. A viabilidade de células infectadas antes da irradiação foi significantemente maior que o controle quando 2 irradiações foram realizadas. Conclusão: Nas condições deste estudo a radiação laser em baixa intensidade é capaz de aumentar o crescimento de células Vero crescidas em déficit, no entanto, não o suficiente para atingir o crescimento característico dessas células crescidas nas suas condições ideais. O número de irradiações influencia o crescimento das células de forma positiva e proporcional ao número de irradiações, exceto para o parâmetro 660 nm/ 3 J/cm2. A radiação laser não altera nem a susceptibilidade das células à infecção, nem a virulência do HSV-1. No entanto, ela prolonga a viabilidade das células infectadas pelo HSV-1. Efeitos positivos da fototerapia que tem sido relatados clinicamente parecem ser devido a efeitos no hospedeiro não relacionados com a replicação viral nas células infectadas. / Purpose: The clinical effects attributed to phototherapy relative to Herpes simplex lesions have included prevention of lesion formation, speeding the healing of lesions, and decreasing the frequency of recurrent lesions. The mechanisms underlying these findings have not been established yet. The aim of this in vitro study was to analyze the effect of phototherapy on epithelial cells, on HSV-1, and on infected epithelial cells in culture. Material and Methods: Cultures of HSV-1 and infected or non-infected monkey epithelial cells (Vero cell line) grown in deficient media (2 % fetal bovine serum-fbs) were used. The laser irradiation was delivered using a GaAlAs laser (660 and 780 nm, focal spot of 3.6 mm2). One, two and three irradiations with 6 hourintervals were done. The experimental groups were: Control: non-irradiated; 660 nm/3 J/cm2 (28 sec); 660 nm/5 J/cm2 (38 sec); 780 nm/3 J/cm2 (19 sec), and 780 nm/5 J/cm2 (25 sec). The HSV-1 cytopatic effects and the cell viability of irradiated cultures and controls were analyzed in four different conditions: 1) irradiation of noninfected epithelial cells; 2) epithelial cells irradiated prior infection; 3) virus irradiated prior infection; and 4) irradiation of HSV-1 infected cells. The cell viability was assessed by the reduction of the MTT test and the cytopatic effects by the light microscopy observation. Results: The cell viability of irradiated cultures grown in nutritional deficit, independently of the irradiation numbers, was always significantly smaller than that of non-irradiated cultures grown at the ideal serum concentration condition (10 %). The cell viability of non-infected cells was similar amongst the groups. The number of irradiations influenced the cell growth positively and proportionally to the number of irradiations, except for the 660 nm/3J/cm2 group. Any variation in cytopatic effects was observed amongst the experimental groups, independently of the irradiation numbers at the 3 conditions analyzed. The cell viability of all experimental groups were not altered either by irradiation of the cells or of the virus prior infection. The viability of infected cells prior irradiation was significantly higher than that of non-irradiated cultures when 2 irradiations were done. Conclusion: The experimental conditions for this study demonstrate that the phototherapy is capable of enhancing the growth of Vero cells grown under nutritional deficit conditions, however, not enough to reach the characteristic cell growth of cells grown at the ideal serum concentration condition. The number of irradiations influences the cell growth positive and proportionally, except when the parameter 660 nm and 3 J/cm2 was used. The laser radiation does not change either the susceptibility of the Vero cell to the HSV-1 infection or the HSV-1 virulence; however, prolongs the cell viability of HSV-1 infected cells. Positive benefits of phototherapy that have been reported clinically would appear to be due to host effects unrelated to viral replication in infected cells.

Orientador: Alexandre Secorun Borges / Banca: João Pessoa Araújo Junior / Banca: Paulo Eduardo Martins Ribolla / Banca: Eduardo Furtado Flores / Banca: Marcelo Mendes Brandão / Resumo: Herpesvirus bovino (BoHV-5) é um alfaherpesvirus que causa meningoencefalite não-supurativa preferencialmente em bovinos jovens. Esta enfermidade ocorre naturalmente em surtos ou casos isolados, apresentando baixa morbidade e alta letalidade. A epidemiologia, as características anatomopatológicas e o diagnóstico do BoHV-5 são bem conhecido. No entanto, as interações moleculares entre a células hospedeiras e o BoHV-5 são pobremente compreendidas. Técnicas moleculares como a PCR quantitativa e o sequenciamento de RNA (RNA-seq) são importantes ferramentas para o estudo de interações entre vírus e células hospedeiras. Neste estudo células MDBK foram infectadas pelo BoHV-5, cepa SV507-99 e o mRNA extraído em 0, 6, 12, 18, e 24 horas após a infecção (pi) foi utilizado para avaliar a expressão de genes do vírus e da célula hospedeira por qPCR e o mRNA de 1h, 6h e 24h pi foram utilizado no estudo por RNA-seq. Células MDBK não infectadas foram usadas como controle. A qPCR demonstrou que os três genes do BoHV-5 estudados (bICP0, UL9 e US4) apresentaram perfil de expressão semelhante nos diferentes momentos após infecção e que o gene bovino GAPDH teve sua expressão diminuída pela infecção viral. Ao menos 900 genes foram diferencialmente expressos para cada momento de infecção na análise por RNA-seq. Estes genes estavam principalmente relacionados a vias celulares de controle de ciclo celular, produção de interleucinas, quimiotaxia para células inflamatórias, reparo e dano ao DNA, resposta a vírus, apoptose, processo oxidativo, e ubiquitização de moléculas. Estes resultados representam novos conhecimentos sobre a interação entre o BoHV-5 e as células bovinas e podem representar um ponto de partida para novas pesquisas e melhor compreensão da patogenia deste vírus / Abstract: Bovine herpesvirus 5 (BoHV-5) is an Alphaherpesvirus that causes nonsuppurative meningoencephalitis mainly in young cattle. This disease occurs naturally in either outbreaks or isolated cases, and exhibits low morbidity and high lethality. Besides BoHV-5 epidemiology, pathological findings and diagnosis were well known, the molecular interactions between host cell and BoHV-5 are poorly understood. Molecular biology techniques such as quantitative PCR (qPCR) and RNA sequencing (RNA-seq) are important tools to study virus and host cell interactions. In this study we infected MDBK cells and use the extracted mRNA at 0, 6, 12, 18 and 24h post infection (pi) to analyse BoHV-5 (bICP0, UL9 e US4) and host cell gene (GAPDH) expression using qPCR and 1h, 6h and 24h pi in the RNA-seq study. Mock-infected cells were used to control purpouse. The qPCR releveled that the three BoHV-5 genes showed the same expression behavior during the infection and the GAPDH gene were down-regulated in the infected group. At least 900 genes were differentially expressed during BoHV-5 in each moment analysed by RNA-seq. These genes were up- or down-regulated and mainly associated with cell cycle, interleukin production, inflammatory cells chemotaxis, DNA damage and repair, response to virus, apoptosis, oxidation-reduction process, and ubiquitination pathways. The results demonstrate new insights about BoHV-5 and bovine cells interactions and could be a starting point to new researches and to better understand the pathology of this virus / Doutor

Herpes simplex virusas sukelia recidyvuojančią burnos-veido ir lytinių organų infekciją. Latentinėje būklėje šis virusas glūdi sensoriniuose ganglijuose. Latencijos metu visi HSV genai yra supresuoti, išskyrus su latencija susijusį geną (LAT). Tyrimais nustatyta, kad tarp HSV LAT promotoriaus mutantų reaktyvacijos dažnis laboratorinių gyvūnėlių modeliuose yra mažesnis nei laukinių virusų. Nėra atlikta tyrimų, kurie nagrinėtų LAT promotoriaus sekų variaciją herpes simplex virusuose, išskirtuose iš žmonių klinikinių mėginių. Šio tyrimo tikslas buvo įvertinti herpes simplex viruso LAT promotoriaus sekų įvairovę molekulinės diagnostikos metodais bei palyginti su infekcijos klinikiniais požymiais. Tuo tikslu buvo sukurtas PGR metodas HSV LAT promotoriaus analizei atlikti. Buvo ištirta Lietuvos ir Švedijos klinikiniuose odos-gleivinių bei cerebrospinalinio skysčio mėginiuose rasto herpes simplex viruso promotoriaus DNR sekų įvairovė. Tyrimo metu rasta, kad 2 tipo herpes simplex virusas buvo pagrindinė lytinių organų HSV infekcijos priežastis tarp Lietuvos pacientų. Visuose veido srities bėrimuose rasta 1 tipo HSV. HSV LAT promotoriaus sekos ištirtos 145 klinikiniuose mėginiuose. Nustatyta, kad HSV LAT promotoriaus sekos yra gausios GC ir turi variabilias homopolimerinių nukleotidų sritis, kurios varijuoja tarp viruso padermių ir pačių padermių viduje. Ši variacija gali turėti įtakos baltymų sintezei, o drauge ir fenotipo pokyčiams. Nenustatytas ryšys tarp HSV LAT promotoriaus... [toliau žr. visą tekstą] / Herpes simplex virusas sukelia recidyvuojančią burnos-veido ir lytinių organų infekciją. Latentinėje būklėje šis virusas glūdi sensoriniuose ganglijuose. Latencijos metu visi HSV genai yra supresuoti, išskyrus su latencija susijusį geną (LAT). Tyrimais nustatyta, kad tarp HSV LAT promotoriaus mutantų reaktyvacijos dažnis laboratorinių gyvūnėlių modeliuose yra mažesnis nei laukinių virusų. Nėra atlikta tyrimų, kurie nagrinėtų LAT promotoriaus sekų variaciją herpes simplex virusuose, išskirtuose iš žmonių klinikinių mėginių. Šio tyrimo tikslas buvo įvertinti herpes simplex viruso LAT promotoriaus sekų įvairovę molekulinės diagnostikos metodais bei palyginti su infekcijos klinikiniais požymiais. Tuo tikslu buvo sukurtas PGR metodas HSV LAT promotoriaus analizei atlikti. Buvo ištirta Lietuvos ir Švedijos klinikiniuose odos-gleivinių bei cerebrospinalinio skysčio mėginiuose rasto herpes simplex viruso promotoriaus DNR sekų įvairovė. Tyrimo metu rasta, kad 2 tipo herpes simplex virusas buvo pagrindinė lytinių organų HSV infekcijos priežastis tarp Lietuvos pacientų. Visuose veido srities bėrimuose rasta 1 tipo HSV. HSV LAT promotoriaus sekos ištirtos 145 klinikiniuose mėginiuose. Nustatyta, kad HSV LAT promotoriaus sekos yra gausios GC ir turi variabilias homopolimerinių nukleotidų sritis, kurios varijuoja tarp viruso padermių ir pačių padermių viduje. Ši variacija gali turėti įtakos baltymų sintezei, o drauge ir fenotipo pokyčiams. Nenustatytas ryšys tarp HSV LAT promotoriaus... [to full text]

Herpes simplex virus (HSV) causes recurrent orofacial and genital infections and establishes latent infection in sensory neurons. During latency all virus genes are supressed except the latency associated transcripts which are transcribed from latency associated gene (LAT). It is established that HSV LAT promoter mutants have lower levels of spontaneous reactivation rates in small animal models compared to wild virus. However, the variation in the LAT promoter has not been studied in viruses from clinical samples in humans. The aim of the sudy was to evaluate the sequence variation in herpes simplex virus latency associated gene promoter from clinical samples by developing and applying molecular methods and correlate with herpes infection clinical features. In this study a new PCR method specific for HSV LAT promoter was developed and HSV LAT promoter DNA sequences from Lithuanian and Swedish mucocutaneous and cerebrospinal fluid clinical samples were analyzed. HSV type 2 was found to be the main cause of genital herpes in the population of the Lithuanian patients. All cases of orofacial herpes simplex infection were caused by HSV type 1. The structure of the LAT promoter region was studied in 145 HSV clinical samples. HSV LAT promoter was found to be G+C rich and contained variable homopolimer tracts. An inter- and intrastrain variability of homopolimer tracts in the promoter region was detected, potentially giving rise to a large variation at the protein level, leading to... [to full text] / Herpes simplex virusas sukelia recidyvuojančią burnos-veido ir lytinių organų infekciją. Latentinėje būklėje šis virusas glūdi sensoriniuose ganglijuose. Latencijos metu visi HSV genai yra supresuoti, išskyrus su latencija susijusį geną (LAT). Tyrimais nustatyta, kad tarp HSV LAT promotoriaus mutantų reaktyvacijos dažnis laboratorinių gyvūnėlių modeliuose yra mažesnis nei laukinių virusų. Nėra atlikta tyrimų, kurie nagrinėtų LAT promotoriaus sekų variaciją herpes simplex virusuose, išskirtuose iš žmonių klinikinių mėginių. Šio tyrimo tikslas buvo įvertinti herpes simplex viruso LAT promotoriaus sekų įvairovę molekulinės diagnostikos metodais bei palyginti su infekcijos klinikiniais požymiais. Tuo tikslu buvo sukurtas PGR metodas HSV LAT promotoriaus analizei atlikti. Buvo ištirta Lietuvos ir Švedijos klinikiniuose odos-gleivinių bei cerebrospinalinio skysčio mėginiuose rasto herpes simplex viruso promotoriaus DNR sekų įvairovė. Tyrimo metu rasta, kad 2 tipo herpes simplex virusas buvo pagrindinė lytinių organų HSV infekcijos priežastis tarp Lietuvos pacientų. Visuose veido srities bėrimuose rasta 1 tipo HSV. HSV LAT promotoriaus sekos ištirtos 145 klinikiniuose mėginiuose. Nustatyta, kad HSV LAT promotoriaus sekos yra gausios GC ir turi variabilias homopolimerinių nukleotidų sritis, kurios varijuoja tarp viruso padermių ir pačių padermių viduje. Ši variacija gali turėti įtakos baltymų sintezei, o drauge ir fenotipo pokyčiams. Nenustatytas ryšys tarp HSV LAT promotoriaus... [toliau žr. visą tekstą]

Made available in DSpace on 2015-10-06T13:02:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-12-12. Added 1 bitstream(s) on 2015-10-06T13:19:26Z : No. of bitstreams: 1 000847194.pdf: 1197011 bytes, checksum: 7209fde66f8374f6f0f630a8a112ded9 (MD5) / Herpesvirus bovino (BoHV-5) é um alfaherpesvirus que causa meningoencefalite não-supurativa preferencialmente em bovinos jovens. Esta enfermidade ocorre naturalmente em surtos ou casos isolados, apresentando baixa morbidade e alta letalidade. A epidemiologia, as características anatomopatológicas e o diagnóstico do BoHV-5 são bem conhecido. No entanto, as interações moleculares entre a células hospedeiras e o BoHV-5 são pobremente compreendidas. Técnicas moleculares como a PCR quantitativa e o sequenciamento de RNA (RNA-seq) são importantes ferramentas para o estudo de interações entre vírus e células hospedeiras. Neste estudo células MDBK foram infectadas pelo BoHV-5, cepa SV507-99 e o mRNA extraído em 0, 6, 12, 18, e 24 horas após a infecção (pi) foi utilizado para avaliar a expressão de genes do vírus e da célula hospedeira por qPCR e o mRNA de 1h, 6h e 24h pi foram utilizado no estudo por RNA-seq. Células MDBK não infectadas foram usadas como controle. A qPCR demonstrou que os três genes do BoHV-5 estudados (bICP0, UL9 e US4) apresentaram perfil de expressão semelhante nos diferentes momentos após infecção e que o gene bovino GAPDH teve sua expressão diminuída pela infecção viral. Ao menos 900 genes foram diferencialmente expressos para cada momento de infecção na análise por RNA-seq. Estes genes estavam principalmente relacionados a vias celulares de controle de ciclo celular, produção de interleucinas, quimiotaxia para células inflamatórias, reparo e dano ao DNA, resposta a vírus, apoptose, processo oxidativo, e ubiquitização de moléculas. Estes resultados representam novos conhecimentos sobre a interação entre o BoHV-5 e as células bovinas e podem representar um ponto de partida para novas pesquisas e melhor compreensão da patogenia deste vírus / Bovine herpesvirus 5 (BoHV-5) is an Alphaherpesvirus that causes nonsuppurative meningoencephalitis mainly in young cattle. This disease occurs naturally in either outbreaks or isolated cases, and exhibits low morbidity and high lethality. Besides BoHV-5 epidemiology, pathological findings and diagnosis were well known, the molecular interactions between host cell and BoHV-5 are poorly understood. Molecular biology techniques such as quantitative PCR (qPCR) and RNA sequencing (RNA-seq) are important tools to study virus and host cell interactions. In this study we infected MDBK cells and use the extracted mRNA at 0, 6, 12, 18 and 24h post infection (pi) to analyse BoHV-5 (bICP0, UL9 e US4) and host cell gene (GAPDH) expression using qPCR and 1h, 6h and 24h pi in the RNA-seq study. Mock-infected cells were used to control purpouse. The qPCR releveled that the three BoHV-5 genes showed the same expression behavior during the infection and the GAPDH gene were down-regulated in the infected group. At least 900 genes were differentially expressed during BoHV-5 in each moment analysed by RNA-seq. These genes were up- or down-regulated and mainly associated with cell cycle, interleukin production, inflammatory cells chemotaxis, DNA damage and repair, response to virus, apoptosis, oxidation-reduction process, and ubiquitination pathways. The results demonstrate new insights about BoHV-5 and bovine cells interactions and could be a starting point to new researches and to better understand the pathology of this virus