Background: Herpes simplex virus type 2 (HSV-2), the primary cause of genital herpes, is one of the most widespread, lifelong sexually transmitted infections (STIs). Incidence is disproportionately higher in women compared to men, so a better understanding of vaginal transmission, the primary mode for HSV-2 infection in women, is crucial for developing preventative strategies. Female sex hormone, estrogen (E2), has been shown to play a protective role against sexually transmitted viral infections and previous studies have shown that vaginal epithelial cells treated with E2 are protected against HSV-2 infection; however, the underlying mechanism of E2 protection remains unclear, so a transcriptome analysis followed by functional studies was performed. Method of Study: In this study, VK2/E6E7 (vaginal epithelial) cells were used to study HSV-2 entry, infection and replication. VK2s were grown in Air-Liquid-Interface (ALI) cultures, allowing for their proliferation and stratified layer formation in transwells; closely mimicking physiological conditions. Media was supplemented with no hormone (NH) or physiological concentrations of E2, P4 and MPA for 7 days. After 24 hours of HSV-2 infection in these cultures, VK2 cells were lysed and processed for RNA isolation. We performed a comprehensive genome-wide microarray to profile gene expression of VK2 cells pre-treated with and without E2, prior to, and following HSV-2 infection. For data analysis, “R” software was used to perform all pre-processing steps and normalization. Gene Set Enrichment Analysis (GSEA) was performed to identify potential cellular pathways regulated by E2 after infection using the Hallmark database, relative to NH conditions. Immunofluorescence staining was used for functional analysis to confirm transcriptomic data. After selecting a pathway for investigation, small-molecule inhibitors and activators of this pathway were used in combination with NH or E2. Vero plaque assay and HSV-2-GFP infection were used to identify examine the protective effects of E2 and the selected pathway. In addition, we also used siRNA to specifically knockdown proteins part of the pathway and investigate the specific effects on protection against HSV-2. Results: Microarray analysis indicated that exposure to HSV-2 in the presence of E2 resulted in differential transcriptional profile compared to NH and P4. GSEA assigned one of the highest enrichment scores to the p53 pathway compared to other pathways under the influence of E2 compared to NH, following HSV-2 infection. Studies to correlate bioinformatic results with functional analysis showed significant increase in p53 protein expression after E2 treatment compared to NH. Vero plaque assay demonstrated 10-fold decrease in viral replication following E2 treatment as well as by direct activation of p53 in absence of E2. In contrast, p53 inhibition even in the presence of E2 resulted in 100-fold increased viral replication compared to E2 alone, suggesting that the p53 is involved in E2-mediated protection. We deduced that E2 particularly affects HSV-2 replication and not entry into VK2 cells. We also found that BST2 is strongly regulated by E2-mediated p53 and also contributes to protection against HSV-2. Lastly, we demonstrated that E2 demonstrates anti-inflammatory effects that correlate with its increase in barrier integrity seen with VK2s. Conclusions: With bioinformatic and functional analysis, we found that E2 provides protection through the p53 pathway, as well as through downstream BST2. Our data provides the first comprehensive overview of host cellular responses to HSV-2 and female sex hormones at a transcriptional level and highlights the protective role of E2-mediated p53 pathway. This study is the first to deduce the antiviral mechanism of E2 against HSV-2 infection in human vaginal epithelial cells. / Thesis / Master of Science (MSc)

Background: Despite the availability of a yearly vaccine and antivirals, the incidence of influenza infections remains high. The genome of the influenza virus can mutate rapidly, therefore novel influenza strains that may be resistant to the current vaccine or antivirals frequently enter the population. Because of the long production time necessary to produce a vaccine, new antivirals must be created to combat early stages of influenza outbreaks. The most effective antivirals will target a highly conserved and essential stage of virus replication. The influenza RNA-dependent RNA polymerase is a heterotrimeric complex composed of three subunits: PA, PB1, and PB2. The three components of the polymerase interact through well-defined domains and are essential for viral replication. Previously, influenza replication has been inhibited using a small synthetic peptide that mimics the interaction domain between PA and PB1 and inhibits the formation of the heterotrimeric complex. Problem and Hypothesis: Although the peptide could inhibit influenza replication, synthetic peptides are costly to produce and are not a viable option for large-scale production. This problem can often be overcome by attaching the peptide to a highly soluble carrier protein. We hypothesize that influenza replication can be inhibited by attaching a peptide, that mimics the binding domain between the PA and PB1 subunits, to a human elastin-like polypeptide (ELP) carrier protein. Methods and Results: The peptide and a nuclear localization sequence was genetically linked to a maltose binding protein (MBP) or ELP carrier protein. The MBP construct was purified by affinity chromatography using FPLC. A high yield of the ELP construct was obtained using inverse transition cycling, a method unique to ELPs because of their temperature-dependent solubility. The ELP construct was designed to be soluble at physiological temperature to limit cellular toxicity due to protein aggregation. The cytotoxicity of the ELP construct was assessed by monitoring the growth of A549 cells, a human lung epithelial cell line. The ELP construct did not have any adverse effects on A549 cell growth. Both constructs could localize to cell nuclei using their respective nuclear localization sequences and could also interact with the PA subunit, demonstrating their potential to inhibit influenza replication. Despite this, only the MBP construct was able to inhibit the replication of influenza. The MBP construct could inhibit the replication of both the H1N1 and H3N2 subtypes of influenza, indicating the recombinant protein had cross-strain activity. Conclusion: Linking a small peptide to carrier protein can result in high protein yields, however a carrier protein must be chosen that will maintain the peptides’ therapeutic activity. In this study, a small anti-influenza peptide inhibited influenza replication when attached to an MBP carrier protein, however was not able to inhibit influenza replication when attached to an ELP carrier protein. Although the peptide was ineffective when attached to this particular ELP carrier protein, different ELP proteins of various lengths and compositions may still be effective carrier proteins for an antiviral peptide. / Thesis / Master of Science (MSc) / The influenza virus causes seasonal outbreaks each year and can have life-threatening symptoms in the young and elderly. In addition, it can rapidly mutate through antigenic drift; therefore, a new vaccine is required each year. Pandemic influenza strains can enter the population when the virus undergoes genetic reassortment by antigenic shift. However, it can take a significant amount of time to formulate a vaccine against pandemic influenzas, which means antiviral drugs are often used as the first line of defense. New antivirals to treat influenza must be developed because resistance to the current influenza antivirals has steadily increased. In this work, we developed an antiviral peptide to disrupt a critical interaction in the influenza RNA-dependent RNA polymerase and inhibit virus replication. This peptide was previously conjugated to an E. coli MBP carrier protein, which would likely not be compatible in vivo. This thesis focused on attaching the antiviral peptide to an elastin-like polypeptide protein, which mimics human tropoelastin, and should be non-immunogenic in humans.

Background: Respiratory syncytial virus (RSV) is one of the leading causes of acute lower respiratory tract infection and childhood hospitalization worldwide. However, there are currently no vaccines or antivirals available to prevent or treat RSV infections. Of the 11 proteins encoded by RSV’s negative-sense single-stranded RNA genome, the nucleoprotein, phosphoprotein, and large polymerase interact through well characterized domains to form the RNA-dependent RNA polymerase complex. This polymerase complex is essential for viral replication and virulence, which makes it an excellent antiviral target. Previous studies have shown that the nucleoprotein-phosphoprotein interaction of the polymerase complex can be disrupted by synthetic peptides of the last 21 C-terminal (P220-241) or the first 29 N-terminal (P1-29) amino acids of the phosphoprotein. Objective: The Mahony lab has also previously demonstrated that P220-241 conjugated to a maltose binding protein (MBP) and HIV-1 Tat cell penetrating peptide (CPP) could inhibit up to 90% of RSV A replication in vitro. However, the bacterial derived MBP is immunogenic. This study builds on these findings by developing and evaluating the efficacy of a P220-241 peptide mimetic conjugated to human thioredoxin (hTrx) carrier protein and a P1-29 peptide mimetic conjugated to MBP. Methods and Results: Inverse PCR and In-Fusion® cloning was used to clone a hTrx-P220-241 plasmid, which was then expressed as a recombinant protein and purified by affinity chromatography for functional analysis. HTrx-P220-241 was shown to specifically interact with RSV nucleoprotein in a glutathione S-transferase (GST) pull down assays and it could successfully enter into LLC-MK2 cells. However, upon challenge with RSV A, LLC-MK2 cells that were incubated with increasing concentrations of hTrx-P220-241 did not inhibit RSV A replication when assessed by indirect immunofluorescence microscopy. The MBP-P1-29 construct did not exhibit any significant cytotoxicity in LLC-MK2 cells nor BEAS-2B cells. Upon challenge with RSV A, LLC-MK2 cells and BEAS-2B cells pre-treated with MBP-P1-29 demonstrated a dose-dependent inhibition of RSV replication in vitro, with a percent inhibition of infection of 80% and 60% respectively. Furthermore, MBP-P1-29 also reduced the release of infectious progeny virion by up to 74% in LLC-MK2 cells and 34% in BEAS-2B cells. Conclusion: Phosphoprotein peptide mimetics targeting essential nucleoprotein-phosphoprotein interaction are a promising approach in the development of therapeutic treatments for RSV. In this study, a P220-241 peptide mimetic conjugated to a human thioredoxin scaffold protein was not able to inhibit RSV A replication while a P1-29 peptide attached to a maltose binding protein was effective in reducing RSV replication in vitro. Thus, further studies are required to evaluate a P1-29 peptide mimetic against different RSV A and B strains and to find an appropriate human carrier protein to attach it to. / Thesis / Master of Science (MSc) / Respiratory syncytial virus is a respiratory illness that is one of the leading causes of childhood hospitalization worldwide. RSV infects almost all infants at least once by the age of two. It can also repeatedly infect individuals throughout their lives, which puts the elderly and individuals with weak immune, cardiac or pulmonary systems at risk. There are also no approved vaccines or antiviral treatments available to prevent or combat a RSV infection, which highlights the pressing need for the development of new antiviral drugs. This thesis focuses on developing and evaluating the efficacy of two different antiviral peptides, which both target and disrupt the formation of the viral machinery required for the replication of the RSV genome.

The emergence of viruses with zoonotic potential, i.e. with the potential ability to cross species barriers to infect unnatural hosts, poses a huge threat to humans. It is therefore essential to develop new methodologies to rapidly and efficiently generate attenuated virus vaccine candidates to attempt to control the threat. Viruses need to be able to at least partially inhibit the host’s innate defence mechanism, known as the interferon (IFN) system, to replicate efficiently in vivo and establish a productive infection. It has been previously reported that viruses that have lost their ability to circumvent the host’s IFN response, or IFN-sensitive viruses, are promising candidates for live attenuated virus vaccines. Here we report on the development of a cell-based method to attempt to rapidly select IFN-sensitive viruses that can not block IFN signalling, from wild-type virus populations. Lentivirus vectors containing selection markers (HSV-tk – Herpes Simplex virus thymidine kinase gene and pac – puromycin resistance gene) under the control of a tight IFN-inducible promoter (the murine Mx1 promoter) were generated and used to specifically engineer HEp2 cell lines, termed Mx GIPSE and Mx TIPSE, for this purpose. The developed methodology relies on the engineered cell lines and a selection procedure using exogenous IFN-α and puromycin: if a cell is infected with IFN-resistant virus, it will die in the presence of IFN-α and puromycin because IFN signalling will be blocked, thereby blocking the activation of the Mx1 promoter and consequent expression of pac; if a cell is infected with an IFN-sensitive virus, it will survive in the presence of IFN-α and puromycin because the Mx1 promoter will become activated through the IFN signalling pathway, leading to the expression of pac. IFN-sensitive viruses can then be rescued from the surviving cells, and amplified using IFN-permissive cell lines expressing viral IFN antagonist proteins (proteins that block the host’s IFN response). When tested on PIV5 strains CPI- (an IFN-sensitive virus) and CPI+ (an IFN-resistant virus), the developed method allowed the survival and amplification of cells infected with CPI-, whilst cell death was observed for cells infected with CPI+. Whilst the developed methodology seems promising, further developments of the system are required. The possibilities of using the developed methodology in combination with other techniques, such as FACS sorting and immune selection, to rapidly select IFN-sensitive mutant viruses from wild-type and mutagenised virus populations are discussed. The potential to use Mx TIPSE cells to select IFN-resistant revertant viruses from IFN-sensitive virus populations is also discussed. In addition, a high throughput screening assay has been developed using the engineered Mx GIPSE and Mx TIPSE cell lines to search for compounds that block IFN signalling or that block the action of viral IFN antagonist proteins. Compounds that block IFN signalling would potentially be useful as anti-inflammatory drugs whilst compounds that block the action of viral IFN antagonist proteins would be valuable as antiviral drugs.

The drug, N,N bis (ethylene)-P (1-adamantyl) phosphonic diamide inhibits focus formation of Rous Sarcoma Virus in tissue culture. Transformation of chick cells was inhibited when the drug was added to chick cells prior to infection. The drug did not inhibit the transformation of Normal Rat Kidney Cells infected with RSV, when the cells were grown at non-permissive temperatures and shifted to permissive temperatures upon addition of the drug. Nor did the drug revert cells transformed at permissive temperatures. These studies indicated that the inhibition of RSV is in the early stage of viral growth, possible penetration or uncoating.

A dengue é a mais importante arbovirose no mundo; aproximadamente 50 milhões de infecções ocorrem anualmente acarretando 500.000 casos de dengue hemorrágica e 22.000 mortes. A febre amarela é uma doença hemorrágica viral com elevada mortalidade que é transmitida por mosquitos. Vacinas eficazes contra a febre amarela já estão disponíveis há quase 70 anos e são responsáveis por uma redução significativa de ocorrências da doença no mundo, no entanto, cerca de 200.000 casos de febre amarela ainda ocorrem anualmente, principalmente na África. Dessa forma, o desenvolvimento de fármacos antivirais contra essas viroses é uma prioridade de saúde pública. Os produtos naturais sejam de origem vegetal ou animal, possuem uma extensa diversidade química, sendo uma fonte inesgotável de compostos com promissoras atividades biológicas. No Brasil, é grande a incidência de animais venenosos ou peçonhentos, tais como serpentes, sapos e escorpiões. Os venenos desses animais são fontes de diversas substâncias químicas que ainda não possuem a sua atividade biológica e farmacológica completamente estudada. Neste trabalho avaliamos a potencial ação antiviral de peçonhas de serpentes (Crotalus durissus terrificus, Bothrops jararacussu, Bothrops jararaca, Bothrops pirajai, Bothrops moojeni, Bothrops brasili e Bothrops fonseca) e escorpião (Tityus serrulatus) contra os virus da febre amarela e dengue usando diferentes estratégias metodológicas (pré-tratamento, pós-tratamento, virucida, adsorção e internalização). Primeiramente realizamos um screening com as peçonhas brutas, observando que a peçonha de Crotalus durissus terrificus inibiu a replicação viral apresentando os maiores índices de seletividade (IS). Crotoxina, crotamina, crotapotina, convulxina, giroxina, PLA2-CB e PLA2-IC, isoladas de Crotalus durissus terrificus, foram então testadas nas diferentes estratégias metodológicas contra os vírus dengue e febre amarela. Foi possível verificar que crotoxina, PLA2-CB e PLA2-IC inibiram a replicação viral com altos índices de seletividade (IS). A ação verificada ocorreu na fase inicial do ciclo de replicação viral (pré-tratamento, virucida, adsorção). A ação antiviral verificada neste estudo foi atribuida a ação da PLA2, visto que a crotoxina é um complexo protéico composto pela crotapotina e pela PLA2-CB. Posteriormente avaliamos uma fosfolipase sem atividade catalítica isolada de Bothrops jararacussu, a BthTX-I. Essa fosfolipase apresentou baixa inibição da replicação viral, sugerindo que a atividade catalítica da fosfolipase é importante, mas possivelmente não a única responsável pela ação antiviral. Os resultados obtidos permitem sugerir também que as fosfolipases apresentam ação tanto sobre a partícula viral quanto sobre receptores celulares, o que justifica os altos índices de seletividade observados. / Dengue is the most important arbovirus disease in the world; nearly 50 million infections occur annually resulting in 500,000 cases of DHF and 22,000 deaths. Yellow fever is a viral haemorrhagic fever with high mortality that is transmitted by mosquitoes. Effective vaccines against yellow fever have been available for almost 70 years and are responsible for a significant reduction of the disease worldwide. However, about 200,000 cases of yellow fever still occur annually, mainly in Africa. Thus, the development of antiviral drugs against these viruses is a public health priority. Natural products of plant or animal origin have an extensive chemical diversity, and an inexhaustible source of compounds with promising biological activities. In Brazil, there is a high incidence of poisonous or venomous animals such as snakes, frogs and scorpions occur. The venoms of these animals are a source of several chemicals that does not possess biological and pharmacological activity completely studied. In this study, we assess the potential antiviral action of snake venom (Crotalus durissus terrificus, Bothrops jararacussu, Bothrops jararaca, Bothrops pirajai, Bothrops moojeni, Bothrops brasili and Bothrops fonseca) and Scorpion (Tityus serrulatus) against yellow fever and dengue viruses using different methodological strategies (pre-treatment, post-treatment, virucidal, adsorption and internalization). First, we performed a screening with the crude venoms, founding that the venom of Crotalus durissus terrificus inhibited viral replication showing the highest selectivity index (SI). Crotoxin crotamin, crotapotin, convulxin, gyroxin, PLA2-CB and PLA2-IC isolated from Crotalus durissus terrificus, were then tested in the different methodological strategies against dengue and yellow fever viruses. We found that crotoxin, PLA2-CB and PLA2-IC inhibited viral replication with high SI. The action of these compounds against the virus was at the first steps of the replication cycle (pre-treatment, virucidal, adsorption). The antiviral action observed in this study was attributed to the action of PLA2, since crotoxin is a protein complex composed of crotapotin and PLA2-CB. Afterwards, we evaluated a phospholipase without catalytic activity isolated from Bothrops jararacussu, the BthTX-I. This phospholipase showed low inhibition of viral replication, showing that the catalytic activity of phospholipase is important, but perhaps not the only one responsible for the antiviral action. Our results also suggest that phospholipases have action on the viral particle and on cell receptors, which explains the high levels of selectivity observed.

A raiva é uma zoonose que afeta todos os mamíferos e causa cerca de 55.000 mortes humanas por ano, causada pelo vírus da raiva. O vírus da raiva pertence à Ordem Mononegavirales, Família Rhabdoviridae e o Gênero Lissavirus. Atualmente, o tratamento humano se baseia no uso do Protocolo de Milwaukee composto de indução do paciente ao coma e uso de massiva terapia antiviral. O protocolo, apesar de ter sido utilizado duas vezes com sucesso, inclusive em um caso brasileiro, ainda requer aprimoramentos. Neste sentido, a interferência por RNA (RNAi) é uma nova abordagem para terapia de doenças virais. O objetivo deste trabalho foi avaliar a inibição da replicação do vírus da raiva in vitro e in vivo utilizando RNAi. Para tanto, foram utilizados três siRNAs (siRNA 124, siRNA 750, siRNA B) com a fita antisenso complementar ao mRNA da nucleoproteína (N) do vírus da raiva. Para o ensaio in vitro foram utilizadas a cepa PV do vírus da raiva e as células de BHK-21 (Baby hamster kidney). As monocamadas celulares foram infectadas com a cepa PV e depois de 2 horas de incubação transfectadas com cada um dos siRNAs em combinação com Lipofectamine 2000®. Depois de 22 horas as placas teste e controle foram submetidas à imunofluorescência direta (IFD) com conjugado globulina de coelho anti-nucleocapsídeo do vírus da raiva/isotiocianato de fluoresceína (BIO-RADTM). Os resultados revelaram títulos de 5,71logTCID50/ml, 5,56logTCID50/ml e 5,65logTCID50/ml para os siRNAs 124, B e 750, respectivamente, enquanto que, para a placa controle, o título foi 6,43logTCID50/ml. Para o ensaio in vivo, foram usados camundongos albino suíços de 21 dias com peso entre 11 e 14g, infectados com a cepa PV via intracerebral. Duas horas depois da infecção foi inoculada por via intracerebral uma solução do \"pool\" dos três siRNAs com Lipofectamine 2000® . Os animais com paralisia foram sacrificados e aqueles sobreviventes foram observados até completar 30 dias de observação quando foram, então, sacrificados. O sistema nervoso central de todos os animas foi recolhido e submetido a IFD. O título viral do grupo teste foi 7.03logLD50%/ml e do grupo controle 7.13logLD50%/ml. O resultado do ensaio in vitro demonstra que os siRNAs utilizados são efetivos em inibir a replicação do vírus da raiva, com eficiências equivalentes. A utilização do \"pool\" dos três siRNA em camundongos resultou em 30% de animais sobreviventes frente a 100 DL50% do vírus PV, enquanto que a mesma dose levou a 100% de mortalidade nos animais não tratados. Pode-se atribuir a menor eficiência em inibir a replicação do vírus da raiva in vivo quando se compara com os resultados in vitro possivelmente às elevadas doses virais utilizadas. Estes resultados, ainda que indiquem a necessidade de mais estudos, permitem concluir que a RNAi é uma tecnologia promissora como antiviral contra a raiva. / Rabies is a zoonotic disease that affects all mammals and causes more than 55.000 human deaths every year, caused by rabies virus (RABV) a virus of the Mononegavirales order, Family Rhabdoviridae and the Lissavirus genus. After the onset of the symptoms, the illness has a fast progression and the patients feel intense physical suffering. Currently, human rabies treatment has been based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. Despite this protocol has been successful in two cases, including a Brazilian case (Recife State), more studies on antiviral for human rabies treatment are required. RNA interference is a new antiviral approach, which gives hope to the possibility of rabies antiviral treatment. The aim of this study was to assess the decrease in the titer of rabies virus in vitro and in vivo using short-interfering RNAs. For this purpose, three siRNAs (siRNA 124, siRNA B, siRNA 750) were used with antisense strands complementary to rabies virus nucleoprotein (N) mRNA. Pasteur virus strain (PV) of rabies virus and BHK-21 cells were used, and the monolayers were transfected with each of tree RNAs with Lipofectamine-2000. After 22 hours, the siRNA-treated and the control plates were tested by direct fluorescent antibody test (DFAT) with anti-rabies virus nucleocapsid antibody conjugate with fluorescein isothiocianate. Virus titers were calculated by the Spearman-Karber method. The results revealed that all three siRNAs reduced the titer of PV strain and a more intense effect was obtained with siRNA B. The titer of the PV strain in the control plate was 6.43lg TCID50%/ml and 5.56lg TCID50%/ml in the plate treated with siRNA B, respectively. The similar result was obtained in plates treated with siRNAs 124 and 750. The title of PV strain of these plates was 5.71lg TCID50%/ml of siRNA 124 and 5.65lg TCID50%/ml of siRNA750, respectively. No cytopathic or cytotoxic effect was observed in the monolayers treated with Lipofectamine-2000®. Swiss albino mice with 21 days weighing between 11 and 14g infected with strain PV by the intracerebral route used in this in vivo essay. After two hours of infection, a pool solution of 3 siRNAs with lipofectamine was also inoculated by the intracerebral route. The animals with paralysis were euthanized and those which survived were observed until the 30th day when they were also euthanized. The central nervous system of all animals were collected and induced to IFD. The title viral test group was 7.03logLD50%/ml and the control group was 7.13logLD50%/ml. The in vitro test results indicate that siRNAS are effective in inhibiting the replication of rabies virus with similar efficiencies. The use of the pool of three siRNA in mice resulted in 30% of survivors for 100 LD50% of PV virus, while the same dose led to 100% mortality in untreated animals. A lower efficiency in inhibiting the replication of rabies virus in vivo when compared with results in vitro could be possibly due to the high viral doses used. These results, although indicative of the need for further studies, show that RNAi is a promising technology as antiviral against rabies.

Ren-Wang Jiang. / "July 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

HBV DNA replicates through a genomic RNA intermediate. The HBV reverse transcriptase lacks proof-reading activity, resulting in a much higher mutation rate for the HBV genome compared with other DNA viruses. HBV DNA thus is often present in quasi-species in an individual. One or more species may be favorably selected by factors like host immune clearance and use of antiviral drugs. / Hepatitis B virus (HBV) infected millions of people worldwide. Chronic HBV infection is the leading cause of liver cirrhosis and hepatocellular carcinoma (HCC). / In summary, this study developed and validated two platforms for (1) HBV mutation discovery; and (2) HBV mutation detection in viral quasi-species. These tools may be useful for research on HBV drug resistant mutations, clinical instructing and monitoring of antiviral treatment. / In this study, I have developed high-throughput methods for (1) discovery of novel HBV mutations; and (2) highly multiplexed detection of known HBV mutations, both in the background of HBV quasi-species. Patients undergoing long-term lamivudine treatment were used for mutation discovery. For mutation discovery in quasi-species, the MassCLEAVE(TM) technology, a method based on base-specific RNA cleavage and automated Matrix Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS), was used. I found that MassCLEAVE(TM) can be used to discover mutations present as minorities. Additionally, a synergistic effect was found between direct sequencing and MassCLEAVE(TM) in identifying minority mutations. Multi-PLEX, a method based on single nucleotide extension and automated MALDI-TOF MS, was used to develop a highly multiplexed assay for simultaneous detection of 60 HBV mutations including all functionally known HBV mutations and other frequently observed mutations during antiviral treatment with unknown functions. This multiplex assay was tested on a large cohort of single and multiple drug-resistant patients and was shown to be highly accurate in detecting HBV viral mutations in quasi-species. / Nucleotide and nucleoside analogues (NAs) are widely used for antiviral therapy by effectively suppressing viral DNA replication. However, long-term administration may select for drug-resistant mutant strains, leading to treatment failure and liver disease progression. A number of HBV mutations such as rtM204V/I, rtN236T and rtL180M within the HBV reverse transcriptase are known to confer drug resistance. Detection of these known mutations is useful genotypic markers for monitoring antiviral treatment. In addition, novel drug resistant mutations continue to be discovered. / by Luan, Ju. / Adviser: Chunming Ding. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 136-149). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Em 2009, foi demonstrado um potente antiviral na hemolinfa de L. obliqua. Esta proteína purificada reduziu o título viral (TCID50 ml-1) do sarampo, pólio e H1N1 em 157, 61 e 64 vezes. Recentemente, expressamos a proteína antiviral em sistema baculovírus, que reduziu o título de vírus do herpes em 106 vezes, da rubéola e EMC em 105 vezes. No entanto, este sistema de expressão é muito caro e trabalhoso. Com isso, clonamos e expressamos esta proteína com atividade antiviral em sistema bacteriano. A sequência antiviral foi clonada em vetor de expressão pET28a. As construções resultantes foram expressas em E. coli Bl21 DE3 pLysS induzidas com IPTG 1,0 mM. Após expressão, o pellet bacteriano foi sonicado e o material foi purificado por afinidade e gel filtração. Testou-se contra o vírus EMC mostrando uma proteção de 104 vezes em relação ao controle. E utilizando qPCR para determinar os níveis de transcrição de RNA do vírus do herpes e da rubéola em células infectadas mostrou uma inibição respectivamente de 106 e 105 vezes, em relação ao controle. / In 2009, we demonstrated a potent antiviral in the hemolymph of L. obliqua. This purified protein has reduced the viral titer (TCID50 ml-1), measles and polio H1N1 157, 61 and 64 times. Recently, the antiviral protein expressed in baculovirus system, which reduced the title of the herpes virus in 106 times, rubella and EMC in 105 times. However, this expression system is very expensive and laborious. Thus, cloned and expressed this protein with antiviral activity in bacterial system. The antiviral sequence was cloned into pET28a expression vector. The resulting constructs were expressed in E. coli BL21 DE3 pLysS cells induced with 1.0 mM IPTG. Following expression, the bacterial pellet was sonicated and the material was purified by affinity and gel filtration. Was tested against EMC virus showing a protection of 104 times compared to the control. And using qPCR to determine the levels of viral RNA transcription of the herpes virus and rubella-infected cells showed an inhibition of respectively 106 and 105 times compared to the control.