Submitted by Amanda Silva (amanda.osilva2@ufpe.br) on 2015-03-13T12:36:29Z No. of bitstreams: 2 TESE Mizia Maria Saboia da Silva.pdf: 10007532 bytes, checksum: 91431b8930bbf3d4ce589ead57ca6b70 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-13T12:36:29Z (GMT). No. of bitstreams: 2 TESE Mizia Maria Saboia da Silva.pdf: 10007532 bytes, checksum: 91431b8930bbf3d4ce589ead57ca6b70 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2014 / CAPES / O diagnóstico laboratorial da Dengue é fundamental para determinar os cuidados clínicos com o paciente, apoiar os programas de vigilância epidemiológica, pesquisar formulação de vacinas e também para a detecção precoce de uma possível epidemia. A proteína não estrutural 1 (NS1) do vírus Dengue é um marcador utilizado durante a fase aguda da enfermidade e tem sido proposto para o diagnóstico da doença. Atualmente, para diagnóstico da NS1 são usados os ensaios imunoenzimáticos e testes imunocromatográficos. Os imunossensores são dispositivos bioanalíticos que convertem a resposta da interação antígeno-anticorpo em um sinal elétrico, passível de quantificação. Recentemente, a contribuição de nanomateriais a estes dispositivos tem possibilitado aumento na reprodutibilidade e alcance de baixos limites de detecção tornando os imunossensores ferramentas promissoras para diagnóstico clínico. Nesta tese foram desenvolvidos dois imunosensores a base de nanomaterias para a detecção NS1, um marcador importante na infeção aguda da dengue. O primeiro imunossensor, constituído por um eletrodo de carbono vítreo (ECV), foi baseado no uso nanotubos de carbono de parede múltiplas carboxilados (NTCPMs-COOH) recoberto por um filme formado por deposição do Hidrocloreto de Polialilamina (PAH). Anticorpos anti-NS1 foram imobilizados de modo orientado via grupos aminos do PAH. De acordo com os resultados, o imunossensor desenvolvido exibiu uma faixa linear variando entre 0,1 μg mL-1 e 2,5 μg mL-1 de NS1, faixa clínica para diagnóstico precoce na fase aguda da doença. Uma boa correlação foi encontrada entre a concentração de NS1 e a mudança da corrente, mostrando um bom limite de detecção (0.035 μg mL-1). O segundo imunossensor foi baseado em eletrodos impressos usando a transdução eletroquímica, visando o desenvolvimento de testes point-of-care. Os eletrodos impressos foram fabricados com um composto de tinta de carbono-Tiofeno seguidos por um filme de nanopartículas de ouro revestidas com proteína A (AuNP-PtnA) que orientaram a imobilização dos anticorpos anti-NS1. Um imunoensaio direto foi realizado, no qual a captura específica da NS1 foi avaliada através das reações da uma sonda redox com a superfície do eletrodo. De acordo com os resultados, foi observado que o uso do tiofeno na tinta de carbono aumentou significativamente a sensibilidade do eletrodo em 70% em relação ao eletrodo sem modificação. A curva de calibração do sensor mostrou uma faixa de resposta linear entre 0.05 – 0.6 μg mL-1 de NS1 e um limite de detecção de 0.015 μg mL-1. Os imunossensores propostos apresentam-se como tecnologias inovadoras ainda não disponíveis no mercado de sensores. Ambos imunossesores apresentaram o uso combinado de tecnologias eletroquímicas com nanomateriais que contribuiu para uniformização da plataforma sensora, melhorara da estabilidade e reprodutibilidade dos eletrodos.

A dengue é uma doença negligenciada que carece de métodos diagnósticos rápidos nos primeiros dias de infecção. São quatro sorotipos diferentes, cuja monitoração é essencial para o controle da ocorrência de casos graves como a dengue hemorrágica. É urgente o desenvolvimento e disponibilização de um dispositivo capaz de suprir essa demanda, de modo que propomos a utilização de imunossensores potenciométricos, devido a facilidade de miniaturização e produção dos dispositivos e seu baixo custo, além da possibilidade de detecção direta (sem marcadores) e simplicidade de manuseio. Dispositivos sensores de pH, como o transistor de efeito de campo de porta estendida e separada (SEGFET) e amplificadores de instrumentação (AI) podem ser utilizados como transdutores de sinal para a reação antígeno-anticorpo, a partir da utilização de materiais não nernstianos, como o ouro, como plataforma sensível. A proteína NS1 do vírus da dengue é um excelente marcador da infecção, pois é secretada em altas concentrações pelo vírus no sangue de pessoas infectadas logo nos primeiros dias, de modo que o sistema preza pelo diagnóstico precoce da doença. Sua detecção é realizada através da imobilização de anticorpos anti-proteína NS1 na plataforma sensível, permitindo sua quantificação através da detecção da alteração local de carga. O eletrodo foi caracterizado por diversas técnicas de microscopia, entre elas de varredura, confocal e de força atômica, além da utilização de espectroscopia de impedância eletroquímica, permitindo um amplo conhecimento da superfície da membrana sensível. Os imunossensores desenvolvidos apresentaram alta sensibilidade, com capacidade de detecção da ordem de ng.mL-1. Na região linear da curva analítica, foram obtidos sensibilidade correspondente a (15.7 ± 4.4) .10-4 μA.μg.mL-1 para o SEGFET e (3.2 ± 0.3) mV.μg.mL-1 para o AI, sendo que este último apresenta uma maior estabilidade de sinal e dispensa a utilização de uma fonte variável de tensão, reduzindo o custo no desenvolvimento de um dispositivo diagnóstico comercial. Estes resultados levaram a um pedido de patente e o prosseguimento do projeto através da miniaturização do sistema e detecção em amostras reais. / Dengue is a neglected disease that lacks fast diagnosis methods in the first days of infection. There are four different serotypes, which monitoring is essential to the occurrence control of severe cases as dengue hemorrhagic fever. The development of a device capable of fulfilling this demand is urgent, so we propose the use of potentiometric immunosensors, since its ease of miniaturization, mass production, low cost and the possibility of direct detection (label-free). pH sensor devices, as the separated extended gate field effect transistors (SEGFET) and instrumentation amplifiers (AI) can be applied as transducers to the antibody-antigen reaction by using non-nernstian materials such as gold as sensitive membrane. The non-structural 1 (NS1) protein is an excellent marker of infection, since its secreted in high concentration in the blood of infected people by the dengue virus in the first days, prioritizing early diagnosis. Its detection is made by immobilization of anti-NS1 protein antibodies, allowing its quantification by local charge changes. The electrode was characterized by many microscopy methods, including scanning electron, confocal and atomic force, besides electrochemistry impedance spectroscopy, providing a wide knowledge of the membrane surface. The developed immunosensors showed high sensitivity with detection capacity in the order of ng.mL-1. In the linear range of the analytic curve, were obtained sensitivities of (15.7 ± 4.4) .10-4 μA.μg.mL-1 for the SEGFET and (3.2 ± 0.3) mV.μg.mL-1 for the AI, whereas the latter has high signal stability sparring the use of a variable voltage source, minimizing the costs in the development of a commercial diagnostic device. These results led to a patent and the project continues by working in miniaturizing and real samples detection.

A dengue é uma doença negligenciada que carece de métodos diagnósticos rápidos nos primeiros dias de infecção. São quatro sorotipos diferentes, cuja monitoração é essencial para o controle da ocorrência de casos graves como a dengue hemorrágica. É urgente o desenvolvimento e disponibilização de um dispositivo capaz de suprir essa demanda, de modo que propomos a utilização de imunossensores potenciométricos, devido a facilidade de miniaturização e produção dos dispositivos e seu baixo custo, além da possibilidade de detecção direta (sem marcadores) e simplicidade de manuseio. Dispositivos sensores de pH, como o transistor de efeito de campo de porta estendida e separada (SEGFET) e amplificadores de instrumentação (AI) podem ser utilizados como transdutores de sinal para a reação antígeno-anticorpo, a partir da utilização de materiais não nernstianos, como o ouro, como plataforma sensível. A proteína NS1 do vírus da dengue é um excelente marcador da infecção, pois é secretada em altas concentrações pelo vírus no sangue de pessoas infectadas logo nos primeiros dias, de modo que o sistema preza pelo diagnóstico precoce da doença. Sua detecção é realizada através da imobilização de anticorpos anti-proteína NS1 na plataforma sensível, permitindo sua quantificação através da detecção da alteração local de carga. O eletrodo foi caracterizado por diversas técnicas de microscopia, entre elas de varredura, confocal e de força atômica, além da utilização de espectroscopia de impedância eletroquímica, permitindo um amplo conhecimento da superfície da membrana sensível. Os imunossensores desenvolvidos apresentaram alta sensibilidade, com capacidade de detecção da ordem de ng.mL-1. Na região linear da curva analítica, foram obtidos sensibilidade correspondente a (15.7 ± 4.4) .10-4 μA.μg.mL-1 para o SEGFET e (3.2 ± 0.3) mV.μg.mL-1 para o AI, sendo que este último apresenta uma maior estabilidade de sinal e dispensa a utilização de uma fonte variável de tensão, reduzindo o custo no desenvolvimento de um dispositivo diagnóstico comercial. Estes resultados levaram a um pedido de patente e o prosseguimento do projeto através da miniaturização do sistema e detecção em amostras reais. / Dengue is a neglected disease that lacks fast diagnosis methods in the first days of infection. There are four different serotypes, which monitoring is essential to the occurrence control of severe cases as dengue hemorrhagic fever. The development of a device capable of fulfilling this demand is urgent, so we propose the use of potentiometric immunosensors, since its ease of miniaturization, mass production, low cost and the possibility of direct detection (label-free). pH sensor devices, as the separated extended gate field effect transistors (SEGFET) and instrumentation amplifiers (AI) can be applied as transducers to the antibody-antigen reaction by using non-nernstian materials such as gold as sensitive membrane. The non-structural 1 (NS1) protein is an excellent marker of infection, since its secreted in high concentration in the blood of infected people by the dengue virus in the first days, prioritizing early diagnosis. Its detection is made by immobilization of anti-NS1 protein antibodies, allowing its quantification by local charge changes. The electrode was characterized by many microscopy methods, including scanning electron, confocal and atomic force, besides electrochemistry impedance spectroscopy, providing a wide knowledge of the membrane surface. The developed immunosensors showed high sensitivity with detection capacity in the order of ng.mL-1. In the linear range of the analytic curve, were obtained sensitivities of (15.7 ± 4.4) .10-4 μA.μg.mL-1 for the SEGFET and (3.2 ± 0.3) mV.μg.mL-1 for the AI, whereas the latter has high signal stability sparring the use of a variable voltage source, minimizing the costs in the development of a commercial diagnostic device. These results led to a patent and the project continues by working in miniaturizing and real samples detection.

The interferon (IFN) system is integral to antiviral innate immunity in vertebrate hosts. Inside a cell, viral pathogen associated molecular patterns (PAMPs) trigger the IFN response, comprised of IFN induction and an IFN-induced antiviral state. However, viruses have evolved strategies to counteract the IFN system. The E3 protein of vaccinia virus (VV), encoded by the E3L gene, impedes cytokine expression and suppresses the activation and function of antiviral proteins. Deletion of the E3L gene (VVΔE3L) produces an IFN sensitive mutant virus that is replication defective in most human cell lines. Due to the limited human cell lines available to support VVΔE3L replication, the capacity of E3 inhibition of human IFN-induced antiviral activities is not well defined. In this study, VVΔE3L was generated and characterized to facilitate the study of other viral IFN antagonists at modulating human IFN-induced antiviral responses. A human liver carcinoma cell line, Huh7, was found to support VVΔE3L replication. A comprehensive analysis of VVΔE3L IFN sensitivity revealed E3 inhibits all human type I and type II IFN-induced antiviral activities by modulation of the protein kinase R (PKR) pathway. Influenza non-structural protein 1 (NS1) is well-known to mediate the suppression of IFN induction and IFN action in influenza virus infections. However, the IFN antagonizing potential of influenza NS1 may be virus subtype and/or isolate specific. VVΔE3L was next applied as an expression vector to study influenza NS1 function in modulating IFN-induced antiviral activities and IFN induction in human cells. Recombinant viruses were generated to express influenza NS1 (from avian H5N1 and pandemic viruses 1918 pH1N1, 1968 pH3N2, and 2009 pH1N1) in replacement of E3. It was found that influenza NS1 inhibits human IFN-induced antiviral activity in a subtype and isolate specific manner. Moreover, influenza NS1 differentially regulates human IFN expression in a virus isolate-dependent manner. Altogether, this work highlights the potential of VVΔE3L as an excellent virus model system to study viral proteins modulating IFN expression and IFN-induced antiviral activities in human cells.

The interferon (IFN) system is integral to antiviral innate immunity in vertebrate hosts. Inside a cell, viral pathogen associated molecular patterns (PAMPs) trigger the IFN response, comprised of IFN induction and an IFN-induced antiviral state. However, viruses have evolved strategies to counteract the IFN system. The E3 protein of vaccinia virus (VV), encoded by the E3L gene, impedes cytokine expression and suppresses the activation and function of antiviral proteins. Deletion of the E3L gene (VVΔE3L) produces an IFN sensitive mutant virus that is replication defective in most human cell lines. Due to the limited human cell lines available to support VVΔE3L replication, the capacity of E3 inhibition of human IFN-induced antiviral activities is not well defined. In this study, VVΔE3L was generated and characterized to facilitate the study of other viral IFN antagonists at modulating human IFN-induced antiviral responses. A human liver carcinoma cell line, Huh7, was found to support VVΔE3L replication. A comprehensive analysis of VVΔE3L IFN sensitivity revealed E3 inhibits all human type I and type II IFN-induced antiviral activities by modulation of the protein kinase R (PKR) pathway. Influenza non-structural protein 1 (NS1) is well-known to mediate the suppression of IFN induction and IFN action in influenza virus infections. However, the IFN antagonizing potential of influenza NS1 may be virus subtype and/or isolate specific. VVΔE3L was next applied as an expression vector to study influenza NS1 function in modulating IFN-induced antiviral activities and IFN induction in human cells. Recombinant viruses were generated to express influenza NS1 (from avian H5N1 and pandemic viruses 1918 pH1N1, 1968 pH3N2, and 2009 pH1N1) in replacement of E3. It was found that influenza NS1 inhibits human IFN-induced antiviral activity in a subtype and isolate specific manner. Moreover, influenza NS1 differentially regulates human IFN expression in a virus isolate-dependent manner. Altogether, this work highlights the potential of VVΔE3L as an excellent virus model system to study viral proteins modulating IFN expression and IFN-induced antiviral activities in human cells.

The genetic requirements for Influenza virus to infect and adapt to new species is largely unknown. To understand the evolutionary steps required by a virus to become virulent, a human virus (A/HK/1/68) (HK), avirulent in mice, was subjected to 20 and 21 serial lung-to-lung passages in mouse. Sequence analysis revealed the emergence of eleven mutations within the NS1 gene of the new virulent strains, many of which occurred in binding sites for transcriptional and translational cellular factors. In the present study we have rescued viruses containing each of the NS1 mouse adapted mutations onto A/PR/8/34 (PR8) backbone. We found 9 of 16 NS1 mutants were adaptive by inducing mortality, body weight loss in BALB/c mice and enhanced virus replication in MDCK cells with properties of host cell interferon transcription inhibition. Sequence comparisons with the highly pathogenic A/Hong Kong/156/1997 (H5N1) and the most severe pandemic A/Brevig Mission/1/1918 (H1N1) NS1 genes showed convergent evolution with some of the mouse adapted viruses for F103L plus M106I and V226I plus R227K mutations respectively. The F103L and M106I mutations in the HK NS1 gene were shown to be adaptive by assessment with respect to replication, early viral protein synthesis, interferon-β antagonism and tropism in the mouse lung. We extended the study and proved increased virulence associated with F103L+M106I mutations in their respective H5N1 NS1 gene on the PR8 and HK backbones, as well as the PR8 NS1 gene and the H9N2 (A/Ck/Bj/1/95) gene in the PR8 and A/WSN/33 backbones respectively. However the V226I and R227K mutations in their respective HK and 1918 NS1 genes slightly enhanced virulence and viral growth at later stages of infection. This study demonstrates that NS1 is a virulence factor; involved in multiple viral processes including interferon antagonism and viral protein synthesis. Furthermore, NS1 mutations acquired during mouse adaptation are proven to be adaptive in human, mouse and avian NS1 genes.

The interferon (lFN) signalling and RNA interference (RNAi) pathways are the major antiviral pathways in animals and plants, respectively. Although the mechanism of RNAi remains to be completely characterised, the genes that encode the proteins involved in this process have been identified in the plant, fungi and animal kingdoms (Fagard et al., 2000, Grishok et aI., 2000, Hall et al., 2003, Kanellopoulou et al., 2005, Kolb et al., 2005); with comparative analyses indicating that RNAi is an evolutionarily conserved mechanism. Several studies have identified RNAi suppressors encoded by animal viruses, suggesting an antiviral role for the RNAi pathway in animals as well as plants (Andersson et al., 2005, Bennasser et al., 2006, Garcia et al., 2006, Li et al., 2004, Lichner et al., 2003, Lingel et al., 2005, Lu & Cullen, 2004, Wang et al., 2006). However, most of these studies were performed in non-mammalian systems and as yet, there is no direct evidence indicating that the RNAi pathway plays a significant antiviral role during the infection of mammalian cells. Interestingly, several viruses have now been shown to express their own microRNA (miRNA) in infected cells (Grey et al., 2005, Pfeffer et al., 2005, Pfeffer et al., 2004, Samols et al., 2005, Sullivan et al., 2005). Further, in the case of hepatitis C virus (HCV), there is evidence that the virus usurps the host cell miRNAs to enhance viral replication (Jopling et al., 2005). The principal aim of this project was to investigate the role of RNAi in mammalian cells during viral infection, particularly infection with the influenza A virus. This thesis is divided into six major chapters followed by a brief general discussion. Chapter 1 contains a general introduction to the RNAi pathway. It describes the history of the discovery of RNAi and summarizes the known and proposed antiviral roles of the RNAi pathway in plants and mammalian cells. Chapter 2 describes the general materials and methods used for this project. There are four main result chapters, each dealing with a specific experimental system. Each chapter is divided into a brief introduction, specific materials and methods used, followed by presentation of the experimental results and a brief discussion. Chapter 3 describes the development of an in vitro Dicer activity assay to study the effect of viral proteins on the activity of the mammalian Dicer protein. It was demonstrated that crude cell lysates derived from influenza A virus infected cells impaired the activity of Dicer and this observation was not due to degradation of the Dicer protein by virally-induced proteases. Chapter 4 describes the use of a GFP reporter assay for screening potential RNAi suppressors. This assay is suitable for studying viral proteins in isolation. The effect of the influenza NS1 protein on the RNAi pathway in HEK293 cells was investigated and it was shown that NS1 could exert modest, but nevertheless significant, suppression of the RNAi pathway. Northern studies, performed to examine the processing of shRNA in the presence of NS1, demonstrated that NSI suppressed the RNAi mechanism through interfering with the maturation ofshRNA into siRNA. Chapter 5 describes the effect of over-expressing components of the RNAi pathway on influenza A virus infection. In these experiments, Exportin 5, which encodes a protein involved in the transport of pre-miRNA/shRNA into the cytoplasm, was over-expressed during influenza A virus infection. Reduced viral infection was observed in cells over-expressing Exportin 5, suggesting that this treatment protects cells from virus infection. Chapter 6 describes the expressed small RNA profile during influenza A virus infection in MDCK cells. Novel canine miRNA homologues were identified through cloning and sequencing. No definitive evidence for virally-derived siRNA/miRNA was found but a general reduction of endogenous miRNA expression was detected.

Proper mRNA nuclear export is essential for harmonious growth and maintenance of a cell. An effective weapon influenza virus employs to hijack a host cell is its ability to inhibit such export. Exactly how influenza virus achieves this inhibition is not fully known. Here, we demonstrate that upon infection, influenza virus degrades two nucleopore proteins (Nup98 and Nup96), which play a key role in mRNA nuclear export. Also, a main virulence factor of influenza virus (non-structural protein 1, NS1) binds directly to NXF1 and E1B-AP5, two key constituents of the mRNA export pathway (NXF1/NXT pathway) responsible for exporting bulk (~70%) mRNA from the nucleus. By increasing the expression levels of members of the NXF1/NXT pathway, we were able to reverse NS1-mediated inhibition of gene expression. On the other hand, by decreasing the levels of members of the NXF1/NXT pathway, we demonstrated that host cells become more sensitive to influenza virus infection and produce more viral particles. These results demonstrate undiscovered influenza-mediated host interactions that may be used to medicinally inhibit influenza virus. To this end, high-throughput screens were designed to identify small molecule antagonists of both NS1-mediated inhibition of gene expression and influenza virus-mediated cell death. Seventy-one compounds were identified, and the most potent molecule (named compound #8) was examined further. We found that compound #8 releases influenza virus-mediated mRNA nuclear export blockage and decreased viral replication and viral gene expression. Thus, the bulk mRNA nuclear export machinery is vital to antiviral response, and compound #8 enhances its ability to fight the cytopathic effects of NS1 and influenza virus. In conclusion, our data demonstrate that the mRNA export machinery is disrupted by influenza virus, and that this machinery also facilitates an antiviral function. We have also shown that these two events can be manipulated chemically to attenuate the negative effect of the virus and enhance the positive antiviral effect of the mRNA export machinery, thereby providing a powerful, new strategy against the ever-present, global threat of influenza virus.

The genetic requirements for Influenza virus to infect and adapt to new species is largely unknown. To understand the evolutionary steps required by a virus to become virulent, a human virus (A/HK/1/68) (HK), avirulent in mice, was subjected to 20 and 21 serial lung-to-lung passages in mouse. Sequence analysis revealed the emergence of eleven mutations within the NS1 gene of the new virulent strains, many of which occurred in binding sites for transcriptional and translational cellular factors. In the present study we have rescued viruses containing each of the NS1 mouse adapted mutations onto A/PR/8/34 (PR8) backbone. We found 9 of 16 NS1 mutants were adaptive by inducing mortality, body weight loss in BALB/c mice and enhanced virus replication in MDCK cells with properties of host cell interferon transcription inhibition. Sequence comparisons with the highly pathogenic A/Hong Kong/156/1997 (H5N1) and the most severe pandemic A/Brevig Mission/1/1918 (H1N1) NS1 genes showed convergent evolution with some of the mouse adapted viruses for F103L plus M106I and V226I plus R227K mutations respectively. The F103L and M106I mutations in the HK NS1 gene were shown to be adaptive by assessment with respect to replication, early viral protein synthesis, interferon-β antagonism and tropism in the mouse lung. We extended the study and proved increased virulence associated with F103L+M106I mutations in their respective H5N1 NS1 gene on the PR8 and HK backbones, as well as the PR8 NS1 gene and the H9N2 (A/Ck/Bj/1/95) gene in the PR8 and A/WSN/33 backbones respectively. However the V226I and R227K mutations in their respective HK and 1918 NS1 genes slightly enhanced virulence and viral growth at later stages of infection. This study demonstrates that NS1 is a virulence factor; involved in multiple viral processes including interferon antagonism and viral protein synthesis. Furthermore, NS1 mutations acquired during mouse adaptation are proven to be adaptive in human, mouse and avian NS1 genes.

A proper vaccination program can play a critical role in prevention and control of avian influenza (AI) in commercial poultry. Low pathogenic avian influenza viruses (LPAIV) of H5 and H7 AI subtypes cause serious economic losses to the poultry industry and have the potential to mutate to highly pathogenic AI (HPAI) strains. Due to trade implications, differentiation of infected from vaccinated animals (DIVA) is an important issue in the control of AI. Therefore, the development and characterization of vaccine candidates with DIVA properties is critical in improving vaccination programs. Keeping these aspects in mind, we investigated the role of an NS1 mutant virus as a potential live attenuated DIVA vaccine. The NS1 protein of influenza virus plays a major role in blocking the host's antiviral response. Using an eight-plasmid reverse genetics system, we recovered the low pathogenic parental (H5N3) and NS1 mutant (H5N3/NS1/144) viruses. H5N3/NS1/144 expresses only the first 144 amino acids of the NS1 protein compared to the 230 of the parental H5N3. The growth properties of H5N3 and H5N3/NS1/144 were compared in cell culture and in different age embryonated chicken eggs. Our results confirmed that NS1 is involved in down regulation of interferon as shown by IFN-beta mRNA expression analysis and by the inability of H5N3/NS1-144 to efficiently grow in older age, interferon competent, chicken embryos. However with regards to safety the virus reverted to virulence within five back passages in chickens and was therefore not a safe vaccine candidate. However the killed form of H5N3/NS1-144 was a safer alternative and it also induced antibody titers and protection not significantly different from the parental H5N3 as vaccine. To further understand the reversion of H5N3/NS1/144 to virulence, we carried out 3 independent serial passages of H5N3/NS1/144 in increasing age of embryonated chicken eggs and examined the NS1 gene for presence of mutations. RT-PCR and sequence analysis of the NS gene in all three lineages showed the presence of a 54 amino acid deletion resulting in the generation of a 87 amino acids long NS1 ORF with a point mutation (L80V) at the site of deletion. In addition, the NS1 ORF in lineages L2 and L3 presented two additional point mutations in the RNA binding domain (Q40R and T73M). To determine if these mutations played a role in increased virulence, recombinant viruses expressing these mutant NS1 proteins in the background of parental virus were generated by reverse genetics and their replication properties and pathogenicity was examined in vitro, in ovo and in vivo systems. Our results showed that the 87 amino acid long NS1 protein clearly increased virus replication and virulence specifically in interferon competent systems. In addition, the two point mutations in the RNA binding domain of NS1 ORF expressing 87 a protein slightly increased the virus virulence. Overall this study reinforces the role of NS1 in influenza virus pathogenicity and supports the use of killed inactivated NS1 mutant virus vaccines as potential DIVA vaccines.