Influenza A virus (IAV) circulates in waterfowl, causing mostly asymptomatic infections. IAV can undergo host adaptation and evolve to cause significant disease and mortality in domestic poultry and mammals, applying an enormous socio-economic burden on society. Sporadically, IAV causes global pandemics in man due to its zoonotic nature, and this can result in millions of deaths worldwide during a single outbreak. Host adaptation of IAV is an incompletely understood phenomenon, but is known to involve both host and viral determinants. It is essential to improve the understanding of the factors governing host range and pathogenicity of avian IAV, especially given the absence of a universal influenza vaccine and a limited weaponry of effective antiviral compounds. This study set out to improve the understanding of host adaptation of avian IAV, focussing on segment 8 (NS segment) of the virus genome. The NS segment of non-chiropteran IAV circulates as two phylogenetically distinct clades – the ‘A-’ and ‘B-alleles’. The A-allele is found in avian and mammalian viruses, but the B-allele is considered to be almost exclusively avian. This might result from one or both of the major NS gene products (NS1 and NEP) being non-functional in mammalian host cells, or from an inability of segment 8 RNA to package into mammalian-adapted strains. To investigate this, the NS segments from a panel of avian A- and B-allele strains were introduced into human H1N1 and H3N2 viruses by reverse genetics. A- and B-allele reassortant viruses replicated equally well in a variety of mammalian cell types in vitro. Surprisingly, the consensus B-allele segment 8 out-competed an A-allele counterpart when reassortant H1N1 viruses were co-infected, with the parental WT segment 8 being most fit in this system. A- and B-allele NS1 proteins were equally efficient at blocking the mammalian IFN response both in the context of viral infection and in transfection-based reporter assays. Consensus A- and B-allele H1N1 viruses also caused disease in mice and replicated to high virus titre in the lung. Interestingly, the B-allele virus induced more weight-loss than the A-allele, although the parental WT virus was most pathogenic in vivo. To re-address the hypothesis that B-allele NS genes really are avian-restricted, the relative rates of independent Aves to Mammalia incursion events of A- and B-allele lineage IAV strains was estimated and compared using phylogenetic analyses of all publically available segment 8 sequences. 32 A-allele introduction events were estimated compared to 6 B-allele incursions, however the total number of avian Aallele sequences outnumbered B-allele sequences by over 3.5 to 1, and the relative rates of introduction were not significantly different across the two lineages suggesting no bias against avian B-allele NS segments entering mammalian hosts in nature. Therefore, this study provides evidence that avian B-allele NS genes are not attenuating in mammalian hosts and are able to cause severe disease. Thus, this lineage of IAV genes, previously assumed to be avian-restricted, should be considered when assessing zoonotic potential and pandemic risk of circulating avian IAVs.

Made available in DSpace on 2015-04-14T13:35:51Z (GMT). No. of bitstreams: 1 455917.pdf: 2015931 bytes, checksum: 9c232dbbf603a1ea4db4fc35a200c003 (MD5) Previous issue date: 2013-12-13 / INTRODUCTION: Studies have shown that LAIV is more effective than TIV for prevention of influenza infection in young children, but the underlying mechanisms associated with protection are still not well defined. OBJECTIVES: To assess differences in cellular immunity and gene expression profiles in children after immunization with LAIV or TIV. METHODS: Peripheral blood of previously healthy children was collected pre and postvaccination and analyzed for B cells measurement and antibodies production. Microarray analyses were applied to assess the gene expression profiles. RESULTS: LAIV vaccinees showed number of na?ve, memory and transitional B cells increased on day 1 compared to baseline (p<0.05). TIV vaccinees showed the number of plasmablasts increased on day 7 post-vaccination (p&#8804;0.01). In addition, the titers for the three strains of the vaccine (H1N1, H3N2 and B) were significantly elevated on the TIV group when compared with the LAIV group. Regarding the transcriptional profiles presented, both vaccines induced interferon signaling, but in different time points, TIV 1 day postvaccination and LAIV 7 days post-vaccination, the last only in children younger than 5 years old. CONCLUSIONS: These results indicate that LAIV and TIV induced different immune responses in peripheral blood of vaccinated children and that both vaccines induce interferon signaling post-vaccination. / INTRODU??O: Estudos t?m demonstrado que a vacina contra os v?rus influenza LAIV ? mais eficiente que a formula??o TIV para a preven??o de infec??o em crian?as, por?m os mecanismos envolvidos ainda n?o est?o bem definidos. OBJETIVOS: Acessar as diferen?as na imunidade celular humoral e de express?o de perfis g?nicos em crian?as imunizadas com LAIV ou TIV. M?TODOS: Sangue perif?rico de crian?as previamente h?gidas foi coletado pr? e p?svacina??o e analisado para mensura??o de c?lulas B e para titula??o de anticorpos. An?lise pela t?cnica de microarranjos foi empregada para avalia??o dos perfis de express?o g?nica. RESULTADOS: Vacina??o com LAIV induziu aumento do n?mero absoluto de c?lulas B na?ve, de mem?ria e transicionais no ap?s 30 dias comparado aos valores medidos antes da vacina??o (p<0.05). Vacina??o com TIV, no entanto, induziu aumento de plasmoblastos no dia 7 p?s-vacina??o (p<0.01). Os t?tulos de anticorpos para os tr?s ant?genos da vacina (H1N1, H3N2 e B) encontraram-se significativamente elevados no grupo vacinado com TIV quando comparado ao grupo que recebeu LAIV. Ambos os grupos vacinados induziram sinaliza??o de genes relacionados ao interferon, por?m em diferentes momentos: TIV induziu-os 1 dia p?s-vacina??o, enquanto LAIV 7 dias p?s-vacina??o, especialmente em crian?as com menos de cinco anos de idade. CONCLUS?ES: Esses resultados sugerem que as vacinas LAIV e TIV induzem resposta celular imune diferente em crian?as e que ambas induzem sinaliza??o de interferon p?s-vacina??o.

Objective: To investigate antiviral activity of maca to reduce viral load in kidney (MDCK) cells infected with influenza type A and B viruses (Flu-A and MFalud-inB-, Dreasrpbeyc ctiavneilny)e. Methods: Maca were extracted with methanol (1:2, v/v). The cell viability and toxicity of the eaxgtariancstts Fwluer-eA e avnaldu aFtleud- oBn v MirDusCeKs cwealsls a usssianyge dm uetshinogd aM TteTs ta sfosar yd. eAtenrtmiviinrainl ga ctthiev itiyn hoifb citoimonp oouf nthdes cytopathic effect on cell culture and multiplex RT-PCR. Results: The methanol extract of maca showed low cytotoxicity and inhibited influenza-induced cytopathic effect significantly, while viral load was reduced via inhibition of viral growth in MDCK infected cells. Maca contains potent inhibitors of Flu-A and Flu-B with a selectivity index [cytotoxic concentration 50%/IC50] of 157.4 and 110.5, respectively. Conclusions: In vitro assays demonstrated that maca has antiviral activity not only against Flu-A (like most antiviral agents) but also Flu-B viruses, providing remarkable therapeutic benefits. / Financial support of this study was provided by AECID grants (PCI: C/033641/10) and AGAUR (MAT2009-11503, MAT2012-36205, 2009SGR-1208). JDVM support was provided by 1st Concurso Incentivo a la Investigación de la Universidad Peruana de Ciencias Aplicadas, Lima, Peru. / Revisión por pares

Influenza A virus (IAV) is a global health threat, causing seasonal epidemics and potential pandemics leading to morbidity, death and economic losses. Currently, there are two main classes of licensed antivirals against IAV available in the US and Europe; adamantanes and neuraminidase inhibitors, both of which are hindered by the generation of resistant virus variants. The viral polymerase has a high error rate leading to mutations that allow the virus to overcome selection pressures directed at its own genome from conventional antivirals. The prospect of inhibiting host proteins that the virus exploits to facilitate its replication is of increasing interest as an antiviral strategy as the emergence of resistance has been predicted to be slower when targeting a host cellular factor. IAV utilizes the host nuclear export protein CRM1 to transport viral ribonucleoproteins (vRNPs) from the nucleus to the cytoplasm of an infected cell, a critical late stage of the influenza lifecycle. Leptomycin B (LMB), a Streptomyces metabolite, has been previously shown to target this pathway, resulting in reduced viral propagation; however, LMB's potent cytotoxicity has limited its use as a therapeutic agent. This thesis examined two novel selective inhibitors of nuclear export (SINE), KPT-335 and KPT-185, with less cytotoxicity. In vitro, KPT-335 inhibited replication of human and animal IAV strains in a dose-dependent manner with minimal cytotoxicity. To assess the resistance potential of KPT-335, IAV viruses were serially passaged in the presence of a sub-optimal concentration of the compound and assayed for the development of resistance. Resistance to KPT-335 became evident at 8-10 rounds of passage. Sequencing analysis of independently derived resistant virus clones identified 4 single amino acid changes on a surface exposed patch of the viral nucleoprotein (NP). Introduction of these amino acid changes, into otherwise wild type viruses by reverse genetics, confirmed that changes Q311R and N309T conferred a drug-resistant phenotype. However, these substitutions came at a fitness cost to virus replication. The molecular basis for resistance was unclear but Q311R and N309T NP-mutant viruses produced increased levels of M1 during infection as well as producing virus particles with increased M1:NP ratios. Furthermore, the KPT-335-resistance mutations were surprisingly similar to NP sequence polymorphisms previously associated with susceptibility to the innate defence protein MxA. Consistent with this, viruses harbouring the Q311R mutation displayed increased susceptibility to MxA inhibition compared to wild-type virus. Altogether this study confirms that SINEs have the potential to be successful therapeutic agents against IAV replication and that although resistance could be generated, it may be difficult for the virus to overcome both drug selection pressures and the human innate immune response restrictions by escape mutations.

Jumonji C (JmjC) domain containing proteins comprise a large family of enzymes that catalyse oxidative reactions. The jumonji domain containing protein 6 (JMJD6) has pleiotropic functions as a lysyl hydroxylase and arginyl demethylase. Previous studies have shown that Jmjd6 is involved in histone modification, mRNA splicing and regulation of polymerase II pause release. A constitutive knockout of Jmjd6 in mice is neonatal lethal and shows defects in macrophage host responses. Recently, JMJD6 was shown to support Foot-and-mouth disease virus replication through interactions with the dead-box RNA helicase Dhx9. This PhD thesis aims to further explore functions of Jmjd6 in macrophages and its roles during viral infections. The hypothesis is that through interactions with RNA helicases, Jmjd6 regulates host responses to foreign nucleic acids and/or has functions as a host factor for replication of DNA and/or RNA viruses. Testing of this hypothesis required the generation of Jmjd6-deficient cell recourses. A new conditional Jmjd6 mouse allele was characterised and a method optimised to knockout the gene in bone marrow-derived macrophages (BMDM) using TAT-Cre recombinase. To study vaccinia (VACV) and influenza A virus (IAV) infections in human cell lines, JMJD6 was depleted using RNA interference or CRISPR-Cas9 gene editing. In BMDM, JMJD6 expression was up-regulated in the late phase of lipopolysaccharide stimulation. The nuclear expression pattern of Jmjd6 in BMDM overlapped with that of DDX41 but not with DHX9, two RNA helicases that have been implicated in sensing of viral DNA and RNA, respectively. Deletion of Jmjd6 in BMDM reduced induction of type I interferon response genes after stimulation with synthetic analogs of viral RNA. To characterise the role of JMJD6 during infection with a DNA virus, Jmjd6-deficient cells were infected with VACV. Knockout of Jmjd6 reduced VACV growth in macrophages but not in HeLa cells. In contrast to HeLa cells, Jmjd6-deficient macrophages displayed abnormal localisations of viral factories and increased cell death, showing that Jmjd6 is specifically required for productive VACV infection in macrophages. To further analyse whether Jmjd6 has pro- or anti-viral functions during RNA virus infection, JMJD6 depleted A549 cells were infected with IAV. JMJD6 depletion in A549 drastically reduced IAV growth from an early stage of infection. Preliminary data indicate that this phenotype is related to a defect in nuclear import of IAV ribonucleoprotein complexes. In summary, this work has identified JMJD6 as a novel pro-viral host factor for VACV and IAV infection and has underpinned its importance for macrophage functions.

Influenza A virus (IAV) infection is a serious respiratory disease associated with significant morbidity and mortality worldwide. Annual vaccination is the most effective way to prevent infection and its potentially severe complications; however, the vaccines currently offered have several drawbacks that limit its availability and protective efficacy. Influenza virus-like particles (VLPs), which lack viral genetic material and are non-infectious, represent a promising vaccine candidate. Previous reports have shown VLPs are more immunogenic than subunit or recombinant proteins, and confer protection upon lethal challenge. A critical component of this protection is mediated by influenza HA-specific neutralizing Abs produced by memory B cells and plasma cells, the cellular products of the germinal center (GC) reaction. While preliminary studies have examined the humoral immune response to VLP vaccination, the current study is the first to characterize the GC response in secondary and tertiary lymphoid tissues. Mice were vaccinated with influenza VLPs using three immunization routes: subcutaneous (s.c.), intramuscular (i.m.), and intranasal (i.n.) and the GC response was assessed over time. Robust GC reactions were induced in the dLNs regardless of vaccination route, though the largest response was generated with VLPs s.c. The pattern of isotype expression was remarkably similar between routes, with IgM+ and IgG2+ B cells representing the majority of the GC B cell population. Mucosal immune responses in the upper (nasal) and lower (lung) airway were measured in mice vaccinated i.n. Marked GC reactions were induced in the nasal-associated lymphoid tissue (NALT), while the pulmonary response was relatively modest and short-lived compared to infection with IAV. Within the GC B cell population, IgM+ and IgG2+ B cells made up the majority, similar to the dLN response. Importantly, the pattern of isotype expression induced by VLPs mimicked the response induced by natural IAV infection, and suggests that VLPs contain the necessary innate immune agonists to induce a TH1 biased response.

Influenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by viral nucleoprotein (NP) and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins (vRNPs) responsible for RNA synthesis. NP is a structural component of the vRNP but also interacts with both viral and host factors to regulate viral RNA expression. NP is conserved among influenza A isolates, making NP interactions compelling antiviral targets. Here I characterize mutations within 5 amino acids of NP that comprise an accessible region of the NP body domain, as determined by NP crystal structure. This region was selected for mutagenesis to target interaction between NP and RdRP. NPbd3 encodes glycine at 5 amino acids within the accessible NP body domain. Cellular fractionation and Western Blot, in addition to NP-GFP fusions and fluorescence, confirm NPbd3 was expressed and localized as WT-NP. Gel shift with purified NP protein confirm NPbd3 bound nucleic acids as WT-NP. Although NPbd3 was expressed, localized, and bound nucleic acid as WT-NP, I found NPbd3 was defective for RNA expression in reconstituted vRNPs, as evaluated by reverse transcription and quantitative polymerase chain reaction (RT-qPCR). To investigate this NP body domain further, single and double amino acid mutations were cloned. Analysis of NP single mutants revealed that all were nearly as functional as WT-NP for RNA expression in reconstituted vRNPs, suggesting these accessible amino acids in the NP body domain play a redundant role. However, four different combinations of two amino acid mutations resulted in NP double mutants that displayed a significant defect in RNA expression in reconstituted vRNPs, confirming these accessible amino acids in the NP body domain play a significant role for viral RNA synthesis. A disruption in an essential NP interaction with the RdRP is likely the explanation for the RNA defect observed. In support of this, avian influenza virus passaged in human cells resulted in virus with one NP amino acid change in this domain consistently paired with specific changes in the PB2 subunit of the RdRP. I reason this accessible body domain of NP is a viable antiviral target. Indeed, two amino acids in this NP body domain comprise a novel groove implicated in binding the small molecule inhibitor nucleozin. My thesis highlights this conserved NP body domain as an important interaction surface essential for viral RNA synthesis and support further investigation of antiviral drugs that target this region of NP.

The influenza viral ribonucleoprotein complexes (vRNPs) are responsible for viral RNA synthesis. Each vRNP is comprised of one vRNA segment, the viral RNA dependent RNA polymerase complex (RdRP), and multiple copies of nucleoprotein (NP). NP serves as scaffold in formation of vRNPs, but also regulates vRNP activity. The N-terminus of NP contains a nonconventional nuclear localization signal (NLS1) essential for initial vRNP nuclear import, but also interacts with host RNA helicases to enhance viral RNA replication in the nucleus. NP contains at least one additional NLS sequence, with bioinformatics revealing a third NLS in some NP proteins. Published yeast-two hybrid results indicate that the first 20 amino acids of NP can sufficiently bind with cellular protein UAP56. Suggesting the interaction of NP-UAP56 can be a major mechanism of how NP involve in viral replication. Thus, to examine the role of the N-terminus of NP aside from its vRNP nuclear localization activity N-terminal 20 amino acid deletion mutants with or without the addition of the conventional NLS from SV-40 T-antigen were constructed, termed del20NLS-NP and del20-NP. Nuclear localization of vRNPs with these constructs was assessed by GFP expression and western blotting. All these constructs exhibit nuclear localization, consistent with NLS1 being utilized for vRNP localization but not NP localization and vRNP formation in the nucleus. Furthermore, qPCR results demonstrated decreased vRNA synthesis activity, exacerbated as the vRNA template is lengthened in both plasmids, consistent with a lack of interaction with host RNA helicases. Interestingly, del20-NP vRNP activity is less severe than del20NLS-NP, suggesting perturbations of the N-terminus disrupt vRNP activity. To narrow down the region responsible for vRNA expression defect, del10-NP was constructed. GFP expression displayed similar activity between del10-NP and WT-NP with del20-NP showing a severe defection, suggesting NP amino acids 11-20 might be the major region responsible for the vRNA synthesis defect. However, sucrose density gradient results do not support the published interaction between NP and UAP56 in 293T cells. These results support the N-terminal region, potentially amino acids 11-20 of NP, is playing the important role in efficient viral gene expression during virus replication especially as vRNA template lengthen, and that the NLS1 of NP is not essential for NP/vRNP nuclear localization in our reconstituted vRNP assay.

Neonates are more susceptible to influenza virus infection than adults, resulting in increased morbidity and mortality as well as delayed clearance of the virus. Efforts to improve influenza infection outcomes in neonates typically center on prevention, although current vaccines fall short of complete protection and can only be administered in humans after 6 months of life. We propose that as the neonatal immune system responds differently than the adult immune system, interventions that are efficacious or tolerable in adults cannot be guaranteed to perform the same in neonates. T cell vaccines that target conserved influenza virus epitopes have been proposed for conferring protection to multiple influenza virus strains, but if T cell vaccines will be used in infants and adults, neonates must be able to respond to the same T cell antigens as adults. Mouse pups responded to influenza virus peptide PA224-233 but not NP366-374 during influenza virus infection in contrast to the codominant adult response. Mice infected as pups also generated diminished T cell memory compared to mice infected as adults and displayed skewed immunodominance during secondary infection. Adult bone marrow derived dendritic cells (BMDCs) improved viral clearance when loaded with influenza virus and promoted NP366-374-specific CD8+ T cell responses in infected pups. BMDC peptide vaccination could stimulate PA224-233-specific but not NP366-374-specific CD8+ T cell responses in pups, but, PA224-233 vaccination offered no protection to pups during lethal infection. These data suggest that altered immunodominance must be considered when stimulating CD8+ T cell responses in adults and neonates. Immaturity and active suppression of immune responses are both factors in neonatal vulnerability to disease. Specifically, active suppression of neonatal immunity by regulatory T cells (Tregs) has been proposed as a driving factor in diminished neonatal immunity, but removing these cells can compromise viral defense or increase deleterious inflammation. Mice that lacked Tregs displayed compromised anti-influenza antibody responses and decreased lymph node responses during influenza virus infection. A high proportion of pup Tregs also expressed Gata3. Transgenic pups with a Treg specific Gata3 knockout displayed an increase in Tbet expression in both conventional and regulatory T cells and an increase in IFNγ producing CD4+ T cells in the lungs during infection. These data suggest that Tregs are required for effective humoral responses to influenza virus and that Gata3 expression influences Treg suppressive function in neonates.

The increasing threat of epidemic and pandemic influenza underscore the need to better-understand the immune response to influenza virus infections and to better understand the factors that contribute to the clearance of virus without complications of immunopathology. A hallmark of the adaptive immune response to primary influenza virus infections is the induction of influenza-specific CD8+ T cell responses. These T cells target and kill influenza-infected epithelial cells in the airway, thereby clearing the virus and allowing recovery of the infected host. Recent reports demonstrated that CD8+ T cells express TNF-related apoptosis-inducing ligand (TRAIL) after influenza virus infection. While roles for perforin/granzyme and Fas:FasL interactions in clearing influenza virus infections had been established, little was known about the role of TRAIL in the CD8+ T cell responses to influenza virus infection. We hypothesized that influenza-specific CD8+ T cells would express TRAIL after influenza infection and could utilize TRAIL to induce the apoptosis of virally-infected cells. We discovered that CD8+ T cells do express TRAIL after influenza infection, and that this expression occurs in an influenza-specific fashion. Further, we demonstrated that these influenza-specific CD8+ T cells utilize this TRAIL to kill virally infected cells and protect the host from death, while T cells lacking TRAIL were unable to kill targets as efficiently and provided reduced protection. These data supported our hypothesis that CD8+ T cells utilize TRAIL to kill infected cells. Unexpectedly, when we increased the initial viral inoculum, the pulmonary cytotoxicity of T cells in TRAIL-/- mice was increased compared to those in TRAIL+/+ mice. Investigation of this phenomenon revealed that changes in cytotoxicity correlated not with changes in effector molecule expression on the T cells, but with increased recruitment of T cells to the lung. T cell recruitment to the lungs of TRAIL-/- mice was dependent on CCR5 and CXCR3, and likely the result of aberrant expression of MIG and MIP-1α in the lungs. Together, these data suggest that TRAIL expression contributes not only to T cell cytotoxicity, but also to the regulation of chemokine expression and associated cell recruitment after influenza virus infections. To confirm the relevance of our animal model to the study of human disease, we examined the potential role for TRAIL in the human immune response to infection. We determined that in vitro influenza infection stimulates upregulation of functional TRAIL on the surface of CD3+, CD14+, CD19+, and CD56+ PBMC populations. This expression was not caused by infection of the cells, but by interferon produced as a result of the infection. Infected (TRAIL-expressing) PBMCs killed influenza-infected lung epithelial cells, revealing that influenza infection sensitizes epithelial cells to TRAIL-induced apoptosis. Surprisingly, blocking TRAIL signaling, but not FasL signaling, was able to abrogate this killing of infected epithelial cells. Together, these data support a role for TRAIL in the human immune response to influenza virus infections. Considered as a whole, the data from these studies suggest an additional, previously-unappreciated mechanism by which CD8+ T cells can kill virally infected cells, TRAIL. They also suggest additional, previously-unappreciated roles for TRAIL in immune responses: in helping clear virally infected cells after infections and in helping control cytokine/chemokine expression, and thus the immune response, after virus infection.