Computational analysis for infectious diseases surveillance and host-pathogen interactions (in the context of Influenza A viruses)

Influenza A virus (IAV) is a major public health problem responsible for the death of half a million people every year worldwide. Zoonotic transmissions of the virus from swine and avian origin have occurred and can in the worst-case result in pandemics. Prediction of the next pandemic strain is still a major challenge as mechanisms of antigenic shift and the zoonotic nature of influenza viruses are still poorly understood. On the other hand, the current vaccination strategy treating seasonal influenza viruses, given its own problems in efficacy, is not well suited to mitigate an impending pandemic. While there are few clinically approved anti-IAV drugs for therapeutic and prophylactic use, these drugs have also been challenged by the emergence of drug resistance, toxicity, adverse effects and low efficacy. Hence, development of a broad range anti-IAV drug that could target all of the IAV strains irrespective of their source is critically important. To discover effective IAV vaccines and alternative anti-IAV therapeutics, it is crucial to understand how IAVs adapt to different species’ host ranges and vice versa, how different species hosts respond to different IAV subtypes. Additionally, it is crucial to understand IAVhost interactions and identifying critical viral or host factors that could support the replication of the virus or induce pathologic conditions. Recently, high throughput technologies such as mRNA microarray-based gene expression, genome-wide siRNA screening, and proteomic analysis are providing in-depth insights into host-pathogen interactions of IAVs. Hence, this study aimed to computationally investigate IAV-host interactions using three data set (transcriptome, genomewide siRNA screens and interactome) and identify 1) virus and host specific responses, 2) host determinants in host adaptations and 3) host targets for IAV therapeutics. This thesis contains four main projects. In the first project (chapter 3), we investigated the host gene expression changes of eight IAVs (H1N1/WSN, pH1N1, H5N2/F118, H5N2/F59, H5N2/F189, H5N3, H7N9 and H9N2 viruses) in A549 cells at different time points of infections. Then we integrated the differentially expressed genes (DEGs) in at least 3 viruses, with 1713 and 1780 host factors comprehensively curated from 11 siRNA and 13 interactome studies respectively. The integration of the three data set highlighted plausible influenza A virus required host factors (IHFs) that could be targeted against IAVs. The up-regulated IHFs (e.g. TRIM21, TRIM26, IRF2, and SAMHD1) might support the replication of IAV through suppression of the innate and adaptive antiviral immune responses. The other up-regulated IHFs could also enhance the replication of IAV at different stages of the virus lifecycle: endocytosis (BTC), prevention of apoptosis (TIMM17A), nuclear import (JAK2), and translation elongation of viral proteins (HEXIM1). Although several of these IHFs have been implicated in other viruses, the detailed mechanisms of how several of these up-regulated IHFs could support IAV replication require further investigation. The second project (chapter 4), explored a comprehensive analysis of host gene expression changes in different IAV infections in different host species. We used host gene expression signatures of cell lines from three species (A549 (human), CEF (chicken), and MDCK (canine) in response to six IAVs (H1N1/WSN, H5N2/F59, H5N2/F118, H5N2/F189, H5N3 and H9N2 viruses). To compare the expression changes between the three species, we performed comprehensive probe set re-annotation and human ortholog mapping. The result showed that the expression signatures of different IAV isolates in a single species cell type are more similar to each other compared to the expression signatures of a single isolate in different species’ cell types. The functional annotations (pathways) and the highly expressed cell-specific signatures indicated that IAVs up-regulated host factors could induce virus infectivity (e.g. OSBPL1A and ARHGAP21), reduce apoptosis (e.g. MRPS27) and increase cell proliferation (e.g. COPS2) in CEF cells. Conversely, increased antiviral, pro-apoptotic and inflammatory signatures have been identified in A549 cells. Except in H5N3 virus infections, generally IAV infections down-regulates genes associated with cell cycle and metabolic pathways with the strongest effect in MDCK cells, followed by A549 cells in a strain dependent manner, but not in CEF cells. Previously our group demonstrated that the replication of the examined viruses was significantly higher in CEF cells than the other cell types. Thus, we hypothesise that this could partially explain the mechanism how infectious LPAI viruses shed by chickens lack the inflammatory response and cellular disruption that may lead to disease conditions. In the third project (chapter 5), we used a systems-based approach to investigate changes to the transcriptome of primary murine lung macrophages (PMФ) in response to infection with the mouse-adapted H1N1/WSN virus and low pathogenic avian influenza (LPAI) viruses H5N2 and H5N3. The results showed that while all viruses induced antiviral responses, the H5N3 infection resulted in higher expression levels of cytokines and chemokines associated with inflammatory responses. Previously, our group showed that the LPAI H5N2 and H5N3 were able to infect murine lung macrophages and together with increased expression of inflammatory mediators particularly in H5N3 virus could impose threats to human health in the future. The IHFs identified by the IAV genome-wide siRNA screening studies could be used as potential anti-IV targets. However, these studies were not consistent (reproducible) mainly due to false negative results. Hence, in the fourth project (chapter 6), we applied computational gene network growing for discovering gene network links that could have been missed by these experimental investigations. Using the known IHFs we compared the network growing function of two free tools GeneMANIA and STRING and the commercial IPA for their performance of recovering other known IHFs previously identified from siRNA screens. The result showed that, given small (~30 genes) or medium (~150 genes) input sets, all three network growing tools detected significantly more known host factors than random human genes with STRING overall performing strongest. Extending the networks with all the three tools significantly improved the detection of GO biological processes of known host factors compared to not growing networks. Notably, the rate of identification of true host factors using computational network growing is equal or better to doing another experimental siRNA screening study which could also be true and applied to other biological pathways/processes. Finally, using the known IHFs and the "new IHF candidates" (genes connected to the IHFs from the network growing analysis), we predicted computational drug-target interactions using MetaCore. We identified 343 US Food and Drug Administration (FDA) approved drugs that had an inhibitory effect on either the known or new candidate IHFs, of which 258 were new predictions. Furthermore, using different criteria, we computationally ranked the 343 FDA approved drugs for further experimental validation.​Doctor of Philosophy (SBS

The magnitude and risk factors of intestinal parasitic infection in relation to human immunodeficiency virus infection and immune status, at ALERT hospital, Addis Ababa, Ethiopia

Human Immunodeficiency Virus (HIV) and intestinal parasitic infections are among the main health problems in developing countries like Ethiopia. Particularly, co-infections of these diseases would worsen the progression of HIV to Acquired Immunodeficiency Syndrome (AIDS). The purpose of this study was to determine the magnitude and risk factors for intestinal parasites in relation to HIV infection and immune status. The study was conducted in (1) HIV positive on antiretroviral therapy (ART) and (2) ART naïve HIV positive patients, and (3) HIV-negative individuals, at All African Leprosy and Tuberculosis (TB) Eradication and Rehabilitation Training Center (ALERT) hospital in Addis Ababa, Ethiopia. Study participants were interviewed using structured questionnaires to obtain socio-demographic characteristics and assess risk factors associated with intestinal parasitic infection. Intestinal parasites were identified from fecal samples by direct wet mount, formol ether concentration, and modified Ziehl-Neelsen staining techniques. The immune status was assessed by measuring whole blood CD4 T-cell count. The overall magnitude of intestinal parasite was 35.08%. This proportion was different among study groups with 39.2% (69/176), 38.83% (40/103) and 27.14% (38/140) in ART naïve HIV positives patients, in HIV negatives, and in HIV positive on ART patients respectively. HIV positive patients on ART had significantly lower magnitude of intestinal parasitic infection compared to HIV negative individuals. Intestinal helminths were significantly lower in HIV positive on ART and ART naïve patients than HIV negatives. Low monthly income, and being married, divorced or widowed were among the socio-demographic characteristics associated with intestinal parasitic infection. No association was observed between the magnitude of intestinal parasites and CD4 T-cell count. However, Cryptosporidium parvum, and Isospora belli were exclusively identified in individuals with CD4 T-cell count of ≤ 350 cells/mm(3). Regular provision of mass preventive chemotherapy and extended health education will curb the burden of intestinal parasitic infection in the community. Emphasis should also be given to laboratory diagnosis and identification of opportunistic intestinal parasites in patients with lower CD4-Tcell count.Accepted versio

Pulmonary tuberculosis preventive practices among Anibessa Bus users at Addis Ababa, Ethiopia: a cross-sectional study

Abstract Objective Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis. Smear positive tuberculosis patients are responsible for up to 90% of transmission occurring in the community. However, little is known about pulmonary tuberculosis preventive practices among bus users in Ethiopia. This study aimed to assess the level of Pulmonary Tuberculosis (PTB) preventive practices and associated factors among bus users at Addis Ababa. Results Community based cross-sectional study was conducted among bus users at Addis Ababa. Participants were selected using systematic sampling technique. Overall, 50.5% of bus users had good practices on prevention of PTB at Addis Ababa. The odds of practicing prevention of PTB among participants who were attended secondary school (AOR = 4.63; 95% CI 2.62, 11.17) and higher education (AOR = 2.86: 95% CI 1.13, 7.73), resided at Addis Ababa (AOR = 2.51; 95% CI 1.61, 5.21), knowledgeable about PTB (AOR = 4.12; 95% CI 3.14, 5.70), and using mass media (AOR = 2.14; 95% CI 1.78, 4.27) as a source of information were higher than the odds of their respective counterparts. The overall practice of pulmonary tuberculosis prevention among city bus users in the study area was low. Therefore, enhancing educational opportunity and increase community awareness about the causes, risk factors and means of transmission using mass media might improve the practices of PTB prevention during bus transportation

Systems-based approach to examine the cytokine responses in primary mouse lung macrophages infected with low pathogenic avian Influenza virus circulating in South East Asia

Abstract Background Influenza A virus (IAV) is a major public health concern, being responsible for the death of approximately half a million people each year. Zoonotic transmissions of the virus from swine and avian origin have occurred in the past, and can potentially lead to the emgergence of new IAV stains in future pandemics. Pulmonary macrophages have been implicated in disease severity in the lower airway, and understanding the host response of macrophages infected with avian influenza viruses should provide new therapeutic strategies. Results We used a systems-based approach to investigate the transcriptome response of primary murine lung macrophages (PMФ) infected with the mouse-adapted H1N1/WSN virus and low pathogenic avian influenza (LPAI) viruses H5N2 and H5N3. The results showed that the LPAI viruses H5N2 and H5N3 can infect PMФ with similar efficiency to the H1N1/WSN virus. While all viruses induced antiviral responses, the H5N3 virus infection resulted in higher expression levels of cytokines and chemokines associated with inflammatory responses. Conclusions The LPAI H5N2 and H5N3 viruses are able to infect murine lung macrophages. However, the H5N3 virus was associated with increased expression of pro-inflammatory mediators. Although the H5N3 virus it is capable of inducing high levels of cytokines that are associated with inflammation, this property is distinct from its inability to efficiently replicate in a mammalian host

Inter-Species Host Gene Expression Differences in Response to Human and Avian Influenza A Virus Strains

Low pathogenic avian influenza (LPAI) viruses are a source of sporadic human infections and could also contribute to future pandemic outbreaks but little is known about inter-species differences in the host responses to these viruses. Here, we studied host gene expression signatures of cell lines from three species (human, chicken, and canine) in response to six different viruses (H1N1/WSN, H5N2/F59, H5N2/F118, H5N2/F189, H5N3 and H9N2). Comprehensive microarray probe set re-annotation and ortholog mapping of the host genes was necessary to allow comparison over extended functionally annotated gene sets and orthologous pathways. The annotations are made available to the community for commonly used microarray chips. We observe a strong tendency of the response being cell type- rather than virus-specific. In chicken cells, we found up-regulation of host factors inducing virus infectivity (e.g., oxysterol binding protein like 1A (OSBPL1A) and Rho GTPase activating protein 21 (ARHGAP21)) while reducing apoptosis (e.g., mitochondrial ribosomal protein S27 (MRPS27)) and increasing cell proliferation (e.g., COP9 signalosome subunit 2 (COPS2)). On the other hand, increased antiviral, pro-apoptotic and inflammatory signatures have been identified in human cells while cell cycle and metabolic pathways were down-regulated. This signature describes how low pathogenic avian influenza (LPAI) viruses are being tolerated and shed from chicken but potentially causing cellular disruption in mammalian cells

Additional file 1: Figure S1. of Systems-based approach to examine the cytokine responses in primary mouse lung macrophages infected with low pathogenic avian Influenza virus circulating in South East Asia

A) Identification of PMФ using ant-F4/80 antibody and ant-CD11b antibody. B) FACS analysis of anti-CD11b and anti-F4/80 stained PMФ. C) Overlap analysis of DEGs in three IAV viruses and RSV (the data for RSV was obtained from our previously published work (Ravi et al., 2013)). D) Top 20 significantly enriched pathways of DEGs in IAVs and RSV infected PMФ. E) Unsupervised hierarchical clustering of DEGs at 2 and 24hpi in H1N1/WSN and H5N2 infections. (PDF 230 kb

Additional file 2: of Systems-based approach to examine the cytokine responses in primary mouse lung macrophages infected with low pathogenic avian Influenza virus circulating in South East Asia

List of differentially expressed probe sets or genes A) List of all DEGs with logFC and adjusted p-values B) Expression levels of cytokines in H5N3 compared to H1N1/WSN and H5N2 C) Cytokine expression levels in H5N3 and RSV. (XLSX 233 kb

A System Based-Approach to Examine Host Response during Infection with Influenza A Virus Subtype H7N9 in Human and Avian Cells

Although the influenza A virus H7N9 subtype circulates within several avian species, it can also infect humans with a severe disease outcome. To better understand the biology of the H7N9 virus we examined the host response to infection in avian and human cells. In this study we used the A/Anhui/1/2013 strain, which was isolated during the first wave of the H7N9 epidemic. The H7N9 virus-infected both human (Airway Epithelial cells) and avian (Chick Embryo Fibroblast) cells, and each infected host transcriptome was examined with bioinformatic tools and compared with other representative avian and human influenza A virus subtypes. The H7N9 virus induced higher expression changes (differentially regulated genes) in both cell lines, with more prominent changes observed in avian cells. Ortholog mapping of differentially expression genes identified significant enriched common and cell-type pathways during H7N9 infections. This data confirmed our previous findings that different influenza A virus subtypes have virus-specific replication characteristics and anti-virus signaling in human and avian cells. In addition, we reported for the first time, the new HIPPO signaling pathway in avian cells, which we hypothesized to play a vital role to maintain the antiviral state of H7N9 virus-infected avian cells. This could explain the absence of disease symptoms in avian species that tested positive for the presence of H7N9 virus

A system based-approach to examine host response during infection with influenza a virus subtype H7N9 in human and avian cells

Although the influenza A virus H7N9 subtype circulates within several avian species, it can also infect humans with a severe disease outcome. To better understand the biology of the H7N9 virus we examined the host response to infection in avian and human cells. In this study we used the A/Anhui/1/2013 strain, which was isolated during the first wave of the H7N9 epidemic. The H7N9 virus-infected both human (Airway Epithelial cells) and avian (Chick Embryo Fibroblast) cells, and each infected host transcriptome was examined with bioinformatic tools and compared with other representative avian and human influenza A virus subtypes. The H7N9 virus induced higher expression changes (differentially regulated genes) in both cell lines, with more prominent changes observed in avian cells. Ortholog mapping of differentially expression genes identified significant enriched common and cell-type pathways during H7N9 infections. This data confirmed our previous findings that different influenza A virus subtypes have virus-specific replication characteristics and anti-virus signaling in human and avian cells. In addition, we reported for the first time, the new HIPPO signaling pathway in avian cells, which we hypothesized to play a vital role to maintain the antiviral state of H7N9 virus-infected avian cells. This could explain the absence of disease symptoms in avian species that tested positive for the presence of H7N9 virus.Agency for Science, Technology and Research (A*STAR)Ministry of Education (MOE)Published versionWe thank the Ministry of Education, and DSO National Laboratories for funding this study. B.T. was awarded by the Singapore International Graduate Award (SINGA) scholarship (A*STAR, Singapore) and CH (MOE scholarship)