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Influenza infection remains constant threat to human health and results in huge financial loss every year. Rapid and accurate detection of influenza can help governments and health organizations monitor influenza activity and take measurements when necessary. In addition, influenza detection in a timingly manner can help doctors make diagnosis and provide effective treatment. On the other hand, novel inhibitors of influenza virus are in high demand because circulating strains have started to develop resistance to currently available anti-viral drugs. Influenza virus has two surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA), which play important roles in the influenza infection. The binding of HA to sialic acid-containing carbohydrates on cell surface initiates virus internalization, while cleavage of terminal sialic acid by NA facilitates viral particle release. In this dissertation, we focus on the development of glycan microarray that is comprised of a panel of NA resistant sialosides, and demonstrate the application of microarray to capture influenza virus at ambient temperature without the addition of NA inhibitors. We also describe a novel electrochemical biosensor for the detection of influenza virus. In addition, we have developed a new class of bivalent NA inhibitors that show promising inhibitory activities against influenza viruses.

The burden and behaviour of influenza in Southeast Asia is poorly charac- terised, leading to uncertainty about the importance of influenza as a local health problem and the role of Southeast Asia in the global epidemiology of influenza. Prospective community-based studies have provided fundamental insights into the epidemiology of influenza in temperate regions; therefore a household-based cohort study was established with the aim of determining the intensity and characteristics of influenza transmission in a semi-rural tropical setting. The primary results of the cohort study are presented, along with the results of a survey of social contact patterns in the cohort and a mathemati- cal model of the spread of pandemic influenza A/H1N1/2009 in Vietnam that utilises data from the cohort. Highly pathogenic avian influenza A/H5N1 remains endemic in poultry in parts of Southeast Asia and continues to infect humans. Marked familial clustering of human H5N1 cases has led to speculation that susceptibility to H5N1 infection may have a host genetic component. The epidemiological data that led to the hypothesis of genetic susceptibility to H5N1 is summarised, whilst the evidence for a role of host genetics in susceptibility to influenza in general is systematically reviewed. A genome-wide case-control genetic association study was conducted in Vietnam and Thailand to test the hypothesis of genetic susceptibility to H5N1 infection, and the results are presented. This work provides new data and understanding of the patterns and deter- minants of inter-pandemic, pandemic, and avian influenza epidemiology. The cohort study has added to the body of knowledge that is accruing on the burden and epidemiology of influenza in the tropics by providing community level data that were previously absent. The genetics study has provided the first direct evidence of genetic loci associated with susceptibility to H5N1 and opens new avenues of research to test these findings and their relevance to the pathogenesis of H5N1 and other types of influenza.

PB2 ist ein essentieller Bestandteil der trimeren RNA abhängigen RNA Polymerase von Influenzaviren und ist bekannt für seine Schlüsselrolle in der Bestimmung des Viruswirtsspektrums. Diese Arbeit diente der Identifizierung neuer Interaktionspartner von PB2 eines saisonalen und eines hochpathogenen Influenzavirus Stammes im Kontext infizierter humaner alveolar Epithelzellen (A549) unter Einsatz massenspektrometrischer Analysen. Die anschließende Untersuchung ausgewählter zellulärer Interaktoren hatte zum Ziel, deren Einfluss auf den Replikationszyklus der Influenzaviren zu bestimmen, sowie Unterschiede in ihrer Relevanz für das saisonale und das hochpathogene Virus aufzuzeigen. Die Erzeugung und Nutzung von Influenzaviren die einen Strep-tag an ihrem PB2 Protein tragen ermöglichte eine Anreicherung von PB2 und seiner Interaktionspartner. Die anschließende massenspektrometrische Analyse identifizierte 22 potentielle PB2 Interaktionspartner. Eine Auswahl an 13 Proteinen wurde tiefer gehend analysiert und eine Komplexbildung mit PB2 konnte für 9 Proteine bestätigt werden. Darüber hinaus zeigten 11 Proteine einen Polymerase stimulierenden bzw. hemmenden Effekt. Das Polymerase stimulierende Protein HSPA8 wurde zur weiteren Untersuchung ausgewählt. Während ein Einfluss von HSPA8 auf den hochpathogenen Influenzastamm nicht abschließend geklärt werden konnte, wurde seine Bedeutung für den Vermehrungszyklus des saisonalen Stammes aufgezeigt. Die Überexpression von HSPA8 führte zu einer Steigerung der Polymerase-Aktivität, wohingegen die Erniedrigung des HSPA8 Spiegels in einer Verringerung der viralen Replikation und der Polymerase-Aktivität resultierte. Interessanterweise führte die Erniedrigung des HSPA8 Spiegels auch zu stark verminderter PB2-Expression, jedoch nur im Falle des saisonalen Influenzastammes. Dieser Befund deutet auf eine Rolle von HSPA8 als PB2-Chaperon, notwendig für Proteinstabilität von saisonalen aber nicht hochpathogenen Influenzaviren, hin. / PB2 is an essential component of the influenza virus trimeric RNA dependent RNA polymerase and is known to play a key role in virus host range determination. Here, a combined affinity-purification/mass spectrometric approach was performed to identify novel interaction partners of PB2 of seasonal and highly pathogenic viral strains in infected human alveolar epithelial cells (A549). The subsequent analysis of selected cellular interaction partners aimed to determine the influence of these proteins on the replication cycle, as well as to determine differences in their relevance for the seasonal and the highly pathogenic influenza virus strain. Generation and use of recombinant influenza viruses carrying a Strep-tag at their PB2 protein allowed for enrichment of PB2 and its interaction partners. The subsequent mass spectrometric analysis identified 22 potential PB2 interaction partners. A selection of 13 proteins was further analyzed, and co-precipitation with PB2 was confirmed for 9 proteins. Moreover, an inhibitory or stimulatory effect on polymerase activity was observed for 11 proteins. The polymerase stimulating protein HSPA8 was selected for further investigation. While the influence of HSPA8 on the highly pathogenic strain remained unclear, its importance for seasonal influenza virus life cycle was demonstrated. Overexpression of HSPA8 resulted in increased polymerase activity while HSP8 knock down resulted in reduction of viral replication and viral polymerase activity. Intriguingly, the knock down of HSPA8 led to a strong decrease of PB2 protein expression. However, this was only observed for seasonal PB2. These results indicate a role of HSPA8 as a PB2 chaperone, necessary for protein stability of seasonal but not highly pathogenic influenza virus.

M Family Medicine research report - Faculty of Health Sciences / Residents of old age homes are at increased risk for the complications of influenza. Studies in developed countries have consistently shown that influenza vaccination of old age home residents and staff can significantly decrease morbidity and mortality rates and that influenza vaccination is one of the most cost effective interventions possible in this population. No studies have been done on the cost benefit of using influenza vaccine in old age home residents in South Africa. The aim of this study was to evaluate the costs of treating influenza and influenza-like illnesses in old age home residents, and to compare the costs in people who had received the influenza vaccine to those who had not. The study population comprised 151 people residing in two old age homes in Johannesburg, namely Sandringham Gardens and Nazareth House. The study population was divided into two groups- those who received influenza vaccine and those who had not been vaccinated. The residents of Nazareth House who gave consent had all been vaccinated. The subjects at Sandringham Gardens were sub- divided into two groups, namely: “Residents” and “Frail care / wards” section. The general health of the “Frail care” people was poorer than that of the “residents”. Medical records were reviewed, and details of the number of doctor consultations, medication and physiotherapy prescribed, special investigations performed and hospital referrals related to influenza and influenza-like infections were recorded. The costs were then calculated using “medical aid rates”. There were no significant differences in the treatment costs, comparing those who had been vaccinated to those who had not been vaccinated. There are a number of possible explanations for this. These include, most importantly, a low to moderate epidemic activity of influenza in the season that the study was conducted. Other explanations are low patient numbers, the use of symptoms for diagnosis and the use of over the counter therapy. Despite the findings in the present study, the international literature supports the view that influenza vaccination is a cost-effective intervention in the older adult population, particularly those at higher risk. These findings have been implemented in the official guidelines of many countries, including the South African Adult Influenza Vaccination Guideline.

Os vírus da Influenza Equina (EIV) (H3N8 e H7N7) pertencem à família Orthomyxoviridae, gênero Influenza A. Apesar de existirem poucos relatos de infecção humana pelo EIV, é conhecido o risco zoonótico e infecção interespécies. Serviços de vigilância epidemiológica da OIE e WHO informam que o subtipo H3N8 é isolado de surtos que ocorrem mundialmente, enquanto o subtipo H7N7, menos patogênico, não é isolado desde 1980, sendo então considerado um vírus extinto. Embora o EIV seja endêmico em nosso meio, há poucos trabalhos nacionais que tenham versado sobre a avaliação atual de anticorpos (Ac) anti-EIV presentes nos equídeos do Estado de São Paulo, o que motivou a realização do presente estudo. Os objetivos do presente trabalho foram: 1) avaliar a ação de diferentes tratamentos de soro descritos pela OIE e WHO para a remoção de inibidores inespecíficos da hemaglutinação em soros de 10 equinos vacinados (H3N8 A/Equi/Kentucky/1/1997), sendo eles: a) TPH: tripsina, metaperiodato de potássio seguido de adsorção de hemácias; b) KH: kaolin 20% seguido de adsorção de hemácias; e c) RDEH: RDE seguido de adsorção de hemácias; 2) avaliar a presença de Ac contra os vírus H3N8 e H7N7, em 84 equídeos não vacinados do Estado de São Paulo; 3) comparar a frequência de Ac contra H3N8 entre equídeos amostrados do estado de SP e de um painel de soros de equídeos do município de Mossoró - RN, região onde não há estudos sobre a circulação do EIV. Constatou-se que não houve diferença estatística entre os tratamentos de soro para a remoção de inibidores inespecíficos da hemaglutinação (p>0,05; confiança de 95%), todavia o tratamento RDEH apresentou resultados mais consistentes, corroborando a recomendação da OIE e da WHO de utilizar preferencialmente este tratamento. O perfil sorológico dos animais amostrados de SP sugere que circule o subtipo H3N8 e que o subtipo H7N7 circule de forma subclínica nos equídeos, o que é sustentado por outros trabalhos realizados no Brasil. Há evidências no Brasil sobre a detecção de anticorpos em equinos contra o subtipo H7N7, mesmo não havendo o isolamento deste no mundo desde 1980. No painel de soros do RN, onde a espécie Equus asinus era maioria, verificou-se a igualdade estatística entre as frequências de Equus caballus e Equus asinus positivos no teste de HI para o subtipo H3N8 (p>0,05; confiança de 95%), dado inédito em nosso meio. A frequência dos equídeos positivos no teste de HI para o subtipo H3N8 foi estatisticamente maior (p<0,05; confiança de 95%) em SP do que em RN. / The Equine influenza Virus (EIV) (H3N8 and H7N7) belong to Orthomyxoviridae family and Influenza A genus. Although there are few reports of human infection with EIV zoonotic and interspecies infection risk is known. OIE and WHO services on epidemiological surveillance report that H3N8 subtypes are isolated and characterized from worldwide outbreaks while H7N7 subtype less pathogenic has not being isolated since 1980 considered an extinct virus. Although the EIV is endemic in our country there are few national studies that had versed on the current evaluation of horses antibodies (Ab) from São Paulo State which motivated the present study. The study objectives were: 1) to evaluate the effects of different serum treatments described by OIE and WHO for the removal of nonspecific inhibitors of hemagglutination in 10 vaccinated (H3N8 - A/Equi/Kentucky/1/1997) horses sera being: a) TPH: trypsin, potassium metaperiodate followed by adsorption of erythrocytes b) KH: kaolin 20% followed by adsorption of erythrocytes and c) RDEH: RDE followed by adsorption of erythrocytes; 2) investigate the presence of antibodies against H3N8 and H7N7 viruses in 84 unvaccinated equines in São Paulo State; 3) compare the frequency of antibodies against H3N8 sampled between São Paulo State and a panel of equines sera from Mossoró - RN where there are no studies on EIV circulation. There was no statistical difference between the treatments for serum nonspecific inhibitors of hemagglutination removal (p>0.05; 95% confidence) however RDEH treatment showed results more consistent confirming OIE and WHO recommendation to use this treatment with priority. The serological profile of SP samples suggests H3N8 subtype circulates in those animals and H7N7 subtype might circulate in a subclinical form in equines, which is supported by other studies conducted in Brazil. There is evidence of antibodies detection against equine H7N7 subtype in Brazil, even without since 1980 isolation in the world. In animals from RN State which had Equus asinus representing a major fraction there was statistical equal frequencies of Equus caballus and Equus asinus positivity in HI test against H3N8 subtype (p>0.05; 95% of confidence), as unprecedented in the world. The frequency of positive equine against H3N8 subtype on HI test in SP was statistically higher (p<0.05; 95% of confidence) than in RN.

Avian influenza viruses can cause devastating outbreaks in domesticated poultry, with rapid transmission of virus between birds and high mortality. Current measures for control of influenza involve surveillance, closure of live poultry markets and mass culling. However, despite implementing these measures, outbreaks continue. Considering the uncertainty over whether certain strains of avian influenza viruses will adapt to human transmission and limitations over how their spread may be controlled, this study considers the use of anti-viral peptides as protective agents against low pathogenicity avian influenza (LPAI) virus. This study investigated the activity and mode of action of two cell-penetrating anti-viral peptide families against influenza A virus infection: ‘FluPep’ (a family of short, hydrophobic peptides related to suppressor of cytokine signaling- 1 proteins) and ‘Entry Blocker’ (derived from the signal sequence of fibroblast growth factor-4). Plaque reduction assays demonstrated dose-dependent anti-viral activity of both peptide families against a panel of influenza viruses with diverse haemagglutinin subtypes. Determination of IC50 values showed strain-specific differences in sensitivity to FluPep but not Entry Blocker. The IC50 of FluPep 4 for A/PR/8/34 was reduced by reassorting in the HA and NA from a relatively sensitive avian strain using a reverse genetics approach, suggesting inhibitory effects on the viral glycoproteins. Accordingly, viral entry assays focusing on binding, internalisation, fusion and import were designed and optimised to dissect the mechanism(s) of action of the peptides. Results indicated that the peptides acted upstream of nuclear import of viral ribonucleoprotein complexes but did not reduce overall virus binding to cells. However, the peptides caused aggregation of the virus particles on the surface of the host cells and reduced their internalisation. Further work evaluated how the peptides may be delivered to the site(s) of viral replication in poultry. A screen of the current literature was completed to allow for the design of an expression cassette for poultry-derived Lactobacillus to express FluPep and Entry Blocker. Though the cassette has been reported to be suitable for expression of heterologous proteins in Lactobacilli, rescue of recombinants for expression of anti-viral peptides or a reporter protein proved challenging, possibly owing to toxicity. A stable construct for the expression of FluPep 4 in Lactobacillus was obtained but culture supernatant did not inhibit virus replication.

Os vírus da Influenza Equina (EIV) (H3N8 e H7N7) pertencem à família Orthomyxoviridae, gênero Influenza A. Apesar de existirem poucos relatos de infecção humana pelo EIV, é conhecido o risco zoonótico e infecção interespécies. Serviços de vigilância epidemiológica da OIE e WHO informam que o subtipo H3N8 é isolado de surtos que ocorrem mundialmente, enquanto o subtipo H7N7, menos patogênico, não é isolado desde 1980, sendo então considerado um vírus extinto. Embora o EIV seja endêmico em nosso meio, há poucos trabalhos nacionais que tenham versado sobre a avaliação atual de anticorpos (Ac) anti-EIV presentes nos equídeos do Estado de São Paulo, o que motivou a realização do presente estudo. Os objetivos do presente trabalho foram: 1) avaliar a ação de diferentes tratamentos de soro descritos pela OIE e WHO para a remoção de inibidores inespecíficos da hemaglutinação em soros de 10 equinos vacinados (H3N8 A/Equi/Kentucky/1/1997), sendo eles: a) TPH: tripsina, metaperiodato de potássio seguido de adsorção de hemácias; b) KH: kaolin 20% seguido de adsorção de hemácias; e c) RDEH: RDE seguido de adsorção de hemácias; 2) avaliar a presença de Ac contra os vírus H3N8 e H7N7, em 84 equídeos não vacinados do Estado de São Paulo; 3) comparar a frequência de Ac contra H3N8 entre equídeos amostrados do estado de SP e de um painel de soros de equídeos do município de Mossoró - RN, região onde não há estudos sobre a circulação do EIV. Constatou-se que não houve diferença estatística entre os tratamentos de soro para a remoção de inibidores inespecíficos da hemaglutinação (p>0,05; confiança de 95%), todavia o tratamento RDEH apresentou resultados mais consistentes, corroborando a recomendação da OIE e da WHO de utilizar preferencialmente este tratamento. O perfil sorológico dos animais amostrados de SP sugere que circule o subtipo H3N8 e que o subtipo H7N7 circule de forma subclínica nos equídeos, o que é sustentado por outros trabalhos realizados no Brasil. Há evidências no Brasil sobre a detecção de anticorpos em equinos contra o subtipo H7N7, mesmo não havendo o isolamento deste no mundo desde 1980. No painel de soros do RN, onde a espécie Equus asinus era maioria, verificou-se a igualdade estatística entre as frequências de Equus caballus e Equus asinus positivos no teste de HI para o subtipo H3N8 (p>0,05; confiança de 95%), dado inédito em nosso meio. A frequência dos equídeos positivos no teste de HI para o subtipo H3N8 foi estatisticamente maior (p<0,05; confiança de 95%) em SP do que em RN. / The Equine influenza Virus (EIV) (H3N8 and H7N7) belong to Orthomyxoviridae family and Influenza A genus. Although there are few reports of human infection with EIV zoonotic and interspecies infection risk is known. OIE and WHO services on epidemiological surveillance report that H3N8 subtypes are isolated and characterized from worldwide outbreaks while H7N7 subtype less pathogenic has not being isolated since 1980 considered an extinct virus. Although the EIV is endemic in our country there are few national studies that had versed on the current evaluation of horses antibodies (Ab) from São Paulo State which motivated the present study. The study objectives were: 1) to evaluate the effects of different serum treatments described by OIE and WHO for the removal of nonspecific inhibitors of hemagglutination in 10 vaccinated (H3N8 - A/Equi/Kentucky/1/1997) horses sera being: a) TPH: trypsin, potassium metaperiodate followed by adsorption of erythrocytes b) KH: kaolin 20% followed by adsorption of erythrocytes and c) RDEH: RDE followed by adsorption of erythrocytes; 2) investigate the presence of antibodies against H3N8 and H7N7 viruses in 84 unvaccinated equines in São Paulo State; 3) compare the frequency of antibodies against H3N8 sampled between São Paulo State and a panel of equines sera from Mossoró - RN where there are no studies on EIV circulation. There was no statistical difference between the treatments for serum nonspecific inhibitors of hemagglutination removal (p>0.05; 95% confidence) however RDEH treatment showed results more consistent confirming OIE and WHO recommendation to use this treatment with priority. The serological profile of SP samples suggests H3N8 subtype circulates in those animals and H7N7 subtype might circulate in a subclinical form in equines, which is supported by other studies conducted in Brazil. There is evidence of antibodies detection against equine H7N7 subtype in Brazil, even without since 1980 isolation in the world. In animals from RN State which had Equus asinus representing a major fraction there was statistical equal frequencies of Equus caballus and Equus asinus positivity in HI test against H3N8 subtype (p>0.05; 95% of confidence), as unprecedented in the world. The frequency of positive equine against H3N8 subtype on HI test in SP was statistically higher (p<0.05; 95% of confidence) than in RN.

This thesis examines the epidemiology of avian influenza viruses (AIVs) in domestic poultry in Pakistan. Major aim of the current research was: to identify risk factors associated with the spread of these viruses; to quantify their prevalence in live bird retail stalls (LBRSs) and backyard poultry in Lahore district and to genetically characterize AIVs circulating in these stalls. Four independent studies were conducted which included (i) a retrospective matched case-control study in commercial poultry farms in Pakistan to identify the risk factors (ii) an estimation of the seroprevalence of AI from a cross-sectional study of backyard poultry flocks in Lahore district (iii) a cross-sectional study of LBRSs to estimate virus prevalence and identify associated risk factors and (iv) the genetic characterization of isolates collected from LBRSs. The retrospective matched case-control study identified five risk factors for AI infection. Multivariate conditional logistic regression model showed that distance of less than 0.5 kilometer of a commercial farm from the nearest case farm (OR= 145.4; 95% CI: 13.6-1553.5), followed by “previous history of infection of flock with infectious bursal disease (OR= 3.77; 95% CI: 1.18-11.97)”, selling of birds/eggs directly to live bird retail stalls from the farm premises (OR= 9.5; 95% CI: 1.7-51.9) have significant influence on spread of AI infection amongst the commercial farms. Other significant potential risk factors are “age of flock at the time of testing (OR= 1.0; 95% CI: 1.00-1.02)” and “a truck entering the farm areas (OR= 30.74; 95% CI: 1.56-604.78)”. Complete fencing of the farm was observed to be a protective factor (OR= 0.12; 95% CI: 0.02-0.63). The cross-sectional survey of backyard poultry flocks for AI (H9, H7 and H5) showed a seroprevalence of 67% (95% CI: 56.9-77.1) for H9 and 21% (95% CI: 13.8-28.1) for H5. Co-infection with both H9 and H5 was observed in 17 villages. Seroprevalence for H9 was significantly associated with the breed of bird. No samples were positive for H7. The cross-sectional survey of LBRSs in 07 towns of Lahore district showed the prevalence of H9N2 virus to be estimated at 10% (95% CI: 6.4-13.6). Subtypes H5N1 and H7N3 were not detected in any sample. Three risk factors showed a strong association with prevalence of H9N2 which are “adding new birds to the cages that already contained birds (OR= 9.2; 95% CI: 2.4-35.1)”, “purchasing birds for sale on the stall from mixed sources (other live poultry markets, auction markets, farm/individual producers) (OR= 3.4; CI 95%: 1.3-8.8)”, and “keeping birds partially inside and outside on the stalls during the day (OR= 1.7; CI 95%: 1.0-3.0)”. Phylogenetic analysis of ten H9N2 viruses isolated from LBRSs of Lahore district showed that four genes (HA, NA, M and NP) of all viruses belonged to G1-lineage and clustered with A/Quail/Hong Kong/G1/97 reference virus while the other four genes (PB2, PB1, NP and NS) from two of the viruses analysed clustered with a group of viruses from Indian subcontinent, Persian Gulf and Middle East. One recently reported H7N3 isolate from Pakistan also clustered with these genes. All H9N2 viruses examined harboured the mutation known to alter the receptor binding profile to one that preferentially binds to human receptors. The analysis shown in this study confirmed that further gene assortment has occurred since its emergence in poultry in Pakistan and Middle East, which could evolve into new genotype. Understanding the epidemiology of avian influenza has always been considered important in formulating and implementing control policies. Results from the current studies illuminate various aspects of epidemiological features of avian influenza viruses within poultry marketing systems in Asia. The current thesis has identified different risk factors and has also reported the prevalence estimates in backyard birds and LBRSs. The presence of reassortants of H9N2 with public health importance in LBRSs has also been reported in the current thesis. These results could be considered to plan future research and appropriate control and prevention strategiess for AIV by the global community. Continued surveillance and monitoring is essential to identify the viral gene pool circulating in live bird retail stalls and backyard poultry and to better understand the public-health risk posed by these viruses.

Death from infectious disease is common heritable, and in many cases a consequence of the host response, rather than direct effects of the pathogen. Since the host response in sepsis is orchestrated by the transmission of a variety of signals, both intra-cellular and inter-cellular, with which we have at least some capacity to intervene, it follows that it should be possible to prevent death through pharmaceutical modulation of inflammatory cascades. So far, it is not. The best candidate therapy for sepsis, activated protein C, failed to live up to initial promise and was ultimately withdrawn from the market in dismal failure. The premise of the work presented here is that a different approach – to develop an understanding of the host response at a genomic level – may yield more tractable insights, specifically into the problem of host susceptibility to influenza, a heritable cause of death in otherwise healthy people and a significant global threat. Since the sequencing of the human genome, it has become possible to identify genomic loci underlying host susceptibility to disease using genome-wide association studies (GWAS), best exemplified by the Wellcome Trust Case Control Consortium. This new technology creates substantial new challenges. The genetic markers associated with a phenotype are rarely causative, frequently in poorly-understood intergenic regions, and tend to have small effect sizes, such that tens or even hundreds of thousands of subjects must be recruited to have sufficient power to detect them. It is therefore not straightforward to translate these genotype-phenotype associations into useful understanding of the role of genes and gene products in disease pathogenesis. Attempts to overcome these challenges in order to discover genomic loci underlying individual susceptibility to infection form the core of this thesis. Ultimately these efforts converge with the development of a new computational method to detect phenotype-associated loci from genome-wide association studies (GWAS) using co-expression at regulatory regions of the genome.