Dengue virus (DENV) is a reemerging infectious agent causing an estimated 50-100 million cases annually and is endemic in more than 100 countries. Recently there has been an increase in the geographic spread, the overall number, and the severity of disease cases, making DENV an increasing threat to public health. The most prevalent of the arboviruses, DENV is introduced into human hosts during a vector mosquitos blood meal; here, virus is deposited into the epidermal and dermal layers of the skin. Therefore, elucidating the cutaneous immune response to DENV is critical to our understanding of the immune mechanisms involved in protection and pathogenesis of DENV and to the development of effective treatments and vaccines. In addition to resident T- and B-cells, the skin immune system includes three major populations of innate immune cells, dermal dendritic cells (dDCs), dermal macrophages (dMØs), and epidermal Langerhans cells (LCs). These immune cells are potential targets of DENV infection. The receptor profile of dDCs and dMØs indicates that they are susceptible to DENV infection, however evidence of this is lacking. LCs are infected following ex vivo inoculation of human skin explants, yet their role in DENV pathogenesis remains unclear. Upon exposure to a pathogen, cutaneous immune cells mature and migrate to the cutaneous draining lymph node via the draining lymphatics; these functions are critical to the induction of adaptive immune responses and to pathogen clearance. It remains unclear how DENV infection affects the maturation, migration and survival of cutaneous immune cells. Herein, we use a combination of in vitro studies and ex vivo inoculation of healthy human skin explants to explore the role of cutaneous immune cells in DENV infection. We investigated the degree to which cutaneous immune cells become infected with DENV and measured upregulation of activation markers, migration out of the skin, and the life span of infected cells. We discovered that populations of cutaneous DCs, as well as dMØs, become infected with DENV upon scarification of skin with virus. In vitro, we confirmed that DENV infection partially inhibits maturation of DCs and potentially MØs. Additionally, DENV infection induces DC apoptosis in a caspase-dependent manner. These results help to clarify the role of cutaneous immune cells in the immunopathogenesis of DENV.

Current antiretroviral therapy (ART) has reduced morbidity and mortality from HIV-1 infection, but the long-term efficacy of ART is limited by selection of HIV-1 drug-resistant variants. Most HIV-1 drug resistance mutations that have been studied are located in the polymerase domain of HIV-1 reverse transcriptase (RT) and this region of RT is sequenced in genotyping tests used clinically to guide ART. Recently, attention has focused on the connection and RNase H domains of RT as locations of drug resistance mutations, but the prevalence, molecular mechanisms, and impact of such mutations on response to ART are uncertain. We therefore performed a series of studies to address this uncertainty, including in vitro selection of HIV-1 resistant to 3-azidothymidine (AZT), drug susceptibility studies, biochemical assays and genotype analysis of clinical samples to identify and characterize resistance mutations in the RT connection and RNase H domains. From this work, we provide several lines of evidence that connection and RNase H domain mutations emerge with ART and impact nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) susceptibility. First, the connection domain mutation A371V and the RNase H domain mutation Q509L are selected in vitro with AZT and confer > 50-fold AZT resistance and low-level cross resistance to lamivudine, abacavir and tenofovir when in the context of thymidine analog mutations (TAMs) in the polymerase domain of RT. Second, we show that mutation Q509L in the RNase H domain promotes dissociation of RT from RNA/DNA template/primer bound in an RNase H competent mode, thereby decreasing secondary RNase H cleavage and destruction of the template/primer. As a consequence, template/primer binds in a polymerase competent mode allowing AZT-monophosphate excision, DNA polymerization and AZT resistance. Third, the connection domain mutation A360V emerges in patients after prolonged exposure to AZT monotherapy and increases resistance to AZT in the context of 3 or more TAMs. Fourth, connection and RNase H domain mutations are not more frequent at virologic failure in HIV-1 subtype B infected patients treated with 2 NRTI plus efavirenz when failure is defined as a small increase in plasma HIV-1 RNA. However, the connection domain mutation N348I emerges frequently at virologic failure in HIV-1 subtype C infected patients in South Africa who were treated with efavirenz/lamivudine/stavudine or nevirapine/lamivudine/stavudine when virologic failure is defined as confirmed plasma HIV-1 RNA > 1,000 copies/mL. This work provides strong evidence that RT connection and RNase H domain mutations emerge in HIV-1 infected patients treated with ART and these mutations are missed with currently available genotype tests. Mutations missed by routine genotyping tests pose a potential public health threat if left undetected and transmitted to others.

Rift Valley Fever virus (RVFV) is an arthropod-borne bunyavirus that causes a zoonotic disease associated with abortion storms, neonatal mortality in livestock and hemorrhagic fever with a high case/fatality ratio in humans. To date, vaccine developments against RVF have been based on inactivated or attenuated strains but their widespread use has been hampered due to deleterious effects or incomplete protection, justifying further studies to improve the existing vaccines or to develop others. To address this, DNA plasmid and alphavirus replicon vector (VEEV) expressing RVFV Gn glycoprotein were constructed and evaluated for their ability to induce protective immune responses in mice against RVFV. An experimental live-attenuated vaccine (MP12) and its inactivated counterpart (WIV MP12) were developed to serve as benchmarks for comparison. Test vaccine candidates efficiently expressed the RVFV glycoprotein in vitro and elicited anti-RVFV antibody responses in immunized mice, as determined by RVFV specific ELISA, IgG isotype ELISA, and virus neutralization. Interestingly, these vaccine strategies elicited cellular immune responses as determined by Gn specific ELISPOT. More importantly these vaccines not only protected immunized mice from virulent RVFV when challenged via intraperitoneal route, but also conferred protection when challenged via aerosol route. This work is of public health significance as it describes the development of safe and effective vaccine candidates that have the ability to protect both livestock and humans against possible routes of exposure to this zoonotic threat.

It has been shown that HIV/SIV preferentially replicates in the gut associated lymphoid tissue (GALT) which leads to immune activation and disease progression. Different sections of the gastrointestinal (GI) tract have unique biological functions with specialized lymphoid tissue composition and distribution, which may contribute differently to viral pathogenesis. We hypothesize that the GALT serves as an active viral replication site during the acute and AIDS stages of HIV/SIV infection and as a viral reservoir in long-term non-progressors (LTNP) and antiretroviral therapy (ART) treated individuals; this viral replication results in local immune activation which contributes to disease progression. The RNA and DNA were isolated from tissues along the GI tract of uninfected and SIV infected rhesus macaques in different disease stages with or without ART. Real-time PCR was utilized to measure viral RNA and DNA, and mRNAs of CD4, TNF-α, IL-6, IL-1β, and MyD88. Our results showed that SIV DNA/RNA were detected in all GI tissues from infected monkeys regardless of the disease stage and drug intervention. However, the viral load distribution profile in the GI tract varied from monkey to monkey. Despite the undetectable viral load in peripheral blood, both viral RNA and DNA were detected in GI tissues of ART treated monkeys. Compared to the uninfected monkeys, low levels of CD4 mRNA were detected in SIV infected monkeys, particularly LTNP. There is a positive association between viral load and mRNA levels of TNF-α, IL-6, and MyD88 in the stomach and duodenum. However, no association was observed between viral loads and IL-1β mRNA levels in any of the GI tissues examined. Data from this study indicates that the entire GI tract serves as a SIV replication site in all stages of infection, which leads to pro-inflammatory cytokine production and local inflammation. This study reveals the importance of the entire GI tract in HIV/SIV pathogenesis. Especially, in ART treated individuals with undetectable viral loads in blood, active viral replication in gut tissue may lead to development of drug resistant variants and faster progression to AIDS. This will have a profound impact on clinical intervention and public health as a whole.

Highly active anti-retroviral treatment (HAART) targets less than a third of the proteins produced during HIV-1 infection. Testing the effectiveness of an anti-retroviral drug requires assays specific for the individual target that take into account its mechanism of action. Most HIV-1 proteins need to undergo dimerization in order to become functional in the viral life cycle. Historically, it has been difficult to visualize and quantify changes in a protein-protein interaction, which has left this characteristic of proteins unexplored as potential antiviral targets. In this study, a bimolecular fluorescence complementation based screening assay is developed that can quantify a change in dimerization, using the HIV-1 accessory protein Vpr as a proof of concept. Results demonstrated that bimolecular fluorescence complementation of Vpr could be competed off in a dose-dependent manner using untagged, full length Vpr as a competitor molecule. The change in signal intensity was measured quantitatively through flow cytometry and fluorescence microscopy in a high content screening assay. High content imaging was used to screen a library of peptides and a library of small molecules for an effect on Vpr dimerization. None of the Vpr peptides were shown to have an effect; however, one of the small molecules was shown to interfere with Vpr dimerization in a dose-dependent manner. Statement of Public Health relevance: Dimerization is a unique property of many HIV-1 viral proteins and is necessary to complete the viral life cycle, thus it has been identified as a potential drug target. By developing an assay that screens for inhibition of HIV-1 protein dimerization, high throughput screening can be performed to detect inhibitors of a new target in HIV-1 replication. Small molecules identified using this screening method could be developed into a novel anti-retroviral drug.

Objectives/Research questions: Epigallocatechin gallate (EGCG) is a major catechin (antioxidant) component of green tea. Recent studies have shown that it possesses many health benefits, including antimicrobial activity against some bacteria and viruses. Due to its known antimicrobial activity, this study focused on exploring the effects of EGCG on the growth of Mycobacterium tuberculosis, the causative agent of tuberculosis, as it remains a major public health concern. Through the use of a model mycobacterial species, Mycobacterium smegmatis, the experiments investigated the effects of varying concentrations of EGCG on mycobacteria. Methods: The effects of EGCG on the growth of M. tuberculosis and M. smegmatis were explored by setting up serial dilutions and carrying out cell counts over certain time periods. High Pressure Liquid Chromatography (HPLC) was used to fractionate the EGCG in order to better understand its behavior in the media. Results: The study has found that medium containing EGCG has anti-mycobacterial activity, but it is not EGCG itself that carries it out but a degradant molecule. This observation was achieved following experiments showing that a 3-day pre-incubation of EGCG in the medium strengthens its anti-mycobacterial activity. However, the HPLC showed that EGCG is fully degraded by day 1. Therefore, by day 3, there are only degradant molecules in the medium. Findings were consistent with an experiment which found that a pre-incubated green tea extract has more activity against the cells than a fresh one. Conclusions: A three day pre-incubation of green tea extract at 37°C enhances anti-mycobacterial activity, which may have implications in using green tea as a prophylactic agent. Implications for public health: If EGCG has antimicrobial activity green tea could be used as a prophylactic agent against Mycobacterium tuberculosis.

Dengue is an emerging infectious disease and is spreading world-wide at exponential levels. Two billion people in over 100 countries are at risk for infection from one of the four serotypes of the dengue virus. Those infected with dengue may develop diseases such as dengue fever and dengue hemorrhagic fever (DHF) and of the 500,000 cases that progress to DHF each year, more than 22,000 will result in fatality. Discovering new antivirals to treat DHF is essential to reducing this disease burden. Here, we have developed a cytopathic effect-based high-throughput screen (HTS) to discover possible inhibitors of Dengue viral infection of hepatocytes in vitro. Dengue virus infection of hepatocytes induces massive cell death, cytopathic effect (CPE), which we converted into a screening assay whereby inhibitors of Dengue infection prevent cells from dying. In this assay, the viral induced CPE is quantitated by monitoring cellular ATP levels, which positively correlates with cellular viability. ATP in the cell culture will drive the oxidation of luciferin resulting in the emission of light that is quantitated using a luminometer. The assay is simple and highly reproducible yielding a screening window coefficient, Z-factor, of 0.78±0.12 between plates. The Z-factor is a statistical parameter commonly accepted as an assay quality assessment and is reported as a value 0 to 1 and anything over 0.5 is considered excellent quality. This assay is advantageous to current methodology as it simultaneously screens possible inhibitory compounds while controlling for any unwanted toxicity triggered by these drugs. Our initial HTS of a 288 small compound library yielded a total of eleven hits that prevented the CPE of dengue infection. Further evaluation with an immunofluorescence assay showed that two of these compounds, Streptovitacin A and Nagilactone C, are highly potent inhibitors of dengue infection. At effective inhibitory doses, they did not appear to be cytotoxic, and therefore both of these compounds are possible antivirals and could be used to elucidate various cellular mechanisms utilized during the dengue life cycle. The discovery of these two inhibitors demonstrates the efficacy of our newly developed assay and the public health significance of this project.

Lymphatic endothelial cells (LECs) line lymphatic vessels and are present at mucosal portals of entry for many pathogens, including simian immunodeficiency virus (SIV) and human immunodeficiency virus type-1 (HIV-1). Recent studies have shown that LECs express pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), capable of recognizing pathogen-associated molecular patterns (PAMPs). PAMPs are structurally similar molecules expressed by groups of pathogens. LECs have also been shown to express chemokines, a group of small molecules secreted by cells that induce chemotaxis in responsive cells, such as CCL21, which is used by CCR7+ mature antigen presenting dendritic cells (DCs) to migrate to draining lymph nodes (LNs). These previous findings indicate that LECs might play an integral role in innate immune responses to a wide variety of microbes. In this study, I set out to characterize the expression of antiviral restriction factors as well as possible viral entry receptors for SIV/HIV-1 within three populations of human LECs. Real-time RT-PCR and immunofluorescent staining techniques were used to determine the relative expression of the restriction factors BST-2/Tetherin, APOBEC3G, and TRIM5-á. All of these factors have been shown to inhibit the replicative cycle of HIV-1 and have othologs present in nonhuman primates (NHPs). Expression of the viral entry receptors CD4, CXCR4, CCR5, DEC-205/CD205, D6/CCBP2, and CD209 as well as the LEC-specific markers podoplanin and LYVE-1 was also investigated. In addition, LEC populations were exposed to SIV, HIV-1, and markers internalization to determine to what extent LECs interact with virus in vitro. Data from populations exposed to HIV-1 as well as other substrates for internalization of extracellular materials illustrate the ability of LECs to actively monitor the extracellular milieu. LECs exposed to SIV showed multi-spliced viral transcripts possibly due to de novo transcription. Taken together, this study provides evidence that LECs are equipped with tools not only to bind and internalize pathogens, but may also serve as a low-level replicative cellular substrate for virus. Further studies to characterize LECs are of great public health relevance, particularly at mucosa sites of microbial exposure, due to their potential roles during transmission/infection.

Highly active anti-retroviral therapy (HAART) has been successful in delaying the progression to AIDS in HIV infected individuals. Exposure to HAART can result in metabolic side effects such as dyslipidemia and lipodystrophy in a subset of treated patients. We used a custom designed Illumina GoldenGate Genotyping assay to investigate the genetic susceptibility to dyslipidemia attributed to HIV infection and HAART treatment. 1,945 men were selected from the Multicenter AIDS Cohort Study (MACS) for genotyping and phenotypic analysis of serum lipid levels. This population was stratified by biogeographical ancestry and HIV/HAART status. Among men of European ancestry, those who were infected with HIV and receiving HAART had significantly lower serum low-density lipoprotein cholesterol (LDL-C, P = 1.90 x10-4) and high-density lipoprotein cholesterol levels (HDL-C, P < 1.00 x10-7), with significantly higher serum triglyceride (TRIG, P < 1.00 x10-7) levels compared to HIV/HAART (-/-) controls. Among men of mixed African and European ancestry, those who were HIV/HAART (+/+) had significantly lower LDL-C (P = 1.80 x10-4) levels compared to HIV/HAART (-/-) controls. Four SNPs; rs1532624 (P = 1.66 x10-5), rs1532625 (P = 2.36 x10-5), rs711752 (P = 4.48 x10-5), and rs708272 (P = 4.59 x10-5), located in the CETP gene region on chromosome 16 had statistically significant associations with serum HDL-C levels in HIV/HAART (+/+) European men. One SNP, rs261334 (P = 6.53 x10-6), located in the LIPC gene region on chromosome 15 was associated with serum LDL-C levels and another SNP, rs4783961 (P = 9.83 x10-6) located in the CETP gene region, was associated with HDL-C levels in HIV/HAART (+/+) men of mixed African and European ancestry. These results show that dyslipidemia attributed to HAART varies depending on biogeographical ancestry and implicates two genes associated with serum lipid levels in these patients. Understanding the mechanism of HAART-associated dyslipidemia is important to global public health because nearly half of the estimated 30 million individuals infected with HIV are receiving or eligible to receive these drugs and are at risk of these HAART related side effects. Our results can also aid in identifying those individuals at greatest risk of developing HAART-associated dyslipidemia, which could improve monitoring and management of care given to these patients.

ADAR1 is an RNA editing enzyme which acts on completely or partially double-stranded RNA. Since HIV-1 RNA has such secondary structures, we have examined whether ADAR1 exhibits antiviral activity against HIV-1. Our results indicated that ADAR1 inhibited viral replication and infectious HIV-1 production in various cell lines including 293T, HeLa and Jurkat T cells, and was active against a number of X4- and R5-tropic HIV-1 of different clades. Analysis of the level of intracellular HIV-1 RNA showed no change in levels of intracellular gag, pol, and env RNA in the presence of ADAR1 despite a significant inhibition of intracellular and virion associated HIV-1 protein production. Furthermore mutational analysis showed that ADAR1 introduced most of the A-to-G mutations in the first exon of rev at positions 5998, 6011, 6017, and 6036 and in the Rev Response Element (RRE) binding region (positions 8413 and 8438) of rev and env RNA. In elucidating the mechanism of ADAR1 inhibition of HIV-1, we observed that A-G mutations in rev have a significant negative effect on the expression of Rev. However, all mutations could be complemented by wild type Rev.Furthermore, these A-G mutations in the RRE binding region of rev inhibited the binding of Rev to the RRE region in env and inhibited transport of primary transcripts like gag, pol and env from the nucleus to the cytoplasm. Introduction of these specific mutations in rev of an infectious molecular clone of HIV-1 by site directed mutagenesis abolished the replication capacity of HIV-1 by inhibiting viral protein synthesis without any effect on viral RNA synthesis, a phenotype exhibited by HIV-1-infected cells exposed to ADAR1. ADAR1 induced mutations in env further attenuated viral infectivity. ADAR1, thus, constitutes a novel class of cellular antiviral proteins with multiple targets in the viral genome thereby providing a new avenue of exploration for therapeutic drugs benefitting public health.