Browsing by Author "Duggal, Nisha K."
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- Adenovirus transduction to express human ACE2 causes obesity-specific morbidity in mice, impeding studies on the effect of host nutritional status on SARS-CoV-2 pathogenesisRai, Pallavi; Chuong, Christina; LeRoith, Tanya; Smyth, James W.; Panov, Julia; Levi, Moshe; Kehn-Hall, Kylene; Duggal, Nisha K.; Weger-Lucarelli, James (Elsevier, 2021-11-01)The COVID-19 pandemic has paralyzed the global economy and resulted in millions of deaths globally. People with co-morbidities like obesity, diabetes and hypertension are at an increased risk for severe COVID-19 illness. This is of overwhelming concern because 42% of Americans are obese, 30% are pre-diabetic and 9.4% have clinical diabetes. Here, we investigated the effect of obesity on disease severity following SARS-CoV-2 infection using a well-established mouse model of diet-induced obesity. Diet-induced obese and lean control C57BL/6 N mice, transduced for ACE2 expression using replication-defective adenovirus, were infected with SARS-CoV-2, and monitored for lung pathology, viral titers, and cytokine expression. No significant differences in tissue pathology or viral replication was observed between AdV transduced lean and obese groups, infected with SARS-CoV-2, but certain cytokines were expressed more significantly in infected obese mice compared to the lean ones. Notably, significant weight loss was observed in obese mice treated with the adenovirus vector, independent of SARS-CoV-2 infection, suggesting an obesity-dependent morbidity induced by the vector. These data indicate that the adenovirus-transduced mouse model of SARS-CoV-2 infection, as described here and elsewhere, may be inappropriate for nutrition studies.
- Determining the Pathogenesis and Enzootic Transmission of Usutu VirusKuchinsky, Sarah (Virginia Tech, 2022-09-02)Usutu virus (USUV) is an emerging zoonotic virus within the Flaviviridae family that can cause neurological disease in humans and wild birds. USUV is maintained in an enzootic cycle between wild birds, primarily passerine species, and ornithophilic mosquitoes, predominantly Culex spp. mosquitoes. Since its first isolation in 1959 in South Africa, USUV has spread throughout sub-Saharan Africa and Europe. Its emergence into Europe was marked by large die-offs, or epizootics, of the Eurasian blackbird (Turdus merula), as well as an increase in human cases. This dissertation sought to understand whether USUV has evolved to become more pathogenic in humans or transmissible in birds. We compared the pathogenesis of five different USUV isolates, four recent isolates: Spain 2009, Netherlands 2016, Senegal 2003, Uganda 2012, and South Africa 1959, in an interferon α/β receptor knockout (Ifnar-/-) mouse model. We observed significant mortality, high viral levels in serum and tissues in all USUV strains except for the Netherlands 2016 strain. Eighteen non-synonymous mutations were identified throughout the genome of Netherlands 2016 strain compared to the other USUV isolates. To further understand USUV infection in wild birds, we developed a physiologically relevant model of infection using juvenile chickens. In juvenile chickens, we found that the European strains were characterized by more pathogenesis and higher viral titers in tissues compared to the African strains. This work established the first viremic bird model of USUV infection. Passerine birds have been suggested to be important for USUV maintenance, however a species competent for transmission has not been identified. We first determined that wild-caught house sparrows (Passer domesticus) and Culex quinquefasciatus mosquitoes were susceptible to Netherlands 2016 and Uganda 2012 USUV strains. Following an infectious feed to assess enzootic transmission, house sparrows were able to transmit both USUV strains to Cx. quinquefasciatus mosquitoes, with the Netherlands 2016 strain being more infectious compared to the Uganda 2012 strain. The collection of these chapters provides great insights on the pathogenesis of distinct USUV strains, disease presentation in birds, and enzootic transmssion of USUV. Additionally, they indicate that USUV emergence in the United States is entirely feasible.
- Differential pathogenesis of Usutu virus isolates in miceKuchinsky, Sarah C.; Hawks, Seth A.; Mossel, Eric C.; Coutermarsh-Ott, Sheryl; Duggal, Nisha K. (PLOS, 2020-10-12)Usutu virus (USUV; Flavivirus), a close phylogenetic and ecological relative of West Nile virus, is a zoonotic virus that can cause neuroinvasive disease in humans. USUV is maintained in an enzootic cycle between Culex mosquitoes and birds. Since the first isolation in 1959 in South Africa, USUV has spread throughout Africa and Europe. Reported human cases have increased over the last few decades, primarily in Europe, with symptoms ranging from mild febrile illness to severe neurological effects. In this study, we investigated whether USUV has become more pathogenic during emergence in Europe. Interferon α/β receptor knockout (Ifnar1-/-) mice were inoculated with recent USUV isolates from Africa and Europe, as well as the historic 1959 South African strain. The three tested African strains and one European strain from Spain caused 100% mortality in inoculated mice, with similar survival times and histopathology in tissues. Unexpectedly, a European strain from the Netherlands caused only 12% mortality and significantly less histopathology in tissues from mice compared to mice inoculated with the other strains. Viremia was highest in mice inoculated with the recent African strains and lowest in mice inoculated with the Netherlands strain. Based on phylogenetics, the USUV isolates from Spain and the Netherlands were derived from separate introductions into Europe, suggesting that disease outcomes may differ for USUV strains circulating in Europe. These results also suggest that while more human USUV disease cases have been reported in Europe recently, circulating African USUV strains are still a potential major health concern.
- Duration of seminal Zika viral RNA shedding in immunocompetent mice inoculated with Asian and African genotype virusesMcDonald, Erin M.; Duggal, Nisha K.; Delorey, Mark J.; Oksanish, James; Ritter, Jana M.; Brault, Aaron C. (Elsevier, 2019-06-20)Prior to the emergence of Asian genotype Zika virus (ZIKV) in the Western hemisphere, sexual transmission in humans was documented. Sexual transmission by African genotype ZIKVs has not been assessed in laboratory animal models, due to rapid and high mortality rates of immunodeficient mice following inoculation. To overcome these limitations, immunocompetent C57Bl/6 mice were used to longitudinally assess Asian and African genotype ZIKV sexual transmission potential. Furthermore, to determine if enhanced pathogenesis of African genotype ZIKVs is due to structural determinants, PRVABC59 prM/E was replaced with African MR766 prM/E (chimeric ZIKV). The African genotype and chimeric ZIKV elicited greater pathogenic effects in the male reproductive tract and generated higher viremias. Yet, the duration, magnitude and efficiency of seminal shedding of infectious virus and viral RNA were similar between chimeric-, African and Asian genotype ZIKVinoculated mice. These data show that increased male reproductive tract pathology does not increase sexual transmission potential.
- Environmental Stability of Enveloped Viruses Is Impacted by Initial Volume and Evaporation Kinetics of DropletsFrench, Andrea J.; Longest, Alexandra K.; Pan, Jin; Vikesland, Peter J.; Duggal, Nisha K.; Marr, Linsey C.; Lakdawala, Seema S. (American Society for Microbiology, 2023-04)Efficient spread of respiratory viruses requires the virus to maintain infectivity in the environment. Environmental stability of viruses can be influenced by many factors, including temperature and humidity. Our study measured the impact of initial droplet volume (50, 5, and 1 mu L) and relative humidity (RH; 40%, 65%, and 85%) on the stability of influenza A virus, bacteriophage Phi6 (a common surrogate for enveloped viruses), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) under a limited set of conditions. Our data suggest that the drying time required for the droplets to reach quasi-equilibrium (i.e., a plateau in mass) varied with RH and initial droplet volume. The macroscale physical characteristics of the droplets at quasi-equilibrium varied with RH but not with the initial droplet volume. Virus decay rates differed between the wet phase, while the droplets were still evaporating, and the dry phase. For Phi6, decay was faster in the wet phase than in the dry phase under most conditions. For H1N1pdm09, decay rates between the two phases were distinct and initial droplet volume had an effect on virus viability within 2 h. Importantly, we observed differences in virus decay characteristics by droplet size and virus. In general, influenza virus and SARS-CoV-2 decayed similarly, whereas Phi6 decayed more rapidly under certain conditions. Overall, this study suggests that virus decay in media is related to the extent of droplet evaporation, which is controlled by RH. Importantly, accurate assessment of transmission risk requires the use of physiologically relevant droplet volumes and careful consideration of the use of surrogates. IMPORTANCE During the COVID-19 pandemic, policy decisions were being driven by virus stability experiments with SARS-CoV-2 in different droplet volumes under various humidity conditions. Our study, the first of its kind, provides a model for the decay of multiple enveloped RNA viruses in cell culture medium deposited in 50-, 5-, and 1-mu L droplets at 40%, 65%, and 85% RH over time. The results of our study indicate that determination of half-lives for emerging pathogens in large droplets may overestimate transmission risk for contaminated surfaces, as observed during the COVID-19 pandemic. Our study implicates the need for the use of physiologically relevant droplet sizes with use of relevant surrogates in addition to what is already known about the importance of physiologically relevant media for risk assessment of future emerging pathogens.
- Genetic Diversity of Newcastle Disease Virus Involved in the 2021 Outbreaks in Backyard Poultry Farms in TanzaniaAmoia, Charlie F.; Hakizimana, Jean N.; Duggal, Nisha K.; Chengula, Augustino A.; Rohaim, Mohammed A.; Munir, Muhammad; Weger-Lucarelli, James; Misinzo, Gerald (MDPI, 2023-07-21)Newcastle disease virus is a significant avian pathogen with the potential to decimate poultry populations all over the world and cause enormous economic losses. Distinct NDV genotypes are currently causing outbreaks worldwide. Due to the high genetic diversity of NDV, virulent strains that may result in a lack of vaccine protection are more likely to emerge and ultimately cause larger epidemics with massive economic losses. Thus, a more comprehensive understanding of the circulating NDV genotypes is critical to reduce Newcastle disease (ND) burden. In this study, NDV strains were isolated and characterized from backyard poultry farms from Tanzania, East Africa in 2021. Reverse-transcription polymerase chain reaction (RT-PCR) based on fusion (F) gene amplification was conducted on 79 cloacal or tracheal swabs collected from chickens during a suspected ND outbreak. Our results revealed that 50 samples out 79 (50/79; 63.3%) were NDV-positive. Sequencing and phylogenetic analyses of the selected NDV isolates showed that 39 isolates belonged to subgenotype VII.2 and only one isolate belonged to subgenotype XIII.1.1. Nucleotide sequences of the NDV F genes from Tanzania were closely related to recent NDV isolates circulating in southern Africa, suggesting that subgenotype VII.2 is the predominant subgenotype throughout Tanzania and southern Africa. Our data confirm the circulation of two NDV subgenotypes in Tanzania, providing important information to design genotype-matched vaccines and to aid ND surveillance. Furthermore, these results highlight the possibility of the spread and emergence of new NDV subgenotypes with the potential of causing future ND epizootics.
- Heartland Virus Epidemiology, Vector Association, and Disease PotentialBrault, Aaron C.; Savage, Harry M.; Duggal, Nisha K.; Eisen, Rebecca J.; Staples, J. Erin (MDPI, 2018-09-14)First identified in two Missouri farmers exhibiting low white-blood-cell and platelet counts in 2009, Heartland virus (HRTV) is genetically closely related to severe fever with thrombocytopenia syndrome virus (SFTSV), a tick-borne phlebovirus producing similar symptoms in China, Korea, and Japan. Field isolations of HRTV from several life stages of unfed, host-seeking Amblyomma americanum, the lone star tick, implicated it as a putative vector capable of transstadial transmission. Laboratory vector competence assessments confirmed transstadial transmission of HRTV, demonstrated vertical infection, and showed co-feeding infection between A. americanum. A vertical infection rate of 33% from adult females to larvae in the laboratory was observed, while only one of 386 pools of molted nymphs (1930) reared from co-feeding larvae was positive for HRTV (maximum-likelihood estimate of infection rate = 0.52/1000). Over 35 human HRTV cases, all within the distribution range of A. americanum, have been documented. Serological testing of wildlife in areas near the index human cases, as well as in widely separated regions of the eastern United States where A. americanum occur, indicated many potential hosts such as raccoons and white-tailed deer. Attempts, however, to experimentally infect mice, rabbits, hamsters, chickens, raccoons, goats, and deer failed to produce detectable viremia. Immune-compromised mice and hamsters are the only susceptible models. Vertical infection augmented by co-feeding transmission could play a role in maintaining the virus in nature. A more complete assessment of the natural transmission cycle of HRTV coupled with serosurveys and enhanced HRTV disease surveillance are needed to better understand transmission dynamics and human health risks.
- Infectious SARS-CoV-2 Is Emitted in Aerosol ParticlesHawks, Seth A.; Prussin, Aaron J. II; Kuchinsky, Sarah C.; Pan, Jin; Marr, Linsey C.; Duggal, Nisha K. (American Society for Microbiology, 2021-10-19)Respiratory viruses such as SARS-CoV-2 are transmitted in respiratory droplets and aerosol particles, which are released during talking, breathing, coughing, and sneezing. Noncontact transmission of SARS-CoV-2 has been demonstrated, suggesting transmission via virus carried through the air. Here, we demonstrate that golden Syrian hamsters produce infectious SARS-CoV-2 in aerosol particles prior to and concurrent with the onset of mild clinical signs of disease. The average emission rate in this study was 25 infectious virions/hour on days 1 and 2 postinoculation, with average viral RNA levels 200-fold higher than infectious virus in aerosol particles. The majority of virus was contained within particles <5 μm in size. Thus, we provide direct evidence that, in hamsters, SARS-CoV-2 is an airborne virus.
- Investigation of flagellotropic phage interactions with their motile host bacteriaGonzalez, Floricel (Virginia Tech, 2021-06-21)Bacteriophages cohabit with their bacterial hosts and shape microbial communities. To initiate infection, phages use bacterial components as receptors to recognize and attach to hosts. Flagellotropic phages utilize bacterial flagella as receptors. Studies focused on uncovering mechanistic details of flagellotropic phage infection are lacking. As the number of phage-based applications grows, it is important to understand these details to predict the potential outcomes of phage therapy. To this end, we studied two flagellotropic phages: Agrobacterium phage 7-7-1 and bacteriophage χ. Phage 7-7-1 infects Agrobacterium spp., while bacteriophage χ infects Salmonella and Escherichia coli. Chapter 1 consists of a literature review. Chapter 2 addresses factors underlying phage-bacteria coexistence. We document the emergence of a sector-shaped lysis pattern following co-inoculation of phage χ and one of its Salmonella hosts on swim plates. We propose that this pattern serves as a reporter for balanced phage-bacteria coexistence. Using a combined experimental and mathematical modelling approach, we discovered that variations to intrinsic factors (i.e., bacterial motility, phage adsorption) skews the pattern towards either bacterial or phage predominance. Thus, this computational model may be used to predict phage therapy application outcomes. Chapter 3 details the identification of cell surface receptors essential for phage 7-7-1 infection using a transposon mutagenesis approach. We identified three Agrobacterium sp. H13-3 genes involved in phage 7-7-1 infection. Using mass spectrometry and other analyses, we determined that the LPS profiles of strains lacking these genes varied compared to the wild type. Thus, LPS is a secondary cell surface receptor for phage 7-7-1. Chapter 4 focuses on the discovery of phage encoded receptor binding proteins (RBPs) in Agrobacterium phage 7-7-1. Using an RBP screen, we discovered three candidate RBPs. We learned that our top candidate, Gp4, inhibits the growth of Agrobacterium sp. H13-3 cells in a motility and glycan dependent manner. Because of its bacteriostatic activities, this protein is a promising candidate for therapeutic use. Overall, the described works contribute to a deepened understanding of flagellotropic phage infection and the factors influencing their coexistence with motile bacteria. These works will contribute towards the development of phage therapies using whole phage or their components.
- Mutations present in a low-passage Zika virus isolate result in attenuated pathogenesis in miceDuggal, Nisha K.; McDonald, Erin M.; Weger-Lucarelli, James; Hawks, Seth A.; Ritter, Jana M.; Romo, Hannah; Ebel, Gregory D.; Brault, Aaron C. (2019-04)Zika virus (ZIKV) infection can result in neurological disorders including Congenital Zika Syndrome in infants exposed to the virus in utero. Pregnant women can be infected by mosquito bite as well as by sexual transmission from infected men. Herein, the variants of ZIKV within the male reproductive tract and ejaculates were assessed in inoculated mice. We identified two non-synonymous variants at positions E-V330L and NS1-W98G. These variants were also present in the passage three PRVABC59 isolate and infectious clone relative to the patient serum PRVABC59 sequence. In subsequent studies, ZIKV E-330L was less pathogenic in mice than ZIKV E-330V as evident by increased average survival times. In Vero cells, ZIKV E-330L/NS1-98G outcompeted ZIKV E-330V/NS1-98W within 3 passages. These results suggest that the E-330L/NS1-98G variants are attenuating in mice and were enriched during cell culture passaging. Cell culture propagation of ZIKV could significantly affect animal model development and vaccine efficacy studies.
- On the Fly: Interactions Between Birds, Mosquitoes, and Environment That Have Molded West Nile Virus Genomic Structure Over Two DecadesDuggal, Nisha K.; Langwig, Kate E.; Ebel, Gregory D.; Brault, Aaron C. (Oxford University Pres, 2019-09-24)West Nile virus (WNV) was first identified in North America almost 20 yr ago. In that time, WNV has crossed the continent and established enzootic transmission cycles, resulting in intermittent outbreaks of human disease that have largely been linked with climatic variables and waning avian seroprevalence. During the transcontinental dissemination of WNV, the original genotype has been displaced by two principal extant genotypes which contain an envelope mutation that has been associated with enhanced vector competence by Culex pipiens L. (Diptera: Culicidae) and Culex tarsalis Coquillett vectors. Analyses of retrospective avian host competence data generated using the founding NY99 genotype strain have demonstrated a steady reduction in viremias of house sparrows over time. Reciprocally, the current genotype strains WN02 and SW03 have demonstrated an inverse correlation between house sparrow viremia magnitude and the time since isolation. These data collectively indicate that WNV has evolved for increased avian viremia while house sparrows have evolved resistance to the virus such that the relative host competence has remained constant. Intrahost analyses of WNV evolution demonstrate that selection pressures are avian species-specific and purifying selection is greater in individual birds compared with individual mosquitoes, suggesting that the avian adaptive and/or innate immune response may impose a selection pressure on WNV. Phylogenomic, experimental evolutionary systems, and models that link viral evolution with climate, host, and vector competence studies will be needed to identify the relative effect of different selective and stochastic mechanisms on viral phenotypes and the capacity of newly evolved WNV genotypes for transmission in continuously changing landscapes.
- Pathogenesis and shedding of Usutu virus in juvenile chickensKuchinsky, Sarah C.; Frere, Francesca; Heitzman-Breen, Nora; Golden, Jacob; Vázquez, Ana; Honaker, Christa F.; Siegel, Paul B.; Ciupe, Stanca M.; LeRoith, Tanya; Duggal, Nisha K. (Taylor & Francis, 2021-01-01)Usutu virus (USUV; family: Flaviviridae, genus: Flavivirus), is an emerging zoonotic arbovirus that causes severe neuroinvasive disease in humans and has been implicated in the loss of breeding bird populations in Europe. USUV is maintained in an enzootic cycle between ornithophilic mosquitos and wild birds. As a member of the Japanese encephalitis serocomplex, USUV is closely related to West Nile virus (WNV) and St. Louis encephalitis virus (SLEV), both neuroinvasive arboviruses endemic in wild bird populations in the United States. An avian model for USUV is essential to understanding zoonotic transmission. Here we describe the first avian models of USUV infection with the development of viremia. Juvenile commercial ISA Brown chickens were susceptible to infection by multiple USUV strains with evidence of cardiac lesions. Juvenile chickens from two chicken lines selected for high (HAS) or low (LAS) antibody production against sheep red blood cells showed markedly different responses to USUV infection. Morbidity and mortality were observed in the LAS chickens, but not HAS chickens. LAS chickens had significantly higher viral titers in blood and other tissues, as well as oral secretions, and significantly lower development of neutralizing antibody responses compared to HAS chickens. Mathematical modelling of virus-host interactions showed that the viral clearance rate is a stronger mitigating factor for USUV viremia than neutralizing antibody response in this avian model. These chicken models provide a tool for further understanding USUV pathogenesis in birds and evaluating transmission dynamics between avian hosts and mosquito vectors.
- Persistence of Zika virus RNA in the epididymis of the murine male reproductive tractVogt, Megan B.; Frere, Francesca; Hawks, Seth A.; Perez, Claudia E.; Coutermarsh-Ott, Sheryl; Duggal, Nisha K. (2021-08)Zika virus (ZIKV) can infect developing fetuses in utero and cause severe congenital defects independent of route of maternal infection. Infected men can shed ZIKV RNA in semen for over six months. Whether prolonged viral RNA shedding in semen indicates a persistent infection in the male reproductive tract is unknown. We hypothesized that if ZIKV establishes a persistent infection in the male reproductive tract (MRT), then immunosuppressant treatment should stimulate ZIKV replication and seminal shedding. Male mice were infected with ZIKV and immunosuppressed when they shed viral RNA but not infectious virus in ejaculates. Following immunosuppression, we did not detect infectious virus in ejaculates. However, we did detect ZIKV positive and negative sense RNA in the epididymal lumens of mice treated with cyclophosphamide, suggesting that ZIKV persists in the epididymis. This study provides insight into the mechanisms behind ZIKV sexual transmission, which may inform public health decisions regarding ZIKV risks.
- The Pro-Inflammatory Chemokines CXCL9, CXCL10 and CXCL11 Are Upregulated Following SARS-CoV-2 Infection in an AKT-Dependent MannerCallahan, Victoria; Hawks, Seth A.; Crawford, Matthew A.; Lehman, Caitlin W.; Morrison, Holly A.; Ivester, Hannah M.; Akhrymuk, Ivan V.; Boghdeh, Niloufar; Flor, Rafaela; Finkielstein, Carla V.; Allen, Irving C.; Weger-Lucarelli, James; Duggal, Nisha K.; Hughes, Molly A.; Kehn-Hall, Kylene (MDPI, 2021-06-03)Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA virus that is the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Patients with severe COVID-19 may develop acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) and require mechanical ventilation. Key features of SARS-CoV-2 induced pulmonary complications include an overexpression of pro-inflammatory chemokines and cytokines that contribute to a ‘cytokine storm.’ In the current study an inflammatory state in Calu-3 human lung epithelial cells was characterized in which significantly elevated transcripts of the immunostimulatory chemokines CXCL9, CXCL10, and CXCL11 were present. Additionally, an increase in gene expression of the cytokines IL-6, TNFα, and IFN-γ was observed. The transcription of CXCL9, CXCL10, IL-6, and IFN-γ was also induced in the lungs of human transgenic angiotensin converting enzyme 2 (ACE2) mice infected with SARS-CoV-2. To elucidate cell signaling pathways responsible for chemokine upregulation in SARS-CoV-2 infected cells, small molecule inhibitors targeting key signaling kinases were used. The induction of CXCL9, CXCL10, and CXCL11 gene expression in response to SARS-CoV-2 infection was markedly reduced by treatment with the AKT inhibitor GSK690693. Samples from COVID-19 positive individuals also displayed marked increases in CXCL9, CXCL10, and CXCL11 transcripts as well as transcripts in the AKT pathway. The current study elucidates potential pathway specific targets for reducing the induction of chemokines that may be contributing to SARS-CoV-2 pathogenesis via hyperinflammation.
- Usutu Virus: An Emerging Arbovirus ThreatBates, Tyler Alexander (Virginia Tech, 2021-02-04)Mosquito-borne viruses, such as dengue virus (DENV), Zika virus (ZIKV), chikungunya virus (CHIKV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), and West Nile virus (WNV) are major threats to global public health resulting in millions of infections and hundreds of thousands of deaths annually. The presence of these viruses and their increasing emergence/spread continues to escalate. Notably, Usutu virus (USUV; Genus: Flavivirus; Family: Flaviviridae) is one such pathogen currently causing mass die-offs of avian hosts throughout Europe. USUV is categorized in the Japanese Encephalitis virus (JEV) antigenic complex and thus shares many antigenic and pathologic characteristics with fellow members, such as JEV and WNV. Respective to human infections, USUV cases are generally asymptomatic; nonetheless, acute cases have been reported. These acute cases typically cause mild symptoms, such as fevers and rashes; however, more severe cases can result in neurologic diseases, such as encephalitis and/or meningoencephalitis. In addition to these pathologic similarities, USUV shares several ecological and geographical traits with WNV, a pathogen responsible for several outbreaks during its spread from Africa, to Europe, and eventually the United States. Currently, WNV is considered endemic in areas across the United States due to its transmission via Culex spp.; mosquitoes that are ubiquitous in the United States. These parallels suggest the possible emergence of USUV into the United States and therefore, it is imperative to broaden our knowledge of USUV and assess its potential to become a major global health concern. The overall goal of this thesis was to characterize USUV and evaluate its emergence potential in the United States by: (1) developing infectious clones of recent European and African USUV isolates as tools for characterization and analysis of USUV and (2) assessing the transmission potential of several species of North American mosquitoes. In Aim 1, we show that the aforementioned infectious clones infect and replicate similarly to their parental strains in vitro in both vertebrate and invertebrate models, as well as in transiently immunocompromised CD-1 and IFNAR-/- murine models, and thus serve as useful tools for future molecular studies focusing on USUV. Furthermore, in Aim 2, we describe the ability of field-caught (Southwest Virginia, USA) Culex spp. and Aedes spp. mosquitoes to become infected with a recent European isolate of USUV; although, we report an overall limited potential for these species to transmit this virus. Altogether, these studies form a foundation for understanding the potential emergence of USUV in the United States as well as provide necessary tools needed to aid future research on USUV emergence, transmission, and pathogenesis.
- West Nile Virus Vaccination Protects against Usutu Virus Disease in MiceSalgado, Rebecca; Hawks, Seth A.; Frere, Francesca; Vázquez, Ana; Huang, Claire Y.-H.; Duggal, Nisha K. (MDPI, 2021-11-23)West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne flaviviruses that can cause neuroinvasive disease in humans. WNV and USUV circulate in both Africa and Europe and are closely related. Due to antigenic similarity, WNV-specific antibodies and USUV-specific antibodies have the potential to bind heterologous viruses; however, it is unclear whether this interaction may offer protection against infection. To investigate how prior WNV exposure would influence USUV infection, we used an attenuated WNV vaccine that contains the surface proteins of WNV in the backbone of a dengue virus 2 vaccine strain and protects against WNV disease. We hypothesized that vaccination with this attenuated WNV vaccine would protect against USUV infection. Neutralizing responses against WNV and USUV were measured in vitro using sera following vaccination. Sera from vaccinated CD-1 and Ifnar1−/− mice cross-neutralized with WNV and USUV. All mice were then subsequently challenged with an African or European USUV strain. In CD-1 mice, there was no difference in USUV titers between vaccinated and mock-vaccinated mice. However, in the Ifnar1−/− model, vaccinated mice had significantly higher survival rates and significantly lower USUV viremia compared to mock-vaccinated mice. Our results indicate that exposure to an attenuated form of WNV protects against severe USUV disease in mice and elicits a neutralizing response to both WNV and USUV. Future studies will investigate the immune mechanisms responsible for the protection against USUV infection induced by WNV vaccination, providing critical insight that will be essential for USUV and WNV vaccine development.