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 pathogenesis
    Rai, 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.
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    Characterization of pro- and anti-inflammatory immune responses in SARS-CoV-2 infection
    Ivester, Hannah Marie (Virginia Tech, 2024-05-14)
    Viral infection stimulates the immune response to produce many cytokines and chemokines, the proteins imperative to fight a brewing infection. This response begins through recognition of pathogen-associated molecular patterns (PAMPs) from the virus, or from other signatures characteristic of tissue damage, damage-associated molecular patterns (DAMPs), by pattern recognition receptors (PRRs) that in turn stimulate pro-inflammatory signaling cascades. The results of these signaling pathways include the release of cytokines and chemokines that work to further upregulate immune responses and attract immune cells to the site of infection, respectively. In the case of SARS-CoV-2 infection, these responses can become problematic if they go unmitigated or unresolved, resulting in the severe COVID-19 manifestation of the 'cytokine storm,' or multisystem inflammatory syndrome in children (MIS-C). One classically increased protein in cytokine storm of COVID-19 patients is C-X-C motif chemokine 10 (CXCL10), which has been explored as a prognostic marker as it is shown to be predictive of disease outcome in hospitalized patients. To prevent severe outcomes like cytokine storm, a delicate balance must be struck, to ensure that this inflammation does not result in high levels of diffuse tissue damage. To achieve this, anti-inflammatory pathways exist within the immune system and help dampen the signals being induced. One such unique anti-inflammatory protein is a pattern recognition receptor known as NLRX1 (Nucleotide binding oligomerization domain, leucine rich repeat containing X1), that can interact with two main pathways involved with anti-viral immunity, the NFB and interferon pathways, downregulating them to keep off-target tissue damage at bay. NLRX1 is also involved in several other cellular processes, including modulating cell death processes and cellular metabolism which can also impact viral replication and clearance indirectly. In this work, we investigated both the pro- and anti-inflammatory arms of the anti-SARS-CoV-2 response focusing on two key proteins – pro-inflammatory chemokine CXCL10 and immunoregulatory PRR NLRX1. The roles of these two proteins were explored utilizing transcriptomic analysis of both human and mouse RNA samples, immortalized cell culture work, humanized mouse models of SARS-CoV-2 infection, and mouse-adapted virus models to be able to utilize deficient mouse models. In this work we better characterize the immune response to SARS-CoV-2 and its related immune-driven pathobiology of disease. The data presented in this work continues to elucidate CXCL10's role as an important driver of viral clearance of SARS-CoV-2, translating data from human patient nasal swabs to the animal model of disease, exploring differential inflammation and immune responses in the absence of CXCL10. Additionally, the work shown here provides further understanding of NLRX1 and its role in antiviral immunity with the context of SARS-CoV-2 infection. The interactions between this protein and the virus remains to be fully characterized, however, it appears they have some degree of mutual inhibition as determined by animal and cell culture models. The culmination of work here emphasizes the importance for both the pro- and anti-inflammatory responses in SARS-CoV-2 infection and offers insight into two possible related targets for future drug development.
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    Cross-protection from St. Louis encephalitis virus and Usutu virus disease by human West Nile virus convalescent plasma in mice
    Hossain, Md Shakhawat (Virginia Tech, 2024-08-21)
    West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Usutu virus (USUV) are emerging mosquito-borne flaviviruses. These viruses are phylogenetically closely related and belong to the Japanese encephalitis serocomplex group. Similar to other flaviviruses, these viruses are enveloped, with genomes comprising positive-sense, single-stranded RNA approximately 11 kb in length. Upon translation, a single polyprotein is produced, consisting of three structural and seven non-structural proteins. These proteins function in virus binding to the cell membrane, entry into cells, replication, immune evasion, and the production of new virus progeny. Typically, these viruses are maintained in a sylvatic cycle involving avian hosts, such as passerine birds, and mosquitoes. However, they can accidentally spill over to humans through mosquito bites or wildlife exposure. Although humans generally remain asymptomatic and do not support sufficient viral replication for transmission, they can develop febrile disease and, in some cases, severe neuroinvasive diseases, especially among the elderly or immunocompromised individuals. Due to their co-circulation in the same geographical areas and sharing similar hosts and vectors, individuals in Italy and Germany have been detected as seropositive for WNV and USUV, while seropositivity for WNV and SLEV has been observed in the Americas. Viruses in the Japanese encephalitis virus serocomplex group exhibit significant antigenic similarity. The envelope protein alone contains 12 distinct epitopes and at least three highly conserved epitopes among the JEV serocomplex. Consequently, infection with one member of the JEV serocomplex group, such as WNV, induces WNV-specific antibodies and heterotypic antibodies that can cross-neutralize other members of the JEV serocomplex group, such as USUV and SLEV. Therefore, cross-reactive epitopes can protect against heterologous virus challenges to varying extents, depending on the accessibility of the antibodies to the epitopes. Prior infection with WNV or its envelope domain III (EDIII) or non-structural protein 1 (NS1) protected mice from lethal JEV challenges. Vaccination against WNV protected mice from lethal USUV challenges, and vice versa. Immunity to JEV or SLEV protected hamsters from lethal WNV challenges. Although human sera immune to WNV cross-neutralized USUV and SLEV in vitro during serodiagnosis, the actual mechanism of cross-protection among WNV, USUV, and SLEV remains poorly characterized. Therefore, this study aims to understand the mechanism of cross-protection. Specifically, this research investigated whether human plasma immune to WNV could cross-protect mice from encephalitis caused by SLEV or USUV. Initially, WNV-specific human convalescent plasma and mouse WNV convalescent serum (as a positive control) neutralized WNV and cross-neutralized USUV and SLEV in vitro in a neutralization test. Subsequently, immunocompetent mice were intraperitoneally injected with human WNV convalescent plasma, human normal plasma, mouse WNV convalescent serum, or mouse normal serum the day before being challenged with WNV, SLEV, or USUV via footpad injection. We found that human WNV convalescent plasma provided mice with strong protection against neuroinvasive encephalitis caused by WNV. Additionally, human WNV convalescent plasma reduced the viremia titers of SLEV and USUV for several days during acute infection. Human WNV convalescent serum also demonstrated a trend towards protecting mice from SLEV-induced encephalitis, as evidenced by lower SLEV titers in the brain and histopathology scores. These findings will aid in decoding the mechanisms of cross-protection among the JEV serovars, developing therapeutic strategies against WNV, SLEV, and USUV, and anticipating potential disease outcomes, especially in regions where multiple viruses of the JEV serocomplex are endemic.
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    Determining the Pathogenesis and Enzootic Transmission of Usutu Virus
    Kuchinsky, 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.
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    Differential pathogenesis of Usutu virus isolates in mice
    Kuchinsky, 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.
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    Differential regulation of herpes simplex virus-1 and herpes simplex virus-2 during latency and post reactivation in response to stress hormones and nerve trauma in primary adult sensory and sympathetic neurons
    Goswami, Poorna (Virginia Tech, 2022-08-18)
    The contrasting infection strategy of herpes simplex virus (HSV) consists of an initial primary lytic infection in epithelial cells, followed by establishment of lifelong latency in sensory and autonomic neurons of the peripheral nervous system that innervate the site of infection. Any cellular stress trigger, ranging from external stimuli such as UV radiation or nerve injury to psychological and physiological stress, can reactivate HSV from latency in the neurons, resulting in recurrent disease episodes. Stress hormones and deprivation of neurotrophic factor (NTF) both have a strong correlation with HSV reactivation from neurons. However, neuronal signaling pathways cardinal to HSV latency and reactivation are still not clear. This dissertation provides new understanding of HSV latency and reactivation in response to two orthogonal stress stimuli, viz. stress hormones epinephrine (EPI) and corticosterone (CORT), as well as NTF deprivation that simulates a nerve injury in primary neuronal cultures. In this dissertation, we demonstrate that physiological stress hormones EPI and CORT differentially regulate HSV-1 and HSV-2 reactivation in adult neurons. Both EPI and CORT treatment reactivated only HSV-1 in sympathetic superior cervical ganglia (SCG) neurons, while HSV-2 was reactivated only by CORT in both sensory trigeminal ganglia (TG) neurons and sympathetic superior cervical (SCG) neurons. EPI utilized the combination of α and β adrenergic receptor complex, while CORT signaled through glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) to reactivate HSV in the neurons. NTFs are tissue-target derived growth factors required for neuronal protection and survival. Neurotrophins are also required for maintaining HSV latency, as NTF deprivation reactivates both HSV-1 and HSV-2 in adult sensory TG and sympathetic SCG neurons. In addition, assessing the temporal kinetics of HSV gene expression showed differential expression profiles of viral immediate-early (IE) genes ICP0, ICP4, ICP27 and trans-activator VP16 following treatment with stress hormones and NTF deprivation in HSV-1 and HSV-2 infected neurons. We also show that different molecular mechanisms are involved in HSV latency and reactivation, which are dependent on the stimuli and the type of neurons. Tyrosine kinase receptor-mediated PI3K-Akt-mTORC signaling cascades have been studied for their role in maintaining HSV latency. Activation of β-catenin signalosome expression has also been implicated during HSV latency and following reactivation. GSK3β is a key effector molecule that inter-connects Akt and β-catenin mediated pathways, forming an Akt-GSK3β-β-catenin signaling axis. Analyzing the Akt-GSK3β-β-catenin signaling in response to stress hormone and NTF deprivation revealed significant differences in protein expression levels between HSV-1 and HSV-2 infected sensory and sympathetic neurons. In HSV-1 infected neurons, the Akt-GSK3β-β-catenin maintains the signal transmission in order to keep the neurons alive, but HSV-2 infections obliterated the entire axis in the adult neurons, particularly in sympathetic neurons. In summary, we demonstrate that HSV-1 and HSV-2 do not have a 'one for all' infection mechanism. Establishment of latency and reactivation by HSV is virus specific, stimulus specific and neuron specific.
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    Duration of seminal Zika viral RNA shedding in immunocompetent mice inoculated with Asian and African genotype viruses
    McDonald, 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.
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    Effective Strategies for Preventing and Mitigating Emerging Viruses
    Chuong, Christina (Virginia Tech, 2023-05-08)
    The world is grappling with an escalating risk of viral outbreaks of pandemic proportion, with zoonotic RNA viruses such as chikungunya virus (CHIKV) and SARS-CoV-2 posing significant threats to global health. Several environmental and evolutionary factors have fueled the emergence and spread of infection, creating a constant arms race against emerging pathogens. Current prevention and mitigation strategies are inadequate, necessitating tools to prevent and control viral infections; innovative strategies are needed in the pipeline to address significant challenges. CHIKV is a mosquito-borne virus that has caused millions of disease cases worldwide and is a reemerging threat with increasing potential to become endemic in the US. Currently, there are no licensed treatments available to protect against CHIK disease, making the development of a vaccine crucial. Live-attenuated vaccines (LAVs) have traditionally been a promising strategy due to their high immunogenicity and cost-effectiveness. However, concerns regarding adverse side effects and the potential for viral replication leading to pathogenic reversions or transmission into mosquitoes have limited their use. To that end, we have developed a new generation of safer vaccines by modifying the standard LAV platform through innovative attenuating strategies. Our dual-attenuated platform utilizes a previously developed chimera of CHIKV and the closely related Semliki Forest virus (SFV) as a vaccine backbone which expresses antiviral mouse cytokines IFN-γ or IL-21, as an additional mechanism to control infection. In several mouse models, both cytokine-expressing candidates showed reduced footpad swelling and minimal to no systemic replication or dissemination capacity compared to the parental vaccine post-vaccination. Importantly, these candidates conferred full protection from wildtype CHIK disease. Our IFNγ-expressing vaccine showed the most significant attenuation of viral replication. To understand the underlying mechanism, we identified three IFNγ-regulated antiviral genes (Gbp1/2 and Ido1) that were highly upregulated in 3T3 mouse fibroblasts post-infection with the IFN-γ-expressing candidate but not the parental backbone. To further investigate the role of these genes in restricting viral replication and enhance the clinical relevance of our vaccine platform, we redesigned our vaccine to express human IFNγ (hIFNγ) and performed viral growth kinetics in MRC5 human lung fibroblasts. Our vaccine showed reduced viral replication compared to controls and high expression of human GBP1/2/3 was observed post-infection. Overexpression of these genes demonstrated a direct impact on viral replication against wildtype CHIKV. These findings shed light on the mechanism of action of our vaccine and highlight the potential of targeting IFNγ-regulated antiviral genes for developing effective vaccines against CHIKV. Our results provided a foundation for investigating the broad-use application of IFN-γ against other alphaviruses for vaccine or therapeutic design. We evaluated the effects of increasing levels of exogenous hIFNγ on Mayaro virus (MAYV), Ross River virus (RRV), and Venezuelan Equine Encephalitis virus (VEEV). We observed a positive dose-dependent relationship between hIFNγ and decreasing viral titers for all three viruses. Interestingly, we also observed similar patterns of GBP upregulation with MAYV and RRV, both Old World alphaviruses, but not with VEEV, a New World alphavirus. This finding may indicate an alternative IFNγ-stimulated pathway responsible for controlling different alphaviruses. Overall, these studies establish a fundamental role of IFNγ in controlling viral infection and highlight its potential use in both vaccine and therapeutic intervention. While LAVs are a gold standard for developing immunity against a virus, the urgency of responding to an active and deadly pandemic has promoted the use of faster strategies such as mRNA vaccines. Once the viral sequence was known, these vaccines were comparatively quick to produce for SARS-CoV-2 and prevented millions of disease cases at the height of their introduction. However, the emergence of variants of concerns bypassing previous immunization efforts has demonstrated the need for complementary treatments such as antivirals to control disease. To that end, we evaluated several rhodium organometallic complexes as potential antivirals against SARS-CoV-2. We show that two pentamethylcyclopentadienyl (Cp*) rhodium piano stool complexes, Cp*Rh(ICy)Cl2 and Cp*Rh(dpvm)Cl are non-toxic in Vero E6 and Calu3 cells and reduce SARS-CoV-2 plaque formation up to 99%. These complexes have previously demonstrated high antimicrobial activity against multiple antibiotic-resistance bacteria and with our results, support their potential application as pharmaceuticals, warranting further investigation into their activity.
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    Environmental Stability of Enveloped Viruses Is Impacted by Initial Volume and Evaporation Kinetics of Droplets
    French, 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.
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    Equine Herpesvirus Type 1: Filling Gaps Toward Improved Outbreak Management
    Saklou, Nadia Talal (Virginia Tech, 2023-09-06)
    Equine herpesvirus type 1 (EHV-1) is a common pathogen of horses that typically causes upper respiratory disease, however is also associated with late-term abortion, neonatal foal death and neurologic disease. Once a horse is infected, the virus concentrates to local lymphoid tissue, where it becomes latent. The virus can recrudesce during times of stress, which can lead to the initiation of devastating outbreaks. Some variants of EHV-1 have been associated with more severe disease outcomes. Appropriate outbreak management focuses on minimizing the movement of potentially exposed horses. This approach lacks a strategy for prevention at the level of latency largely due to a knowledge paucity in regards to carriage rate of latent EHV-1. Biosecurity decisions are also dependent on awaiting currently-available diagnostic testing that often take several days for results. Thus, our work has been focused on understanding the carriage rate of the latent virus in different geographic regions as well as improving diagnostic efficiency, both of which are essential for improving the management of EHV-1 disease. Loop mediated isothermal amplification (LAMP) is a method that amplifies nucleic acid rapidly at a constant temperature and is minimally affected by inhibitors that are often found in clinical samples. This procedure can be followed by multiple detection methods. A new, efficient sequencing method, called nanopore sequencing, has been developed in a handheld device, called MinION, that provides thorough output in a timely manner. When combined with LAMP, it has been referred to as LAMPore. The first objective of our work was to estimate the prevalence of latent EHV-1 and compare the frequency of each variant in the submandibular lymph nodes from horses in Virginia. Our second objective was to perform direct DNA sequencing of EHV-1 using the mobile MinION sequencer in combination with LAMP viral enrichment. Our findings demonstrated a low apparent prevalence of latent EHV-1 DNA in submandibular lymph nodes in this population of horses in Virginia as well as successful detection and identification of EHV-1 in equine nasal swab samples using LAMPore sequencing.
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    Evolutionary History of Immunomodulatory Genes of Giant Viruses
    Perez, Claudia Elizabeth (Virginia Tech, 2022-05-20)
    Nucleocytoplasmic large DNA viruses (NCLDVs) have genome sizes that range from around 100 kilobases (kb) to up to 2.5 megabases, and virion sizes that can reach up to 1.5 μm. Their large size in both of these contexts is atypical and defies the traditional view that viruses are streamlined, "filterable infectious agents". NCLDVs include many diverse groups, including Poxviruses, Asfarviruses, Iridoviruses, Mimiviruses, and Marseilleviruses. Poxviruses are perhaps the most well-studied; these viruses have 135-360 kbp genomes with about half of the genes encoding essential replication genes and the other half encoding genes related to host-virus interactions. Many of the genes involved in host-virus interactions are involved in immunomodulatory processes and have homology to proteins encoded by the host. These viral genes, often referred to as "mimics", are therefore believed to be the result of host-to-virus gene transfer. In this study I sought to examine if common poxvirus immunomodulatory genes were found in other NCLDV lineages, and if so, to analyze the evolutionary history of these genes. I identified 5 protein families of immunomodulatory genes that were found in both poxviruses and other NCLDV lineages, and I used phylogenetic tools to compare viral immunomodulatory genes of NCLDVs to their eukaryotic orthologs to evaluate the number of times different NCLDV lineages have acquired these genes. Our phylogenetic analyses showed that several viral immunomodulatory genes were acquired multiple times by different NCLDV lineages, while others appear to have been transferred between viral groups. Interestingly, some NCLDV genes clustered together with homologs from the unrelated Herpesviridae family, suggesting that inter-viral gene exchange can traverse vast evolutionary distances. The vast diversity of hosts infected by different NCLDV lineages suggests that these immunomodulatory genes play key roles that are useful to viruses in a variety of contexts. This research provides insight into how giant viruses acquire host genes, which contribute to their large genome size, and how those genes evolved to subvert antiviral defenses.
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    Exploring the drivers and consequences of emerging infectious disease of wildlife
    Grimaudo, Alexander Thomas (Virginia Tech, 2024-04-22)
    Emerging infectious diseases of wildlife have threatened host populations of diverse taxa in recent history, which is largely attributable to anthropogenic global change. In three data chapters, this dissertation examines the drivers of individual- to population-level variation in how host populations respond to novel and emerging pathogens. Each chapter explores these processes in bat populations of North America, predominantly the Northeast and Midwest regions of the United States, impacted by the emerging fungal pathogen that causes white-nose syndrome, Pseudogymnoascus destructans. In Chapter 2, I disentangle the effects of adaptive host traits and environmental influences in driving host population stabilization of the little brown bat (Myotis lucifugus), finding that host-pathogen coexistence in this system is the product of their complex interaction. In Chapter 3, I characterize the range-wide variation in white-nose syndrome impacts on a federally endangered and poorly studied species, the Indiana bat (Myotis sodalis), as well as environmental and demographic determinants of its declines over epidemic time. In Chapter 4, I explore the role of individual variation in roosting microclimate selection of little brown bats in driving their infection severity, yielding important insights into the pathophysiology and environmental dependence of white-nose syndrome. Ultimately, this dissertation characterizes complex drivers of variation in host responses to emerging and invading pathogens, yielding insights essential to the successful mitigation of their impacts.
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    Genetic Diversity of Newcastle Disease Virus Involved in the 2021 Outbreaks in Backyard Poultry Farms in Tanzania
    Amoia, 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.
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    Gnotobiotic Pig Models for the Study of Enteric Pathogen Replication and Pathogenesis
    Nyblade, Charlotte June (Virginia Tech, 2024-10-09)
    Clostridioides difficile (C. difficile) and human rotavirus (HRV) are leading causes of bacterial and viral gastroenteritis worldwide. Treatment and vaccination options for both pathogens have significant limitations. C. difficile infections are treated with antibiotics, which is paradoxical as C. difficile itself is associated with antibiotic usage. In the United States, two live oral attenuated vaccines (Rotarix and RotaTeq) are licensed for protection against HRV. Since receiving approval from the World Health Organization (WHO), Rotarix and RotaTeq have been widely implemented into global national childhood immunization schedules, with one report finding 59 countries using Rotarix and 25 using RotaTeq. However, these vaccines have much lower efficacy rates in low- and middle-income countries. Because of these caveats, there is an urgent need to generate novel prophylaxes and treatments for C. difficile and HRV. In order to address this need, animal models that replicate the nuances of each infection are imperative. We have developed gnotobiotic (Gn) pig models for each pathogen. Gn pigs infected with spores of the hypervirulent UK1 strain of C. difficile develop classical signs of infection, including watery diarrhea and weight loss. Gross necropsy reveals colonic distention and discoloration, and histopathological evaluation shows volcano lesions, pseudo membrane formation, and epithelial cell erosion. Gn pigs infected with a G4P[6] strain of HRV also display pathogen specific signs of infection, including diarrhea, fecal rotavirus shedding, and damaged intestinal villi. A dose response study of the G4P[6] strain revealed diarrhea and virus shedding occurred at all tested doses, however the most severe diarrhea and virus shedding, measured by cumulative diarrhea score, area under the curve (AUC) of diarrhea, peak virus titer, and AUC of virus shedding, were all detected in the highest dose group. Based on the presentation of clinical signs of infection, 105 fluorescent focus units was selected as the optimal challenge dose for future studies. These models enable us to test candidate therapeutics, but also elucidate unique replicative features of the pathogens. For example, we found that HRV can replicate in the salivary glands and nasal cavity of Gn pigs in addition to the small intestine. HRV infection primed immune responses in the ileum, tonsils, and facial lymph nodes; infection also induced high levels of systemic and mucosal rotavirus specific antibody responses. Moving forward, we hope to expand upon this replication study to identify what cell types within the glands are infected as well as look at local cellular immune responses to HRV infection. Additional future directions include determining the protective efficacy of next generation HRV vaccines and evaluating effectiveness of an engineered probiotic yeast in reducing severity of C. difficile infection and disease. The Gn pig models of C. difficile and G4P[6] HRV are clinically relevant, and they will continue to serve as useful tools to better our understanding of pathogenesis, infection, and prevention of these pathogens.
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    Heartland Virus Epidemiology, Vector Association, and Disease Potential
    Brault, 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.
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    Infectious SARS-CoV-2 Is Emitted in Aerosol Particles
    Hawks, 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.
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    Intersecting Threats: Exploring Obesity's Impact on Viral Pathogenesis and Transmission
    Rai, Pallavi (Virginia Tech, 2024-05-28)
    Malnutrition, including both undernutrition and obesity, affects millions of people globally and is persistently on the rise. Obesity affects ~13% of adults globally and was identified as a risk factor for worse disease outcomes after the H1N1 influenza pandemic of 2009 and has since been shown to aggravate disease outcomes of respiratory viruses like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mosquito-borne viruses like West Nile Virus (WNV), chikungunya virus (CHIKV) and Mayaro virus (MAYV) and reduce the vaccine efficacy for influenza and SARS-CoV-2. Obesity is associated with a chronic state of inflammation and dysregulated immune response which has been proposed to be one of the mechanisms driving the severity of coronavirus disease 2019 (COVID-19). These altered signatures or biomarkers might be associated with disease outcome and prognosis. Therefore, animal models reflecting the clinical outcomes and natural immune responses observed in humans are crucial to identifying reliable biomarkers. Using mouse hepatitis virus 1 (MHV-1) as a model for SARS-CoV-2, we established obesity as a risk factor and identified biomarkers and pathways associated with worse disease outcomes. Obesity rates in low and middle-income countries (LMICs) are approaching levels found in high-income countries (HICs). Mosquito-borne viral diseases like dengue, chikungunya, and Zika pose a significant threat to LMICs and cause huge health and economic losses. Obesity was shown to worsen alphavirus pathogenesis, but interestingly, it also reduced their transmission by mosquitoes. Given the global prevalence of obesity and mosquito-borne viruses, it is critical to understand how obesity drives reduced alphavirus transmission. Using a natural transmission cycle between lean and obese mice and mosquitoes, we confirmed that obesity reduced the transmission potential of alphaviruses like CHIKV and MAYV and activated the Toll pathway in mosquito midguts. Various genes and other pathways were also altered in response to obese bloodmeal at various time-points post-bloodmeal; however, one gene, AAEL009965, was downregulated in the mosquito midguts 1-day-post-bloodmeal and its knockdown led to reduced infection rates and titers in mosquitoes. Through this thesis, we aimed to utilize obesity as a tool to identify biomarkers to predict coronavirus disease outcomes and design effective alphavirus transmission control strategies.
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    Investigation of flagellotropic phage interactions with their motile host bacteria
    Gonzalez, 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.
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    Investigation of Novel Prophylactics Against Human Rotavirus Using Gnotobiotic Pig Models
    Hensley, Casey (Virginia Tech, 2023-06-22)
    Human rotavirus (HRV) is a major causative agent of acute gastroenteritis (AGE), which causes severe dehydrating diarrhea in children under the age of five and results in up to 215,000 deaths worldwide each year. There are two live oral attenuated vaccines licensed for use in the United States that are highly effective in high-income countries but much less so in low-and middle-income countries (LMICs). Several factors contributing to decreased efficacy in these areas include chronic malnutrition, gut dysbiosis, and concurrent viral infection. Along with this, currently used vaccines require constant cold-chain storage to maintain vaccine stability, and those resources can be scarce in LMICs. These areas continue to maintain a high burden of HRV morbidity and mortality, and more efficacious vaccines are needed. The gnotobiotic (Gn) pig model of HRV infection and diarrhea has long been used in the evaluation of novel HRV vaccines due to Gn pigs' susceptibility to HRV infection, development of clinical signs, histopathological changes in the intestine, and the infection kinetics that mimic those seen in human infants. The first project in this dissertation used the Gn pig model to evaluate a thermostable live oral attenuated vaccine administered as a dissolvable film. Two doses of the tetravalent dissolvable film vaccine conferred significant protection from virus shedding by delaying its onset and reducing peak titers in feces. It also significantly delayed the onset of diarrhea and reduced the duration and area under the curve (AUC) of diarrhea. The dissolvable film was highly immunogenic, inducing high titers of serum virus neutralizing (VN) antibodies specific to each of the four G-types included in the vaccine formulation, HRV-specific serum IgA and IgG, and intestinal IgA. These data confirm the thermostable platform as a useful alternative to liquid vaccines that require cold-chain. The second project evaluated three mRNA-based nonreplicating vaccine candidates in the Gn pig model. All three mRNA candidates encoded a universal CD4+ T cell epitope, P2, derived from tetanus toxoid, fused with the encoded VP8* from P[4], P[6], and P[8] HRVs. Two candidates also encoded for a lumazine synthase (LS) domain fused with the P2-VP8*. A dose response study of the LS-P2-VP8* candidates was conducted simultaneously. Significant protection against virus shedding was induced by all three candidates, with LS-P2-VP8* candidates inducing significantly higher VP8*-specific serum IgG. All three candidates induced significantly higher numbers of P[8]-VP8*-specific IgG antibody-secreting cells (ASCs) and IFN-γ-producing T cells in the ileum, spleen and blood. These data provide guidance for further development of the relatively new mRNA-based technology for use in HRV vaccine development. In the final study of this dissertation, we used the Gn pig model of both P[8] and P[6] HRV infection to evaluate a cocktail nanoparticle-based HRV vaccine. This vaccine was made up of an S60 nanoparticle, self-assembled from the S domain of the human norovirus capsid protein. The exposed C-termini on the S60 nanoparticle were utilized as an antigen display platform, where VP8* from P[4], P[6] and P[8] HRVs was fused. This vaccine was tested as both a two-dose intramuscular (IM) regimen, or as an IM booster preceded by an oral priming immunization with commercial monovalent Rotarix®. Pigs were challenged with either P[6] or P[8] HRV to evaluate cross-protection of the nanoparticle vaccine. Both regimens were highly immunogenic, inducing high titers of serum VN, IgG and IgA antibodies. Furthermore, the prime-boost regimen conferred significant protection against virus shedding in P[8] HRV-challenged pigs as evidenced by the shortened duration of fecal virus shedding. There was also significant protection in P[6] HRV-challenged pigs vaccinated with the prime-boost regimen, as evidenced by the shortened duration, reduced mean peak titer and AUC of virus shedding. Prime-boost-vaccinated pigs challenged with P[8] HRV had significantly higher P[8]-specific IgG ASCs in the spleen post-challenge. Prime-boost-vaccinated pigs challenged with P[6] HRV had significantly higher numbers of P[6] and P[8]-specific IgG ASCs in the ileum, as well as significantly higher numbers of P[8]-specific IgA ASCs in the spleen post-challenge. Oral priming followed by parenteral boosting appears to be a promising vaccination strategy for HRV and these data warrant further investigation into this regimen. Through these studies, we improved our understanding of the effect of different vaccination routes and formulations in the effectiveness of conferring protection against an enteric virus. The knowledge will facilitate the development of more effective vaccination strategies against HRV, the leading cause of infantile diarrhea in LMICs, as well as other enteric viruses.
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    Mutations present in a low-passage Zika virus isolate result in attenuated pathogenesis in mice
    Duggal, 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.