Browsing by Author "Weger-Lucarelli, James"

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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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    Adventitious viruses persistently infect three commonly used mosquito cell lines
    Weger-Lucarelli, James; Rückert, Claudia; Grubaugh, Nathan D.; Misencik, Michael J.; Armstrong, Philip M.; Stenglein, Mark D.; Ebel, Gregory D.; Brackney, Doug E. (2018-08)
    Mosquito cell lines have been used extensively in research to isolate and propagate arthropod-borne viruses and understand virus-vector interactions. Despite their utility as an in vitro tool, these cell lines are poorly defined and may harbor insect-specific viruses. Accordingly, we screened four commonly-used mosquito cell lines, C6/36 and U4.4 cells from Aedes albopictus, Aag2 cells from Aedes aegypti, and Hsu cells from Culex quinquefasciatus, for the presence of adventitious (i.e. exogenous) viruses. All four cell lines stained positive for double-stranded RNA, indicative of RNA virus replication. We subsequently identified viruses infecting Aag2, U4.4 and Hsu cell lines using untargeted next-generation sequencing, but not C6/36 cells. PCR confirmation revealed that these sequences stem from active viral replication and/or integration into the cellular genome. Our results show that these commonly-used mosquito cell lines are persistently-infected with several viruses. This finding may be critical to interpreting data generated in these systems.
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    Battle of the Viruses: Aripo Virus Induced Superinfection Exclusion of Mosquito-borne Viruses
    Carver, James A. (Virginia Tech, 2021-01-06)
    Flaviviruses are a single-stranded, positive sense, RNA virus that affect around 400 million people annually. Flaviviruses are transmitted by arthropod vectors, the most common vector being the mosquito. Currently, many mosquito control strategies are in use, these control strategies are diverse in both efficiency and cost. However, developing new vector control strategies is becoming increasingly important, due to climate changing affecting vector population distribution and the current limitations conventional control strategies face. Although many different control strategies exist, there is limited research pertaining to reducing viral infection in the vector. Reducing the transmission capabilities of vectors could help relieve the disease burden felt around the world. Aripo virus (ARPV), an insect-specific flavivirus, has ushered in opportunities to discover a novel approach to arbovirus control. The exclusionary effects of ARPV were explored as a means to eventually understanding superinfection exclusion (SIE) and utilizing it as a calculated defense against mosquito-borne disease. Aripo virus was evaluated for its SIE potential in vitro and experiments were performed to explore the possible mechanisms underlying SIE. Aripo virus showed significant exclusion against the flaviviruses tested, as well as an alphavirus. Additionally, West Nile virus was unable to adapt and overcome SIE barriers over 9 serial passages. Lastly, ARPV was superinfected with chimeric viruses to asses replication kinetics, and possible exclusionary bias was seen with non-structural genes. These data show ARPV is capable of reducing viral titer, as well as possible leads into understanding the underlying mechanism of SIE, a critical step in utilizing SIE as a strategy to combat vector-borne disease.
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    Bivalent single domain antibody constructs for effective neutralization of Venezuelan equine encephalitis
    Liu, Jinny L.; Zabetakis, Dan; Gardner, Christina L.; Burke, Crystal W.; Glass, Pamela J.; Webb, Emily M.; Shriver-Lake, Lisa C.; Anderson, George P.; Weger-Lucarelli, James; Goldman, Ellen R. (Nature Portfolio, 2022-01-13)
    Venezuelan equine encephalitis virus (VEEV) is a mosquito borne alphavirus which leads to high viremia in equines followed by lethal encephalitis and lateral spread to humans. In addition to naturally occurring outbreaks, VEEV is a potential biothreat agent with no approved human vaccine or therapeutic currently available. Single domain antibodies (sdAb), also known as nanobodies, have the potential to be effective therapeutic agents. Using an immune phage display library derived from a llama immunized with an equine vaccine that included inactivated VEEV, five sdAb sequence families were identified that showed varying ability to neutralize VEEV. One of the sequence families had been identified previously in selections against chikungunya virus, a related alphavirus of public health concern. A key advantage of sdAb is the ability to optimize properties such as neutralization capacity through protein engineering. Neutralization of VEEV was improved by two orders of magnitude by genetically linking sdAb. One of the bivalent constructs showed effective neutralization of both VEEV and chikungunya virus. Several of the bivalent constructs neutralized VEEV in cell-based assays with reductions in the number of plaques by 50% at protein concentrations of 1 ng/mL or lower, making future evaluation of their therapeutic potential compelling.
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    Chikungunya virus superinfection exclusion is mediated by a block in viral replication and does not rely on non-structural protein 2
    Boussier, Jeremy; Levi, Laura I.; Weger-Lucarelli, James; Poirier, Enzo Z.; Vignuzzi, Marco; Albert, Matthew L. (2020-11-12)
    Superinfection exclusion (SIE) is a process by which a virally infected cell is protected from subsequent infection by the same or a closely related virus. By preventing cell coinfection, SIE favors preservation of genome integrity of a viral strain and limits its recombination potential with other viral genomes, thereby impacting viral evolution. Although described in virtually all viral families, the precise step(s) impacted by SIE during the viral life cycle have not been systematically explored. Here, we describe for the first time SIE triggered by chikungunya virus (CHIKV), an alphavirus of public health importance. Using single-cell technologies, we demonstrate that CHIKV excludes subsequent infection with: CHIKV; Sindbis virus, a related alphavirus; and influenza A, an unrelated RNA virus. We further demonstrate that SIE does not depend on the action of type I interferon, nor does it rely on host cell transcription. Moreover, exclusion is not mediated by the action of a single CHIKV protein; in particular, we observed no role for non-structural protein 2 (nsP2), making CHIKV unique among characterized alphaviruses. By stepping through the viral life cycle, we show that CHIKV exclusion occurs at the level of replication, but does not directly influence virus binding, nor viral structural protein translation. In sum, we characterized co-infection during CHIKV replication, which likely influences the rate of viral diversification and evolution.
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    Co-Infection Patterns in Individual Ixodes scapularis Ticks Reveal Associations between Viral, Eukaryotic and Bacterial Microorganisms
    Cross, Shaun T.; Kapuscinski, Marylee L.; Perino, Jacquelyn; Maertens, Bernadette L.; Weger-Lucarelli, James; Ebel, Gregory D.; Stenglein, Mark D. (MDPI, 2018-07-22)
    Ixodes scapularis ticks harbor a variety of microorganisms, including eukaryotes, bacteria and viruses. Some of these can be transmitted to and cause disease in humans and other vertebrates. Others are not pathogenic, but may impact the ability of the tick to harbor and transmit pathogens. A growing number of studies have examined the influence of bacteria on tick vector competence but the influence of the tick virome remains less clear, despite a surge in the discovery of tick-associated viruses. In this study, we performed shotgun RNA sequencing on 112 individual adult I. scapularis collected in Wisconsin, USA. We characterized the abundance, prevalence and co-infection rates of viruses, bacteria and eukaryotic microorganisms. We identified pairs of tick-infecting microorganisms whose observed co-infection rates were higher or lower than would be expected, or whose RNA levels were positively correlated in co-infected ticks. Many of these co-occurrence and correlation relationships involved two bunyaviruses, South Bay virus and blacklegged tick phlebovirus-1. These viruses were also the most prevalent microorganisms in the ticks we sampled, and had the highest average RNA levels. Evidence of associations between microbes included a positive correlation between RNA levels of South Bay virus and Borrelia burgdorferi, the Lyme disease agent. These findings contribute to the rationale for experimental studies on the impact of viruses on tick biology and vector competence.
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    Comparison of two DNA extraction methods from larvae, pupae, and adults of Aedes aegypti
    de la Cruz-Ramos, Josue M.; Hernandez-Triana, Luis M.; Garcia-De la Pena, Cristina; Gonzalez-Alvarez, Vicente H.; Weger-Lucarelli, James; Karmy Siller-Rodriguez, Quetzaly; Sanchez Ramos, Francisco J.; Rodriguez, Americo D.; Ortega-Morales, Aldo, I. (2019-10)
    Mosquitoes are the most important arthropods from the point of view of public health, due to the fact that they can transmit a large number of pathogens which can cause diseases to humans and animals. Aedes aegypti (L.) is one of the most important vector species in the world, since it can transmit numerous pathogens such as dengue, Zika, and chikungunya. Therefore, studies involving the molecular aspects of this and other mosquitoes species are currently increasing. In this report, we describe the comparison between two DNA extraction techniques, Chelex and cetyltrimethylammonium bromide (CTAB), for carrying out DNA extraction in larvae, pupae and adult female of Ae. aegypti. The Chelex technique was superior in the amount and purity of DNA as compared to the CTAB technique in the three life stages we tested.
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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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    Cross-reactivity among alphaviruses provides insight into viral emergence and novel defense strategies
    Webb, Emily Morgan (Virginia Tech, 2022-04-13)
    Alphaviruses are a group of medically relevant arthropod-borne viruses (arboviruses) belonging to the Togaviridae family that are maintained by mosquito vectors. These zoonotic viruses are clustered into two groups: New World and Old World, depending on their geographical origin/distribution and clinical manifestations. Both of these groups cause disease symptoms of an acute febrile illness; however, each group has a distinct, hallmark disease symptom; New World alphaviruses, such as Eastern, Western, and Venezuelan equine encephalitis viruses (EEEV, WEEV, and VEEV, respectively), present with severe encephalitis while Old World alphaviruses, such as Sindbis, chikungunya, and Mayaro viruses (SINV, CHIKV, and MAYV, respectively) present with an incapacitating polyarthralgia that can persist for years following initial infection. To date, the most effective means of controlling these arboviral infections is through mosquito control programs. However, these programs have crucial limitations in their effectiveness; therefore, novel approaches are necessary to control the spread of these crippling pathogens and lessen their disease burden. Given the close phylogenetic and antigenic relationship between MAYV and CHIKV, we hypothesized that prior CHIKV immunity may affect the outcome of MAYV disease and/or limit its emergence in humans. Our work has shown that anti-CHIKV neutralizing antibodies can provide cross-protective immunity against MAYV disease. Alongside these studies, we have characterized the potency of a camelid-derived single-domain antibody (sdAb) that neutralizes a breadth of alphaviruses, including CHIKV and MAYV. With these data, we have designed and generated transgenic Aedes aegypti mosquitoes that express two anti-CHIKV sdAbs to target infection, dissemination, and transmission of MAYV and CHIKV within this deadly vector. These findings are particularly significant because they highlight the ability to co-target two emerging alphaviruses that are crippling public health and obliterating quality of life around the globe within a single defense strategy.
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    Defective viral genomes from chikungunya virus are broad-spectrum antivirals and prevent virus dissemination in mosquitoes
    Levi, Laura I.; Rezelj, Veronica V.; Henrion-Lacritick, Annabelle; Erazo, Diana; Boussier, Jeremy; Vallet, Thomas; Bernhauerova, Veronika; Suzuki, Yasutsugu; Carrau, Lucia; Weger-Lucarelli, James; Saleh, Maria-Carla; Vignuzzi, Marco (2021-02)
    Defective viral genomes (DVGs) are truncated and/or rearranged viral genomes produced during virus replication. Described in many RNA virus families, some of them have interfering activity on their parental virus and/or strong immunostimulatory potential, and are being considered in antiviral approaches. Chikungunya virus (CHIKV) is an alphavirus transmitted by Aedes spp. that infected millions of humans in the last 15 years. Here, we describe the DVGs arising during CHIKV infection in vitro in mammalian and mosquito cells, and in vivo in experimentally infected Aedes aegypti mosquitoes. We combined experimental and computational approaches to select DVG candidates most likely to have inhibitory activity and showed that, indeed, they strongly interfere with CHIKV replication both in mammalian and mosquito cells. We further demonstrated that some DVGs present broad-spectrum activity, inhibiting several CHIKV strains and other alphaviruses. Finally, we showed that pre-treating Aedes aegypti with DVGs prevented viral dissemination in vivo. Author summary Defective viral genomes (DVGs) are produced during virus replication. On their own they cannot replicate, but some of them can compete with wild-type virus for viral and/or cellular resources. For chikungunya virus, interference by DVGs has not been described. Here, we use a new approach based on experimental evolution and computational analysis to characterize all DVGs generated in a virus population and identify those with the highest antiviral potential. We confirm their antiviral activity in both mammalian and mosquito host environments and show that some can broadly interfere with other strains or related alphaviruses. Finally, we show that DVGs can inhibit virus dissemination in mosquitoes.
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    Development, Characterization, and Use of Molecular Tools to Study Immune-Driven Zika Virus Evolution
    Marano, Jeffrey Matthew (Virginia Tech, 2023-02-16)
    Emerging viruses represent a significant threat to human health. Understanding the drivers of emergence, such as viral evolution, is a critical avenue to combat these pathogens. One specific group of emerging pathogens of interest is flaviviruses. Flaviviruses are arthropod-borne viruses (arbovirus) in the family Flaviviridae. The medically relevant flaviviruses can be divided into two groups – tick-borne and mosquito-borne. Included within the mosquito-borne flaviviruses group are dengue viruses 1-4 (DENV 1-4), which causes 400 million infections annually, and Zika virus (ZIKV), which caused over 128 million infections from 2013-2018. These viruses, which are cocirculating, share high sequence similarity in key antigenic regions. Because of these similarities, pre-existing immunity to DENV has been correlated with altered pathogenesis of subsequent ZIKV infections. Despite this, there has been little analysis of the effects of pre-existing DENV immunity on the evolution of subsequent flavivirus infection, despite being characterized for many other viruses. Given that mutation that could arise from cross-reactive immune selection could alter pathology or transmissibility, it is critical to assess the role of cross-reactive immune selection as an evolutionary driver. However, this line of research has historically been difficult due to the inherent toxicity of flavivirus infectious clones in bacteria. To mitigate the toxic nature of flavivirus clones, we developed several entirely in vitro workflows using a combination of rolling circle amplification (RCA) and replication cycle reaction (RCR). We demonstrated that RCA was a comparable substitute to traditional plasmid propagation using an alphavirus infection clone. We further demonstrated that RCR could be used to generate infectious clones by producing infectious clones of DENV2 and SARS-CoV-2, as well as demonstrating it could be used to introduce mutations into infectious clones by producing a D614G SARS-CoV-2 mutations. With this technology in place, we used in vitro directed evolution system, where we passaged ZIKV in convalescent patient serum to assess the role of cross-reactive immune selection as an evolutionary driver. After passaging, we performed next-generation sequencing to assess the impacts of cross-reactive immune selection on the viral populations and to identify mutations that arose post-passaging. We observed that ZIKV passaged in convalescent DENV serum had reduced diversity and divergence in the premembrane region. Within the convalescent DENV passaged population, we identified two mutations of interest with the dominant antibody binding region – E-V335I and NS1-T139A. These mutations were then introduced using our in vitro workflows. The resulting mutant viruses were then assessed for their replicative fitness in mammalian cell culture and mosquito models and their sensitivity to neutralization. We observed that while both E-V355I and NS1-T139A have increased fitness in mammalian cells, they had reduced fitness in mosquitoes. These results align with the trade-off hypothesis, which states that in a multi-host system, adaptation to one host reduces fitness in the other hosts. When we assessed the neutralization sensitivity of the mutants, we observed that while NS1-T193A was resistant to neutralization, E-V355I was more sensitive to neutralization. These results indicate that neutralization escape is not necessary for enhanced post-passaging in convalescent DENV serum. Our findings demonstrate that cross-reactive immune selection can generate several mutations with altered fitness in mammalian cells and mosquitos. This research is significant for both highlighting novel technologies to facilitate molecular virology and demonstrating that cross-reactive immune selection has the potential to alter the evolutionary trajectory of flaviviruses. This work provides critical information to understand how flaviviruses are evolving and emerging, and therefore critical information to address their threat to human health.
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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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    Enemy of My Enemy: A Novel Insect-Specific Flavivirus Offers a Promising Platform for a Zika Virus Vaccine
    Porier, Danielle L.; Wilson, Sarah N.; Auguste, Dawn I.; Leber, Andrew; Coutermarsh-Ott, Sheryl; Allen, Irving C.; Caswell, Clayton C.; Budnick, James A.; Bassaganya-Riera, Josep; Hontecillas, Raquel; Weger-Lucarelli, James; Weaver, Scott C.; Auguste, A. Jonathan (MDPI, 2021-10-07)
    Vaccination remains critical for viral disease outbreak prevention and control, but conventional vaccine development typically involves trade-offs between safety and immunogenicity. We used a recently discovered insect-specific flavivirus as a vector in order to develop an exceptionally safe, flavivirus vaccine candidate with single-dose efficacy. To evaluate the safety and efficacy of this platform, we created a chimeric Zika virus (ZIKV) vaccine candidate, designated Aripo/Zika virus (ARPV/ZIKV). ZIKV has caused immense economic and public health impacts throughout the Americas and remains a significant public health threat. ARPV/ZIKV vaccination showed exceptional safety due to ARPV/ZIKV’s inherent vertebrate host-restriction. ARPV/ZIKV showed no evidence of replication or translation in vitro and showed no hematological, histological or pathogenic effects in vivo. A single-dose immunization with ARPV/ZIKV induced rapid and robust neutralizing antibody and cellular responses, which offered complete protection against ZIKV-induced morbidity, mortality and in utero transmission in immune-competent and -compromised murine models. Splenocytes derived from vaccinated mice demonstrated significant CD4+ and CD8+ responses and significant cytokine production post-antigen exposure. Altogether, our results further support that chimeric insect-specific flaviviruses are a promising strategy to restrict flavivirus emergence via vaccine development.
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    Expression of anti-chikungunya single-domain antibodies in transgenic Aedes aegypti reduces vector competence for chikungunya virus and Mayaro virus
    Webb, Emily M.; Compton, Austin; Rai, Pallavi; Chuong, Christina; Paulson, Sally L.; Tu, Zhijian; Weger-Lucarelli, James (Frontiers, 2023-06-12)
    Chikungunya virus (CHIKV) and Mayaro virus (MAYV) are closely related alphaviruses that cause acute febrile illness accompanied by an incapacitating polyarthralgia that can persist for years following initial infection. In conjunction with sporadic outbreaks throughout the sub-tropical regions of the Americas, increased global travel to CHIKV- and MAYV-endemic areas has resulted in imported cases of MAYV, as well as imported cases and autochthonous transmission of CHIKV, within the United States and Europe. With increasing prevalence of CHIKV worldwide and MAYV throughout the Americas within the last decade, a heavy focus has been placed on control and prevention programs. To date, the most effective means of controlling the spread of these viruses is through mosquito control programs. However, current programs have limitations in their effectiveness; therefore, novel approaches are necessary to control the spread of these crippling pathogens and lessen their disease burden. We have previously identified and characterized an anti-CHIKV single-domain antibody (sdAb) that potently neutralizes several alphaviruses including Ross River virus and Mayaro virus. Given the close antigenic relationship between MAYV and CHIKV, we formulated a single defense strategy to combat both emerging arboviruses: we generated transgenic Aedes aegypti mosquitoes that express two camelid-derived anti-CHIKV sdAbs. Following an infectious bloodmeal, we observed significant reduction in CHIKV and MAYV replication and transmission potential in sdAb-expressing transgenic compared to wild-type mosquitoes; thus, this strategy provides a novel approach to controlling and preventing outbreaks of these pathogens that reduce quality of life throughout the tropical regions of the world.
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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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    Genome Number and Size Polymorphism in Zika Virus Infectious Units
    Sexton, Nicole R.; Bellis, Eric D.; Murrieta, Reyes A.; Spangler, Mark Cole; Cline, Parker J.; Weger-Lucarelli, James; Ebel, Gregory D. (2021-03)
    Zika virus (ZIKV; Flaviviridae, Flavivirus) is an arthropod-borne infection that can result in severe outcomes, particularly in fetuses infected in utero. It has been assumed that infection by ZIKV, as well as other viruses, is largely initiated by individual virus particles binding to and entering a cell. However, recent studies have demonstrated that multiple virus particles are frequently delivered to a cell simultaneously and that this collective particle delivery enhances infection. ZIKV is maintained in nature between Aedes aegypti mosquitos and vertebrate hosts, including humans. Human infection is initiated through the injection of a relatively small initial inoculum comprised of a genetically complex virus population. Since most mutations decrease virus fitness, collective particle transmission could benefit ZIKV and other arthropod-borne diseases by facilitating the maintenance of genetic complexity and adaptability during infection or through other mechanisms. Therefore, we utilized a barcoded ZIKV to quantify the number of virus genomes that initiate a plaque. We found that individual plaques contain a mean of 10 infecting viral genomes (range, 1 to 212). Few plaques contained more than two dominant genomes. To determine whether multigenome infectious units consist of collectively transmitting virions, infectious units of ZIKV were then separated mechanically by centrifugation, and heavier fractions were found to contain more genomes per plaque-forming unit, with larger diameters. Finally, larger/heavier infectious units reformed after removal. These data suggest that ZIKV populations consist of a variety of infectious unit sizes, likely mostly made up of aggregates, and only rarely begin with a single virus genome. IMPORTANCE The arthropod-borne Zika virus (ZIKV) infects humans and can cause severe neurological sequelae, particularly in fetuses infected in utero. How this virus has been able to spread across vast geological ranges and evolve in new host populations is not yet understood. This research demonstrates a novel mechanism of ZIKV transmission through multigenome aggregates, providing insight into ZIKV evolution, immunologic evasion, and better future therapeutic design. This study shows that ZIKV plaques result from collections of genomes rather than individual genomes, increasing the potential for interactions between ZIKV genotypes.
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    Genomic and phenotypic analyses suggest moderate fitness differences among Zika virus lineages
    Oliveira, Glenn; Vogels, Chantal B. F.; Zolfaghari, Ashley; Saraf, Sharada; Klitting, Raphaelle; Weger-Lucarelli, James; Leon, Karla P.; Ontiveros, Carlos O.; Agarwal, Rimjhim; Tsetsarkin, Konstantin A.; Harris, Eva; Ebel, Gregory D.; Wohl, Shirlee; Grubaugh, Nathan D.; Andersen, Kristian G. (Public Library of Science, 2023-02)
    Zika virus was introduced to the Western Hemisphere, spread rapidly, and led to the 2015-2016 Zika epidemic and a rise in congenital microcephaly. It remains unclear whether Zika virus evolved to become more transmissible directly before or during the epidemic. To investigate whether Zika evolved to become more transmissible, we engineered a library of recombinant viruses that represent twelve major Zika virus lineages that circulated throughout the Americas. We measured the replicative fitness of each of these lineages by infecting live mosquitoes and human cells that are relevant for disease or transmission. We found that two of the lineages, one that dominated Central America and another that existed mostly in the Caribbean, appear to replicate more efficiently in human cells. While the fitness changes do not appear to have significant effects on the 2015-2016 Zika epidemic, our analysis suggests Zika virus evolved at least twice during this outbreak. Monitoring the phenotypic evolution during the course of an outbreak can help control spread and mitigate disease. We believe this framework can be applied to study phenotypic evolution during future epidemics caused by emerging RNA viruses. RNA viruses have short generation times and high mutation rates, allowing them to undergo rapid molecular evolution during epidemics. However, the extent of RNA virus phenotypic evolution within epidemics and the resulting effects on fitness and virulence remain mostly unknown. Here, we screened the 2015-2016 Zika epidemic in the Americas for lineage-specific fitness differences. We engineered a library of recombinant viruses representing twelve major Zika virus lineages and used them to measure replicative fitness within disease-relevant human primary cells and live mosquitoes. We found that two of these lineages conferred significant in vitro replicative fitness changes among human primary cells, but we did not find fitness changes in Aedes aegypti mosquitoes. Additionally, we found evidence for elevated levels of positive selection among five amino acid sites that define major Zika virus lineages. While our work suggests that Zika virus may have acquired several phenotypic changes during a short time scale, these changes were relatively moderate and do not appear to have enhanced transmission during the epidemic.
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    Genomic Diversity and Geographic Distribution of Newcastle Disease Virus Genotypes in Africa: Implications for Diagnosis, Vaccination, and Regional Collaboration
    Amoia, Charlie F.; Hakizimana, Jean N.; Chengula, Augustino A.; Munir, Muhammad; Misinzo, Gerald; Weger-Lucarelli, James (MDPI, 2024-05-16)
    The emergence of new virulent genotypes and the continued genetic drift of Newcastle disease virus (NDV) implies that distinct genotypes of NDV are simultaneously evolving in different geographic locations across the globe, including throughout Africa, where NDV is an important veterinary pathogen. Expanding the genomic diversity of NDV increases the possibility of diagnostic and vaccine failures. In this review, we systematically analyzed the genetic diversity of NDV genotypes in Africa using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Information published between 1999 and 2022 were used to obtain the genetic background of different genotypes of NDV and their geographic distributions in Africa. The following genotypes were reported in Africa: I, II, III, IV, V, VI, VII, VIII, XI, XIII, XIV, XVII, XVIII, XX, and XXI. A new putative genotype has been detected in the Democratic Republic of the Congo. However, of 54 African countries, only 26 countries regularly report information on NDV outbreaks, suggesting that this number may be vastly underestimated. With eight different genotypes, Nigeria is the country with the greatest genotypic diversity of NDV among African countries. Genotype VII is the most prevalent group of NDV in Africa, which was reported in 15 countries. A phylogeographic analysis of NDV sequences revealed transboundary transmission of the virus in Eastern Africa, Western and Central Africa, and in Southern Africa. A regional and continental collaboration is recommended for improved NDV risk management in Africa.
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    Host nutritional status affects alphavirus virulence, transmission, and evolution
    Weger-Lucarelli, James; Carrau, Lucia; Levi, Laura I.; Rezelj, Veronica V.; Vallet, Thomas; Blanc, Herve; Boussier, Jeremy; Megrian, Daniela; Coutermarsh-Ott, Sheryl; LeRoith, Tanya; Vignuzzi, Marco (2019-11)
    Malnourishment, specifically overweight/obesity and undernourishment, affects more than 2.5 billion people worldwide, with the number affected ever-increasing. Concurrently, emerging viral diseases, particularly those that are mosquito-borne, have spread dramatically in the past several decades, culminating in outbreaks of several viruses worldwide. Both forms of malnourishment are known to lead to an aberrant immune response, which can worsen disease outcomes and reduce vaccination efficacy for viral pathogens such as influenza and measles. Given the increasing rates of malnutrition and spread of arthropod-borne viruses (arboviruses), there is an urgent need to understand the role of host nutrition on the infection, virulence, and transmission of these viruses. To address this gap in knowledge, we infected lean, obese, and undernourished mice with arthritogenic arboviruses from the genus Alphavirus and assessed morbidity, virus replication, transmission, and evolution. Obesity and undernourishment did not consistently influence virus replication in the blood of infected animals except for reductions in virus in obese mice late in infection. However, morbidity was increased in obese mice under all conditions. Using Mayaro virus (MAYV) as a model arthritogenic alphavirus, we determined that both obese and undernourished mice transmit virus less efficiently to mosquitoes than control (lean) mice. In addition, viral genetic diversity and replicative fitness were reduced in virus isolated from obese compared to lean controls. Taken together, nutrition appears to alter the course of alphavirus infection and should be considered as a critical environmental factor during outbreaks.
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    Impact of extrinsic incubation temperature on natural selection during Zika virus infection of Aedes aegypti and Aedes albopictus
    Murrieta, Reyes A.; Garcia-Luna, Selene M.; Murrieta, Deedra J.; Halladay, Gareth; Young, Michael C.; Fauver, Joseph R.; Gendernalik, Alex; Weger-Lucarelli, James; Rückert, Claudia; Ebel, Gregory D. (PLoS, 2021-11-09)
    Arthropod-borne viruses (arboviruses) require replication across a wide range of temperatures to perpetuate. While vertebrate hosts tend to maintain temperatures of approximately 37°C-40°C, arthropods are subject to ambient temperatures which can have a daily fluctuation of > 10°C. Temperatures impact vector competence, extrinsic incubation period, and mosquito survival unimodally, with optimal conditions occurring at some intermediate temperature. In addition, the mean and range of daily temperature fluctuations influence arbovirus perpetuation and vector competence. The impact of temperature on arbovirus genetic diversity during systemic mosquito infection, however, is poorly understood. Therefore, we determined how constant extrinsic incubation temperatures of 25°C, 28°C, 32°C, and 35°C control Zika virus (ZIKV) vector competence and population dynamics within Aedes aegypti and Aedes albopictus mosquitoes. We also examined fluctuating temperatures which better mimic field conditions in the tropics. We found that vector competence varied in a unimodal manner for constant temperatures peaking between 28°C and 32°C for both Aedes species. Transmission peaked at 10 days post-infection for Aedes aegypti and 14 days for Aedes albopictus. Conversely, fluctuating temperature decreased vector competence. Using RNA-seq to characterize ZIKV population structure, we identified that temperature alters the selective environment in unexpected ways. During mosquito infection, constant temperatures more often elicited positive selection whereas fluctuating temperatures led to strong purifying selection in both Aedes species. These findings demonstrate that temperature has multiple impacts on ZIKV biology, including major effects on the selective environment within mosquitoes.