Search Results (37)

Chalcones are flavonoid compounds containing an α,β-unsaturated ketone core that are often found in plants and have diverse biological activities including antiviral activity. For example, chalcone 8o was previously shown to have antiviral activity against human cytomegalovirus (HCMV) and human immunodeficiency virus (HIV); two viruses that use a nuclear phase to complete their growth cycle. Here, we synthesized ten new derivatives of 8o and tested them for antiviral activity against four RNA viruses that replicate exclusively in the cytoplasm, including prototype members of the paramyxovirus, flavivirus, bunyavirus, and coronavirus families. For example, chalcones 8o and 8p showed potent inhibition of PIV5 replication with minimal cytotoxicity in human fibroblast cultures. Time-of-addition studies showed that these chalcones inhibit an early stage of viral replication and prevent viral spread through cell cultures. Most importantly, our top performing chalcones showed potent in vitro antiviral activity against Zika virus, La Crosse Virus, and the coronavirus OC43. These studies offer mechanistic insight into chalcone-mediated inhibition of viral replication, demonstrate the influence of functional group changes of chalcone scaffolds on their efficacy as antivirals, and support the development of chalcones as broad-spectrum antiviral compounds. Full article

Feline parvovirus (FPV) causes feline panleukopenia, a highly contagious disease in cats, marked by severe leukopenia, biphasic fever, diarrhea, vomiting, and hemorrhagic enteritis. Recently, FPV infection in giant pandas has increased, causing diarrhea and ultimately fatal outcomes, thereby threatening their survival and reproduction. Here, we investigated the transmission of FPV in giant pandas and its interaction with cellular receptors using an FPV strain (pFPV-sc) isolated from giant panda feces. Recombinant feline transferrin receptor 1 (fTfR1) and the giant panda ortholog (gpTfR1) were expressed in non-susceptible HEK293T and HeLa cells, while viral infection levels were measured to determine the effect of gpTfR1 on pFPV-sc replication. The findings indicated that gpTfR1 overexpression in non-susceptible cells significantly enhanced pFPV-sc replication, particularly influencing the viral attachment and internalization stages. Our data further revealed early-stage colocalization between gpTfR1 expression and virus infection, suggesting that gpTfR1 facilitates early viral infection and replication. Taken together, our study provides the first evidence on the mechanism of FPV cross-species infection in giant pandas and elucidates the interaction between gpTfR1 and FPV, which establishes a theoretical basis for the development of preventive and therapeutic strategies, thereby safeguarding the health and survival of giant panda populations from FPV. Full article

Arboviruses, transmitted by blood-sucking arthropods, are responsible for significant human and animal diseases, including fever, hemorrhagic fever, and encephalitis, posing a serious threat to global public health. Nevertheless, research on the mechanisms of arbovirus infection and the development of therapeutic interventions has been impeded. This delay is primarily due to the limitations inherent in current in vitro research models, including cell cultures and animal models. The simplicity of cell types and interspecies differences present significant obstacles to advancing our understanding of arbovirus infection mechanisms and the development of effective drugs. Human brain organoids, derived from human pluripotent stem cells or human embryonic stem cells and cultured in three-dimensional systems, more accurately replicate the extensive neuronal cellular diversity and key characteristics of human neurodevelopment. These organoids serve as an ideal model for investigating the intricate interactions between viruses and human hosts, and providing a novel platform for the development of antiviral drugs. In this review, we summarize how brain organoid models complement classical approaches to accelerate research into the infection mechanisms of arboviruses, with a particular focus on the types of neural cells, key factors, and cellular signaling pathways involved in the arbovirus infection of brain organoids that have been reported. Furthermore, we examine the development of brain organoids, address their current limitations, and propose future directions to enhance the application of brain organoids in the study of arboviral infectious diseases. Full article

La Crosse virus (LACV) is a rare cause of pediatric encephalitis, yet identifying and mitigating transmission foci is critical to detecting additional cases. Neurologic disease disproportionately occurs among children, and survivors often experience substantial, life-altering chronic disability. Despite its severe clinical impact, public health resources to detect and mitigate transmission are lacking. This study aimed to design a Bayesian modelling approach to effectively identify and predict LACV incidence for geospatially informed public health interventions. A Bayesian negative binomial spatio-temporal regression model best fit the data and demonstrated high accuracy. Nine variables were statistically significant in predicting LACV incidence for the Appalachian Mountain Region. Proportion of children, proportion of developed open space, and proportion of barren land were positively associated with LACV incidence, while vapor pressure deficit index, year, and proportions of developed high intensity land, evergreen forest, hay pasture, and woody wetland were negatively associated with LACV incidence. Model prediction error was low, less than 2%, indicating high accuracy in predicting annual LACV human incidence at the county level. In summary, this study demonstrates the utility of Bayesian negative binomial spatio-temporal regression models for predicting rare but medically important LACV human cases. Future studies could examine more granular models for predicting LACV cases from localized variables such as mosquito control efforts, local reservoir host density and local weather fluctuations. Full article

Tomato chlorosis virus (ToCV) is an emerging plant virus that poses a substantial threat to the cultivation of economically vital vegetable crops, particularly tomato (Solanum lycopersicum). Despite its substantial impact on crop yield, resistant or tolerant tomato germplasms have not been well documented, and the genetic basis of resistance to ToCV remains poorly understood. In this study, two wild accessions that were immune to ToCV and five accessions that were highly resistant to ToCV were identified from 58 tomato accessions. Additionally, a novel method was developed for evaluating resistance to ToCV in tomatoes, and it was observed that tomatoes exhibited typical pathological features on days 15 and 30 after ToCV inoculation, referred to as Stage 1 and Stage 2, respectively. Using quantitative trait locus (QTL) sequencing in conjunction with classical QTL approaches, ToCV resistance loci were identified in two F2 populations derived from the crosses between SG11 (susceptible) and LA1028 (resistant) and between SP15 (susceptible) and LA0444 (resistant). Genetic analysis indicated that resistance to ToCV in the wild-type ToCV-resistant tomato accessions LA1028 and LA0444 was quantitative and mainly governed by four loci (Qtc1.1 and Qtc11.1 from LA1028 and Qtc7.1 and Qtc9.1 from LA0444). Subsequently, transcriptome analysis of three resistant accessions (LA2157, LA0444, and LA1028) and two susceptible accessions (SG11 and SP15) revealed unique differentially expressed genes and specific biological processes in the two stages of ToCV infection. This study provides new resistant germplasms and potential genetic resources for ToCV resistance, which can be valuable in tomato molecular breeding programs in obtaining resistant varieties. Full article

Rift Valley fever phlebovirus (RVFV) is a zoonotic pathogen that causes Rift Valley fever (RVF) in livestock and humans. Currently, there is no licensed human vaccine or antiviral drug to control RVF. Although multiple species of animals and humans are vulnerable to RVFV infection, host factors affecting susceptibility are not well understood. To identify the host factors or genes essential for RVFV replication, we conducted CRISPR-Cas9 knockout screening in human A549 cells. We then validated the putative genes using siRNA-mediated knock-downs and CRISPR-Cas9-mediated knock-out studies. The role of a candidate gene in the virus replication cycle was assessed by measuring intracellular viral RNA accumulation, and the virus titers were analyzed using plaque assay or TCID₅₀ assay. We identified approximately 900 genes with potential involvement in RVFV infection and replication. Further evaluation of the effect of six genes on viral replication using siRNA-mediated knock-downs revealed that silencing two genes (WDR7 and LRP1) significantly impaired RVFV replication. For further analysis, we focused on the WDR7 gene since the role of the LRP1 gene in RVFV replication was previously described in detail. WDR7 knockout A549 cell lines were generated and used to dissect the effect of WRD7 on a bunyavirus, RVFV, and an orthobunyavirus, La Crosse encephalitis virus (LACV). We observed significant effects of WDR7 knockout cells on both intracellular RVFV RNA levels and viral titers. At the intracellular RNA level, WRD7 affected RVFV replication at a later phase of its replication cycle (24 h) when compared with the LACV replication, which was affected in an earlier replication phase (12 h). In summary, we identified WDR7 as an essential host factor for the replication of two different viruses, RVFV and LACV, both of which belong to the Bunyavirales order. Future studies will investigate the mechanistic role through which WDR7 facilitates phlebovirus replication. Full article

La Crosse virus (LACV) is circulating in the midwestern and southeastern states of the United States and can cause human encephalitis. The main vector of the virus is the eastern tree-hole mosquito, Aedes triseriatus. Ae. albopictus has been also described as a natural LACV vector, while Ae. aegypti has been infected with the virus under laboratory conditions. Here, we compare the vertical transmission potential of LACV in Ae. albopictus and Ae. aegypti, with emphasis given to the ovarian infection patterns that the virus generates in both species. Both mosquito species received artificial bloodmeals containing LACV. At defined time points post-infection/bloodmeal, midguts, head tissue, and ovaries were analyzed for the presence of virus. Viral infection patterns in the ovaries were visualized via immunofluorescence confocal microscopy and immunohistopathology assays using an LACV-specific monoclonal antibody. In Ae. aegypti, LACV was confronted with midgut infection and escape barriers, which were much less pronounced in Ae. albopictus, resulting in a significantly higher prevalence of infection in the latter. Following the ingestion of a single virus-containing bloodmeal, no progeny larvae were found to be virus-infected. Regardless, females of both species showed the presence of LACV antigen in their ovariole sheaths. Furthermore, in a single Ae. albopictus female, viral antigen was associated with the nurse cells inside the primary follicles. Following the ingestion of a second non-infectious bloodmeal at 7- or 10-days post-ingestion of an LACV-containing bloodmeal, more progeny larvae of Ae. albopictus than of Ae. aegypti were virus-infected. LACV antigen was detected in the egg chambers and ovariole sheaths of both mosquito species. Traces of viral antigen were also detected in a few oocytes from Ae. albopictus. The low level of vertical transmission and the majority of the ovarian infection patterns suggested the transovum rather than transovarial transmission (TOT) of the virus in both vector species. However, based on the detection of LACV antigen in follicular tissue and oocytes, there was the potential for TOT among several Ae. albopictus females. Thus, TOT is not a general feature of LACV infection in mosquitoes. Instead, the TOT of LACV seems to be dependent on its particular interaction with the reproductive tissues of a female. Full article

Mosquito-borne La Crosse virus (LACV; family: Peribunyaviridae) is the leading cause of pediatric arboviral encephalitis in the United States, with clinical cases generally centered in the Midwest and Appalachian regions. Incidence of LACV cases in Appalachian states has increased, such that the region currently represents the majority of reported LACV cases in the USA. The amount of reported LACV cases from Virginia, however, is minimal compared to neighboring states such as North Carolina, West Virginia, and Tennessee, and non-Appalachian regions of Virginia are understudied. Here we examine the hypothesis that LACV is circulating widely in Virginia, despite a low clinical case report rate, and that the virus is circulating in areas not associated with LACV disease. In this study, we screened local mammalian wildlife in northwestern counties of Virginia using passive surveillance via patients submitted to wildlife rehabilitation centers. Blood sera (527 samples; 9 species, 8 genera) collected between October 2019 and December 2022 were screened for neutralizing antibodies against LACV, indicating prior exposure to the virus. We found an overall LACV seroprevalence of 1.90% among all wild mammals examined and reveal evidence of LACV exposure in several wild species not generally associated with LACV, including eastern cottontails and red foxes, along with established reservoirs, eastern gray squirrels, although there was no serological evidence in chipmunks. These data indicate the circulation of LACV in Virginia outside of Appalachian counties, however, at a lower rate than reported for endemic areas within the state and in other states. Full article

Aedes japonicus (Diptera: Culicidae), or the Asian rock pool mosquito, is an invasive mosquito in Europe and America. It was first detected outside of Asia in 1990 in Oceania. It has since expanded to North America and Europe in 1998 and 2000, respectively. Even though it is classified as a secondary vector of pathogens, it is competent to several arboviruses and filarial worms, and it is contributing to the transmission of La Crosse virus (LACV) and West Nile virus (WNV). In this study, CDC light, BG-sentinel, and gravid traps were used to collect mosquitoes between June and October 2021, in Wooster, Northeastern Ohio, USA. Morphological identification or/and Sanger sequencing were performed to identify the collected mosquitoes. Our results revealed that (adult) Ae. japonicus mosquitoes were the most abundant mosquito species collected with gravid traps in Wooster in 2021, confirming its establishment in Ohio. Molecular analyses of Ae. japonicus showed 100% nucleotide similarity with Ae. japonicus collected in Iowa (USA) and Canada, suggesting multiple introductions. Its presence may increase the risk of future arbovirus outbreaks in Wooster, Ohio. This study stresses the importance of actively monitoring the density and distribution of all members of the Ae. japonicus complex. Full article

Culex spp. mosquitoes transmit several pathogens concerning public health, including West Nile virus and Saint Louis encephalitis virus. Understanding the antiviral immune system of Culex spp. mosquitoes is important for reducing the transmission of these viruses. Mosquitoes rely on RNA interference (RNAi) to control viral replication. While the siRNA pathway in mosquitoes is heavily studied, less is known about the piRNA pathway. The piRNA pathway in mosquitoes has recently been connected to mosquito antiviral immunity. In Aedes aegypti, Piwi4 has been implicated in antiviral responses. The antiviral role of the piRNA pathway in Culex spp. mosquitoes is understudied compared to Ae. aegypti. Here, we aimed to identify the role of PIWI genes and piRNAs in Culex quinquefasciatus and Culex tarsalis cells during virus infection. We examined the effect of PIWI gene silencing on virus replication of two arboviruses and three insect-specific viruses in Cx. quinquefasciatus derived cells (Hsu) and Cx. tarsalis derived (CT) cells. We show that Piwi4 is antiviral against the La Crosse orthobunyavirus (LACV) in Hsu and CT cells, and the insect-specific rhabdovirus Merida virus (MERDV) in Hsu cells. None of the silenced PIWI genes impacted replication of the two flaviviruses Usutu virus (USUV) and Calbertado virus, or the phasivirus Phasi-Charoen-like virus. We further used small RNA sequencing to determine that LACV-derived piRNAs, but not USUV-derived piRNAs were generated in Hsu cells and that PIWI gene silencing resulted in a small reduction in vpiRNAs. Finally, we determined that LACV-derived DNA was produced in Hsu cells during infection, but whether this viral DNA is required for vpiRNA production remains unclear. Overall, we expanded our knowledge on the piRNA pathway and how it relates to the antiviral response in Culex spp mosquitoes. Full article

La Crosse virus (LACV) is a major cause of pediatric encephalitis and aseptic meningitis in the Midwestern, Mid-Atlantic, and Southern United States, where it is an emerging pathogen. The LACV Gc glycoprotein plays a critical role in the neuropathogenesis of LACV encephalitis as the putative virus attachment protein. Previously, we identified and experimentally confirmed the location of the LACV fusion peptide within Gc and generated a panel of recombinant LACVs (rLACVs) containing mutations in the fusion peptide as well as the wild-type sequence. These rLACVs retained their ability to cause neuronal death in a primary embryonic rat neuronal culture system, despite decreased replication and fusion phenotypes. To test the role of the fusion peptide in vivo, we tested rLACVs in an age-dependent murine model of LACV encephalitis. When inoculated directly into the CNS of young adult mice (P28), the rLACV fusion peptide mutants were as neurovirulent as the rLACV engineered with a wild-type sequence, confirming the results obtained in tissue culture. In contrast, the fusion peptide mutant rLACVs were less neuroinvasive when suckling (P3) or weanling (P21) mice were inoculated peripherally, demonstrating that the LACV fusion peptide is a determinant of neuroinvasion, but not of neurovirulence. In a challenge experiment, we found that peripheral challenge of weanling (P21) mice with fusion peptide mutant rLACVs protected from a subsequent WT-LACV challenge, suggesting that mutations in the fusion peptide are an attractive target for generating live-attenuated virus vaccines. Importantly, the high degree of conservation of the fusion peptide amongst the Bunyavirales and, structurally, other arboviruses suggests that these findings are broadly applicable to viruses that use a class II fusion mechanism and cause neurologic disease. Full article

The neuroinvasive disease caused by Jamestown Canyon virus (JCV) infection is rare. However, increasing incidence and widespread occurrence of the infection make JCV a growing public health concern. Presently, clinical diagnosis is achieved through serological testing, and mosquito pool surveillance requires virus isolation and identification. A rapid molecular detection test, such as real-time RT-PCR, for diagnosis and surveillance of JCV has not been widely utilized. To enhance testing and surveillance, here, we describe the development and validation of a real-time RT-PCR test for the detection of JCV RNA. Three primer and probe sets were evaluated for analytical sensitivity and specificity. One probe set, JCV132FAM, was found to be the most sensitive test detecting 7.2 genomic equivalents/µL. While less sensitive, a second probe set JCV231cFAM was the most specific test with limited detection of Keystone virus at high RNA loads. Taken together, these data indicate both probe sets can be utilized for a primary sensitive screening assay and a secondary specific confirmatory assay. While both primer and probe sets detected high viral loads of Keystone virus, these assays did not detect any virus in the California encephalitis virus clade, including negative detection of the medically important La Crosse virus (LACV) and snowshoe hare virus (SSHV). The real-time RT-PCR assay described herein could be utilized in diagnosis and surveillance in regions with co-circulation of JCV and LACV or SSHV to inform public health action. Full article

Largemouth bass virus (LMBV) is a major viral pathogen in largemouth bass culture, usually causing high mortality and heavy economic losses. Accurate and early detection of LMBV is crucial for diagnosis and control of the diseases caused by LMBV. Previously, we selected the specific aptamers, LA38 and LA13, targeting LMBV by systematic evolution of ligands by exponential enrichment (SELEX). In this study, we further generated truncated LA38 and LA13 (named as LA38s and LA13s) with high specificity and affinities and developed an aptamer-based sandwich enzyme-linked apta-sorbent assay (ELASA) for LMBV diagnosis. The sandwich ELASA showed high specificity and sensitivity for the LMBV detection, without cross reaction with other viruses. The detection limit of the ELASA was as low as 1.25 × 10² LMBV-infected cells, and the incubation time of the lysate and biotin labeled aptamer was as short as 10 min. The ELASA could still detect LMBV infection in spleen lysates at dilutions of 1/25, with good consistency of qRT-PCR. For the fish samples collected from the field, the sensitivity of ELASA was 13.3% less than PCR, but the ELASA was much more convenient and less time consuming. The procedure of ELASA mainly requires washing and incubation, with completion in approximately 4 h. The sandwich ELASA offers a useful tool to rapidly detect LMBV rapidly, contributing to control and prevention of LMBV infection. Full article

Pathogenesis of viral infections of the central nervous system (CNS) is poorly understood, and this is partly due to the limitations of currently used preclinical models. Brain organoid models can overcome some of these limitations, as they are generated from human derived stem cells, differentiated in three dimensions (3D), and can mimic human neurodevelopmental characteristics. Therefore, brain organoids have been increasingly used as brain models in research on various viruses, such as Zika virus, severe acute respiratory syndrome coronavirus 2, human cytomegalovirus, and herpes simplex virus. Brain organoids allow for the study of viral tropism, the effect of infection on organoid function, size, and cytoarchitecture, as well as innate immune response; therefore, they provide valuable insight into the pathogenesis of neurotropic viral infections and testing of antivirals in a physiological model. In this review, we summarize the results of studies on viral CNS infection in brain organoids, and we demonstrate the broad application and benefits of using a human 3D model in virology research. At the same time, we describe the limitations of the studies in brain organoids, such as the heterogeneity in organoid generation protocols and age at infection, which result in differences in results between studies, as well as the lack of microglia and a blood brain barrier. Full article

The fields of entomology, geospatial science, and science communication are understaffed in many areas, resulting in poor community awareness and heightened risks of vector-borne diseases. This is especially true in East Tennessee, where La Crosse encephalitis (LACE) causes pediatric illness each year. In response to these problems, we created a community engagement program that includes a yearlong academy for secondary STEM educators in the 6–12 grade classroom. The objectives of this program were to support inquiry-driven classroom learning to foster student interest in STEM fields, produce community-driven mosquito surveillance, and enhance community awareness of LACE. We trained educators in medical entomology, geospatial science, and science communication, and they incorporated those skills into lesson plans for a mosquito oviposition experiment that tested hypotheses developed in the classroom. Here, we share results from the first two years of the MEGA:BITESS academy, tailored for our community by having students ask questions directly related to Aedes mosquito oviposition biology and La Crosse encephalitis. In year one, we recruited 17 educators to participate in the project, and 15 of those educators returned in year two. All participating educators completed the academy, conducted the oviposition experiment, and informed over 400 students about a variety of careers and disciplines for their students. Here, we present a community-based program that helps to address the problems associated with long-term mosquito surveillance, health and science education and communication, career opportunities, and the community needs of Appalachia, as well as the initial data on the effectiveness of two years of an educator-targeted professional-development program. Full article