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Herein we describe a previously uninvestigated salivary gland escape barrier (SEB) in Culex tarsalis mosquitoes infected with two different strains of Western equine encephalitis virus (WEEV). The WEEV strains were originally isolated either from mosquitoes (IMP181) or a human patient (McMillan). Both IMP181 and McMillan viruses were fully able to infect the salivary glands of Culex tarsalis after intrathoracic injection as determined by expression of mCherry fluorescent protein. IMP181, however, was better adapted to transmission as measured by virus titer in saliva as well as transmission rates in infected mosquitoes. We used chimeric recombinant WEEV strains to show that inclusion of IMP181-derived structural genes partially circumvents the SEB.

Objectives: Barriers to healthcare in rural areas can delay treatment in pediatric patients who have experienced poisoning. We compared emergency medical services (EMS) response times and EMS-reported delays in responding to pediatric poisoning incidents between rural and urban settings using the 2021 National Emergency Medical Services Information System (NEMSIS). Methods: The NEMESIS defines rural areas as locations with a population of <50,000, not part of metropolitan areas, while all other locations are classified as urban (metropolitan) areas. In this study we included 11,911 patients (12% rural) <18 years of age who were transported by EMS with a first-responder primary impression of poisoning. We compared study variables using rank-sum tests and chi-square tests. Multivariable analysis of outcomes included quantile regression and logistic regression for continuous data and categorical data, respectively. Results: The median total prehospital time by EMS was 40 minutes (interquartile range 29-57), and the most common type of delay was scene delay (6%). On multivariable quantile regression, patients transported by rural EMS agencies experienced 6.6 minutes (95% confidence interval 5-8, P<0.001) longer prehospital time than those transported by urban agencies. There were no differences between rural and urban EMS agencies in the occurrence of dispatch, response, scene, and transportation delays. Conclusion: These results elucidate the need for equitable allocation of resources and training to enhance rural EMS responders. The additional nearly seven minutes translates into greater risk for the human body to remain physiologically unstable and not be optimally treated. Therefore, by integrating targeted interventions to rural pediatric populations, better care can be achieved across all geographic regions. Further research must be conducted to ascertain the specific factors, aside from delays, that result in the disparity between rural and urban prehospital response time.

Background Arthropod-borne viruses (arboviruses) can persistently infect and cause limited damage to mosquito vectors. RNA interference (RNAi) is a mosquito antiviral response important in restricting RNA virus replication and has been shown to be active against some arboviruses. The goal of this study was to use a recombinant Sindbis virus (SINV; family Togaviridae ; genus Alphavirus ) that expresses B2 protein of Flock House virus (FHV; family Nodaviridae ; genus Alphanodavirus ), a protein that inhibits RNAi, to determine the effects of linking arbovirus infection with RNAi inhibition. Results B2 protein expression from SINV (TE/3'2J) inhibited the accumulation of non-specific small RNAs in Aedes aegypti mosquito cell culture and virus-specific small RNAs both in infected cell culture and Ae. aegypti mosquitoes. More viral genomic and subgenomic RNA accumulated in cells and mosquitoes infected with TE/3'2J virus expressing B2 (TE/3'2J/B2) compared to TE/3'2J and TE/3'2J virus expressing GFP. TE/3'2J/B2 exhibited increased infection rates, dissemination rates, and infectious virus titers in mosquitoes following oral bloodmeal. Following infectious oral bloodmeal, significantly more mosquitoes died when TE/3'2J/B2 was ingested. The virus was 100% lethal following intrathoracic inoculation of multiple mosquito species and lethality was dose-dependent in Ae. aegypti . Conclusion We show that RNAi is active in Ae. aegypti cell culture and that B2 protein inhibits RNAi in mosquito cells when expressed by a recombinant SINV. Also, SINV more efficiently replicates in mosquito cells when RNAi is inhibited. Finally, TE/3'2J/B2 kills mosquitoes in a dose-dependent manner independent of infection route and mosquito species.

Western equine encephalitis virus (WEEV; Alphavirus) is a mosquito-borne virus that can cause severe encephalitis in humans and equids. Previous studies have shown that intranasal infection of outbred CD-1 mice with the WEEV McMillan (McM) strain result in high mortality within 4 days of infection. Here in vivo and ex vivo bioluminescence (BLM) imaging was applied on mice intranasally infected with a recombinant McM virus expressing firefly luciferase (FLUC) to track viral neuroinvasion by FLUC detection and determine any correlation between BLM and viral titer. Immunological markers of disease (MCP-1 and IP-10) were measured and compared to wild type virus infection. Histopathology was guided by corresponding BLM images, and showed that neuroinvasion occurred primarily through cranial nerves, mainly in the olfactory tract. Olfactory bulb neurons were initially infected with subsequent spread of the infection into different regions of the brain. WEEV distribution was confirmed by immunohistochemistry as having marked neuronal infection but very few infected glial cells. Axons displayed infection patterns consistent with viral dissemination along the neuronal axis. The trigeminal nerve served as an additional route of neuroinvasion showing significant FLUC expression within the brainstem. The recombinant virus WEEV.McM.FLUC had attenuated replication kinetics and induced a weaker immunological response than WEEV.McM but produced comparable pathologies. Immunohistochemistry staining for FLUC and WEEV antigen showed that transgene expression was present in all areas of the CNS where virus was observed. BLM provides a quantifiable measure of alphaviral neural disease progression and a method for evaluating antiviral strategies.

Western equine encephalitis virus (WEEV) is a naturally occurring recombinant virus derived from ancestral Sindbis and Eastern equine encephalitis viruses. We previously showed that infection by WEEV isolates McMillan (McM) and IMP-181 (IMP) results in high (∼90-100%) and low (0%) mortality, respectively, in outbred CD-1 mice when virus is delivered by either subcutaneous or aerosol routes. However, relatively little is known about specific virulence determinants of WEEV. We previously observed that IMP infected Culex tarsalis mosquitoes at a high rate (app. 80%) following ingestion of an infected bloodmeal but these mosquitoes were infected by McM at a much lower rate (10%). To understand the viral role in these phenotypic differences, we characterized the pathogenic phenotypes of McM/IMP chimeras. Chimeras encoding the E2 of McM on an IMP backbone (or the reciprocal) had the most significant effect on infection phenotypes in mice or mosquitoes. Furthermore, exchanging the arginine, present on IMP E2 glycoprotein at position 214, for the glutamine present at the same position on McM, ablated mouse mortality. Curiously, the reciprocal exchange did not confer mouse virulence to the IMP virus. Mosquito infectivity was also determined and significantly, one of the important loci was the same as the mouse virulence determinant identified above. Replacing either IMP E2 amino acid 181 or 214 with the corresponding McM amino acid lowered mosquito infection rates to McM-like levels. As with the mouse neurovirulence, reciprocal exchange of amino acids did not confer mosquito infectivity. The identification of WEEV E2 amino acid 214 as necessary for both IMP mosquito infectivity and McM mouse virulence indicates that they are mutually exclusive phenotypes and suggests an explanation for the lack of human or equine WEE cases even in the presence of active transmission.

New World arenaviruses cause fatal hemorrhagic disease in South America. Pirital virus (PIRV), a mammarenavirus hosted by Alston’s cotton rat (Sigmodon alstoni), causes a disease in Syrian golden hamsters (Mesocricetus auratus) (biosafety level-3, BSL-3) that has many pathologic similarities to the South American hemorrhagic fevers (BSL-4) and, thus, is considered among the best small-animal models for human arenavirus disease. Here, we extend in greater detail previously described clinical and pathological findings in Syrian hamsters and provide evidence for a pro-inflammatory macrophage response during PIRV infection. The liver was the principal target organ of the disease, and signs of Kupffer cell involvement were identified in mortally infected hamster histopathology data. Differential expression analysis of liver mRNA revealed signatures of the pro-inflammatory response, hematologic dysregulation, interferon pathway and other host response pathways, including 17 key transcripts that were also reported in two non-human primate (NHP) arenavirus liver-infection models, representing both Old and New World mammarenavirus infections. Although antigen presentation may differ among rodent and NHP species, key hemostatic and innate immune-response components showed expression parallels. Signatures of pro-inflammatory macrophage involvement in PIRV-infected livers included enrichment of Ifng, Nfkb2, Stat1, Irf1, Klf6, Il1b, Cxcl10, and Cxcl11 transcripts. Together, these data indicate that pro-inflammatory macrophage M1 responses likely contribute to the pathogenesis of acute PIRV infection.

Herein we describe a previously uninvestigated salivary gland escape barrier (SEB) in Culex tarsalis mosquitoes infected with two different strains of Western equine encephalitis virus (WEEV). The WEEV strains were originally isolated either from mosquitoes (IMP181) or a human patient (McMillan). Both IMP181 and McMillan viruses were fully able to infect the salivary glands of Culex tarsalis after intrathoracic injection as determined by expression of mCherry fluorescent protein. IMP181, however, was better adapted to transmission as measured by virus titer in saliva as well as transmission rates in infected mosquitoes. We used chimeric recombinant WEEV strains to show that inclusion of IMP181-derived structural genes partially circumvents the SEB.

Neuroinvasive infections with mosquito-borne alphaviruses such as Western equine encephalitis virus (WEEV) can cause post-encephalitic parkinsonism. To understand the mechanisms underlying these neurological effects, we examined the capacity of WEEV to induce progressive neurodegeneration in outbred CD-1 mice following non-lethal encephalitic infection. Animals were experientally infected with recombinant WEEV expressing firefly luciferase or dsRed (RFP) reporters and the extent of viral replication was controlled using passive immunotherapy. WEEV spread along the neuronal axis from the olfactory bulb to the entorhinal cortex, hippocampus and basal midbrain by 4 days post infection (DPI). Infection caused activation of microglia and astrocytes, selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and neurobehavioral abnormalities. After 8 weeks, surviving mice displayed continued loss of dopamine neurons in the SNpc, lingering glial cell activation and gene expression profiles consistent with a neurodegenerative phenotype. Strikingly, prominent proteinase K-resistant protein aggregates were present in the the entorhinal cortex, hippocampus and basal midbrain that stained positively for phospho-serine129 α-synuclein (SNCA). These results indicate that WEEV may cause lasting neurological deficits through a severe neuroinflammatory response promoting both neuronal injury and protein aggregation in surviving individuals.