The 2014 chikungunya outbreak in the Dominican Republic resulted in intense local transmission, with high postoutbreak seroprevalence. The resulting population immunity will likely minimize risk for another large outbreak through 2035, but changes in population behavior or environmental conditions or emergence of different virus strains could lead to increased transmission.

Oropouche virus has recently caused outbreaks in South America and the Caribbean, expanding into areas to which the virus was previously not endemic. This geographic range expansion, in conjunction with the identification of vertical transmission and reports of deaths, has raised concerns about the broader threat this virus represents to the Americas. We review information on Oropouche virus, factors influencing its spread, transmission risk in the United States, and current status of public health response tools. On the basis of available data, the risk for sustained local transmission in the continental United States is considered low because of differences in vector ecology and in human-vector interactions when compared with Oropouche virus-endemic areas. However, more information is needed about the drivers for the current outbreak to clarify the risk for further expansion of this virus. Timely detection and control of this emerging pathogen should be prioritized to mitigate disease burden and stop its spread.

West Nile virus (WNV) is the most common cause of human arboviral disease in the contiguous United States, where only lineage 1 (L1) WNV had been found. In 2023, an immunocompetent patient was hospitalized in Nebraska with West Nile neuroinvasive disease and multisystem organ failure. Testing at the Centers for Disease Control and Prevention indicated an unusually high viral load and acute antibody response. Upon sequencing of serum and cerebrospinal fluid, we detected lineage 3 (L3) and L1 WNV genomes. L3 WNV had previously only been found in Central Europe in mosquitoes. The identification of L3 WNV in the United States and the observed clinical and laboratory features raise questions about the potential effect of L3 WNV on the transmission dynamics and pathogenicity of WNV infections. Determining the distribution and prevalence of L3 WNV in the United States and any public health and clinical implications is critical.

Beginning in late 2023, Oropouche virus was identified as the cause of large outbreaks in Amazon regions with known endemic transmission and in new areas in South America and the Caribbean. The virus is spread to humans by infected biting midges and some mosquito species. Although infection typically causes a self-limited febrile illness, reports of two deaths in patients with Oropouche virus infection and vertical transmission associated with adverse pregnancy outcomes have raised concerns about the threat of this virus to human health. In addition to approximately 8,000 locally acquired cases in the Americas, travel-associated Oropouche virus disease cases have recently been identified in European travelers returning from Cuba and Brazil. As of August 16, 2024, a total of 21 Oropouche virus disease cases were identified among U.S. travelers returning from Cuba. Most patients initially experienced fever, myalgia, and headache, often with other symptoms including arthralgia, diarrhea, nausea or vomiting, and rash. At least three patients had recurrent symptoms after the initial illness, a common characteristic of Oropouche virus disease. Clinicians and public health jurisdictions should be aware of the occurrence of Oropouche virus disease in U.S. travelers and request testing for suspected cases. Travelers should prevent insect bites when traveling, and pregnant persons should consider deferring travel to areas experiencing outbreaks of Oropouche virus disease.

We describe the isolation of a novel flavivirus, isolated from a pool of mosquitoes identified as Culex (Culex) chidesteri collected in 2010 in the Pantanal region of west-central Brazil. The virus is herein designated Nhumirim virus (NHUV) after the name of the ranch from which the mosquito pool was collected. Flavivirus RNA was detected by real-time RT-PCR of homogenized mosquitoes and from the corresponding C6/36 culture supernatant. Based on full-genome sequencing, the virus isolate was genetically distinct from but most closely related to Barkedji virus (BJV), a newly described flavivirus from Senegal. Phylogenetic analysis demonstrated that NHUV grouped with mosquito-borne flaviviruses forming a clade with BJV. This clade may be genetically intermediate between the Culex-borne flaviviruses amplified by birds and the insect-only flaviviruses.

In May 2021, an agricultural worker originally from Trementinal, Argentina, sought treatment for febrile illness in Tarija, Bolivia, where he resided at the time of illness onset. The patient tested negative for hantavirus RNA, but next-generation sequencing of a serum sample yielded a complete genome for Rio Negro virus.

BACKGROUND: In 2021, four patients who had received solid organ transplants in the USA developed encephalitis beginning 2-6 weeks after transplantation from a common organ donor. We describe an investigation into the cause of encephalitis in these patients. METHODS: From Nov 7, 2021, to Feb 24, 2022, we conducted a public health investigation involving 15 agencies and medical centres in the USA. We tested various specimens (blood, cerebrospinal fluid, intraocular fluid, serum, and tissues) from the organ donor and recipients by serology, RT-PCR, immunohistochemistry, metagenomic next-generation sequencing, and host gene expression, and conducted a traceback of blood transfusions received by the organ donor. FINDINGS: We identified one read from yellow fever virus in cerebrospinal fluid from the recipient of a kidney using metagenomic next-generation sequencing. Recent infection with yellow fever virus was confirmed in all four organ recipients by identification of yellow fever virus RNA consistent with the 17D vaccine strain in brain tissue from one recipient and seroconversion after transplantation in three recipients. Two patients recovered and two patients had no neurological recovery and died. 3 days before organ procurement, the organ donor received a blood transfusion from a donor who had received a yellow fever vaccine 6 days before blood donation. INTERPRETATION: This investigation substantiates the use of metagenomic next-generation sequencing for the broad-based detection of rare or unexpected pathogens. Health-care workers providing vaccinations should inform patients of the need to defer blood donation for at least 2 weeks after receiving a yellow fever vaccine. Despite mitigation strategies and safety interventions, a low risk of transfusion-transmitted infections remains. FUNDING: US Centers for Disease Control and Prevention (CDC), the Biomedical Advanced Research and Development Authority, and the CDC Epidemiology and Laboratory Capacity Cooperative Agreement for Infectious Diseases.

Error-prone replication of RNA viruses generates the genetic diversity required for adaptation within rapidly changing environments. Thus, arthropod-borne virus (arbovirus) populations exist in nature as mutant swarms that are maintained between arthropods and vertebrates. Previous studies have demonstrated that West Nile virus (WNV) population dynamics are host dependent: In American crows, which experience extremely high viremia, purifying selection is weak and population diversity is high compared to American robins, which have 100 to 1000-fold lower viremia. WNV passed in robins experiences fitness gains, whereas that passed in crows does not. Therefore, we tested the hypothesis that high crow viremia allows higher genetic diversity within individual avian peripheral-blood mononuclear cells (PBMCs), reasoning that this could have produced the previously observed host-specific differences in genetic diversity and fitness. Specifically, we infected cells and birds with a novel, barcoded version of WNV and sequenced viral RNA from single cells to quantify the number of WNV barcodes that each contained. Our results demonstrate that the richness of WNV populations within crows far exceeds that in robins. Similarly, rare WNV variants were maintained by crows more frequently than by robins. Our results suggest that increased viremia in crows relative to robins leads to maintenance of defective genomes and less prevalent variants, presumably through complementation. Our findings further suggest that weaker purifying selection in highly susceptible crows is attributable to this higher viremia, polyinfections and complementation. These studies further document the role of particular, ecologically relevant hosts in shaping virus population structure. Copyright The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.

Arthropod-borne virus (arbovirus) populations exist as mutant swarms that are maintained between arthropods and vertebrates. West Nile virus (WNV) population dynamics are host-dependent. In American crows, purifying selection is weak and population diversity is high compared to American robins, which have 100- to 1000-fold lower viremia. WNV passed in robins leads to fitness gains, whereas that passed in crows does not. Therefore, we tested the hypothesis that high crow viremia allows for higher genetic diversity within individual avian peripheral blood mononuclear cells (PBMCs), reasoning that this could have produced the previously observed host-specific differences in genetic diversity and fitness. Specifically, we infected cells and birds with a molecularly barcoded WNV and sequenced viral RNA from single cells to quantify the number of WNV barcodes in each. Our results demonstrate that the richness of WNV populations within crows far exceeds that in robins. Similarly, rare WNV variants were maintained by crows more frequently than by robins. Our results suggest that increased viremia in crows relative to robins leads to the maintenance of defective genomes and less prevalent variants, presumably through complementation. Our findings further suggest that weaker purifying selection in highly susceptible crows is attributable to this higher viremia, polyinfections and complementation.

It is time to revisit the need for human West Nile virus (WNV) vaccines. Since its initial detection in the United States in 1999, WNV has become the leading cause of domestic arthropod-borne viral (arboviral) disease. Spread by infected culex-species mosquitoes, WNV has caused more than 55,000 reported cases of human disease, more than 27,000 of them neuroinvasive, and 2600 deaths between 1999 and 2021, according to data from the Centers for Disease Control and Prevention (CDC). WNV is also an ongoing public health threat in many areas of the world; the largest recorded outbreak in Europe occurred in 2018.

In 2020, Montana, USA, reported a large increase in Colorado tick fever (CTF) cases. To investigate potential causes of the increase, we conducted a case-control study of Montana residents who tested positive or negative for CTF during 2020, assessed healthcare providers' CTF awareness and testing practices, and reviewed CTF testing methods. Case-patients reported more time recreating outdoors on weekends, and all reported finding a tick on themselves before illness. No consistent changes were identified in provider practices. Previously, only CTF serologic testing was used in Montana. In 2020, because of SARS-CoV-2 testing needs, the state laboratory sent specimens for CTF testing to the Centers for Disease Control and Prevention, where more sensitive molecular methods are used. This change in testing probably increased the number of CTF cases detected. Molecular testing is optimal for CTF diagnosis during acute illness. Tick bite prevention measures should continue to be advised for persons doing outdoor activities.

With increasing use of rituximab and other B-cell depleting monoclonal antibodies for multiple indications, infectious complications are being recognized. We summarize clinical findings of patients on rituximab with arboviral diseases identified through literature review or consultation with the Centers for Disease Control and Prevention. We identified 21 patients on recent rituximab therapy who were diagnosed with an arboviral disease caused by West Nile, tick-borne encephalitis, eastern equine encephalitis, Cache Valley, Jamestown Canyon, and Powassan viruses. All reported patients had neuroinvasive disease. The diagnosis of arboviral infection required molecular testing in 20 (95%) patients. Median illness duration was 36 days (range, 12 days-1 year) and 15/19 (79%) patients died from their illness. Patients on rituximab with arboviral disease can have a severe or prolonged course with an absence of serologic response. Patients should be counseled about mosquito and tick bite prevention when receiving rituximab and other B-cell depleting therapies.

BACKGROUND: Early detection of human yellow fever (YF) infection in YF-endemic regions is critical to timely outbreak mitigation. African National Laboratories chiefly rely on serological assays that require confirmation at Regional Reference Laboratories, thus delaying results, which themselves are not always definitive often due to antibody cross-reactivity. A positive molecular test result is confirmatory for YF; therefore, a standardized YF molecular assay would facilitate immediate confirmation at National Laboratories. The WHO-coordinated global Eliminate Yellow Fever Epidemics Laboratory Technical Working Group sought to independently evaluate the quality and performance of commercial YF molecular assays relevant to use in countries with endemic YF, in the absence of stringent premarket assessments. This report details a limited laboratory WHO-coordinated evaluation of the altona Diagnostics RealStar Yellow Fever Virus RT-PCR kit 1.0. METHODOLOGY AND PRINCIPAL FINDINGS: Specific objectives were to assess the assay's ability to detect YF virus strains in human serum from YF-endemic regions, determine the potential for interference and cross-reactions, verify the performance claims as stated by the manufacturer, and assess usability. RNA extracted from normal human serum spiked with YF virus showed the assay to be precise with minimal lot-to-lot variation. The 95% limit of detection calculated was approximately 1,245 RNA copies/ml [95% confidence interval 497 to 1,640 copies/ml]. Positive results were obtained with spatially and temporally diverse YF strains. The assay was specific for YF virus, was not subject to endogenous or exogenous interferents, and was clinically sensitive and specific. A review of operational characteristics revealed that a positivity cutoff was not defined in the instructions for use, but otherwise the assay was user-friendly. CONCLUSIONS AND SIGNIFICANCE: The RealStar Yellow Fever Virus RT-PCR kit 1.0 has performance characteristics consistent with the manufacturer's claims and is suitable for use in YF-endemic regions. Its use is expected to decrease YF outbreak detection times and be instrumental in saving lives.

Zika virus (ZIKV) is a mosquito-borne flavivirus that causes congenital defects. Sexual transmission of ZIKV was confirmed in a recent epidemic; however, mechanisms behind ZIKV infection and persistence in the male reproductive tract are unknown. Previously, we found that ∼33% of men with symptomatic ZIKV infections shed ZIKV RNA in semen, and some men shed ZIKV RNA for >3 months. Here, we evaluated the semen of 49 ZIKV-infected men to identify immune factors correlating with long-term ZIKV shedding in semen and ZIKV-infected cell types in semen. We found prolonged ZIKV RNA shedding in semen was associated with male reproductive tract inflammation, indicated by higher leukocyte counts and inflammatory cytokine concentrations in semen of long-term versus short-term shedders. Additionally, we found ZIKV RNA in seminal leukocytes and epithelial cells. This study of human semen from ZIKV-infected men provides critical insights into impacts of ZIKV on male reproductive tract health.

BACKGROUND: Cache Valley virus (CVV) is a mosquito-borne virus that is a rare cause of disease in humans. In the Fall of 2020, a patient developed encephalitis six weeks following kidney transplantation and receipt of multiple blood transfusions. METHODS: After ruling out more common etiologies, metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) was performed. We reviewed the medical histories of the index kidney recipient, organ donor, and recipients of other organs from the same donor and conducted a blood traceback investigation to evaluate blood transfusion as a possible source of infection in the kidney recipient. We tested patient specimens by reverse transcription-polymerase chain reaction (RT-PCR), plaque reduction neutralization test (PRNT), cell culture, and whole genome sequencing. RESULTS: CVV was detected in CSF from the index patient by mNGS, and this result was confirmed by RT-PCR, viral culture, and additional whole genome sequencing. The organ donor and other organ recipients had no evidence of infection with CVV by molecular or serologic testing. Neutralizing antibodies against CVV were detected in serum from a donor of red blood cells received by the index patient immediately prior to transplant. CVV neutralizing antibodies were also detected in serum from a patient who received the co-component plasma from the same blood donation. CONCLUSION: Our investigation demonstrates probable CVV transmission through blood transfusion. Clinicians should consider arboviral infections in unexplained meningoencephalitis after blood transfusion or organ transplantation. The use of mNGS testing might facilitate detection of rare, unexpected infections, particularly in immunocompromised patients.

Yellow fever virus (YFV) is a flavivirus that frequently causes outbreaks of hemorrhagic fever in Africa and South America and is considered a reemerging public health threat. Accurate diagnosis of yellow fever (YF) disease is critical as one confirmed case constitutes an outbreak and may trigger a mass vaccination campaign. Highly sensitive and specific molecular diagnostics have been developed; however, these assays require maintenance of cold-chain during transport of specimens to prevent the degradation of viral RNA prior to testing. Such cold-chain requirements are difficult to meet in some regions. In this study, we investigated Whatman FTA cards as an alternative stabilization method of YFV RNA for use in molecular diagnosis. Using contrived specimens, linear regression analysis showed that RNA detection from a single 6mm FTA card punch was significantly less sensitive than traditional RNA extraction; however, pooling RNA extracted from two FTA punches significantly lowered the limit of detection to be equal to that of the traditional RNA extraction gold standard. In experiments addressing the ability of FTA card methodology to stabilize YFV RNA at variable temperature, RNA could be detected for more than two weeks following storage at 25°C. Even more promising, YFV RNA was detectable on cards held at 37°C from two days to over two weeks depending on viral input. FTA cards were also shown to stabilize YFV RNA at high humidity if cards were desiccated prior to inoculation. These results support that FTA cards could be cost effective and easy to use in molecular diagnosis of YF, preserving viral RNA to allow for positive diagnoses in situations where maintaining cold-chain is not feasible.

The type species of the genus Coltivirus, Colorado tick fever virus (CTFV), was discovered in 1943 and is the most common tick-borne viral infection in the Western US. Despite its long history, very little is known about the molecular diversity of viruses classified within the species Colorado tick fever coltivirus. Previous studies have suggested genetic variants and potential serotypes of CTFV, but limited genetic sequence information is available for CTFV strains. To address this knowledge gap, we report herein the full-length genomes of five strains of CTFV, including Salmon River virus and California hare coltivirus (CTFV-Ca). The sequence from the full-length genome of Salmon River virus identified a high genetic identity to the CTFV prototype strain with >90% amino acid identity in all the segments except segment four, suggesting Salmon River virus is a strain of the species Colorado tick fever coltivirus. Additionally, analysis suggests that segment four has been associated with reassortment in at least one strain. The CTFV-Ca full-length genomic sequence was highly variable from the prototype CTFV in all the segments. The genome of CTFV-Ca was most similar to the Eyach virus, including similar segments six and seven. These data suggest that CTFV-Ca is not a strain of CTFV but a unique species. Additional sequence information of CTFV strains will improve the molecular surveillance tools and provide additional taxonomic resolution to this understudied virus.

Most flaviviruses are transmitted horizontally between vertebrate hosts by haematophagous arthropods. Others exhibit host ranges restricted to vertebrates or arthropods. Vertebrate-specific flaviviruses are commonly referred to as no-known-vector (NKV) flaviviruses and can be separated into bat- and rodent-associated NKV flaviviruses. Rio Bravo virus (RBV) is one of eight recognized bat-associated NKV (B-NKV) flaviviruses. Studies designed to identify the genetic determinants that condition the host range restriction of B-NKV flaviviruses have never been performed. To investigate whether the host range restriction occurs at the level of attachment or entry, chimeric flaviviruses were created by inserting the pre-membrane and envelope protein genes of RBV into the genetic backbones of yellow fever virus (YFV) and Zika virus (ZIKV), two mosquito-borne flaviviruses associated with human disease. The chimeric viruses infected both vertebrate and mosquito cells. In vertebrate cells, all viruses produced similar mean peak titres, but the chimeric viruses grew more slowly than their parental viruses during early infection. In mosquito cells, the chimeric virus of YFV and RBV grew more slowly than YFV at early post-inoculation time points, but reached a similar mean peak titre. In contrast, the chimeric virus of ZIKV and RBV produced a mean peak titre that was approximately 10-fold lower than ZIKV. The chimeric virus of YFV and RBV produced an intermediate plaque phenotype, while the chimeric virus of ZIKV and RBV produced smaller plaques than both parental viruses. To conclude, we provide evidence that the structural glycoproteins of RBV permit entry into both mosquito and vertebrate cells, indicating that the host range restriction of B-NKV flaviviruses is mediated by a post-attachment/entry event.

Eastern equine encephalitis virus (EEEV) is an arbovirus in the family Togaviridae, genus Alphavirus, found in North America and associated with freshwater/hardwood swamps in the Atlantic, Gulf Coast, and Great Lakes regions. EEEV disease in humans is rare but causes substantial illness and death. To investigate the molecular epidemiology and microevolution of EEEV from a fatal case in Alabama, USA, in 2019, we used next-generation sequencing of serum and cerebrospinal fluid (CSF). Phylogenetic inference indicated that the infecting strain may be closely related to isolates from Florida detected during 2010-2014, suggesting potential seeding from Florida. EEEV detected in serum displayed a higher degree of variability with more single-nucleotide variants than that detected in the CSF. These data refine our knowledge of EEEV molecular epidemiologic dynamics in the Gulf Coast region and demonstrate potential quasispecies bottlenecking within the central nervous system of a human host.

Long Pine Key virus (LPKV) and Lammi virus are insect-specific flaviviruses that phylogenetically affiliate with dual-host flaviviruses. The goal of this study was to provide insight into the genetic determinants that condition this host range restriction. Chimeras were initially created by replacing select regions of the Zika virus genome, including the premembrane and envelope protein (prM-E) genes, with the corresponding regions of the LPKV genome. Of the four chimeras produced, one (the prM-E swap) yielded virus that replicated in mosquito cells. Another chimeric virus with a mosquito replication-competent phenotype was created by inserting the prM-E genes of Lammi virus into a Zika virus genetic background. Vertebrate cells did not support the replication of either chimeric virus although trace to modest amounts of viral antigen were produced, consistent with suboptimal viral entry. These data suggest that dual-host affiliated insect-specific flaviviruses cannot replicate in vertebrate cells due to entry and post-translational restrictions.

Prenatal exposure to Zika virus (ZIKV) is associated with congenital anomalies of the brain and the eye and neurodevelopmental sequelae. The spectrum of disease outcomes may relate to timing of infection as well as genetic and environmental factors. Congenital infections occurring in twin pregnancies can inform the clinical spectrum of these conditions and provide unique information regarding timing of infection and in utero environment with disease pathophysiology. Herein, we report a monozygotic dichorionic-diamniotic twin pregnancy with probable prenatal ZIKV exposure identified through the Colombian ZIKV disease surveillance system. Multidisciplinary clinical evaluations were provided to the twins during their first three years of life through a national program for children with in utero ZIKV exposure. Laboratory evidence of congenital infection as well as microcephaly, brain, eye, and neurodevelopmental compromise related to prenatal ZIKV infection were identified in only one infant of the twin pregnancy. This is the first report of monozygotic twins discordant for Zika-associated birth defects. The evaluation of the pathophysiology of discordance in disease outcome for congenital infections in twin pregnancies may lead to a better understanding of potential complex environmental and genetic interactions between the mother, her offspring, and an infectious exposure.

BACKGROUND: Heartland virus (HRTV), a recently reclassified member of the genus Bandavirus, family Phenuiviridae, was first isolated in 2009 from a Missouri farmer exhibiting leukopenia and thrombocytopenia with suspected ehrlichiosis. Since then, more HRTV cases have been diagnosed, and firstline laboratory diagnostic assays are needed to identify future infections Objectives. We sought to develop rapid and reliable IgM and IgG microsphere immunoassays (MIAs) to test sera of patients suspected of having HRTV infection, and to distinguish between recent and past infections. STUDY DESIGN: Heartland virus antigen was captured by an anti-HRTV monoclonal antibody covalently bound to microspheres. Antibodies in human sera from confirmed HRTV-positive and negative cases were reacted with the microsphere complexes and detected using a BioPlex® 200 instrument. Assay cutoffs were determined by receiver operator characteristic analysis of the normalized test output values, equivocal zones for each assay were defined, and sensitivities, specificities, accuracies, and imprecision values were calculated. RESULTS: Sensitivities, specificities and accuracies of the IgM and IgG MIAs were all >95 %. Both tests were precise within and between assay plates, and cross-reactivity with other arboviruses was not observed. CONCLUSIONS: HRTV IgM and IgG MIAs are accurate and rapid first-line methods to serologically identify recent and past HRTV infections.

Japanese encephalitis (JE) is a vaccine-preventable, mosquito-borne disease. Substantial progress with JE control in Asia has been made during the past decade, with most endemic countries now having JE vaccination programs, commonly using live attenuated SA14-14-2 JE vaccine (CD-JEV). If a child develops encephalitis during the weeks to months following CD-JEV vaccination and anti-JE virus IgM (JE IgM) antibody is detected in serum, the question arises if this is JE virus infection indicating vaccine failure, or persistent JE IgM antibody postvaccination. To better understand JE IgM seropositivity following vaccination, sera from 268 children from a previous CD-JEV study were tested by two different JE IgM assays to determine JE IgM antibody frequency on days 28, 180, and 365 postvaccination. With the CDC JE IgM antibody capture ELISA (MAC-ELISA), 110 children (41%) had JE IgM positive or equivocal results on their day 28 sample, and eight (3%) and two (1%) had positive or equivocal results on day 180 and day 365 samples, respectively. With the Inbios JE Detect™ MAC-ELISA, 118 (44%) children had positive or equivocal results on day 28 sample, and three (1%) and one (0.4%) had positive or equivocal results on day 180 and day 365 samples, respectively. Our results indicate that more than 40% children vaccinated with CD-JEV can have JE IgM antibodies in their serum at 1 month postvaccination but JE IgM antibody is rare by 6 months. These data will help healthcare workers assess the likelihood that JE IgM antibodies in the serum of a child with encephalitis after vaccination are vaccine related.

When this special issue was first conceived in early 2019, we never anticipated that the publication of this collection of articles would be happening during a pandemic. While this outbreak collection is focused on viruses transmitted by arthropods, its release concurrent with the SARS-CoV-2 pandemic and international health emergency provides an appropriate context in which to draw attention to research focused on other high-consequence, epidemic-causing viruses that may be next to emerge on the global stage.

Zika virus (ZIKV) can establish infection in immune privileged sites such as the testes, eye and placenta. Whether ZIKV infection of white blood cells is required for dissemination of the virus to immune privileged sites has not been definitively shown. To assess whether initial ZIKV replication in myeloid cell populations is critical for dissemination during acute infection, recombinant ZIKVs were generated that could not replicate in these specific cells. ZIKV was cell-restricted by insertion of a complementary sequence to a myeloid-specific microRNA in the 3'untranslated region. Following inoculation of a highly sensitive immunodeficient mouse model, crucial immune parameters, such as quantification of leukocyte cell subsets, cytokine and chemokine secretion, and viremia were assessed. Decreased neutrophil numbers in the spleen were observed during acute infection with myeloid-restricted ZIKV that precluded the generation of viremia and viral dissemination to peripheral organs. Mice inoculated with a non-target microRNA control ZIKV demonstrated increased expression of key cytokines and chemokines critical for neutrophil and monocyte recruitment and increased neutrophil influx in the spleen. In addition, ZIKV-infected Ly6C(hi) monocytes were identified in vivo in the spleen. Mice inoculated with myeloid-restricted ZIKV exhibited similar trends as PBS-sham inoculated mice, including a lack of inflammatory cytokine production and a decrease in Ly6C(hi) ZIKV RNA positive monocytes.ImportanceMyeloid cells, including monocytes, play a crucial role in immune responses to pathogens. Monocytes have also been implicated as "Trojan horses" during viral infections, carrying infectious virus particles to immune privileged sites and/or to sites protected by physical blood-tissue barriers, such as the blood-testis barrier and the blood-brain barrier. In this study, we found that myeloid cells are crucial to Zika virus (ZIKV) pathogenesis. By engineering ZIKV clones to encode myeloid-specific microRNA target sequences, viral replication was inhibited in myeloid cells by harnessing the RNAi pathway. Severely immunodeficient mice inoculated with myeloid-restricted ZIKV did not demonstrate clinical signs of disease and survived infection. Furthermore, viral dissemination to peripheral organs was not observed in these mice. Lastly, we identified Ly6C(mid/hi) murine monocytes as the major myeloid cell population that disseminates ZIKV.

St. Louis encephalitis virus (SLEV) is a flavivirus that circulates in an enzootic cycle between birds and mosquitoes and can also infect humans to cause febrile disease and sometimes encephalitis. Although SLEV is endemic to the United States, no activity was detected in California during the years 2004 through 2014, despite continuous surveillance in mosquitoes and sentinel chickens. In 2015, SLEV-positive mosquito pools were detected in Maricopa County, Arizona, concurrent with an outbreak of human SLEV disease. SLEV-positive mosquito pools were also detected in southeastern California and Nevada in summer 2015. From 2016 to 2018, SLEV was detected in mosquito pools throughout southern and central California, Oregon, Idaho, and Texas. To understand genetic relatedness and geographic dispersal of SLEV in the western United States since 2015, we sequenced four historical genomes (3 from California and 1 from Louisiana) and 26 contemporary SLEV genomes from mosquito pools from locations across the western US. Bayesian phylogeographic approaches were then applied to map the recent spread of SLEV. Three routes of SLEV dispersal in the western United States were identified: Arizona to southern California, Arizona to Central California, and Arizona to all locations east of the Sierra Nevada mountains. Given the topography of the Western United States, these routes may have been limited by mountain ranges that influence the movement of avian reservoirs and mosquito vectors, which probably represents the primary mechanism of SLEV dispersal. Our analysis detected repeated SLEV introductions from Arizona into southern California and limited evidence of year-to-year persistence of genomes of the same ancestry. By contrast, genetic tracing suggests that all SLEV activity since 2015 in central California is the result of a single persistent SLEV introduction. The identification of natural barriers that influence SLEV dispersal enhances our understanding of arbovirus ecology in the western United States and may also support regional public health agencies in implementing more targeted vector mitigation efforts to protect their communities more effectively.

Understanding the circumstances under which arboviruses emerge is critical for the development of targeted control and prevention strategies. This is highlighted by the emergence of chikungunya and Zika viruses in the New World. However, to comprehensively understand the ways in which viruses emerge and persist, factors influencing reductions in virus activity must also be understood. Western equine encephalitis virus (WEEV), which declined during the late 20th century in apparent enzootic circulation as well as equine and human disease incidence, provides a unique case study on how reductions in virus activity can be understood by studying evolutionary trends and mechanisms. Previously, we showed using phylogenetics that during this period of decline, six amino acid residues appeared to be positively selected. To assess more directly the effect of these mutations, we utilized reverse genetics and competition fitness assays in the enzootic host and vector (house sparrows and Culex tarsalis mosquitoes). We observed that the mutations contemporary with reductions in WEEV circulation and disease that were non-conserved with respect to amino acid properties had a positive effect on enzootic fitness. We also assessed the effects of these mutations on virulence in the Syrian-Golden hamster model in relation to a general trend of increased virulence in older isolates. However, no change effect on virulence was observed based on these mutations. Thus, while WEEV apparently underwent positive selection for infection of enzootic hosts, residues associated with mammalian virulence were likely eliminated from the population by genetic drift or negative selection. These findings suggest that ecologic factors rather than fitness for natural transmission likely caused decreased levels of enzootic WEEV circulation during the late 20th century.

Since its recent discovery, Bourbon virus has been isolated from a human and ticks. To assess exposure of potential vertebrate reservoirs, we assayed banked serum and plasma samples from wildlife and domestic animals in Missouri, USA, for Bourbon virus-neutralizing antibodies. We detected high seroprevalence in raccoons (50%) and white-tailed deer (86%).

West Nile virus (WNV) was first identified in North America almost 20 yr ago. In that time, WNV has crossed the continent and established enzootic transmission cycles, resulting in intermittent outbreaks of human disease that have largely been linked with climatic variables and waning avian seroprevalence. During the transcontinental dissemination of WNV, the original genotype has been displaced by two principal extant genotypes which contain an envelope mutation that has been associated with enhanced vector competence by Culex pipiens L. (Diptera: Culicidae) and Culex tarsalis Coquillett vectors. Analyses of retrospective avian host competence data generated using the founding NY99 genotype strain have demonstrated a steady reduction in viremias of house sparrows over time. Reciprocally, the current genotype strains WN02 and SW03 have demonstrated an inverse correlation between house sparrow viremia magnitude and the time since isolation. These data collectively indicate that WNV has evolved for increased avian viremia while house sparrows have evolved resistance to the virus such that the relative host competence has remained constant. Intrahost analyses of WNV evolution demonstrate that selection pressures are avian species-specific and purifying selection is greater in individual birds compared with individual mosquitoes, suggesting that the avian adaptive and/or innate immune response may impose a selection pressure on WNV. Phylogenomic, experimental evolutionary systems, and models that link viral evolution with climate, host, and vector competence studies will be needed to identify the relative effect of different selective and stochastic mechanisms on viral phenotypes and the capacity of newly evolved WNV genotypes for transmission in continuously changing landscapes.

The N-linked glycosylation motif at amino acid position 154-156 of the envelope (E) protein of West Nile virus (WNV) is linked to enhanced murine neuroinvasiveness, avian pathogenicity and vector competence. Naturally occurring isolates with altered E protein glycosylation patterns have been observed in WNV isolates; however, the specific effects of these polymorphisms on avian host pathogenesis and vector competence have not been investigated before. In the present study, amino acid polymorphisms, NYT, NYP, NYF, SYP, SYS, KYS and deletion (A'DEL), were reverse engineered into a parental WNV (NYS) cDNA infectious clone to generate WNV glycosylation mutant viruses. These WNV glycosylation mutant viruses were characterized for in vitro growth, pH-sensitivity, temperature-sensitivity and host competence in American crows (AMCR), house sparrows (HOSP) and Culex quinquefasciatus. The NYS and NYT glycosylated viruses showed higher viral replication, pH and temperature sensitivity than NYP, NYF, SYP, SYS, KYS and A'DEL viruses in vitro. Interestingly, in vivo results demonstrated asymmetric effects in avian and mosquito competence that were independent of the E-protein glycosylation status. In AMCRs and HOSPs, all viruses showed comparable viremias with the exception of NYP and KYS viruses that showed attenuated phenotypes. Only NYP showed reduced vector competence in both Cx. quinquefasciatus and Cx. tarsalis. Glycosylated NYT exhibited similar avian virulence properties as NYS, but resulted in higher mosquito oral infectivity than glycosylated NYS and nonglycosylated, NYP, NYF, SYP and KYS mutants. These data demonstrated that amino acid polymorphisms at E154/156 dictate differential avian host and vector competence phenotypes independent of E-protein glycosylation status.