Emerging outbreaks of the arbovirus chikungunya virus affect millions of individuals globally, causing debilitating arthralgia and fever. A study on the burden and etiologies of acute febrile illnesses (AFI) in Kenya reported an increase in CHIKV cases in Mombasa between December 2017 and December 2019. In January and February 2020, another outbreak of CHIKV occurred in Dadaab-Hagadera. Using next-generation sequencing on the Illumina MiSeq platform, we established molecular characteristics and phylogenetic differences of the CHIKVs collected from the two epidemiologically distinct settings. Sequenced viruses belonged to the Indian Ocean Lineage and clustered according to geographic region, with E1:K211E and E2:V264A mutations present in both settings. Additional mutations previously reported during Kenyan CHIKV outbreaks and suggested to influence CHIKV adaptation to Aedes albopictus, including E1:T82I and E1:V84D, were also identified. These molecular markers provide useful information on CHIKV spread and can help strengthen outbreak preparedness and response. Clinical features were described only for laboratory-confirmed CHIKV cases from the AFI surveillance cohort and compared with AFI cases testing negative for CHIKV. Sore muscles, headache, and convulsions were more frequently reported among CHIKV-positive participants, whereas diarrhea was less common. No differences were observed in the frequency of cough, skin rash, conjunctivitis, or vomiting. Given the limited number of confirmed CHIKV cases and the predominance of young children in the cohort, the reported clinical symptom associations should only be interpreted as exploratory and warrant confirmation in larger and more representative populations.

After the introduction of West Nile virus (WNV) in 1999, the virus became endemic in the United States, causing approximately 1,300 severe neurological disease cases and 130 deaths annually. WNV is also responsible for a substantial disease burden in Canada, Europe, and countries around the Mediterranean Sea. WNV prevention currently relies on personal protective behaviors (PPBs) and mosquito control, but these measures have been inadequate to substantially reduce WNV disease cases or deaths. Additional measures are also necessary to diagnose, treat, prevent, and control WNV disease. Human WNV vaccine, anti-WNV monoclonal antibody used as preexposure prophylaxis in patients at higher risk for severe WNV disease, and more rapidly detecting WNV infection, among other measures, will be critical to help combat WNV disease morbidity and mortality. Partners in government, academia, and private industry should collaborate to ensure optimal use and availability of current and additional lifesaving measures.

Background:Oropouche virus (OROV) is an emerging vector-borne pathogen endemic to the Americas, which causes acute febrile illness (AFI) in humans. Starting in late 2023, surges in OROV infections were reported across Latin America, including an outbreak in Iquitos, a city in the Eastern Peruvian Amazon, where RIVERA, an ongoing AFI surveillance program detected and characterized incident OROV cases. Methods:AFI cases presenting to health facilities were screened for OROV using PCR. OROV-positive subjects were compared to AFI OROV negative cases to describe the principal features of clinical disease. Genomes from OROV strains were sequenced and compared using phylogenetic analysis with those from extant samples isolated from other locations in the Americas. Findings:In early 2024, an 8.6% OROV-positivity rate (29 detections in 339 samples) in RIVERA subjects was recorded, a more than 20-fold increase compared with pre-outbreak levels. Illness was characterized by fever, arthralgia, myalgia and dysuria. Genome sequences from strains in this outbreak were phylogenetically distinct from those from a concurrent one in Brazil, but resembled strains from Colombia and Ecuador. The last common ancestor of outbreak strains from Peru and Brazil was 226 years prior to sampling, and that of Peru and Ecuador and Colombia approximately 10 and 8 years prior to sampling, respectively. Interpretation:Genomic analysis suggests that the current outbreak in South America is multifocal in origin and not the result of geographic spread from Brazil. An existing AFI surveillance program successfully documented the emergence and characterized the symptom profile of this emerging arboviral disease. Funding:CDC/HHS U01GH002270; NIH D43TW010913, K43TW012298, K01AI168493, 5T32AI007046-48.

The tools available to vector control districts (VCDs) to collect mosquito surveillance data are constantly evolving. As more VCDs obtain real-time polymerase chain reaction (PCR) instruments and the costs associated with computing power and next-generation sequencing continue to decrease, the option of generating useful molecular data in-house becomes more viable. Measures such as arbovirus testing and genotyping for insecticide resistance mutations using RT-qPCR, and identifying species used for mosquito bloodmeals with next-generation sequencing or Sanger sequencing are examples. In this study we identify mosquito host bloodmeal species using Nanopore sequencing from Oxford Nanopore Technologies. We used MinION and Flongle flow cells and a Mk1C device to sequence 96 barcoded amplicon samples in a single sequencing run, and share details of data analysis using the free-to-use Galaxy bioinformatics platform. After sequencing the same samples with Sanger sequencing, we conclude that Nanopore sequencing is better at identifying species in mixed bloodmeals. This work demonstrates a potential use of nanopore sequencing by VCDs with basic biology laboratory and computing equipment.

The 2015-16 Zika virus epidemic emerged in the Americas and rapidly spread throughout the region and beyond, showing the epidemic potential of this mosquito-borne Orthoflavivirus and its capacity to cause severe congenital malformations and neurological sequelae. WHO declared the Zika virus epidemic a public health emergency of international concern in 2016. Despite this declaration, there are no licensed Zika virus vaccines, therapeutics, or diagnostic tests appropriate for routine antenatal screening. To address this absence of essential tools to detect and mitigate the threat of future Zika virus outbreaks, a group of global experts developed a priority agenda for Zika virus research and development. This Series paper summarises crucial challenges and knowledge gaps and outlines a comprehensive strategy to advance research, surveillance, global capacity, policy, and investment for Zika virus preparedness and response. Lancet 25: Published February https://doi.org/10.1016/ S1473-3099(24)00794-1 This of to with All

In December 2023, infections of western equine encephalitis virus (WEEV) within Argentina were reported to the World Health Organization (WHO). By April 2024, more than 250 human infections, 12 of which were fatal, and 2500 equine infections were identified in South America. Laboratory diagnosis and surveillance in affected countries were hindered by a lack of facilities equipped with BSL-3 laboratories, as confirmatory serodiagnosis for WEEV requires live virus in the plaque reduction neutralization test (PRNT). To expand serodiagnosis for WEEV in the Americas, we developed a virus chimera composed of vesicular stomatitis virus (VSV) engineered to display the E2-E1 glycoproteins of WEEV (VSV/WEEV) in place of the VSV glycoprotein (G). PRNT90 and IC90 values of parental WEEV and VSV/WEEV were analogous using sera collected from mice, horses, and chickens. VSV/WEEV rapidly formed plaques with clear borders and reduced the assay readout time by approximately 8 h compared to the parental virus. Overall, we demonstrate that chimeric VSV/WEEV is a suitable surrogate for WEEV in a diagnostic PRNT. Use of chimeric VSV/WEEV in place of authentic WEEV will dramatically expand testing capacity by enabling PRNTs to be performed at BSL-2 containment, while simultaneously decreasing the health risk to testing personnel.

West Nile virus lineage 1 (WNVL1) has been the only lineage known to circulate within the United States; however, multiple other lineages have been identified around the world. West Nile virus lineage 3 (WNVL3) was previously only isolated from mosquitoes in Czechia, but no human cases had been observed. West Nile virus lineage 3 was identified alongside WNVL1 in 2023 in a severely ill Nebraska patient with no history of international travel. To determine the extent of WNVL3 transmission, 1,199 historical mosquito pools collected from the state of Nebraska from 2012 through 2024 totaling >40,000 Culex spp. mosquitoes were tested. All pools tested negative for WNVL3 RNA. This study suggests that WNVL3 may circulate at very low levels or in alternative vector species in Nebraska.

Powassan virus (POWV) and Eastern equine encephalitis virus (EEEV) are regionally endemic arboviruses in the United States that can cause neuroinvasive disease and death. Recent identification of EEEV transmission through organ transplantation and POWV transmission through blood transfusion have increased concerns about infection risk. After historically high numbers of cases of both viruses were reported in 2019, we conducted a seroprevalence survey using blood donation samples from selected endemic counties. Specimens were screened for virus-specific neutralizing antibodies, and population seroprevalence was estimated using weights calibrated to county population census data. For POWV, median county seroprevalence in 4 states was 0.84%, ranging from 0% (95% CI 0%-2.28%) to 11.5% (95% CI 0.82%-40.9%). EEEV infection was identified in a single county (estimated seroprevalence 1.62% [95% CI 0.04%-8.75%]). Although seroprevalence estimates in sampled areas were generally low, additional investigation of higher-prevalence areas could inform risk for transmission from asymptomatic blood and organ donors.

West Nile virus (WNV) causes thousands of arboviral infections in the United States each year. Patients with immune-compromising conditions and elderly people are at higher risk of severe WNV neuroinvasive disease (WNND). Despite its broad endemicity nationwide, no U.S. Food and Drug Administration-approved vaccine or therapeutic treatments exist. We summarized existing peer-reviewed literature on the preclinical development of monoclonal antibody (MAb) prophylaxis and therapeutics for the prevention and treatment of WNND. Five bibliographical databases (CINAHL, Cochrane Library, Embase, MEDLINE, and Scopus) were searched for applicable research studies performed from 1 January 1998 to 1 May 2025. In total, 2347 titles and abstracts were screened, 263 full-text publications reviewed, and 25 studies included. Studies included detailed preclinical development and evaluations of MAbs targeting the envelope (E) protein (n = 13), other viral proteins (n = 3), flaviviral cross-protective monoclonal antibodies (n = 4), and novel antibody configurations or delivery methods (n = 5). The most well-studied MAb, E16, targeting E- Domain III (E-DIII), was effective at inhibiting and treating WNND in experimental animal models. No work investigated ways to traffic therapeutic antibodies across the blood–brain barrier. This review summarizes the current research in the development of monoclonal antibody therapeutics for WNV and addresses gaps in the knowledge for future consideration.

Powassan virus (POWV) is an emerging tick-borne neurotropic human pathogen. Currently, there are no approved medications or vaccines available. The goal of this study is to investigate the receptor usage for POWV, which can be a potential therapeutic target. For this purpose, we used cell culture-based models including neuronal cells, 293T cells stably expressing different viral entry receptors, and induced pluripotent stem cell (iPSC)-derived cortical organoids, as well as in vivo studies in wild-type C57BL/6 mice. Among the receptors studied, phosphatidylserine (PtdSer)-recognizing TIM-1 and AXL receptors facilitated higher infection. To further validate our findings, a neutralization assay was performed in which the soluble form of the TIM-1 receptor efficiently blocked infection. In addition, we demonstrated that PtdSer receptor-expressing cells in cortical organoids and mouse brain tissues were infected with the virus. We conclude that PtsSer moieties on POWVs’ surface facilitate viral entry through the cellular TIM-1 and AXL receptors.

West Nile virus (WNV) is a neurotropic mosquito-borne virus that re-emerged in the last 25 years to cause substantial short- and long-term morbidity and mortality, particularly in the United States and Europe. Attempts to mitigate the impact of WNV by vector control have been largely unsuccessful. A WNV vaccine could be effective in decreasing disease burden. To identify and address important barriers to licensing human WNV vaccines, U.S. Centers for Disease Control and Prevention convened a meeting of key stakeholders from the U.S. federal and municipal governments, industry, academia, and regulatory agencies in April 2024. Topics discussed included epidemiology, impact of outbreaks, rationale for human vaccines, animal models and correlates of protection, diagnostic considerations, candidate human vaccines in clinical development, and regulatory considerations for vaccine development. Several barriers to human WNV vaccine licensure were identified, including 1) episodic transmission with substantial geographic and temporal variability in disease occurrence impeding the feasibility of phase III clinical trials evaluating vaccine efficacy against WNV disease, 2) high asymptomatic attack rate affecting potential clinical trial endpoint considerations, 3) lack of a surrogate endpoint to predict clinical benefit, 4) need for standardized reagents and assays to quantitate antibodies, and 5) minimal economic support for vaccine development and commercial manufacturing. Future activities to support advancing WNV vaccine development and licensure involve developing a target product profile, establishing suitable animal models and surrogate endpoints to predict clinical benefit, developing a diagnostic test to differentiate immunologic response to infection versus vaccination, conducting a sales forecast analysis, exploring partnerships that could advance vaccine development and licensure, and considering alternative approaches for licensure such as the accelerated approval pathway. We need to work collaboratively as a public health and scientific community to ensure human vaccines are available to decrease the ongoing morbidity and mortality caused by WNV.

BACKGROUND:Jamestown Canyon virus, a mosquito-borne virus, can cause asymptomatic infection, febrile illness, or neuroinvasive disease in humans. Previous studies have found Jamestown Canyon virus-specific antibodies in a 4%-54% of people in various U.S. regions. To understand baseline seroprevalence in regions with the highest number of reported disease cases, we performed a serosurvey among blood donors. METHODS:We randomly selected blood donation specimens collected during December 2019-April 2020 from residents of counties reporting ≥2 disease cases in 2019 or 1 case in 2019 and ≥1 case during 2010-2018. Specimens were screened for Jamestown Canyon virus-specific neutralizing antibodies and, if positive, tested for immunoglobulin M (IgM) antibodies. We estimated county population seroprevalence by calibrating sample weights to population census data. RESULTS:Fourteen counties in 3 states, Massachusetts, Minnesota, and Wisconsin, met the inclusion criteria. Within each state, average county seroprevalence ranged from 16.8% (95% confidence interval [CI], 9.3-27.0) to 18.8% (95% CI, 14.0-24.4) for Jamestown Canyon virus-neutralizing antibodies and from 7.6% (95% CI, 4.2-12.5) to 13.5% (95% CI, 9.6-18.3) for both neutralizing and IgM antibodies. CONCLUSIONS:Estimated Jamestown Canyon virus seroprevalence, including for IgM antibodies, is elevated in endemic areas, complicating the interpretation of serologic testing in diagnosing acute disease in symptomatic individuals. Diagnosing Jamestown Canyon virus disease requires a high degree of clinical suspicion, ruling out other possible causes of illness, and if possible, collecting acute and convalescent samples. New assays to detect acute infection could improve diagnosis and public health surveillance for Jamestown Canyon virus disease.

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.

On August 27, 2024, this report was posted as an MMWR Early Release on the MMWR website (https://www.cdc.gov/mmwr). 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.

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.

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.

Oropouche virus is an arbovirus endemic to the Americas. Periodic outbreaks have occurred since its description in 1955. In late 2023, an outbreak occurred in Peru, centered in and around Iquitos in the Eastern Peruvian Amazon. An existing acute febrile illness (AFI) surveillance program was able to document its emergence and characterize arthralgia and dysuria and the absence of diarrhea as distinctive clinical features of Oropouche virus-associated febrile illness relative to other causes of AFI. Sequencing of isolates from the outbreak demonstrated that strains from this region were distinct from those causing disease in Brazil, despite the large-scale movement of people along the Amazon corridor, but highly similar to strains from Colombia and Ecuador. Our findings suggest that the current outbreak in South America is fundamentally multifocal in origin and not the result of geographic spread from Brazil, which experienced an outbreak between 2022 and 2024.

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.

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.Author Summary:WNV mutational diversity in vertebrates is species-dependent. In crows, low frequency variants are common, and viral populations are more diverse. In robins, fewer mutations become permanent fixtures of the overall viral population. We infected crows, robins and a chicken cell line with a genetically marked (barcoded) WNV. Higher levels of virus led to multiple unique WNV genomes infecting individual cells, even when a genotype was present at low levels in the input viral stock. Our findings suggest that higher levels of circulating virus in natural hosts allow less fit viruses to survive in RNA virus populations through complementation by more fit viruses. This is significant as it allows less represented and less fit viruses to be maintained at low levels until they potentially emerge when virus environments change. Overall our data reveal new insights on the relationships between host susceptibility to high viremia and virus evolution.