Regional molecular epidemiology of dengue and the potential optimization of its control through the use of vaccines. Report of the arbovirus committee of the latin american society of pediatric infectious diseases, SLIPE.

INTRODUCTION: Dengue disease represents a large and growing global threat to public health, accounting for a significant burden to health systems of endemic countries. The World Health Organization's (WHO) Strategic Advisory Group of Experts (SAGE) and the European Medicines Agency (EMA) currently recommend the use of TAK-003 dengue vaccine in high dengue burden and transmission settings for countries considering vaccination as part of their integrated management strategy for prevention and control of Dengue. AREAS COVERED: This paper describes the main conclusions of a workshop held by the Arbovirus Committee of the Latin American Society of Pediatric Infectious Diseases (SLIPE) in November 2024, to generate consensus recommendations on the introduction of this new vaccine in the region. Considerations were made regarding the molecular epidemiology of dengue infection in the Americas and the need for more precise phylogenetic classification and correlation with clinical outcome and disease severity. EXPERT OPINION: Introduction of dengue vaccine should be considered as an strategy for health entities in the region, with participation of social sectors, scientific societies, and ministries of health that could be able to create a successful vaccination program.

Publicly available surveillance data on tick-borne encephalitis in Europe, 2023.

The European Centre for Disease Prevention and Control (ECDC) defines a case of tick-borne encephalitis (TBE) as an infection by the TBE virus (TBEV) with clinical manifestations of central nervous system inflammation (e.g., meningitis, encephalitis). To better understand the TBE surveillance landscape, online searches were conducted to determine if cases of TBE, TBEV infection, acute meningitis or encephalitis, or viral meningitis or encephalitis were subject to statutory reporting in European countries in 2023. In countries with statutory reporting, notification responsibility and available information on surveillance-reported cases were determined. The number of TBE cases reported to ECDC were compared with the number of cases recorded in national surveillance reports. Of 44 countries of the Europe Region of the United Nations, 37 (84 %) mandated statutory reporting of cases of TBE, TBEV infection, or acute/viral meningitis/encephalitis. Twenty-six (87 %) of 30 countries with identified surveillance reports recorded TBE cases in 2020-2023. Of these countries, 17 (65 %) required TBE reporting by clinicians and laboratories, 5 (19 %) by clinicians only, and 4 (15 %) by laboratories only. Twenty-four countries reported on TBE cases to ECDC in 2020; however, surveillance for TBE in Europe is heterogeneous. Standardization of TBE surveillance would enhance the understanding of TBE disease burden in Europe.

Western equine encephalitis, a report of two cases in pediatric patients. / Encefalitis equina del oeste, reporte de dos casos en pacientes pediátricos.

Western equine encephalitis (WEE) is vector-borne infection caused by an RNA virus of the genus Alphavirus, disseminated by mosquitoes that can cause WEE in humans. There are two cycles of transmission, a maintenance cycle and an occasional amplification with vector augmentation, where equines and humans are terminal hosts. In Argentina, no human cases had been reported since 1983. Here we describe 2 pediatric patients with brain symptoms and serological diagnosis of WEE. Both samples of cerebrospinal fluid (CSF) showed pleocytosis, while the neuroimaging test showed alterations in the basal ganglia. The serological diagnosis was based on the detection of specific IgM in serum and CSF and neutralizing antibodies 14 days after symptom onset. The patients were managed with supportive treatment. One patient recovered his normal neurological status without seizures before discharge, while the other was discharged with right hemiparesis, which resolved after 2 months, and continued with anticonvulsants due to a pathological EEG.

La encefalitis equina del oeste (EEO) es una infección causada por un virus ARN del género Alphavirus, de transmisión vectorial por mosquitos que pueden causar la enfermedad en humanos. Hay dos ciclos de transmisión, de mantenimiento y de amplificación ocasional con aumento de vectores, donde equinos y seres humanos son huéspedes terminales. En Argentina no se reportaban casos humanos desde 1983. Se presentan dos pacientes pediátricos con síntomas encefálicos y diagnóstico por serología de EEO. Ambos líquidos cefalorraquídeos (LCR) evidenciaron pleocitosis y las neuroimágenes, alteraciones en ganglios de la base. Se arribó al diagnóstico por serología con detección de IgM específica en suero y LCR, y anticuerpos neutralizantes 14 días después del inicio de síntomas. El tratamiento fue de sostén. Un paciente recuperó el estado neurológico habitual previo al alta sin crisis comiciales y el otro egresó con hemiparesia derecha, que se resolvió luego de dos meses, y continuó con anticonvulsivantes por EEG patológico.

Pathogenicity and virulence of chikungunya virus.

Chikungunya virus (CHIKV) is a mosquito-transmitted, RNA virus that causes an often-severe musculoskeletal illness characterized by fever, joint pain, and a range of debilitating symptoms. The virus has re-emerged as a global health threat in recent decades, spreading from its origin in Africa across Asia and the Americas, leading to widespread outbreaks impacting millions of people. Despite more than fifty years of research into the pathogenesis of CHIKV, there is still no curative treatment available. Current management of CHIKV infections primarily involves providing supportive care to alleviate symptoms and improve the patient's quality of life. Given the ongoing threat of CHIKV, there is an urgent need to better understand its pathogenesis. This understanding is crucial for deciphering the mechanisms underlying the disease and for developing effective strategies for both prevention and management. This review aims to provide a comprehensive overview of CHIKV and its pathogenesis, shedding light on the complex interactions of viral genetics, host factors, immune responses, and vector-related factors. By exploring these intricate connections, the review seeks to contribute to the knowledge base surrounding CHIKV, offering insights that may ultimately lead to more effective prevention and management strategies for this re-emerging global health threat.

Climate, demography, immunology, and virology combine to drive two decades of dengue virus dynamics in Cambodia.

The incidence of dengue virus disease has increased globally across the past half-century, with highest number of cases ever reported in 2019 and again in 2023. We analyzed climatological, epidemiological, and phylogenomic data to investigate drivers of two decades of dengue in Cambodia, an understudied endemic setting. Using epidemiological models fit to a 19-y dataset, we first demonstrate that climate-driven transmission alone is insufficient to explain three epidemics across the time series. We then use wavelet decomposition to highlight enhanced annual and multiannual synchronicity in dengue cycles between provinces in epidemic years, suggesting a role for climate in homogenizing dynamics across space and time. Assuming reported cases correspond to symptomatic secondary infections, we next use an age-structured catalytic model to estimate a declining force of infection for dengue through time, which elevates the mean age of reported cases in Cambodia. Reported cases in >70-y-old individuals in the 2019 epidemic are best explained when also allowing for waning multitypic immunity and repeat symptomatic infections in older patients. We support this work with phylogenetic analysis of 192 dengue virus (DENV) genomes that we sequenced between 2019 and 2022, which document emergence of DENV-2 Cosmopolitan Genotype-II into Cambodia. This lineage demonstrates phylogenetic homogeneity across wide geographic areas, consistent with invasion behavior and in contrast to high phylogenetic diversity exhibited by endemic DENV-1. Finally, we simulate an age-structured, mechanistic model of dengue dynamics to demonstrate how expansion of an antigenically distinct lineage that evades preexisting multitypic immunity effectively reproduces the older-age infections witnessed in our data.

Molecular Epidemiology of Western Equine Encephalitis Virus, South America, 2023-2024.

Western equine encephalitis virus (WEEV) is a mosquitoborne virus that reemerged in December 2023 in Argentina and Uruguay, causing a major outbreak. We investigated the outbreak using epidemiologic, entomological, and genomic analyses, focusing on WEEV circulation near the Argentina‒Uruguay border in Rio Grande do Sul state, Brazil. During November 2023‒April 2024, the outbreak in Argentina and Uruguay resulted in 217 human cases, 12 of which were fatal, and 2,548 equine cases. We determined cases on the basis of laboratory and clinical epidemiologic criteria. We characterized 3 fatal equine cases caused by a novel WEEV lineage identified through a nearly complete coding sequence analysis, which we propose as lineage C. Our findings highlight the importance of continued surveillance and equine vaccination to control future WEEV outbreaks in South America.

Low humidity enhances Zika virus infection and dissemination in Aedes aegypti mosquitoes.

As climate change alters Earth's biomes, it is expected the transmission dynamics of mosquito-borne viruses will change. While the effects of temperature changes on mosquito-virus interactions and the spread of the pathogens have been elucidated over the last decade, the impact of relative humidity changes is still relatively unknown. To overcome this knowledge gap, we exposed Aedes aegypti females to various humidity conditions. We measured different components of vectorial capacity such as survival, blood-feeding rates, and changes in infection and dissemination of Zika virus. Survival decreased as the humidity level decreased, while infection rates increased as the humidity level decreased. Alternatively, blood feeding rates and disseminated infection rates peaked at the intermediate 50% relative humidity treatment but were the same in the 30% and 80% relative humidity treatments. These results provide empirical evidence that Ae. aegypti exposure to low humidity can enhance Zika virus infection in the mosquito, which has important implications in predicting how climate change will impact mosquito-borne viruses.IMPORTANCEViruses transmitted by mosquitoes to humans are a major public health burden and are expected to increase under climate change. While we know that temperature is an important driver of variation in arbovirus replication in the mosquito, very little is known about how other relevant climate variables such as humidity will influence the interaction between mosquitoes and the viruses they transmit. Given the variability in humidity across environments, and the predicted changes in humidity under climate change, it is imperative that we also study the impact that it has on mosquito infection and transmission of arboviruses.

Characterization of mosquito host-biting networks of potential Rift Valley fever virus vectors in north-eastern KwaZulu-Natal province, South Africa.

BACKGROUND: Rift Valley fever virus (RVFV) is a zoonotic mosquito-borne virus with serious implications for livestock health, human health, and the economy in Africa, and is suspected to be endemic in north-eastern KwaZulu-Natal (KZN), South Africa. The vectors of RVFV in this area are poorly known, although several species, such as Aedes (Neomelaniconion) mcintoshi, Aedes (Neomelaniconion) circumluteolus, Aedes (Aedimorphus) durbanensis, and Culex (Lasioconops) poicilipes may be involved. The aim of the study was to determine the vertebrate blood meal sources of potential RVFV mosquito vectors in north-eastern KZN and to characterize the host-biting network. METHODS: Blood-fed mosquitoes were collected monthly from November 2019 to February 2023 using a backpack aspirator, CO2-baited Centers for Disease Control and Prevention (CDC) miniature light traps and tent traps, in the vicinity of water bodies and livestock farming households. The mosquitoes were morphologically identified. DNA was extracted from individual mosquitoes and used as templates to amplify the vertebrate cytochrome c oxidase I (COI) and cytochrome b (cytb) genes using conventional polymerase chain reaction (PCR). Amplicons were sequenced and queried in GenBank and the Barcode of Life Data systems to identify the vertebrate blood meal sources and confirm mosquito identifications. All mosquitoes were screened for RVFV using real time reverse transcription (RT)-PCR. RESULTS: We identified the mammalian (88.8%) and avian (11.3%) blood meal sources from 409 blood-fed mosquitoes. Aedes circumluteolus (n = 128) made up the largest proportion of collected mosquitoes. Cattle (n = 195) and nyala (n = 61) were the most frequent domestic and wild hosts, respectively. Bipartite network analysis showed that the rural network consisted of more host-biting interactions than the reserve network. All mosquitoes tested negative for RVFV. CONCLUSIONS: Several mosquito species, including Ae. circumluteolus, and vertebrate host species, including cattle and nyala, could play a central role in RVFV transmission. Future research in this region should focus on these species to better understand RVFV amplification.

Viral and cellular determinants of polarized trafficking of viral envelope proteins from insect-specific and insect-vectored viruses in insect midgut and salivary gland cells.

Systemic viral infection of insects typically begins with the primary infection of midgut epithelial cells (enterocytes) and subsequent transit of the progeny virus in an apical-to-basal orientation into the hemocoel. For insect-vectored viruses, an oppositely oriented process (basal-to-apical transit) occurs upon secondary infection of salivary glands and is necessary for virus transmission to non-insect hosts. To examine this inversely oriented virus transit in these polarized tissues, we assessed the intracellular trafficking of two model viral envelope proteins (baculovirus GP64 and vesicular stomatitis virus G) in the midgut and salivary gland cells of the model insect, Drosophila melanogaster. Using fly lines that inducibly express either GP64 or VSV G, we found that each protein, expressed alone, was trafficked basally in midgut enterocytes. In salivary gland cells, VSV G was trafficked apically in most but not all cells, whereas GP64 was consistently trafficked basally. We demonstrated that a YxxØ motif present in both proteins was critical for basal trafficking in midgut enterocytes but dispensable for trafficking in salivary gland cells. Using RNAi, we found that clathrin adaptor protein complexes AP-1 and AP-3, as well as seven Rab GTPases, were involved in polarized VSV G trafficking in midgut enterocytes. Our results indicate that these viral envelope proteins encode the requisite information and require no other viral factors for appropriately polarized trafficking. In addition, they exploit tissue-specific differences in protein trafficking pathways to facilitate virus egress in the appropriate orientation for establishing systemic infections and vectoring infection to other hosts. IMPORTANCE: Viruses that use insects as hosts must navigate specific routes through different insect tissues to complete their life cycles. The routes may differ substantially depending on the life cycle of the virus. Both insect pathogenic viruses and insect-vectored viruses must navigate through the polarized cells of the midgut epithelium to establish a systemic infection. In addition, insect-vectored viruses must also navigate through the polarized salivary gland epithelium for transmission. Thus, insect-vectored viruses appear to traffic in opposite directions in these two tissues. In this study, we asked whether two viral envelope proteins (VSV G and baculovirus GP64) alone encode the signals necessary for the polarized trafficking associated with their respective life cycles. Using Drosophila as a model to examine tissue-specific polarized trafficking of these viral envelope proteins, we identified one of the virus-encoded signals and several host proteins associated with regulating the polarized trafficking in the midgut epithelium.

Unexpected arboviruses found in an epidemiological surveillance of acute tropical febrile syndrome in the department of Meta, Eastern Colombia.

BACKGROUND: Nonspecific acute tropical febrile illnesses (NEATFI) are common in the Latin American tropics. Dengue, Chikungunya, Zika, Mayaro, and Usutu, among others, can coexist in the American tropics. This study aimed to surveil the arboviruses that cause| acute febrile syndrome in patients in the Meta department, Colombia. METHODS: Between June 2021 and February 2023, an epidemiological surveillance study was conducted in the Llanos of the Meta department in Eastern Colombia. RESULTS: One hundred patients in the acute phase with typical prodromal symptoms of NEATFI infection who attended the emergency department of the Villavicencio Departmental Hospital were included. ELISA tests were performed for Dengue, Usutu, Chikungunya, and Mayaro. RT-qPCR was performed to detect the arboviruses Usutu, Dengue, Zika, Mayaro, and Oropouche. The seroprevalence for the Chikungunya, Mayaro, and Usutu viruses was 41 % (28/68), 40 % (27/67), and 62 % (47/75), respectively. Seroconversion for Chikungunya was observed in one patient; two seroconverted to Mayaro and one to Usutu. The NS5 gene fragment of the Usutu virus was detected in nine febrile patients. RT-qPCR of the remaining arboviruses was negative. The clinical symptoms of the nine Usutu-positive patients were very similar to those of Dengue, Chikungunya, Zika, and Mayaro infections. CONCLUSIONS: The pervasive detection of unexpected viruses such as Usutu and Mayaro demonstrated the importance of searching for other viruses different from Dengue. Because Usutu infection and Mayaro fever have clinical features like Dengue, a new algorithm should be proposed to improve the accuracy of acute tropical fevers.

Exploring fine-scale urban landscapes using satellite data to predict the distribution of Aedes mosquito breeding sites.

BACKGROUND: The spread of mosquito-transmitted diseases such as dengue is a major public health issue worldwide. The Aedes aegypti mosquito, a primary vector for dengue, thrives in urban environments and breeds mainly in artificial or natural water containers. While the relationship between urban landscapes and potential breeding sites remains poorly understood, such a knowledge could help mitigate the risks associated with these diseases. This study aimed to analyze the relationships between urban landscape characteristics and potential breeding site abundance and type in cities of French Guiana (South America), and to evaluate the potential of such variables to be used in predictive models. METHODS: We use Multifactorial Analysis to explore the relationship between urban landscape characteristics derived from very high resolution satellite imagery, and potential breeding sites recorded from in-situ surveys. We then applied Random Forest models with different sets of urban variables to predict the number of potential breeding sites where entomological data are not available. RESULTS: Landscape analyses applied to satellite images showed that urban types can be clearly identified using texture indices. The Multiple Factor Analysis helped identify variables related to the distribution of potential breeding sites, such as buildings class area, landscape shape index, building number, and the first component of texture indices. Models predicting the number of potential breeding sites using the entire dataset provided an R² of 0.90, possibly influenced by overfitting, but allowing the prediction over all the study sites. Predictions of potential breeding sites varied highly depending on their type, with better results on breeding sites types commonly found in urban landscapes, such as containers of less than 200 L, large volumes and barrels. The study also outlined the limitation offered by the entomological data, whose sampling was not specifically designed for this study. Model outputs could be used as input to a mosquito dynamics model when no accurate field data are available. CONCLUSION: This study offers a first use of routinely collected data on potential breeding sites in a research study. It highlights the potential benefits of including satellite-based characterizations of the urban environment to improve vector control strategies.

Paris 2024 Olympic Games: A risk enhancer for autochthonous arboviral diseases epidemics?

OBJECTIVES: The anticipated increase in international tourist flows and the first locally acquired dengue cases in the Paris region in October 2023 have raised concerns about potential arbovirus outbreaks during the 2024 Olympics. Unlike previous mass sporting events at risk of arbovirus outbreaks, Paris is a nonendemic arbovirus area, requiring a unique investigation. METHODS: Therefore, we analyzed factors conducive to possible arbovirus epidemics in temperate regions: vector distribution in the Paris area, seasonal global arboviral disease patterns, projected visitor demographics, and international flight bookings. RESULTS AND CONCLUSION: Our results suggest that the expected visitors' profile for the summer of 2024 should not increase the risk of arbovirus importation into the Paris region compared to a typical year. Conversely, the primary risk of arbovirus outbreaks is likely to come from within France, particularly from the French West Indies, where a notable, albeit declining, dengue outbreak is underway. Vigilant surveillance by French health authorities will ensure that this trend continues.

Pooled Cohort Profile: ReCoDID Consortium's Harmonized Acute Febrile Illness Arbovirus Meta-Cohort.

Infectious disease (ID) cohorts are key to advancing public health surveillance, public policies, and pandemic responses. Unfortunately, ID cohorts often lack funding to store and share clinical-epidemiological (CE) data and high-dimensional laboratory (HDL) data long term, which is evident when the link between these data elements is not kept up to date. This becomes particularly apparent when smaller cohorts fail to successfully address the initial scientific objectives due to limited case numbers, which also limits the potential to pool these studies to monitor long-term cross-disease interactions within and across populations. CE data from 9 arbovirus (arthropod-borne viruses) cohorts in Latin America were retrospectively harmonized using the Maelstrom Research methodology and standardized to Clinical Data Interchange Standards Consortium (CDISC). We created a harmonized and standardized meta-cohort that contains CE and HDL data from 9 arbovirus studies from Latin America. To facilitate advancements in cross-population inference and reuse of cohort data, the Reconciliation of Cohort Data for Infectious Diseases (ReCoDID) Consortium harmonized and standardized CE and HDL from 9 arbovirus cohorts into 1 meta-cohort. Interested parties will be able to access data dictionaries that include information on variables across the data sets via Bio Studies. After consultation with each cohort, linked harmonized and curated human cohort data (CE and HDL) will be made accessible through the European Genome-phenome Archive platform to data users after their requests are evaluated by the ReCoDID Data Access Committee. This meta-cohort can facilitate various joint research projects (eg, on immunological interactions between sequential flavivirus infections and for the evaluation of potential biomarkers for severe arboviral disease).

Executive Summary of the Spanish Guidelines for the Diagnosis and Management of Imported Febrile Illnesses from the Spanish Society of Tropical Medicine and International Health (SEMTSI), the Imported Pathology Group of the Spanish Society of Infectious Diseases and Clinical Microbiology (GEPI-SEIMC), the Spanish Society of Family and Community Medicine (SEMFYC), the Spanish Society of Primary Care Physicians (SEMERGEN) and the Spanish Society of Emergency Medicine (SEMES).

The Spanish Society of Tropical Medicine and International Health (SEMTSI), the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), the Spanish Society of Emergency Medicine (SEMES), the Spanish Society of Primary Care Physicians (SEMERGEN) and the Spanish Society of Family and Community Medicine (SEMFYC) have prepared a consensus statement on the diagnosis and management of patients with imported febrile illnesses. Twenty authors with different backgrounds and representing different healthcare perspectives (ambulatory primary care, travel and tropical medicine specialists, emergency medicine, hospital care, microbiology and parasitology and public health), identified 39 relevant questions, which were organised in 7 thematic blocks. After a systematic review of the literature and a thoughtful discussion, the authors prepared 125 recommendations, as well as several tables and figures to be used as a consulting tool. The present executive summary shows a selection of some of the most relevant questions and recommendations included in the guidelines.

Aedes aegypti Vector Competence Assay for Rift Valley Fever Virus Using Artificial Blood Meal.

Vector competence assays allow to measure, in the laboratory, the ability of a mosquito to get infected and then retransmit an arbovirus while mimicking natural vector infection route. Aedes aegypti is a major vector of arboviruses worldwide and thus a reference species used in vector competence assays. Rift Valley fever virus (RVFV) is a major public health threat, mostly in Africa, that infects humans and animals through the bite of mosquito vectors. Here, we describe vector competence assay of Aedes aegypti mosquitoes for RVFV, from mosquito exposure to the virus through an infectious artificial blood meal to the measurement of virus prevalence in the mosquito's body, head, and saliva.

Cross-Neutralizing Anti-Chikungunya and Anti-Dengue 2 IgG Antibodies from Patients and BALB/c Mice against Dengue and Chikungunya Viruses.

Dengue (DENV) and Chikungunya (CHIKV) viruses can be transmitted simultaneously by Aedes mosquitoes, and there may be co-infections in humans. However, how the adaptive immune response is modified in the host has yet to be known entirely. In this study, we analyzed the cross-reactivity and neutralizing activity of IgG antibodies against DENV and CHIKV in sera of patients from the Mexican Institute of Social Security in Veracruz, Mexico, collected in 2013 and 2015 and using IgG antibodies of BALB/c mice inoculated with DENV and/or CHIKV. Mice first inoculated with DENV and then with CHIKV produced IgG antibodies that neutralized both viruses. Mice were inoculated with CHIKV, and then with DENV; they had IgG antibodies with more significant anti-CHIKV IgG antibody neutralizing activity. However, the inoculation only with CHIKV resulted in better neutralization of DENV2. In sera obtained from patients in 2013, significant cross-reactivity and low anti-CHIKV IgG antibody neutralizing activity were observed. In CHIKV-positive 2015 sera, the anti-DENV IgG antibody neutralizing activity was high. These results suggest that CHIKV stimulates DENV2-induced memory responses and vice versa. Furthermore, cross-reactivity between the two viruses generated neutralizing antibodies, but exchanging CHIKV for DENV2 generated a better anti-CHIKV neutralizing response.

Transovarial Transmission of Cell-Fusing Agent Virus in Naturally Infected Aedes aegypti Mosquitoes.

Mosquito-borne arboviruses include several pathogens that are responsible for many diseases of significant public health burden. Mosquitoes also host many insect-specific viruses that cannot replicate in vertebrate cells. These insect-specific viruses persist in nature predominantly via vertical transmission (VT), and they exhibit high VT rates (VTRs). Cell-fusing agent virus (CFAV), an insect-specific orthoflavivirus, shows high VTRs in naturally infected mosquitoes but not in artificially infected mosquitoes. To determine whether the high VTRs are due to transovarial transmission, we investigated VT and ovary infection patterns in naturally CFAV-infected Aedes aegypti (Bangkok) mosquitoes. VT was monitored by detecting CFAV among the progeny by reverse-transcription polymerase chain reaction and ovary infection was determined by in situ hybridization using a virus-specific probe. We showed that in CFAV-positive mosquitoes, ovarian follicles were infected, suggesting that VT occurs by transovarial transmission in naturally infected mosquitoes. Additionally, mosquitoes harbored dormant, non-replicative CFAV that remained below the detection level. These results suggested that CFAV persists via VT in nature and has the potential to remain dormant in diapausing mosquitoes during unfavorable conditions. Understanding this VT mechanism is crucial for comprehending the persistence of insect-specific viruses (and potentially dual-host arboviruses) in their natural environment.

Is Culex modestus a New Usutu virus vector?

Usutu virus is an emerging pathogen transmitted by mosquitoes. Culex modestus mosquitoes are widespread in Europe, but their role in disease transmission is poorly understood. Recent data from a single infectious mosquito suggested that Culex modestus could be an unrecognized vector for Usutu virus. In this study, our aim was to corroborate this finding using a larger sample size. We collected immature Culex modestus from a reedbed pond in Flemish Brabant, Belgium, and reared them in the laboratory until the third generation. Adult females were then experimentally infected with Usutu virus in a blood meal and incubated at 25 °C for 14 days. The presence of Usutu virus in the saliva, head and body of each female was determined by plaque assay and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). The transmission efficiency was 54% (n = 15/28), confirming that Belgian Culex modestus can experimentally transmit Usutu virus.

Characterization and arbovirus susceptibility of cultured CERNI cells derived from sika deer (Cervus nippon).

Cervus nippon (sika deer) are widely distributed throughout eastern Asia. Deer possess a variety of antibodies against several zoonotic pathogens, indicating that they act as reservoir of zoonoses. In this study, we reported the characterization of cultured cells derived from sika deer and evaluated their susceptibility to arthropod-borne viruses to clarify their usefulness in virological studies. Cells derived from testicular tissue in Dulbecco's modified eagle medium with 16% fetal bovine serum started growing as primary cultured cells. The diploid cells consisted of 68 chromosomes, consistent with those of Japanese sika deer previously reported. The phylogenetic analysis showed the cells formed a robust clade with Japanese population of C. nippon, indicating that the cultured cells established in this study were originated from the Japanese sika deer. The cells immortalized by the simian virus 40 T-antigen were predominantly spindle-shaped cells exhibiting adhesive properties, and cultivated at 37°C and 5% CO2, which are common culture conditions for many mammalian cell lines. Western blotting analysis indicated that the cultured cells were multiple types of cells that coexist, including at least epithelial, fibroblast, and also Leydig cells. We confirmed that the cells have susceptibility to several arboviruses distributed in Japan: Getah virus, Japanese encephalitis virus, Oz virus, and severe fever with thrombocytopenia syndrome virus, but not to Tarumiz tick virus. From these results, the cells contribute to clarify the role of sika deer as a reservoir of zoonoses in nature and deer-associated experimental research at the cellular and molecular levels.