Recurrent Occupational Hantavirus Infections Linked to Feeder Rodent Breeding Farm, Taiwan, 2022.

We investigated 2 acute cases and 1 previous case of Seoul hantavirus infection in workers in a feeder rodent breeding farm in Taiwan. Prevalence of hantavirus IgG among the tested feeder rats was 37.5%. Appropriate prevention measures, including using disinfection protocols and personal protective equipment, are crucial to lowering risk.

Facile quantitative diagnostic testing for neutralizing antibodies against Chikungunya virus.

BACKGROUND: Viral neutralization (NT) assays can be used to determine the immune status of patients or assess the potency of candidate vaccines or therapeutic monoclonal antibodies (mAbs). Focus reduction neutralization test (FRNT) is a conventional neutralization test (cVNT) with superior specificity for measurement of neutralizing antibodies against a specific virus. Unfortunately, the application of FRNT to the chikungunya virus (CHIKV) involves a highly pathogenic bio-agent requiring biosafety level 3 (BSL3) facilities, which inevitably imposes high costs and limits accessibility. In this study, we evaluated a safe surrogate virus neutralization test (sVNT) that uses novel CHIKV replicon particles (VRPs) expressing eGFP and luciferase (Luc) to enable the rapid detection and quantification of neutralizing activity in clinical human serum samples. METHODS: This unmatched case-control validation study used serum samples from laboratory-confirmed cases of CHIKV (n = 19), dengue virus (DENV; n = 9), Japanese encephalitis virus (JEV; n = 5), and normal individuals (n = 20). We evaluated the effectiveness of sVNT, based on mosquito cell-derived CHIK VRPs (mos-CHIK VRPs), in detecting (eGFP) and quantifying (Luc) neutralizing activity, considering specificity, sensitivity, and reproducibility. We conducted correlation analysis between the proposed rapid method (20 h) versus FRNT assay (72 h). We also investigated the correlation between sVNT and FRNT in NT titrations in terms of Pearson's correlation coefficient (r) and sigmoidal curve fitting. RESULTS: In NT screening assays, sVNT-eGFP screening achieved sensitivity and specificity of 100%. In quantitative neutralization assays, we observed a Pearson's correlation coefficient of 0.83 for NT50 values between sVNT-Luc and FRNT. CONCLUSIONS: Facile VRP-based sVNT within 24 h proved highly reliable in the identification and quantification of neutralizing activity against CHIKV in clinical serum samples.

Aggressive organ penetration and high vector transmissibility of epidemic dengue virus-2 Cosmopolitan genotype in a transmission mouse model.

Dengue virus (DENV) causes dengue fever and severe hemorrhagic fever in humans and is primarily transmitted by Aedes aegypti and A. albopictus mosquitoes. The incidence of DENV infection has been gradually increasing in recent years due to global urbanization and international travel. Understanding the virulence determinants in host and vector transmissibility of emerging epidemic DENV will be critical to combat potential outbreaks. The DENV serotype 2 (DENV-2), which caused a widespread outbreak in Taiwan in 2015 (TW2015), is of the Cosmopolitan genotype and is phylogenetically related to the virus strain linked to another large outbreak in Indonesia in 2015. We found that the TW2015 virus was highly virulent in type I and type II interferon-deficient mice, with robust replication in spleen, lung, and intestine. The TW2015 virus also had high transmissibility to Aedes mosquitoes and could be effectively spread in a continuous mosquitoes-mouse-mosquitoes-mouse transmission cycle. By making 16681-based mutants carrying different segments of the TW2015 virus, we identified the structural pre-membrane (prM) and envelope (E) genes as key virulence determinants in the host, with involvement in the high transmissibility of the TW2015 virus in mosquitoes. The transmission mouse model will make a useful platform for evaluation of DENV with high epidemic potential and development of new strategies against dengue outbreaks.

Accuracy of Dengue, Chikungunya, and Zika diagnoses by primary healthcare physicians in Tegucigalpa, Honduras.

BACKGROUND: Dengue, Chikungunya, and Zika are co-endemic in Honduras and are often misdiagnosed due to similar clinical and epidemiological behavior. Most arboviral infections reported in primary care are based on clinical diagnoses without laboratory confirmation. Therefore, the accuracy of physicians' diagnoses and the factors that affect them needs to be evaluated. METHODS: A cross-sectional study with convenience sampling at primary healthcare centers was conducted from June to September 2016 and 2017. Clinical data and dried blood spots on Whatman 903 filter paper from 415 arboviral cases and 248 non-arboviral febrile cases were collected. Viral RNA was extracted from a 6-mm DBS paper disc and confirmed by RT-qPCR and sequencing. RESULTS: Only 30.84% of diagnostic accuracy was observed in physicians in primary care when comparing arboviral clinical diagnosis with RT-qPCR detection. Moreover, in Dengue and Zika clinical cases, only 8.23% and 27.08% were RT-qPCR confirmed, respectively. No Chikungunya cases were confirmed. In 2017, 20.96% of febrile cases were RT-qPCR confirmed arboviral infections. The symptoms of 45.5% of arboviral cases can fit more than one case definition for arboviruses. The "symptom compliance" and "patient with suspected close contact" were the criteria most utilized by physicians for arboviral diagnosis. The pattern of the epidemiological curves of the arboviral clinical cases didn't match the one of the RT-qPCR confirmed cases. CONCLUSIONS: Low diagnostic accuracy for overall and individual arboviral infections was observed in physicians. Unspecific symptomatology, overlapping case definitions, and reported close contact to an arboviral patient might contribute to misdiagnosis. Without laboratory confirmation, surveillance data may not reflect the real behavior of these diseases and could impact health interventions.

Antigenicity and immunogenicity of chikungunya virus-like particles from mosquito cells.

The spread of chikungunya virus (CHIKV) is reaching pandemic levels, and vaccines and antivirals to control CHIKV infection have yet to be approved. Virus-like particles (VLPs), a self-assembled native multi-subunit protein structure, could potentially be used as an antigen for serological detection and vaccine development. In the current study, we describe the production of novel CHIKV VLPs from mosquitoes using a Baculovirus/Mosquito (BacMos) system in a simple Biosafety Level-2 laboratory. Substantial envelope and capsid protein secretions were detected in culture medium. Co-fractionation of CHIKV E2, E1, and capsid proteins via sucrose gradient ultracentrifugation provided evidence of VLP formation. Transmission electron microscopy and dynamic light scattering analysis revealed the formation of VLPs in the form of spherical particles with a diameter of roughly 40 nm in transduced cells and culture medium. VLP-based IgM capture ELISA in CHIKV patient sera revealed native epitopes on the VLPs. These non-purified VLPs were shown to act as an antigen in CHIKV-specific IgM capture ELISA. The immunization of CHIKV-VLPs alone in mice induced a balance CHIKV-specific IgG2a/IgG1 antibodies and neutralized antibody responses. The study provides support for the hypothesis that mosquito cell-derived CHIKV VLPs could serve as a novel antigen for serological detection and the development of vaccines against CHIKV infection. KEY POINTS: • CHIKV VLPs secreted from BacMos-CHIKV 26S-transduced mosquito cell. • This CHIKV VLPs potentially serve as an alternative capture antigen for MAC-ELISA. • Unadjuvanted CHIK VLPs induce CHIKV-specific IgG and NT responses in mice.

Human case of Anaplasma phagocytophilum infection in Eastern Taiwan.

Human granulocytic anaplasmosis (HGA) is a tick-borne infection caused by the bacterium Anaplasma phagocytophilum. In this study, we report an indigenous case of clinically diagnosed HGA. The patient was a 41-year-old man who experienced a tick bite and later developed fever, chills, myalgia, malaise, thrombocytopenia, leukocytosis with a left shift, elevated hepatic transaminase levels, and splenomegaly upon admission to the hospital. Immunofluorescence assays detected seroconversion against A. phagocytophilum, whereas tests for spotted fever group rickettsiae, murine typhus, scrub typhus, Q fever, and ehrlichiosis were negative. ELISA and Western blot analysis using recombinant MSP2 protein confirmed the exposure to A. phagocytophilum. Oral doxycycline and intravenous ceftriaxone were prescribed, and the patient made a full recovery. Our findings indicate the presence of HGA on the main island of Taiwan. Precautions against tick bites should be taken when engaging in outdoor activities, and HGA should be considered by physicians in the differential diagnosis.

Dengue Virus Ribonucleic Acid Detection Rates in Blood Donors Correlate With Local Infection Incidences During a Dengue Outbreak in Taiwan.

BACKGROUND: Evidence for mitigation of transfusion-transmitted dengue informed by surveillance data is lacking. In this study, we evaluated the risk of positive dengue viral (DENV) ribonucleic acid (RNA) from blood transfusions during a large outbreak in Taiwan. METHODS: Serum collected from blood donors living in districts experiencing the dengue epidemic were tested for DENV RNA using a qualitative transcription-mediated nucleic acid amplification assay (TMA). The TMA-reactive specimens were further tested for immunoglobulin (Ig)M and IgG antibodies, nonstructural protein 1 (NS1) antigen, and viral RNA by reverse-transcription polymerase chain reaction. We estimated DENV RNA prevalence and the number of DENV infections among blood donors. RESULTS: A total of 4976 specimens were tested for DENV RNA, and 21 were TMA-reactive. The detection rate was 0.84 (95% confidence interval [CI], 0.15-4.73), 3.36 (95% CI, 1.31-8.60), and 6.19 (95% CI, 3.14-12.17) per 1000 donors in districts where the weekly dengue incidence was 5-50, 50-200, and 200 or more per 100 000 residents, respectively. Alanine aminotransferase screening only detected 4.4% of TMA-reactive donations. A total of 143 transfusion-transmitted DENV infections probably occurred during this outbreak, accounting for 9.2 in 10 000 dengue infections. CONCLUSIONS: Approximately 0.5%-1% of blood donations were DENV RNA positive in epidemic districts. The correlation of DENV RNA rates with dengue incidence may inform the design of effective control measures.

Chikungunya infection: First autochthonous cases in Taiwan.

The first autochthonous case and the first outbreak of chikungunya in Taiwan occurred during July-October 2019, with a total of 21 cases confirmed. Genetic analysis revealed the strains belonged to East/Central/South African genotype and had 99.95%-100% identity with the strains from the imported cases from Myanmar in 2019. This event confirmed that the imported chikungunya cases has the potential to cause autochthonous transmission in Taiwan; intensified surveillance and vector control measures are essential to contain the outbreak.

Human Case of Severe Fever with Thrombocytopenia Syndrome Virus Infection, Taiwan, 2019.

We report on a 70-year-old man with fever, leukopenia, thrombocytopenia, vomiting, malaise, dyspnea, and consciousness disturbance who was infected with severe fever with thrombocytopenia syndrome virus in northern Taiwan, 2019. This autochthonous case was confirmed by reverse transcription PCR, virus isolation, and genomic sequencing.

Dengue viruses circulating in Indonesia: A systematic review and phylogenetic analysis of data from five decades.

Although epidemiological and molecular epidemiological (serotype, genotype, and lineage information) data are available for several major cities in Indonesia, there is yet to be a comprehensive national study of dengue in Indonesia over time. This study was conducted to provide a comprehensive epidemiology of circulating dengue viruses (DENV) in Indonesia between 1973 and 2016. This was conducted through a systematic review of the literature and phylogenetic analysis of available DENV sequences. Available data from National Disease Surveillance System have indicated an increasing trend of dengue incidence in Indonesia over the past 50 years. Incidence rates appear to be cyclic, peaking approximately every 6 to 8 years. In contrast, the case fatality rate has decreased approximately by half with each decade since 1980. Over this 50-year time span, serotype shifts, genotype displacement within DENV-1 and DENV-2, and introduction of DENV-1 and DENV-3 genotype from other countries occurred. These events were associated with increased incidence of dengue cases. Our study also provides a valuable national snapshot of DENV genetic diversity in Indonesia that may contribute to development of more effective dengue vaccine compositions for the region.

Epidemiological analysis of the Kaohsiung city strategy for dengue fever quarantine and epidemic prevention.

BACKGROUND: Dengue is endemic in over 100 countries and is an important public health problem worldwide. Dengue fever is not endemic in Taiwan; the importation of dengue viruses from neighboring countries via close commercial links and air travel is considered to be the cause of local outbreaks. Therefore, efforts toward disease control have focused on preventing the importation of dengue into Taiwan. In this study, we investigated the relationships between the numbers of imported and indigenous dengue cases to test the validity of this strategy. METHODS: Data on cases of dengue fever that occurred between 2013 and 2018 were obtained from the surveillance systems of the Taiwan Center for Disease Control and Kaohsiung City Health Department. Standard epidemiological data, including the monthly numbers of indigenous and imported cases of dengue, were calculated. Potential associations between the numbers of indigenous and imported cases were investigated using correlation analyses. RESULTS: We identified a possible relationship between the period of disease concealment and the number of imported dengue cases, which resulted in epidemics of indigenous dengue fever within local communities. Further analysis of confirmed cases during previous epidemics in Kaohsiung City found that the risk of indigenous dengue fever may be related to the likelihood that patients with imported dengue fever will stay within local communities. CONCLUSION: Given the correlations found between imported and indigenous cases of dengue fever, as well as the relationship between the disease concealment period and the risk of indigenous dengue fever, prevention of disease importation and efficient identification of dengue cases within high-risk communities remain the major priorities for disease control.

Human Case of Ehrlichia chaffeensis Infection, Taiwan.

In 2018, an immunosuppressed woman in southern Taiwan had onset of fever, chills, myalgia, malaise, thrombocytopenia, lymphocytopenia, and elevated hepatic transaminases. Investigation revealed infection with Ehrlichia chaffeensis. This autochthonous case of human monocytotropic ehrlichiosis was confirmed by PCR, DNA sequencing, and seroconversion.

Development of an Enzyme-Linked Immunosorbent Assay for Rapid Detection of Dengue Virus (DENV) NS1 and Differentiation of DENV Serotypes during Early Infection.

Dengue fever, caused by infections with the dengue virus (DENV), affects nearly 400 million people globally every year. Early diagnosis and management can reduce the morbidity and mortality rates of severe forms of dengue disease as well as decrease the risk of wider outbreaks. Although the early diagnosis of dengue can be achieved using a number of commercial NS1 detection kits, none of these can differentiate among the four dengue virus serotypes. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) for the detection of dengue virus (DENV) NS1 by pairing a serotype-cross-reactive monoclonal antibody (MAb) with one of four serotype-specific MAbs in order to facilitate the rapid detection of NS1 antigens and the simultaneous differentiation of DENV serotypes. A total of 146 serum samples obtained from patients suspected to be in the acute phase of DENV infection were used to evaluate the clinical application of our novel test for the detection and serotyping of DENV. The overall sensitivity rate of our test was 84.85%, and the sensitivity rates for serotyping were as follows: 88.2% (15/17) for DENV serotype 1 (DENV1), 94.7% (18/19) for DENV2, 75% (12/16) for DENV3, and 66.6% (6/9) for DENV4. Moreover, there was no cross-reactivity among serotypes, and no cross-reactivity was observed in sera from nondengue patients. Thus, our test not only enables the rapid detection of the dengue virus but also can distinguish among the specific serotypes during the early stages of infection. These results indicate that our ELISA for DENV NS1 is a convenient tool that may help elucidate the epidemiology of DENV outbreaks and facilitate the clinical management of DENV infections.

Imported Chikungunya Virus Strains, Taiwan, 2006-2014.

We identified 78 imported chikungunya cases in Taiwan during 2006-2014. Sixty-six (84.6%) cases were initially suspected to be dengue, which indicates the necessity for laboratory diagnostics in differentiation between dengue and chikungunya. Results also emphasize the need for active surveillance of febrile illness at points of entry.

A new reportable disease is born: Taiwan Centers for Disease Control's response to emerging Zika virus infection.

Zika virus infection, usually a mild disease transmitted through the bite of Aedes mosquitos, has been reported to be possibly associated with microcephaly and neurologic complications. Taiwan's first imported case of Zika virus infection was found through fever screening at airport entry in January 2016. No virus was isolated from patient's blood taken during acute illness; however, PCR products showed that the virus was of Asian lineage closely related to virus from Cambodia. To prevent Zika virus from spreading in Taiwan, the Taiwan Centers for Disease Control has strengthened efforts in quarantine and surveillance, increased Zika virus infection diagnostic capacity, implemented healthcare system preparedness plans, and enhanced vector control program through community mobilization and education. Besides the first imported case, no additional cases of Zika virus infection have been identified. Furthermore, no significant increase in the number of microcephaly or Guillain- Barré Syndrome has been observed in Taiwan. To date, there have been no autochthonous transmissions of Zika virus infection.

Widespread Rickettsia spp. Infections in Ticks (Acari: Ixodoidea) in Taiwan.

Ticks are second to mosquitoes as the most important disease vectors, and recent decades have witnessed the emergence of many novel tick-borne rickettsial diseases, but systematic surveys of ticks and tick-borne rickettsioses are generally lacking in Asia. We collected and identified ticks from small mammal hosts between 2006 and 2010 in different parts of Taiwan. Rickettsia spp. infections in ticks were identified by targeting ompB and gltA genes with nested polymerase chain reaction. In total, 2,732 ticks were collected from 1,356 small mammals. Rhipicephalus haemaphysaloides Supino (51.8% of total ticks), Haemaphysalis bandicota Hoogstraal & Kohls (28.0%), and Ixodes granulatus Supino (20.0%) were the most common tick species, and Rattus losea Swinhoe (44.7% of total ticks) and Bandicota indica Bechstein (39.9%) were the primary hosts. The average Rickettsia infective rate in 329 assayed ticks was 31.9% and eight Rickettsia spp. or closely related species were identified. This study shows that rickettsiae-infected ticks are widespread in Taiwan, with a high diversity of Rickettsia spp. circulating in the ticks. Because notifiable rickettsial diseases in Taiwan only include mite-borne scrub typhus and flea-borne murine typhus, more studies are warranted for a better understanding of the real extent of human risks to rickettsioses in Taiwan.

Susceptibility of Aedes albopictus and Aedes aegypti to three imported Chikungunya virus strains, including the E1/226V variant in Taiwan.

BACKGROUND/PURPOSE: An E1/226V variant Chikungunya virus (CHIKV) efficiently transmitted by Aedes albopictus to humans poses a significant threat to public health for those areas with the presence of Aedes albopictus, including Taiwan. METHODS: We infected three imported CHIKV isolates including the E1/226V variant with Ae. albopictus and Aedes aegypti in the laboratory to understand the disease risk. Viral RNA was measured by real time reverse transcription polymerase chain reaction. RESULTS: The viral susceptibility varied by virus strain and mosquito species and strain. The Asian virus strain started to replicate at 5-6 days post infection (dpi) with the maximum virus yield, ranging from 10(3.63) to 10(3.87) at 5-10 dpi in both species. The variant CHIKV Central/East/South African (CESA) virus genotype replicated earlier at 1 dpi with the maximum virus yield ranging from 10(5.63) to 10(6.52) at 3-6 dpi in Ae. albopictus females while the nonvariant virus strain replicated at 1-2 dpi with the maximum virus yield ranging from 10(5.51) to 10(6.27) at 6-12 dpi. In Ae. aegypti, these viruses replicated at 1-2 dpi, with maximum yields at 4-5 dpi (range from 10(5.38) to 10(5.62)). CONCLUSION: We concluded that the risk of CHIKV in Taiwan is high in all distribution areas of Ae. aegypti and Ae. albopictus for the CESA genotype and that the E1/226V variant virus strain presents an even higher risk.

Concatenated ScaA and TSA56 Surface Antigen Sequences Reflect Genome-Scale Phylogeny of Orientia tsutsugamushi: An Analysis Including Two Genomes from Taiwan.

Orientia tsutsugamushi is an obligate intracellular bacterium associated with trombiculid mites and is the causative agent of scrub typhus, a life-threatening febrile disease. Strain typing of O. tsutsugamushi is based on its immunodominant surface antigen, 56-kDa type-specific antigen (TSA56). However, TSA56 gene sequence-based phylogenetic analysis is only partially congruent with core genome-based phylogenetic analysis. Thus, this study investigated whether concatenated surface antigen sequences, including surface cell antigen (Sca) proteins, can reflect the genome-scale phylogeny of O. tsutsugamushi. Complete genomes were obtained for two common O. tsutsugamushi strains in Taiwan, TW-1 and TW-22, and the core genome/proteome was identified for 11 O. tsutsugamushi strains. Phylogenetic analysis was performed using maximum likelihood (ML) and neighbor-joining (NJ) methods, and the congruence between trees was assessed using a quartet similarity measure. Phylogenetic analysis based on 691 concatenated core protein sequences produced identical tree topologies with ML and NJ methods. Among TSA56 and core Sca proteins (ScaA, ScaC, ScaD, and ScaE), TSA56 trees were most similar to the core protein tree, and ScaA trees were the least similar. However, concatenated ScaA and TSA56 sequences produced trees that were highly similar to the core protein tree, the NJ tree being more similar. Strain-level characterization of O. tsutsugamushi may be improved by coanalyzing ScaA and TSA56 sequences, which are also important targets for their combined immunogenicity.

Epidemiology of Murine Typhus in Taiwan from 2013 to 2020.

Murine typhus is a flea-borne disease caused by Rickettsia typhi infection. The disease is a notifiable infectious disease in Taiwan. Specimens from suspected cases are required to be sent to the Taiwan Centers for Disease Control and Prevention for laboratory diagnosis. In this study, 204 cases of murine typhus were identified by bacterial isolation, real-time polymerase chain reaction, or indirect immunofluorescence assay between 2013 and 2020. The average incidence rate was 0.11/100,000 person-years (95% CI: 0.08-0.13). Murine typhus occurred throughout the year, but it was most prevalent in summer (May to August). The majority of patients were males (75%), residents of Kaohsiung city (31%), and worked in agriculture, forestry, fishing, and animal husbandry (27%). Fever was the most common symptom, present in 95.6% of patients, followed by headache (41%), myalgia (33%), and liver dysfunction (33%). Only 13% of patients had a rash. Up to 80% of cases were among hospitalized patients, and 43% of patients developed severe manifestations. Serological assays also indicated coinfection events. Seven patients showed a 4-fold increase in antibody titers against Orientia tsutsugamushi (N = 2), Coxiella burnetii (n = 2), and Leptospira (N = 3). In conclusion, murine typhus is an endemic and important zoonotic rickettsial disease in Taiwan that cannot be ignored. Further epidemiological surveillance and clinical characteristics should be continuously investigated to prevent and control murine typhus.

Development of a Novel Chikungunya Virus-Like Replicon Particle for Rapid Quantification and Screening of Neutralizing Antibodies and Antivirals.

Chikungunya fever is a mosquito-transmitted infectious disease that induces rash, myalgia, and persistent incapacitating arthralgia. At present, no vaccines or antiviral therapies specific to Chikungunya virus (CHIKV) infection have been approved, and research is currently restricted to biosafety level 3 containment. CHIKV-like replicon particles (VRPs) are single-cycle infectious particles containing viral structure proteins, as well as a defective genome to provide a safe surrogate for living CHIKV to facilitate the testing of vaccines and antivirals. However, inefficient RNA transfection and the potential emergence of the competent virus through recombination in mammalian cells limit VRP usability. This study describes a transfection-free system for the safe packaging of CHIK VRP with all necessary components via transduction of mosquito cell lines using a single baculovirus vector. We observed the release of substantial quantities of mosquito cell-derived CHIK VRP (mos-CHIK VRP) from baculovirus-transduced mosquito cell lines. The VRPs were shown to recapitulate viral replication and subgenomic dual reporter expression (enhanced green fluorescent protein [eGFP] and luciferase) in infected host cells. Interestingly, the rapid expression kinetics of the VRP-expressing luciferase reporter (6 h) makes it possible to use mos-CHIK VRPs for the rapid quantification of VRP infection. Treatment with antivirals (suramin or 6-azauridine) or neutralizing antibodies (monoclonal antibodies [MAbs] or patient sera) was shown to inhibit mos-CHIK VRP infection in a dose-dependent manner. Ease of manufacture, safety, scalability, and high throughput make mos-CHIK VRPs a highly valuable vehicle for the study of CHIKV biology, the detection of neutralizing (NT) antibody activity, and the screening of antivirals against CHIKV. IMPORTANCE This study proposes a transfection-free system that enables the safe packaging of CHIK VRPs with all necessary components via baculovirus transduction. Those mosquito cell-derived CHIK VRP (mos-CHIK VRPs) were shown to recapitulate viral replication and subgenomic dual reporter (enhanced green fluorescent protein [eGFP] and luciferase) expression in infected host cells. Rapid expression kinetics of the VRP-expressing luciferase reporter (within hours) opens the door to using mos-CHIK VRPs for the rapid quantification of neutralizing antibody and antiviral activity against CHIKV. To the best of our knowledge, this is the first study to report a mosquito cell-derived alphavirus VRP system. Note that this system could also be applied to other arboviruses to model the earliest event in arboviral infection in vertebrates.