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Infections caused by arthropod-borne RNA viruses are overrepresented among emerging infectious diseases. Effective methods for collecting, storing, and transporting clinical or biological specimens are needed worldwide for disease surveillance. However, many tropical regions where these diseases are endemic lack analytical facilities and possibility of continuous cold chains, which presents challenges from both a biosafety and material preservation perspective. Whatman (R) FTA (R) Classic Cards may serve as an effective and safe option for transporting hazardous samples at room temperature, particularly for RNA viruses classified as biosafety level (BSL) 2 and 3 pathogens, from sampling sites to laboratories. In this study, we investigated the biosafety and perseverance of representative alpha-and flaviviruses stored on FTA (R) cards. To evaluate the virus inactivation capacity of FTA (R) cards, we used Sindbis virus (SINV), chikungunya virus (CHIKV), and Japanese encephalitis virus (JEV). We inoculated susceptible cells with dilution series of eluates from viral samples stored on the FTA (R) cards and observed for cytopathic effect to evaluate the ability of the cards to inactivate viruses. All tested viruses were inactivated after storage on FTA (R) cards. In addition, we quantified viral RNA of JEV, SINV, and tick-borne encephalitis virus (TBEV) stored on FTA (R) cards at 4 degrees C, 25 degrees C, and 37 degrees C for 30 days using two reverse transcriptase quantitative PCR assays. Viral RNA of SINV stored on FTA (R) cards was not reduced at either 4 degrees C or 25 degrees C over a 30-day period, but degraded rapidly at 37 degrees C. For JEV and TBEV, degradation was observed at all temperatures, with the most rapid degradation occurring at 37 degrees C. Therefore, the use of FTA (R) cards provides a safe and effective workflow for the collection, storage, and analysis of BSL 2- and 3-virus RNA samples, but there is a risk of false negative results if the cards are stored at higher temperatures for long periods of time. Conscious usage of the cards can be useful in disease surveillance and research, especially in tropical areas where transportation and cold chains are problematic.

Background: Dengue virus and Japanese encephalitis virus are two common flaviviruses that are spread widely by Aedes and Culex mosquitoes. Livestock keeping is vital for cities, however, it can pose the risk of increasing the mosquito population. Our study explored how livestock keeping in and around a large city is associated with the presence of mosquitoes and the risk of them spreading flaviviruses. Methods: An entomological study was conducted in 6 districts with 233 households with livestock, and 280 households without livestock, in Hanoi city. BG-Sentinel traps and CDC light traps were used to collect mosquitoes close to animal farms and human habitats. Adult mosquitoes were counted, identified to species level, and grouped into 385 pools, which were screened for flaviviruses using a pan-flavivirus qPCR protocol and sequencing. Results: A total of 12,861 adult mosquitoes were collected at the 513 households, with 5 different genera collected, of which the Culex genus was the most abundant. Our study found that there was a positive association between livestock keeping and the size of the mosquito population—most predominantly between pig rearing and Culex species (p &lt, 0.001). One pool of Cx. tritaeniorhynchus, collected in a peri-urban district, was found to be positive for Japanese encephalitis virus. Conclusions: The risk of flavivirus transmission in urban areas of Hanoi city due to the spread of Culex and Aedes mosquitoes could be facilitated by livestock keeping.

Additional File 1. RT-qPCR validation results and individual Ct values.

Bird-hosted viruses have the potential to be transported over large areas of the world and to be transmitted in distant geographical regions. Sindbis virus (SINV) is a mosquito-borne alphavirus that is locally amplified in a bird-mosquito enzootic cycle and distributed all over the Old World and Australia/Oceania. Sindbis virus genotype I (SINV-I) is the cause of disease outbreaks in humans in South Africa as well as in northern Europe. To trace the evolutionary history and potential strain-disease association of SINV-I, we sequenced 36 complete genomes isolated from field material in Europe, as well as in Africa and the Middle East, collected over 58 years. These were analyzed together with 30 additional published whole SINV-I genomes using Bayesian analysis. Our results suggested that SINV-I was introduced only once to northern Europe from central Africa, in the 1920s. After its first introduction to Sweden, it spread east and southward on two separate occasions in the 1960s and 1970s. Another introduction from central Africa to southern/central Europe seems to have occurred, and where these two introductions meet, one recombination event was detected in central Europe. In addition, another recombinant strain was found in central Africa, where the most divergent SINV-I strains also originated. IMPORTANCE This study shows that only a single introduction of SINV into a new geographical area is required for spread and establishment, provided that the requisite vector(s) and reservoir(s) of epizootological and epidemiological importance are present. Furthermore, we present the first report of recombination between two strains of SINV in nature. Our study increases the knowledge on new introductions and dispersal of arboviruses in general and of SINV in particular.