Usage of FTA® Classic Cards for Safe Storage, Shipment, and Detection of Arboviruses

Abstract

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^® FTA^® 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^® cards. To evaluate the virus inactivation capacity of FTA^® 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^® 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^® cards. In addition, we quantified viral RNA of JEV, SINV, and tick-borne encephalitis virus (TBEV) stored on FTA^® cards at 4 °C, 25 °C, and 37 °C for 30 days using two reverse transcriptase quantitative PCR assays. Viral RNA of SINV stored on FTA^® cards was not reduced at either 4 °C or 25 °C over a 30-day period, but degraded rapidly at 37 °C. For JEV and TBEV, degradation was observed at all temperatures, with the most rapid degradation occurring at 37 °C. Therefore, the use of FTA^® 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.

Keywords: FTA® Classic Card; RNA stability; alphavirus; arboviruses; biosafety; flavivirus.

Figure 1

Overview of workflow. For biosafety, virus stocks were inoculated onto FTA^® cards and eluted, and then eluates were placed on cell monolayers to observe development of cell cytopathic effect. RNA stability was tested by inoculating stocks onto FTA^® cards, which were stored at different temperatures for different periods of time. RNA was then extracted and quantified in a reverse transcriptase quantitative PCR assay. Not all viruses were used in all assays, but representatives of each virus family were selected based on biosafety and availability. Workflow figure created in BioRender.com (accessed on 19 May 2022).

Figure 2

Cell cytopathic effect (CPE) development in Vero E6 cells infected with infectious and FTA^® inactivated Japanese encephalitis virus (JEV) or chikungunya virus (CHIKV). Virus was inoculated at various concentrations on Vero E6 monolayers and incubated for 48 h. Virus-induced CPE could be observed down to a dilution of 10⁻⁴. Chemically induced CPE could be observed in the FTA^® control down to a concentration of 10⁻¹. The inactivated virus samples (JEV,FTA^® and CHIKV,FTA^®) showed CPE development identical to the CPE in the FTA^® control. The supernatant was additionally collected and extracted to verify whether virus replication underlay the inactivated virus samples. No replication was detected. The wells shown are representative of all replicates, and the total magnification used to observe the cells was 100×.

Figure 3

Temporal and temperature-based degradation of Sindbis virus (SINV), tickborne encephalitis virus (TBEV), and Japanese encephalitis virus (JEV) RNA stored on FTA^® cards, visualized by the change in cycle threshold (Ct) value over time. Points are average Ct values of all replicates and the error bars are the standard deviation.