10th International Aerosol Conference September 2 - September 7, 2018 America's Center Convention Complex St. Louis, Missouri, USA
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Viability of Aerosolized Murine Noroviruses in Experimental Setup
MALIN ALSVED, Anders Widell, Mats Bohgard, Patrik Medstrand, Jakob Löndahl, Lund University, Sweden
Abstract Number: 1452 Working Group: Infectious Bioaerosol
Abstract Introduction Norovirus is the major cause of acute gastroenteritis in the world and it is considered to spread by food, contact, by the fecal–oral route and by droplets from splashes. However, there are studies showing disease transmission between individuals without contact, indicating airborne transmission [1]. One previous study successfully detected NoV RNA in air samples from hospitals [2]. However, important issues that remain to be understood are for instance what sources that generate airborne NoV and to what extent airborne NoV are infectious.
In order to understand what parameters that affect the infectivity of airborne NoV, we have developed an experimental setup for aerosolization and collection of murine noroviruses (MNV), a cultivable model virus for human NoV.
The genomes of NoV and MNV are of positive sense strand RNA. During replication, the intermediary, complementary minus strand RNA is formed inside the infected cells. By specifically detecting the minus strand RNA in cells, infection can be identified with high sensitivity and specificity [3].
Methodology MNVs in cell culture medium were aerosolized by an atomizer (Model 3076, TSI inc.) into a 1 m long stainless steel flow tube, and collected at the end of the flow tube into phosphate buffer saline (PBS, Life Technologies) solution with a BioSampler® impinger (SKC inc.). The particle number concentration and size distribution of the aerosol was monitored by a scanning mobility particle sizer (SMPS, built in-house, Lund University) and an aerodynamic particle sizer (APS, Model 3321, TSI inc.).
The liquid in the BioSampler® was analyzed by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) targeting the viral genome (positive RNA) to determine the number of collected viral RNA genomes. For infectivity analysis, inoculation of the collection liquid on RAW 264.74 cells was performed during 72 h. After the incubation time, the cells and the supernatant were separated for analysis. Intra and extracellular RNA was obtained by RNeasy Mini Kit (Qiagen) and detected by strand specific plus and minus RT-qPCR as described by Vashist et al. [3].
Results and Discussion The aerosolized and collected MNV that were inoculated on cell cultures showed cytopathic effect (CPE) on the cells by visual observation in a microscope. Infections were confirmed by the minus RNA RT-qPCR analysis on the cells after the incubation time. The lower limit of detection of minus RNA was 102 genomes/mL and the concentrations of intracellular minus RNA after incubation were in the range of 1.5·103 to 3.9·106 genomes/mL. This infectivity assay, thus serves as a sensitive qualitative determination of MNV infection. This is useful since visual CPE observations need a trained observer to be correctly analyzed. Our future goal is to develop the assay to give relative quantitative information on virus infectivity.
Conclusions In this study we have successfully developed a setup for experimental studies on airborne MNV. From our results, we saw that infectivity was preserved after aerosolization. This experimental setup allows further studies to be made to gain insight into what factors that affect infectivity of airborne MNV.
Acknowledgements This work was financed by The Swedish Research Council FORMAS and AFA Insurance.
References [1] Marks P.J. et al. 2000, Evidence for airborne transmission of Norwalk-like virus (NLV) in a hotel restaurant, Epidemiol. Infect., 124, 481-487. [2] Bonifait L. et al. 2015, Detection and quantification of airborne norovirus during outbreaks in healthcare facilities, Clin Infect Dis, 61(3), 299-304. [3] Vashist S et al. 2012, ”Development of a strand specific real-time RT-qPCR assay for the detection and quantitation of murine norovirus RNA”, J Virol Methods, 69-76.