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Enhanced detection of infectious airborne influenza virus

https://doi.org/10.1016/j.jviromet.2011.05.030Get rights and content

Abstract

Current screening methodologies for detecting infectious airborne influenza virus are limited and lack sensitivity. To increase the sensitivity for detecting infectious influenza virus in an aerosol sample, the viral replication assay was developed. With this assay, influenza virus is first amplified by replication in Madin–Darby canine kidney (MDCK) cells followed by detection with quantitative PCR (qPCR). Spanning a 20-h replication period, matrix gene expression levels from infectious virus were measured at several time points using qPCR and found to exponentially increase. Compared with the traditional culture-based viral plaque assay, the viral replication assay resulted in a 4.6 × 105 fold increase in influenza virus detection. Furthermore, viral replication assay results were obtained in half the time of the viral plaque assay. To demonstrate that the viral replication assay is capable of detecting airborne influenza virus, dilute preparations of strain A/WS/33 were loaded into a nebulizer, aerosolized within a calm-air settling chamber and subsequently collected using NIOSH Two-Stage Bioaerosol Samplers. At the most diluted concentration corresponding to a chicken embryo infectious dose 50% endpoint (CEID50) of 2.8E+02/ml, the viral replication assay was able to detect infectious influenza virus that was otherwise undetectable by viral plaque assay. The results obtained demonstrate that the viral replication assay is highly sensitive at detecting infectious influenza virus from aerosol samples.

Introduction

Influenza is a highly contagious respiratory virus that causes severe morbidity and extensive mortality each year world-wide. A wide body of evidence shows that influenza spreads at least in part by contact and droplet transmission; however the amount of airborne transmission that occurs remains to be determined. Reviews on aerosol transmission by Tellier (2009) and Weber & Stilianakis (2008) suggest that airborne transmission plays a critical role in the spread of influenza. In contrast, Brankston et al. (2007) conclude that airborne transmission is an unlikely route in the spread of influenza virus. Despite conflicting reports, the role of airborne transmission cannot be over-looked.

Small airborne particles and/or droplet nuclei that are generated during respiratory activities such as coughing, breathing or sneezing can remain airborne for prolonged periods and dispersed by circulating air currents. Several studies have reported that influenza virus RNA is detectable in the exhaled breath and coughs of influenza patients (Fabian et al., 2008, Huynh et al., 2008, Stelzer-Braid et al., 2009, Lindsley et al., 2010a). The airborne distribution of influenza virus RNA was also examined in a hospital emergency department (Blachere et al., 2009) and in an urgent care clinic (Lindsley et al., 2010b). Results established that influenza virus was present and over half of the collected particles were detected within the respirable aerosol fraction. However, the question remains as to whether these airborne particles have the ability to cause acute infection in susceptible individuals. Due to inherent difficulties of stress-sensitivity, small particle size and low concentration, there is limited research that demonstrates the likelihood of airborne influenza transmission. Further studies are greatly needed to resolve the debate of short and long-range airborne influenza virus transmission in healthcare and other indoor settings.

Methods of detection play a critical role in evaluating airborne transmission. Although a number of detection methods are available, limitations such as specimen type, turnaround time, sensitivity and specificity exist. As with most bioaerosols, influenza-laden aerosol samples pose a particular challenge and require optimal methods of detection. Real-time quantitative PCR (qPCR) analysis has proven to be a sensitive method for quickly and accurately detecting influenza virus from aerosol samples (Blachere et al., 2009, Lindsley et al., 2010a, Lindsley et al., 2010b, Hermann et al., 2006, Chen et al., 2009). However, qPCR alone does not distinguish between infectious and non-infectious virus. On the other hand, the viral plaque assay, tissue culture infectious dose 50% endpoint (TCID50) assay, and chicken embryo infectious dose 50% endpoint (CEID50) assay are important methods for establishing viral titers but are time consuming, labor intensive and limited with regards to detection threshold levels (Cottey et al., 2001, LaBarre and Lowy, 2001, Matrosovich et al., 2006). In order to enhance detection levels and reduce turnaround time, the purpose of this study was to develop a sensitive and specific assay that would detect infectious influenza virus from aerosol samples.

Section snippets

Cell and virus stock

Madin–Darby canine kidney (MDCK) cells (ATCC CCL-34) were purchased from the American Type Tissue Culture (ATCC), Manassas, VA. Cells were propagated and maintained in 75-cm2 Corning CellBind Surface flasks (Corning, NY). Complete growth medium for MDCK cells consisted of Eagle's Minimum Essential Medium (EMEM) (ATCC) containing 10% fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT), 200 units/ml penicillin G/200 μg/ml streptomycin (Invitrogen, Carlsbad, CA). Cells were incubated at 35 °C

Optimization of infection

In the viral replication assay, influenza virus is analyzed after initially incubating the viral sample with MDCK cells for 45 min to allow adsorption and entry of the virus. Following infection, viral RNA replicates to several orders of magnitude within the cell. A time course experiment analyzing matrix gene expression was, therefore, performed to determine the optimal incubation time for maximum amplification of viral copy number. During the initial 2 h of sampling (Fig. 1), the total matrix

Discussion

The extent by which airborne transmission plays a role in the spread of influenza remains to be determined. A number of factors limit the recovery and detection of infectious influenza virus from aerosol samples. In this study, an enhanced method of viral detection that employs both standard culture techniques and quantitative real-time PCR is reported. Prior to RNA isolation and qPCR analysis, MDCK cells are washed to remove residual virus and therefore, detection of the matrix gene by qPCR is

Acknowledgements

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. This work was supported, in part, by an interagency agreement (DW7592259701) with the Environmental Protection Agency.

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