Analysis of SARS-CoV-2 RNA Stability and Infectivity in Oropharyngeal Swab Samples

The reliable detection of SARS-CoV-2 genomic RNA and infectious virus particles from patient samples requires a good sample quality. This is especially critical when the sample has to be transported to the analysing laboratory which can take several days. To determine optimal transport conditions, we simulated oropharyngeal swab samples using dened virus amounts and stored the samples at 4 °C or at room temperature for up to four days. Moreover, we analysed the inuence of dry swabs in comparison to swabs stored in transport medium. Our results show that care should be taken when analysing samples for infectious SARS-CoV-2 particles since infectivity is strongly inuenced by sample storage. the stability of SARS-CoV-2 genomic RNA and infectious virus imitating a sample from an Using oropharyngeal swabs from healthy volunteers more the during transport than plain transport or plain dry without the saliva components present in patient samples. that for virus be transported in medium, e.g. amies during transport to 4°C and analysed within two to ensure a correct diagnosis.


Introduction
The gold-standard method for the detection of SARS-CoV-2 in clinical specimens is real-time PCR. In contrast to other methods, like lateral-ow assay-based rapid antigen test, real-time PCR is highly sensitive, detecting less than 10 copies of SARS-CoV-2 RNA per reaction [1,2]. There is a certain correlation of the amount of viral RNA detected with real-time PCR and infectivity; e.g. from samples with low genome copy numbers the isolation of infectious virus is highly unlikely [3]. Although a potential infectivity of the sample can be deduced from a cT value, re ecting a de ned number of RNA genome molecules, it remains important to test for infectious virus particles using virus isolation in cell culture in certain scenarios. For example, a basis must be provided for ending the isolation of long-term positive patients who are often immunocompromised and can have positive PCR results for up to several months after the rst positive test [4,5]. Additionally, upon the detection of new variants of concern (VOC), infectivity testing in cell culture might be performed to elucidate how infectious virus shedding is in uenced by the new variant [6]. The ability to isolate genomic viral RNA and infectious virus particles from naso-or oropharyngeal swab samples generally depends on the sampling itself, the sample transport time and temperature and, moreover, the swab type, e.g. dry or in transport medium.
Additionally, patient-speci c features can have an in uence on the detection, e.g. the isolation of infectious virus particles likely depends on the presence of neutralizing antibodies [7,8]. The stability of SARS-CoV-2 has been analysed under different conditions, showing that genomic RNA and infectious virus particles are sensitive to heat and less stable on rough surfaces like wood and cloth. Additionally, the stability in aerosols has been analysed demonstrating the presence of infectious virus particles in aerosols for up to 3h [9,10]. However, the stability of SARS-CoV-2 genomic RNA and infectious virus particles can hardly be determined in naso-and oropharyngeal swabs, because the starting amount present in the specimen is simply unknown. Therefore, in the present study, we simulated patient samples with a de ned number of infectious particles to study the stability of SARS-CoV-2 genomic RNA and infectious virus particles in oropharyngeal swab samples.

Materials And Methods
Simulation of SARS-CoV-2-positive oropharyngeal swabs and storage To ensure reproducibility with a de ned amount of virus in the samples, SARS-CoV-2-positive oropharyngeal swabs were simulated. For this purpose, healthy laboratory staff members swabbed themselves using two common swab types: wet eSwab with amies medium (Copan, Brescia, IT, no. 490CE) and dry FLOQSwabs (Copan, Brescia, IT, no. 552c). The following steps were performed in a BSL-3 laboratory. After self-testing, the swabs were transferred into 2-ml microcentrifuge tubes (Eppendorf, Hamburg, DE) containing 500 µl of cell culture supernatant with a total of 5x10 3 PFU of SARS-2-CoV (strain BetaCoV/Germany/BavPat1/2020, kindly provided by the Institute for Microbiology of the German Armed Forces). Swabs were wiped around in the virus suspension for approximately 10 seconds and subsequently transferred into their associated storage vessels (transport medium or dry tube). Swabs in storage vessels were either stored at room temperature (RT) or in a refrigerator at 4 °C. Samples for realtime PCR and virus cultivation were taken immediately, after two and four days of storage. For each time point and each swab type, samples were prepared and analysed in biological triplicate.

Real-time PCR analysis
For nucleic acid extraction the QIAamp® Viral RNA Mini Kit (Qiagen, Hilden, DE) was used. Brie y, dry swabs were vortexed in 1 ml of PBS and 140 µl were transferred into 560 µl of AVL buffer. For wet swabs 140 µl of storage medium were used for extraction. Viral and cellular nucleic acids were analysed using an in-house real-time PCR as described by Michel et al. [2]. In a duplex assay, both the SARS-CoV-2 orf1ab gene and the cellular MYC gene were targeted. Brie y, 50 µl of each simulated swab sample were diluted by addition of 150 µl of infection medium. The diluted sample was used to infect one well of VeroE6 cells cultivated in 24-well cell culture plates. All samples were tested in triplicate. After 1 h of incubation at 37°C and 5 % CO 2 to allow adhesion of SARS-CoV-2, the diluted sample was removed and the cells were washed once in PBS, before adding 500 µl of fresh medium. Subsequently, the cells were incubated at 37°C and 5 % CO 2 for three days. At day 3 post infection (p.i.), cells were examined for signs of cytopathic effect (CPE). Furthermore, supernatant from two of the three replicate wells was harvested on day 3 and subjected to RNA extraction and real-time PCR analysis. The remaining replicate was cultivated until day 7 p.i. before it was processed equally.

Virus titration (TCID 50 )
A total of 2x10 4 VeroE6 cells per well in 100 µl of medium (DMEM + 10 % FCS + 2 mM L-Gln) were seeded in 96-well plates and incubated overnight at 37°C, 5% CO 2. Samples were serially diluted in medium (10 -1 to 10 -10 ) and 100 µl of each dilution were added in eight replicates to the cells. After incubation for ve days at 37°C, 5% CO 2 the CPE was analysed by light microscopy and the tissue culture infectious dose 50 (TCID 50 ) was calculated.

Results
We simulated SARS-CoV-2 oropharyngeal swab samples and analysed the stability of viral and cellular nucleic acids after 0, 2 and 4 days of storage at RT or 4 °C using wet or dry swabs (Figure 1).
At RT, a decrease of the viral genomic RNA amount was observed over time, while it was not affected after storage at 4 °C (Figure 2A). This was also true for cellular nucleic acids which were used as a control for effective sampling ( Figure 2B). The decrease in viral genomic RNA at RT was moreover associated with a strong decrease in infectivity. No infectivity was detectable after two days of storage at RT. In contrast, infectivity was better preserved in oropharyngeal swabs stored at 4°C for up to four days (Table 1). At a temperature of 4°C no signi cant differences were detected between wet and dry swabs in PCR analysis for a storage time of up to four days. However, wet swabs stored at 4°C for up to four days showed slightly better results in the infectivity test with 67% positive culture rate compared to 22% positive culture rate for dry swabs.
The successful isolation of infectious virus particles is displayed by "+", no isolation of infectious virus particles is displayed by a "-". For samples stored at 4°C, the number of infectious particles was calculated (TCID 50 /ml) and shown as mean of three replicates.

Discussion
For the detection of SARS-CoV-2 genomic RNA and the isolation of infectious virus particles, naso-or oropharyngeal swabs have to be transported from the patient to the laboratory. During transport, the majority of samples is stored at RT or, in rare cases, at 4°C. Hence, we analysed the stability of viral genomic RNA and virus particles at these two temperatures. We used a storage time of up to four days to simulate a sample shipment taking several days. In agreement with the literature, viral nucleic acids and infectivity were better preserved at a storage temperature of 4°C compared to RT [9]. Moreover, at 4°C the amount of nucleic acids in wet and dry swabs was comparable. Surprisingly, the detection of infectious virus particles in the samples was already dramatically reduced after storage at RT for two days. From these results we recommend SARS-CoV-2 oropharyngeal swab samples designated for virus isolation in cell culture to be cooled at 4°C during sample transport to ensure preservation of infectious virus particles. A possibly reason for the reduction of infectivity in the samples is the presence of proteases from saliva rendering viruses non-infectious by degradation of virion proteins. Saliva is a digestive uid and contains a mixture of diverse proteases, and even low amounts in oropharyngeal swab samples can potentially reduce the number of infectious virus particles [11]. Additionally, RNases present in salivaand hence in oropharyngeal swabs -can degrade SARS-CoV-2 RNA [12]. Using dry swabs, the virus particles could also be prone to dry-out, potentially explaining the slightly better preservation of infectious virus particles in wet compared to dry swabs.
The present study analyses the stability of SARS-CoV-2 genomic RNA and infectious virus particles, imitating a sample from an infected patient as closely as possible. Using oropharyngeal swabs from healthy volunteers more closely re ects the natural conditions during sample transport than plain transport medium or plain dry swabs without the saliva components present in patient samples. We conclude that samples designated for virus cultivation should be transported in medium, e.g. amies medium, cooled during transport to 4°C and analysed within two days to ensure a correct diagnosis. Authors' contributions MN conceptualized the study and analysed the data. MN and MG prepared the gures and wrote the original manuscript. DB was responsible for the infectivity experiments in the BLS-3 lab. AN and LS supervised the study. All authors read and approved the nal manuscript. Figure 1 Experimental setup to analyse the stability of SARS-CoV-2 genomic RNA and infectivity in oropharyngeal swab samples.