ABSTRACT
Aerosols or saliva containing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can contaminate living environments, and viruses can be indirectly transmitted. To understand the survival potential of the virus, the viral titers of bovine coronavirus (BCoV), as a model virus, and SARS-CoV-2 were measured on porous and non-porous surfaces. The amount of infectious BCoV recovered remained relatively high on non-porous substrates. However, it quickly decreased on several non-porous surfaces such as nitrile rubber. The time taken to reach the limit of detection on non-woven masks, as a porous substrate, was longer than that of non-porous substrates. On porous substrates other than non-woven masks, the amount of virus recovered quickly decreased, and then remained at a low level. Representative substrates were tested with SARS-CoV-2. The decrease in the amount of infectious virus recovered was similar to that of BCoV, although that of SARS-CoV-2 was more rapid. RNA derived from SARS-CoV-2 was also detected using real-time PCR, and it remained on surfaces much longer than infectious virus, on all substrates. Therefore, it is important to measure the viral titer to avoid the overestimation of infectious virus contamination in the environments. Our results suggest that the surface structure was not directly related to viral survivability.
Subject(s)
COVID-19 , Coronavirus, Bovine , Aerosols , Humans , Masks , SARS-CoV-2ABSTRACT
The most common diagnostic method used for coronavirus disease-2019 (COVID-19) is real-time reverse transcription polymerase chain reaction (PCR). However, it requires complex and labor-intensive procedures and involves excessive positive results derived from viral debris. We developed a method for the direct detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs, which uses matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-ToF MS) to identify specific peptides from the SARS-CoV-2 nucleocapsid phosphoprotein (NP). SARS-CoV-2 viral particles were separated from biological molecules in nasopharyngeal swabs by an ultrafiltration cartridge. Further purification was performed by an anion exchange resin, and purified NP was digested into peptides using trypsin. The peptides from SARS-CoV-2 that were inoculated into nasopharyngeal swabs were detected by MALDI-ToF MS, and the limit of detection was 106.7 viral copies. This value equates to 107.9 viral copies per swab and is approximately equivalent to the viral load of contagious patients. Seven NP-derived peptides were selected as the target molecules for the detection of SARS-CoV-2 in clinical specimens. The method detected between two and seven NP-derived peptides in 19 nasopharyngeal swab specimens from contagious COVID-19 patients. These peptides were not detected in four specimens in which SARS-CoV-2 RNA was not detected by PCR. Mutated NP-derived peptides were found in some specimens, and their patterns of amino acid replacement were estimated by accurate mass. Our results provide evidence that the developed MALDI-ToF MS-based method in a combination of straightforward purification steps and a rapid detection step directly detect SARS-CoV-2-specific peptides in nasopharyngeal swabs and can be a reliable high-throughput diagnostic method for COVID-19.