A startling paper by a team of French scientists published in the preprint journal bioRxiv in April 2020 suggests that the novel coronavirus is capable of surviving at high temperatures.
How was the study done?
The team led by Professor Remi Charrel at the Aix-Marseille University in southern France aimed to find out at what temperature the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could survive. They subjected the viral culture within the host cells, from an African green monkey kidney, to slow heating for an hour, reaching a temperature of 60 degrees Celsius. The cells used for culture are standard for viral activity. The viral strain used came from a patient with COVID-19 in Berlin, Germany.
Novel Coronavirus SARS-CoV-2: This scanning electron microscope image shows SARS-CoV-2 (round gold objects) emerging from the surface of cells cultured in the lab. SARS-CoV-2, also known as 2019-nCoV, is the virus that causes COVID-19. The virus shown was isolated from a patient in the U.S. Credit: NIAID-RML
The study included 10 protocols for virus inactivation, including 3 lysis buffers and 6 heat inactivation protocols on the supernatant of SARS-CoV-2 cultures.
The infected cells were then placed within two different types of tubes, to mimic exposure to clean and dirty environments in real life. The ‘dirty’ tube contained animal protein contaminants, the aim being to produce a setting somewhat like that found in the real world, where an oral swab, for instance, may contain traces of other proteins.
When they tested the residual culture material for any surviving virus, they found that the infectivity was markedly reduced, but some strains could still replicate – enough to begin another round of infection. While the strains in the clean environment showed zero survival, some strains in the dirty environments survived.
They then went on to heat the culture to almost 100 degrees Celsius before they could confirm that the virus had been killed.
Why is this study important?
Testing for COVID-19 infection is happening the world over on a grand scale, due to the rapid spread of the pandemic. Some of these tests are being performed in laboratories with lower levels of biosecurity. Therefore, laboratory workers processing clinical samples can be exposed to the infectious SARS-CoV-2 virus.
SARS-CoV-2 direct diagnosis is based on RNA detection by RT-qPCR. The methods for nucleic acid (NA) extraction use buffers, which formulation intends to obtain high-quality NAs. They are not primarily developed for inactivation. Automated NA extraction is generally performed outside of biosafety cabinets, which demands that only non-infectious samples must be loaded. To achieve this objective, a prior inactivation step under appropriate biosafety conditions is an absolute requirement.
Previous studies have addressed the ability of lysis buffers added to the samples in the initial step of NA extraction to act as inactivation agents of several pathogenic viruses (including coronaviruses). However, discrepant results observed with dissimilar protocols led to controversial conclusions.
Very soon, widespread antibody testing will be used to determine if people have already been infected. This testing will require the use of blood samples, which again means that complete viral inactivation must be first accomplished.
The most common protocol for virus deactivation is the one-hour 60-degree Celsius heating technique. In combination with the use of Triton X-100, it has been recommended by the Centers for Disease Control and Prevention (CDC), and used in many laboratories to inactivate viruses with high case fatality ratios, like the Ebola virus.
However, the new study shows that this may be inadequate to kill all the virus particles in samples with a high viral load, the researchers say. If only a small amount of the virus is present, though, this protocol will kill a high percentage of the strains, causing almost complete inactivation.
The investigators found that when they heated the samples to higher temperatures, namely, 92 degrees Celsius for 15 minutes, viruses were denatured entirely, and the sample became non-infectious. However, this is not a feasible solution because, at such temperatures, the viral RNA becomes fragmented, causing the number of false negatives to rise.
A better way is to combine the more extended lower-heat protocol with chemical sterilization to improve laboratory safety while preserving the optimal efficiency of virus detection. The researchers recommend the use of buffers with the chemicals sodium-dodecyl-sulfate and Triton-X100 to obtain an almost complete reduction in the infectious virus load. This is equivalent to a less than 6-log drop in the viral load even with large amounts of the virus.
In the words of the authors, “The results presented in this study should help to choose the best-suited protocol for inactivation in order to prevent exposure of laboratory personnel in charge of direct and indirect detection of Sars-CoV-2 for diagnostic purpose.”
Evaluation of heating and chemical protocols for inactivating SARS-CoV-2, Boris Pastorino, Franck Touret, Magali Gilles, Xavier de Lamballerie, Remi N. Charrel, bioRxiv 2020.04.11.036855; doi: https://doi.org/10.1101/2020.04.11.036855