An experimental study involving Inaf also assessed the virucid effects of UV-C irradiation on Sars-Cov-2 virus, for different doses of illumination and virus concentrations, establishing the level of illumination that ensures both the inactivation of the virus and the inhibition of its replication. Media Inaf interviewed Andrea Bianco of the 'Inaf Astronomical Observatory of Brera'.
Andrea Bianco, researcher at the INAF Astronomical Observatory of Brera.
Thanks to a study by the National Institute of Astrophysics and University of Milan, carried out in collaboration with the National Cancer Institute of Milan and the Irccs Don Gnocchi Foundation of Milan, The virucid effects of UV-C irradiation on Sars-Cov-2 virus have been evaluated experimentally for different doses of illumination and virus concentrations. The experimental study has allowed to establish which level of illumination ensures both the inactivation of the virus and the inhibition of its replication. After a careful testing campaign conducted at the Sacco Hospital in Milan, researchers found that a UV-doseC of only 3.7 mj/cm2 is sufficient to achieve inactivation by a factor of one thousand on a sample whose virus density is comparable to that observed in the Sars-infectionCov-2, while complete inhibition of all viral concentrations was observed with 16.9 mj/cm2.
Media Inaf has joined Andrea Bianco of the Inaf Astronomical Observatory of Brera - first author of the study at the time available in a preprint on the international archive medrxiv, awaiting acceptance for publication - which told us some details of the experiment and helped to understand its results and their importance for the development of new sterilization methods to contain the infection with Sars-Cov-2.
What was the purpose of the measures presented in the study?
The goal was to understand the extent to which UV-C radiation is capable of inhibiting and inactivating replication of the Sars-Cov-2 virus. I make a premise: when you make these kinds of measurements, there are many variables to consider. For example, what form the virus is in: if it is deposited on a surface, if it is an aerosol that is depositing on a target, or if it is suspended in an aqueous medium that is the classic one in which the cells grow, and therefore is in a more favorable environment than a wooden or plastic surface. This is also evident from all the reading works and the results of the measurement can depend a lot on how the experiment was designed and performed. So much so that for the first Sars-Cov1, if you go to consider the data in the literature, you see that they are very dispersed: there are very low values for aerosols and higher values for organic components, such as blood. So, being able to do a "clean" experiment was one of the goals we had».
What did you do?
Here at the Astronomical Observatory in Brera, we designed an UV-C lamp so that it had an evenly lit and stable area over time, where we placed our sample, and we made these measurements. We performed a calibration of the intensity because, since it is necessary to provide a certain dose of UV-C rays, we must know the intensity and the time of irradiation. The intensity of the lamp was calibrated with a spectrum-radiometer: from the spectrum of the lamp, we selected the intensity of the peak in UV-C, which was what we were interested in. This was made possible thanks to the collaboration with the National Cancer Institute of Milan, which has calibrated and certified instrumentation to do so. We then did some tests in our optics laboratories and then it was taken to the laboratories of the University of Milan, at the Sacco Hospital in Milan, where all the tests on the virus were carried out. We instructed the operators on how to use the lamp and then of course all the tests were done by the staff, on the question of danger and regulation. Another thing we’ve been working on, very important, was to consider the UV-C absorption of the solution in which the virus was suspended. This is because if this solution partially absorbs light, the dose that actually reaches the virus is less than what we nominally sent to the sample. We found that the light that was being transmitted was about 70 percent, so we did this correction, along with other optical corrections that we have expertise on, so that we had a fairly accurate dose assessment, with a certain error due to the source, which has its own instability. We have considered all the variables and I believe that this is an important aspect of the tests carried out».
How was the experiment performed?
We chose three doses of UV-C rays, quite different from each other: 3.7, 16.9 and 84.4 mj/cm2, just to go and see, in these three conditions, how the virus behaved. In the biological counterpart, three concentrations of virus have been used: the intermediate concentration is the typical concentration in which the virus can be found in a sick person emitting water particles (aerosol of a sick person, emitted when, for example, sneezes), a much higher concentration, typical of an extremely ill person, and a very low concentration, typical of a surface that may have been mildly contaminated. So they were three representative conditions of very specific cases».
How was the presence of the virus assessed in the samples?
'I obviously have no specific competence in this area. It assessed how the virus replicated in the environment, in the supernatant - this is the measure we took as a reference - and also its intracellular concentration. Basically, you take the virus that’s been illuminated with UV-C and you put it on cells. After a certain time (24 hours, 48 hours, 6 days) we see if the virus has replicated. I can go and see the virus replicated, and so it’s around in the supernatant, in the culture fluid, or what’s inside the cells. These are two slightly different assessments, which give different biological information. The concentration of the virus is evaluated after certain times because the longer it is left to the virus, the easier it is to replicate in the cells, which means that it is still active».
What did you see?
There have been very distinct behaviors. At the lowest concentration, we had to wait six days for the virus to replicate and infect the cells, without being subjected to UV-C illumination. In the intermediate concentration, which we assumed as a reference, Even with the lowest dose of irradiation, after 24 hours we had a reduction of more than 1000. Waiting for more time, we have seen that the virus no longer grows back, that is, it is no longer able to replicate: it is said that it has been completely inhibited. While in the highest concentration, at the lowest dose of irradiation, at the beginning there is a fairly significant decrease in the amount of virus active but later rises, which means that the virus has been inactivated but the concentration is so high that it manages to restart (because it was not completely inhibited). When we gave him the second dose - 16.7 mj/cm2 - it was seen that it is sufficient to inhibit it completely: even after 72 hours it is not able to "regain forces". In this high concentration of the virus this aspect emerged: at low doses I manage to have a considerable decrease of the active virus but he is still able to infect the cells, so after some time he starts again with his activity»
What does that mean?
'We must always consider that we are assessing the virus which is, in this case, in a favourable environment, while if I have a virus which is deposited on a surface such as plastic, it is not such a favorable condition. But in the condition in which I have a high concentration in the cells, the lower dose is sufficient to kill it consistently (even by about a factor of 1000) but then the virus is able to regain vitality».
A factor of 1000 is a good result?
'The levels of abatement required in disinfection are always quite high: the factor 1000, which we obtain with the minimum dose UV-C, means having eliminated 99.9 percent of the virus which is an excellent result for many applications, but in some hospital settings it is not enough. You have to reach 99.99 percent, then 10 thousand. In these cases you will have to increase the UV-C dose provided. Generally, it depends on the application. The result we obtained is very positive especially because the UV-C dose is not very high. This means that in a disinfection system of this type, a few seconds of treatment may be enough to have a good disinfection of the surface using lamps of adequate power».
Will you perform the same measurements for UV-A and UV-B?
The measurements will be done in the coming weeks, both with UV-B and UV-A taking into account some considerations. It has been known for decades, since the practice and technology of disinfection on UV has spread, that the effectiveness of UV-C compared to UV-B is much greater, up to a factor of 1000. And even more than UV-A. This is especially true for viruses, because UV-C radiation is able to be absorbed directly by RNA - as in the case of this virus - or by DNA, in the case of other viruses, rather than by capsid proteins. There is therefore a direct modification of the vital structures of the virus, there are photoreactions that prevent the virus from surviving. If you go to see the absorption spectrum of RNA and DNA, you notice that it falls right into the UV-C. Then it falls into the UV-B and so you definitely need higher intensities to inhibit it. We started with UV-C because all disinfection systems on the market are based on UV-C lamps. What we used in this test is a mercury lamp, which are very important but at the same time, for environmental reasons, we try not to use them. The next step will be to try high power UV-C leds, currently proposed for the realization of plants or disinfection systems».
So you prefer UV-C disinfection?
«The UV-B aspect is more related to a disinfection aspect due to the Sun, as predicted by the models according to which the Sun has a disinfectant action. We also consider the fact that UV-B leds are difficult to produce at high power. If I had to choose a disinfection system I would still use a UV-C led for a matter of available technology. Different is the case for UV-A, for which there are Leds with very high power and efficiency. However, UV-A does not directly affect the virus. The mechanisms involved in this case are no longer endogenous: the radiation is not absorbed by the virus, which something happens. Maybe it is absorbed by another component that is in the environment where the virus is located, and at that point this component is activated that reacts with the virus itself».
So there could be a correlation between the effect of the Sun and the spread of the virus?
As I see them, such a correlation is more related to UV-B than to UV-A from the Sun. On the effects of UV-A on viruses, in the literature there is not much (different for bacteria and more complex structures that have cell walls with other components inside that absorb at higher wavelengths). The effects of solar UV-B have also been reported in the literature on viruses. So we can expect that prolonged solar radiation can have a disinfectant effect».
And by the way, do UV-Cs remain the most effective?
«If I fix the disinfection effectiveness of UV-C at 1 (254 nm), with UV-B we are at a factor of 100-1000, with UV-A we are at a factor of 10 thousand, 100 thousand, 1 million less effective. From my point of view, I certainly expect from the Sun the greatest contribution from UV-B. We will however make measurements with both UV-B and UV-A and actually we will also do them with the visible, because it has been seen (on bacteria and pathogenic microorganisms) that violet radiation at 405 nm can have an effect. Also in this case will be made lamps ad hoc».
Are these rays dangerous to man?
«Going from UV-A to UV-C, the danger for humans increases, in the sense that UV-C radiation causes mutations, thus increasing the likelihood of having skin tumors in a considerable way. So much so that, by law, the maximum dose of UV-C that one can take daily is very low. UV-B and UV-A are much less so, so much so that we tan, protecting ourselves with creams to avoid the risk of tumors and rashes to the skin. The risk is there but with much less probability».
Could these UV-C lamps be used in hospitals?
«UV-C systems have always been used for disinfecting surgeons' tools. It is a technology known for more than 50 years. What I do not explain is why a technology known for years, widespread and accepted at the regulatory level, has not been used immediately even simply for the disinfection of documents, accessories and objects of patients. Even with a limited investment. We hope that now it can find a marked diffusion mainly because being a physical and non-contact technique, independent from the operator, it is easier to use and allows to guarantee the levels of disinfection required. For example, here near the Observatory of Brera, in Merate, we are in contact with the hospital Valduce "Villa Beretta", a hospital known for rehabilitation and which also opened a department Covid-19, because all the equipment they will use for rehabilitative persons must be disinfected after each treatment. Having UV systems that, automatically, finish the treatment of a patient and before the next, can sanitize the equipment would be fundamental for safety issues and maintenance of the number of daily treatments».
Other applications? Precautions for use?
I imagine that these systems could be deployed in airport environments, in the wards and common areas of hospitals, in large-scale distribution, in toilets and in elevators. Where it’s reasonable to think that a person can contaminate the environment. Another important aspect of UV-C, being so energetic, is that they can also have effects on objects: plastic materials can discolor or crack. So we mustn’t exaggerate. It is important to design the UV-C lighting system for the specific application that you want to achieve, in order to maximize the effectiveness, trying to minimize the undesirable effects».
To learn more read the preprint of the medrxiv international archive "UV-C Irradiation is highly effective in inactivating and inhibiting SARS-Cov-2 replication" by Andrea Bianco, Mara Biasin, Giovanni Pareschi, Adalberto Cavalleri, Claudia Cavatorta, Claudio Fenizia, Paola Galli, Luigi Lessio, Manuela Lualdi, Edoardo Redaelli, Irma Saulle, Daria Trabattoni, Alessio Zanutta, Mario Clerici.
- Credits: Andrea Bianco
- Written by: Maura Sandri