To disinfect a surface, you can shine it with a beam of ultraviolet (UV) light, which is bluer than what the human eye sees. But which wavelength is best to specifically inactivate SARS-CoV-2, the virus that causes COVID-19? How much radiation is enough? Answering these questions requires scientists to overcome two major obstacles. First, they need to completely separate the virus from foreign substances in the environment. Second, they needed to irradiate the virus with a single wavelength of ultraviolet light at a time, with little variation in experimental setup between tests. A recent collaboration between the National Institute of Standards and Technology (NIST) and the National Center for Biodefense Analysis and Countermeasures (NBACC), a laboratory of the U.S. Department of Homeland Security’s Science and Technology Council, overcame both obstacles and completed perhaps the most thorough ever tested for how several different wavelengths of ultraviolet and visible light affect SARS-CoV-2. In a new paper published this week in Applied Optics, the collaborators describe their new system that projects a single wavelength of light onto a sample of the COVID-19 virus at a time in a security lab. The laboratory is classified as Biosafety Level 3 (BSL-3) and is designed to study microorganisms that can be lethal when inhaled. To date, their experiments have tested more ultraviolet and visible wavelengths than any other study on the virus that causes COVID-19. So, what is kryptonite for SARS-CoV-2? As it turned out, there was nothing special: the virus was as susceptible to ultraviolet light of the same wavelength as other viruses, such as the one that caused the flu. The most effective wavelengths are those in the “UVC” range between 222 and 280 nanometers (nm). UVC light (the full range from 200 to 280 nm) is shorter than the UVB wavelength (280 to 315 nm) that causes sunburn. The researchers also showed that the surrounding environment of the virus can have a protective effect on the virus. In this study, a smaller dose of ultraviolet light was required to place the virus in pure water than in simulated saliva, which contains salts, proteins, and other substances found in actual human saliva. Suspension of the virus in simulated saliva creates a situation similar to a real-life scenario of sneezing and coughing. This may allow the results to be more directly informative than previous studies. “I think one of the big contributions of this study is that we’re able to demonstrate that the kind of idealized outcomes that we see in most studies don’t always predict what happens when there’s a more realistic scenario at play,” Michael Schuit said NBACC. “When the virus is surrounded by materials such as simulated saliva, it reduces the efficacy of UV purification methods.” Manufacturers of UV disinfection equipment and regulatory agencies can use these results to help inform medical settings, aircraft, and even surfaces in liquids for how long they should be irradiated to achieve inactivation of the SARS-CoV-2 virus. NIST Fellow Cameron Miller said, “There is now a big push to bring UVC disinfection into the commercial environment. “In the long term, it is hoped that this study will develop standards and other methods to measure the UV dose required to inactivate SARS-CoV-2 and other harmful viruses.” The project builds on the NIST team’s earlier work with another collaborator on inactivating microorganisms in water. Depending on the wavelength, UV light destroys pathogens in different ways. Certain wavelengths damage the RNA or DNA of microorganisms, causing them to lose their ability to replicate. Other wavelengths can break down proteins, destroying the virus itself. Although the disinfection power of UV light has been known for more than a hundred years, research on UV disinfection has exploded over the past decade. One reason is that traditional UV light sources sometimes contain toxic substances such as mercury. Recently, the use of non-toxic LED lights as UV light sources has mitigated some of these issues. In this study, NIST’s collaborators collaborated with biologists at NBACC, and their research informed biodefense planning for biological threats such as anthrax and Ebola. “What NBACC was able to do was grow the virus, concentrate it, and then get rid of everything else,” Miller said. “We’re trying to get a clear message about how much light we need to inactivate the SARS-CoV-2 virus.” In this study, the team tested the virus in different suspensions. In addition to using saliva mimics, scientists also put the virus in water to see what happens in a “pure” environment, with no ingredients that can protect it. They tested their viral suspension as a liquid and dry droplets on a steel surface, which represented something that an infected person might sneeze or cough on. NIST’s job is to direct ultraviolet light from the laser onto the sample. They are looking for the dose needed to kill 90% of the virus. With this setup, the collaboration is able to measure the response of the virus to 16 different wavelengths, ranging from 222 nm, the very low end of the UVC, all the way to the middle of the visible wavelength range, which is 488 nm. The researchers included longer wavelengths because some blue light has been shown to have disinfecting properties. Irradiating a laser onto a sample in a safety lab is not an easy task. Researchers in the BSL-3 lab wear scrubs with respirators and bandanas. Leaving the lab requires taking a shower and then changing back into casual clothes. The team’s expensive lasers and other equipment had to go through quite strict sterilization procedures. “It’s a one-way door,” Miller said. “Anything that comes out of that lab has to be incinerate, autoclaved [heat sterilized], or chemically sterilized with hydrogen peroxide vapor. So we didn’t want to use a $120,000 laser. Instead, NIST researchers designed a system in which the lasers and some optics were located in a corridor outside the lab. They transmit the light through a 4-meter fiber optic cable that passes through a seal under the laboratory door. The negative pressure allows air to flow from the hallway into the lab and prevents anything from leaking out. The laser produces one wavelength at a time and is fully tunable so researchers can produce any wavelength they like. But because light bends at different angles depending on its wavelength, they have to create a prism system that changes the angle at which light enters the fiber so that it is aligned correctly. Changing the exit angle involves manually turning the knob they created to adjust the position of the prism. They tried to make it all as simple as possible with a minimal number of moving parts. “The equipment proposed by the NIST team allows us to quickly test a wide range of different wavelengths in very controllable and precise bands,” says Schuit. “If we were to try to achieve the same number of wavelengths without the system, we would have to use a bunch of different types of devices at the same time, each of which would produce a different width of the band. They will need different configurations, and there will be a lot of extra variables in the mix. “Manipulating light requires mirrors and lenses, but the researchers designed it to be used as little as possible, as each one results in a loss of UV intensity. For materials that must go into the lab to project light from fiber optics onto COVID virus samples, the team tried to use inexpensive parts. “We 3D print a lot of things,” says NIST physicist Steve Grantham, a key member of NIST’s Thomas Larason team. “So, there’s nothing really expensive, and it’s not a big deal if we don’t use it anymore.” Even communication between the laser area and the inside of the lab was difficult because people couldn’t come and go at will, so they used a wired intercom system. Despite the challenges, the system worked very well, especially considering they only had a few months to assemble it, Miller said. “There are a couple of areas where we might be able to improve, but I think our gains are going to be minimal,” Miller said. The NIST team plans to use the system for future research on other viruses and microorganisms that biologists in high-security labs may want to do. “When a virus or themWhen any pathogen of interest comes along, all we have to do is roll the laser system up, push the fiber there, and they’ll connect it to their projector system,” Miller said. “So now we’re ready for the next one.”