The mixture is prepared with detergent and water or kitchen oil, to create films that inactivate the avian virus, similar to that of Covid-19. Fiocruz will begin testing the film against Sars-Cov-2, the causative agent of Covid-19. If effective against the new coronavirus, the films will be a simple and accessible solution to sanitize hands and surfaces. Brazilian researchers from three institutions found a new use for an old cleaning product, the detergent. When tested, the substances based on this product have shown to be promising in forming an antiviral film over hands and inanimate surfaces, capable of inactivating in a few minutes a coronavirus similar to that of Covid-19, with a more lasting effect than other sanitizers such as alcohol, which evaporates faster. The formula is simple, of low cost, and easy to prepare. For use on hands, the detergent was mixed with a small amount of vegetable oil to create a more flexible film and also to reduce skin dehydration. For inanimate surfaces, the detergent was diluted in water. In both cases, the virus was inactivated thousands of times in up to 10 minutes. As a biological material, the study used the avian coronavirus, ACoV, since it can be grown in a laboratory with a lower biosafety level than that required to work with the new coronavirus, it does not cause diseases in humans, and it is chemically and morphologically similar to Sars-SoV-2, the causative agent of Covid-19. Both films were effective against the ACoV coronavirus, confirming their use as a possible alternative to prevent the disease, especially among the least favored part of the population of developing countries, without access to commercial sanitizers. The results obtained in the study also open the way for several alternative uses and applications, aiming to mitigate the contamination by other pathogenic microorganisms. The research involved three units of Embrapa: Embrapa Instrumentation and Embrapa Southeast Livestock, located in São Carlos, in the state of São Paulo; and Embrapa Swine & Poultry, located in Concórdia, in the state of Santa Catarina. The study was also supported by the Laboratory of Compared and Environmental Virology of Oswaldo Cruz Foundation (Fiocruz), in Rio de Janeiro, as well as by Instituto de Zootecnia (IZ) and by a doctoral student of Sao Carlos Institute of Chemistry of University of Sao Paulo (IQSC-USP). “We believe that the antiviral activity of the film is due, in great part, to the biocidal action of the surfactants – the main constituents of the formulas of cleaning and personal hygiene products – present in the detergent”, points out Embrapa’s researcher Luiz Alberto Colnago, coordinator of the study, together with researcher Lucimara Aparecida Forato, and advisor of doctoral student Cirlei Igreja do Nascimento Mitre. According to those involved in the study, several biocidal agents are capable of inactivating in minutes the coronavirus on inanimate surfaces. However, these products are quick to evaporate, flammable, toxic, or soon ineffective, allowing the cleaned surfaces to become a new source of transmission after being contaminated again. The proposal is to develop detergent-based films that can be applied to inanimate surfaces, such as doorknobs, handrails, trash cans, glass doors, mirrors, among others, and to hands, as an effective alternative to prevent Sars-CoV-2 from spreading, especially in places where the known prophylactic measures are not easily accessible. According to virologist José Paulo Gagliardi Leite, director of Fiocruz, the results of the assays are promising, with interesting and important socioeconomic perspectives due to the low cost of the formula and its possible use in inactivating viruses that cause other diseases with high mortality rates. Biologist Rodrigo Giglioti, of Instituto de Zootecnia, evaluated the reliability of the data obtained in the trials with the antiviral film, using a statistical model as a control. He pointed out that the used model (nonparametric) allowed comparing the performed analyses, confirming the robustness of their results for these type of data. The formula that worked against the avian virus The application of the detergent, combined with kitchen oil or water, depending on its proposed use, was effective in inactivating the avian coronavirus, CoV. However, although this virus is from the same family as that of the causative agent of Covid-19, the formula still needs to be tested by Fiocruz against Sars-CoV-2. In a laboratory experiment, the researchers prepared the formula to be applied to hands using 20 spoons of detergent for one of oil, which was added as a plasticizer to promote flexibility and reduce skin dryness. The film for inanimate surfaces was prepared by adding one cup of diluted detergent to half a cup of water. In both cases, the ingredients should be mixed to obtain a homogeneous solution before being applied. The protective film for the hands was emulsified until a white solution was formed, to be applied as a lotion and reapplied as needed. The film for surfaces was applied using a cleaning cloth or kitchen sponge. Photo: Adriana Ibelli The next phase of the research will be carried out at Fiocruz, in a biosafety level 3 (BSL-3) laboratory – an environment destined for the manipulation of extremely pathogenic agents. “The originality of the idea of researcher Colnago is very important, particularly due to the characteristics of the film, which is not flammable and does not pose risks to its users. Although we still have to test it against the new coronavirus, Sars-CoV-2, very promising results are already expected”, declares the director of Fiocruz. He informs that there is no set date to start testing at Fiocruz, since the institution is still preparing the BSL-3 laboratory to carry out the trials, but he believes that it should be soon. Embrapa’s researcher highlighted the importance of the partnership among the institutions involved in the study. “This is how it should be done, by seeking collaborations”, he points out. Remains stable for days According to the World Health Organization (WHO), current evidence suggests that the most frequent mode of transmission of the virus is from person to person and through contaminated surfaces, where the virus remains active from hours to days. Among the procedures recommended by the health authorities are washing hands with soap and sanitizing them with alcohol to prevent transmission by contact. However, these measures are not enough. Colnago explains that recently reviewed studies have shown that the coronavirus remains infectious for up to several days on inanimate surfaces made of plastic, such as PVC, as well as on other materials, including: Teflon, silicone, latex, disposable aprons, steel, aluminum, wood, paper, glass, and ceramics. According to Embrapa’s researcher, the dried film can inactivate the virus from hours to days, contributing to reduce the use of dangerous chemical products and the cleaning frequency with these compounds, which are usually dissolved in water, generating a large amount of toxic residues that can contaminate ecosystems. The detergents, according to Embrapa’s specialist in immunology and virology, Cíntia Hiromi Okino, have high concentrations of surfactants, which have the capacity of removing fat from water and are capable of modifying the viral structural protein. “If the surfactants modify the structure of the viral protein that attaches to human cells, they are capable of reducing its infection capacity. Since the coronavirus is surrounded by an envelope, the substances present in the detergent can also act on this lipid membrane, inactivating the virus”, explains the specialist. The surfactants of the detergent used in the experiment are: sodium dodecyl sulfate (SDS), also called sodium lauryl ether sulfate (SLES) and sodium lauryl sulfate (SLS), a known protein denaturing agent – which alters the structure of a substance or of a product –, as well as linear alkylbenzene sulfonate (LAS). According to Colnago, the results of the evaluation of the chemical stability of the films, tested through high-resolution nuclear magnetic resonance spectroscopy (MRS), showed that the SDS and LAS compounds are highly stable in the dried film on plastic surfaces for up to, at least, seven days. “The chemical stability of the film indicates that the properties of its compounds, including antiviral activity, will be preserved for the same amount of time, maintaining a residual protection effect, still to be tested”, he adds. The avian coronavirus is the viral challenge model for the research The objective of the research was to investigate the chemical stability of two detergent-based films and their capacity of inactivating, in up to a million times and in 10 minutes, the avian coronavirus, ACoV. The study, supported by São Paulo Research Foundation (Fapesp), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Embrapa, began in February when Covid-19 was spreading around the world and had already reached Europe. Among the measures recommended by the health authorities to minimize the spread of the virus were sanitizing hands with alcohol or sanitizer gel (70% alcohol) and washing hands with soap and water. However, the effectiveness of these procedures was only confirmed and published at the end of March 2020. “Due to this recommendation, in Brazil and around the world, there was a great search for 70% alcohol, especially in the form of gel, which is less flammable than the liquid one. Consequently, there was a high and sudden demand for this product, which practically disappeared from the market, causing its prices to rise”, the researcher recalls. He says that it was at that time that he had the idea of developing an antiviral agent that could be applied to hands, of easy access and low cost, and with a more lasting activity, preventing contamination for a longer period. “The first idea was to change the way of washing ours hands with soap. Instead of washing and rinsing, I thought about skipping the last step, to keep a layer of soap over the skin, which could continue inactivating the virus for a longer time”, he clarifies. Colnago and Forato only found patents for antiviral films in the form of lotion and based on chemical products, tested on the flu and cold viruses. “It is well known that the active components (called surfactants) of soaps, detergents, and other cleaning and hygiene products have antiviral properties, effective against several human and animal viruses”, explains Colnago. With this in mind, they chose the house-cleaning detergent, a product with a high concentration of surfactants, but whose effectiveness still needed to be proven experimentally for it to be endorsed. The researcher adds that Fiocruz had already shown interest in testing the film. However, the institution still needed to prepare the procedures to grown the new coronavirus in laboratory conditions. For this reason, the researchers decided to use a virus similar to that of Covid-19. Okino suggested using the avian coronavirus as a viral challenge model for the trials. “I proposed isolating the avian coronavirus in embryonated eggs and detecting it through real-time PCR, which is also the main method used to diagnose Covid-19. After that, the team of Embrapa Swine & Poultry improved, optimized, and performed the experimental protocols”, clarifies the specialist. Colnago explains that using a microorganism similar to the targeted one is a common practice in science. “The viral challenge model is a procedure used in researches when the virus is extremely dangerous, the laboratory does not have the required biosafety level, or there is no knowledge of how to grow it. The type of virus used is called a ‘surrogate virus’”, he points out. According to Okino, the two protective films were applied to plastic surfaces until completely dried. Then, the avian coronavirus was added at three different doses (high, medium, and low) for 10 minutes. The virus was recovered and inoculated in embryonated chicken eggs, which act as a culture medium and allow the multiplication of the organism. During seven days, the eggs were evaluated daily, checking for mortality or characteristic lesions induced by the virus on the embryos, that is, looking for indicators of the presence of the active virus. The viral load in the biological material of the embryos was also estimated. The use of a similar virus The main advantage of using ACoV as a model is the nonzoonotic nature of this virus, that is, the fact that it does not infect humans, only poultry, and can, therefore, be grown in a laboratory with lower biosafety levels than those required for the new coronavirus. Another advantage is that the new detergent-based film can be applied to inanimate surfaces without removing prior layers, reducing the need of water use and making it harder to contaminate the environment. ACoV was the first coronavirus to be identified. In 1937, it was found in chicken with respiratory disease. It is one of the most studied viruses of the last decades due to the great economic impact it causes. “The avian coronavirus belongs to the same family as Sars-CoV-2, which is Coronaviridae. Both of them have a similar structural and chemical composition. Moreover, a similar resistance was observed for the two viruses. Under 56 degrees, Sars-CoV-2 is viable for up to 10 minutes and was inactivated after 30 minutes, whereas nine lines of the avian coronavirus were inactivated after 15 minutes. Besides, both are stable in a wide pH range (3–10) under ambient temperature”, the specialist compares. At Embrapa Swine & Poultry, a vaccine strain of the avian coronavirus, ACoV – popularly known as infectious bronchitis virus of chicken –, from the Collection of Microorganisms of Interest for Poultry and Swine Culture (CMISEA), was exposed to a surface that had been covered with the homemade film, under previously evaluated and determined conditions. The intense work at the laboratory was carried out by researcher Iara Maria Trevisol, as well as by assistant Tânia Alvina Potter Klein and analyst Daiane VossRech, with the support from a distance of Cíntia Okino. Trevisol works in the classic avian virology area and has experience in similar trials for veterinary input. “To evaluate the neutralization of viral activity, the material was inoculated in an adequate substrate and the viral load was quantified using the real-time PCR molecular technique. This method is widely known and adopted in researches with animal or human viruses. With this procedure, we were able to check the effectiveness of the product, comparing the changes in the substrate to those caused only by the virus (positive control) or by its absence (negative control)”, explains Trevisol. The testing phase of the avian coronavirus was concluded in July, but the research team continues working on this project, testing the films against other viruses. Additional analyses will be carried out to determine the life span of the films when manually applied to different inanimate materials and using different viral challenge models. “The proposal of researcher Luiz Colnago opens the possibility of Embrapa’s Unit collaborating even more in the fight against the Sars-CoV-2 pandemics. Although our researches in the avian virology laboratory focus on the virus that causes diseases in chicken, we expect the results obtained for the avian coronavirus may be a model for the human coronavirus, since both belong to the same family”, informs the researcher. Challenge It takes approximately ten days for each trial to be prepared, the experiments executed, and the data evaluated. The first trial showed that the film was working, but that it was also being toxic to the embryo, which made it difficult to reach conclusions about its effectiveness. Colnago reports that only in the seventh experiment, about 70 days after the first, did the film reach conditions in which it was not toxic to the embryos and was effective in inactivating ACoV, even at high doses ““In addition, we also evaluated the use of the detergent-based film without oil, which can be applied to inanimate surfaces such as plastics, metals, glass, doorknobs, handrails, trash cans, glass doors, and mirrors, to reduce the survival time of the virus from days to minutes”, he concludes. Photo: Adriana Ubelli