Six of ANSES's nine laboratories, working in the fields of animal health, food and hydrology, took part in the COVRIN project, which was funded by the "One Health European Joint Programme (EJP)", coordinated by ANSES. Launched in the midst of the COVID-19 pandemic, the project sought to gain a better understanding of the factors behind the emergence and spread of SARS-CoV-2 and build models to better assess the conditions of virus transmission. It was coordinated by Wageningen Bioveterinary Research (WBVR) in the Netherlands and the University of Surrey in England.
Multiple coronaviruses in animals
Animal coronaviruses are good models for studying human coronaviruses, because they carry multiple viruses in this family, some of which have been known for a long time. "The first coronavirus was discovered in poultry in the 1930s," observes Paul Brown of the Ploufragan-Plouzané-Niort Laboratory. The presence of several coronaviruses in the same individual implies that recombination is possible, i.e. the exchange of genes between viruses. This ability increases the risk of a novel virus emerging, for example one with a greater ability to infect a new species or with new characteristics that could affect the immune system of the infected animal or human. One of the project's objectives was to assess the possibility of recombination between coronaviruses and estimate the frequency of these events, using pig coronaviruses as a model.
Work on the ability of coronaviruses to jump from one species to another
Scientists at the Ploufragan Laboratory also examined whether coronaviruses usually detected in one species could infect related species. To do this they focused on the three main coronaviruses affecting poultry: avian infectious bronchitis virus (which mainly affects hens), turkey coronavirus and guinea fowl enteric coronavirus. All three viruses have a comparable genetic structure, apart from the gene of the virus's "spike" (S) surface protein, which allows the virus to enter the host cell. To find out how this difference affects their infectivity, the scientists inoculated groups of hens with the three viruses, and planned to do the same with guinea fowl and turkeys. Their results have not yet been published.
Still on the issue of the virus jumping from one species to another, the Nancy Laboratory for Rabies and Wildlife undertook to test coronavirus transmission from one wildlife species to another. The team used the hedgehog coronavirus as a model. "This is a very common coronavirus, carried by about 50% of hedgehogs brought to animal health centres" explains Élodie Monchatre-Leroy, Director of the laboratory. The first step was to isolate the virus in order to test its ability to infect other animal species. This proved to be easier said than done, as this coronavirus, found in the digestive tract and excrement, is difficult to isolate and culture. Work is still in progress to achieve this.
Improving detection of coronaviruses in wildlife
As COVID-19 has shown, when viruses carried by wild animals are transmitted to humans, they can potentially cause epidemics. Detecting viruses circulating in wildlife is therefore essential to try and pre-empt those posing an emerging risk. However, it is important to be able to detect them reliably. Some substances, such as blood or stools, contain inhibitors that prevent PCR tests from being performed correctly, making it difficult to detect viruses from animal carcasses. Evelyne Picard-Meyer, from the Nancy Laboratory for Rabies and Wildlife, has therefore improved the detection test by using "housekeeping genes": "these are genes that are found in all cells," she explains. "If they are detected, then the PCR analysis is valid. It can therefore be concluded that if the virus detection test is negative, the animal is not a carrier. Otherwise, it means that the sample is too damaged to be included in the monitoring statistics."
At the ANSES Lyon Laboratory, Viviane Hénaux was involved in documenting SARS-CoV-2 monitoring activities in pets, livestock and wildlife. The purpose was to compile monitoring data collected from April 2020 to December 2021 in each participating country. These data were then used to build models of the risk of virus transmission from animals to humans. The Laboratory for Rabies and Wildlife provided the data it had collected from bats and pets as part of another project.
Study of a deadly coronavirus in cats
A common coronavirus in cats can cause a deadly disease, feline infectious peritonitis. The genetic sequences of the circulating strains vary. The Virology Joint Research Unit, which is a shared venture between the ANSES Laboratory for Animal Health, the Alfort National Veterinary School and INRAE, conducted a study on the variability of these sequences, in order to determine whether some are linked to symptoms and higher mortality.
The unexpected ability of SARS-CoV-2 to survive freezing
In addition to the risk of transmission through animals, the possibility of SARS-CoV-2 being spread through contact with the environment or sewage has also been raised. "To consider these alternative routes of entry, it is necessary to demonstrate the presence of viruses able to infect a human on surfaces or in water" explains Sandra Martin-Latil. This scientist from the Laboratory for Food Safety looked at plastic and stainless steel surfaces in contact with food. "The problem arose when, in the summer of 2020, cases were being reported in China even though the country was free of the virus," she recalls. "The assumption was that contamination had occurred through contact with plastic packaging from imported frozen products." This possibility had not previously been considered, because enveloped viruses such as coronavirus are usually too sensitive to remain infectious for very long on the surface of an object. It is now known that it takes a week at 4°C to reduce the amount of virus by 90% and that it is resistant for even longer at -20°C.
Virus spread through wastewater: an unresolved issue
As for contamination of water by SARS-CoV-2, it is already known that the virus genome can be detected in wastewater and sewage sludge. This is in fact used to monitor the evolution of the pandemic in the population. However, as pointed out by Ali Atoui, a researcher at the Nancy Laboratory for Hydrology, "just because a fragment of the virus is detected, doesn't mean it is infectious. To be sure, you need to isolate a whole virus." The laboratory participated in an analysis of published studies on the subject. So far, none of them has provided evidence of infectious viruses in water or sludge from sewage treatment plants. It should be noted that few studies on this subject have been carried out. This can be explained by the complexity of the methods employed, which need to be closely supervised and carried out in a containment level-3 laboratory, which is not available to all scientific teams. In addition, chemicals or micro-organisms present in environmental samples can bias the results and limit their relevance.
Not all of the questions asked in the COVRIN project could be answered, given the huge scale of this issue. Nevertheless, the project helped consolidate or initiate collaborations between European specialists and enabled discussions to be held on study methods and protocols. More of the project findings are expected in the coming months. Research on SARS-CoV-2 and coronaviruses is ongoing, with a view to improving knowledge and detection methods to better pre-empt any future pandemic.