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The SARS-CoV-2 pandemic and its unprecedented global societal and economic disruptive impact has marked the third zoonotic introduction of a highly pathogenic coronavirus into the human population. Although the previous coronavirus SARS-CoV and MERS-CoV epidemics raised awareness of the need for clinically available therapeutic or preventive interventions, to date, no treatments with proven efficacy are available. The development of effective intervention strategies relies on the knowledge of molecular and cellular mechanisms of coronavirus infections, which highlights the significance of studying virus–host interactions at the molecular level to identify targets for antiviral intervention and to elucidate critical viral and host determinants that are decisive for the development of severe disease. In this Review, we summarize the first discoveries that shape our current understanding of SARS-CoV-2 infection throughout the intracellular viral life cycle and relate that to our knowledge of coronavirus biology. The elucidation of similarities and differences between SARS-CoV-2 and other coronaviruses will support future preparedness and strategies to combat coronavirus infections.In this Review, Thiel and colleagues discuss the key aspects of coronavirus biology and their implications for SARS-CoV-2 infections as well as for treatment and prevention strategies.

Positive-sense RNA viruses hijack intracellular membranes that provide niches for viral RNA synthesis and a platform for interactions with host proteins. However, little is known about host factors at the interface between replicase complexes and the host cytoplasm. We engineered a biotin ligase into a coronaviral replication/transcription complex (RTC) and identified >500 host proteins constituting the RTC microenvironment. siRNA-silencing of each RTC-proximal host factor demonstrated importance of vesicular trafficking pathways, ubiquitin-dependent and autophagy-related processes, and translation initiation factors. Notably, detection of translation initiation factors at the RTC was instrumental to visualize and demonstrate active translation proximal to replication complexes of several coronaviruses. Collectively, we establish a spatial link between viral RNA synthesis and diverse host factors of unprecedented breadth. Our data may serve as a paradigm for other positive-strand RNA viruses and provide a starting point for a comprehensive analysis of critical virus-host interactions that represent targets for therapeutic intervention. Coronaviruses can infect the nose and throat and are a main cause of the common cold. Infections are usually mild and short-lived, but sometimes they can turn nasty. In 2002 and 2012, two dangerous new coronaviruses emerged and caused diseases known as SARS and MERS. These viruses caused much more serious symptoms and in some cases proved deadly. The question is, why are some coronaviruses more dangerous than others? Scientists know that the body's response to virus infection can make a difference to whether someone had mild or severe disease. So, to understand why some coronaviruses cause a cold and others kill, they also need to learn how people react to virus infection. Coronaviruses hijack membranes inside cells and turn them into virus factories. Within these factories, the viruses build molecular machinery called replicase complexes to copy their genetic code, which is needed for the next generation of virus particles. The viruses steal and repurpose proteins from their host cell that will assist in the copying process. However, scientists do not yet know which host proteins are essential for the virus to multiply. So, to find out, V’kovski et al. developed a way to tag any host protein that came near the virus factories. The new technique involved attaching an enzyme called a biotin ligase to the replicase complex. This enzyme acts as a molecular label gun, attaching a chemical tag to any protein that comes within ten nanometres. The label gun revealed that more than 500 different proteins come into contact with the replicase complex. To find out what these proteins were doing, the next step was to switch off their genes one by one. This revealed the key cell machinery that coronaviruses hijack when they are replicating. It included the cell's cargo transport system, the waste disposal system, and the protein production system. Using these systems allows the viruses to copy their genetic code next to machines that can turn it straight into viral proteins. These new results provide clues about which proteins viruses actually need from their host cells. They also do not just apply to coronaviruses. Other viruses use similar strategies to complete their infection cycle. These findings could help researchers to understand more generally about how viruses multiply. In the future, this knowledge could lead to new ways to combat virus infections.

Reverse genetics has been an indispensable tool to gain insights into viral pathogenesis and vaccine development. The genomes of large RNA viruses, such as those from coronaviruses, are cumbersome to clone and manipulate in Escherichia coli owing to the size and occasional instability of the genome 1 – 3 . Therefore, an alternative rapid and robust reverse-genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform to genetically reconstruct diverse RNA viruses, including members of the Coronaviridae , Flaviviridae and Pneumoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples or synthetic DNA, and these fragments were then reassembled in one step in Saccharomyces cerevisiae using transformation-associated recombination cloning to maintain the genome as a yeast artificial chromosome. T7 RNA polymerase was then used to generate infectious RNA to rescue viable virus. Using this platform, we were able to engineer and generate chemically synthesized clones of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 4 , which has caused the recent pandemic of coronavirus disease (COVID-19), in only a week after receipt of the synthetic DNA fragments. The technical advance that we describe here facilitates rapid responses to emerging viruses as it enables the real-time generation and functional characterization of evolving RNA virus variants during an outbreak. A yeast-based synthetic genomics platform is used to reconstruct and characterize large RNA viruses from synthetic DNA fragments; this technique will facilitate the rapid analysis of RNA viruses, such as SARS-CoV-2, during an outbreak.

Zoonotic coronaviruses (CoVs) are substantial threats to global health, as exemplified by the emergence of two severe acute respiratory syndrome CoVs (SARS-CoV and SARS-CoV-2) and Middle East respiratory syndrome CoV (MERS-CoV) within two decades 1 – 3 . Host immune responses to CoVs are complex and regulated in part through antiviral interferons. However, interferon-stimulated gene products that inhibit CoVs are not well characterized 4 . Here, we show that lymphocyte antigen 6 complex, locus E (LY6E) potently restricts infection by multiple CoVs, including SARS-CoV, SARS-CoV-2 and MERS-CoV. Mechanistic studies revealed that LY6E inhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in immune cells were highly susceptible to a murine CoV—mouse hepatitis virus. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic immune cells, higher splenic viral burden and reduction in global antiviral gene pathways. Accordingly, we found that constitutive Ly6e directly protects primary B cells from murine CoV infection. Our results show that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo—knowledge that could help inform strategies to combat infection by emerging CoVs. Here, the authors identify lymphocyte antigen 6E (LY6E) as a coronavirus (CoV) restriction factor that prevents infection of B cells and dendritic cells. LY6E inhibits both human and mouse CoV entry into cells by interfering with viral spike protein-mediated membrane fusion. It facilitates an antiviral immune response that prevents liver disease and reduces death in the mouse model of MHV-A59 CoV infection.

Since its emergence in December 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally and become a major public health burden. Despite its close phylogenetic relationship to SARS-CoV, SARS-CoV-2 exhibits increased human-to-human transmission dynamics, likely due to efficient early replication in the upper respiratory epithelium of infected individuals. Since different temperatures encountered in the human upper and lower respiratory tract (33°C and 37°C, respectively) have been shown to affect the replication kinetics of several respiratory viruses, as well as host innate immune response dynamics, we investigated the impact of temperature on SARS-CoV-2 and SARS-CoV infection using the primary human airway epithelial cell culture model. SARS-CoV-2, in contrast to SARS-CoV, replicated to higher titers when infections were performed at 33°C rather than 37°C. Although both viruses were highly sensitive to type I and type III interferon pretreatment, a detailed time-resolved transcriptome analysis revealed temperature-dependent interferon and pro-inflammatory responses induced by SARS-CoV-2 that were inversely proportional to its replication efficiency at 33°C or 37°C. These data provide crucial insight on pivotal virus–host interaction dynamics and are in line with characteristic clinical features of SARS-CoV-2 and SARS-CoV, as well as their respective transmission efficiencies.

Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I). This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU) activity is key to prevent early induction of double-stranded RNA (dsRNA) host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis-within the replicase complex-suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses.

Since the emergence of coronavirus disease (COVID-19) in late 2019, domestic cats have been demonstrated to be susceptible to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) under natural and experimental conditions. As pet cats often live in very close contact with their owners, it is essential to investigate SARS-CoV-2 infections in cats in a One-Health context. This study reports the first SARS-CoV-2 infection in a cat in a COVID-19-affected household in Switzerland. The cat (Cat 1) demonstrated signs of an upper respiratory tract infection, including sneezing, inappetence, and apathy, while the cohabiting cat (Cat 2) remained asymptomatic. Nasal, oral, fecal, fur, and environmental swab samples were collected twice from both cats and analyzed by RT-qPCR for the presence of SARS-CoV-2 viral RNA. Both nasal swabs from Cat 1 tested positive. In addition, the first oral swab from Cat 2 and fur and bedding swabs from both cats were RT-qPCR positive. The fecal swabs tested negative. The infection of Cat 1 was confirmed by positive SARS-CoV-2 S1 receptor binding domain (RBD) antibody testing and neutralizing activity in a surrogate assay. The viral genome sequence from Cat 1, obtained by next generation sequencing, showed the closest relation to a human sequence from the B.1.1.39 lineage, with one single nucleotide polymorphism (SNP) difference. This study demonstrates not only SARS-CoV-2 infection of a cat from a COVID-19-affected household but also contamination of the cats’ fur and bed with viral RNA. Our results are important to create awareness that SARS-CoV-2 infected people should observe hygienic measures to avoid infection and contamination of animal cohabitants.

Bovine viral diarrhoea virus (BVDV) and related ruminant pestiviruses occur worldwide and cause considerable economic losses in livestock and severely impair animal welfare. Switzerland started a national mandatory control programme in 2008 aiming to eradicate BVD from the Swiss cattle population. The peculiar biology of pestiviruses with the birth of persistently infected (PI) animals upon in utero infection in addition to transient infection of naïve animals requires vertical and horizontal transmission to be taken into account. Initially, every animal was tested for PI within the first year, followed by testing for the presence of virus in all newborn calves for the next four years. Prevalence of calves being born PI thus diminished substantially from around 1.4% to <0.02%, which enabled broad testing for the virus to be abandoned and switching to economically more favourable serological surveillance with vaccination being prohibited. By the end of 2020, more than 99.5% of all cattle farms in Switzerland were free of BVDV but eliminating the last remaining PI animals turned out to be a tougher nut to crack. In this review, we describe the Swiss BVD eradication scheme and the hurdles that were encountered and still remain during the implementation of the programme. The main challenge is to rapidly identify the source of infection in case of a positive result during antibody surveillance, and to efficiently protect the cattle population from re-infection, particularly in light of the endemic presence of the related pestivirus border disease virus (BDV) in sheep. As a consequence of these measures, complete eradication will (hopefully) soon be achieved, and the final step will then be the continuous documentation of freedom of disease.

The first records of smallpox and rabies date back thousands of years and foot-and-mouth disease in cattle was described in the 16th century. These diseases stood out by their distinct signs, dramatic way of transmission from rabid dogs to humans, and sudden appearance in cattle herds. By contrast, infectious diseases that show variable signs and affect few individuals were identified only much later. Bovine viral diarrhea (BVD), endemic in cattle worldwide, was first described in 1946, together with the eponymous RNA virus as its cause. There is general agreement that BVD was not newly emerging at that time, but its history remains unknown. A search for associations between the nucleotide sequences of over 7,000 BVD viral strains obtained during a national campaign to eradicate BVD and features common to the hosts of these strains enabled us to trace back in time the presence of BVD in the Swiss cattle population. We found that animals of the two major traditional cattle breeds, Fleckvieh and Swiss Brown, were infected with strains of only four different subgenotypes of BVDV-1. The history of these cattle breeds and the events that determined the current distribution of the two populations are well documented. Specifically, Fleckvieh originates from the Bernese and Swiss Brown from the central Alps. The spread to their current geographic distribution was determined by historic events during a major expansion of the Swiss Confederation during the 15th and 16th centuries. The association of the two cattle populations with different BVD viral subgenotypes may have been preserved by a lack of cattle imports, trade barriers within the country, and unique virus-host interactions. The congruent traces of history in the distribution of the two cattle breeds and distinct viral subgenotypes suggests that BVD may have been endemic in Switzerland for at least 600 years.

Bovine viral diarrhea virus (BVDV), a pestivirus in the family Flaviviridae, is a major livestock pathogen. Horizontal transmission leads to acute transient infections via the oronasal route, whereas vertical transmission might lead to the birth of immunotolerant, persistently infected animals. In both cases, BVDV exerts an immunosuppressive effect, predisposing infected animals to secondary infections. Erns, an immunomodulatory viral protein, is present on the envelope of the virus and is released as a soluble protein. In this form, it is taken up by cells and, with its RNase activity, degrades single- and double-stranded (ds) RNA, thus preventing activation of the host’s interferon system. Here, we show that Erns of the pestiviruses BVDV and Bungowannah virus effectively inhibit dsRNA-induced IFN synthesis in well-differentiated airway epithelial cells cultured at the air–liquid interface. This activity was observed independently of the side of entry, apical or basolateral, of the pseudostratified, polarized cell layer. Virus infection was successful from both surfaces but was inefficient, requiring several days of incubation. Virus release was almost exclusively restricted to the apical side. This confirms that primary, well-differentiated respiratory epithelial cells cultured at the air–liquid interface are an appropriate model to study viral infection and innate immunotolerance in the bovine respiratory tract. Furthermore, evidence is presented that Erns might contribute to the immunosuppressive effect observed after BVDV infections, especially in persistently infected animals.