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. Coronaviruses (CoVs) are enveloped RNA viruses with broad host tropism and documented capability to cross the species barrier to infect humans 5. The mammalian innate immune response controls CoV infection partly through the action of interferons (IFNs) 4,6,7. IFNs inhibit viral infection by inducing hundreds of genes, many of which encode antiviral effectors 8. The IFN-stimulated genes (ISGs) that inhibit CoV infection are not well

Type I interferons (IFN) comprise a family of cytokines that signal through a common cellular receptor to induce a plethora of genes with antiviral and other activities. Recombinant IFNs are used for the treatment of hepatitis C virus infection, multiple sclerosis, and certain malignancies. The capability of type I IFN to suppress virus replication and resultant cytopathic effects is frequently used to measure their bioactivity. However, these assays are time-consuming and require appropriate biosafety containment. In this study, an improved IFN assay is presented which is based on a recombinant vesicular stomatitis virus (VSV) replicon encoding two reporter proteins, firefly luciferase and green fluorescent protein. The vector lacks the essential envelope glycoprotein (G) gene of VSV and is propagated on a G protein-expressing transgenic cell line. Several mammalian and avian cells turned out to be susceptible to infection with the complemented replicon particles. Infected cells readily expressed the reporter proteins at high levels five hours post infection. When human fibroblasts were treated with serial dilutions of human IFN-β prior to infection, reporter expression was accordingly suppressed. This method was more sensitive and faster than a classical IFN bioassay based on VSV cytopathic effects. In addition, the antiviral activity of human IFN-λ (interleukin-29), a type III IFN, was determined on Calu-3 cells. Both IFN-β and IFN-λ were acid-stable, but only IFN-β was resistant to alkaline treatment. The antiviral activities of canine, porcine, and avian type I IFN were analysed with cell lines derived from the corresponding species. This safe bioassay will be useful for the rapid and sensitive quantification of multi-species type I IFN and potentially other antiviral cytokines.

Objectives: To validate the diagnostic accuracy of a Euroimmun SARS-CoV-2 IgG and IgA immunoassay for COVID-19. Methods: In this unmatched (1:2) case-control validation study, we used sera of 181 laboratoryconfirmed SARS-CoV-2 cases and 326 controls collected before SARS-CoV-2 emergence. Diagnostic accuracy of the immunoassay was assessed against a whole spike protein-based recombinant immunofluorescence assay (rIFA) by receiver operating characteristic (ROC) analyses. Discrepant cases between ELISA and rIFA were further tested by pseudo-neutralization assay. Results: COVID-19 patients were more likely to be male and older than controls, and 50.3% were hospitalized. ROC curve analyses indicated that IgG and IgA had high diagnostic accuracies with AUCs of 0.990 (95% Confidence Interval [95%CI]: 0.983-0.996) and 0.978 (95%CI: 0.967-0.989), respectively. IgG assays outperformed IgA assays (p¼0.01). Taking an assessed 15% inter-assay imprecision into account, an optimized IgG ratio cutoff > 2.5 displayed a 100% specificity (95%CI: 99-100) and a 100% positive predictive value (95%CI: 96-100). A 0.8 cutoff displayed a 94% sensitivity (95%CI: 88-97) and a 97% negative predictive value (95%CI: 95-99). Substituting the upper threshold for the manufacturer's, improved assay performance, leaving 8.9% of IgG ratios indeterminate between 0.8-2.5. Conclusions: The Euroimmun assay displays a nearly optimal diagnostic accuracy using IgG against SARS-CoV-2 in patient samples, with no obvious gains from IgA serology. The optimized cutoffs are fit for rulein and rule-out purposes, allowing determination of whether individuals in our study population have

Avian influenza viruses (AIV) raise worldwide veterinary and public health concerns due to their potential for zoonotic transmission. While infection with highly pathogenic AIV results in high mortality in chickens, this is not necessarily the case in wild birds and ducks. It is known that innate immune factors can contribute to the outcome of infection. In this context, retinoic acid-inducible gene I (RIG-I) is the main cytosolic pattern recognition receptor known for detecting influenza A virus infection in mammalian cells. Chickens, unlike ducks, lack RIG-I, yet chicken cells do produce type I interferon (IFN) in response to AIV infection. Consequently, we sought to identify the cytosolic recognition elements in chicken cells. Chicken mRNA encoding the putative chicken analogs of CARDIF and LGP2 (chCARDIF and chLGP2, respectively) were identified. HT7-tagged chCARDIF was observed to associate with mitochondria in chicken DF-1 fibroblasts. The exogenous expression of chCARDIF, as well as of the caspase activation and recruitment domains (CARDs) of the chicken melanoma differentiation-associated protein 5 (chMDA5), strongly activated the chicken IFN-␤ (chIFN-␤) promoter. The silencing of chMDA5, chCARDIF, and chIRF3 reduced chIFN-␤ levels induced by AIV, indicating their involvement in AIV sensing. As with mammalian cells, chLGP2 had opposing effects. While overexpression decreased the activation of the chIFN-␤ promoter, the silencing of endogenous chLGP2 reduced chIFN-␤ induced by AIV. We finally demonstrate that the chMDA5 signaling pathway is inhibited by the viral nonstructural protein 1. In conclusion, chicken cells, including DF-1 fibroblasts and HD-11 macrophage-like cells, employ chMDA5 for sensing AIV.

Canine distemper virus (CDV) and measles virus (MV) cause severe illnesses in their respective hosts. The viruses display a characteristic cytopathic effect by forming syncytia in susceptible cells. For CDV, the proficiency of syncytium formation varies among different strains and correlates with the degree of viral attenuation. In this study, we examined the determinants for the differential fusogenicity of the wild-type CDV isolate 5804Han89 (CDV 5804 ), the small-and large-plaque-forming variants of the CDV vaccine strain Onderstepoort (CDV OS and CDV OL , respectively), and the MV vaccine strain Edmonston B (MV Edm ). The cotransfection of different combinations of fusion (F) and hemagglutinin (H) genes in Vero cells indicated that the H protein is the main determinant of fusion efficiency. To verify the significance of this observation in the viral context, a reverse genetic system to generate recombinant CDVs was established. This system is based on a plasmid containing the full-length antigenomic sequence of CDV OS . The coding regions of the H proteins of all CDV strains and MV Edm were introduced into the CDV and MV genetic backgrounds, and recombinant viruses rCDV-H 5804 , rCDV-H OL , rCDV-H Edm , rMV-H 5804 , rMV-H OL , and rMV-H OS were recovered. Thus, the H proteins of the two morbilliviruses are interchangeable and fully functional in a heterologous complex. This is in contrast with the glycoproteins of other members of the family Paramyxoviridae, which do not function efficiently with heterologous partners. The fusogenicity, growth characteristics, and tropism of the recombinant viruses were examined and compared with those of the parental strains. All these characteristics were found to be predominantly mediated by the H protein regardless of the viral backbone used. Canine distemper virus (CDV) and Measles virus (MV) are closely related members of the Morbillivirus genus in theParamyxoviridae family in the order Mononegavirales (29). The disease caused by CDV in susceptible animals, like dogs and ferrets, strongly resembles the course of MV infection in humans and is characterized by fever, rash, and leukocytopenia. CDV frequently spreads in the central nervous system and can lead to different neuropathological alterations (48).The genome organizations of CDV and MV are very similar, with both consisting of single-stranded negative-sense RNA of 15,690 nucleotides (nt) (CDV vaccine strain Onderstepoort) or 15,894 nt (MV vaccine strain Edmonston B [MV Edm ]), respectively (30, 37). The genomic RNA that is tightly encapsidated by the nucleocapsid (N) protein serves as a template for transcription and replication by the viral polymerase (L) protein and its cofactor phosphoprotein (P). The N, P, and L proteins together with the viral RNA constitute the ribonucleoprotein (RNP) complex (36), which directs the sequential synthesis of capped and polyadenylated mRNAs from six transcription units or the replication of full-length encapsidated antigenomes (19). The viral envelope contains two integral memb...