The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the most pressing medical and socioeconomic challenge. Constituting important correlates of protection, the determination of virus-neutralizing antibodies (NAbs) is indispensable for convalescent plasma selection, vaccine candidate evaluation, and immunity certificates. In contrast to standard serological ELISAs, plaque reduction neutralization tests (PRNTs) are laborious, time-consuming, expensive, and restricted to specialized laboratories. To replace microscopic counting-based SARS-CoV-2 PRNTs by a novel assay exempt from genetically modified viruses, which are inapplicable in most diagnostics departments, we established a simple, rapid, and automated SARS-CoV-2 neutralization assay employing an in-cell ELISA (icELISA) approach. After optimization of various parameters such as virus-specific antibodies, cell lines, virus doses, and duration of infection, SARS-CoV-2-infected cells became amenable as direct antigen source for quantitative icELISA. Antiviral agents such as human sera containing NAbs or antiviral interferons dose dependently reduced the SARS-CoV-2-specific signal. Applying increased infectious doses, the icELISA-based neutralization test (icNT) was superior to PRNT in discriminating convalescent sera with high from those with intermediate neutralizing capacities. In addition, the icNT was found to be specific, discriminating between SARS-CoV-2-specific NAbs and those raised against other coronaviruses. Altogether, the SARS-CoV-2 icELISA test allows rapid (<48 h in total, read-out in seconds) and automated quantification of virus infection in cell culture to evaluate the efficacy of NAbs and antiviral drugs using reagents and equipment present in most routine diagnostics departments.

Viruses and hosts are situated in a molecular arms race. To avoid morbidity and mortality, hosts evolved antiviral restriction factors. These restriction factors exert selection pressure on the viruses and drive viral evolution toward increasingly efficient immune antagonists. Numerous viruses exploit cellular DNA damage-binding protein 1 (DDB1)-containing Cullin RocA ubiquitin ligases (CRLs) to induce the ubiquitination and subsequent proteasomal degradation of antiviral factors expressed by their hosts. To establish a comprehensive understanding of the underlying protein interaction networks, we performed immuno-affinity precipitations for a panel of DDB1-interacting proteins derived from viruses such as mouse cytomegalovirus (MCMV, Murid herpesvirus [MuHV] 1), rat cytomegalovirus Maastricht MuHV2, rat cytomegalovirus English MuHV8, human cytomegalovirus (HCMV), hepatitis B virus (HBV), and human immunodeficiency virus (HIV). Cellular interaction partners were identified and quantified by mass spectrometry (MS) and validated by classical biochemistry. The comparative approach enabled us to separate unspecific interactions from specific binding partners and revealed remarkable differences in the strength of interaction with DDB1. Our analysis confirmed several previously described interactions like the interaction of the MCMV-encoded interferon antagonist pM27 with STAT2. We extended known interactions to paralogous proteins like the interaction of the HBV-encoded HBx with different Spindlin proteins and documented interactions for the first time, which explain functional data like the interaction of the HIV-2-encoded Vpr with Bax. Additionally, several novel interactions were identified, such as the association of the HIV-2-encoded Vpx with the transcription factor RelA (also called p65). For the latter interaction, we documented a functional relevance in antagonizing NF-κB-driven gene expression. The mutation of the DDB1 binding interface of Vpx significantly impaired NF-κB inhibition, indicating that Vpx counteracts NF-κB signaling by a DDB1- and CRL-dependent mechanism. In summary, our findings improve the understanding of how viral pathogens hijack cellular DDB1 and CRLs to ensure efficient replication despite the expression of host restriction factors.

Brief summary: Knowledge concerning SARS-CoV-2-neutralizing antibodies (NAbs) is 14 indispensable for COVID-19 convalescent plasma selection, evaluation of vaccines, and immunity 15 certificates. Our in-cell ELISA (icELISA) test allows rapid (<48h) and high-throughput detection 16 and quantification of SARS-CoV-2-specific NAbs and antiviral activity of drug candidates. 17 18 # Correspondence to 19 Mirko Trilling; Mirko.Trilling@uk-essen.de; ORCID: M Trilling Abstract 28The coronavirus disease 2019 caused by the severe acute respiratory syndrome 29 coronavirus 2 (SARS-CoV-2) is currently the most pressing medical and socioeconomic challenge. 30 Constituting important correlates of protection, determination of virus-neutralizing antibodies 31 (NAbs) is indispensable for convalescent plasma selection, vaccine candidate evaluation, and 32 immunity certificates. In contrast to standard serology ELISAs, plaque reduction neutralization 33 tests (PRNTs) are laborious, time-consuming, expensive, and restricted to specialized laboratories. 34To replace microscopic counting-based SARS-CoV-2 PRNTs by a novel assay exempt from 35 genetically modified viruses, which are inapplicable in most diagnostics departments, we 36 established a simple, rapid, and automated SARS-CoV-2 neutralization assay employing an in-cell 37 ELISA (icELISA) approach. 38 After optimization of various parameters such as virus-specific antibodies, cell lines, virus doses, 39 and duration of infection, SARS-CoV-2-infected cells became amenable as direct antigen source 40 for quantitative icELISA. Using commercially available nucleocapsid protein-specific antibodies, 41 viral infection could easily be quantified in human and highly permissive Vero E6 cells by 42 icELISA. Antiviral agents such as human sera containing NAbs or antiviral interferons dose-43 dependently reduced the SARS-CoV-2-specific signal. Applying increased infectious doses, the 44 icNT was superior to PRNT in discriminating convalescent sera with high from those with 45 intermediate neutralizing capacities.46The SARS-CoV-2 icELISA test allows rapid (<48h in total, read-out in seconds) and automated 47 quantification of virus infection in cell culture to evaluate the efficacy of NAbs as well as antiviral 48 drugs, using reagents and equipment present in most routine diagnostics departments. We propose 49 the icELISA and the icNT for COVID-19 research and diagnostics. 50 65 the outbreak a global pandemic. Since its beginning, the centre of the pandemic shifted from China, 66 via Europe and Northern Americas to Central and Southern Americas. This dynamic nature of the 67 pandemic poses an inherent danger of repetitive local and temporal reintroduction circles. Thus, 68 even countries which coped relatively well with the first wave must prepare in terms of diagnostics 69 capacities for potential future re-emergences.70Most SARS-CoV-2 infections lead to mild or moderate illnesses. However, a considerable fraction 71 of cases proceeds to severe pneumonia or life-threatening acute respir...

virus devoid of detectable pM34HA protein. The replication of MCMVs lacking M34 was found to be 10-to 100-fold reduced as compared to wt-MCMV which might explain previous unsuccessful reconstitution attempts conducted by others. Taken together, our findings reveal that MCMV remains replication competent despite the absence of M34, enabling functional studies in the infection context.

Human cytomegalovirus (HCMV) frequently causes congenital infections, resulting in birth defects and developmental disorders. A vaccine is needed, but unavailable. We analyzed the potential of CMV mutants, lacking their STAT2 antagonists to serve as live attenuated vaccine viruses in mice. Infections with attenuated viruses elicited strong ELISA-reactive binding IgG responses and induced neutralizing antibodies as well as antibodies stimulating cellular Fcγ receptors, including the antibody-dependent cellular cytotoxicity (ADCC)-eliciting receptors FcγRIII/CD16 and FcγRIV. Accordingly, vaccinated mice were fully protected against challenge infections. Female mice vaccinated prior to gestation transmitted CMV-specific IgG to their offspring, which protected the progeny from perinatal infections in a mouse model for congenital CMV disease. To define the role of maternal antibodies, female mice either capable or incapable of producing antibodies were vaccinated and subsequently bred to males of the opposite genotype. Challenge infections of the genotypically identical F1 generation revealed the indispensability of maternal antibodies for vaccine-induced protection against cytomegaloviruses.