Herpesviruses are large DNA viruses and include important human and veterinary pathogens. Their genomes can be cloned as bacterial artificial chromosomes (BACs) and genetically engineered in Escherichia coli using BAC recombineering methods. While the recombineering methods are efficient, the initial BAC-cloning step remains laborious. To overcome this limitation, we have developed a simple, rapid, and efficient BAC-cloning method based on single-step transformation-associated recombination (STAR) in Saccharomyces cerevisiae. The linear viral genome is directly integrated into a vector comprising a yeast centromeric plasmid and a BAC replicon. Following transfer into E. coli, the viral genome can be modified using standard BAC recombineering techniques. We demonstrate the speed, fidelity, and broad applicability of STAR by cloning two strains of both rat cytomegalovirus (a betaherpesvirus) and Kaposi’s sarcoma-associated herpesvirus (a gammaherpesvirus). STAR cloning facilitates the functional genetic analysis of herpesviruses and other large DNA viruses and their use as vaccines and therapeutic vectors.

Norovirus capsids are icosahedral particles composed of 90 dimers of the major capsid protein VP1. The C-terminus of the VP1 proteins forms a protruding (P)-domain, mediating receptor attachment, and providing a target for neutralizing antibodies. NMR and native mass spectrometry directly detect P-domain monomers in solution for murine (MNV) but not for human norovirus (HuNoV). We report that the binding of glycochenodeoxycholic acid (GCDCA) stabilizes MNV-1 P-domain dimers (P-dimers) and induces long-range NMR chemical shift perturbations (CSPs) within loops involved in antibody and receptor binding, likely reflecting corresponding conformational changes. Global line shape analysis of monomer and dimer cross-peaks in concentration-dependent methyl TROSY NMR spectra yields a dissociation rate constant k off of about 1 s −1 for MNV-1 P-dimers. For structurally closely related HuNoV GII.4 Saga P-dimers a value of about 10 −6 s −1 is obtained from ion-exchange chromatography, suggesting essential differences in the role of GCDCA as a cofactor for MNV and HuNoV infection.

With newly rising coronavirus disease 2019 (COVID-19) cases, important data gaps remain on (i) long-term dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection rates in fixed cohorts (ii) identification of risk factors, and (iii) establishment of effective surveillance strategies. By polymerase chain reaction and antibody testing of 1% of the local population and >90,000 app-based datasets, the present study surveilled a catchment area of 300,000 inhabitants from March 2020 to February 2021. Cohort (56% female; mean age, 45.6 years) retention was 75 to 98%. Increased risk for seropositivity was detected in several high-exposure groups, especially nurses. Unreported infections dropped from 92 to 29% during the study. "Contact to COVID-19-affected" was the strongest risk factor, whereas public transportation, having children in school, or tourism did not affect infection rates. With the first SARS-CoV-2 cohort study, we provide a transferable model for effective surveillance, enabling monitoring of reinfection rates and increased preparedness for future pandemics.

Background More than one year into the COVID-19 pandemic, important data gaps remain on longitudinal prevalence of SARS-CoV-2 infection at the population level and in defined risk groups, efficacy of specific lockdown measures, and on (cost-)effective surveillance. Methods The ELISA (Luebeck Longitudinal Investigation of SARS-CoV-2 Infection) study invited adult inhabitants (n=~300,000) from the Luebeck area (Northern Germany) and enrolled 3051 participants (~1%); 1929 population-matched and 1645 with high-exposure based on profession. The one-year study period (03/2020-02/2021) covered massive influx of tourism in the summer, rise of infection rates in the fall/winter 2020/2021, and two lockdowns. Participants were screened seven times for SARS-CoV-2 infection using PCR and antibody testing and monitored with an app-based questionnaire (n=~91,000). Results Cohort (56% female; mean age: 45.6 years) retention was 75%-98%; 92 persons (3.5%) were antibody- and/or PCR-positive. Seropositivity was almost 2-fold higher in men and increased risk detected in several high-exposure groups (highest for nurses, followed by police, army, firemen, and students). In May 2020, 92% of the infections were missed by PCR testing; by February 2021, only 29% remained undiagnosed. Contact to COVID-19-affected was the most relevant risk factor. Other factors, such as frequent use of public transportation, shopping, close contacts at work, and extensive tourism in the summer did not impact infection rates. Conclusions We i) provide a model for effective, regional surveillance; ii) identify infection risk factors informing public health measures; iii) demonstrate that easing of lockdown measures appears safe at times of low prevalence in the presence of continuous monitoring.

We have recently established that human norovirus (HuNoV) replicates efficiently in zebrafish larvae after inoculation of a clinical sample into the yolk, providing a simple and robust in vivo system in which to study HuNoV. In this Protocol Extension, we present a detailed description of virus inoculation by microinjection, subsequent daily monitoring and harvesting of larvae, followed by viral RNA quantification. This protocol can be used to study viral replication of genogroup (G)I and GII HuNoVs in vivo within 3–4 d. Additionally, we describe how to evaluate the in vivo antiviral effect and toxicity of small molecules using HuNoV-infected zebrafish larvae, in multi-well plates and without the need for specific formulations. This constitutes a great advantage for drug discovery efforts, as no specific antivirals or vaccines currently exist to treat or prevent norovirus gastroenteritis. This Protocol Extension details the use of zebrafish larvae as a simple and robust in vivo system for studying human norovirus infection, enabling evaluation of the antiviral effects and toxicities of small molecules.

The murine norovirus (MNV) capsid protein is the target for various neutralizing antibodies binding to distal tips of its protruding (P)-domain. The bile acid glycochenodeoxycholic acid (GCDCA), an important co-factor for murine norovirus (MNV) infection, has recently been shown to induce conformational changes in surface-loops and a contraction of the virion. Here, we employ protein NMR experiments using stable isotope labeled MNV P-domains to shed light on underlying molecular mechanisms. We observe two separate sets of NMR resonance signals for P-domain monomers and dimers, permitting analysis of the corresponding exchange kinetics. Unlike human norovirus GII.4 P-dimers, which exhibit a half-life in the range of several days, MNV P-dimers are very short lived with a half-life of about 17 s. Addition of GCDCA shifts the equilibrium towards the dimeric form by tightly binding to the P-dimers. In MNV virions GCDCA-mediated stabilization of the dimeric arrangement of P-domains generates a more ordered state, which in turn may entropically assist capsid contraction. Numerous long-range chemical shift perturbations (CSPs) upon addition of GCDCA reflect allosteric conformational changes as a feature accompanying dimer stabilization. In particular, CSPs indicate rearrangement of the E’F’ loop, a target for various neutralizing antibodies. Indeed, treating MNV virions with GCDCA prior to neutralizing antibody exposure abolishes neutralization. These findings advance our understanding of GCDCA-induced structural changes of MNV capsids and experimentally support an intriguing viral immune escape mechanism relying on GCDCA-triggered conformational changes of the P-dimer. Significance Statement This study sheds light on the role of glycochenodeoxycholic acid (GCDCA) in promoting murine norovirus (MNV) infection and immune escape. Binding of GCDCA to the dimeric P-domain has been well characterized by crystallography and cryo EM studies, showing that upon GCDCA binding, a 90° rotation of the P-domain occurs, which results in its collapse onto the underlying shell of the virus. Our NMR experiments now reveal P-dimer stability as a new dimension of plasticity of MNV capsids and suggest that capsid contraction is entropically assisted. Conformational changes as a feature of P-dimer stabilization eliminate recognition by neutralizing antibodies, no longer being able to prevent infection. These findings highlight key differences between human and MNV capsid structures, promote our understanding of MNV infection on a molecular level, and reveal a novel immune escape mechanism.

Cell–cell fusion is a fundamental and complex process that occurs during reproduction, organ and tissue growth, cancer metastasis, immune response, and infection. All enveloped viruses express one or more proteins that drive the fusion of the viral envelope with cellular membranes. The same proteins can mediate the fusion of the plasma membranes of adjacent cells, leading to the formation of multinucleated syncytia. While cell–cell fusion triggered by alpha- and gammaherpesviruses is well-studied, much less is known about the fusogenic potential of betaherpesviruses such as human cytomegalovirus (HCMV) and human herpesviruses 6 and 7 (HHV-6 and HHV-7). These are slow-growing viruses that are highly prevalent in the human population and associated with several diseases, particularly in individuals with an immature or impaired immune system such as fetuses and transplant recipients. While HHV-6 and HHV-7 are strictly lymphotropic, HCMV infects a very broad range of cell types including epithelial, endothelial, mesenchymal, and myeloid cells. Syncytia have been observed occasionally for all three betaherpesviruses, both during in vitro and in vivo infection. Since cell–cell fusion may allow efficient spread to neighboring cells without exposure to neutralizing antibodies and other host immune factors, viral-induced syncytia may be important for viral dissemination, long-term persistence, and pathogenicity. In this review, we provide an overview of the viral and cellular factors and mechanisms identified so far in the process of cell–cell fusion induced by betaherpesviruses and discuss the possible consequences for cellular dysfunction and pathogenesis.