The ongoing pandemic coronavirus (CoV) disease 2019 (COVID-19) by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has already caused substantial morbidity, mortality, and economic devastation. Reverse genetic approaches to generate recombinant viruses are a powerful tool to characterize and understand newly emerging viruses. To contribute to the global efforts for countermeasures to control the spread of SARS-CoV-2, we developed a passage-free SARS-CoV-2 clone based on a bacterial artificial chromosome (BAC). Moreover, using a Lambda-based Red recombination, we successfully generated different reporter and marker viruses, which replicated similar to a clinical isolate in a cell culture. Moreover, we designed a full-length reporter virus encoding an additional artificial open reading frame with wild-type-like replication features. The virus-encoded reporters were successfully applied to ease antiviral testing in cell culture models. Furthermore, we designed a new marker virus encoding 3xFLAG-tagged nucleocapsid that allows the detection of incoming viral particles and, in combination with bio-orthogonal labeling for the visualization of viral RNA synthesis via click chemistry, the spatiotemporal tracking of viral replication on the single-cell level. In summary, by applying BAC-based Red recombination, we developed a powerful, reliable, and convenient platform that will facilitate studies answering numerous questions concerning the biology of SARS-CoV-2.

Mutations in the spike protein of SARS-CoV-2 can lead to evasion from neutralizing antibodies and affect the efficacy of passive and active immunization strategies. Immunization of mice harboring an entire set of human immunoglobulin variable region gene segments allowed to identify nine neutralizing monoclonal antibodies, which either belong to a cluster of clonally related RBD or NTD binding antibodies. To better understand the genetic barrier to emergence of SARS-CoV-2 variants resistant to these antibodies, escape mutants were selected in cell culture to one antibody from each cluster and a combination of the two antibodies. Three independently derived escape mutants to the RBD antibody harbored mutations in the RBD at the position T478 or S477. These mutations impaired the binding of the RBD antibodies to the spike protein and conferred resistance in a pseudotype neutralization assay. Although the binding of the NTD cluster antibodies were not affected by the RBD mutations, the RBD mutations also reduced the neutralization efficacy of the NTD cluster antibodies. The mutations found in the escape variants to the NTD antibody conferred resistance to the NTD, but not to the RBD cluster antibodies. A variant resistant to both antibodies was more difficult to select and only emerged after longer passages and higher inoculation volumes. VOC carrying the same mutations as the ones identified in the escape variants were also resistant to neutralization. This study further underlines the rapid emergence of escape mutants to neutralizing monoclonal antibodies in cell culture and indicates the need for thorough investigation of escape mutations to select the most potent combination of monoclonal antibodies for clinical use.

Even with its endemic transition, the COVID-19 pandemic remains a public health threat, particularly in the light of emerging variants of concern (VoCs) and the need for pandemic preparedness in the future. In November 2021, the SARS-CoV-2 VoC Omicron emerged and its subvariants BA.1, BA.2 and BA.5 became predominant. Although the protease inhibitor Paxlovid^® and the polymerase inhibitors Molnupiravir and Remdesivir were approved as specific antiviral treatment options for COVID-19 patients in the early stages after infection, effective prophylactically acting substances without adverse effects are not available yet. In a recent study, we demonstrated that iota-carrageenan, a sulfated polysaccharide extracted from red seaweed, efficiently inhibits the replication of the SARS-CoV-2 Wuhan Type and the VoCs Alpha, Beta, Gamma and Delta. Now, we extended this study by investigating the antiviral effects of iota-, lambda- and kappa-carrageenans on the VoC Omicron subvariants BA.1, BA.2 and BA.5. Using a VoC Omicron BA.1 spike pseudotyped murine leukemia virus (BA.1 MLV_OMVLP) as well as patient-derived SARS-CoV-2 Omicron isolates BA.1, BA.2 and BA.5 (SARS-CoV-2_{OM BA.1}, SARS-CoV-2_{OM BA.2} and SARS-CoV-2_{OM BA.5}), we demonstrate that iota-carrageenan exhibits similar antiviral activity against all analyzed Omicron subvariants. As with other VoCs shown before, the biologically inert iota-carrageenan was more efficient than kappa- and lambda-carrageenan. Altogether, these results confirm that, independent of the current and potential future variants, the physical barrier provided by iota-carrageenan might be applicable for prophylaxis and early treatment of SARS-CoV-2 infections.

The COVID-19 pandemic caused by SARS-CoV-2 has lasted for more than two years. Despite the presence of very effective vaccines, the number of virus variants that escape neutralizing antibodies is growing. Thus, there is still a need for effective antiviral treatments that target virus replication independently of the circulating variant. Here, we show for the first time that deficiency or pharmacological inhibition of the cellular lysine-methyltransferase SMYD2 decreases TMPRSS2 expression on both mRNA and protein levels. SARS-CoV-2 uses TMPRSS2 for priming its spike protein to infect target cells. Treatment of cultured cells with the SMYD2 inhibitors AZ505 or BAY598 significantly inhibited viral replication. In contrast, treatment of Vero E6 cells, which do not express detectable amounts of TMPRSS2, had no effect on SARS-CoV-2 infection. Moreover, by generating a recombinant reporter virus that expresses the spike protein of the Delta variant of SARS-CoV-2, we demonstrate that BAY598 exhibits similar antiviral activity against this variant of concern. In summary, SMYD2 inhibition downregulates TMPRSS2 and blocks viral replication. Targeting cellular SMYD2 represents a promising tool to curtail SARS-CoV-2 infection.

Monkeypox virus (MPXV) has caused a large pandemic outbreak in 2022 with more than 90.000 confirmed cases and 181 deaths. Notably, signs of microevolution and host adaption have been observed. Here, we demonstrate that viral genomes from Franconia, Bavaria acquired different mutations. Three isolates obtained from diagnostic samples, submitted from suspected Mpox cases, show differences in their replication capacities. One MPXV isolate which shows the fastest replication kinetics and higher viral loads, possesses a unique non-synonymous mutation (D616L) in the A11L protein (gene OPG136), which encodes for a protein that is part of a major viral core structure. In regard to pandemic preparedness and future outbreaks, we analyzed the antiviral activity of dihydroorotate dehydrogenase (DHODH) inhibitors, and show that they are active against MPXV, vaccinia virus (VACV), and cowpox virus (CPXV) and therefore likely against orthopoxviruses in general. In agreement with that, we also demonstrated that chemical optimization leads to compounds with EC₅₀ values in the sub-nanomolar range, associated with low cytotoxicity, which forms a good basis for future drug development from this chemical series.

The infection of human cytomegalovirus (HCMV) is strongly determined by the host–cell interaction in a way that the efficiency of HCMV lytic replication is dependent on the regulatory interplay between viral and cellular proteins. In particular, the activities of protein kinases, such as cyclin-dependent kinases (CDKs) and the viral CDK ortholog (vCDK/pUL97), play an important role in both viral reproduction and virus–host interaction. Very recently, we reported on the complexes formed between vCDK/pUL97, human cyclin H, and CDK7. Major hallmarks of this interplay are the interaction between cyclin H and vCDK/pUL97, which is consistently detectable across various conditions and host cell types of infection, the decrease or increase in pUL97 kinase activity resulting from cyclin H knock-down or elevated levels, respectively, and significant trans-stimulation of human CDK7 activity by pUL97 in vitro. Due to the fact that even a ternary complex of vCDK/pUL97–cyclin H–CDK7 can be detected by coimmunoprecipitation and visualized by bioinformatic structural modeling, we postulated a putative impact of the respective kinase activities on the patterns of transcription in HCMV-infected cells. Here, we undertook a first vCDK/pUL97-specific transcriptomic analysis, which combined conditions of fully lytic HCMV replication with those under specific vCDK/pUL97 or CDK7 drug-mediated inhibition or transient cyclin H knockout. The novel results were further strengthened using bioinformatic modeling of the involved multi-protein complexes. Our data underline the importance of these kinase activities for the C-terminal domain (CTD) phosphorylation-driven activation of host RNA polymerase in HCMV-infected cells. The impact of the individual experimental conditions on differentially expressed gene profiles is described in detail and discussed.