Abstract Background Orthohantaviruses are zoonotic RNA viruses that cause hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. Their slow replication, lack of cytopathic effects, and variable biosafety requirements have long hindered the standardization of infection assays and antiviral testing. While serological and pseudotype-based systems enable high-throughput screening, scalable assays that directly quantify full-cycle Orthohantavirus infection remain limited. Results Here, we present a modular BSL-2 workflow that integrates standardized virus stock generation with complementary infection-quantification readouts. The platform combines (i) titration-guided pooling of daily supernatants to produce high-titer virus stocks without ultracentrifugation, (ii) a cross-reactive monoclonal antibody recognizing conserved nucleocapsid epitopes across multiple Orthohantavirus species, and (iii) three quantitative detection methods: qPCR, in-cell ELISA, and intracellular flow cytometry. Flow cytometry was used as the primary readout because it enables rapid, quantitative, single-cell detection of infection and can be readily integrated with multiparametric analyses. Using this system, we quantified the antiviral effects of rottlerin and observed distinct dose‒response profiles among Puumala, Tula, and Prospect Hill viruses. Conclusion Our workflow delivers a practical, reproducible, and scalable toolkit for comparative Orthohantavirus studies, enabling quantitative infection analysis and small-molecule testing under standard laboratory conditions.
Schwarzer-Sperber et al. (2026) studied this question.