One of the principal obstacles to studying SFTSV has always been biosafety. Live virus work demands containment at biosafety level 3 or higher, a requirement that dramatically limits the number of laboratories able to investigate the pathogen’s infection biology. The infrastructure, personnel training, and regulatory oversight associated with BSL-3 facilities make routine experimentation slow and expensive, and they create a bottleneck for drug discovery pipelines that depend on high-throughput screening. A team of researchers led by Chenxiao Huang, Wei Li, and Jianfeng Dai at Soochow University in Suzhou, China, has now developed a solution to this problem, publishing their findings in Cellular and Molecular Life Sciences.

The strategy the team pursued centers on a concept known as conditional replication deficiency. Rather than creating a pseudovirus that can perform only a single round of entry, as is common with HIV-based or VSV-based surrogate systems, the researchers engineered an SFTSV genome that retains the capacity for genuine viral replication but only under specific laboratory conditions. They accomplished this by removing the gene encoding the nucleoprotein (NP) from the viral S segment and replacing it with a reporter gene. The nucleoprotein is essential for viral genome encapsidation and replication, so without it, the engineered virus cannot complete its life cycle unless the missing protein is supplied in trans by the host cell.

This design creates a powerful safety barrier. The SFTSV ΔN pseudovirus can only replicate in cells or tissues that have been deliberately engineered to express the nucleoprotein from a separate plasmid or transgene. In any other environment, including a laboratory worker’s body or a non-complementing cell line, the virus enters but cannot produce infectious progeny. The approach mirrors strategies used in other conditional replication systems, such as the ΔN systems developed for influenza A virus, where the M2 or NS1 gene is deleted and complemented in trans. The elegance of the design lies in the fact that the pseudovirus retains the full complement of viral structural proteins except for the one that has been removed, meaning that entry, genome replication, transcription, and assembly all proceed normally as long as the missing component is provided.

To make the system useful for drug screening, the researchers created two versions of the ΔN genome, each carrying a different reporter gene in place of the nucleoprotein. One version carried the gene for Gaussia luciferase (Gluc), a secreted luciferase that produces a bright and quantifiable luminescent signal. The other carried the gene for green fluorescent protein (GFP), which allows direct visualization of infected cells under a fluorescence microscope. Using a reverse genetics system, the team successfully rescued both reporter pseudoviruses, confirming that the engineered genomes could be packaged into infectious particles and that the reporter genes were expressed upon infection of complementing cells.

The Gaussia luciferase version proved particularly valuable for high-throughput applications. Because Gluc is secreted into the culture medium, the researchers could measure infection levels simply by sampling the supernatant and adding luciferin substrate, a process that is compatible with automated plate readers and robotic liquid handling systems. This means that hundreds or thousands of compounds can be tested in parallel in a single experiment, with luminescence serving as a direct readout of viral replication. Any compound that reduces the luminescent signal relative to untreated controls is a candidate inhibitor of SFTSV replication.

Using this platform, the researchers performed a screen of a natural product library and identified several compounds that effectively inhibited SFTSV replication. Among the hits was sesamin, a lignan compound found in sesame seeds and sesame oil that has previously been reported to possess anti-inflammatory and antioxidant properties. The identification of sesamin as an inhibitor of SFTSV replication demonstrates the practical utility of the ΔN pseudovirus system for discovering antiviral candidates from complex chemical libraries. The researchers note that the platform can be adapted to screen diverse compound collections, including FDA-approved drugs, natural product extracts, and synthetic small molecules.

Beyond drug screening, the SFTSV ΔN pseudovirus offers a safe and versatile tool for studying viral infection mechanisms and tissue tropism. Because the virus can replicate in cells that provide the nucleoprotein in trans, researchers can investigate host factors that influence viral entry, genome replication, and assembly without the need for BSL-3 containment. The system can also be used in mouse models where the nucleoprotein is expressed in specific tissues, allowing researchers to map which organs and cell types are susceptible to SFTSV infection. This capability is particularly important for understanding the pathogenesis of severe fever with thrombocytopenia syndrome, which involves complex interactions between the virus and the host immune system, including dysregulated cytokine responses and impaired platelet function.

The work represents a significant advance in the toolkit available for studying emerging bunyaviruses. As climate change and expanding tick populations continue to push SFTSV into new geographic areas, the need for effective antiviral therapies and vaccines grows more urgent. The conditional replication-deficient pseudovirus platform developed by the Soochow University team provides a bridge between the safety constraints of high-containment research and the practical demands of drug discovery and mechanistic investigation. By enabling routine experimentation with a virus that retains its authentic replication machinery but cannot spread beyond engineered complementing cells, the system opens the door to broader participation in SFTSV research and accelerates the pace at which candidate therapeutics can be identified and validated.

Subject of Research: Development of a conditional replication-deficient SFTSV reporter pseudovirus for antiviral drug screening and viral infection studies

Article Title: Construction of an SFTSV ΔN reporter pseudovirus for antiviral drug screening

Article References: Huang, C., Li, W., Xu, Y., Pan, W., Zhou, X., Dong, C., & Dai, J. (2026). Construction of an SFTSV ΔN reporter pseudovirus for antiviral drug screening. Cellular and Molecular Life Sciences. https://doi.org/10.1007/s00018-026-06485-7

Image Credits: AI Generated

DOI: 10.1007/s00018-026-06485-7

Keywords: SFTSV, Dabie bandavirus, pseudovirus, conditional replication deficiency, reporter gene, antiviral screening, Gaussia luciferase, green fluorescent protein, reverse genetics, sesamin, biosafety level 3, nucleoprotein

The stakes of this kind of work are hard to overstate. In the past two decades, three zoonotic coronaviruses—SARS-CoV-1, MERS-CoV, and SARS-CoV-2—have caused major outbreaks, and each emergence began with a virus crossing a species barrier. For sarbecoviruses, that barrier is defined largely by compatibility between the viral receptor-binding domain (RBD) on the spike protein and the host’s angiotensin-converting enzyme 2 (ACE2) receptor. If a virus’s RBD can engage a species’ ACE2 efficiently, that species becomes a plausible host; if it cannot, the chain of transmission breaks at the very first step. Understanding which receptors are vulnerable to which viruses is therefore central to anticipating where the next spillover might come from.

To build the atlas, the researchers first established an evolutionary framework. Phylogenetic analysis of the RBD sequences divided the 46 viruses—31 from bats, 10 from humans, three from civets, and two from pangolins—into three major clades, with clade 1 further splitting into subclades 1a and 1b. Clade 1a contains SARS-CoV-1 and related bat and civet strains; clade 1b contains SARS-CoV-2 and its closest relatives, including the bat virus RaTG13 and pangolin coronaviruses. Clade 2, which comprises most known sarbecoviruses, carries a distinctive double-segment deletion in the receptor-binding motif, while clade 3 viruses from African and European bats carry a single-segment deletion in the same region. These structural differences in the RBM, the loop that physically contacts ACE2, turned out to predict much of the functional variation the team later observed.

The functional screen itself relied on pseudotyped reporter viruses: vesicular stomatitis virus particles decorated with each sarbecovirus spike protein, whose entry into cells expressing a given ACE2 ortholog could be quantified by luminescence. The team confirmed comparable spike expression and incorporation across pseudovirus preparations by western blot and normalized viral input by quantitative RT-PCR, allowing a fair comparison across the roughly 3,000 possible virus-receptor pairings. The result is a heatmap of cross-species infectivity that functions as a kind of vulnerability index for each species’ receptor.

Clade 1 viruses emerged as the clear standouts in receptor breadth. Within clade 1b, the pangolin viruses PCoV-GD and PCoV-GX, which share 89.6 and 92.3 percent spike identity with SARS-CoV-2 respectively, showed broad ACE2 compatibility, with PCoV-GD efficiently infecting nearly all tested non-bat mammalian ACE2s and most bat ACE2s. Intriguingly, RaTG13—despite sharing 97.4 percent identity with SARS-CoV-2 across the full spike protein—displayed a more restricted host range, implying that subtle differences within the receptor-binding motif underlie the expanded tropism of SARS-CoV-2. Within clade 1a, human SARS-CoV-1 isolates used ACE2 orthologs from a wide range of bat families, whereas most bat- and civet-derived relatives showed narrower usage, with the bat virus WIV1 bridging human and bat lineages through its ability to engage ACE2 from multiple Rhinolophus and Myotis species.

Clade 2 and clade 3 viruses told very different stories. None of the clade 2 pseudoviruses, which represent the majority of known sarbecoviruses, mediated entry through any of the 66 ACE2 orthologs, consistent with their double RBM deletion and previously reported ACE2-independent entry mechanisms. Clade 3 viruses showed intermediate phenotypes: BtKY72 used ACE2 from only a few Rhinolophus species and did not engage human ACE2, while Khosta-2 showed broader tropism, infecting cells expressing rabbit ACE2 more than ten-fold more efficiently than those expressing the human receptor—a striking example of lineage-specific adaptation.

The study also delivered a decisive verdict on several proposed alternative receptors. Host factors including NRP1, CD147, AXL, and TMEM106B have each been suggested to facilitate SARS-CoV-2 entry in various experimental contexts. Yet across the entire 46-virus panel, none of these factors supported detectable infection when overexpressed in the assay cells, indicating that none functions as an independent entry receptor for any sarbecovirus tested. The authors are careful to note that context-dependent accessory roles in specific SARS-CoV-2 settings cannot be excluded, but the message is clear: ACE2 remains the only demonstrated primary entry receptor across this viral diversity.

Among the most consequential findings is the identification of two bat species whose receptors behave remarkably like the human version. Only the ACE2 orthologs of Rhinolophus affinis and Miniopterus schreibersii correlated strongly with human ACE2 usage across the viral panel, with correlation coefficients of 0.8314 and 0.8777 respectively. Surface plasmon resonance confirmed that M. schreibersii ACE2 binds the SARS-CoV-1 and SARS-CoV-2 RBDs with high affinity—dissociation constants of 8.14 × 10⁻⁸ M and 8.06 × 10⁻⁷ M, lower than human ACE2’s nanomolar binding but fully consistent with efficient entry. Because both species range across Southeast Asia, Europe, and North Africa in regions overlapping human habitats, the authors argue they should be priorities for ongoing surveillance. The data also flagged rabbits, cattle, sheep, raccoon dogs, badgers, Siberian chipmunks, and pigs as mammalian species whose ACE2 usage correlates strongly with human ACE2, marking them as candidate intermediate hosts worth investigating.

Perhaps the most mechanistically revealing part of the study concerns two closely related Miniopterus bats whose receptors sit at opposite ends of the permissiveness spectrum. M. schreibersii ACE2 supported entry by nearly all ACE2-dependent pseudoviruses, while the ACE2 of M. natalensis was entirely non-permissive—despite the two proteins differing by only seven amino acids. Three of those substitutions, at positions 27, 31, and 42, sit directly at the predicted RBD-binding interface. Molecular dynamics simulations using the molecular mechanics generalized Born surface area method showed that mutating M. schreibersii ACE2 residue I27 to lysine raised the binding free energy by roughly 5 kcal/mol, while the K42E substitution raised it by nearly 7 kcal/mol, disrupting a salt-bridge network involving residue D38 and the SARS-CoV-1 RBD residue Y442. Entry assays confirmed the simulations: the I27K mutation selectively impaired civet-derived SARS-CoV-1 entry, N31G had minimal impact, and K42E broadly abolished usage by nearly all pseudotypes. Reciprocally, introducing the corresponding M. schreibersii residues into M. natalensis ACE2 restored entry competence for multiple SARS-CoV-1 pseudotypes. Residues 27 and 42, in short, are the principal molecular switches governing Miniopterus receptor recognition.

The authors are candid about the limits of their approach. Pseudoviruses built from available sequences may not capture the full diversity of natural sarbecoviruses, and receptor compatibility is only one barrier to cross-species transmission; protease availability, replication competence, and immune evasion all matter for productive infection in a living animal. Efficient ACE2 engagement alone does not guarantee disease. Still, the atlas provides exactly the kind of mechanistic framework that field surveillance has lacked: a way to prioritize which of the world’s more than 1,400 bat species, and which of the hundreds of circulating sarbecovirus sequences, warrant the closest monitoring. As the authors put it, identifying species with ACE2 structures resembling the human receptor may help focus receptor-based risk assessment where it matters most—before, rather than after, the next spillover.

Subject of Research: Cross-species ACE2 receptor usage and host-range determinants of sarbecoviruses

Article Title: An atlas of sarbecovirus infectivity across diverse ACE2 orthologs

Article References: Sun, Y., Cheng, Z., Wu, X., Liu, K., Wang, L., Yang, D., Huang, W., & Nie, J. (2026). An atlas of sarbecovirus infectivity across diverse ACE2 orthologs. iScience, 29(10), Article 117769. https://doi.org/10.1016/j.isci.2026.117769

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117769

Keywords: sarbecovirus, ACE2, SARS-CoV-2, SARS-CoV-1, bats, spillover, pseudovirus, receptor-binding domain, host range, surveillance, Miniopterus, Rhinolophus