Epithelial cell lines of the cotton rat (Sigmodon hispidus) are highly susceptible in vitro models to zoonotic Bunya-, Rhabdo-, and Flaviviruses.

BACKGROUND: Small mammals such as bats and rodents have been increasingly recognized as reservoirs of novel potentially zoonotic pathogens. However, few in vitro model systems to date allow assessment of zoonotic viruses in a relevant host context. The cotton rat (Sigmodon hispidus) is a New World rodent species that has a long-standing history as an experimental animal model due to its unique susceptibility to human viruses. Furthermore, wild cotton rats are associated with a large variety of known or potentially zoonotic pathogens. METHODS: A method for the isolation and culture of airway epithelial cell lines recently developed for bats was applied for the generation of rodent airway and renal epithelial cell lines from the cotton rat. Continuous cell lines were characterized for their epithelial properties as well as for their interferon competence. Susceptibility to members of zoonotic Bunya-, Rhabdo-, and Flaviviridae, in particular Rift Valley fever virus (RVFV), vesicular stomatitis virus (VSV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV) was tested. Furthermore, novel arthropod-derived viruses belonging to the families Bunya-, Rhabdo-, and Mesoniviridae were tested. RESULTS: We successfully established airway and kidney epithelial cell lines from the cotton rat, and characterized their epithelial properties. Cells were shown to be interferon-competent. Viral infection assays showed high-titre viral replication of RVFV, VSV, WNV, and TBEV, as well as production of infectious virus particles. No viral replication was observed for novel arthropod-derived members of the Bunya-, Rhabdo-, and Mesoniviridae families in these cell lines. CONCLUSION: In the current study, we showed that newly established cell lines from the cotton rat can serve as host-specific in vitro models for viral infection experiments. These cell lines may also serve as novel tools for virus isolation, as well as for the investigation of virus-host interactions in a relevant host species.

Nanobodies dismantle post-pyroptotic ASC specks and counteract inflammation in vivo.

Inflammasomes sense intracellular clues of infection, damage, or metabolic imbalances. Activated inflammasome sensors polymerize the adaptor ASC into micron-sized "specks" to maximize caspase-1 activation and the maturation of IL-1 cytokines. Caspase-1 also drives pyroptosis, a lytic cell death characterized by leakage of intracellular content to the extracellular space. ASC specks are released among cytosolic content, and accumulate in tissues of patients with chronic inflammation. However, if extracellular ASC specks contribute to disease, or are merely inert remnants of cell death remains unknown. Here, we show that camelid-derived nanobodies against ASC (VHHASC ) target and disassemble post-pyroptotic inflammasomes, neutralizing their prionoid, and inflammatory functions. Notably, pyroptosis-driven membrane perforation and exposure of ASC specks to the extracellular environment allowed VHHASC to target inflammasomes while preserving pre-pyroptotic IL-1ß release, essential to host defense. Systemically administrated mouse-specific VHHASC attenuated inflammation and clinical gout, and antigen-induced arthritis disease. Hence, VHHASC neutralized post-pyroptotic inflammasomes revealing a previously unappreciated role for these complexes in disease. VHHASC are the first biologicals that disassemble pre-formed inflammasomes while preserving their functions in host defense.

Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo-electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.