The Orthobunyavirus Germiston Enters Host Cells from Late Endosomes.

With more than 80 members worldwide, the Orthobunyavirus genus in the Peribunyaviridae family is a large genus of enveloped RNA viruses, many of which are emerging pathogens in humans and livestock. How orthobunyaviruses (OBVs) penetrate and infect mammalian host cells remains poorly characterized. Here, we investigated the entry mechanisms of the OBV Germiston (GERV). Viral particles were visualized by cryo-electron microscopy and appeared roughly spherical with an average diameter of 98 nm. Labeling of the virus with fluorescent dyes did not adversely affect its infectivity and allowed the monitoring of single particles in fixed and live cells. Using this approach, we found that endocytic internalization of bound viruses was asynchronous and occurred within 30 to 40 min. The virus entered Rab5a-positive (Rab5a+) early endosomes and, subsequently, late endosomal vacuoles containing Rab7a but not LAMP-1. Infectious entry did not require proteolytic cleavage, and endosomal acidification was sufficient and necessary for viral fusion. Acid-activated penetration began 15 to 25 min after initiation of virus internalization and relied on maturation of early endosomes to late endosomes. The optimal pH for viral membrane fusion was slightly below 6.0, and penetration was hampered when the potassium influx was abolished. Overall, our study provides real-time visualization of GERV entry into host cells and demonstrates the importance of late endosomal maturation in facilitating OBV penetration. IMPORTANCE Orthobunyaviruses (OBVs), which include La Crosse, Oropouche, and Schmallenberg viruses, represent a growing threat to humans and domestic animals worldwide. Ideally, preventing OBV spread requires approaches that target early stages of infection, i.e., virus entry. However, little is known about the molecular and cellular mechanisms by which OBVs enter and infect host cells. Here, we developed accurate, sensitive tools and assays to investigate the penetration process of GERV. Our data emphasize the central role of late endosomal maturation in GERV entry, providing a comprehensive overview of the early stages of an OBV infection. Our study also brings a complete toolbox of innovative methods to study each step of the OBV entry program in fixed and living cells, from virus binding and endocytosis to fusion and penetration. The information gained herein lays the foundation for the development of antiviral strategies aiming to block OBV entry.

Tecovirimat for the treatment of severe Mpox in Germany.

BACKGROUND: In May 2022, a multi-national mpox outbreak was reported in several non-endemic countries. The only licensed treatment for mpox in the European Union is the orally available small molecule tecovirimat, which in Orthopox viruses inhibits the function of a major envelope protein required for the production of extracellular virus. METHODS: We identified presumably all patients with mpox that were treated with tecovirimat in Germany between the onset of the outbreak in May 2022 and March 2023 and obtained demographic and clinical characteristics by standardized case report forms. RESULTS: A total of twelve patients with mpox were treated with tecovirimat in Germany in the study period. All but one patient identified as men who have sex with men (MSM) who were most likely infected with mpox virus (MPXV) through sexual contact. Eight of them were people living with HIV (PLWH), one of whom was newly diagnosed with HIV at the time of mpox, and four had CD4+ counts below 200/µl. Criteria for treatment with tecovirimat included severe immunosuppression, severe generalized and/or protracted symptoms, a high or increasing number of lesions, and the type and location of lesions (e.g., facial or oral soft tissue involvement, imminent epiglottitis, or tonsillar swelling). Patients were treated with tecovirimat for between six and 28 days. Therapy was generally well-tolerated, and all patients showed clinical resolution. CONCLUSIONS: In this cohort of twelve patients with severe mpox, treatment with tecovirimat was well tolerated and all individuals showed clinical improvement.

Orthobunyaviruses: From Virus Binding to Penetration into Mammalian Host Cells.

With over 80 members worldwide, Orthobunyavirus is the largest genus in the Peribunyaviridae family. Orthobunyaviruses (OBVs) are arthropod-borne viruses that are structurally simple, with a trisegmented, negative-sense RNA genome and only four structural proteins. OBVs are potential agents of emerging and re-emerging diseases and overall represent a global threat to both public and veterinary health. The focus of this review is on the very first steps of OBV infection in mammalian hosts, from virus binding to penetration and release of the viral genome into the cytosol. Here, we address the most current knowledge and advances regarding OBV receptors, endocytosis, and fusion.

Novel Toscana Virus Reverse Genetics System Establishes NSs as an Antagonist of Type I Interferon Responses.

The sand fly-borne Toscana virus (TOSV) is the major cause of human meningoencephalitis in the Mediterranean basin during the summer season. In this work, we have developed a T7 RNA polymerase-driven reverse genetics system to recover infectious particles of a lineage B strain of TOSV. The viral protein pattern and growth properties of the rescued virus (rTOSV) were found to be similar to those of the corresponding wild-type (wt) virus. Using this system, we genetically engineered a TOSV mutant lacking expression of the non-structural protein NSs (rTOSVɸNSs). Unlike rTOSV and the wt virus, rTOSVɸNSs was unable to (i) suppress interferon (IFN)-b messenger RNA induction; and (ii) grow efficiently in cells producing IFN-b. Together, our results highlight the importance of NSs for TOSV in evading the IFN response and provide a comprehensive toolbox to investigate the TOSV life cycle in mammalian and insect host cells, including several novel polyclonal antibodies.