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Vaccines of outstanding efficiency, safety, and public acceptance are needed to halt the current SARS-CoV-2 pandemic. Concerns include potential side effects caused by the antigen itself and safety of viral DNA and RNA delivery vectors. The large SARS-CoV-2 spike (S) protein is the main target of current COVID-19 vaccine candidates but can induce non-neutralizing antibodies, which might cause vaccination-induced complications or enhancement of COVID-19 disease. Besides, encoding of a functional S in replication-competent virus vector vaccines may result in the emergence of viruses with altered or expanded tropism. Here, we have developed a safe single round rhabdovirus replicon vaccine platform for enhanced presentation of the S receptor-binding domain (RBD). Structure-guided design was employed to build a chimeric minispike comprising the globular RBD linked to a transmembrane stem-anchor sequence derived from rabies virus (RABV) glycoprotein (G). Vesicular stomatitis virus (VSV) and RABV replicons encoding the minispike not only allowed expression of the antigen at the cell surface but also incorporation into the envelope of secreted non-infectious particles, thus combining classic vector-driven antigen expression and particulate virus-like particle (VLP) presentation. A single dose of a prototype replicon vaccine complemented with VSV G, VSVΔG-minispike-eGFP (G), stimulated high titers of SARS-CoV-2 neutralizing antibodies in mice, equivalent to those found in COVID-19 patients, and protected transgenic K18-hACE2 mice from COVID-19-like disease. Homologous boost immunization further enhanced virus neutralizing activity. The results demonstrate that non-spreading rhabdovirus RNA replicons expressing minispike proteins represent effective and safe alternatives to vaccination approaches using replication-competent viruses and/or the entire S antigen. Author summary: Two critical problems are associated with replicating paramyxo- and rhabdovirus vaccines expressing SARS-CoV-2 spike (S) protein such as VSVΔG(S). One is eliciting of potentially disease-enhancing non-neutralizing antibodies, the other the S-mediated spread in humans. In view of the multi-organ tropism of SARS-CoV-2 in humans, their pathogenic outcome is not predictable. Here, we address and resolve both issues. We describe an innovative VSV vaccine, which is safe both in terms of virus propagation and immune response, as it is a non-spreading single round replicon vector, and the immunogen is limited to the spike's receptor binding domain (RBD), which emerged as the antigen eliciting the desired virus-neutralizing antibodies in humans. An excellent protective immune response in animals is achieved by the design of a chimeric RBD-minispike, which allows a combined "2-in-1" approach, meaning that the optimized antigen is simultaneously presented on cells and on noninfectious virus-like particles (VLPs). With such enhanced RBD antigen presentation it is thus not necessary to use replication-competent virus or entire S antigen. [ABSTRACT FROM AUTHOR]

Formation of cytoplasmic inclusion bodies (IBs) is a hallmark of infections with non-segmented negative-strand RNA viruses (order Mononegavirales). We show here that Nipah virus (NiV), a bat-derived highly pathogenic member of the Paramyxoviridae family, differs from mononegaviruses of the Rhabdo-, Filo- and Pneumoviridae families by forming two types of IBs with distinct localizations, formation kinetics, and protein compositions. IBs in the perinuclear region form rapidly upon expression of the nucleocapsid proteins. These IBperi are highly mobile and associate with the aggresome marker y-tubulin. IBperi can recruit unrelated overexpressed cytosolic proteins but do not contain the viral matrix (M) protein. Additionally, NiV forms an as yet undescribed IB population at the plasma membrane (IBPM) that is y-tubulin-negative but contains the M protein. Infection studies with recombinant NiV revealed that IBPM require the M protein for their formation, and most likely represent sites of NiV assembly and budding. The identification of this novel type of plasma membrane-associated IBs not only provides new insights into NiV biology and may open new avenues to develop novel antiviral approaches to treat these highly pathogenic viruses, it also provides a basis for a more detailed characterization of IBs and their role in virus assembly and replication in infections with other Mononegavirales. [ABSTRACT FROM AUTHOR]

Kyasanur Forest disease virus (KFDV) and Alkhumra hemorrhagic fever virus (AHFV) are genetically closely-related, tick-borne flaviviruses that cause severe, often fatal disease in humans. Flaviviruses in the tick-borne encephalitis (TBE) complex typically cause neurological disease in humans whereas patients infected with KFDV and AHFV predominately present with hemorrhagic fever. A small animal model for KFDV and AHFV to study the pathogenesis and evaluate countermeasures has been lacking mostly due to the need of a high biocontainment laboratory to work with the viruses. To evaluate the utility of an existing mouse model for tick-borne flavivirus pathogenesis, we performed serial sacrifice studies in BALB/c mice infected with either KFDV strain P9605 or AHFV strain Zaki-1. Strikingly, infection with KFDV was completely lethal in mice, while AHFV caused no clinical signs of disease and no animals succumbed to infection. KFDV and high levels of pro-inflammatory cytokines were detected in the brain at later time points, but no virus was found in visceral organs; conversely, AHFV Zaki-1 and elevated levels of cytokines were found in the visceral organs at earlier time points, but were not detected in the brain. While infection with either virus caused a generalized leukopenia, only AHFV Zaki-1 induced hematologic abnormalities in infected animals. Our data suggest that KFDV P9605 may have lost its ability to cause hemorrhagic disease as the result of multiple passages in suckling mouse brains. However, likely by virtue of fewer mouse passages, AHFV Zaki-1 has retained the ability to replicate in visceral organs, cause hematologic abnormalities, and induce pro-inflammatory cytokines without causing overt disease. Given these striking differences, the use of inbred mice and the virus passage history need to be carefully considered in the interpretation of animal studies using these viruses. Author Summary: Kyasanur Forest disease virus (KFDV) and Alkhumra hemorrhagic fever virus (AHFV) are tick-borne flaviviruses that cause severe hemorrhagic disease in humans. The pathogenesis of the disease is still not very well understood mostly due to the lack of suitable animal models. Despite sharing a high degree of genetic sequence similarity, KFDV replicates primarily in the brain and is uniformly lethal for BALB/c mice. In contrast, AHFV does not cause clinically overt signs in mice, replicates in the visceral organs, and induces pro-inflammatory cytokines and hematological changes. Given the striking differences in pathogenesis and tissue tropism, the use of inbred mice as well as the passage history of the virus needs to be carefully considered in the interpretation of animal studies using these viruses. [ABSTRACT FROM AUTHOR]