Work with infectious Ebola viruses is restricted to biosafety level 4 (BSL4) laboratories, presenting a significant barrier for studying these viruses. Life cycle modeling systems, including minigenome systems and transcription- and replication-competent virus-like particle (trVLP) systems, allow modeling of the virus life cycle under BSL2 conditions; however, all current systems model only certain aspects of the virus life cycle, rely on plasmid-based viral protein expression, and have been used to model only single infectious cycles. We have developed a novel life cycle modeling system allowing continuous passaging of infectious trVLPs containing a tetracistronic minigenome that encodes a reporter and the viral proteins VP40, VP24, and GP _1,2 . This system is ideally suited for studying morphogenesis, budding, and entry, in addition to genome replication and transcription. Importantly, the specific infectivity of trVLPs in this system was ∼500-fold higher than that in previous systems. Using this system for functional studies of VP24, we showed that, contrary to previous reports, VP24 only very modestly inhibits genome replication and transcription when expressed in a regulated fashion, which we confirmed using infectious Ebola viruses. Interestingly, we also discovered a genome length-dependent effect of VP24 on particle infectivity, which was previously undetected due to the short length of monocistronic minigenomes and which is due at least partially to a previously unknown function of VP24 in RNA packaging. Based on our findings, we propose a model for the function of VP24 that reconciles all currently available data regarding the role of VP24 in nucleocapsid assembly as well as genome replication and transcription. IMPORTANCE Ebola viruses cause severe hemorrhagic fevers in humans, with no countermeasures currently being available, and must be studied in maximum-containment laboratories. Only a few of these laboratories exist worldwide, limiting our ability to study Ebola viruses and develop countermeasures. Here we report the development of a novel reverse genetics-based system that allows the study of Ebola viruses without maximum-containment laboratories. We used this system to investigate the Ebola virus protein VP24, showing that, contrary to previous reports, it only modestly inhibits virus genome replication and transcription but is important for packaging of genomes into virus particles, which constitutes a previously unknown function of VP24 and a potential antiviral target. We further propose a comprehensive model for the function of VP24 in nucleocapsid assembly. Importantly, on the basis of this approach, it should easily be possible to develop similar experimental systems for other viruses that are currently restricted to maximum-containment laboratories.

Widespread circulation of SARS-CoV-2 in humans raises the theoretical risk of reverse zoonosis events with wildlife, reintroductions of SARS-CoV-2 into permissive nondomesticated animals. Here we report that North American deer mice (Peromyscus maniculatus) are susceptible to SARS-CoV-2 infection following intranasal exposure to a human isolate, resulting in viral replication in the upper and lower respiratory tract with little or no signs of disease. Further, shed infectious virus is detectable in nasal washes, oropharyngeal and rectal swabs, and viral RNA is detectable in feces and occasionally urine. We further show that deer mice are capable of transmitting SARS-CoV-2 to naïve deer mice through direct contact. The extent to which these observations may translate to wild deer mouse populations remains unclear, and the risk of reverse zoonosis and/or the potential for the establishment ofPeromyscusrodents as a North American reservoir for SARS-CoV-2 remains unknown.

Vesicular stomatitis virus (VSV), which belongs to the Vesiculovirus genus of the family Rhabdoviridae, is a well studied livestock pathogen and prototypic non-segmented, negative-sense RNA virus. Although VSV is responsible for causing economically significant outbreaks of vesicular stomatitis in cattle, horses, and swine, the virus also represents a valuable research tool for molecular biologists and virologists. Indeed, the establishment of a reverse genetics system for the recovery of infectious VSV from cDNA transformed the utility of this virus and paved the way for its use as a vaccine vector. A highly effective VSV-based vaccine against Ebola virus recently received clinical approval, and many other VSV-based vaccines have been developed, particularly for high-consequence viruses. This review seeks to provide a holistic but concise overview of VSV, covering the virus’s ascension from perennial agricultural scourge to promising medical countermeasure, with a particular focus on vaccines.

Many viruses have evolved strategies to counteract cellular immune responses, including apoptosis. Vaccinia virus, a member of the poxvirus family, encodes an antiapoptotic protein, F1L. F1L localizes to mitochondria and inhibits apoptosis by preventing the release of cytochrome c by an undetermined mechanism (S. T. Wasilenko, T. L. Stewart, A. F. Meyers, and M. Barry, Proc. Natl. Acad. Sci. USA 100: 14345-14350, 2003; T. L. Stewart, S. T. Wasilenko, and M. Barry, J. Virol. 79: 1084-1098, 2005). Here, we show that in the absence of an apoptotic stimulus, F1L associates with Bak, a proapoptotic member of the Bcl-2 family that plays a pivotal role in the release of cytochrome c . Cells infected with vaccinia virus were resistant to Bak oligomerization and the initial N-terminal exposure of Bak following the induction of apoptosis with staurosporine. A mutant vaccinia virus missing F1L was no longer able to inhibit apoptosis or Bak activation. In addition, the expression of F1L was essential to inhibit tBid-induced cytochrome c release in both wild-type murine embryonic fibroblasts (MEFs) and Bax-deficient MEFs, indicating that F1L could inhibit apoptosis in the presence and absence of Bax. tBid-induced Bak oligomerization and N-terminal exposure of Bak in Bax-deficient MEFs were inhibited during virus infection, as assessed by cross-linking and limited trypsin proteolysis. Infection with the F1L deletion virus no longer provided protection from tBid-induced Bak activation and apoptosis. Additionally, infection of Jurkat cells with the F1L deletion virus resulted in cellular apoptosis, as measured by loss of the inner mitochondrial membrane potential, caspase 3 activation, and cytochrome c release, indicating that the presence of F1L was pivotal for inhibiting vaccinia virus-induced apoptosis. Our data indicate that F1L expression during infection inhibits apoptosis and interferes with the activation of Bak.

The recent emergence of the monkeypox virus (MPXV) in non-endemic countries has been designated a Public Health Emergency of International Concern by the World Health Organization. There are currently no approved treatments for MPXV infection in the United States or Canada. The antiviral drug tecovirimat (commonly called TPOXX), previously approved for smallpox treatment, is currently being deployed for treatment of MPXV infections where available based on previously accrued data. We tested the efficacy of TPOXX both in vitro and in vivo against a clade 2 Canadian 2022 isolate of MPXV isolated during the current outbreak. TPOXX prevented MPXV replication in vitro with an effective concentration in the nanomolar range. To evaluate TPOXX efficacy in vivo, we first characterized the CAST/EiJ mouse model with the same 2022 Canadian isolate. Unlike previous descriptions of this model, the Canadian isolate was not lethal in CAST/EiJ mice, although it replicated efficiently in the respiratory tract after intranasal infection. Subsequent experiments demonstrated that daily oral TPOXX treatment markedly reduced viral titers in the tissues 1 and 2 weeks after infection. Our data indicate that TPOXX is highly effective against currently circulating MPXV strains and could be an important contributor to curbing the ongoing outbreak.