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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of human coronavirus disease 2019 (COVID-19), emerged in Wuhan, China, in December 2019. The virus rapidly spread globally, resulting in a public health crisis including almost 5 million cases and 323,256 deaths as of May 21, 2020. Here, we describe the identification and evaluation of commercially available reagents and assays for the molecular detection of SARS-CoV-2 in infected FFPE cell pellets. We identified a suitable rabbit polyclonal anti-SARS-CoV spike protein antibody and a mouse monoclonal anti-SARS-CoV nucleocapsid protein (NP) antibody for cross-detection of the respective SARS-CoV-2 proteins by IHC and immunofluorescence assay (IFA). Next, we established RNAscope in situ hybridization (ISH) to detect SARS-CoV-2 RNA. Furthermore, we established a multiplex FISH (mFISH) to detect positive-sense SARS-CoV-2 RNA and negative-sense SARS-CoV-2 RNA (a replicative intermediate indicating viral replication). Finally, we developed a dual staining assay using IHC and ISH to detect SARS-CoV-2 antigen and RNA in the same FFPE section. It is hoped that these reagents and assays will accelerate COVID-19 pathogenesis studies in humans and in COVID-19 animal models.

Ebola virus (EBOV), a member of the mononegaviral family Filoviridae , causes severe disease associated with high lethality in humans. Despite enormous progress in development of EBOV medical countermeasures, no anti-EBOV treatment has been approved. We designed an immunotoxin in which a single-chain variable region fragment of the EBOV glycoprotein-specific monoclonal antibody 6D8 was fused to the effector domains of Pseudomonas aeruginosa exotoxin A (PE38). This immunotoxin, 6D8-PE38, bound specifically to cells expressing EBOV glycoproteins. Importantly, 6D8-PE38 targeted EBOV-infected cells, as evidenced by inhibition of infectious EBOV production from infected cells, including primary human macrophages. The data presented here provide a proof of concept for immunotoxin-based targeted killing of infected cells as a potential antiviral intervention for Ebola virus disease.

Viral receptor identified The transferrin receptor 1 (TfR1) has been identified as the cellular receptor for four New World arenaviruses — the Junin, Machupo, Guanarito and Sabia viruses. This class of arenaviruses is important because they cause fatal haemorrhagic fevers. Treating cultured cells with an antibody against TfR1 blocks viral entry and replication. Antibodies that limit arenavirus replication without interfering with host iron metabolism may be effective in controlling outbreaks of New World haemorrhagic fever. At least five arenaviruses cause viral haemorrhagic fevers in humans. Lassa virus, an Old World arenavirus, uses the cellular receptor α-dystroglycan to infect cells 1 . Machupo, Guanarito, Junin and Sabia viruses are New World haemorrhagic fever viruses that do not use α-dystroglycan 2 . Here we show a specific, high-affinity association between transferrin receptor 1 (TfR1) and the entry glycoprotein (GP) of Machupo virus. Expression of human TfR1, but not human transferrin receptor 2, in hamster cell lines markedly enhanced the infection of viruses pseudotyped with the GP of Machupo, Guanarito and Junin viruses, but not with those of Lassa or lymphocytic choriomeningitis viruses. An anti-TfR1 antibody efficiently inhibited the replication of Machupo, Guanarito, Junin and Sabia viruses, but not that of Lassa virus. Iron depletion of culture medium enhanced, and iron supplementation decreased, the efficiency of infection by Junin and Machupo but not Lassa pseudoviruses. These data indicate that TfR1 is a cellular receptor for New World haemorrhagic fever arenaviruses.

The ability of a New World (NW) clade B arenavirus to enter cells using human transferrin receptor 1 (TfR1) strictly correlates with its ability to cause hemorrhagic fever. Amapari (AMAV) and Tacaribe (TCRV), two nonpathogenic NW clade B arenaviruses that do not use human TfR1, are closely related to the NW arenaviruses that cause hemorrhagic fevers. Here we show that pseudotyped viruses bearing the surface glycoprotein (GP) of AMAV or TCRV can infect cells using the TfR1 orthologs of several mammalian species, including those of their respective natural hosts, the small rodent Neacomys spinosus and the fruit bat Artibeus jamaicensis. Mutation of one residue in human TfR1 makes it a functional receptor for TCRV, and mutation of four residues makes it a functional receptor for AMAV. Our data support an in vivo role for TfR1 in the replication of most, if not all, NW clade B arenaviruses, and suggest that with modest changes in their GPs the nonpathogenic arenaviruses could use human TfR1 and emerge as human pathogens.

The endosomal sorting complex ESCRT-III, which is formed by the structurally related CHMP proteins, is engaged by HIV-1 to promote viral budding. Here we show that progressive truncations into the C-terminal acidic domains of CHMP proteins trigger an increasingly robust anti-HIV budding activity. Together with biochemical evidence for specific intramolecular interactions between the basic and acidic halves of CHMP3 and CHMP4B, these results suggest that the acidic domains are autoinhibitory. The acidic half of CHMP3 also interacts with the endosome-associated ubiquitin isopeptidase AMSH, and the coexpression of AMSH or its CHMP3-binding domain converts wild-type CHMP3 into a potent inhibitor of HIV-1 release. Point mutations in CHMP3 that prevent binding to AMSH abrogate this effect, suggesting that binding to AMSH relieves the autoinhibition of CHMP3. Collectively, our results indicate that CHMP proteins are regulated through an autoinhibitory switch mechanism that allows tight control of ESCRT-III assembly.

Ebola virus (EBOV) persistence in asymptomatic humans and Ebola virus disease (EVD) sequelae have emerged as significant public health concerns since the 2013–2016 EVD outbreak in Western Africa. Until now, studying how EBOV disseminates into and persists in immune-privileged sites was impossible due to the absence of a suitable animal model. Here, we detect persistent EBOV replication coinciding with systematic inflammatory responses in otherwise asymptomatic rhesus monkeys that had survived infection in the absence of or after treatment with candidate medical countermeasures. We document progressive EBOV dissemination into the eyes, brain and testes through vascular structures, similar to observations in humans. We identify CD68 + cells (macrophages/monocytes) as the cryptic EBOV reservoir cells in the vitreous humour and its immediately adjacent tissue, in the tubular lumina of the epididymides, and in foci of histiocytic inflammation in the brain, but not in organs typically affected during acute infection. In conclusion, our data suggest that persistent EBOV infection in rhesus monkeys could serve as a model for persistent EBOV infection in humans, and we demonstrate that promising candidate medical countermeasures may not completely clear EBOV infection. A rhesus monkey model may lay the foundation to study EVD sequelae and to develop therapies to abolish EBOV persistence. A lack of models has hindered the study of Ebola virus (EBOV) persistence and sequelae, which are now shown to occur in rhesus monkeys. Progressive EBOV spread into eyes, brain and testes with CD68 + cells as the cryptic EBOV reservoir.

Lassa virus (LASV), a mammarenavirus, infects an estimated 100,000–300,000 individuals yearly in western Africa and frequently causes lethal disease. Currently, no LASV-specific antivirals or vaccines are commercially available for prevention or treatment of Lassa fever, the disease caused by LASV. The development of medical countermeasure screening platforms is a crucial step to yield licensable products. Using reverse genetics, we generated a recombinant wild-type LASV (rLASV-WT) and a modified version thereof encoding a cleavable green fluorescent protein (GFP) as a reporter for rapid and quantitative detection of infection (rLASV-GFP). Both rLASV-WT and wild-type LASV exhibited similar growth kinetics in cultured cells, whereas growth of rLASV-GFP was slightly impaired. GFP reporter expression by rLASV-GFP remained stable over several serial passages in Vero cells. Using two well-characterized broad-spectrum antivirals known to inhibit LASV infection, favipiravir and ribavirin, we demonstrate that rLASV-GFP is a suitable screening tool for the identification of LASV infection inhibitors. Building on these findings, we established a rLASV-GFP-based high-throughput drug discovery screen and an rLASV-GFP-based antibody neutralization assay. Both platforms, now available as a standard tool at the IRF-Frederick (an international resource), will accelerate anti-LASV medical countermeasure discovery and reduce costs of antiviral screens in maximum containment laboratories.

Little is known about the repertoire of cellular factors involved in the replication of pathogenic alphaviruses. To uncover molecular regulators of alphavirus infection, and to identify candidate drug targets, we performed a high-content imaging-based siRNA screen. We revealed an actin-remodeling pathway involving Rac1, PIP5K1- α, and Arp3, as essential for infection by pathogenic alphaviruses. Infection causes cellular actin rearrangements into large bundles of actin filaments termed actin foci. Actin foci are generated late in infection concomitantly with alphavirus envelope (E2) expression and are dependent on the activities of Rac1 and Arp3. E2 associates with actin in alphavirus-infected cells and co-localizes with Rac1-PIP5K1-α along actin filaments in the context of actin foci. Finally, Rac1, Arp3, and actin polymerization inhibitors interfere with E2 trafficking from the trans-Golgi network to the cell surface, suggesting a plausible model in which transport of E2 to the cell surface is mediated via Rac1- and Arp3-dependent actin remodeling.

Segmented negative-sense viruses of the family Arenaviridae encode a large polymerase (L) protein that contains all of the enzymatic activities required for RNA synthesis. These activities include an RNA-dependent RNA polymerase (RdRP) and an RNA endonuclease that cleaves capped primers from cellular mRNAs to prime transcription. Using purified catalytically active Machupo virus L, we provide a view of the overall architecture of this multifunctional polymerase and reconstitute complex formation with an RNA template in vitro. The L protein contains a central ring domain that is similar in appearance to the RdRP of dsRNA viruses and multiple accessory appendages that may be responsible for 5' cap formation. RNA template recognition by L requires a sequence-specific motif located at positions 2-5 in the 3' terminus of the viral genome. Moreover, L-RNA complex formation depends on single-stranded RNA, indicating that inter-termini dsRNA interactions must be partially broken for complex assembly to occur. Our results provide a model for arenavirus polymerase-template interactions and reveal the structural organization of a negative-strand RNA virus L protein.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a recently isolated betacoronavirus identified as the etiologic agent of a frequently fatal disease in Western Asia, Middle East respiratory syndrome. Attempts to identify the natural reservoirs of MERS-CoV have focused in part on dromedaries. Bats are also suspected to be reservoirs based on frequent detection of other betacoronaviruses in these mammals. For this study, ten distinct cell lines derived from bats of divergent species were exposed to MERS-CoV. Plaque assays, immunofluorescence assays, and transmission electron microscopy confirmed that six bat cell lines can be productively infected. We found that the susceptibility or resistance of these bat cell lines directly correlates with the presence or absence of cell surface-expressed CD26/DPP4, the functional human receptor for MERS-CoV. Human anti-CD26/DPP4 antibodies inhibited infection of susceptible bat cells in a dose-dependent manner. Overexpression of human CD26/DPP4 receptor conferred MERS-CoV susceptibility to resistant bat cell lines. Finally, sequential passage of MERS-CoV in permissive bat cells established persistent infection with concomitant downregulation of CD26/DPP4 surface expression. Together, these results imply that bats indeed could be among the MERS-CoV host spectrum, and that cellular restriction of MERS-CoV is determined by CD26/DPP4 expression rather than by downstream restriction factors.