Depletion of Bone Marrow Hematopoietic Cells in Ebolavirus-Infected Rhesus Macaques: A Possible Cause of Hematologic Abnormalities in Ebolavirus Disease.

The pathophysiology of long-recognized hematologic abnormalities in Ebolavirus (EBOV) disease (EVD) is unknown. From limited human sampling (of peripheral blood), it has been postulated that emergency hematopoiesis plays a role in severe EVD, but the systematic characterization of the bone marrow (BM) has not occurred in human disease or in nonhuman primate models. In a lethal rhesus macaque model of EVD, 18 sternal BM samples exposed to the Kikwit strain of EBOV were compared to those from uninfected controls (n = 3). Immunohistochemistry, RNAscope in situ hybridization, transmission electron microscopy, and confocal microscopy showed that EBOV infects BM monocytes/macrophages and megakaryocytes. EBOV exposure was associated with severe BM hypocellularity, including depletion of myeloid, erythroid, and megakaryocyte hematopoietic cells. These depletions were negatively correlated with cell proliferation (Ki67 expression) and were not associated with BM apoptosis during disease progression. In EBOV-infected rhesus macaques with terminal disease, BM showed marked hemophagocytosis, megakaryocyte emperipolesis, and the release of immature hematopoietic cells into the sinusoids. Collectively, these data demonstrate not only direct EBOV infection of BM monocytes/macrophages and megakaryocytes but also that disease progression is associated with hematopoietic failure, notably in peripheral cytopenia. These findings inform current pathophysiologic unknowns and suggest a crucial role for BM dysfunction and/or failure, including emergency hematopoiesis, as part of the natural history of severe human disease.

Ebola virus infection induces a delayed type I IFN response in bystander cells and the shutdown of key liver genes in human iPSC-derived hepatocytes.

Liver damage and an exacerbated inflammatory response are hallmarks of Ebola virus (EBOV) infection. Little is known about the intrinsic response to infection in human hepatocytes and their contribution to inflammation. Here, we present an induced pluripotent stem cell (iPSC)-derived hepatocyte-like cell (HLC) platform to define the hepato-intrinsic response to EBOV infection. We used this platform to show robust EBOV infection, with characteristic ultrastructural changes and evidence for viral replication. Transcriptomics analysis revealed a delayed response with minimal early transcriptomic changes, followed by a general downregulation of hepatic function and upregulation of interferon signaling, providing a potential mechanism by which hepatocytes participate in disease severity and liver damage. Using RNA-fluorescence in situ hybridization (FISH), we showed that IFNB1 and CXCL10 were mainly expressed in non-infected bystander cells. We did not observe an inflammatory signature during infection. In conclusion, iPSC-HLCs are an immune competent platform to study responses to EBOV infection.

Natural Occurring Polymorphisms in HIV-1 Integrase and RNase H Regulate Viral Release and Autoprocessing.

Recently, a genome-wide association study using plasma HIV RNA from antiretroviral therapy-naive patients reported that 14 naturally occurring nonsynonymous single-nucleotide polymorphisms (SNPs) in HIV derived from antiretrovirus drug-naive patients were associated with virus load (VL). Those SNPs were detected in reverse transcriptase, RNase H, integrase, envelope, and Nef. However, the impact of each mutation on viral fitness was not investigated. Here, we constructed a series of HIV variants encoding each SNP and examined their replicative abilities. An HIV variant containing a Met-to-Ile change at codon 50 in integrase [HIV(IN:M50I)] was found as an impaired virus. Despite the mutation being in integrase, the virus release was significantly suppressed (P < 0.001). Transmission electron microscopy analysis revealed that abnormal bud accumulation on the plasma membrane and the released virus particles retained immature forms. Western blot analysis demonstrated a defect in autoprocessing of GagPol and Gag polyproteins' autoprocessing in the HIV(IN:M50I) particles, although Förster resonance energy transfer (FRET) assay displayed that GagPol containing IN:M50I forms a homodimer with a similar efficiency with GagPol (wild type). The impaired maturation and replication were rescued by two other VL-associated SNPs, Ser-to-Asn change at codon 17 of integrase and Asn-to-Ser change at codon 79 of RNase H. These data demonstrate that Gag and GagPol assembly, virus release, and autoprocessing are regulated by not only integrase but also RNase H. IMPORTANCE Nascent HIV-1 is a noninfectious viral particle. Cleaving Gag and GagPol polyproteins in the particle by mature HIV protease (PR), the nascent virus becomes an infectious virus. PR is initially translated as an inactive embedded enzyme in a GagPol polyprotein. The embedded PR in homodimerized GagPol polyproteins catalyzes a proteolytic reaction to release the mature PR. This excision step by self-cleavage is called autoprocessing. Here, during the evaluation of the roles of naturally emerging nonsynonymous SNPs in HIV RNA, we found that autoprocessing is inhibited by Met-to-Ile change at codon 50 in integrase GagPol. Other coexisting SNPs, Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser mutation at codon 79 in RNase H, recovered this defect, suggesting that autoprocessing is regulated by not only integrase but also RNase H in GagPol polyprotein.

Acute Late-Stage Myocarditis in the Crab-Eating Macaque Model of Hemorrhagic Smallpox.

Hemorrhagic smallpox, caused by variola virus (VARV), was a rare but nearly 100% lethal human disease manifestation. Hemorrhagic smallpox is frequently characterized by secondary bacterial infection, coagulopathy, and myocardial and subendocardial hemorrhages. Previous experiments have demonstrated that intravenous (IV) cowpox virus (CPXV) exposure of macaques mimics human hemorrhagic smallpox. The goal of this experiment was to further understand the onset, nature, and severity of cardiac pathology and how it may contribute to disease. The findings support an acute late-stage myocarditis with lymphohistiocytic infiltrates in the CPXV model of hemorrhagic smallpox.

FIB-SEM as a Volume Electron Microscopy Approach to Study Cellular Architectures in SARS-CoV-2 and Other Viral Infections: A Practical Primer for a Virologist.

The visualization of cellular ultrastructure over a wide range of volumes is becoming possible by increasingly powerful techniques grouped under the rubric "volume electron microscopy" or volume EM (vEM). Focused ion beam scanning electron microscopy (FIB-SEM) occupies a "Goldilocks zone" in vEM: iterative and automated cycles of milling and imaging allow the interrogation of microns-thick specimens in 3-D at resolutions of tens of nanometers or less. This bestows on FIB-SEM the unique ability to aid the accurate and precise study of architectures of virus-cell interactions. Here we give the virologist or cell biologist a primer on FIB-SEM imaging in the context of vEM and discuss practical aspects of a room temperature FIB-SEM experiment. In an in vitro study of SARS-CoV-2 infection, we show that accurate quantitation of viral densities and surface curvatures enabled by FIB-SEM imaging reveals SARS-CoV-2 viruses preferentially located at areas of plasma membrane that have positive mean curvatures.

Peripheral Neuronopathy Associated With Ebola Virus Infection in Rhesus Macaques: A Possible Cause of Neurological Signs and Symptoms in Human Ebola Patients.

Neurological signs and symptoms are the most common complications of Ebola virus disease. However, the mechanisms underlying the neurologic manifestations in Ebola patients are not known. In this study, peripheral ganglia were collected from 12 rhesus macaques that succumbed to Ebola virus (EBOV) disease from 5 to 8 days post exposure. Ganglionitis, characterized by neuronal degeneration, necrosis, and mononuclear leukocyte infiltrates, was observed in the dorsal root, autonomic, and enteric ganglia. By immunohistochemistry, RNAscope in situ hybridization, transmission electron microscopy, and confocal microscopy, we confirmed that CD68+ macrophages are the target cells for EBOV in affected ganglia. Further, we demonstrated that EBOV can induce satellite cell and neuronal apoptosis and microglial activation in infected ganglia. Our results demonstrate that EBOV can infect peripheral ganglia and results in ganglionopathy in rhesus macaques, which may contribute to the neurological signs and symptoms observed in acute and convalescent Ebola virus disease in human patients.

Filoviruses Infect Rhesus Macaque Synoviocytes in Vivo and Primary Human Synoviocytes in Vitro.

The most commonly reported symptom of post-Ebola virus disease syndrome in survivors is arthralgia, yet involvement of the joints in acute or convalescent Ebola virus infection is not well characterized in human patients or animal models. Through immunohistochemistry, we found that the lining synovial intima of the stifle (knee) is a target for acute infection by Ebola virus/Kikwit, Ebola virus/Makona-C05, and Marburg virus/Angola in the rhesus macaque model. Furthermore, histologic analysis, immunohistochemistry, RNAscope in situ hybridization, and transmission electron microscopy showed that synoviocytes of the stifle, shoulder, and hip are a target for mouse-adapted Ebola virus/Yambuku-Mayinga infection during acute disease in rhesus macaques. A time course of infection study with Ebola virus/Kikwit found that the large joint synovium became immunopositive beginning on postinfection day 6. In total, the synovium of 28 of 30 rhesus macaques with terminal filovirus disease had evidence of infection (64 of 96 joints examined). On the basis of immunofluorescence, infected cell types included CD68+ type A (macrophage-like) synoviocytes and CD44+ type B (fibroblast-like) synoviocytes. Cultured primary human fibroblast-like synoviocytes were permissive to infection with Ebola and Marburg viruses in vitro. Because synovial joints include immune privileged sites, these findings are significant for future investigations of filovirus pathogenesis and persistence as well as arthralgias in acute and convalescent filovirus disease.

Clinical Characterization of Host Response to Simian Hemorrhagic Fever Virus Infection in Permissive and Refractory Hosts: A Model for Determining Mechanisms of VHF Pathogenesis.

Simian hemorrhagic fever virus (SHFV) causes a fulminant and typically lethal viral hemorrhagic fever (VHF) in macaques (Cercopithecinae: Macaca spp.) but causes subclinical infections in patas monkeys (Cercopithecinae: Erythrocebus patas). This difference in disease course offers a unique opportunity to compare host responses to infection by a VHF-causing virus in biologically similar susceptible and refractory animals. Patas and rhesus monkeys were inoculated side-by-side with SHFV. Unlike the severe disease observed in rhesus monkeys, patas monkeys developed a limited clinical disease characterized by changes in complete blood counts, serum chemistries, and development of lymphadenopathy. Viral RNA was measurable in circulating blood 2 days after exposure, and its duration varied by species. Infectious virus was detected in terminal tissues of both patas and rhesus monkeys. Varying degrees of overlap in changes in serum concentrations of interferon (IFN)-γ, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-6 were observed between patas and rhesus monkeys, suggesting the presence of common and species-specific cytokine responses to infection. Similarly, quantitative immunohistochemistry of livers from terminal monkeys and whole blood flow cytometry revealed varying degrees of overlap in changes in macrophages, natural killer cells, and T-cells. The unexpected degree of overlap in host response suggests that relatively small subsets of a host's response to infection may be responsible for driving hemorrhagic fever pathogenesis. Furthermore, comparative SHFV infection in patas and rhesus monkeys offers an experimental model to characterize host⁻response mechanisms associated with viral hemorrhagic fever and evaluate pan-viral hemorrhagic fever countermeasures.

Persistence of Lassa Virus Associated With Severe Systemic Arteritis in Convalescing Guinea Pigs (Cavia porcellus).

Lassa fever (LF) survivors develop various clinical manifestations including polyserositis, myalgia, epididymitis, and hearing loss weeks to months after recovery from acute infection. We demonstrate a systemic lymphoplasmacytic and histiocytic arteritis and periarteritis in guinea pigs more than 2 months after recovery from acute Lassa virus (LASV) infection. LASV was detected in the arterial tunica media smooth muscle cells by immunohistochemistry, in situ hybridization, and transmission electron microscopy. Our results suggest that the sequelae of LASV infection may be due to virus persistence resulting in systemic vascular damage. These findings shed light on the pathogenesis of LASV sequelae in convalescent human survivors.

New Insights Into Marburg Virus Disease Pathogenesis in the Rhesus Macaque Model.

Previously, several studies have been performed to delineate the development and progression of Marburg virus infection in nonhuman primates (NHPs), primarily to clarify the mechanisms of severe (fatal) disease. After the 2013-2016 Ebola virus disease (EVD) epidemic in Western Africa, there has been a reassessment of the available filovirus animal models and the utility of these to faithfully recapitulate human disease. The high lethality of the NHP models has raised doubts as to their ability to provide meaningful data for the full spectrum of disease observed in humans. Of particular interest are the etiologic and pathophysiologic mechanisms underlying postconvalescent sequelae observed in human survivors of EVD and Marburg virus disease (MVD). In the current study, we evaluated the lesions of MVD in NHPs; however, in contrast to previous studies, we focused on the potential for development of sequelae similar to those reported in human survivors of MVD and EVD. We found that during acute MVD in the macaque model, there is frequent inflammation of peripheral nerves, autonomic ganglia, and the iris of the eye. Furthermore, we demonstrate viral infection of the ocular ciliary body and retina, testis, epididymis, ovary, oviduct, uterine endometrium, prostate, and mammary gland. These findings are relevant for both development of postconvalescent sequelae and the natural transmission of virus.

Development of a novel real-time polymerase chain reaction assay for the quantitative detection of Nipah virus replicative viral RNA.

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus that can result in severe pulmonary disease and fatal encephalitis in humans and is responsible for outbreaks in Bangladesh, Malaysia, Singapore, India and possibly the Philippines. NiV has a negative-sense RNA genome that contains six genes and serves as a template for production of viral mRNA transcripts. NiV mRNA transcripts are subsequently translated into viral proteins. Traditionally, NiV quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) assays have relied on using primer sets that amplify a target (N that encodes the nucleocapsid) within the coding region of the viral gene that also amplifies viral mRNA. Here we describe a novel one-step qRT-PCR assay targeting the intergenic region separating the viral F and G proteins, thereby eliminating amplification of the viral mRNA. This assay is more accurate than the traditional qRT-PCR in quantifying concentrations of viral genomic RNA.

Ebola Virus Localization in the Macaque Reproductive Tract during Acute Ebola Virus Disease.

Sexual transmission of Ebola virus (EBOV) has been demonstrated more than a year after recovery from the acute phase of Ebola virus disease (EVD). The mechanisms underlying EBOV persistence and sexual transmission are not currently understood. Using the acute macaque model of EVD, we hypothesized EBOV would infect the reproductive tissues and sought to localize the infection in these tissues using immunohistochemistry and transmission electron microscopy. In four female and eight male macaques that succumbed to EVD between 6 and 9 days after EBOV challenge, we demonstrate widespread EBOV infection of the interstitial tissues and endothelium in the ovary, uterus, testis, seminal vesicle, epididymis, and prostate gland, with minimal associated tissue immune response or organ pathology. Given the widespread involvement of EBOV in the reproductive tracts of both male and female macaques, it is reasonable to surmise that our understanding of the mechanisms underlying sexual transmission of EVD and persistence of EBOV in immune-privileged sites would be facilitated by the development of a nonhuman primate model in which the macaques survived past the acute stage into convalescence.

One-Health: a Safe, Efficient, Dual-Use Vaccine for Humans and Animals against Middle East Respiratory Syndrome Coronavirus and Rabies Virus.

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012 and is a highly pathogenic respiratory virus. There are no treatment options against MERS-CoV for humans or animals, and there are no large-scale clinical trials for therapies against MERS-CoV. To address this need, we developed an inactivated rabies virus (RABV) that contains the MERS-CoV spike (S) protein expressed on its surface. Our initial recombinant vaccine, BNSP333-S, expresses a full-length wild-type MERS-CoV S protein; however, it showed significantly reduced viral titers compared to those of the parental RABV strain and only low-level incorporation of full-length MERS-CoV S into RABV particles. Therefore, we developed a RABV-MERS vector that contained the MERS-CoV S1 domain of the MERS-CoV S protein fused to the RABV G protein C terminus (BNSP333-S1). BNSP333-S1 grew to titers similar to those of the parental vaccine vector BNSP333, and the RABV G-MERS-CoV S1 fusion protein was efficiently expressed and incorporated into RABV particles. When we vaccinated mice, chemically inactivated BNSP333-S1 induced high-titer neutralizing antibodies. Next, we challenged both vaccinated mice and control mice with MERS-CoV after adenovirus transduction of the human dipeptidyl peptidase 4 (hDPP4) receptor and then analyzed the ability of mice to control MERS-CoV infection. Our results demonstrated that vaccinated mice were fully protected from the MERS-CoV challenge, as indicated by the significantly lower MERS-CoV titers and MERS-CoV and mRNA levels in challenged mice than those in unvaccinated controls. These data establish that an inactivated RABV-MERS S-based vaccine may be effective for use in animals and humans in areas where MERS-CoV is endemic. IMPORTANCE: Rabies virus-based vectors have been proven to be efficient dual vaccines against rabies and emergent infectious diseases such as Ebola virus. Here we show that inactivated rabies virus particles containing the MERS-CoV S1 protein induce potent immune responses against MERS-CoV and RABV. This novel vaccine is easy to produce and may be useful to protect target animals, such as camels, as well as humans from deadly MERS-CoV and RABV infections. Our results indicate that this vaccine approach can prevent disease, and the RABV-based vaccine platform may be a valuable tool for timely vaccine development against emerging infectious diseases.

West Nile Virus Spreads Transsynaptically within the Pathways of Motor Control: Anatomical and Ultrastructural Mapping of Neuronal Virus Infection in the Primate Central Nervous System.

BACKGROUND: During recent West Nile virus (WNV) outbreaks in the US, half of the reported cases were classified as neuroinvasive disease. WNV neuroinvasion is proposed to follow two major routes: hematogenous and/or axonal transport along the peripheral nerves. How virus spreads once within the central nervous system (CNS) remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: Using immunohistochemistry, we examined the expression of viral antigens in the CNS of rhesus monkeys that were intrathalamically inoculated with a wild-type WNV. The localization of WNV within the CNS was mapped to specific neuronal groups and anatomical structures. The neurological functions related to structures containing WNV-labeled neurons were reviewed and summarized. Intraneuronal localization of WNV was investigated by electron microscopy. The known anatomical connectivity of WNV-labeled neurons was used to reconstruct the directionality of WNV spread within the CNS using a connectogram design. Anatomical mapping revealed that all structures identified as containing WNV-labeled neurons belonged to the pathways of motor control. Ultrastructurally, virions were found predominantly within vesicular structures (including autophagosomes) in close vicinity to the axodendritic synapses, either at pre- or post-synaptic positions (axonal terminals and dendritic spines, respectively), strongly indicating transsynaptic spread of the virus between connected neurons. Neuronal connectivity-based reconstruction of the directionality of transsynaptic virus spread suggests that, within the CNS, WNV can utilize both anterograde and retrograde axonal transport to infect connected neurons. CONCLUSIONS/SIGNIFICANCE: This study offers a new insight into the neuropathogenesis of WNV infection in a primate model that closely mimics WNV encephalomyelitis in humans. We show that within the primate CNS, WNV primarily infects the anatomical structures and pathways responsible for the control of movement. Our findings also suggest that WNV most likely propagates within the CNS transsynaptically, by both, anterograde and retrograde axonal transport.

Divergent Simian Arteriviruses Cause Simian Hemorrhagic Fever of Differing Severities in Macaques.

UNLABELLED: Simian hemorrhagic fever (SHF) is a highly lethal disease in captive macaques. Three distinct arteriviruses are known etiological agents of past SHF epizootics, but only one, simian hemorrhagic fever virus (SHFV), has been isolated in cell culture. The natural reservoir(s) of the three viruses have yet to be identified, but African nonhuman primates are suspected. Eleven additional divergent simian arteriviruses have been detected recently in diverse and apparently healthy African cercopithecid monkeys. Here, we report the successful isolation in MARC-145 cell culture of one of these viruses, Kibale red colobus virus 1 (KRCV-1), from serum of a naturally infected red colobus (Procolobus [Piliocolobus] rufomitratus tephrosceles) sampled in Kibale National Park, Uganda. Intramuscular (i.m.) injection of KRCV-1 into four cynomolgus macaques (Macaca fascicularis) resulted in a self-limiting nonlethal disease characterized by depressive behavioral changes, disturbance in coagulation parameters, and liver enzyme elevations. In contrast, i.m. injection of SHFV resulted in typical lethal SHF characterized by mild fever, lethargy, lymphoid depletion, lymphoid and hepatocellular necrosis, low platelet counts, increased liver enzyme concentrations, coagulation abnormalities, and increasing viral loads. As hypothesized based on the genetic and presumed antigenic distance between KRCV-1 and SHFV, all four macaques that had survived KRCV-1 injection died of SHF after subsequent SHFV injection, indicating a lack of protective heterotypic immunity. Our data indicate that SHF is a disease of macaques that in all likelihood can be caused by a number of distinct simian arteriviruses, although with different severity depending on the specific arterivirus involved. Consequently, we recommend that current screening procedures for SHFV in primate-holding facilities be modified to detect all known simian arteriviruses. IMPORTANCE: Outbreaks of simian hemorrhagic fever (SHF) have devastated captive Asian macaque colonies in the past. SHF is caused by at least three viruses of the family Arteriviridae: simian hemorrhagic fever virus (SHFV), simian hemorrhagic encephalitis virus (SHEV), and Pebjah virus (PBJV). Nine additional distant relatives of these three viruses were recently discovered in apparently healthy African nonhuman primates. We hypothesized that all simian arteriviruses are potential causes of SHF. To test this hypothesis, we inoculated cynomolgus macaques with a highly divergent simian arterivirus (Kibale red colobus virus 1 [KRCV-1]) from a wild Ugandan red colobus. Despite being only distantly related to red colobuses, all of the macaques developed disease. In contrast to SHFV-infected animals, KRCV-1-infected animals survived after a mild disease presentation. Our study advances the understanding of an important primate disease. Furthermore, our data indicate a need to include the full diversity of simian arteriviruses in nonhuman primate SHF screening assays.

Severe Acute Respiratory Syndrome Coronavirus ORF7a Inhibits Bone Marrow Stromal Antigen 2 Virion Tethering through a Novel Mechanism of Glycosylation Interference.

UNLABELLED: Severe acute respiratory syndrome (SARS) emerged in November 2002 as a case of atypical pneumonia in China, and the causative agent of SARS was identified to be a novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV). Bone marrow stromal antigen 2 (BST-2; also known as CD317 or tetherin) was initially identified to be a pre-B-cell growth promoter, but it also inhibits the release of virions of the retrovirus human immunodeficiency virus type 1 (HIV-1) by tethering budding virions to the host cell membrane. Further work has shown that BST-2 restricts the release of many other viruses, including the human coronavirus 229E (hCoV-229E), and the genomes of many of these viruses encode BST-2 antagonists to overcome BST-2 restriction. Given the previous studies on BST-2, we aimed to determine if BST-2 has the ability to restrict SARS-CoV and if the SARS-CoV genome encodes any proteins that modulate BST-2's antiviral function. Through an in vitro screen, we identified four potential BST-2 modulators encoded by the SARS-CoV genome: the papain-like protease (PLPro), nonstructural protein 1 (nsp1), ORF6, and ORF7a. As the function of ORF7a in SARS-CoV replication was previously unknown, we focused our study on ORF7a. We found that BST-2 does restrict SARS-CoV, but the loss of ORF7a leads to a much greater restriction, confirming the role of ORF7a as an inhibitor of BST-2. We further characterized the mechanism of BST-2 inhibition by ORF7a and found that ORF7a localization changes when BST-2 is overexpressed and ORF7a binds directly to BST-2. Finally, we also show that SARS-CoV ORF7a blocks the restriction activity of BST-2 by blocking the glycosylation of BST-2. IMPORTANCE: The severe acute respiratory syndrome coronavirus (SARS-CoV) emerged from zoonotic sources in 2002 and caused over 8,000 infections and 800 deaths in 37 countries around the world. Identifying host factors that regulate SARS-CoV pathogenesis is critical to understanding how this lethal virus causes disease. We have found that BST-2 is capable of restricting SARS-CoV release from cells; however, we also identified a SARS-CoV protein that inhibits BST-2 function. We show that the SARS-CoV protein ORF7a inhibits BST-2 glycosylation, leading to a loss of BST-2's antiviral function.

Simian hemorrhagic fever virus cell entry is dependent on CD163 and uses a clathrin-mediated endocytosis-like pathway.

UNLABELLED: Simian hemorrhagic fever virus (SHFV) causes a severe and almost uniformly fatal viral hemorrhagic fever in Asian macaques but is thought to be nonpathogenic for humans. To date, the SHFV life cycle is almost completely uncharacterized on the molecular level. Here, we describe the first steps of the SHFV life cycle. Our experiments indicate that SHFV enters target cells by low-pH-dependent endocytosis. Dynamin inhibitors, chlorpromazine, methyl-ß-cyclodextrin, chloroquine, and concanamycin A dramatically reduced SHFV entry efficiency, whereas the macropinocytosis inhibitors EIPA, blebbistatin, and wortmannin and the caveolin-mediated endocytosis inhibitors nystatin and filipin III had no effect. Furthermore, overexpression and knockout study and electron microscopy results indicate that SHFV entry occurs by a dynamin-dependent clathrin-mediated endocytosis-like pathway. Experiments utilizing latrunculin B, cytochalasin B, and cytochalasin D indicate that SHFV does not hijack the actin polymerization pathway. Treatment of target cells with proteases (proteinase K, papain, α-chymotrypsin, and trypsin) abrogated entry, indicating that the SHFV cell surface receptor is a protein. Phospholipases A2 and D had no effect on SHFV entry. Finally, treatment of cells with antibodies targeting CD163, a cell surface molecule identified as an entry factor for the SHFV-related porcine reproductive and respiratory syndrome virus, diminished SHFV replication, identifying CD163 as an important SHFV entry component. IMPORTANCE: Simian hemorrhagic fever virus (SHFV) causes highly lethal disease in Asian macaques resembling human illness caused by Ebola or Lassa virus. However, little is known about SHFV's ecology and molecular biology and the mechanism by which it causes disease. The results of this study shed light on how SHFV enters its target cells. Using electron microscopy and inhibitors for various cellular pathways, we demonstrate that SHFV invades cells by low-pH-dependent, actin-independent endocytosis, likely with the help of a cellular surface protein.

Controlled viral glycoprotein expression as a safety feature in a bivalent rabies-ebola vaccine.

Using a recombinant rabies (RABV) vaccine platform, we have developed several safe and effective vaccines. Most recently, we have developed a RABV-based ebolavirus (EBOV) vaccine that is efficacious in nonhuman primates. One safety feature of this vaccine is the utilization of a live but replication-deficient RABV construct. In this construct, the RABV glycoprotein (G) has been deleted from the genome, requiring G trans complementation in order for new infectious viruses to be released from the initial infected cell. Here we analyze this safety feature of the bivalent RABV-based EBOV vaccine comprised of the G-deleted RABV backbone expressing EBOV glycoprotein (GP). We found that, while the level of RABV genome in infected cells is equivalent regardless of G supplementation, the production of infectious virus is indeed restricted by the lack of G, and most importantly, that the presence of EBOV GP does not substitute for G. These findings further support the safety profile of this replication-deficient RABV-EBOV bivalent vaccine.

CD26/DPP4 cell-surface expression in bat cells correlates with bat cell susceptibility to Middle East respiratory syndrome coronavirus (MERS-CoV) infection and evolution of persistent infection.

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.

Activation of virus uptake through induction of macropinocytosis with a novel polymerizing peptide.

A 27-aa peptide (P27) was previously shown to decrease the accumulation of human immunodeficiency virus type 1 (HIV-1) in the supernatant of chronically infected cells; however, the mechanism was not understood. Here, we show that P27 prevents virus accumulation by inducing macropinocytosis (MPC). Treatment of HIV-1- and human T-cell lymphotropic virus type 1 (HTLV-1)-infected cells with 2-10 µM P27 caused cell membrane ruffling and uptake of virus and polymerized forms of the peptide into large vacuoles. As demonstrated by electron microscopy, activation of MPC did not require virus or cells infected with virus, as P27 initiated its own uptake in the absence of virus. Inhibitors of MPC, Cytochalasin D and amiloride, decreased P27-mediated uptake of soluble dextran and inhibited P27-induced virus uptake by >60%, which provides further evidence that P27 induces MPC. In CD4(+) HeLa cells, HIV-1 infection was enhanced by P27 up to 4-fold, and P27 increased infection at concentrations as low as 20 nM. The 5-aa C-terminal domain of P27 was necessary for virus uptake and may be responsible for the polymerization of P27 into fibrils. These forms of P27 may play a key role in triggering MPC, making this peptide a useful tool for studying virus uptake and infection, as well as MPC of other macromolecules.