Coordinated inflammatory responses dictate Marburg virus control by reservoir bats.

Bats are increasingly recognized as reservoirs of emerging zoonotic pathogens. Egyptian rousette bats (ERBs) are the known reservoir of Marburg virus (MARV), a filovirus that causes deadly Marburg virus disease (MVD) in humans. However, ERBs harbor MARV asymptomatically, likely due to a coadapted and specific host immunity-pathogen relationship. Recently, we measured transcriptional responses in MARV-infected ERB whole tissues, showing that these bats possess a disease tolerant strategy that limits pro-inflammatory gene induction, presumably averting MVD-linked immunopathology. However, the host resistant strategy by which ERBs actively limit MARV burden remains elusive, which we hypothesize requires localized inflammatory responses unresolvable at bulk-tissue scale. Here, we use dexamethasone to attenuate ERB pro-inflammatory responses and assess MARV replication, shedding and disease. We show that MARV-infected ERBs naturally mount coordinated pro-inflammatory responses at liver foci of infection, comprised of recruited mononuclear phagocytes and T cells, the latter of which proliferate with likely MARV-specificity. When pro-inflammatory responses are diminished, ERBs display heightened MARV replication, oral/rectal shedding and severe MVD-like liver pathology, demonstrating that ERBs balance immunoprotective tolerance with discreet MARV-resistant pro-inflammatory responses. These data further suggest that natural ERB immunomodulatory stressors like food scarcity and habitat disruption may potentiate viral shedding, transmission and therefore outbreak risk.

Human-to-Human Transmission of Andes Virus Modeled in Syrian Hamsters.

Several occurrences of human-to-human transmission of Andes virus, an etiological agent of hantavirus cardiopulmonary syndrome, are documented. Syrian hamsters consistently model human hantavirus cardiopulmonary syndrome, yet neither transmission nor shedding has been investigated. We demonstrate horizontal virus transmission and show that Andes virus is shed efficiently from both inoculated and contact-infected hamsters.

Peripheral immune responses to filoviruses in a reservoir versus spillover hosts reveal transcriptional correlates of disease.

Several filoviruses, including Marburg virus (MARV), cause severe disease in humans and nonhuman primates (NHPs). However, the Egyptian rousette bat (ERB, Rousettus aegyptiacus), the only known MARV reservoir, shows no overt illness upon natural or experimental infection, which, like other bat hosts of zoonoses, is due to well-adapted, likely species-specific immune features. Despite advances in understanding reservoir immune responses to filoviruses, ERB peripheral blood responses to MARV and how they compare to those of diseased filovirus-infected spillover hosts remain ill-defined. We thus conducted a longitudinal analysis of ERB blood gene responses during acute MARV infection. These data were then contrasted with a compilation of published primate blood response studies to elucidate gene correlates of filovirus protection versus disease. Our work expands on previous findings in MARV-infected ERBs by supporting both host resistance and disease tolerance mechanisms, offers insight into the peripheral immunocellular repertoire during infection, and provides the most direct known cross-examination between reservoir and spillover hosts of the most prevalently-regulated response genes, pathways and activities associated with differences in filovirus pathogenesis and pathogenicity.

Generating human artery and vein cells from pluripotent stem cells highlights the arterial tropism of Nipah and Hendra viruses.

Stem cell research endeavors to generate specific subtypes of classically defined "cell types." Here, we generate >90% pure human artery or vein endothelial cells from pluripotent stem cells within 3-4 days. We specified artery cells by inhibiting vein-specifying signals and vice versa. These cells modeled viral infection of human vasculature by Nipah and Hendra viruses, which are extraordinarily deadly (∼57%-59% fatality rate) and require biosafety-level-4 containment. Generating pure populations of artery and vein cells highlighted that Nipah and Hendra viruses preferentially infected arteries; arteries expressed higher levels of their viral-entry receptor. Virally infected artery cells fused into syncytia containing up to 23 nuclei, which rapidly died. Despite infecting arteries and occupying ∼6%-17% of their transcriptome, Nipah and Hendra largely eluded innate immune detection, minimally eliciting interferon signaling. We thus efficiently generate artery and vein cells, introduce stem-cell-based toolkits for biosafety-level-4 virology, and explore the arterial tropism and cellular effects of Nipah and Hendra viruses.

Tolerance and Persistence of Ebola Virus in Primary Cells from Mops condylurus, a Potential Ebola Virus Reservoir.

Although there have been documented Ebola virus disease outbreaks for more than 40 years, the natural reservoir host has not been identified. Recent studies provide evidence that the Angolan free-tailed bat (Mops condylurus), an insectivorous microbat, is a possible ebolavirus reservoir. To investigate the potential role of this bat species in the ecology of ebolaviruses, replication, tolerance, and persistence of Ebola virus (EBOV) were investigated in 10 different primary bat cell isolates from M. condylurus. Varying EBOV replication kinetics corresponded to the expression levels of the integral membrane protein NPC1. All primary cells were highly tolerant to EBOV infection without cytopathic effects. The observed persistent EBOV infection for 150 days in lung primary cells, without resultant selective pressure leading to virus mutation, indicate the intrinsic ability of EBOV to persist in this bat species. These results provide further evidence for this bat species to be a likely reservoir of ebolaviruses.

Therapeutic Efficacy of Human Monoclonal Antibodies against Andes Virus Infection in Syrian Hamsters.

Andes virus, an orthohantavirus endemic to South America, causes severe hantavirus cardiopulmonary syndrome associated with human-to-human transmission. No approved treatments or vaccines against this virus are available. We show that a combined treatment with 2 monoclonal antibodies protected Syrian hamsters when administered at midstage or late-stage disease.

Nipah Virus Efficiently Replicates in Human Smooth Muscle Cells without Cytopathic Effect.

Nipah virus (NiV) is a highly pathogenic zoonotic virus with a broad species tropism, originating in pteropid bats. Human outbreaks of NiV disease occur almost annually, often with high case-fatality rates. The specific events that lead to pathogenesis are not well defined, but the disease has both respiratory and encephalitic components, with relapsing encephalitis occurring in some cases more than a year after initial infection. Several cell types are targets of NiV, dictated by the expression of the ephrin-B2/3 ligand on the cell's outer membrane, which interact with the NiV surface proteins. Vascular endothelial cells (ECs) are major targets of infection. Cytopathic effects (CPE), characterized by syncytia formation and cell death, and an ensuing vasculitis, are a major feature of the disease. Smooth muscle cells (SMCs) of the tunica media that line small blood vessels are infected in humans and animal models of NiV disease, although pathology or histologic changes associated with antigen-positive SMCs have not been reported. To gain an understanding of the possible contributions that SMCs might have in the development of NiV disease, we investigated the susceptibility and potential cytopathogenic changes of human SMCs to NiV infection in vitro. SMCs were permissive for NiV infection and resulted in high titers and prolonged NiV production, despite a lack of cytopathogenicity, and in the absence of detectable ephrin-B2/3. These results indicate that SMC might be important contributors to disease by producing progeny NiV during an infection, without suffering cytopathogenic consequences.

Hematology and Clinical Chemistry Reference Ranges for Laboratory-Bred Natal Multimammate Mice (Mastomys natalensis).

Laboratory-controlled physiological data for the multimammate rat (Mastomys natalensis) are scarce, despite this species being a known reservoir and vector for zoonotic viruses, including the highly pathogenic Lassa virus, as well as other arenaviruses and many species of bacteria. For this reason, M. natalensis is an important rodent for the study of host-virus interactions within laboratory settings. Herein, we provide basic blood parameters for age- and sex-distributed animals in regards to blood counts, cell phenotypes and serum chemistry of a specific-pathogen-monitored M.natalensis breeding colony, to facilitate scientific insight into this important and widespread rodent species.

Asymptomatic Infection of Marburg Virus Reservoir Bats Is Explained by a Strategy of Immunoprotective Disease Tolerance.

Marburg virus (MARV) is among the most virulent pathogens of primates, including humans. Contributors to severe MARV disease include immune response suppression and inflammatory gene dysregulation ("cytokine storm"), leading to systemic damage and often death. Conversely, MARV causes little to no clinical disease in its reservoir host, the Egyptian rousette bat (ERB). Previous genomic and in vitro data suggest that a tolerant ERB immune response may underlie MARV avirulence, but no significant examination of this response in vivo yet exists. Here, using colony-bred ERBs inoculated with a bat isolate of MARV, we use species-specific antibodies and an immune gene probe array (NanoString) to temporally characterize the transcriptional host response at sites of MARV replication relevant to primate pathogenesis and immunity, including CD14+ monocytes/macrophages, critical immune response mediators, primary MARV targets, and skin at the inoculation site, where highest viral loads and initial engagement of antiviral defenses are expected. Our analysis shows that ERBs upregulate canonical antiviral genes typical of mammalian systems, such as ISG15, IFIT1, and OAS3, yet demonstrate a remarkable lack of significant induction of proinflammatory genes classically implicated in primate filoviral pathogenesis, including CCL8, FAS, and IL6. Together, these findings offer the first in vivo functional evidence for disease tolerance as an immunological mechanism by which the bat reservoir asymptomatically hosts MARV. More broadly, these data highlight factors determining disparate outcomes between reservoir and spillover hosts and defensive strategies likely utilized by bat hosts of other emerging pathogens, knowledge that may guide development of effective antiviral therapies.

The Utility of Human Immune System Mice for High-Containment Viral Hemorrhagic Fever Research.

Human immune system (HIS) mice are a subset of humanized mice that are generated by xenoengraftment of human immune cells or tissues and/or their progenitors into immunodeficient mice. Viral hemorrhagic fevers (VHFs) cause severe disease in humans, typically with high case fatality rates. HIS mouse studies have been performed to investigate the pathogenesis and immune responses to VHFs that must be handled in high-containment laboratory facilities. Here, we summarize studies on filoviruses, nairoviruses, phenuiviruses, and hantaviruses, and discuss the knowledge gained from using various HIS mouse models. Furthermore, we discuss the complexities of designing and interpreting studies utilizing HIS mice while highlighting additional questions about VHFs that can still be addressed using HIS mouse models.

Rousette Bat Dendritic Cells Overcome Marburg Virus-Mediated Antiviral Responses by Upregulation of Interferon-Related Genes While Downregulating Proinflammatory Disease Mediators.

Dysregulated and maladaptive immune responses are at the forefront of human diseases caused by infection with zoonotic viral hemorrhagic fever viruses. Elucidating mechanisms of how the natural animal reservoirs of these viruses coexist with these agents without overt disease, while permitting sufficient replication to allow for transmission and maintenance in a population, is important for understanding the viral ecology and spillover to humans. The Egyptian rousette bat (ERB) has been identified as a reservoir for Marburg virus (MARV), a filovirus and the etiological agent of the highly lethal Marburg virus disease. Little is known regarding how these bats immunologically respond to MARV infection. In humans, macrophages and dendritic cells (DCs) are primary targets of infection, and their dysregulation is thought to play a central role in filovirus diseases, by disturbing their normal functions as innate sensors and adaptive immune response facilitators while serving as amplification and dissemination agents for the virus. The infection status and responses to MARV in bat myeloid-lineage cells are uncharacterized and likely represent an important modulator of the bat's immune response to MARV infection. Here, we generate DCs from the bone marrow of rousette bats. Infection with a bat isolate of MARV resulted in a low level of transcription in these cells and significantly downregulated DC maturation and adaptive immune-stimulatory pathways while simultaneously upregulating interferon-related pathogen-sensing pathways. This study provides a first insight into how the bat immune response is directed toward preventing aberrant inflammatory responses while mounting an antiviral response to defend against MARV infection.IMPORTANCE Marburg viruses (MARVs) cause severe human disease resulting from aberrant immune responses. Dendritic cells (DCs) are primary targets of infection and are dysregulated by MARV. Dysregulation of DCs facilitates MARV replication and virus dissemination and influences downstream immune responses that result in immunopathology. Egyptian rousette bats (ERBs) are natural reservoirs of MARV, and infection results in virus replication and shedding, with asymptomatic control of the virus within weeks. The mechanisms that bats employ to appropriately respond to infection while avoiding disease are unknown. Because DC infection and modulation are important early events in human disease, we measured the transcriptional responses of ERB DCs to MARV. The significance of this work is in identifying cell type-specific coevolved responses between ERBs and MARV, which gives insight into how bat reservoirs are able to harbor MARV and permit viral replication, allowing transmission and maintenance in the population while simultaneously preventing immunopathogenesis.

Dendritic Cells Generated From Mops condylurus, a Likely Filovirus Reservoir Host, Are Susceptible to and Activated by Zaire Ebolavirus Infection.

Ebola virus infection of human dendritic cells (DCs) induces atypical adaptive immune responses and thereby exacerbates Ebola virus disease (EVD). Human DCs, infected with Ebola virus aberrantly express low levels of the DC activation markers CD80, CD86, and MHC class II. The T cell responses ensuing are commonly anergic rather than protective against EVD. We hypothesize that DCs derived from potential reservoir hosts such as bats, which do not develop disease signs in response to Ebola virus infection, would exhibit features associated with activation. In this study, we have examined Zaire ebolavirus (EBOV) infection of DCs derived from the Angolan free-tailed bat species, Mops condylurus. This species was previously identified as permissive to EBOV infection in vivo, in the absence of disease signs. M. condylurus has also been recently implicated as the reservoir host for Bombali ebolavirus, a virus species that is closely related to EBOV. Due to the absence of pre-existing M. condylurus species-specific reagents, we characterized its de novo assembled transcriptome and defined its phylogenetic similarity to other mammals, which enabled the identification of cross-reactive reagents for M. condylurus bone marrow-derived DC (bat-BMDC) differentiation and immune cell phenotyping. Our results reveal that bat-BMDCs are susceptible to EBOV infection as determined by detection of EBOV specific viral RNA (vRNA). vRNA increased significantly 72 h after EBOV-infection and was detected in both cells and in culture supernatants. Bat-BMDC infection was further confirmed by the observation of GFP expression in DC cultures infected with a recombinant GFP-EBOV. Bat-BMDCs upregulated CD80 and chemokine ligand 3 (CCL3) transcripts in response to EBOV infection, which positively correlated with the expression levels of EBOV vRNA. In contrast to the aberrant responses to EBOV infection that are typical for human-DC, our findings from bat-BMDCs provide evidence for an immunological basis of asymptomatic EBOV infection outcomes.

Differential Innate Immune Responses Elicited by Nipah Virus and Cedar Virus Correlate with Disparate In Vivo Pathogenesis in Hamsters.

Syrian hamsters (Mesocricetus auratus) are a pathogenesis model for the Nipah virus (NiV), and we sought to determine if they are also susceptible to the Cedar virus (CedPV). Following intranasal inoculation with CedPV, virus replication occurred in the lungs and spleens of infected hamsters, a neutralizing antibody was produced in some hamsters within 8 days post-challenge, and no conspicuous signs of disease occurred. CedPV replicated to a similar magnitude as NiV-Bangladesh in type I IFN-deficient BHK-21 Syrian hamster fibroblasts but replicated 4 logs lower in type I IFN-competent primary Syrian hamster and human pulmonary endothelial cells, a principal target of henipaviruses. The coinfection of these cells with CedPV and NiV failed to rescue CedPV titers and did not diminish NiV titers, suggesting the replication machinery is virus-specific. Type I IFN response transcripts Ifna7, Ddx58, Stat1, Stat2, Ccl5, Cxcl10, Isg20, Irf7, and Iigp1 were all significantly elevated in CedPV-infected hamster endothelial cells, whereas Ifna7 and Iigp1 expression were significantly repressed during NiV infection. These results are consistent with the hypothesis that CedPV's inability to counter the host type I IFN response may, in part, contribute to its lack of pathogenicity. Because NiV causes a fatal disease in Syrian hamsters with similarities to human disease, this model will provide valuable information about the pathogenic mechanisms of henipaviruses.

Two recombinant human monoclonal antibodies that protect against lethal Andes hantavirus infection in vivo.

Andes hantavirus (ANDV) is an etiologic agent of hantavirus cardiopulmonary syndrome (HCPS), a severe disease characterized by fever, headache, and gastrointestinal symptoms that may progress to hypotension, pulmonary failure, and cardiac shock that results in a 25 to 40% case-fatality rate. Currently, there is no specific treatment or vaccine; however, several studies have shown that the generation of neutralizing antibody (Ab) responses strongly correlates with survival from HCPS in humans. In this study, we screened 27 ANDV convalescent HCPS patient sera for their capacity to bind and neutralize ANDV in vitro. One patient who showed high neutralizing titer was selected to isolate ANDV-glycoprotein (GP) Abs. ANDV-GP-specific memory B cells were single cell sorted, and recombinant immunoglobulin G antibodies were cloned and produced. Two monoclonal Abs (mAbs), JL16 and MIB22, potently recognized ANDV-GPs and neutralized ANDV. We examined the post-exposure efficacy of these two mAbs as a monotherapy or in combination therapy in a Syrian hamster model of ANDV-induced HCPS, and both mAbs protected 100% of animals from a lethal challenge dose. These data suggest that monotherapy with mAb JL16 or MIB22, or a cocktail of both, could be an effective post-exposure treatment for patients infected with ANDV-induced HCPS.

Pathogenicity of Ebola and Marburg Viruses Is Associated With Differential Activation of the Myeloid Compartment in Humanized Triple Knockout-Bone Marrow, Liver, and Thymus Mice.

Ebola virus (EBOV) and Marburg virus (MARV) outbreaks are highly lethal, and infection results in a hemorrhagic fever with complex etiology. These zoonotic viruses dysregulate the immune system to cause disease, in part by replicating within myeloid cells that would normally innately control viral infection and shape the adaptive immune response. We used triple knockout (TKO)-bone marrow, liver, thymus (BLT) humanized mice to recapitulate the early in vivo human immune response to filovirus infection. Disease severity in TKO-BLT mice was dissimilar between EBOV and MARV with greater severity observed during EBOV infection. Disease severity was related to increased Kupffer cell infection in the liver, higher levels of myeloid dysfunction, and skewing of macrophage subtypes in EBOV compared with MARV-infected mice. Overall, the TKO-BLT model provided a practical in vivo platform to study the human immune response to filovirus infection and generated a better understanding of how these viruses modulate specific components of the immune system.

Pathogenicity and Viral Shedding of MERS-CoV in Immunocompromised Rhesus Macaques.

Middle East respiratory syndrome coronavirus (MERS-CoV) has recently emerged in the Middle East. Since 2012, there have been approximately 2,100 confirmed cases, with a 35% case fatality rate. Disease severity has been linked to patient health status, as people with chronic diseases or an immunocompromised status fare worse, although the mechanisms of disease have yet to be elucidated. We used the rhesus macaque model of mild MERS to investigate whether the immune response plays a role in the pathogenicity in relation to MERS-CoV shedding. Immunosuppressed macaques were inoculated with MERS-CoV and sampled daily for 6 days to assess their immune statues and to measure viral shedding and replication. Immunosuppressed macaques supported significantly higher levels of MERS-CoV replication in respiratory tissues and shed more virus, and virus disseminated to tissues outside of the respiratory tract, whereas viral RNA was confined to respiratory tissues in non-immunosuppressed animals. Despite increased viral replication, pathology in the lungs was significantly lower in immunosuppressed animals. The observation that the virus was less pathogenic in these animals suggests that disease has an immunopathogenic component and shows that inflammatory responses elicited by the virus contribute to disease.

Severity of Disease in Humanized Mice Infected With Ebola Virus or Reston Virus Is Associated With Magnitude of Early Viral Replication in Liver.

Both Ebola virus (EBOV) and Reston virus (RESTV) cause disease in nonhuman primates, yet only EBOV causes disease in humans. To investigate differences in viral pathogenicity, humanized mice (hu-NSG-SGM3) were inoculated with EBOV or RESTV. Consistent with differences in disease in human infection, pronounced weight loss and markers of hepatic damage and disease were observed exclusively in EBOV-infected mice. These abnormalities were associated with significantly higher EBOV replication in the liver but not in the spleen, suggesting that in this model, efficiency of viral replication in select tissues early in infection may contribute to differences in viral pathogenicity.

Human immune system mouse models of Ebola virus infection.

Human immune system (HIS) mice, immunodeficient mice engrafted with human cells (with or without donor-matched tissue), offer a unique opportunity to study pathogens that cause disease predominantly or exclusively in humans. Several HIS mouse models have recently been used to study Ebola virus (EBOV) infection and disease. The results of these studies are encouraging and support further development and use of these models in Ebola research. HIS mice provide a small animal model to study EBOV isolates, investigate early viral interactions with human immune cells, screen vaccines and therapeutics that modulate the immune system, and investigate sequelae in survivors. Here we review existing models, discuss their use in pathogenesis studies and therapeutic screening, and highlight considerations for study design and analysis. Finally, we point out caveats to current models, and recommend future efforts for modeling EBOV infection in HIS mice.

Immunobiology of Ebola and Lassa virus infections.

Two of the most important contemporary emerging viruses that affect human health in Africa are Ebola virus (EBOV) and Lassa virus (LASV). The 2013-2016 West African outbreak of EBOV was responsible for more than 11,000 deaths, primarily in Guinea, Sierra Leone and Liberia. LASV is constantly emerging in these and surrounding West African countries, with an estimate of more than 500,000 cases of Lassa fever, and approximately 5,000 deaths, annually. Both EBOV and LASV are zoonotic, and human infection often results in a severe haemorrhagic fever in both cases. However, the contribution of specific immune responses to disease differs between EBOV and LASV. This Review examines innate and adaptive immune responses to these viruses with the goal of delineating responses that are associated with protective versus pathogenic outcomes.

Amending Koch's postulates for viral disease: When "growth in pure culture" leads to a loss of virulence.

It is a common laboratory practice to propagate viruses in cell culture. While convenient, these methodologies often result in unintentional genetic alterations, which have led to adaptation and even attenuation in animal models of disease. An example is the attenuation of hantaviruses (family: Bunyaviridae, genus: Hantavirus) when cultured in vitro. In this case, viruses propagated in the natural reservoir species cause disease in nonhuman primates that closely mimics the human disease, but passaging in cell culture attenuates these viruses to the extent that do not cause any measurable disease in nonhuman primates. As efforts to develop animal models progress, it will be important to take into account the influences that culture in vitro may have on the virulence of viruses. In this review we discuss this phenomenon in the context of past and recent examples in the published literature.