Asymmetric and non-stoichiometric glycoprotein recognition by two distinct antibodies results in broad protection against ebolaviruses.

Several ebolaviruses cause outbreaks of severe disease. Vaccines and monoclonal antibody cocktails are available to treat Ebola virus (EBOV) infections, but not Sudan virus (SUDV) or other ebolaviruses. Current cocktails contain antibodies that cross-react with the secreted soluble glycoprotein (sGP) that absorbs virus-neutralizing antibodies. By sorting memory B cells from EBOV infection survivors, we isolated two broadly reactive anti-GP monoclonal antibodies, 1C3 and 1C11, that potently neutralize, protect rodents from disease, and lack sGP cross-reactivity. Both antibodies recognize quaternary epitopes in trimeric ebolavirus GP. 1C11 bridges adjacent protomers via the fusion loop. 1C3 has a tripartite epitope in the center of the trimer apex. One 1C3 antigen-binding fragment anchors simultaneously to the three receptor-binding sites in the GP trimer, and separate 1C3 paratope regions interact differently with identical residues on the three protomers. A cocktail of both antibodies completely protected nonhuman primates from EBOV and SUDV infections, indicating their potential clinical value.

Repeated mRNA vaccination sequentially boosts SARS-CoV-2-specific CD8+ T cells in persons with previous COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hybrid immunity is more protective than vaccination or previous infection alone. To investigate the kinetics of spike-reactive T (TS) cells from SARS-CoV-2 infection through messenger RNA vaccination in persons with hybrid immunity, we identified the T cell receptor (TCR) sequences of thousands of index TS cells and tracked their frequency in bulk TCRß repertoires sampled longitudinally from the peripheral blood of persons who had recovered from coronavirus disease 2019 (COVID-19). Vaccinations led to large expansions in memory TS cell clonotypes, most of which were CD8+ T cells, while also eliciting diverse TS cell clonotypes not observed before vaccination. TCR sequence similarity clustering identified public CD8+ and CD4+ TCR motifs associated with spike (S) specificity. Synthesis of longitudinal bulk ex vivo single-chain TCRß repertoires and paired-chain TCRÉ‘ß sequences from droplet sequencing of TS cells provides a roadmap for the rapid assessment of T cell responses to vaccines and emerging pathogens.

Defining the risk of SARS-CoV-2 variants on immune protection.

The global emergence of many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants jeopardizes the protective antiviral immunity induced after infection or vaccination. To address the public health threat caused by the increasing SARS-CoV-2 genomic diversity, the National Institute of Allergy and Infectious Diseases within the National Institutes of Health established the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme. This effort was designed to provide a real-time risk assessment of SARS-CoV-2 variants that could potentially affect the transmission, virulence, and resistance to infection- and vaccine-induced immunity. The SAVE programme is a critical data-generating component of the US Government SARS-CoV-2 Interagency Group to assess implications of SARS-CoV-2 variants on diagnostics, vaccines and therapeutics, and for communicating public health risk. Here we describe the coordinated approach used to identify and curate data about emerging variants, their impact on immunity and effects on vaccine protection using animal models. We report the development of reagents, methodologies, models and notable findings facilitated by this collaborative approach and identify future challenges. This programme is a template for the response to rapidly evolving pathogens with pandemic potential by monitoring viral evolution in the human population to identify variants that could reduce the effectiveness of countermeasures.

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.

Clinical and Immunologic Correlates of Vasodilatory Shock Among Ebola Virus-Infected Nonhuman Primates in a Critical Care Model.

BACKGROUND: Existing models of Ebola virus infection have not fully characterized the pathophysiology of shock in connection with daily virologic, clinical, and immunologic parameters. We implemented a nonhuman primate critical care model to investigate these associations. METHODS: Two rhesus macaques received a target dose of 1000 plaque-forming units of Ebola virus intramuscularly with supportive care initiated on day 3. High-dimensional spectral cytometry was used to phenotype neutrophils and peripheral blood mononuclear cells daily. RESULTS: We observed progressive vasodilatory shock with preserved cardiac function following viremia onset on day 5. Multiorgan dysfunction began on day 6 coincident with the nadir of circulating neutrophils. Consumptive coagulopathy and anemia occurred on days 7 to 8 along with irreversible shock, followed by death. The monocyte repertoire began shifting on day 4 with a decline in classical and expansion of double-negative monocytes. A selective loss of CXCR3-positive B and T cells, expansion of naive B cells, and activation of natural killer cells followed viremia onset. CONCLUSIONS: Our model allows for high-fidelity characterization of the pathophysiology of acute Ebola virus infection with host innate and adaptive immune responses, which may advance host-targeted therapy design and evaluation for use after the onset of multiorgan failure.

Ebola Virus Disease Features Hemophagocytic Lymphohistiocytosis/Macrophage Activation Syndrome in the Rhesus Macaque Model.

BACKGROUND: Ebola virus (EBOV) disease (EVD) is one of the most severe and fatal viral hemorrhagic fevers and appears to mimic many clinical and laboratory manifestations of hemophagocytic lymphohistiocytosis syndrome (HLS), also known as macrophage activation syndrome. However, a clear association is yet to be firmly established for effective host-targeted, immunomodulatory therapeutic approaches to improve outcomes in patients with severe EVD. METHODS: Twenty-four rhesus monkeys were exposed intramuscularly to the EBOV Kikwit isolate and euthanized at prescheduled time points or when they reached the end-stage disease criteria. Three additional monkeys were mock-exposed and used as uninfected controls. RESULTS: EBOV-exposed monkeys presented with clinicopathologic features of HLS, including fever, multiple organomegaly, pancytopenia, hemophagocytosis, hyperfibrinogenemia with disseminated intravascular coagulation, hypertriglyceridemia, hypercytokinemia, increased concentrations of soluble CD163 and CD25 in serum, and the loss of activated natural killer cells. CONCLUSIONS: Our data suggest that EVD in the rhesus macaque model mimics pathophysiologic features of HLS/macrophage activation syndrome. Hence, regulating inflammation and immune function might provide an effective treatment for controlling the pathogenesis of acute EVD.

Clinical, Virologic, and Immunologic Evaluation of Symptomatic Coronavirus Disease 2019 Rebound Following Nirmatrelvir/Ritonavir Treatment.

BACKGROUND: Nirmatrelvir/ritonavir, the first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protease inhibitor, reduces the risk of hospitalization and death by coronavirus disease 2019 (COVID-19) but has been associated with symptomatic rebound after therapy completion. METHODS: Six individuals with relapse of COVID-19 symptoms after treatment with nirmatrelvir/ritonavir, 2 individuals with rebound symptoms without prior antiviral therapy and 7 patients with acute Omicron infection (controls) were studied. Soluble biomarkers and serum SARS-CoV-2 nucleocapsid protein were measured. Nasal swabs positive for SARS-CoV-2 underwent viral isolation and targeted viral sequencing. SARS-CoV-2 anti-spike, anti-receptor-binding domain, and anti-nucleocapsid antibodies were measured. Surrogate viral neutralization tests against wild-type and Omicron spike protein, as well as T-cell stimulation assays, were performed. RESULTS: High levels of SARS-CoV-2 anti-spike immunoglobulin G (IgG) antibodies were found in all participants. Anti-nucleocapsid IgG and Omicron-specific neutralizing antibodies increased in patients with rebound. Robust SARS-CoV-2-specific T-cell responses were observed, higher in rebound compared with early acute COVID-19 patients. Inflammatory markers mostly decreased during rebound. Two patients sampled longitudinally demonstrated an increase in activated cytokine-producing CD4+ T cells against viral proteins. No characteristic resistance mutations were identified. SARS-CoV-2 was isolated by culture from 1 of 8 rebound patients; Polybrene addition increased this to 5 of 8. CONCLUSIONS: Nirmatrelvir/ritonavir treatment does not impede adaptive immune responses to SARS-CoV-2. Clinical rebound corresponds to development of a robust antibody and T-cell immune response, arguing against a high risk of disease progression. The presence of infectious virus supports the need for isolation and assessment of longer treatment courses. CLINICAL TRIALS REGISTRATION: NCT04401436.

Expanded Histopathology and Tropism of Ebola Virus in the Rhesus Macaque Model: Potential for Sexual Transmission, Altered Adrenomedullary Hormone Production, and Early Viral Replication in Liver.

The pathogenesis of Ebola virus disease (EVD) is still incomplete, in spite of the availability of a nonhuman primate modelfor more than 4 decades. To further investigate EVD pathogenesis, a natural history study was conducted using 27 Chinese-origin rhesus macaques. Of these, 24 macaques were exposed intramuscularly to Kikwit Ebola virus and euthanized at predetermined time points or when end-stage clinical disease criteria were met, and 3 sham-exposed macaques were euthanized on study day 0. This study showed for the first time that Ebola virus causes uterine cervicitis, vaginitis, posthitis, and medullary adrenalitis. Not only was Ebola virus detected in the interstitial stromal cells of the genital tract, but it was also present in the epididymal and seminal vesicular tubular epithelial cells, ectocervical and vaginal squamous epithelial cells, and seminal fluid. Furthermore, as early as day 3 after exposure, Ebola virus replicative intermediate RNA was detected in Kupffer cells and hepatocytes. These findings in the nonhuman model provide additional insight into potential sexual transmission, possible disruption of sympathetic hormone production, and early virus replication sites in human EVD patients.

The SKI complex is a broad-spectrum, host-directed antiviral drug target for coronaviruses, influenza, and filoviruses.

The SARS-CoV-2 pandemic has made it clear that we have a desperate need for antivirals. We present work that the mammalian SKI complex is a broad-spectrum, host-directed, antiviral drug target. Yeast suppressor screening was utilized to find a functional genetic interaction between proteins from influenza A virus (IAV) and Middle East respiratory syndrome coronavirus (MERS-CoV) with eukaryotic proteins that may be potential host factors involved in replication. This screening identified the SKI complex as a potential host factor for both viruses. In mammalian systems siRNA-mediated knockdown of SKI genes inhibited replication of IAV and MERS-CoV. In silico modeling and database screening identified a binding pocket on the SKI complex and compounds predicted to bind. Experimental assays of those compounds identified three chemical structures that were antiviral against IAV and MERS-CoV along with the filoviruses Ebola and Marburg and two further coronaviruses, SARS-CoV and SARS-CoV-2. The mechanism of antiviral activity is through inhibition of viral RNA production. This work defines the mammalian SKI complex as a broad-spectrum antiviral drug target and identifies lead compounds for further development.

Naturally Acquired SARS-CoV-2 Immunity Persists for Up to 11 Months Following Infection.

BACKGROUND: Characterizing the kinetics of the antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of critical importance to developing strategies that may mitigate the public health burden of coronavirus disease 2019 (COVID-19). We conducted a prospective, longitudinal analysis of COVID-19 convalescent plasma donors at multiple time points over an 11-month period to determine how circulating antibody levels change over time following natural infection. METHODS: From April 2020 to February 2021, we enrolled 228 donors. At each study visit, subjects either donated plasma or had study samples drawn only. Anti-SARS-CoV-2 donor testing was performed using the VITROS Anti-SARS-CoV-2 Total and IgG assays and an in-house fluorescence reduction neutralization assay. RESULTS: Anti-SARS-CoV-2 antibodies were identified in 97% of COVID-19 convalescent donors at initial presentation. In follow-up analyses, of 116 donors presenting at repeat time points, 91.4% had detectable IgG levels up to 11 months after symptom recovery, while 63% had detectable neutralizing titers; however, 25% of donors had neutralizing levels that dropped to an undetectable titer over time. CONCLUSIONS: Our data suggest that immunological memory is acquired in most individuals infected with SARS-CoV-2 and is sustained in a majority of patients for up to 11 months after recovery. Clinical Trials Registration. NCT04360278.

How do I facilitate a rapid response to a public health emergency requiring plasma collection with a public-private partnership?

In March 2020, there were no treatment options for COVID-19. Passive immune therapy including anti-SARS-CoV-2 hyperimmune globulin (hIVIG) was a logical candidate for COVID-19 therapeutic trials, given past success treating emerging pathogens with endogenous neutralizing antibodies. We established a plasma collection protocol for persons recovered from COVID-19. To speed recruitment in the first U.S. hotspot, Seattle, Washington, federal and state public health agencies collaborated with Bloodworks Northwest to collect convalescent plasma (CP) for manufacturing hIVIG. During March-December 2020, we identified and recruited prospective CP donors via letters to persons recovered from COVID-19 with laboratory-confirmed SARS-CoV-2 infection. Prospective donors were pre-screened and administered a medical history survey. Anti-SARS-CoV-2 neutralizing antibody (NAb) titers were classified as qualifying (≥1:80) or non-qualifying (<1:80) for enrollment based on a live virus neutralization assay. Generalized estimating equations were used to identify characteristics of donors associated with qualifying versus nonqualifying NAb titers. Overall, 21,359 letters resulted in 3207 inquiries, 2159 prescreenings with laboratory-confirmed SARS-CoV-2 infection, and 573 donors (27% of all pre-screenings with confirmed infection) who provided a screening plasma donation. Of 573 donors screened, 254 (44%) provided plasma with qualifying NAb titers, resulting in 1284 units for hIVIG manufacture. In a multivariable model, after adjusting for other factors, time (60 days) from COVID-19 symptom onset to screening was associated with lower odds of qualifying NAb (adjusted odds ratio = 0.67, 95% CI: 0.48-0.94). The collaboration facilitated a rapid response to develop and provide hIVIG for clinical trials and CP for transfusion. Only 1 in 12 donor inquiries resulted in a qualifying plasma donation. Challenges included recruitment and the relatively low percentage of persons with high NAb titers and limited screening capacity. This resource-intensive collaboration may not be scalable but informs preparedness and response strategies for plasma collection in future epidemics. Operational readiness plans with templates for screening, consent, and data collection forms are recommended.

The Use of Large-Particle Aerosol Exposure to Nipah Virus to Mimic Human Neurological Disease Manifestations in the African Green Monkey.

Nipah virus (NiV) is an emerging virus associated with outbreaks of acute respiratory disease and encephalitis. To develop a neurological model for NiV infection, we exposed 6 adult African green monkeys to a large-particle (approximately 12 µm) aerosol containing NiV (Malaysian isolate). Brain magnetic resonance images were obtained at baseline, every 3 days after exposure for 2 weeks, and then weekly until week 8 after exposure. Four of six animals showed abnormalities reminiscent of human disease in brain magnetic resonance images. Abnormalities ranged from cytotoxic edema to vasogenic edema. The majority of lesions were small infarcts, and a few showed inflammatory or encephalitic changes. Resolution or decreased size in some lesions resembled findings reported in patients with NiV infection. Histological lesions in the brain included multifocal areas of encephalomalacia, corresponding to known ischemic foci. In other regions of the brain there was evidence of vasculitis, with perivascular infiltrates of inflammatory cells and rare intravascular fibrin thrombi. This animal model will help us better understand the acute neurological features of NiV infection and develop therapeutic approaches for managing disease caused by NiV infection.

Delayed diagnosis of splenic injuries: A case series.

INTRODUCTION: Injury of the spleen may result in significant morbidity and mortality, often related to blood loss. Splenic injuries may be missed on the initial Emergency Department (ED) presentation. This study was undertaken to describe cases of delayed diagnosis, and to identify factors associated with delayed diagnosis, treatment, and outcomes. METHODS: This retrospective study examined eligible participants with injury to the spleen who were admitted between July 2015-December 2017. Eligible participants included patients age 16 and over with injury to the spleen, with two or more ED presentations prior to admission and inpatient management. Data collected included age, gender, ethnicity, trauma triage category, vital signs, mechanism of injury, CT diagnosis, time from injury to diagnosis, toxicologic test results, inpatient management, outcome, and days of hospitalization. RESULTS: Among 210 patients with splenic injury, the mean age was 36. Most participants were male (N = 132; 63%) and White (N = 165; 79%). A small percentage (6%) was not diagnosed with splenic injury during the initial ED encounter. Missed diagnosis on the initial ED visit was not associated with age, gender, ethnicity, mechanism of injury, vital signs, grade of injury, intervention, or days of hospitalization. Most patients were discharged home (N = 9); a minority died (N = 1) or were discharged to a rehabilitation facility (N = 1). CONCLUSIONS: In this study, 6% of patients with splenic injury were not diagnosed during the initial ED encounter. These patients with delayed diagnosis had similar grade of injury, need for intervention, days of hospitalization, and outcome.

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.

The Calcium Channel Blocker Bepridil Demonstrates Efficacy in the Murine Model of Marburg Virus Disease.

No therapeutics are approved for the treatment of filovirus infections. Bepridil, a calcium channel blocker developed for treating angina, was identified as a potent inhibitor of filoviruses in vitro, including Ebola and Marburg viruses, and Ebola virus in vivo. We evaluated the efficacy of bepridil in a lethal mouse model of Marburg virus disease. A dose of 12 mg/kg bepridil once or twice daily resulted in 80% or 90% survival, respectively. These data confirm bepridil's broad-spectrum anti-filovirus activity warranting further investigation of bepridil, or improved compounds with a similar mechanism, as a pan-filovirus therapeutic agent.

Identification of Combinations of Approved Drugs With Synergistic Activity Against Ebola Virus in Cell Cultures.

Background: A need to develop therapeutics to treat Ebola virus disease patients in remote and resource-challenged settings remains in the wake of the 2013-2016 epidemic in West Africa. Toward this goal, we screened drugs under consideration as treatment options and other drugs of interest, most being small molecules approved by the Food and Drug Administration. Drugs demonstrating in vitro antiviral activity were advanced for evaluation in combinations because of advantages often provided by drug cocktails. Methods: Drugs were screened for blockade of Ebola virus infection in cultured cells. Twelve drugs were tested in all (78 pair-wise) combinations, and 3 were tested in a subset of combinations. Results: Multiple synergistic drug pairs emerged, with the majority comprising 2 entry inhibitors. For the pairs of entry inhibitors studied, synergy was demonstrated at the level of virus entry into host cells. Highly synergistic pairs included aripiprazole/piperacetazine, sertraline/toremifene, sertraline/bepridil, and amodiaquine/clomiphene. Conclusions: Our study shows the feasibility of identifying pairs of approved drugs that synergistically block Ebola virus infection in cell cultures. We discuss our findings in terms of the theoretic ability of these or alternate combinations to reach therapeutic levels. Future research will assess selected combinations in small-animal models of Ebola virus disease.

Effects of opioid medications on cognitive skills among Emergency Department patients.

INTRODUCTION: Treatment for pain and related conditions has been identified as the most common reason for Emergency Department (ED) visits. Concerns exist regarding the effects of opioid pain medications on cognition and patient ability to consent for procedures, hospital admission, or to refuse recommended medical interventions. This study was undertaken to identify cognitive skills before and after opioid pain medication in the ED setting. METHODS: This was a prospective study comparing performance on the Mini-Mental Status Examination (MMSE) and Montreal Cognitive Assessment (MoCA) before and after administration of opioid pain medications. Eligible participants included ED patients with pain, who received opioid treatment. Participants were randomized to receive either the MMSE before pain medication and the MoCA after medication, or the reverse. MoCA scores were converted to MMSE equivalent scores for comparison. RESULTS: Among 65 participants, the median age was 36 and median triage pain score was 8. 35% of patients were considered cognitively impaired based on their MMSE score prior to any opioid medication (MMSE<27). There was a median decrease in pain scores of 1 point following pain medication, p-value<0.001. There was a median decrease in MMSE scores of 1 point following pain medication, p-value=0.003. The range of change in scores (post minus pre) on the MMSE-equivalent was -7 to 3. 35 patients (56%) had a decrease in scores, 6 (10%) had no change, and 21 (34%) had an increase. After medication, 31 (48%) were abnormal (MMSE score<27). No differences in MMSE scores were identified by gender, ethnicity, mode of arrival, insurance, age, triage pain scores, opioid agent given, or ED diagnosis. CONCLUSIONS: There is an association between opioid pain medication and decrease in cognitive performance on the MMSE. Because of the wide range of cognitive performance following opioid pain medication, assessment of individual patients' cognitive function is indicated.

Antiviral potential of ERK/MAPK and PI3K/AKT/mTOR signaling modulation for Middle East respiratory syndrome coronavirus infection as identified by temporal kinome analysis.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a lineage C betacoronavirus, and infections with this virus can result in acute respiratory syndrome with renal failure. Globally, MERS-CoV has been responsible for 877 laboratory-confirmed infections, including 317 deaths, since September 2012. As there is a paucity of information regarding the molecular pathogenesis associated with this virus or the identities of novel antiviral drug targets, we performed temporal kinome analysis on human hepatocytes infected with the Erasmus isolate of MERS-CoV with peptide kinome arrays. bioinformatics analysis of our kinome data, including pathway overrepresentation analysis (ORA) and functional network analysis, suggested that extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K)/serine-threonine kinase (AKT)/mammalian target of rapamycin (mTOR) signaling responses were specifically modulated in response to MERS-CoV infection in vitro throughout the course of infection. The overrepresentation of specific intermediates within these pathways determined by pathway and functional network analysis of our kinome data correlated with similar patterns of phosphorylation determined through Western blot array analysis. In addition, analysis of the effects of specific kinase inhibitors on MERS-CoV infection in tissue culture models confirmed these cellular response observations. Further, we have demonstrated that a subset of licensed kinase inhibitors targeting the ERK/MAPK and PI3K/AKT/mTOR pathways significantly inhibited MERS-CoV replication in vitro whether they were added before or after viral infection. Taken together, our data suggest that ERK/MAPK and PI3K/AKT/mTOR signaling responses play important roles in MERS-CoV infection and may represent novel drug targets for therapeutic intervention strategies.

Cytokine response in mouse bone marrow derived macrophages after infection with pathogenic and non-pathogenic Rift Valley fever virus.

Rift Valley fever virus (RVFV) is the most pathogenic member of the genus Phlebovirus within the family Bunyaviridae, and can cause severe disease in humans and livestock. Until recently, limited information has been published on the cellular host response elicited by RVFV, particularly in macrophages and dendritic cells, which play critical roles in stimulating adaptive and innate immune responses to viral infection. In an effort to define the initial response of host immunomodulatory cells to infection, primary mouse bone marrow derived macrophages (BMDM) were infected with the pathogenic RVFV strain ZH501, or attenuated strains MP-12 or MP-12 based Clone13 type (rMP12-C13 type), and cytokine secretion profiles examined. The secretion of T helper (Th)1-associated antiviral cytokines, chemokines and various interleukins increased rapidly after infection with the attenuated rMP12-C13 type RVFV, which lacks a functional NSs virulence gene. In comparison, infection with live-attenuated MP-12 encoding a functional NSs gene appeared to cause a delayed immune response, while pathogenic ZH501 ablates the immune response almost entirely. These data demonstrate that NSs can inhibit components of the BMDM antiviral response and supports previous work indicating that NSs can specifically regulate the type I interferon response in macrophages. Furthermore, our data demonstrate that genetic differences between ZH501 and MP-12 reduce the ability of MP-12 to inhibit antiviral signalling and subsequently reduce virulence in BMDM, demonstrating that viral components other than NSs play a critical role in regulating the host response to RVFV infection.

A critical determinant of neurological disease associated with highly pathogenic tick-borne flavivirus in mice.

UNLABELLED: Tick-borne encephalitis virus (TBEV) and Omsk hemorrhagic fever virus (OHFV) are highly pathogenic tick-borne flaviviruses; TBEV causes neurological disease in humans, while OHFV causes a disease typically identified with hemorrhagic fever. Although TBEV and OHFV are closely related genetically, the viral determinants responsible for these distinct disease phenotypes have not been identified. In this study, chimeric viruses incorporating components of TBEV and OHFV were generated using infectious clone technology, and their pathological characteristics were analyzed in a mouse model to identify virus-specific determinants of disease. We found that only four amino acids near the C terminus of the NS5 protein were primarily responsible for the development of neurological disease. Mutation of these four amino acids had no effect on viral replication or histopathological features, including inflammatory responses, in mice. These findings suggest a critical role for NS5 in stimulating neuronal dysfunction and degeneration following TBEV infection and provide new insights into the molecular mechanisms underlying the pathogenesis of tick-borne flaviviruses. IMPORTANCE: Tick-borne encephalitis virus (TBEV) and Omsk hemorrhagic fever virus (OHFV) belong to the tick-borne encephalitis serocomplex, genus Flavivirus, family Flaviviridae. Although TBEV causes neurological disease in humans while OHFV causes a disease typically identified with hemorrhagic fever. In this study, we investigated the viral determinants responsible for the different disease phenotypes using reverse genetics technology. We identified a cluster of only four amino acids in nonstructural protein 5 primarily involved in the development of neurological disease in a mouse model. Moreover, the effect of these four amino acids was independent of viral replication property and did not affect the formation of virus-induced lesions in the brain directly. These data suggest that these amino acids may be involved in the induction of neuronal dysfunction and degeneration in virus-infected neurons, ultimately leading to the neurological disease phenotype. These findings provide new insight into the molecular mechanisms of tick-borne flavivirus pathogenesis.