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.

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.

Persistence of Severe Acute Respiratory Syndrome Coronavirus 2 in Aerosol Suspensions.

We aerosolized severe acute respiratory syndrome coronavirus 2 and determined that its dynamic aerosol efficiency surpassed those of severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome. Although we performed experiment only once across several laboratories, our findings suggest retained infectivity and virion integrity for up to 16 hours in respirable-sized aerosols.

Comparison of respiratory inductive plethysmography versus head-out plethysmography for anesthetized nonhuman primates in an animal biosafety level 4 facility.

For inhalational studies and aerosol exposures to viruses, head-out plethysmography acquisition has been traditionally used for the determination of estimated inhaled dose in anesthetized nonhuman primates prior to or during an aerosol exposure. A pressure drop across a pneumotachograph is measured within a sealed chamber during inspiration/exhalation of the nonhuman primate, generating respiratory values and breathing frequencies. Due to the fluctuation of depth of anesthesia, pre-exposure respiratory values can be variable, leading to less precise and accurate dosing calculations downstream. Although an anesthesia infusion pump may help stabilize the depth of sedation, pumps are difficult to use within a sealed head-out plethysmography chamber. Real-time, head-out plethysmography acquisition could increase precision and accuracy of the measurements, but the bulky equipment needed for head-out plethysmography precludes real-time use inside a Class III biological safety cabinet, where most aerosol exposures occur. However, the respiratory inductive plethysmography (RIP) acquisition method measures the same respiratory parameters by detecting movement of the chest and abdomen during breathing using two elastic bands within the Class III biological safety cabinet. As respiratory values are relayed to a computer for software integration and analysis real-time, adjustment of aerosol exposure duration is based on the depth of sedation of the animal. The objective of this study was to compare values obtained using two methodologies (pre-exposure head-out plethysmography and real-time RIP). Transitioning to RIP technology with real-time acquisition provides more consistent, precise, and accurate aerosol dosing by reducing reported errors in respiratory values from anesthesia variability when using pre-exposure head-out plethysmography acquisition.

Generation and characterization of large-particle aerosols using a center flow tangential aerosol generator with a non-human-primate, head-only aerosol chamber.

Aerosol droplets or particles produced from infected respiratory secretions have the potential to infect another host through inhalation. These respiratory particles can be polydisperse and range from 0.05 to 500 µm in diameter. Animal models of infection are generally established to facilitate the potential licensure of candidate prophylactics and/or therapeutics. Consequently, aerosol-based animal infection models are needed to properly study and counter airborne infections. Ideally, experimental aerosol exposure should reliably result in animal disease that faithfully reproduces the modeled human disease. Few studies have been performed to explore the relationship between exposure particle size and induced disease course for infectious aerosol particles. The center flow tangential aerosol generator (CenTAG™) produces large-particle aerosols capable of safely delivering a variety of infectious aerosols to non-human primates (NHPs) within a Class III Biological Safety Cabinet (BSC) for establishment or refinement of NHP infectious disease models. Here, we report the adaptation of this technology to the Animal Biosafety Level 4 (ABSL-4) environment for the future study of high-consequence viral pathogens and the characterization of CenTAG™-created sham (no animal, no virus) aerosols using a variety of viral growth media and media supplements.

Comparison of the effectiveness of antibody and cell-mediated immunity against inhaled and instilled influenza virus challenge.

BACKGROUND: To evaluate immunity against influenza, mouse challenge studies are typically performed by intranasal instillation of a virus suspension to anesthetized animals. This results in an unnatural environment in the lower respiratory tract during infection, and therefore there is some concern that immune mechanisms identified in this model may not reflect those that protect against infectious virus particles delivered directly to the lower respiratory tract as an aerosol. METHOD: To evaluate differences in protection against instilled and inhaled virus, mice were immunized with influenza antigens known to induce antibody or cell-mediated responses and then challenged with 100 LD50 A/PR/8/34 (PR8) in the form of aerosol (inhaled) or liquid suspension (instilled). RESULTS: Mice immunized with recombinant adenovirus (Ad) expressing hemagglutinin were protected against weight loss and death in both challenge models, however immunization with Ad expressing nucleoprotein of influenza A (NPA) or M2 resulted in greater protection against inhaled aerosolized virus than virus instilled in liquid suspension. Ad-M2, but not Ad-NPA-immunized mice were protected against a lower instillation challenge dose. CONCLUSIONS: These results demonstrate differences in protection that are dependent on challenge method, and suggest that cell-mediated immunity may be more accurately demonstrated in mouse inhalation studies. Furthermore, the data suggest immune mechanisms generally characterized as incomplete or weak in mouse models using liquid intranasal challenge may offer greater immunity against influenza infection than previously thought.

Nipah Virus Aerosol Challenge of Three Distinct Particle Sizes in Nonhuman Primates.

Aerosol and inhalational studies of high-consequence pathogens allow researchers to study the disease course and effects of biologicals transmitted through aerosol in a laboratory-controlled environment. Inhalational studies involving Nipah virus with small (1-3 µm), intermediate (6-8 µm), and large particles (10-14 µm) were explored in African green nonhuman primates to determine if the subsequent disease course more closely recapitulated what is observed in Nipah virus human disease. The aerosol procedures outlined describe the different equipment/techniques used to generate the three particle sizes and control the site of particle deposition within this animal model.

Development and Clinical Evaluation of a Rapid Point of Care Test for Ebola Virus Infection in Humans.

The genus Ebolavirus contains multiple species of viruses that are highly contagious and lethal, often causing severe hemorrhagic fever. To minimize the global threat from Ebola virus disease (EVD), sustainable, field-appropriate tools are needed to quickly screen and triage symptomatic patients and conduct rapid screening of cadavers to ensure proper handling of human remains. The OraQuick® Ebola Rapid Antigen Test is an in vitro diagnostic single-use immunoassay for the qualitative detection of Ebola virus antigens that detects all known species within the genus Ebolavirus. Here, we report the performance of the OraQuick® Ebola Rapid Antigen Test and provide a comparison of its performance with other rapid diagnostic tests (RDTs) for EVD. OraQuick® Ebola demonstrated clinical sensitivity of 84.0% in archived EVD patient venous whole-blood (WB) samples, 90.9% in Ebola virus-infected monkey fingerstick samples, and 97.1% in EVD patient cadaver buccal swabs, as well as clinical specificity of 98.0-100% in venous WB samples and 99.1-100% in contrived saliva samples. It is the only 510(k)-cleared Ebola rapid test, has analytical sensitivity as good as or better than all RDT comparators for EVD, and can detect the Sudan virus. Our data demonstrate that the OraQuick® Ebola Rapid Antigen Test is a sensitive and specific assay that can be used for rapid detection of EBOV in humans and could support efforts for EVD-specific interventions and control over outbreaks.

Previremic Identification of Ebola or Marburg Virus Infection Using Integrated Host-Transcriptome and Viral Genome Detection.

Outbreaks of filoviruses, such as those caused by the Ebola (EBOV) and Marburg (MARV) virus, are difficult to detect and control. The initial clinical symptoms of these diseases are nonspecific and can mimic other endemic pathogens. This makes confident diagnosis based on clinical symptoms alone impossible. Molecular diagnostics for these diseases that rely on the detection of viral RNA in the blood are only effective after significant disease progression. As an approach to identify these infections earlier in the disease course, we tested the effectiveness of viral RNA detection combined with an assessment of sentinel host mRNAs that are upregulated following filovirus infection. RNAseq analysis of EBOV-infected nonhuman primates identified host RNAs that are upregulated at early stages of infection. NanoString probes that recognized these host-response RNAs were combined with probes that recognized viral RNA and were used to classify viral infection both prior to viremia and postviremia. This approach was highly successful at identifying samples from nonhuman primate subjects and correctly distinguished the causative agent in a previremic stage in 10 EBOV and 5 MARV samples. This work suggests that unified host response/viral fingerprint assays can enable diagnosis of disease earlier than testing for viral nucleic acid alone, which could decrease transmission events and increase therapeutic effectiveness.IMPORTANCE Current molecular tests that identify infection with high-consequence viruses such as Ebola virus and Marburg virus are based on the detection of virus material in the blood. These viruses do not undergo significant early replication in the blood and, instead, replicate in organs such as the liver and spleen. Thus, virus begins to accumulate in the blood only after significant replication has already occurred in those organs, making viremia an indicator of infection only after initial stages have become established. Here, we show that a multianalyte assay can correctly identify the infectious agent in nonhuman primates (NHPs) prior to viremia through tracking host infection response transcripts. This illustrates that a single-tube, sample-to-answer format assay could be used to advance the time at which the type of infection can be determined and thereby improve outcomes.

The Human Sodium Iodide Symporter as a Reporter Gene for Studying Middle East Respiratory Syndrome Coronavirus Pathogenesis.

Single photon emission computed tomography (SPECT) is frequently used in oncology and cardiology to evaluate disease progression and/or treatment efficacy. Such technology allows for real-time evaluation of disease progression and when applied to studying infectious diseases may provide insight into pathogenesis. Insertion of a SPECT-compatible reporter gene into a virus may provide insight into mechanisms of pathogenesis and viral tropism. The human sodium iodide symporter (hNIS), a SPECT and positron emission tomography reporter gene, was inserted into Middle East respiratory syndrome coronavirus (MERS-CoV), a recently emerged virus that can cause severe respiratory disease and death in afflicted humans to obtain a quantifiable and sensitive marker for viral replication to further MERS-CoV animal model development. The recombinant virus was evaluated for fitness, stability, and reporter gene functionality. The recombinant and parental viruses demonstrated equal fitness in terms of peak titer and replication kinetics, were stable for up to six in vitro passages, and were functional. Further in vivo evaluation indicated variable stability, but resolution limits hampered in vivo functional evaluation. These data support the further development of hNIS for monitoring infection in animal models of viral disease.IMPORTANCE Advanced medical imaging such as single photon emission computed tomography with computed tomography (SPECT/CT) enhances fields such as oncology and cardiology. Application of SPECT/CT, magnetic resonance imaging, and positron emission tomography to infectious disease may enhance pathogenesis studies and provide alternate biomarkers of disease progression. The experiments described in this article focus on insertion of a SPECT/CT-compatible reporter gene into MERS-CoV to demonstrate that a functional SPECT/CT reporter gene can be inserted into a virus.

Loss in lung volume and changes in the immune response demonstrate disease progression in African green monkeys infected by small-particle aerosol and intratracheal exposure to Nipah virus.

Nipah virus (NiV) is a paramyxovirus (genus Henipavirus) that emerged in the late 1990s in Malaysia and has since been identified as the cause of sporadic outbreaks of severe febrile disease in Bangladesh and India. NiV infection is frequently associated with severe respiratory or neurological disease in infected humans with transmission to humans through inhalation, contact or consumption of NiV contaminated foods. In the work presented here, the development of disease was investigated in the African Green Monkey (AGM) model following intratracheal (IT) and, for the first time, small-particle aerosol administration of NiV. This study utilized computed tomography (CT) and magnetic resonance imaging (MRI) to temporally assess disease progression. The host immune response and changes in immune cell populations over the course of disease were also evaluated. This study found that IT and small-particle administration of NiV caused similar disease progression, but that IT inoculation induced significant congestion in the lungs while disease following small-particle aerosol inoculation was largely confined to the lower respiratory tract. Quantitative assessment of changes in lung volume found up to a 45% loss in IT inoculated animals. None of the subjects in this study developed overt neurological disease, a finding that was supported by MRI analysis. The development of neutralizing antibodies was not apparent over the 8-10 day course of disease, but changes in cytokine response in all animals and activated CD8+ T cell numbers suggest the onset of cell-mediated immunity. These studies demonstrate that IT and small-particle aerosol infection with NiV in the AGM model leads to a severe respiratory disease devoid of neurological indications. This work also suggests that extending the disease course or minimizing the impact of the respiratory component is critical to developing a model that has a neurological component and more accurately reflects the human condition.

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology.

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol releases or unexpected natural aerosol transmission. Specific laboratories designed to perform these experiments require tremendous engineering controls to provide a safe and secure working environment and constant systems maintenance to sustain functionality. Class III biosafety cabinets, also referred to as gloveboxes, are gas-tight enclosures with non-opening windows. These cabinets are maintained under negative pressure by double high-efficiency-particulate-air (HEPA)-filtered exhaust systems and are the ideal primary containment for housing aerosolization equipment. A well planned workflow between staff members within high containment from, for instance, an animal biosafety level-4 (ABSL-4) suit laboratory to the ABSL-4 cabinet laboratory is a crucial component for successful experimentation. For smooth study execution, establishing a communication network, moving equipment and subjects, and setting up and placing equipment, requires staff members to meticulously plan procedures prior to study initiation. Here, we provide an overview and a visual representation of how aerobiology research is conducted at the National Institutes of Health, National Institute of Allergy and Infectious Diseases Integrated Research Facility at Fort Detrick, Maryland, USA, within an ABSL-4 environment.

Modeling [(18)F]-FDG lymphoid tissue kinetics to characterize nonhuman primate immune response to Middle East respiratory syndrome-coronavirus aerosol challenge.

BACKGROUND: The pathogenesis and immune response to Middle East respiratory syndrome (MERS) caused by a recently discovered coronavirus, MERS-CoV, have not been fully characterized because a suitable animal model is currently not available. (18)F-Fluorodeoxyglucose ([(18)F]-FDG)-positron emission tomography/computed tomography (PET/CT) as a longitudinal noninvasive approach can be beneficial in providing biomarkers for host immune response. [(18)F]-FDG uptake is increased in activated immune cells in response to virus entry and can be localized by PET imaging. We used [(18)F]-FDG-PET/CT to investigate the host response developing in nonhuman primates after MERS-CoV exposure and applied kinetic modeling to monitor the influx rate constant (K i ) in responsive lymphoid tissue. METHODS: Multiple [(18)F]-FDG-PET and CT images were acquired on a PET/CT clinical scanner modified to operate in a biosafety level 4 environment prior to and up to 29 days after MERS-CoV aerosol exposure. Time activity curves of various lymphoid tissues were reconstructed to follow the [(18)F]-FDG uptake for approximately 60 min (3,600 s). Image-derived input function was used to calculate K i for lymphoid tissues by Patlak plot. RESULTS: Two-way repeated measures analysis of variance revealed alterations in K i that was associated with the time point (p < 0.001) after virus exposure and the location of lymphoid tissue (p = 0.0004). As revealed by a statistically significant interaction (p < 0.0001) between these two factors, the pattern of K i changes over time differed between three locations but not between subjects. A distinguished pattern of statistically significant elevation in K i was observed in mediastinal lymph nodes (LNs) that correlated to K i changes in axillary LNs. Changes in LNs K i were concurrent with elevations of monocytes in peripheral blood. CONCLUSIONS: [(18)F]-FDG-PET is able to detect subtle changes in host immune response to contain a subclinical virus infection. Full quantitative analysis is the preferred approach rather than semiquantitative analysis using standardized uptake value for detection of the immune response to the virus.

Development of a murine nose-only inhalation model of influenza: comparison of disease caused by instilled and inhaled A/PR/8/34.

Influenza continues to cause widespread disease and death during winter months. In preclinical studies to evaluate the potential efficacy of drugs and vaccines, influenza challenge virus is usually instilled into the noses of animals in the form of large liquid drops. Since inhalation of aerosolized influenza is commonly associated with human transmission, instillation of challenge virus raises uncertainty about the applicability of results. In order to compare the challenge methods, we established conditions to generate influenza aerosols with a mass median aerodynamic diameter (MMAD) of 1 µm that were delivered to mice in a nose-only inhalation system. In this report, we describe the system and compare the 50% lethal dose (LD(50)) of instilled and inhaled A/PR/8/34 (PR8) in BALB/c mice. The estimated LD(50) for inhaled virus was 8.7 plaque forming units (PFU) and the mean time to death was 7.7 days, whereas the estimated LD(50) for instilled virus was 51.6 PFU and the mean time to death was 8.2 days. Our results show that mice are more sensitive to inhaled virus than virus delivered by intranasal instillation. The murine nose-only inhalation model of influenza infection can be used to infect large numbers of animals simultaneously with well-characterized, homogenous PR8 bioaerosol in a controlled and reproducible manner. This model provides the means to evaluate the efficacy of drug and vaccine candidates against the relevant route of challenge, thereby providing data that may better predict clinical outcome.