Seroconversion and fever are dose-dependent in a nonhuman primate model of inhalational COVID-19.

While evidence exists supporting the potential for aerosol transmission of SARS-CoV-2, the infectious dose by inhalation remains unknown. In the present study, the probability of infection following inhalation of SARS-CoV-2 was dose-dependent in a nonhuman primate model of inhalational COVID-19. The median infectious dose, assessed by seroconversion, was 52 TCID50 (95% CI: 23-363 TCID50), and was significantly lower than the median dose for fever (256 TCID50, 95% CI: 102-603 TCID50), resulting in a group of animals that developed an immune response post-exposure but did not develop fever or other clinical signs of infection. In a subset of these animals, virus was detected in nasopharyngeal and/or oropharyngeal swabs, suggesting that infected animals without signs of disease are able to shed virus and may be infectious, which is consistent with reports of asymptomatic spread in human cases of COVID-19. These results suggest that differences in exposure dose may be a factor influencing disease presentation in humans, and reinforce the importance of public health measures that limit exposure dose, such as social distancing, masking, and increased ventilation. The dose-response data provided by this study are important to inform disease transmission and hazard modeling, and, ultimately, mitigation strategies. Additionally, these data will be useful to inform dose selection in future studies examining the efficacy of therapeutics and vaccines against inhalational COVID-19, and as a baseline in healthy, young adult animals for assessment of the importance of other factors, such as age, comorbidities, and viral variant, on the infectious dose and disease presentation.

SARS-CoV-2 is rapidly inactivated at high temperature.

In the absence of a vaccine, preventing the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the primary means to reduce the impact of the 2019 coronavirus disease (COVID-19). Multiple studies have reported the presence of SARS-CoV-2 genetic material on surfaces suggesting that fomite transmission of SARS-CoV-2 is feasible. High temperature inactivation of virus has been previously suggested, but not shown. In the present study, we investigated the environmental stability of SARS-CoV-2 in a clinically relevant matrix dried onto stainless steel at a high temperature. The results show that at 54.5 °C, the virus half-life was 10.8 ± 3.0 min and the time for a 90% decrease in infectivity was 35.4 ± 9.0 min. These findings suggest that in instances where the environment can reach temperatures of at least 54.5 °C, such as in vehicle interior cabins when parked in warmer ambient air, that the potential for exposure to infectious virus on surfaces could be decreased substantially in under an hour.

Simulated Sunlight Rapidly Inactivates SARS-CoV-2 on Surfaces.

Previous studies have demonstrated that SARS-CoV-2 is stable on surfaces for extended periods under indoor conditions. In the present study, simulated sunlight rapidly inactivated SARS-CoV-2 suspended in either simulated saliva or culture media and dried on stainless steel coupons. Ninety percent of infectious virus was inactivated every 6.8 minutes in simulated saliva and every 14.3 minutes in culture media when exposed to simulated sunlight representative of the summer solstice at 40°N latitude at sea level on a clear day. Significant inactivation also occurred, albeit at a slower rate, under lower simulated sunlight levels. The present study provides the first evidence that sunlight may rapidly inactivate SARS-CoV-2 on surfaces, suggesting that persistence, and subsequently exposure risk, may vary significantly between indoor and outdoor environments. Additionally, these data indicate that natural sunlight may be effective as a disinfectant for contaminated nonporous materials.

Airborne SARS-CoV-2 Is Rapidly Inactivated by Simulated Sunlight.

Aerosols represent a potential transmission route of COVID-19. This study examined effect of simulated sunlight, relative humidity, and suspension matrix on stability of SARS-CoV-2 in aerosols. Simulated sunlight and matrix significantly affected decay rate of the virus. Relative humidity alone did not affect the decay rate; however, minor interactions between relative humidity and other factors were observed. Mean decay rates (± SD) in simulated saliva, under simulated sunlight levels representative of late winter/early fall and summer were 0.121 ±â€…0.017 min-1 (90% loss, 19 minutes) and 0.306 ±â€…0.097 min-1 (90% loss, 8 minutes), respectively. Mean decay rate without simulated sunlight across all relative humidity levels was 0.008 ±â€…0.011 min-1 (90% loss, 286 minutes). These results suggest that the potential for aerosol transmission of SARS-CoV-2 may be dependent on environmental conditions, particularly sunlight. These data may be useful to inform mitigation strategies to minimize the potential for aerosol transmission.

Exploring Crimean-Congo Hemorrhagic Fever Virus-Induced Hepatic Injury Using Antibody-Mediated Type I Interferon Blockade in Mice.

Crimean-Congo hemorrhagic fever virus (CCHFV) can cause severe hepatic injury in humans. However, the mechanism(s) causing this damage is poorly characterized. CCHFV produces an acute disease, including liver damage, in mice lacking type I interferon (IFN-I) signaling due to either STAT-1 gene deletion or disruption of the IFN-I receptor 1 gene. Here, we explored CCHFV-induced liver pathogenesis in mice using an antibody to disrupt IFN-I signaling. When IFN-I blockade was induced within 24 h postexposure to CCHFV, mice developed severe disease with greater than 95% mortality by 6 days postexposure. In addition, we observed increased proinflammatory cytokines, chemoattractants, and liver enzymes in these mice. Extensive liver damage was evident by 4 days postexposure and was characterized by hepatocyte necrosis and the loss of CLEC4F-positive Kupffer cells. Similar experiments in CCHFV-exposed NOD-SCID-γ (NSG), Rag2-deficient, and perforin-deficient mice also demonstrated liver injury, suggesting that cytotoxic immune cells are dispensable for hepatic damage. Some apoptotic liver cells contained viral RNA, while other apoptotic liver cells were negative, suggesting that cell death occurred by both intrinsic and extrinsic mechanisms. Protein and transcriptional analysis of livers revealed that activation of tumor necrosis factor superfamily members occurred by day 4 postexposure, implicating these molecules as factors in liver cell death. These data provide insights into CCHFV-induced hepatic injury and demonstrate the utility of antibody-mediated IFN-I blockade in the study of CCHFV pathogenesis in mice.IMPORTANCE CCHFV is an important human pathogen that is both endemic and emerging throughout Asia, Africa, and Europe. A common feature of acute disease is liver injury ranging from mild to fulminant hepatic failure. The processes through which CCHFV induces severe liver injury are unclear, mostly due to the limitations of existing small-animal systems. The only small-animal model in which CCHFV consistently produces severe liver damage is mice lacking IFN-I signaling. In this study, we used antibody-mediated blockade of IFN-I signaling in mice to study CCHFV liver pathogenesis in various transgenic mouse systems. We found that liver injury did not depend on cytotoxic immune cells and observed extensive activation of death receptor signaling pathways in the liver during acute disease. Furthermore, acute CCHFV infection resulted in a nearly complete loss of Kupffer cells. Our model system provides insight into both the molecular and the cellular features of CCHFV hepatic injury.

A Haploid Genetic Screen Identifies Heparan Sulfate Proteoglycans Supporting Rift Valley Fever Virus Infection.

UNLABELLED: Rift Valley fever virus (RVFV) causes recurrent insect-borne epizootics throughout the African continent, and infection of humans can lead to a lethal hemorrhagic fever syndrome. Deep mutagenesis of haploid human cells was used to identify host factors required for RVFV infection. This screen identified a suite of enzymes involved in glycosaminoglycan (GAG) biogenesis and transport, including several components of the cis-oligomeric Golgi (COG) complex, one of the central components of Golgi complex trafficking. In addition, disruption of PTAR1 led to RVFV resistance as well as reduced heparan sulfate surface levels, consistent with recent observations that PTAR1-deficient cells exhibit altered Golgi complex morphology and glycosylation defects. A variety of biochemical and genetic approaches were utilized to show that both pathogenic and attenuated RVFV strains require GAGs for efficient infection on some, but not all, cell types, with the block to infection being at the level of virion attachment. Examination of other members of the Bunyaviridae family for GAG-dependent infection suggested that the interaction with GAGs is not universal among bunyaviruses, indicating that these viruses, as well as RVFV on certain cell types, employ additional unidentified virion attachment factors and/or receptors. IMPORTANCE: Rift Valley fever virus (RVFV) is an emerging pathogen that can cause severe disease in humans and animals. Epizootics among livestock populations lead to high mortality rates and can be economically devastating. Human epidemics of Rift Valley fever, often initiated by contact with infected animals, are characterized by a febrile disease that sometimes leads to encephalitis or hemorrhagic fever. The global burden of the pathogen is increasing because it has recently disseminated beyond Africa, which is of particular concern because the virus can be transmitted by widely distributed mosquito species. There are no FDA-licensed vaccines or antiviral agents with activity against RVFV, and details of its life cycle and interaction with host cells are not well characterized. We used the power of genetic screening in human cells and found that RVFV utilizes glycosaminoglycans to attach to host cells. This furthers our understanding of the virus and informs the development of antiviral therapeutics.

Nuclear relocalization of polyadenylate binding protein during rift valley fever virus infection involves expression of the NSs gene.

Rift Valley fever virus (RVFV), an ambisense member of the family Bunyaviridae, genus Phlebovirus, is the causative agent of Rift Valley fever, an important zoonotic infection in Africa and the Middle East. Phlebovirus proteins are translated from virally transcribed mRNAs that, like host mRNA, are capped but, unlike host mRNAs, are not polyadenylated. Here, we investigated the role of PABP1 during RVFV infection of HeLa cells. Immunofluorescence studies of infected cells demonstrated a gross relocalization of PABP1 to the nucleus late in infection. Immunofluorescence microscopy studies of nuclear proteins revealed costaining between PABP1 and markers of nuclear speckles. PABP1 relocalization was sharply decreased in cells infected with a strain of RVFV lacking the gene encoding the RVFV nonstructural protein S (NSs). To determine whether PABP1 was required for RVFV infection, we measured the production of nucleocapsid protein (N) in cells transfected with small interfering RNAs (siRNAs) targeting PABP1. We found that the overall percentage of RVFV N-positive cells was not changed by siRNA treatment, indicating that PABP1 was not required for RVFV infection. However, when we analyzed populations of cells producing high versus low levels of PABP1, we found that the percentage of RVFV N-positive cells was decreased in cell populations producing physiologic levels of PABP1 and increased in cells with reduced levels of PABP1. Together, these results suggest that production of the NSs protein during RVFV infection leads to sequestration of PABP1 in the nuclear speckles, creating a state within the cell that favors viral protein production.

IFITM-2 and IFITM-3 but not IFITM-1 restrict Rift Valley fever virus.

We show that interferon-induced transmembrane protein 1 (IFITM-1), IFITM-2, and IFITM-3 exhibit a broad spectrum of antiviral activity against several members of the Bunyaviridae family, including Rift Valley fever virus (RVFV), La Crosse virus, Andes virus, and Hantaan virus, all of which can cause severe disease in humans and animals. We found that RVFV was restricted by IFITM-2 and -3 but not by IFITM-1, whereas the remaining viruses were equally restricted by all IFITMs. Indeed, at low doses of alpha interferon (IFN-α), IFITM-2 and -3 mediated more than half of the antiviral activity of IFN-α against RVFV. IFITM-2 and -3 restricted RVFV infection mostly by preventing virus membrane fusion with endosomes, while they had no effect on virion attachment to cells, endocytosis, or viral replication kinetics. We found that large fractions of IFITM-2 and IFITM-3 occupy vesicular compartments that are distinct from the vesicles coated by IFITM-1. In addition, although overexpression of all IFITMs expanded vesicular and acidified compartments within cells, there were marked phenotypic differences among the vesicular compartments occupied by IFITMs. Collectively, our data provide new insights into the possible mechanisms by which the IFITM family members restrict distinct viruses.

Junin Virus Activates p38 MAPK and HSP27 Upon Entry.

Junín virus (JUNV), a New World arenavirus, is a rodent-borne virus and the causative agent of Argentine hemorrhagic fever. Humans become infected through exposure to rodent host secreta and excreta and the resulting infection can lead to an acute inflammatory disease with significant morbidity and mortality. Little is understood about the molecular pathogenesis of arenavirus hemorrhagic fever infections. We utilized Reverse Phase Protein Microarrays (RPPA) to compare global alterations in the host proteome following infection with an attenuated vaccine strain, Candid#1 (CD1), and the most parental virulent strain, XJ13, of JUNV in a human cell culture line. Human small airway epithelial cells were infected with CD1 or XJ13 at an MOI of 10, or mock infected. To determine proteomic changes at early timepoints (T = 1, 3, 8 and 24 h), the JUNV infected or mock infected cells were lysed in compatible buffers for RPPA. Out of 113 proteins that were examined by RPPA, 14 proteins were significantly altered following JUNV infection. Several proteins were commonly phosphorylated between the two strains and these correspond to entry and early replication events, to include p38 mitogen-activated protein kinase (MAPK), heat shock protein 27 (HSP27), and nuclear factor kappa B (NFκB). We qualitatively confirmed the alterations of these three proteins following infection by western blot analysis. We also determined that the inhibition of either p38 MAPK, with the small molecule inhibitor SB 203580 or siRNA knockdown, or HSP27, by siRNA knockdown, significantly decreases JUNV replication. Our data suggests that HSP27 phosphorylation at S82 upon virus infection is dependent on p38 MAPK activity. This work sheds light on the nuances of arenavirus replication.

Comparison of Dose-Response Relationships for Two Isolates of SARS-CoV-2 in a Nonhuman Primate Model of Inhalational COVID-19.

Background: As the COVID-19 pandemic has progressed, numerous variants of SARS-CoV-2 have arisen, with several displaying increased transmissibility. Methods: The present study compared dose-response relationships and disease presentation in nonhuman primates infected with aerosols containing an isolate of the Gamma variant of SARS-CoV-2 to the results of our previous study with the earlier WA-1 isolate of SARS-CoV-2. Results: Disease in Gamma-infected animals was mild, characterized by dose-dependent fever and oronasal shedding of virus. Differences were observed in shedding in the upper respiratory tract between Gamma- and WA-1-infected animals that have the potential to influence disease transmission. Specifically, the estimated median doses for shedding of viral RNA or infectious virus in nasal swabs were approximately 10-fold lower for the Gamma variant than the WA-1 isolate. Given that the median doses for fever were similar, this suggests that there is a greater difference between the median doses for viral shedding and fever for Gamma than for WA-1 and potentially an increased range of doses for Gamma over which asymptomatic shedding and disease transmission are possible. Conclusions: These results complement those of previous studies, which suggested that differences in exposure dose may help to explain the range of clinical disease presentations observed in individuals with COVID-19, highlighting the importance of public health measures designed to limit exposure dose, such as masking and social distancing. The dose-response data provided by this study are important to inform disease transmission and hazard modeling, as well as to inform dose selection in future studies examining the efficacy of therapeutics and vaccines in animal models of inhalational COVID-19.

Comparison of transcriptional responses between pathogenic and nonpathogenic hantavirus infections in Syrian hamsters using NanoString.

BACKGROUND: Syrian hamsters infected with Andes virus (ANDV) develop a disease that recapitulates many of the salient features of human hantavirus pulmonary syndrome (HPS), including lethality. Infection of hamsters with Hantaan virus (HTNV) results in an asymptomatic, disseminated infection. In order to explore this dichotomy, we examined the transcriptome of ANDV- and HTNV-infected hamsters. RESULTS: Using NanoString technology, we examined kinetic transcriptional responses in whole blood collected from ANDV- and HTNV-infected hamsters. Of the 770 genes analyzed, key differences were noted in the kinetics of type I interferon sensing and signaling responses, complement activation, and apoptosis pathways between ANDV- and HTNV-infected hamsters. CONCLUSIONS: Delayed activation of type I interferon responses in ANDV-infected hamsters represents a potential mechanism that ANDV uses to subvert host immune responses and enhance disease. This is the first genome-wide analysis of hantavirus-infected hamsters and provides insight into potential avenues for therapeutics to hantavirus disease.

Transcriptomic Analysis Reveals Host miRNAs Correlated with Immune Gene Dysregulation during Fatal Disease Progression in the Ebola Virus Cynomolgus Macaque Disease Model.

Ebola virus is a continuing threat to human populations, causing a virulent hemorrhagic fever disease characterized by dysregulation of both the innate and adaptive host immune responses. Severe cases are distinguished by an early, elevated pro-inflammatory response followed by a pronounced lymphopenia with B and T cells unable to mount an effective anti-viral response. The precise mechanisms underlying the dysregulation of the host immune system are poorly understood. In recent years, focus on host-derived miRNAs showed these molecules to play an important role in the host gene regulation arsenal. Here, we describe an investigation of RNA biomarkers in the fatal Ebola virus disease (EVD) cynomolgus macaque model. We monitored both host mRNA and miRNA responses in whole blood longitudinally over the disease course in these non-human primates (NHPs). Analysis of the interactions between these classes of RNAs revealed several miRNA markers significantly correlated with downregulation of genes; specifically, the analysis revealed those involved in dysregulated immune pathways associated with EVD. In particular, we noted strong interactions between the miRNAs hsa-miR-122-5p and hsa-miR-125b-5p with immunological genes regulating both B and T-cell activation. This promising set of biomarkers will be useful in future studies of severe EVD pathogenesis in both NHPs and humans and may serve as potential prognostic targets.

Strategies for Validation of Inactivation of Viruses with Trizol® LS and Formalin Solutions.

Introduction: Inactivation of biological agents and particularly select agents has come under increased scrutiny since the US Army inadvertently shipped live anthrax both inside and outside the US, leading to more stringent regulations regarding inactivation. Methods: Formalin and Trizol® LS were used to inactivate virus samples in complex matrices. Cytotoxic chemicals were removed using either desalting or concentrating columns or through dilution using HYPERFlasks. Efficacy of inactivation was evaluated either through plaque assay or immunofluorescence assay. Results: All virus samples and tissue specimens were successfully inactivated using either formalin or Trizol® LS. Both the desalting columns and concentrating columns were able to remove cytotoxic chemicals to facilitate viral amplification in controls. Dilution of cytotoxic chemicals through HYPERFlasks was also successful provided that media was changed completely within 48 hours of first cell passage. Discussion: All inactivation testing demonstrates that both formalin and Trizol® LS successfully inactivate virus-infected cell lines and tissues, which is consistent with previously published literature. Each sample cleanup method has its benefits and pitfalls. Desalting columns can process the largest sample size but are also susceptible to plugging and degradation, whereas concentrating columns are not as vulnerable but can only process 5% of the sample load per run. Conclusion: Based on our results along with those of our colleagues, it is recommended that the regulatory authorities re-evaluate the requirements for each entity to validate well-established inactivation methods in house because there would be limited benefits despite the considerable resources required for this effort.

Increasing Temperature and Relative Humidity Accelerates Inactivation of SARS-CoV-2 on Surfaces.

Coronavirus disease 2019 (COVID-19) was first identified in China in late 2019 and is caused by newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Previous studies had reported the stability of SARS-CoV-2 in cell culture media and deposited onto surfaces under a limited set of environmental conditions. Here, we broadly investigated the effects of relative humidity, temperature, and droplet size on the stability of SARS-CoV-2 in a simulated clinically relevant matrix dried on nonporous surfaces. The results show that SARS-CoV-2 decayed more rapidly when either humidity or temperature was increased but that droplet volume (1 to 50 µl) and surface type (stainless steel, plastic, or nitrile glove) did not significantly impact decay rate. At room temperature (24°C), virus half-life ranged from 6.3 to 18.6 h depending on the relative humidity but was reduced to 1.0 to 8.9 h when the temperature was increased to 35°C. These findings suggest that a potential for fomite transmission may persist for hours to days in indoor environments and have implications for assessment of the risk posed by surface contamination in indoor environments.IMPORTANCE Mitigating the transmission of SARS-CoV-2 in clinical settings and public spaces is critically important to reduce the number of COVID-19 cases while effective vaccines and therapeutics are under development. SARS-CoV-2 transmission is thought to primarily occur through direct person-to-person transfer of infectious respiratory droplets or through aerosol-generating medical procedures. However, contact with contaminated surfaces may also play a significant role. In this context, understanding the factors contributing to SARS-CoV-2 persistence on surfaces will enable a more accurate estimation of the risk of contact transmission and inform mitigation strategies. To this end, we have developed a simple mathematical model that can be used to estimate virus decay on nonporous surfaces under a range of conditions and which may be utilized operationally to identify indoor environments in which the virus is most persistent.

GP38-targeting monoclonal antibodies protect adult mice against lethal Crimean-Congo hemorrhagic fever virus infection.

Crimean-Congo hemorrhagic fever virus (CCHFV) is an important human pathogen. Limited evidence suggests that antibodies can protect humans against lethal CCHFV disease but the protective efficacy of antibodies has never been evaluated in adult animal models. Here, we used adult mice to investigate the protection provided against CCHFV infection by glycoprotein-targeting neutralizing and non-neutralizing monoclonal antibodies (mAbs). We identified a single non-neutralizing antibody (mAb-13G8) that protected adult type I interferon-deficient mice >90% when treatment was initiated before virus exposure and >60% when administered after virus exposure. Neutralizing antibodies known to protect neonatal mice from lethal CCHFV infection failed to confer protection regardless of immunoglobulin G subclass. The target of mAb-13G8 was identified as GP38, one of multiple proteolytically cleaved glycoproteins derived from the CCHFV glycoprotein precursor polyprotein. This study reveals GP38 as an important antibody target for limiting CCHFV pathogenesis and lays the foundation to develop immunotherapeutics against CCHFV in humans.

Transcriptomics Reveal Antiviral Gene Induction in the Egyptian Rousette Bat Is Antagonized In Vitro by Marburg Virus Infection.

The Egyptian rousette bat (ERB) is the only known Marburg virus (MARV) reservoir host. ERBs develop a productive MARV infection with low viremia and shedding but no overt disease, suggesting this virus is efficiently controlled by ERB antiviral responses. This dynamic would contrast with humans, where MARV-mediated interferon (IFN) antagonism early in infection is thought to contribute to the severe, often fatal disease. The newly-annotated ERB genome and transcriptome have now enabled us to use a custom-designed NanoString nCounter ERB CodeSet in conjunction with RNA-seq to investigate responses in a MARV-infected ERB cell line. Both transcriptomic platforms correlated well and showed that MARV inhibited the antiviral program in ERB cells, while an IFN antagonism-impaired MARV was less efficient at suppressing the response gene induction, phenotypes previously reported for primate cells. Interestingly, and despite the expansion of IFN loci in the ERB genome, neither MARV showed specific induction of almost any IFN gene. However, we detected an upregulation of putative, unannotated ERB antiviral paralogs, as well as an elevated basal expression in uninfected ERB cells of key antiviral genes.

Sequence Optimized Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Detection of Crimean-Congo Hemorrhagic Fever Virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus of the genus Nairovirus within the family Bunyaviridae. Infection can result in general myalgia, fever, and headache with some patients developing hemorrhagic fever with mortality rates ranging from 5% to 30%. CCHFV has a wide geographic range that includes Africa, Asia, the Middle East, and Europe with nucleotide sequence variation approaching 20% across the three negative-sense RNA genome segments. While phylogenetic clustering generally aligns with geographic origin of individual strains, distribution can be wide due to tick/CCHFV dispersion via migrating birds. This sequence diversity negatively impacts existing molecular diagnostic assays, leading to false negative diagnostic results. Here, we updated a previously developed CCHFV real-time reverse transcription polymerase chain reaction (RT-PCR) assay to include strains not detected using that original assay. Deep sequencing of eight different CCHFV strains, including three that were not detectable using the original assay, identified sequence variants within this assay target region. New primers and probe based on the sequencing results and newly deposited sequences in GenBank greatly improved assay sensitivity and inclusivity with the exception of the genetically diverse strain AP92. For example, we observed a four log improvement in IbAr10200 detection with a new limit of detection of 256 PFU/mL. Subsequent comparison of this assay to another commonly used CCHFV real-time RT-PCR assay targeting a different region of the viral genome showed improved detection, and both assays could be used to mitigate CCHFV diversity for diagnostics. Overall, this work demonstrated the importance of continued viral sequencing efforts for robust diagnostic assay development.

A conserved transcriptional response to intranasal Ebola virus exposure in nonhuman primates prior to onset of fever.

Ebola virus disease (EVD), caused by Ebola virus (EBOV), is a severe illness characterized by case fatality rates of up to 90%. The sporadic nature of outbreaks in resource-limited areas has hindered the ability to characterize the pathogenesis of EVD at all stages of infection but particularly early host responses. Pathogenesis is often studied in nonhuman primate (NHP) models of disease that replicate major aspects of human EVD. Typically, NHP models use a large infectious dose, are carried out through intramuscular or aerosol exposure, and have a fairly uniform disease course. By contrast, we report our analysis of the host response to EBOV after intranasal exposure. Twelve cynomolgus macaques were infected with 100 plaque-forming units of EBOV/Makona through intranasal exposure and presented with varying times to onset of EVD. We used RNA sequencing and a newly developed NanoString CodeSet to monitor the host response via changes in RNA transcripts over time. When individual animal gene expression data were phased based on the onset of sustained fever, the first clinical sign of severe disease, mathematical models indicated that interferon-stimulated genes appeared as early as 4 days before fever onset. This demonstrates that lethal EVD has a uniform and predictable response to infection regardless of time to onset. Furthermore, expression of a subset of genes could predict disease development before other host-based indications of infection such as fever.

Virus-encoded miRNAs in Ebola virus disease.

Ebola virus (EBOV) is a negative-strand RNA virus that replicates in the cytoplasm and causes an often-fatal hemorrhagic fever. EBOV, like other viruses, can reportedly encode its own microRNAs (miRNAs) to subvert host immune defenses. miRNAs are short noncoding RNAs that can regulate gene expression by hybridizing to multiple mRNAs, and viral miRNAs can enhance viral replication and infectivity by regulating host or viral genes. To date, only one EBOV miRNA has been examined in human infection. Here, we assayed mouse, rhesus macaque, cynomolgus macaque, and human samples infected with three EBOV variants for twelve computationally predicted viral miRNAs using RT-qPCR. Ten miRNAs aligned to EBOV variants and were detectable in the four species during disease with several viral miRNAs showing presymptomatic amplification in animal models. miRNA abundances in both the mouse and nonhuman primate models mirrored the human cohort, with miR-1-5p, miR-1-3p, and miR-T3-3p consistently at the highest levels. These striking similarities in the most abundant miRNAs during infection with different EBOV variants and hosts indicate that these miRNAs are potential valuable diagnostic markers and key effectors of EBOV pathogenesis.

Comparison of Saliva Collection Methods for the Determination of Salivary Cortisol Levels in Rhesus Macaques (Macaca mulatta), Cynomolgus Macaques (Macaca fascicularis), and African Green Monkeys (Chlorocebus aethiops).

The ability to quickly and accurately determine cortisol as a biomarker for stress is a valuable tool in assessing the wellbeing of NHP. In this study, 2 methods of collecting saliva (a commercial collection device and passive drool) and the resulting free salivary cortisol levels were compared with total serum cortisol concentration in rhesus macaques (Macaca mulatta), cynomolgus macaques (Macaca fascicularis) and African green monkeys (Chlorocebus aethiops) at 2 collection time points. Serum and salivary cortisol levels were determined using a competitive quantitative ELISA. In addition, both saliva collection methods were evaluated for volume collected and ease of use. Compared with passive drool, the experimental collection device was more reliable in collecting sufficient volumes of saliva, and the resulting salivary cortisol values demonstrated stronger correlation with serum cortisol concentration in all species and collection days except cynomolgus macaques on day 1. This saliva collection device allows quick and reliable sample collection for the determination of salivary cortisol levels. In addition, the results might provide a useful tool for evaluating hypothalamic-pituitary-adrenal axis activity or the physiologic stress reaction in NHP as well as a biomarker of psychologic stress states in a variety of situations.