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Crimean-Congo hemorrhagic fever virus (CCHFV) is endemic in Asia, infecting many animal hosts, but CCHFV has not been reported in Myanmar. We conducted a seroepidemiologic survey of logging communities in Myanmar and found CCHFV exposure was common (9.8%) and exposure to wild animal blood and body fluids was associated with seropositivity.

Purpose of Review This review is aimed at highlighting recent research and articles on the complicated relationship between virus, vector, and host and how biosurveillance at each level informs disease spread and risk. Recent Findings While human cases of CCHFV and tick identification in non-endemic areas in 2019–2020 were reported to sites such as ProMed, there is a gap in recent published literature on these and broader CCHFV surveillance efforts from the late 2010s. Summary A review of the complex aspects of CCHFV maintenance in the environment coupled with high fatality rate and lack of vaccines and therapeutics warrants the need for a One-Health approach toward detection and increased biosurveillance programs for CCHFV.

SARS-CoV-2 is the causative agent of COVID-19 and human infections have resulted in a global health emergency. Small animal models that reproduce key elements of SARS-CoV-2 human infections are needed to rigorously screen candidate drugs to mitigate severe disease and prevent the spread of SARS-CoV-2. We and others have reported that transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE2) viral receptor under the control of the Keratin 18 (K18) promoter develop severe and lethal respiratory disease subsequent to SARS-CoV-2 intranasal challenge. Here we report that some infected mice that survive challenge have residual pulmonary damages and persistent brain infection on day 28 post-infection despite the presence of anti-SARS-COV-2 neutralizing antibodies. Because of the hypersensitivity of K18-hACE2 mice to SARS-CoV-2 and the propensity of virus to infect the brain, we sought to determine if anti-infective biologics could protect against disease in this model system. We demonstrate that anti-SARS-CoV-2 human convalescent plasma protects K18-hACE2 against severe disease. All control mice succumbed to disease by day 7; however, all treated mice survived infection without observable signs of disease. In marked contrast to control mice, viral antigen and lesions were reduced or absent from lungs and absent in brains of antibody-treated mice. Our findings support the use of K18-hACE2 mice for protective efficacy studies of anti-SARS-CoV-2 medical countermeasures (MCMs). They also support the use of this system to study SARS-CoV-2 persistence and host recovery.

The emergence of SARS-CoV-2 pandemic has highlighted the need for animal models that faithfully recapitulate the salient features of COVID-19 disease in humans; these models are necessary for the rapid down-selection, testing, and evaluation of medical countermeasures. Here we performed a direct comparison of two distinct routes of SARS-CoV-2 exposure, combined intratracheal/intranasal and small particle aerosol, in two nonhuman primate species: rhesus and cynomolgus macaques. While all four experimental groups displayed very few outward clinical signs, evidence of mild to moderate respiratory disease was present on radiographs and at the time of necropsy. Cynomolgus macaques exposed via the aerosol route also developed the most consistent fever responses and had the most severe respiratory disease and pathology. This study demonstrates that while all four models were suitable representations of mild COVID-like illness, aerosol exposure of cynomolgus macaques to SARS-CoV-2 produced the most severe disease, which may provide additional clinical endpoints for evaluating therapeutics and vaccines.

Airborne transmission is predicted to be a prevalent route of human exposure with SARS-CoV-2. Aside from African green monkeys, nonhuman primate models that replicate airborne transmission of SARS-CoV-2 have not been investigated. A comparative evaluation of COVID-19 in African green monkeys, rhesus macaques, and cynomolgus macaques following airborne exposure to SARS-CoV-2 was performed to determine critical disease parameters associated with disease progression, and establish correlations between primate and human COVID-19. Respiratory abnormalities and viral shedding were noted for all animals, indicating successful infection. Cynomolgus macaques developed fever, and thrombocytopenia was measured for African green monkeys and rhesus macaques. Type II pneumocyte hyperplasia and alveolar fibrosis were more frequently observed in lung tissue from cynomolgus macaques and African green monkeys. The data indicate that, in addition to African green monkeys, macaques can be successfully infected by airborne SARS-CoV-2, providing viable macaque natural transmission models for medical countermeasure evaluation.

COVID-19 presents herculean challenges to research and scientific communities for producing diagnostic and treatment solutions. Any return to normalcy requires rapid development of countermeasures, with animal models serving as a critical tool in testing vaccines and therapeutics. Animal disease status and potential COVID-19 exposure prior to study execution may severely bias efficacy testing. We developed a toolbox of immunological and molecular tests to monitor countermeasure impact on disease outcome and evaluate pre-challenge COVID-19 status. Assay application showed critical necessity for animal pre-screening. Specifically, real-time PCR results documented pre-exposure of an African Green Monkey prior to SARS-CoV-2 challenge with sequence confirmation as a community-acquired exposure. Longitudinal monitoring of nasopharyngeal swabs and serum showed pre-exposure impacted both viral disease course and resulting immunological response. This study demonstrates utility in a comprehensive pre-screening strategy for animal models, which captured the first documented case of community-acquired, non-human primate infection.One Sentence SummaryPre-exposure to SARS-CoV-2 affects biomarker responses in animal models, highlighting a need for robust pre-screening protocols prior to medical countermeasure studies.

Crimean-Congo hemorrhagic fever (CCHF) is the most medically important tick-borne viral disease of humans and tuberculosis is the leading cause of death worldwide by a bacterial pathogen. These two diseases overlap geographically, however, concurrent infection of CCHF virus (CCHFV) with mycobacterial infection has not been assessed nor has the ability of virus to persist and cause long-term sequela in a primate model. In this study, we compared the disease progression of two diverse strains of CCHFV in the recently described cynomolgus macaque model. All animals demonstrated signs of clinical illness, viremia, significant changes in clinical chemistry and hematology values, and serum cytokine profiles consistent with CCHF in humans. The European and Asian CCHFV strains caused very similar disease profiles in monkeys, which demonstrates that medical countermeasures can be evaluated in this animal model against multiple CCHFV strains. We identified evidence of CCHFV persistence in the testes of three male monkeys that survived infection. Furthermore, the histopathology unexpectedly revealed that six additional animals had evidence of a latent mycobacterial infection with granulomatous lesions. Interestingly, CCHFV persisted within the granulomas of two animals. This study is the first to demonstrate the persistence of CCHFV in the testes and within the granulomas of non-human primates with concurrent latent tuberculosis. Our results have important public health implications in overlapping endemic regions for these emerging pathogens., Author summary CCHF is an emerging tick-borne viral disease that is endemic across much of Africa and Asia, and parts of Europe where its range and exposure risk to human populations is expanding. Tuberculosis threatens millions of lives world-wide and is the leading cause of death due to a bacterial pathogen. Concurrent mycobacterial infection with other infectious diseases has been described, but not for CCHFV despite the geographic overlap of these two pathogens. During our study we unexpectedly determined that some of the animals had latent tuberculosis and that CCHFV can persist within the granulomas. Furthermore, our study provides the first direct evidence that CCHFV can replicate and persist in the male genital tract, which has important implications for human sexual transmission. The ability of viral RNA to persist in immune-privileged sites or fluids has been described with increasing frequency for other emerging infectious diseases and can cause a burden on public health. This provides the impetus to utilize the model described here to better understand the mechanisms of CCHFV persistence and its effect on the development of long-term sequelae.

There is a pressing need for sustainable and sensitive immunodiagnostics for use in public health efforts to understand and combat the threat of endemic and emerging infectious diseases. In this proof-of-concept work, we describe an immunodiagnostic approach based on the utilization of virus-like particles (VLPs) in a magnetic bead-based platform for multiplexed detection of antiviral humoral response. A retroviral-based VLP, that presents Venezuelan equine encephalitis virus E1/E2 glycoprotein antigen on its surface, was synthesized and coupled to magnetic beads to create VLP-conjugated microspheres (VCMs). Using these VCMs, IgM and IgG antibodies were detectable in nonhuman primate (NHP) and human clinical serum samples at dilutions of 1 × 10 Basile et al. [4] and greater. We also extended the VCM methodology to an Old World alphavirus, chikungunya virus, demonstrating the flexibility of this approach toward different VLP architectures. When multiplexed on the MAGPIX® platform, this method provided differential detection between Old World and New World alphaviral IgM. This flexible, immunodiagnostic method, based on the MAGPIX® platform, demonstrates compatibility of particulate antigens with bead-based assays, improves sensitivity by up to 2-logs, and has faster sample-to-answer time over traditional methods.

There is a pressing need for sustainable and sensitive immunodiagnostics for use in public health efforts to understand and combat the threat of endemic and emerging infectious diseases. We describe a novel approach to immunodiagnostics based on virus-like particles (VLPs) attached to magnetic beads. This flexible, innovative immunoassay system, based on the MAGPIX® platform, improves sensitivity by up to 2-logs and has faster sample-to-answer time over traditional methods. As a proof of concept, a retroviral-based VLP, that presents the Venezuelan equine encephalitis virus E1/E2 glycoprotein antigen on its surface, was generated and coupled to magnetic beads to create VLP-conjugated microspheres (VCMs). Using these VCMs, IgG and IgM antibodies were detectable in nonhuman primate (NHP) and human clinical serum samples at dilutions of 1 × 104 and greater. We extended the VCM methodology to two other New-World alphaviruses, eastern and western equine encephalitis viruses, as well as an Old-World alphavirus, Chikungunya virus, demonstrating the flexibility of this approach toward different VLP architectures. When multiplexed on the MAGPIX® platform, the VCMs provided differential diagnosis between Old-World and New-World alphaviruses and well as a route toward assessing the humoral response to both natural infection and vaccination. This VCM system will allow more rapid and efficient detection of endemic and emerging viral pathogens in human populations.

Clinical diagnostics play an important role in infectious disease monitoring. Often these diseases are endemic in settings that lack laboratory resources, which limits the types of tools that are available. Mixing is a common procedure in clinical diagnostic assays and is not immune to these challenges. In this report, we characterize the efficacy of a handheld therapeutic massager that has been repurposed into a mixer. Using metal affinity functionalized magnetic beads and a histidine-tagged fluorescent label, we find that this inexpensive orbital mixer is effective over a range of clinically relevant sample volumes and viscosities. Even with larger sample volumes (>200 μL) and more viscous sample matrices (3× ηH2O), the mixer binds more than 90% of the biomarkers in solution in less than 90 seconds. In a clinical assay for the detection of Plasmodium falciparum histidine-rich protein 2, we find there is no statistical difference in performance between our inexpensive and portable orbital mixer and a standard laboratory benchtop vortexer.

Rapid diagnostic tests (RDTs) designed to function at the point of care are becoming more prevalent in malaria diagnostics because of their low cost and simplicity. While many of these tests function effectively with high parasite density samples, their poor sensitivity can often lead to misdiagnosis when parasitemia falls below 100 parasites/μl. In this study, a flow-through pipette-based column was explored as a cost-effective means to capture and elute more Plasmodium falciparum histidine-rich protein II (HRPII) antigen, concentrating the biomarker available in large-volume lysed whole blood samples into volumes compatible with Plasmodium falciparum-specific RDTs. A systematic investigation of immobilized metal affinity chromatography divalent metal species and solid phase supports established the optimal design parameters necessary to create a flow-through column incorporated into a standard pipette tip. The bidirectional flow inherent to this format maximizes mixing efficiency so that in less than 5 min of sample processing, the test band signal intensity was increased up to a factor of twelve from HRPII concentrations as low as 25 pM. In addition, the limit of detection per sample was decreased by a factor of five when compared to the RDT manufacturer's suggested protocol. Both the development process and commercial viability of this application are explored, serving as a potential model for future applications.

Urine samples are increasingly used for diagnosing infections including Escherichia coli, Ebola virus, and Zika virus. However, extraction and concentration of nucleic acid biomarkers from urine is necessary for many molecular detection strategies such as polymerase chain reaction (PCR). Since urine samples typically have large volumes with dilute biomarker concentrations making them prone to false negatives, another impediment for urine-based diagnostics is the establishment of appropriate controls particularly to rule out false negatives. In this study, a mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) DNA target was added to retrospectively collected urine samples from tuberculosis (TB)-infected and TB-uninfected patients to indicate extraction of intact DNA and removal of PCR inhibitors from urine samples. We tested this design on surrogate urine samples, retrospective 1 milliliter (mL) urine samples from patients in Lima, Peru and retrospective 5 mL urine samples from patients in Cape Town, South Africa. Extraction/PCR control DNA was detectable in 97% of clinical samples with no statistically significant differences among groups. Despite the inclusion of this control, there was no difference in the amount of TB IS6110 Tr-DNA detected between TB-infected and TB-uninfected groups except for samples from known HIV-infected patients. We found a increase in TB IS6110 Tr-DNA between TB/HIV co-infected patients compared to TB-uninfected/HIV-infected patients (N=18, p=0.037). The inclusion of an extraction/PCR control DNA to indicate successful DNA extraction and removal of PCR inhibitors should be easily adaptable as a sample preparation control for other acellular sample types.

Background The characteristic ease of use, rapid time to result, and low cost of malaria rapid diagnostic tests (RDTs) promote their widespread use at the point-of-care for malaria detection and surveillance. However, in many settings, the success of malaria elimination campaigns depends on point-of-care diagnostics with greater sensitivity than currently available RDTs. To address this need, a sample preparation method was developed to deliver more biomarkers onto a malaria RDT by concentrating the biomarker from blood sample volumes that are too large to be directly applied to a lateral flow strip. Methods In this design, Ni–NTA-functionalized magnetic beads captured the Plasmodium falciparum biomarker HRPII from a P. falciparum D6 culture spiked blood sample. This transfer of magnetic beads to the RDT was facilitated by an inexpensive 3D-printed apparatus that aligned the sample tube with the sample deposition pad and a magnet beneath the RDT. Biomarkers were released from the bead surface onto the lateral flow strip using imidazole-spiked running buffer. Kinetics of HRPII binding to the Ni–NTA beads as a function of blood sample volume were explored prior to determining the effect of the proposed method on the limit of detection of Paracheck RDTs. Results More than 80 % of HRPII biomarkers were extracted from blood sample volumes ranging from 25 to 250 µL. The time required to reach 80 % binding ranged from 5 to 60 min, depending on sample volume. Using 250 μL of blood and a 30-min biomarker binding time, the limit of detection of the Paracheck Pf RDT brand was improved by 21-fold, resulting in a limit of detection below 1 parasite/μL. Conclusions This approach has the sensitivity and simplicity required to assist in malaria elimination campaigns in settings with limited access to clinical and laboratory resources. Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1448-6) contains supplementary material, which is available to authorized users.