TRIM7 ubiquitinates SARS-CoV-2 membrane protein to limit apoptosis and viral replication.

SARS-CoV-2 is a highly transmissible virus that causes COVID-19 disease. Mechanisms of viral pathogenesis include excessive inflammation and viral-induced cell death, resulting in tissue damage. We identified the host E3-ubiquitin ligase TRIM7 as an inhibitor of apoptosis and SARS-CoV-2 replication via ubiquitination of the viral membrane (M) protein. Trim7 -/- mice exhibited increased pathology and virus titers associated with epithelial apoptosis and dysregulated immune responses. Mechanistically, TRIM7 ubiquitinates M on K14, which protects cells from cell death. Longitudinal SARS-CoV-2 sequence analysis from infected patients revealed that mutations on M-K14 appeared in circulating variants during the pandemic. The relevance of these mutations was tested in a mouse model. A recombinant M- K14/K15R virus showed reduced viral replication, consistent with the role of K15 in virus assembly, and increased levels of apoptosis associated with the loss of ubiquitination on K14. TRIM7 antiviral activity requires caspase-6 inhibition, linking apoptosis with viral replication and pathology.

Smartphone-readable RPA-LFA for the high-sensitivity detection of Leishmania kDNA using nanophosphor reporters.

Early diagnosis of infectious diseases improves outcomes by enabling earlier delivery of effective treatment, and helps prevent further transmission by undiagnosed persons. We demonstrated a proof-of-concept assay combining isothermal amplification and lateral flow assay (LFA) for early diagnosis of cutaneous leishmaniasis, a vector-borne infectious disease that affects ca. 700,000 to 1.2 million people annually. Conventional molecular diagnostic techniques based on polymerase chain reaction (PCR) require complex apparatus for temperature cycling. Recombinase polymerase amplification (RPA) is an isothermal DNA amplification method that has shown promise for use in low-resource settings. Combined with lateral flow assay as the readout, RPA-LFA can be used as a point-of-care diagnostic tool with high sensitivity and specificity, but reagent costs can be problematic. In this work, we developed a highly-sensitive smartphone-based RPA-LFA for the detection of Leishmania panamensis DNA using blue-emitting [(Sr0.625Ba0.375)1.96Eu0.01Dy0.03]MgSi2O7 (SBMSO) persistent luminescent nanophosphors as LFA reporters. The greater detectability of nanophosphors allows the use of a reduced volume of RPA reagents, potentially reducing the cost of RPA-LFA. The limit of detection (LOD) of RPA with gold nanoparticle-based LFA readout is estimated at 1 parasite per reaction, but LOD can be 100-fold better, 0.01 parasites per reaction, for LFA based on SBMSO. This approach may be useful for sensitive and cost-effective point-of-care diagnosis and contribute to improved clinical and economic outcomes, especially in resource-limited settings.

A Preliminary Study to Compare Recombinase Polymerase Amplification-Lateral Flow and Quantitative PCR in the Detection of Cutaneous Leishmania in Communities from the Volta Region of Ghana.

Background: Leishmaniasis is a parasitic disease that mostly affects populations in tropical and subtropical countries. In Ghana, cutaneous leishmaniasis (CL) is the most common form of the disease affecting communities of the Volta Region. Conventional parasitological method (microscopy) is the commonly used test for CL diagnosis in many endemic countries, but has low sensitivity in chronic cases. Therefore, there is a clear need for a sensitive and easy-to-use point-of-care diagnostic method like an isothermal recombinase polymerase amplification-lateral flow (RPA-LF) test, suitable for use in austere and low-resource settings for the identification of CL cases. This study compared the efficacy of RPA-LF test with quantitative PCR (qPCR) in detecting Leishmania in suspected CL cases from the Volta Region. Methods: Twenty-five participants between 5 and 14 years were enrolled in the study from whom a total of 26 samples were obtained. Lesion samples were collected using FTA® filter papers applied to ulcerated lesions for molecular diagnosis. DNA isolated from filter papers was used for both the RPA-LF test and qPCR. Results: Twenty-two participants (88%) presented with one or two ulcerated active lesions per individual, while the rest of them had plaques or dried lesions. Among the 26 samples, 19/26 (73%) had concordant results when comparing the two diagnostic methods. Conclusion: Data from this study suggest that the RPA-LF test can be used in addition to a conventional parasitological diagnostic test (microscopy) to detect CL cases in communities of the Volta Region.

A Murine Model of Waning Scrub Typhus Cross-Protection between Heterologous Strains of Orientia tsutsugamushi.

Orientia tsutsugamushi, the etiologic agent of the life-threatening febrile disease scrub typhus, is an obligately intracellular small coccobacillary bacterium belonging to the family Rickettsiaceae and is transmitted by the parasitic larval stage of trombiculid mites. Progress towards a vaccine for protection against scrub typhus has been impeded by characteristics of the pathogen and the infection. There are numerous strains of O. tsutsugamushi in the Asia-Pacific region with geographical overlap. In human cases immunity has been described as poor against heterologous strains of the pathogen, as well as short-lived against the homologous strain, with a mean antibody reversion rate of less than one year. Animal models of cross-protection as well as of deterioration of this cross-protection are needed to enhance understanding of transient immunity to scrub typhus. To build upon current understanding of this ineffective protection we sought to utilize our recently developed models, sublethal intradermal infection followed by challenge via ordinarily lethal hematogenous dissemination. Mice that were initially infected sublethally with O. tsutsugamushi Gilliam strain and were challenged with an ordinarily lethal dose of heterologous Karp strain were protected from death by a robust immune response at one month after the primary infection as evidenced by an abundance of mononuclear cellular infiltrates in target organs such as lung, liver, and kidney; maintenance of body weight; and low bacterial loads in the organs. Waning protection from lethal Karp strain challenge indicated by weight loss mirroring that observed in naïve mice was observed as early as 9 months after primary Gilliam strain infection, and higher bacterial loads, severe disease, and eventual death in some mice was observed after challenge with Karp strain at 14 months post-initial heterologous infection.

Host EPAC1 Modulates Rickettsial Adhesion to Vascular Endothelial Cells via Regulation of ANXA2 Y23 Phosphorylation.

INTRODUCTION: Intracellular cAMP receptor exchange proteins directly activated by cAMP 1 (EPAC1) regulate obligate intracellular parasitic bacterium rickettsial adherence to and invasion into vascular endothelial cells (ECs). However, underlying precise mechanism(s) remain unclear. The aim of the study is to dissect the functional role of the EPAC1-ANXA2 signaling pathway during initial adhesion of rickettsiae to EC surfaces. METHODS: In the present study, an established system that is anatomically based and quantifies bacterial adhesion to ECs in vivo was combined with novel fluidic force microscopy (FluidFM) to dissect the functional role of the EPAC1-ANXA2 signaling pathway in rickettsiae-EC adhesion. RESULTS: The deletion of the EPAC1 gene impedes rickettsial binding to endothelium in vivo. Rickettsial OmpB shows a host EPAC1-dependent binding strength on the surface of a living brain microvascular EC (BMEC). Furthermore, ectopic expression of phosphodefective and phosphomimic mutants replacing tyrosine (Y) 23 of ANXA2 in ANXA2-knock out BMECs results in different binding force to reOmpB in response to the activation of EPAC1. CONCLUSIONS: EPAC1 modulates rickettsial adhesion, in association with Y23 phosphorylation of the binding receptor ANXA2. Underlying mechanism(s) should be further explored to delineate the accurate role of cAMP-EPAC system during rickettsial infection.

Intracellular receptor EPAC regulates von Willebrand factor secretion from endothelial cells in a PI3K-/eNOS-dependent manner during inflammation.

Coagulopathy is associated with both inflammation and infection, including infections with novel severe acute respiratory syndrome coronavirus-2, the causative agent Coagulopathy is associated with both inflammation and infection, including infection with novel severe acute respiratory syndrome coronavirus-2, the causative agent of COVID-19. Clot formation is promoted via cAMP-mediated secretion of von Willebrand factor (vWF), which fine-tunes the process of hemostasis. The exchange protein directly activated by cAMP (EPAC) is a ubiquitously expressed intracellular cAMP receptor that plays a regulatory role in suppressing inflammation. To assess whether EPAC could regulate vWF release during inflammation, we utilized our EPAC1-null mouse model and revealed increased secretion of vWF in endotoxemic mice in the absence of the EPAC1 gene. Pharmacological inhibition of EPAC1 in vitro mimicked the EPAC1-/- phenotype. In addition, EPAC1 regulated tumor necrosis factor-α-triggered vWF secretion from human umbilical vein endothelial cells in a manner dependent upon inflammatory effector molecules PI3K and endothelial nitric oxide synthase. Furthermore, EPAC1 activation reduced inflammation-triggered vWF release, both in vivo and in vitro. Our data delineate a novel regulatory role for EPAC1 in vWF secretion and shed light on the potential development of new strategies to control thrombosis during inflammation.

Diagnostic Efficacy of Recombinase-Polymerase-Amplification Coupled with Lateral Flow Strip Reading in Patients with Cutaneous Leishmaniasis from the Amazonas Rainforest of Perú.

Cutaneous leishmaniasis (CL) is highly prevalent in rural and sylvatic regions of Latin America, with an estimated 55,000 annual cases. Diagnosis in resource-limited areas still relies on microscopy of dermal scrapings, while more sensitive methods like PCR are not attainable due to costs and lack of adequate health infrastructure. Isothermal amplification of Leishmania DNA can be performed without sophisticated equipment and training and may become a point of care (POC) test for health care centers with scarce resources. We evaluated the efficacy of recombinase-polymerase-amplification (RPA-LF) to diagnose CL in 226 patients attending a clinic in Puerto Maldonado within the Peruvian Amazon basin. Conventional PCR targeting kinetoplast DNA (kDNA-PCR) was used as the gold standard. Eight of 226 patients were considered true negatives (microscopy, kDNA-PCR, and RPA-LF negative), while RPA-LF resulted positive in 186 of 204 kDNA-PCR positive patients, yielding 91.2% (confidence interval [CI] = 86.5-94.4%) sensitivity and 93% (CI 88.6-95.8%) positive predictive value. There were 14% (32/226) discrepant samples alternating positive and negative results in similar proportions between both tests. Quantitative PCR used to resolve the discrepancies suggested that they occurred in samples with scarce parasite numbers as determined by high cycle threshold (Ct) values (≥32; cutoff 35.5). Microscopy had the lowest sensitivity of all methods (45.4%). Nested real-time PCR performed in 71 samples determined that Leishmania (Viannia) braziliensis was highly prevalent (69/71), and Leishmania (Viannia) lainsoni was present in only two isolates. Results indicated that RPA-LF has POC potential for CL endemic areas, yet further simplification and optimization coupled with field validation will be necessary to confirm its broad applicability.

Isothermal recombinase polymerase amplification-lateral flow detection of SARS-CoV-2, the etiological agent of COVID-19.

The rapid detection of novel pathogens including SARS-CoV-2 necessitates the development of easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. Delay in diagnosis has facilitated the rapid spread of this novel virus throughout the world resulting in global mortality that will surpass 2.5 million people. Development of point-of-care diagnostic assays that can be performed in rural or decentralized health care centers to expand testing capacity is needed. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of SARS-CoV-2. The RPA-LF detected SARS-CoV-2 with a limit of detection of 35.4 viral cDNA nucleocapsid (N) gene copies/µL. Additionally, the RPA-LF was able to detect 0.25-2.5 copies/µL of SARS-CoV-2 N gene containing plasmid. We evaluated 37 nasopharyngeal samples using CDC's N3, N1 and N2 RT-real-time PCR assays for SARS-CoV-2 as reference test. We found a 100 % concordance between RPA-LF and RT-qPCR reference test as determined by 18/18 positive and 19/19 negative samples. All positive samples had Ct values between 19-37 by RT-qPCR. The RPA-LF primers and probe did not cross react with other relevant betacoronaviruses such as SARS and MERS. This is the first isothermal amplification test paired with lateral flow developed for qualitative detection of COVID-19 allowing rapid viral detection and with prospective applicability in resource limited and decentralized laboratories.

Direct RT-PCR amplification of SARS-CoV-2 from clinical samples using a concentrated viral lysis-amplification buffer prepared with IGEPAL-630.

The pandemic of 2019 caused by the novel coronavirus (SARS-CoV-2) is still rapidly spreading worldwide. Nucleic acid amplification serves as the gold standard method for confirmation of COVID-19 infection. However, challenges faced for diagnostic laboratories from undeveloped countries includes shortage of kits and supplies to purify viral RNA. Therefore, it is urgent to validate alternative nucleic acid isolation methods for SARS-CoV-2. Our results demonstrate that a concentrated viral lysis amplification buffer (vLAB) prepared with the nonionic detergent IGEPAL enables qualitative detection of SARS-CoV-2 by direct Reverse Transcriptase-Polymerase Chain Reaction (dRT-PCR). Furthermore, vLAB was effective in inactivating SARS-CoV-2. Since this method is inexpensive and no RNA purification equipment or additional cDNA synthesis is required, this dRT-PCR with vLAB should be considered as an alternative method for qualitative detection of SARS-CoV-2.

Diagnostic performance of a Recombinant Polymerase Amplification Test-Lateral Flow (RPA-LF) for cutaneous leishmaniasis in an endemic setting of Colombia.

BACKGROUND: Control of cutaneous leishmaniasis by public health systems in the Americas relies on case identification and treatment. Point-of-care diagnostics that can be performed by health workers within or near affected communities could effectively bring the health system to the resource-limited sites providing early diagnosis and treatment, reducing morbidity and the burden of disease. METHODOLOGY/PRINCIPAL FINDINGS: A cross-sectional study was undertaken to evaluate the diagnostic test performance of Isothermal Recombinase Polymerase Amplification (RPA) targeting Leishmania kinetoplast DNA, coupled with a lateral flow (LF) immunochromatographic strip, in a field setting and a laboratory reference center. Minimally invasive swab and FTA filter paper samples were obtained by community health workers and highly trained technicians from ulcerated lesions of > 2 weeks' evolution from 118 patients' ≥ 2 years of age in the municipality of Tumaco, Nariño. Extracted DNA was processed by RPA-LF at a reference center or in a primary health facility in the field. Evaluation was based on a composite "gold standard" that included microscopy, culture, biopsy and real-time polymerase chain reaction detection of Leishmania 18S rDNA. Standard of care routine diagnostic tests were explored as comparators. Sensitivity and specificity of RPA-LF in the reference lab scenario were 87% (95%CI 74-94) and 86% (95%CI 74-97), respectively. In the field scenario, the sensitivity was 75% (95%CI 65-84) and specificity 89% (95%CI 78-99). Positive likelihood ratios in both scenarios were higher than 6 while negative likelihood ratios ranged to 0.2-0.3 supporting the usefulness of RPA-LF to rule-in and potentially to rule-out infection. CONCLUSIONS/SIGNIFICANCE: The low complexity requirements of RPA-LF combined with non-invasive sampling support the feasibility of its utilization by community health workers with the goal of strengthening the diagnostic capacity for cutaneous leishmaniasis in Colombia. TRIAL REGISTRATION: ClinicalTrials.gov NCT04500873.

Isothermal Recombinase Polymerase Amplification-Lateral Flow Point-of-Care Diagnostic Test for Heartland Virus.

The detection of novel or re-emergent pathogens necessitates the development of rapid, easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. Heartland virus (HRTV) is the first pathogenic Phlebovirus responsible for serious and fatal cases in the United States. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of HRTV. The RPA-LF detected HRTV with a limit of detection of 1.19-1.54 plaque-forming unit equivalents/reaction. In addition, the RPA-LF was able to detect 0.6075 copies/µL of HRTV nucleoprotein gene-containing plasmid. We evaluated six clinical samples that were previously found to be real-time PCR positive for HRTV and found five out of six samples to be positive by RPA-LF, yielding 83.3% concordance with real-time PCR. All six samples had Ct values between 29 and 39 by real-time PCR. We also determined that the HRTV primers and probe do not cross-react with other tick-transmitted viruses such as Bourbon and Powassan, or other related viruses, including Lonestar tick virus and Sunday canyon virus (100% specificity). This is the first isothermal amplification test developed for a tick-borne virus, which will allow for rapid differentiation between HRTV and other pathogens producing similar clinical manifestations.

EPAC regulates von Willebrand factor secretion from endothelial cells in a PI3K/eNOS-dependent manner during inflammation.

Coagulopathy is associated with both inflammation and infection, including infection with the novel SARS-CoV-2 (COVID-19). Endothelial cells (ECs) fine tune hemostasis via cAMP-mediated secretion of von Willebrand factor (vWF), which promote the process of clot formation. The e xchange p rotein directly a ctivated by c AMP (EPAC) is a ubiquitously expressed intracellular cAMP receptor that plays a key role in stabilizing ECs and suppressing inflammation. To assess whether EPAC could regulate vWF release during inflammation, we utilized our EPAC1 -null mouse model and revealed an increased secretion of vWF in endotoxemic mice in the absence of the EPAC1 gene. Pharmacological inhibition of EPAC1 in vitro mimicked the EPAC1 -/- phenotype. EPAC1 regulated TNFα-triggered vWF secretion from human umbilical vein endothelial cells (HUVECs) in a phosphoinositide 3-kinases (PI3K)/endothelial nitric oxide synthase (eNOS)-dependent manner. Furthermore, EPAC1 activation reduced inflammation-triggered vWF release, both in vivo and in vitro . Our data delineate a novel regulatory role of EPAC1 in vWF secretion and shed light on potential development of new strategies to controlling thrombosis during inflammation. KEY POINT: PI3K/eNOS pathway-mediated, inflammation-triggered vWF secretion is the target of the pharmacological manipulation of the cAMP-EPAC system.

Increased talin-vinculin spatial proximities in livers in response to spotted fever group rickettsial and Ebola virus infections.

Talin and vinculin, both actin-cytoskeleton-related proteins, have been documented to participate in establishing bacterial infections, respectively, as the adapter protein to mediate cytoskeleton-driven dynamics of the plasma membrane. However, little is known regarding the potential role of the talin-vinculin complex during spotted fever group rickettsial and Ebola virus infections, two dreadful infectious diseases in humans. Many functional properties of proteins are determined by their participation in protein-protein complexes, in a temporal and/or spatial manner. To resolve the limitation of application in using mouse primary antibodies on archival, multiple formalin-fixed mouse tissue samples, which were collected from experiments requiring high biocontainment, we developed a practical strategic proximity ligation assay (PLA) capable of employing one primary antibody raised in mouse to probe talin-vinculin spatial proximal complex in mouse tissue. We observed an increase of talin-vinculin spatial proximities in the livers of spotted fever Rickettsia australis or Ebola virus-infected mice when compared with mock mice. Furthermore, using EPAC1-knockout mice, we found that deletion of EPAC1 could suppress the formation of spatial proximal complex of talin-vinculin in rickettsial infections. In addition, we observed increased colocalization between spatial proximity of talin-vinculin and filamentous actin-specific phalloidin staining in single survival mouse from an ordinarily lethal dose of rickettsial or Ebola virus infection. These findings may help to delineate a fresh insight into the mechanisms underlying liver specific pathogenesis during infection with spotted fever rickettsia or Ebola virus in the mouse model.

Polarized lung inflammation and Tie2/angiopoietin-mediated endothelial dysfunction during severe Orientia tsutsugamushi infection.

Orientia tsutsugamushi infection can cause acute lung injury and high mortality in humans; however, the underlying mechanisms are unclear. Here, we tested a hypothesis that dysregulated pulmonary inflammation and Tie2-mediated endothelial malfunction contribute to lung damage. Using a murine model of lethal O. tsutsugamushi infection, we demonstrated pathological characteristics of vascular activation and tissue damage: 1) a significant increase of ICAM-1 and angiopoietin-2 (Ang2) proteins in inflamed tissues and lung-derived endothelial cells (EC), 2) a progressive loss of endothelial quiescent and junction proteins (Ang1, VE-cadherin/CD144, occuludin), and 3) a profound impairment of Tie2 receptor at the transcriptional and functional levels. In vitro infection of primary human EC cultures and serum Ang2 proteins in scrub typhus patients support our animal studies, implying endothelial dysfunction in severe scrub typhus. Flow cytometric analyses of lung-recovered cells further revealed that pulmonary macrophages (MΦ) were polarized toward an M1-like phenotype (CD80+CD64+CD11b+Ly6G-) during the onset of disease and prior to host death, which correlated with the significant loss of CD31+CD45- ECs and M2-like (CD206+CD64+CD11b+Ly6G-) cells. In vitro studies indicated extensive bacterial replication in M2-type, but not M1-type, MΦs, implying the protective and pathogenic roles of M1-skewed responses. This is the first detailed investigation of lung cellular immune responses during acute O. tsutsugamushi infection. It uncovers specific biomarkers for vascular dysfunction and M1-skewed inflammatory responses, highlighting future therapeutic research for the control of this neglected tropical disease.

Correction: CD8+ T cells provide immune protection against murine disseminated endotheliotropic Orientia tsutsugamushi infection.

[This corrects the article DOI: 10.1371/journal.pntd.0005763.].

Type 1-skewed neuroinflammation and vascular damage associated with Orientia tsutsugamushi infection in mice.

BACKGROUND: Scrub typhus is a life-threatening disease, due to infection with O. tsutsugamushi, a Gram-negative bacterium that preferentially replicates in endothelial cells and professional phagocytes. Meningoencephalitis has been reported in scrub typhus patients and experimentally-infected animals; however, the neurological manifestation and its underlying mechanisms remain poorly understood. To address this issue, we focused on Orientia tsutsugamushi Karp strain (OtK), and examined host responses in the brain during lethal versus self-healing scrub typhus disease in our newly established murine models. PRINCIPLE FINDINGS: Following inoculation with a lethal dose of OtK, mice had a significant increase in brain transcripts related to pathogen-pattern recognition receptors (TLR2, TLR4, TLR9), type-1 responses (IFN-γ, TNF-α, CXCL9, CXCR3), and endothelial stress/damage such as angiopoietins, but a rapid down-regulation of Tie2. Sublethal infection displayed similar trends, implying the development of type 1-skewed proinflammatory responses in infected brains, independent of time and disease outcomes. Focal hemorrhagic lesions and meningitis were evident in both infection groups, but pathological changes were more diffuse and frequent in lethal infection. At 6-10 days of lethal infection, the cortex and cerebellum sections had increased ICAM-1-positive staining in vascular cells, as well as increased detection of CD45+ leukocytes, CD3+ T cells, IBA1+ phagocytes, and GFAP+ astrocytes, but a marked loss of occludin-positive tight junction staining, implying progressive endothelial activation/damage and cellular recruitment in inflamed brains. Orientia were sparse in the brains, but readily detectable within lectin+ vascular and IBA-1+ phagocytic cells. These CNS alterations were consistent with type 1-skewed, IL-13-suppressed responses in lethally-infected mouse lungs. SIGNIFICANCE: This is the first report of type 1-skewed neuroinflammation and cellular activation, accompanied with vascular activation/damage, during OtK infection in C57BL/6 mice. This study not only enhances our understanding of the pathophysiological mechanisms of scrub typhus, but also correlates the impact of immune and vascular dysfunction on disease pathogenesis.

CD8+ T cells provide immune protection against murine disseminated endotheliotropic Orientia tsutsugamushi infection.

Scrub typhus, caused by a Gram-negative obligately intracellular coccobacillus, Orientia tsutsugamushi, is a long neglected but important tropical disease. Orientia tsutsugamushi causes illness in one million people each year, and 1 billion people are at risk. Without appropriate diagnosis and treatment, the disease can cause severe multiorgan failure with a case fatality rate of 7-15%. The current gaps in knowledge of immunity include the unknown mechanisms of host immunity to O. tsutsugamushi. Using an intravenous (i.v.) disseminated infection mouse model, we observed that more CD8+ T cells than CD4+ T cells were present in the spleen of infected mice at 12 dpi. We also determined that Treg cells and the proportion of T cells producing IL-10 were significantly increased from 6 dpi, which correlated with the onset of illness, body weight loss, and increased bacterial loads. We further studied CD8-/-, MHC I-/- and wild type control (WT) C57BL/6J mice to determine the importance of CD8+ T cells and MHC I molecules. After infection with an ordinarily sub-lethal dose of O. tsutsugamushi, all CD8-/- and MHC I-/- mice were moribund between 12 and 15 dpi, whereas all WT mice survived. Bacterial loads in the lung, kidney, liver and spleen of CD8-/- and MHC I-/- mice were significantly greater than those in WT mice. Interferon-γ (IFN-γ) and granzyme B mRNA levels in the liver of CD8-/- and MHC I-/- mice were significantly greater than in WT mice. In addition, more severe histopathologic lesions were observed in CD8-/- mice. Finally, adoptive transfer confirmed a major role of immune CD8+ T cells as well as a less effective contribution by immune CD8 T cell-depleted splenocytes in protection against O. tsutsugamushi infection. These studies demonstrated the critical importance of CD8+ T cells in the host immune response during O. tsutsugamushi infection.

An Intradermal Inoculation Mouse Model for Immunological Investigations of Acute Scrub Typhus and Persistent Infection.

Scrub typhus is a neglected tropical disease, caused by Orientia tsutsugamushi, a Gram-negative bacterium that is transmitted to mammalian hosts during feeding by Leptotrombidium mites and replicates predominantly within endothelial cells. Most studies of scrub typhus in animal models have utilized either intraperitoneal or intravenous inoculation; however, there is limited information on infection by the natural route in murine model skin or its related early host responses. Here, we developed an intradermal (i.d.) inoculation model of scrub typhus and focused on the kinetics of the host responses in the blood and major infected organs. Following ear inoculation with 6 x 104 O. tsutsugamushi, mice developed fever at 11-12 days post-infection (dpi), followed by marked hypothermia and body weight loss at 14-19 dpi. Bacteria in blood and tissues and histopathological changes were detected around 9 dpi and peaked around 14 dpi. Serum cytokine analyses revealed a mixed Th1/Th2 response, with marked elevations of MCP-1/CCL2, MIP-1α/CCL3 and IL-10 at 9 dpi, followed by increased concentrations of pro-inflammatory markers (IL-6, IL-12, IFN-γ, G-CSF, RANTES/CCL5, KC/CCL11, IL-1α/ß, IL-2, TNF-α, GM-CSF), as well as modulatory cytokines (IL-9, IL-13). Cytokine levels in lungs had similar elevation patterns, except for a marked reduction of IL-9. The Orientia 47-kDa gene and infectious bacteria were detected in several organs for up to 84 dpi, indicating persistent infection. This is the first comprehensive report of acute scrub typhus and persistent infection in i.d.-inoculated C57BL/6 mice. This is a significant improvement over current murine models for Orientia infection and will permit detailed studies of host immune responses and infection control interventions.

IL-33-Dependent Endothelial Activation Contributes to Apoptosis and Renal Injury in Orientia tsutsugamushi-Infected Mice.

Endothelial cells (EC) are the main target for Orientia tsutsugamushi infection and EC dysfunction is a hallmark of severe scrub typhus in patients. However, the molecular basis of EC dysfunction and its impact on infection outcome are poorly understood. We found that C57BL/6 mice that received a lethal dose of O. tsutsugamushi Karp strain had a significant increase in the expression of IL-33 and its receptor ST2L in the kidneys and liver, but a rapid reduction of IL-33 in the lungs. We also found exacerbated EC stress and activation in the kidneys of infected mice, as evidenced by elevated angiopoietin (Ang) 2/Ang1 ratio, increased endothelin 1 (ET-1) and endothelial nitric oxide synthase (eNOS) expression. Such responses were significantly attenuated in the IL-33-/- mice. Importantly, IL-33-/- mice also had markedly attenuated disease due to reduced EC stress and cellular apoptosis. To confirm the biological role of IL-33, we challenged wild-type (WT) mice with a sub-lethal dose of O. tsutsugamushi and gave mice recombinant IL-33 (rIL-33) every 2 days for 10 days. Exogenous IL-33 significantly increased disease severity and lethality, which correlated with increased EC stress and activation, increased CXCL1 and CXCL2 chemokines, but decreased anti-apoptotic gene BCL-2 in the kidneys. To further examine the role of EC stress, we infected human umbilical vein endothelial cells (HUVEC) in vitro. We found an infection dose-dependent increase in the expression of IL-33, ST2L soluble ST2 (sST2), and the Ang2/Ang1 ratio at 24 and 48 hours post-infection. This study indicates a pathogenic role of alarmin IL-33 in a murine model of scrub typhus and highlights infection-triggered EC damage and IL-33-mediated pathological changes during the course of Orientia infection.

An animal model of a newly emerging human ehrlichiosis.

BACKGROUND: Human ehrlichioses are emerging life-threatening diseases transmitted by ticks. Animal models have been developed to study disease development; however, there is no valid small animal model that uses a human ehrlichial pathogen. The objective of this study was to develop a mouse model for ehrlichiosis with the newly discovered human pathogen, Ehrlichia muris-like agent (EMLA). METHODS: Three strains of mice were inoculated with different doses of EMLA by the intravenous, intraperitoneal, or intradermal route and evaluated for clinical and pathologic changes during the course of infection. RESULTS: EMLA infected C57Bl/6, BALB/c, and C3H/HeN mice and induced lethal or persistent infection in a route- and dose-dependent manner. The clinical chemistry and hematologic changes were similar to those of human infection by Ehrlichia chaffeensis or EMLA. Bacterial distribution in tissues differed after intradermal infection, compared with the distribution after intravenous or intraperitoneal injection. Lethal infection did not cause remarkable pathologic changes, but it caused fluid imbalance. EMLA infection of endothelium and mononuclear cells likely plays a role in the severe outcome. CONCLUSIONS: The EMLA mouse model mimics human infection and can be used to study pathogenesis and immunity and for development of a vector transmission model of ehrlichiosis.