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/β, 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.     

A Hematogenously Disseminated Orientia tsutsugamsushi-Infected Murine Model of Scrub Typhus

Orientia tsutsugamushi, the etiologic agent of scrub typhus, is a mite-borne rickettsia transmitted by the parasitic larval stage of trombiculid mites. Approximately one-third of the world''s population is at risk of infection with Orientia tsutsugamushi, emphasizing its importance in global health. In order to study scrub typhus, Orientia tsutsugamushi Karp strain has been used extensively in mouse studies with various inoculation strategies and little success in inducing disease progression similar to that of human scrub typhus. The objective of this project was to develop a disease model with pathology and target cells similar to those of severe human scrub typhus. This study reports an intravenous infection model of scrub typhus in C57BL/6 mice. This mouse strain was susceptible to intravenous challenge, and lethal infection occurred after intravenous inoculation of 1.25×10⁶ focus (FFU) forming units. Signs of illness in lethally infected mice appeared on day 6 with death occurring ∼6 days later. Immunohistochemical staining for Orientia antigens demonstrated extensive endothelial infection, most notably in the lungs and brain. Histopathological analysis revealed cerebral perivascular, lymphohistiocytic infiltrates, focal hemorrhages, meningoencephalitis, and interstitial pneumonia. Disseminated infection of endothelial cells with Orientia in C57BL/6 mice resulted in pathology resembling that of human scrub typhus. The use of this model will allow detailed characterization of the mechanisms of immunity to and pathogenesis of O. tsutsugamushi infection.     

Strong Type 1, but Impaired Type 2, Immune Responses Contribute to Orientia tsutsugamushi-Induced Pathology in Mice

Scrub typhus is a neglected, but important, tropical disease, which puts one-third of the world''s population at risk. The disease is caused by Orientia tsutsugamushi, an obligately intracellular Gram-negative bacterium. Dysregulation in immune responses is known to contribute to disease pathogenesis; however, the nature and molecular basis of immune alterations are poorly defined. This study made use of a newly developed murine model of severe scrub typhus and focused on innate regulators and vascular growth factors in O. tsutsugamushi-infected liver, lungs and spleen. We found no activation or even reduction in base-line expression for multiple molecules (IL-7, IL-4, IL-13, GATA3, ROR-γt, and CXCL12) at 2, 6 and 10 days post-infection. This selective impairment in type 2-related immune responses correlated with a significant activation of the genes for IL-1β, IL-6, IL-10, TNF-α, IFN-γ, as well as CXCR3- and CXCR1-related chemokines in inflamed tissues. The elevated angiopoietin (Ang)-2 expression and Ang-2/Ang-1 ratios suggested excessive inflammation and the loss of endothelial integrity. These alterations, together with extensive recruitment of myeloperoxidase (MPO)-expressing neutrophils and the influx of CD3⁺ T cells, contributed to acute tissue damage and animal death. This is the first report of selective alterations in a panel of immune regulators during early O. tsutsugamushi infection in intravenously inoculated C57BL/6 mice. Our findings shed new light on the pathogenic mechanisms associated with severe scrub typhus and suggest potential targets for therapeutic investigation.     

Dissemination of Orientia tsutsugamushi and Inflammatory Responses in a Murine Model of Scrub Typhus

Central aspects in the pathogenesis of scrub typhus, an infection caused by Orientia (O.) tsutsugamushi, have remained obscure. Its organ and cellular tropism are poorly understood.The purpose of this study was to analyze the kinetics of bacterial dissemination and associated inflammatory responses in infected tissues in an experimental scrub typhus mouse model, following infection with the human pathogenic strain Karp. We provide a thorough analysis of O. tsutsugamushi infection in inbred Balb/c mice using footpad inoculation, which is close to the natural way of infection. By a novel, highly sensitive qPCR targeting the multi copy traD genes, we quantitatively monitored the spread of O. tsutsugamushi Karp from the skin inoculation site via the regional lymph node to the internal target organs. The highest bacterial loads were measured in the lung. Using confocal imaging, we also detected O. tsutsugamushi at the single cell level in the lung and found a predominant macrophage rather than endothelial localization. Immunohistochemical analysis of infiltrates in lung and brain revealed differently composed lesions with specific localizations: iNOS-expressing macrophages were frequent in infiltrative parenchymal noduli, but uncommon in perivascular lesions within these organs. Quantitative analysis of the macrophage response by immunohistochemistry in liver, heart, lung and brain demonstrated an early onset of macrophage activation in the liver. Serum levels of interferon (IFN)-γ were increased during the acute infection, and we showed that IFN-γ contributed to iNOS-dependent bacterial growth control.Our data show that upon inoculation to the skin, O. tsutsugamushi spreads systemically to a large number of organs and gives rise to organ-specific inflammation patterns. The findings suggest an essential role for the lung in the pathogenesis of scrub typhus. The model will allow detailed studies on host-pathogen interaction and provide further insight into the pathogenesis of O. tsutsugamushi infection.     

Urbanization of Scrub Typhus Disease in South Korea

Background

Scrub typhus is an endemic disease in Asia. It has been a rural disease, but indigenous urban cases have been observed in Seoul, South Korea. Urban scrub typhus may have a significant impact because of the large population.

Methods

Indigenous urban scrub typhus was epidemiologically identified in Seoul, the largest metropolitan city in South Korea, using national notifiable disease data from 2010 to 2013. For detailed analysis of clinical features, patients from one hospital that reported the majority of cases were selected and compared to a historic control group. Chigger mites were prospectively collected in the city using a direct chigger mite-collecting trap, and identified using both phenotypic and 18S rDNA sequencing analyses. Their infection with Orientia tsutsugamushi was confirmed by sequencing the 56-kDa antigen gene.

Results

Eighty-eight cases of urban scrub typhus were determined in Seoul. The possible sites of infection were mountainous areas (56.8%), city parks (20.5%), the vicinity of one’s own residence (17.0%), and riversides (5.7%). Eighty-seven chigger mites were collected in Gwanak mountain, one of the suspected infection sites in southern Seoul, and seventy-six (87.4%) of them were identified as Helenicula miyagawai and eight (9.2%) as Leptotrombidium scutellare. Pooled DNA extracted from H. miyagawai mites yielded O. tsutsugamushi Boryong strain. Twenty-six patients from one hospital showed low APACHE II score (3.4 ± 2.7), low complication rate (3.8%), and no hypokalemia.

Conclusions

We identified the presence of indigenous urban scrub typhus in Seoul, and a subgroup of them had mild clinical features. The chigger mite H. miyagawai infected with O. tsutsugamushi within the city was found. In endemic area, urban scrub typhus needs to be considered as one of the differential febrile diseases and a target for prevention.     

High Rates of Homologous Recombination in the Mite Endosymbiont and Opportunistic Human Pathogen Orientia tsutsugamushi

Orientia tsutsugamushi is an intracellular α-proteobacterium which resides in trombiculid mites, and is the causative agent of scrub typhus in East Asia. The genome sequence of this species has revealed an unprecedented number of repeat sequences, most notably of the genes encoding the conjugative properties of a type IV secretion system (T4SS). Although this observation is consistent with frequent intragenomic recombination, the extent of homologous recombination (gene conversion) in this species is unknown. To address this question, and to provide a protocol for the epidemiological surveillance of this important pathogen, we have developed a multilocus sequence typing (MLST) scheme based on 7 housekeeping genes (gpsA, mdh, nrdB, nuoF, ppdK, sucD, sucB). We applied this scheme to the two published genomes, and to DNA extracted from blood taken from 84 Thai scrub typhus patients, from 20 cultured Thai patient isolates, 1 Australian patient sample, and from 3 cultured type strains. These data demonstrated that the O. tsutsugamushi population was both highly diverse [Simpson''s index (95% CI) = 0.95 (0.92–0.98)], and highly recombinogenic. These results are surprising given the intracellular life-style of this species, but are broadly consistent with results obtained for Wolbachia, which is an α-proteobacterial reproductive parasite of arthropods. We also compared the MLST data with ompA sequence data and noted low levels of consistency and much higher discrimination by MLST. Finally, twenty-five percent of patients in this study were simultaneously infected with multiple sequence types, suggesting multiple infection caused by either multiple mite bites, or multiple strains co-existing within individual mites.     

Secreted Wnt antagonists in scrub typhus

BackgroundThe mechanisms that control local and systemic inflammation in scrub typhus have only been partially elucidated. The wingless (Wnt) signaling pathways are emerging as important regulators of inflammation and infection, but have not been investigated in scrub typhus.Methodology/Principal findingsPlasma levels of secreted Wnt antagonists (i.e. DKK-1, sFRP-3, WIF-1 and SOST) were analyzed in patients with scrub typhus (n = 129), patients with similar febrile illness without O. tsutsugamushi infection (n = 31), febrile infectious disease controls, and in healthy controls (n = 31) from the same area of South India, and were correlated to markers of inflammation, immune and endothelial cell activation as well as for their association with organ specific dysfunction and mortality in these patients. We found i) Levels of SOST and in particular sFRP-3 and WIF-1 were markedly increased and DKK-1 decreased in scrub typhus patients at admission to the hospital compared to healthy controls. ii) In recovering scrub typhus patients, SOST, sFRP-3 and WIF-1 decreased and DKK-1 increased. iii) SOST was positively correlated with markers of monocyte/macrophage and endothelial/vascular activation as well as with renal dysfunction and poor outcome iv) Finally, regulation of Wnt pathways by O. tsutsugamushi in vitro in monocytes and ex vivo in mononuclear cells isolated from patients with scrub typhus, as evaluated by gene expression studies available in public repositories, revealed markedly attenuated canonical Wnt signaling.Conclusions/SignificanceOur findings suggest that scrub typhus is characterized by attenuated Wnt signaling possibly involving dysregulated levels of several secreted pathway antagonists. The secreted Wnt antagonist SOST was strongly associated with renal dysfunction and poor prognosis in these patients.     

High initial IgG antibody levels against Orientia tsutsugamushi are associated with an increased risk of severe scrub typhus infection

BackgroundScrub typhus is a dominant cause of febrile illness in many parts of Asia. Immunity is limited by the great strain diversity of Orientia tsutsugamushi. It is unclear whether previous infection protects from severe infection or enhances the risk.Methods/principal findingsWe studied IgG antibody levels against O. tsutsugamushi at presentation in 636 scrub typhus patients using enzyme-linked immunosorbent assays (ELISA). The association between ELISA optical density (OD) and risk of severe infection was modelled using Poisson regression. OD was categorised as low (<1.0), intermediate (1.0 to 2.9), and high (≥3.0). OD was also modelled as a continuous variable (cubic spline). Median age of cases was 41 years (range 0–85), with 37% having severe infection. Compared to the low category, the age-adjusted risk of severe infection was 1.5 times higher in the intermediate category (95%CI 1.2, 1.9), and 1.3 times higher in the high category (95%CI 1.0, 1.7). The effect was stronger in cases <40 years, doubling the risk in the intermediate and high categories compared to the low category. The effect was more pronounced in cases tested within 7 days of fever onset when IgG ODs are more likely to reflect pre-infection levels.Conclusions/SignificanceIntermediate and high IgG antibody levels at the time of diagnosis are associated with a higher risk of severe scrub typhus infection. The findings may be explained by severe infection eliciting an accelerated IgG response or by previous scrub typhus infection enhancing the severity of subsequent episodes.     

Immunization with an Autotransporter Protein of Orientia tsutsugamushi Provides Protective Immunity against Scrub Typhus

BackgroundScrub typhus is an acute febrile disease caused by Orientia tsutsugamushi infection. Recently, the rapid increase of scrub typhus incidence in several countries within the endemic region has become a serious public health issue. Despite the wide range of preventative approaches that have been attempted in the past 70 years, all have failed to develop an effective prophylactic vaccine. Currently, the selection of the proper antigens is one of the critical barriers to generating cross-protective immunity against antigenically-variable strains of O. tsutsugamushi.Conclusions/SignificanceImmunization of ScaA proteins provides protective immunity in mice when challenged with the homologous strain and significantly enhanced protective immunity against infection with heterologous strains. To our knowledge, this is the most promising result of scrub typhus vaccination trials against infection of heterologous strains in mouse models thus far.     

A model for the transmission dynamics of Orientia tsutsugamushi among its natural reservoirs

The bacteria Orientia tsutsugamushi is the causative agent of scrub typhus, a prevalent disease in Asian countries that can affect humans and which shows an alarming increase of cases during the last years, especially in rural areas. Unfortunately, there is no vaccine for scrub typhus, and antibiotic treatments successfully used in the past appear to be inefficient to treat some strains of O. tsutsugamushi. We introduce a mathematical model that approximates the dynamics of the bacteria among its natural reservoirs. After computing the basic reproductive number from the proposed model, we explore its sensitivity to the parameter values that may be affected by application of control measures. This theoretical model may be of interest to pest managers as well as health authorities interested in gaining insight into the public management of the disease, through a better understanding of its qualitative dynamics.     

Misdiagnosis of scrub typhus as hemorrhagic fever with renal syndrome and potential co-infection of both diseases in patients in Shandong Province,China, 2013–2014

BackgroundScrub typhus, caused by Orientia tsutsugamushi, an obligate intracellular gram-negative bacterium, along with hemorrhagic fever with renal syndrome (HFRS), caused by hantaviruses, are natural-focus infectious diseases prevalent in Shandong Province, China. Both diseases have similar clinical manifestations in certain disease stages and similar epidemic seasons, which has caused difficulties for physicians in distinguishing them. The aim of this study was to investigate whether misdiagnosis of scrub typhus as HFRS occurred in patients in Shandong Province.MethodsSerum samples (N = 112) of clinically suspected HFRS patients from 2013 to 2014 in Shandong Province were analyzed with enzyme-linked immunosorbent assay (ELISA) for antibodies to both hantavirus and Orientia tsutsugamushi.ResultsELISA showed that 56.3% (63/112) and 8.0% (9/112) of clinically suspected HFRS patients were IgM antibody positive to hantavirus and O. tsutsugamushi, respectively. Among the hantavirus IgM antibody positive patients, 7.9% (5/63) were also IgM antibody positive to O. tsutsugamushi. Among the hantavirus IgM antibody negative sera, 8.2% (4/49) of sera were positive to O. tsutsugamushi.ConclusionsWe concluded that some scrub typhus patients were misdiagnosed as HFRS and co-infection of scrub typhus and HFRS might exist in China. Due to the different treatments for scrub typhus and HFRS, physicians should carefully differentiate between scrub typhus and HFRS and consider administering anti-rickettsia antibiotics if treatment for HFRS alone does not work.     

C-type Lectin Receptor Dectin-3 Mediates Trehalose 6,6′-Dimycolate (TDM)-induced Mincle Expression through CARD9/Bcl10/MALT1-dependent Nuclear Factor (NF)-κB Activation

Previous studies indicate that both Dectin-3 (also called MCL or Clec4d) and Mincle (also called Clec4e), two C-type lectin receptors, can recognize trehalose 6,6′-dimycolate (TDM), a cell wall component from mycobacteria, and induce potent innate immune responses. Interestingly, stimulation of Dectin-3 by TDM can also induce Mincle expression, which may enhance the host innate immune system to sense Mycobacterium infection. However, the mechanism by which Dectin-3 induces Mincle expression is not fully defined. Here, we show that TDM-induced Mincle expression is dependent on Dectin-3-mediated NF-κB, but not nuclear factor of activated T-cells (NFAT), activation, and Dectin-3 induces NF-κB activation through the CARD9-BCL10-MALT1 complex. We found that bone marrow-derived macrophages from Dectin-3-deficient mice were severely defective in the induction of Mincle expression in response to TDM stimulation. This defect is correlated with the failure of TDM-induced NF-κB activation in Dectin-3-deficient bone marrow-derived macrophages. Consistently, inhibition of NF-κB, but not NFAT, impaired TDM-induced Mincle expression, whereas NF-κB, but not NFAT, binds to the Mincle promoter. Dectin-3-mediated NF-κB activation is dependent on the CARD9-Bcl10-MALT1 complex. Finally, mice deficient for Dectin-3 or CARD9 produced much less proinflammatory cytokines and keyhole limpet hemocyanin (KLH)-specific antibodies after immunization with an adjuvant containing TDM. Overall, this study provides the mechanism by which Dectin-3 induces Mincle expression in response to Mycobacterium infection, which will have significant impact to improve adjuvant and design vaccine for antimicrobial infection.     

A Systematic Review of Mortality from Untreated Scrub Typhus (Orientia tsutsugamushi)

Background

Scrub typhus, a bacterial infection caused by Orientia tsutsugamushi, is increasingly recognized as an important cause of fever in Asia, with an estimated one million infections occurring each year. Limited access to health care and the disease’s non-specific symptoms mean that many patients are undiagnosed and untreated, but the mortality from untreated scrub typhus is unknown. This review systematically summarizes the literature on the untreated mortality from scrub typhus and disease outcomes.

Methodology/Principal Findings

A literature search was performed to identify patient series containing untreated patients. Patients were included if they were symptomatic and had a clinical or laboratory diagnosis of scrub typhus and excluded if they were treated with antibiotics. The primary outcome was mortality from untreated scrub typhus and secondary outcomes were total days of fever, clinical symptoms, and laboratory results. A total of 76 studies containing 89 patient series and 19,644 patients were included in the final analysis. The median mortality of all patient series was 6.0% with a wide range (min-max) of 0–70%. Many studies used clinical diagnosis alone and had incomplete data on secondary outcomes. Mortality varied by location and increased with age and in patients with myocarditis, delirium, pneumonitis, or signs of hemorrhage, but not according to sex or the presence of an eschar or meningitis. Duration of fever was shown to be long (median 14.4 days Range (9–19)).

Conclusions

Results show that the untreated mortality from scrub typhus appears lower than previously reported estimates. More data are required to clarify mortality according to location and host factors, clinical syndromes including myocarditis and central nervous system disease, and in vulnerable mother-child populations. Increased surveillance and improved access to diagnostic tests are required to accurately estimate the untreated mortality of scrub typhus. This information would facilitate reliable quantification of DALYs and guide empirical treatment strategies.     

Orientia tsutsugamushi Subverts Dendritic Cell Functions by Escaping from Autophagy and Impairing Their Migration

Background

Dendritic cells (DCs) are the most potent antigen-presenting cells that link innate and adaptive immune responses, playing a pivotal role in triggering antigen-specific immunity. Antigen uptake by DCs induces maturational changes that include increased surface expression of major histocompatibility complex (MHC) and costimulatory molecules. In addition, DCs actively migrate to regional lymph nodes and activate antigen-specific naive T cells after capturing antigens. We characterize the functional changes of DCs infected with Orientia tsutsugamushi, the causative agent of scrub typhus, since there is limited knowledge of the role played by DCs in O. tsutsugamushi infection.

Methodology/Principal Finding

O. tsutsugamushi efficiently infected bone marrow-derived DCs and induced surface expression of MHC II and costimulatory molecules. In addition, O. tsutsugamushi induced autophagy activation, but actively escaped from this innate defense system. Infected DCs also secreted cytokines and chemokines such as IL-6, IL-12, MCP5, MIP-1α, and RANTES. Furthermore, in vitro migration of DCs in the presence of a CCL19 gradient within a 3D collagen matrix was drastically impaired when infected with O. tsutsugamushi. The infected cells migrated much less efficiently into lymphatic vessels of ear dermis ex vivo when compared to LPS-stimulated DCs. In vivo migration of O. tsutsugamushi-infected DCs to regional lymph nodes was significantly impaired and similar to that of immature DCs. Finally, we found that MAP kinases involved in chemotactic signaling were differentially activated in O. tsutsugamushi-infected DCs.

Conclusion/Significance

These results suggest that O. tsutsugamushi can target DCs to exploit these sentinel cells as replication reservoirs and delay or impair the functional maturation of DCs during the bacterial infection in mammals.     

Cytokine Network in Scrub Typhus: High Levels of Interleukin-8 Are Associated with Disease Severity and Mortality

Background

Scrub typhus, caused by Orientia tsutsugamushi, is endemic in the Asia-Pacific region. Mortality is high if untreated, and even with treatment as high as 10–20%, further knowledge of the immune response during scrub typhus is needed. The current study was aimed at comparing plasma levels of a variety of inflammatory mediators in scrub typhus patients and controls in South India in order to map the broader cytokine profile and their relation to disease severity and clinical outcome.

Methodology/Principal Findings

We examined plasma levels of several cytokines in scrub typhus patients (n = 129) compared to healthy controls (n = 31) and infectious disease controls (n = 31), both in the acute phase and after recovery, by multiplex technology and enzyme immunoassays. Scrub typhus patients were characterized by marked changes in the cytokine network during the acute phase, differing not only from healthy controls but also from infectious disease controls. While most of the inflammatory markers were raised in scrub typhus, platelet-derived mediators such as RANTES were markedly decreased, probably reflecting enhanced platelet activation. Some of the inflammatory markers, including various chemokines (e.g., interleukin-8, monocyte chemoattractant peptide-1 and macrophage inflammatory protein-1β) and downstream markers of inflammation (e.g., C-reactive protein and pentraxin-3), were also associated with disease severity and mortality during follow-up, with a particular strong association with interleukin-8.

Conclusions/Significance

Our findings suggest that scrub typhus is characterized by a certain cytokine profile that includes dysregulated levels of a wide range of mediators, and that this enhanced inflammation could contribute to disease severity and clinical outcome.     

The burden of scrub typhus in India: A systematic review

BackgroundScrub typhus, a vector-borne zoonotic infection caused by the bacteria Orientia tsutsugamushi, is one of the most common and clinically important rickettsial infections worldwide. An estimated one million cases occur annually with a high case fatality rate. Although scrub typhus is a major public health threat in India, the burden and distribution remains unclear. We aimed to estimate the burden of scrub typhus in India.MethodologyWe performed a systematic review of published literature on scrub typhus from India to extract information on epidemiology, morbidity, and mortality. Important databases were searched using keywords and appropriate combinations. We identified observational, interventional, and population-based studies and extracted the data to evaluate the number of cases diagnosed using serology or PCR and the number of deaths due to scrub typhus. We conducted a systematic narrative synthesis to summarize included studies.Principal findingsIn the last decade, there were 18,781 confirmed scrub typhus cases reported in 138 hospital-based studies and two community-based studies. IgM ELISA was used in 122 studies to confirm the cases in majority (89%). The proportion of scrub typhus among acute undifferentiated febrile illness (AUFI) studies was 25.3%, and community seroprevalence was 34.2%. Ninety studies had data published on multiple organ involvement out of which 17.4% of cases had multiple organ dysfunction syndromes, 20.4% patients required ICU admission, and 19.1% needed ventilation. The overall case-fatality rate was 6.3%, and the mortality among those with multi-organ dysfunction syndrome was as high as 38.9%.Conclusion/significanceScrub typhus, a common acute febrile illness in India causing severe morbidity, accounts for a large number of deaths. The burden of the disease has been underappreciated. Early diagnosis and prompt treatment can significantly reduce complications and mortality. Establishing good surveillance and instituting appropriate control measures are urgently needed.     

Multiplexed Digital mRNA Profiling of the Inflammatory Response in the West Nile Swiss Webster Mouse Model

Background and purpose

The ability to track changes in gene expression following viral infection is paramount to understanding viral pathogenesis. This study was undertaken to evaluate the nCounter, a high throughput digital gene expression system, as a means to better understand West Nile virus (WNV) dissemination and the inflammatory response against WNV in the outbred Swiss Webster (SW) mouse model over the course of infection.

Methodology

The nCounter Mouse Inflammation gene expression kit containing 179 inflammation related genes was used to analyze gene expression changes in multiple tissues over a nine day course of infection in SW mice following intraperitoneal injection with WNV. Protein expression levels for a subset of these cytokine/chemokine genes were determined using a multiplex protein detection system (BioPlex) and comparisons of protein/RNA expression levels made.

Results

Expression analysis of spleen, lung, liver, kidney and brain of SW mice infected with WNV revealed that Cxcl10 and Il12b are differentially expressed in all tissues tested except kidney. Data stratification of positively confirmed infected (WNV (+)) versus non-infected (WNV (−) tissues allowed differentiation of the systemic inflammatory gene response from tissue-specific responses arising from WNV infection. Significant (p<0.05) decrease in C3ar1 was found in WNV (−) spleen. Il23a was significantly upregulated, while Il10rb was down-regulated in WNV (−) lung. Il3 and Mbl2 were down-regulated in WNV (−) liver. In WNV (+) livers, Stat1, Tlr2, chemokines Cxcl1, Cxcl3, Cxcl9, Cxcl10, cytokines Il6, Il18, cytokine-related gene Il1r and cytokine agonist Ilrn were significantly upregulated. In WNV (−) brain tissues, Csf2 and Cxcl10 were significantly upregulated. Similar gene and protein expression kinetics were found for Ccl2, Ccl3, Ccl4 and Ccl5 and correlated with the presence of infectious virus. In summary, the utility of the nCounter platform for rapid identification of gene expression changes in SW mice associated with WNV infection was demonstrated.