Na-Young Ha

Comparative and kinetic analysis of viral shedding and immunological responses in MERS patients representing a broad spectrum of disease severity

Despite the ongoing spread of MERS, there is limited knowledge of the factors affecting its severity and outcomes. We analyzed clinical data and specimens from fourteen MERS patients treated in a hospital who collectively represent a wide spectrum of disease severity, ranging from mild febrile illness to fatal pneumonia, and classified the patients into four groups based on severity and mortality. Comparative and kinetic analyses revealed that high viral loads, weak antibody responses, and lymphopenia accompanying thrombocytopenia were associated with disease mortality, whereas persistent and gradual increases in lymphocyte responses might be required for effective immunity against MERS-CoV infection. Leukocytosis, primarily due to increased neutrophils and monocytes, was generally observed in more severe and fatal cases. The blood levels of cytokines such as IL-10, IL-15, TGF-β, and EGF were either positively or negatively correlated with disease mortality. Robust induction of various chemokines with differential kinetics was more prominent in patients that recovered from pneumonia than in patients with mild febrile illness or deceased patients. The correlation of the virological and immunological responses with disease severity and mortality, as well as their responses to current antiviral therapy, may have prognostic significance during the early phase of MERS.

Spread of Mutant Middle East Respiratory Syndrome Coronavirus with Reduced Affinity to Human CD26 during the South Korean Outbreak

ABSTRACT The newly emerging Middle East respiratory syndrome coronavirus (MERS-CoV) causes a severe respiratory infection with a high mortality rate (~35%). MERS-CoV has been a global threat due to continuous outbreaks in the Arabian peninsula and international spread by infected travelers since 2012. From May to July 2015, a large outbreak initiated by an infected traveler from the Arabian peninsula swept South Korea and resulted in 186 confirmed cases with 38 deaths (case fatality rate, 20.4%). Here, we show the rapid emergence and spread of a mutant MERS-CoV with reduced affinity to the human CD26 receptor during the South Korean outbreak. We isolated 13 new viral genomes from 14 infected patients treated at a hospital and found that 12 of these genomes possess a point mutation in the receptor-binding domain (RBD) of viral spike (S) protein. Specifically, 11 of these genomes have an I529T mutation in RBD, and 1 has a D510G mutation. Strikingly, both mutations result in reduced affinity of RBD to human CD26 compared to wild-type RBD, as measured by surface plasmon resonance analysis and cellular binding assay. Additionally, pseudotyped virus bearing an I529T mutation in S protein showed reduced entry into host cells compared to virus with wild-type S protein. These unexpected findings suggest that MERS-CoV adaptation during human-to-human spread may be driven by host immunological pressure such as neutralizing antibodies, resulting in reduced affinity to host receptor, and thereby impairs viral fitness and virulence, rather than positive selection for a better affinity to CD26. IMPORTANCE Recently, a large outbreak initiated by an MERS-CoV-infected traveler from the Middle East swept South Korea and resulted in 186 confirmed cases with 38 deaths. This is the largest outbreak outside the Middle East, and it raised strong concerns about the possible emergence of MERS-CoV mutations. Here, we isolated 13 new viral genomes and found that 12 of them possess a point mutation in the receptor-binding domain of viral spike protein, resulting in reduced affinity to the human cognate receptor, CD26, compared to the wild-type virus. These unexpected findings suggest that MERS-CoV adaptation in humans may be driven by host immunological pressure. Recently, a large outbreak initiated by an MERS-CoV-infected traveler from the Middle East swept South Korea and resulted in 186 confirmed cases with 38 deaths. This is the largest outbreak outside the Middle East, and it raised strong concerns about the possible emergence of MERS-CoV mutations. Here, we isolated 13 new viral genomes and found that 12 of them possess a point mutation in the receptor-binding domain of viral spike protein, resulting in reduced affinity to the human cognate receptor, CD26, compared to the wild-type virus. These unexpected findings suggest that MERS-CoV adaptation in humans may be driven by host immunological pressure.

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.

An Autotransporter Protein from Orientia tsutsugamushi Mediates Adherence to Nonphagocytic Host Cells

ABSTRACT Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular pathogen whose mechanism of cellular adhesion and invasion is poorly characterized. Bioinformatic analyses of two O. tsutsugamushi genomes revealed the presence of a group of genes that encode autotransporter proteins. In this study, we identified 10 autotransporter gene products and categorized them into five groups of orthologs (ScaA to ScaE) based on their sequence similarities. Sequence homology was highest between members of ScaC group, suggesting the functional conservation of bacterium-host interactions. ScaC was actively expressed on the surface of O. tsutsugamushi and induced antibody responses in scrub typhus patients. Experiments using microbeads conjugated to recombinant ScaC or a surrogate Escherichia coli expression system showed that ScaC was sufficient to mediate attachment to, but not invasion of, nonphagocytic mammalian cells. In addition, preincubation of host cells with recombinant ScaC significantly inhibited their interaction with O. tsutsugamushi. Finally, fibronectin was identified as a potential receptor for ScaC by using yeast two-hybrid screening, and this was confirmed using a glutathione S-transferase (GST) pulldown assay. Taken together, these results demonstrate that ScaC is involved in the interaction of O. tsutsugamushi with mammalian host cells and suggest that ScaC may play a critical role in bacterial pathogenesis.

Active Escape of Orientia tsutsugamushi from Cellular Autophagy

ABSTRACT Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular pathogen. After entry into host cells, the bacterium rapidly escapes from the endosomal pathway and replicates in the cytosol of eukaryotic host cells. Here we show that O. tsutsugamushi infection efficiently promotes cellular autophagy, a cell-autonomous defense mechanism of innate immunity. However, most of the internalized bacteria barely colocalized with the induced autophagosomes, even when stimulated with rapamycin, a chemical inducer of autophagy. Treatment of infected cells with tetracycline suppressed bacterial evasion from autophagy and facilitated O. tsutsugamushi targeting to autophagosomes, suggesting that the intracellular pathogen may be equipped with a bacterial factor or factors that block autophagic recognition. Finally, we also found that chemical modulators of cellular autophagy or genetic knockout of the atg3 gene does not significantly affect the intracellular growth of O. tsutsugamushi in vitro. These results suggest that O. tsutsugamushi has evolved to block autophagic microbicidal defense by evading autophagic recognition even though it activates the autophagy pathway during the early phase of 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.

Multiple Orientia tsutsugamushi Ankyrin Repeat Proteins Interact with SCF1 Ubiquitin Ligase Complex and Eukaryotic Elongation Factor 1 α

Background Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium. Previously, a large number of genes that encode proteins containing eukaryotic protein-protein interaction motifs such as ankyrin-repeat (Ank) domains were identified in the O. tsutsugamushi genome. However, little is known about the Ank protein function in O. tsutsugamushi. Methodology/Principal Findings To characterize the function of Ank proteins, we investigated a group of Ank proteins containing an F-box–like domain in the C-terminus in addition to the Ank domains. All nine selected ank genes were expressed at the transcriptional level in host cells infected with O. tsutsugamushi, and specific antibody responses against three Ank proteins were detected in the serum from human patients, indicating an active expression of the bacterial Ank proteins post infection. When ectopically expressed in HeLa cells, the Ank proteins of O. tsutsugamushi were consistently found in the nucleus and/or cytoplasm. In GST pull-down assays, multiple Ank proteins specifically interacted with Cullin1 and Skp1, core components of the SCF1 ubiquitin ligase complex, as well as the eukaryotic elongation factor 1 α (EF1α). Moreover, one Ank protein co-localized with the identified host targets and induced downregulation of EF1α potentially via enhanced ubiquitination. The downregulation of EF1α was observed consistently in diverse host cell types infected with O. tsutsugamushi. Conclusion/Significance These results suggest that conserved targeting and subsequent degradation of EF1α by multiple O. tsutsugamushi Ank proteins could be a novel bacterial strategy for replication and/or pathogenesis during mammalian host infection.

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

Background Scrub 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. Methodology/Principal Findings We examined the potential role of ScaA protein, an autotransporter protein of O. tsutsugamushi, in bacterial pathogenesis and evaluated the protective attributes of ScaA immunization in lethal O. tsutsugamushi infection in mice. Our findings demonstrate that ScaA functions as a bacterial adhesion factor, and anti-ScaA antibody significantly neutralizes bacterial infection of host cells. In addition, immunization with ScaA not only provides protective immunity against lethal challenges with the homologous strain, but also confers significant protection against heterologous strains when combined with TSA56, a major outer membrane protein of O. tsutsugamushi. Conclusions/Significance Immunization 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.

Detection of antibodies against Orientia tsutsugamushi Sca proteins in scrub typhus patients and genetic variation of sca genes of different strains.

ABSTRACT Scrub typhus, caused by Orientia tsutsugamushi infection, is one of the main causes of acute febrile illness in the Asian-Pacific region. Although early diagnosis and immediate antibiotic treatment are critical for reducing disease severity and mortality, current diagnostic methods using serological and molecular approaches have some limitations in sensitivity and applicability in clinical laboratories. In this study, we identified and characterized O. tsutsugamushi surface cell antigen (sca) family genes encoding autotransporter proteins in order to test them as novel diagnostic targets. We evaluated antibody responses against the Sca proteins in scrub typhus patient sera and examined the genetic diversity of these genes in different strains after PCR amplification. Specific antibody responses against ScaA and ScaC were observed in patients with high indirect immunofluorescence assay titers (≥1:640), whereas specific responses against ScaB and ScaE were relatively low. Genetic analysis using genomic DNAs revealed the sca genes to be quite variable among the different strains. In contrast to scaA, scaC, and scaD, which were detected in all of the tested strains, scaB and scaE were amplified differentially from the different strains, suggesting a differential presence of the genes in the genomes. Among the members of the gene family, the sequence of scaC is the most highly conserved between the different strains, and the size of scaD is the most variable due to the presence of different numbers of internal repeat sequences. These results suggest that the sca genes of O. tsutsugamushi may be valuable targets for use in combination with classical assay methods for scrub typhus diagnosis.

Diversification of Orientia tsutsugamushi genotypes by intragenic recombination and their potential expansion in endemic areas

Background Scrub typhus is a mite-borne febrile disease caused by O. tsutsugamushi infection. Recently, emergence of scrub typhus has attracted considerable attention in several endemic countries in Asia and the western Pacific. In addition, the antigenic diversity of the intracellular pathogen has been a serious obstacle for developing effective diagnostics and vaccine. Methodology/Principal findings To understand the evolutionary pathway of genotypic diversification of O. tsutsugamushi and the environmental factors associated with the epidemiological features of scrub typhus, we analyzed sequence data, including spatiotemporal information, of the tsa56 gene encoding a major outer membrane protein responsible for antigenic variation. A total of 324 tsa56 sequences covering more than 85% of its open reading frame were analyzed and classified into 17 genotypes based on phylogenetic relationship. Extensive sequence analysis of tsa56 genes using diverse informatics tools revealed multiple intragenic recombination events, as well as a substantially higher mutation rate than other house-keeping genes. This suggests that genetic diversification occurred via frequent point mutations and subsequent genetic recombination. Interestingly, more diverse bacterial genotypes and dominant vector species prevail in Taiwan compared to other endemic regions. Furthermore, the co-presence of identical and sub-identical clones of tsa56 gene in geographically distant areas implies potential spread of O. tsutsugamushi genotypes. Conclusions/Significance Fluctuation and diversification of vector species harboring O. tsutsugamushi in local endemic areas may facilitate genetic recombination among diverse genotypes. Therefore, careful monitoring of dominant vector species, as well as the prevalence of O. tsutsugamushi genotypes may be advisable to enable proper anticipation of epidemiological changes of scrub typhus.