Intraperitoneal administration of synthetic microRNA-214 elicits tumor suppression in an intraperitoneal dissemination mouse model of canine hemangiosarcoma.

Canine hemangiosarcoma (HSA) has an extremely poor prognosis, making it necessary to develop new systemic treatment methods. MicroRNA-214 (miR-214) is one of many microRNAs (miRNA) that can induce apoptosis in HSA cell lines. Synthetic miR-214 (miR-214/5AE), which showed higher cytotoxicity and greater nuclease resistance than mature miR-214, has been developed for clinical application. In this study, we evaluated the effects of miR-214/5AE on stage 2 HSA in a mouse model. Mice intraperitoneally administered with miR-214/5AE (5AE group) had significantly fewer intraperitoneal dissemination tumor foci (median number: 72.5 vs. 237.5; p < 0.05) and a lower median foci weight (0.26 g vs. 0.61 g; p < 0.05). Mice in the 5AE group had increased expression of p53 and cleaved caspase-3, and a significantly lower proportion of Ki-67-positive cells, than those in the non-specific miR group. Notably, no significant side effects were observed. These results indicate that intraperitoneal administration of miR-214/5AE exhibits antitumor effects in an intraperitoneal dissemination mouse model of HSA by inducing apoptosis and suppressing cell proliferation. These results provide a basis for future studies on the antitumor effect of miR-214/5AE for HSA.

Protein digestibility and bioavailability in an F2 mouse cross between the selected fat mouse line and an M2 congenic line carrying the anti-obesity and anti-diabetic Tst allele.

The study was performed to investigate protein digestibility and utilization in an F2 cross (M2-F2 cross) between the selected Fat (F) line and an M2 congenic line. The congenic M2 line carried the Fob3b2 quantitative trait locus (QTL) from the selected Lean (L) line previously shown to contain the Tst gene with leanness, anti-diabetic and resistance to diet-induced obesity effects. The main objective of the study was to test if some of the effects on leanness and obesity resistance of the L-line Fob3b2 could also be due to the effect of this QTL on nutrient digestibility and bioavailability. The F2 littermates carrying either the Fat line segment within the Fob3b2 region or the L-line were compared when fed the high-fat diet. Eleven mice per genotype were individually housed in metabolic cages. In 5-day experimental period, body mass and diet intake were measured. The part of study was done on the F and L line and tested the difference in apparent protein digestibility on low-fat (LFD) and high-fat (HFD) diet. The nitrogen content was determined in the diet, faeces, and urine based on which, the apparent protein digestibility, apparent protein biological value and apparent net protein utilization were calculated There were no significant differences in any of these parameters on congenic line, confirming that the phenotypic effect on adiposity between the genotypes in the M2-F2 population was not due to the differential effect of the Fob3b2 locus carrying the Tst gene on protein utilization. We conclude that the observed phenotypic effects of this gene region are due to direct metabolic actions rather than the effects on nutrient absorption and nitrogen utilization since there were no differences in apparent protein digestibility between L and F lines, irrespective to HFD or LFD. The age of animals had significant effect on the level of digestibility.

C1qa deficiency in mice increases susceptibility to mouse hepatitis virus A59 infection.

BACKGROUND: Mouse hepatitis virus (MHV) A59 is a highly infectious pathogen and starts in the respiratory tract and progresses to systemic infection in laboratory mice. The complement system is an important part of the host immune response to viral infection. It is not clear the role of the classical complement pathway in MHV infection. OBJECTIVES: The purpose of this study was to determine the importance of the classical pathway in coronavirus pathogenesis by comparing C1qa KO mice and wild-type mice. METHODS: We generated a C1qa KO mouse using CRISPR/Cas9 technology and compared the susceptibility to MHV A59 infection between C1qa KO and wild-type mice. Histopathological and immunohistochemical changes, viral loads, and chemokine expressions in both mice were measured. RESULTS: MHV A59-infected C1qa KO mice showed severe histopathological changes, such as hepatocellular necrosis and interstitial pneumonia, compared to MHV A59-infected wild-type mice. Virus copy numbers in the olfactory bulb, liver, and lungs of C1qa KO mice were significantly higher than those of wild-type mice. The increase in viral copy numbers in C1qa KO mice was consistent with the histopathologic changes in organs. These results indicate that C1qa deficiency enhances susceptibility to MHV A59 systemic infection in mice. In addition, this enhanced susceptibility effect is associated with dramatic elevations in spleen IFN-γ, MIP-1 α, and MCP-1 in C1qa KO mice. CONCLUSIONS: These data suggest that C1qa deficiency enhances susceptibility to MHV A59 systemic infection, and activation of the classical complement pathway may be important for protecting the host against MHV A59 infection.

53BP1 Repair Kinetics for Prediction of In Vivo Radiation Susceptibility in 15 Mouse Strains.

We present a novel mathematical formalism to predict the kinetics of DNA damage repair after exposure to both low- and high-LET radiation (X rays; 350 MeV/n 40Ar; 600 MeV/n 56Fe). Our method is based on monitoring DNA damage repair protein 53BP1 that forms radiation-induced foci (RIF) at locations of DNA double-strand breaks (DSB) in the nucleus and comparing its expression in primary skin fibroblasts isolated from 15 mice strains. We previously reported strong evidence for clustering of nearby DSB into single repair units as opposed to the classic "contact-first" model where DSB are considered immobile. Here we apply this clustering model to evaluate the number of remaining RIF over time. We also show that the newly introduced kinetic metrics can be used as surrogate biomarkers for in vivo radiation toxicity, with potential applications in radiotherapy and human space exploration. In particular, we observed an association between the characteristic time constant of RIF repair measured in vitro and survival levels of immune cells collected from irradiated mice. Moreover, the speed of DNA damage repair correlated not only with radiation-induced cellular survival in vivo, but also with spontaneous cancer incidence data collected from the Mouse Tumor Biology database, suggesting a relationship between the efficiency of DSB repair after irradiation and cancer risk.

Guinea pig cytomegalovirus trimer complex gH/gL/gO uses PDGFRA as universal receptor for cell fusion and entry.

Species-specific guinea pig cytomegalovirus (GPCMV) causes congenital CMV and the virus encodes homolog glycoprotein complexes to human CMV, including gH-based trimer (gH/gL/gO) and pentamer-complex (PC). Platelet-derived growth factor receptor alpha (gpPDGFRA), only present on fibroblast cells, was identified via CRISPR as the putative receptor for PC-independent GPCMV infection. Immunoprecipitation assays demonstrated direct interaction of gH/gL/gO with gpPDGFRA but not in absence of gO. Expression of viral gB also resulted in precipitation of gB/gH/gL/gO/gpPDGFRA complex. Cell-cell fusion assays determined that expression of gpPDGFRA and gH/gL/gO in adjacent cells enabled cell fusion, which was not enhanced by gB. N-linked gpPDGFRA glycosylation inhibition had limited effect and blocking tyrosine kinase (TK) transduction had no impact on infection. Ectopically expressed gpPDGFRA or TK-domain mutant in trophoblast or epithelial cells previously non-susceptible to GPCMV(PC-) enabled viral infection. In contrast, transient human PDGFRA expression did not complement GPCMV(PC-) infection, a potential basis for viral species specificity.

Murine gammaherpesvirus infection is skewed toward Igλ+ B cells expressing a specific heavy chain V-segment.

One of the defining characteristics of the B cell receptor (BCR) is the extensive diversity in the repertoire of immunoglobulin genes that make up the BCR, resulting in broad range of specificity. Gammaherpesviruses are B lymphotropic viruses that establish life-long infection in B cells, and although the B cell receptor plays a central role in B cell biology, very little is known about the immunoglobulin repertoire of gammaherpesvirus infected cells. To begin to characterize the Ig genes expressed by murine gammaherpesvirus 68 (MHV68) infected cells, we utilized single cell sorting to sequence and clone the Ig variable regions of infected germinal center (GC) B cells and plasma cells. We show that MHV68 infection is biased towards cells that express the Igλ light chain along with a single heavy chain variable gene, IGHV10-1*01. This population arises through clonal expansion but is not viral antigen specific. Furthermore, we show that class-switching in MHV68 infected cells differs from that of uninfected cells. Fewer infected GC B cells are class-switched compared to uninfected GC B cells, while more infected plasma cells are class-switched compared to uninfected plasma cells. Additionally, although they are germinal center derived, the majority of class switched plasma cells display no somatic hypermutation regardless of infection status. Taken together, these data indicate that selection of infected B cells with a specific BCR, as well as virus mediated manipulation of class switching and somatic hypermutation, are critical aspects in establishing life-long gammaherpesvirus infection.

Nest Carbon Dioxide Masks GABA-Dependent Seizure Susceptibility in the Naked Mole-Rat.

African naked mole-rats were likely the first mammals to evolve eusociality, and thus required adaptations to conserve energy and tolerate the low oxygen (O2) and high carbon dioxide (CO2) of a densely populated fossorial nest. As hypercapnia is known to suppress neuronal activity, we studied whether naked mole-rats might demonstrate energy savings in GABAergic inhibition. Using whole-colony behavioral monitoring of captive naked mole-rats, we found a durable nest, characterized by high CO2 levels, where all colony members spent the majority of their time. Analysis of the naked mole-rat genome revealed, uniquely among mammals, a histidine point variation in the neuronal potassium-chloride cotransporter 2 (KCC2). A histidine missense substitution mutation at this locus in the human ortholog of KCC2, found previously in patients with febrile seizures and epilepsy, has been demonstrated to diminish neuronal Cl- extrusion capacity, and thus impairs GABAergic inhibition. Seizures were observed, without pharmacological intervention, in adult naked mole-rats exposed to a simulated hyperthermic surface environment, causing systemic hypocapnic alkalosis. Consistent with the diminished function of KCC2, adult naked mole-rats demonstrate a reduced efficacy of inhibition that manifests as triggering of seizures at room temperature by the GABAA receptor (GABAAR) positive allosteric modulator diazepam. These seizures are blocked in the presence of nest-like levels of CO2 and likely to be mediated through GABAAR activity, based on in vitro recordings. Thus, altered GABAergic inhibition adds to a growing list of adaptations in the naked mole-rat and provides a plausible proximate mechanism for nesting behavior, where a return to the colony nest restores GABA-mediated inhibition.

Megaesophagus Is a Major Pathological Condition in Rats With a Large Deletion in the Rbm20 Gene.

A spontaneously arising, loss-of-function mutation in the RNA binding motif protein 20 (Rbm20) gene, which encodes a nuclear splicing protein, was previously identified as the underlying reason for expression of an abnormally large TITIN (TTN) protein in a rat model of cardiomyopathy. An outbreak of Pseudomonas aeruginosa led to submission of rats with dyspnea, sneezing, lethargy, nasal discharge, and/or unexpected death for diagnostic evaluation. Necropsy revealed underlying megaesophagus in Rbm20-/- rats. Further phenotyping of this rat strain and determination of the size of esophageal TTN was undertaken. The Rbm20-defective rats developed megaesophagus at an early age (26 weeks) with high frequency (13/32, 41%). They also often exhibited secondary rhinitis (9/32, 28%), aspiration pneumonia (8/32, 25%), and otitis media/interna (6/32, 19%). In addition, these rats had a high prevalence of hydronephrosis (13/32, 41%). RBM20 is involved in splicing multiple RNA transcripts, one of which is the muscle-specific protein TTN. Rbm20 mutations are a significant cause of dilated cardiomyopathy in humans. In Rbm20-defective rats, TTN size was significantly increased in the skeletal muscle of the esophagus. Megaesophagus in this rat strain (maintained on a mixed genetic background) is hypothesized to result from altered TTN stretch signaling in esophageal skeletal muscle. This study describes a novel mechanism for the development of megaesophagus, which may be useful for understanding the pathogenesis of megaesophagus in humans and offers insights into potential myogenic causes of this condition. This is the first report of megaesophagus and other noncardiac pathogenic changes associated with mutation of Rbm20 in any species.

Field vole-associated Traemmersee hantavirus from Germany represents a novel hantavirus species.

Vole-associated hantaviruses occur in the Old and New World. Tula orthohantavirus (TULV) is widely distributed throughout the European continent in its reservoir, the common vole (Microtus arvalis), but the virus was also frequently detected in field voles (Microtus agrestis) and other vole species. TULV and common voles are absent from Great Britain. However, field voles there harbor Tatenale and Kielder hantaviruses. Here we screened 126 field voles and 13 common voles from Brandenburg, Germany, for hantavirus infections. One common vole and four field voles were anti-TULV antibody and/or TULV RNA positive. In one additional, seropositive field vole a novel hantavirus sequence was detected. The partial S and L segment nucleotide sequences were only 61.1% and 75.6% identical to sympatrically occurring TULV sequences, but showed highest similarity of approximately 80% to British Tatenale and Kielder hantaviruses. Subsequent determination of the entire nucleocapsid (N), glycoprotein (GPC), and RNA-dependent RNA polymerase encoding sequences and determination of the pairwise evolutionary distance (PED) value for the concatenated N and GPC amino acid sequences confirmed a novel orthohantavirus species, tentatively named Traemmersee orthohantavirus. The identification of this novel hantavirus in a field vole from eastern Germany underlines the necessity of a large-scale, broad geographical hantavirus screening of voles to understand evolutionary processes of virus-host associations and host switches.

Detection of Murine Astrovirus and Myocoptes musculinus in individually ventilated caging systems: Investigations to expose suitable detection methods for routine hygienic monitoring.

Murine Astrovirus is one of the most prevalent viral agents in laboratory rodent facilities worldwide, but its influence on biomedical research results is poorly examined. Due to possible influence on research results and high seroprevalence rates in mice, it appears useful to include this virus into routine health monitoring programs. In order to establish exhaust air particle PCR as a reliable detection method for Murine Astrovirus infections in mice kept in individually ventilated cages (IVC) and compare the method to sentinel mice monitoring regarding reproducibility and detection limit, we conducted a study with defined Murine Astrovirus cage prevalence. In parallel, the efficacy of both detection strategies (soiled-bedding sentinel (SBS) and exhaust air dust (EAD) analysis) was tested for Myocoptes musculinus. The fur mite was used as a reference organism during the whole study period to ensure the validity of this method. Because some publications already demonstrated successful detection of several pathogens, including murine fur mite species, via EAP-PCR. Detection of Murine Astrovirus infections at low prevalence is possible with both methods tested. Detection by exhaust air particles (EAP) is faster, more sensitive and more reliable compared to soiled bedding sentinels (SBS). Exhaust air particle PCR also detected the reference organism Myocoptes musculinus, which was not detected at all by sentinel mice, not even by high sensitivity fur swab qPCR. In conclusion, Murine Astrovirus can be detected by both exhaust air particle PCR and soiled bedding sentinels. We recommend exhaust air particle PCR as the better detection technique for Murine Astrovirus, because it is more reliable. Environmental samples are the method of choice for detection of Myocoptes musculinus because relying on soiled bedding sentinels harbors a big risk of missing existing infestations.

STING induces early IFN-ß in the liver and constrains myeloid cell-mediated dissemination of murine cytomegalovirus.

Cytomegalovirus is a DNA-encoded ß-herpesvirus that induces STING-dependent type 1 interferon responses in macrophages and uses myeloid cells as a vehicle for dissemination. Here we report that STING knockout mice are as resistant to murine cytomegalovirus (MCMV) infection as wild-type controls, whereas mice with a combined Toll-like receptor/RIG-I-like receptor/STING signaling deficiency do not mount type 1 interferon responses and succumb to the infection. Although STING alone is dispensable for survival, early IFN-ß induction in Kupffer cells is STING-dependent and controls early hepatic virus propagation. Infection experiments with an inducible reporter MCMV show that STING constrains MCMV replication in myeloid cells and limits viral dissemination via these cells. By contrast, restriction of viral dissemination from hepatocytes to other organs is independent of STING. Thus, during MCMV infection STING is involved in early IFN-ß induction in Kupffer cells and the restriction of viral dissemination via myeloid cells, whereas it is dispensable for survival.

Hyaline Arteriolosclerosis in 30 Strains of Aged Inbred Mice.

During a screen for vascular phenotypes in aged laboratory mice, a unique discrete phenotype of hyaline arteriolosclerosis of the intertubular arteries and arterioles of the testes was identified in several inbred strains. Lesions were limited to the testes and did not occur as part of any renal, systemic, or pulmonary arteriopathy or vasculitis phenotype. There was no evidence of systemic or pulmonary hypertension, and lesions did not occur in ovaries of females. Frequency was highest in males of the SM/J (27/30, 90%) and WSB/EiJ (19/26, 73%) strains, aged 383 to 847 days. Lesions were sporadically present in males from several other inbred strains at a much lower (<20%) frequency. The risk of testicular hyaline arteriolosclerosis is at least partially underpinned by a genetic predisposition that is not associated with other vascular lesions (including vasculitis), separating out the etiology of this form and site of arteriolosclerosis from other related conditions that often co-occur in other strains of mice and in humans. Because of their genetic uniformity and controlled dietary and environmental conditions, mice are an excellent model to dissect the pathogenesis of human disease conditions. In this study, a discrete genetically driven phenotype of testicular hyaline arteriolosclerosis in aging mice was identified. These observations open the possibility of identifying the underlying genetic variant(s) associated with the predisposition and therefore allowing future interrogation of the pathogenesis of this condition.

Mouse NC/Jic strain provides novel insights into host genetic factors for malaria research.

Malaria is caused by Plasmodium parasites and is one of the most life-threatening infectious diseases in humans. Infection can result in severe complications such as cerebral malaria, acute lung injury/acute respiratory distress syndrome, and acute renal injury. These complications are mainly caused by P. falciparum infection and are major causes of death associated with malaria. There are a few species of rodent-infective malaria parasites, and mice infected with such parasites are now widely used for screening candidate drugs and vaccines and for studying host immune responses and pathogenesis associated with disease-related complications. We found that mice of the NC/Jic strain infected with rodent malarial parasites exhibit distinctive disease-related complications such as cerebral malaria and nephrotic syndrome, in addition to a rapid increase in parasitemia. Here, we focus on the analysis of host genetic factors that affect malarial pathogenesis and describe the characteristic features, utility, and future prospects for exploitation of the NC/Jic strain as a novel mouse model for malaria research.

Secondary contact between diverged host lineages entails ecological speciation in a European hantavirus.

The diversity of viruses probably exceeds biodiversity of eukaryotes, but little is known about the origin and emergence of novel virus species. Experimentation and disease outbreak investigations have allowed the characterization of rapid molecular virus adaptation. However, the processes leading to the establishment of functionally distinct virus taxa in nature remain obscure. Here, we demonstrate that incipient speciation in a natural host species has generated distinct ecological niches leading to adaptive isolation in an RNA virus. We found a very strong association between the distributions of two major phylogenetic clades in Tula orthohantavirus (TULV) and the rodent host lineages in a natural hybrid zone of the European common vole (Microtus arvalis). The spatial transition between the virus clades in replicated geographic clines is at least eight times narrower than between the hybridizing host lineages. This suggests a strong barrier for effective virus transmission despite frequent dispersal and gene flow among local host populations, and translates to a complete turnover of the adaptive background of TULV within a few hundred meters in the open, unobstructed landscape. Genetic differences between TULV clades are homogenously distributed in the genomes and mostly synonymous (93.1%), except for a cluster of nonsynonymous changes in the 5' region of the viral envelope glycoprotein gene, potentially involved in host-driven isolation. Evolutionary relationships between TULV clades indicate an emergence of these viruses through rapid differential adaptation to the previously diverged host lineages that resulted in levels of ecological isolation exceeding the progress of speciation in their vertebrate hosts.

A Comparison of Spontaneous Tumors in Tg.rasH2 Mice in 26-week Carcinogenicity Studies Conducted at a Single Test Facility during 2004 to 2012 and 2013 to 2018.

This article presents the historical control data of spontaneous tumors in Tg.rasH2 published in 2013 (2004-2012) and compares and contrasts it to more recent data collected from 2013 to 2018, reporting differences in the average percentage incidences or incidence ranges as well as the incidence of new tumors. In 2013, we published a comprehensive review of spontaneous tumors in Tg.rasH2 mice used in 26-week carcinogenicity studies, which included data from control dose groups from 26 studies and a total of 710 mice per sex. The total database, now including the more recent data, has nearly doubled the number of animals, completing to date a total of 52 studies in males and 51 studies in females for a total of 1,615 male mice and 1,560 female mice, respectively. In this article, we compare the data collected from 2004 to 2012 against the data collected from 2013 to 2018 and the overall tumor incidence change.

Worm expulsion of Gymnophalloides seoi from C57BL/6 mice: role of metacercarial exosomes in upregulating TLR2 and MUC2 expression in intestinal tissues.

Gymnophalloides seoi worms were rapidly expelled from C57BL/6 mice within days 3-6 post-infection probably due to operation of mucosal innate immunity. To understand better the mucosal immunity related to worm expulsion from the host, we isolated exosomes of G. seoi metacercariae and investigated their role in induction of mRNA and protein expression of several Toll-like receptors and mucin-related factors in vitro. G. seoi-secreted exosomes were collected using differential ultracentrifugation, and cellular internalization of the exosomes into HT-29 intestinal epithelial cells was visualized by confocal microscopy. The expression of TLR2 and MUC2 in HT-29 cells was up-regulated in stimulation with the exosomes. We suggest that G. seoi-secreted exosomes offer a new point of view in the mechanism of worm expulsion from the host through enhancement of TLR2 and MUC2 expression.

Selective Packaging in Murine Coronavirus Promotes Virulence by Limiting Type I Interferon Responses.

Selective packaging is a mechanism used by multiple virus families to specifically incorporate genomic RNA (gRNA) into virions and exclude other types of RNA. Lineage A betacoronaviruses incorporate a 95-bp stem-loop structure, the packaging signal (PS), into the nsp15 locus of ORF1b that is both necessary and sufficient for the packaging of RNAs. However, unlike other viral PSs, where mutations generally resulted in viral replication defects, mutation of the coronavirus (CoV) PS results in large increases in subgenomic RNA packaging with minimal effects on gRNA packaging in vitro and on viral titers. Here, we show that selective packaging is also required for viral evasion of the innate immune response and optimal pathogenicity. We engineered two distinct PS mutants in two different strains of murine hepatitis virus (MHV) that packaged increased levels of subgenomic RNAs, negative-sense genomic RNA, and even cellular RNAs. All PS mutant viruses replicated normally in vitro but caused dramatically reduced lethality and weight loss in vivo PS mutant virus infection of bone marrow-derived macrophages resulted in increased interferon (IFN) production, indicating that the innate immune system limited the replication and/or pathogenesis of PS mutant viruses in vivo PS mutant viruses remained attenuated in MAVS-/- and Toll-like receptor 7-knockout (TLR7-/-) mice, two well-known RNA sensors for CoVs, but virulence was restored in interferon alpha/beta receptor-knockout (IFNAR-/-) mice or in MAVS-/- mice treated with IFNAR-blocking antibodies. Together, these data indicate that coronaviruses promote virulence by utilizing selective packaging to avoid innate immune detection.IMPORTANCE Coronaviruses (CoVs) produce many types of RNA molecules during their replication cycle, including both positive- and negative-sense genomic and subgenomic RNAs. Despite this, coronaviruses selectively package only positive-sense genomic RNA into their virions. Why CoVs selectively package their genomic RNA is not clear, as disruption of the packaging signal in MHV, which leads to loss of selective packaging, does not affect genomic RNA packaging or virus replication in cultured cells. This contrasts with other viruses, where disruption of selective packaging generally leads to altered replication. Here, we demonstrate that in the absence of selective packaging, the virulence of MHV was significantly reduced. Importantly, virulence was restored in the absence of interferon signaling, indicating that selective packaging is a mechanism used by CoVs to escape innate immune detection.

Characterization of interaction between Trim28 and YY1 in silencing proviral DNA of Moloney murine leukemia virus.

Moloney Murine Leukemia Virus (M-MLV) proviral DNA is transcriptionally silenced in embryonic cells by a large repressor complex tethered to the provirus by two sequence-specific DNA binding proteins, ZFP809 and YY1. A central component of the complex is Trim28, a scaffold protein that regulates many target genes involved in cell cycle progression, DNA damage responses, and viral gene expression. The silencing activity of Trim28, and its interactions with corepressors are often regulated by post-translational modifications such as sumoylation and phosphorylation. We defined the interaction domains of Trim28 and YY1, and investigated the role of sumoylation and phosphorylation of Trim28 in mediating M-MLV silencing. The RBCC domain of Trim28 was sufficient for interaction with YY1, and acidic region 1 and zinc fingers of YY1 were necessary and sufficient for its interaction with Trim28. Additionally, we found that residue K779 was critical for Trim28-mediated silencing of M-MLV in embryonic cells.

Molecular Epidemiology of an Orientia tsutsugamushi Gene Encoding a 56-kDa Type-Specific Antigen in Chiggers, Small Mammals, and Patients from the Southwest Region of Korea.

A phylogenetic analysis of Orientia tsutsugamushi was performed to elucidate its antigenic diversity in chiggers, small mammals, and patients. Between September 2014 and December 2016, a total of 3,816 chiggers were identified within nine species of four genera in the southwest region of Korea: Leptotrombidium scutellare (49.9%; 1,907/3,816), Leptotrombidium orientale (21.1%; 804/3,816), Leptotrombidium pallidum (12.4%; 474/3,816), Euchoengastia koreaensis (7.2%; 273/3,816), Leptotrombidium palpale (6.7%; 256/3,816), Neotrombicular gardellai (1.3%; 50/3,816), Leptotrombidium zetum (0.8%; 32/3,816), Walchia fragilis (0.5%; 18/3,816), and Neotrombicular japonica (> 0.1%; 2/3,816). Twelve chiggers (11 L. scutellare and one L. palpale) tested positive for O. tsutsugamushi by polymerase chain reaction and, except for 1 chigger (KY266830), were part of the Boryong strain cluster. Of the 413 small mammals that were analyzed for O. tsutsugamushi, Apodemus agrarius was the most common rodent species (89.5%; 370/413), followed by Crocidura lasiura (6.8%; 28/413) and Myodes regulus (3.6%; 15/413). The sequence identity of an O. tsutsugamushi sample obtained from the A. agrarius sample population belonged to the Saitama strain cluster. Furthermore, a phylogenetic analysis in 125 patients revealed four clusters (Boryong cluster: 82.4% [103/125], Karp: 13.6% [17/125], Kawasaki: 3.2% [4/125], and Saitama: 0.8% [1/125]). This study clarified the phylogenetic relationship for O. tsutsugamushi in chiggers, small mammals, and patients. The Boryong strain was the most common strain in chiggers and patients. In addition, various strains were identified, except for the Boryong strain, in the southwest region of Korea. Overall, the data presented here will be helpful for the establishment of prevention strategies for scrub typhus.