Epidemiology of Murine Typhus in Taiwan from 2013 to 2020.

Murine typhus is a flea-borne disease caused by Rickettsia typhi infection. The disease is a notifiable infectious disease in Taiwan. Specimens from suspected cases are required to be sent to the Taiwan Centers for Disease Control and Prevention for laboratory diagnosis. In this study, 204 cases of murine typhus were identified by bacterial isolation, real-time polymerase chain reaction, or indirect immunofluorescence assay between 2013 and 2020. The average incidence rate was 0.11/100,000 person-years (95% CI: 0.08-0.13). Murine typhus occurred throughout the year, but it was most prevalent in summer (May to August). The majority of patients were males (75%), residents of Kaohsiung city (31%), and worked in agriculture, forestry, fishing, and animal husbandry (27%). Fever was the most common symptom, present in 95.6% of patients, followed by headache (41%), myalgia (33%), and liver dysfunction (33%). Only 13% of patients had a rash. Up to 80% of cases were among hospitalized patients, and 43% of patients developed severe manifestations. Serological assays also indicated coinfection events. Seven patients showed a 4-fold increase in antibody titers against Orientia tsutsugamushi (N = 2), Coxiella burnetii (n = 2), and Leptospira (N = 3). In conclusion, murine typhus is an endemic and important zoonotic rickettsial disease in Taiwan that cannot be ignored. Further epidemiological surveillance and clinical characteristics should be continuously investigated to prevent and control murine typhus.

Human case of Anaplasma phagocytophilum infection in Eastern Taiwan.

Human granulocytic anaplasmosis (HGA) is a tick-borne infection caused by the bacterium Anaplasma phagocytophilum. In this study, we report an indigenous case of clinically diagnosed HGA. The patient was a 41-year-old man who experienced a tick bite and later developed fever, chills, myalgia, malaise, thrombocytopenia, leukocytosis with a left shift, elevated hepatic transaminase levels, and splenomegaly upon admission to the hospital. Immunofluorescence assays detected seroconversion against A. phagocytophilum, whereas tests for spotted fever group rickettsiae, murine typhus, scrub typhus, Q fever, and ehrlichiosis were negative. ELISA and Western blot analysis using recombinant MSP2 protein confirmed the exposure to A. phagocytophilum. Oral doxycycline and intravenous ceftriaxone were prescribed, and the patient made a full recovery. Our findings indicate the presence of HGA on the main island of Taiwan. Precautions against tick bites should be taken when engaging in outdoor activities, and HGA should be considered by physicians in the differential diagnosis.

Correction: Molecular epidemiology of scrub typhus in Taiwan during 2006-2016.

[This corrects the article DOI: 10.1371/journal.pntd.0010369.].

Delayed correlation between the incidence rate of indigenous murine typhus in humans and the seropositive rate of Rickettsia typhi infection in small mammals in Taiwan from 2007-2019.

Murine typhus is a flea-borne zoonotic disease with acute febrile illness caused by Rickettsia typhi and is distributed widely throughout the world, particularly in port cities and coastal regions. We observed that murine typhus was an endemic disease (number of annual indigenous cases = 29.23±8.76) with a low incidence rate (0.13±2.03*10-4 per 100,000 person-years) in Taiwan from 2007-2019. Most (45.79%, 174/380) indigenous infections were reported in May, June, and July. The incidence rates in both May and June were statistically higher than those in other months (p<0.05). Correspondingly, sera collected from small mammals (rodents and shrews) trapped in airports and harbors demonstrated anti-R. typhi antibody responses (seropositive rate = 8.24±0.33%). Interestingly, the ports with the highest seropositivity rates in small mammals are all inside/near the areas with the highest incidence rates of indigenous murine typhus. In addition, incidence rates in humans were positively correlated with the 1-month and 2-month prior seropositive rates in small mammals (R = 0.31 and 0.37, respectively). As early treatment with appropriate antibiotics for murine typhus could effectively shorten the duration of illness and reduce the risk of hospitalization and fatality, flea-related exposure experience should be considered in clinics during peak seasons and the months after a rise in seropositivity rates in small mammals. Surveillance in small mammals might be helpful for the development of real-time reporting or even early reminders for physicians of sporadic murine typhus cases based on the delayed correlation observed in this study.

Molecular epidemiology of scrub typhus in Taiwan during 2006-2016.

Scrub typhus is the most common endemic vector-borne disease in Taiwan. We identified a total of 4,857 laboratory-confirmed cases during 2006-2016 with hyperendemic foci on offshore islands, including Penghu (778 cases, 16.0%) and Kinmen (716 cases, 14.7%), and eastern Taiwan, including Taitung (628 cases, 12.9%) and Hualien (508 cases, 10.5%). Scrub typhus cases occur year-round throughout Taiwan, with a summer peak in June and July. A total of 545 O. tsutsugamushi isolates were successfully obtained from patients infected in diverse geographic areas, including Taiwan and three offshore islands, and the complete open reading frame of the 56 kDa type-specific antigen gene (tsa56) sequence of these isolates was examined. High phylogenetic diversity was found in these isolates, which could be grouped into 36 distinct sequence types. Most isolates belonged to the Karp (49.9%; 272/545), followed by the TW-22 (17.8%; 97/454) and Kawasaki (14.7%; 80/545) genotypes. In conclusion, our data indicate the widespread presence of tsa56 genotypes closely related to Thailand and Korean strains and the presence of the unique endemic strains TW-12, TW-22, TW-29, and TW-36 in Taiwan.

Human Case of Severe Fever with Thrombocytopenia Syndrome Virus Infection, Taiwan, 2019.

We report on a 70-year-old man with fever, leukopenia, thrombocytopenia, vomiting, malaise, dyspnea, and consciousness disturbance who was infected with severe fever with thrombocytopenia syndrome virus in northern Taiwan, 2019. This autochthonous case was confirmed by reverse transcription PCR, virus isolation, and genomic sequencing.

Genome Analysis of a Novel Tembusu Virus in Taiwan.

We identified and isolated a novel Tembusu virus (TMUV) strain TP1906 (TMUV-TP1906) from a Culexannulus mosquito pool collected from the northern part of Taiwan in 2019. The TMUV-TP1906 genome is a 10,990-nucleotide-long, positive-sense, single-stranded RNA, consisting of a single open reading frame (ORF) encoding a polyprotein of 3425 amino acids, with 5' and 3' untranslated regions (UTRs) of 94 and 618 nucleotides, respectively. The nucleotide sequence of the TMUV-TP1906 of ORF exhibited 93.71% and 91.27% similarity with Sitiawan virus (STWV) and the TMUV prototype strain MM1775, respectively. The 3'-UTR variable region of TMUV-TP1906 showed nucleotide sequence divergence with other TMUV strains. Phylogenetic analysis of the complete ORF and polyprotein sequences revealed that TMUV-TP1906 is most closely related to STWV which causes encephalitis and retarded growth in chickens. We found that the TMUV-TP1906 caused a cytopathic effect (CPE) in the DF-1 chicken fibroblast cell line, while no apparent CPE was observed in Vero and C6/36 cells. In this study, we first identified and isolated a novel TMUV strain in Taiwan. In addition, to our knowledge, it is the first time that the TMUV strain was isolated from the Cx. annulus mosquitoes. Further study is warranted to investigate the host range and virulence of TMUV-TP1906.

Human Case of Ehrlichia chaffeensis Infection, Taiwan.

In 2018, an immunosuppressed woman in southern Taiwan had onset of fever, chills, myalgia, malaise, thrombocytopenia, lymphocytopenia, and elevated hepatic transaminases. Investigation revealed infection with Ehrlichia chaffeensis. This autochthonous case of human monocytotropic ehrlichiosis was confirmed by PCR, DNA sequencing, and seroconversion.

Evaluation of Enzyme-Linked Immunosorbent Assay Using Recombinant 56-kDa Type-Specific Antigens Derived from Multiple Orientia tsutsugamushi Strains for Detection of Scrub Typhus Infection.

Scrub typhus is caused by the intracellular bacterium Orientia tsutsugamushi. The 56-kDa type-specific antigen (TSA) displays a significant antigenic variation across different O. tsutsugamushi strains. To minimize the influence of the antigenic diversity of TSA on assay sensitivity, we developed a mixed-TSA enzyme-linked immunosorbent assay (mixed-TSA ELISA) using a mixture of recombinant TSAs of prototype (Karp, Gilliam, and Kato) and local (TW-1, TW-10, TW-19, and TW-22) O. tsutsugamushi strains as antigens to detect immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against O. tsutsugamushi. These four local strains covered a major part of the total genetic diversity of TSA gene of O. tsutsugamushi in Taiwan. A total of 109 acute-phase serum samples from O. tsutsugamushi polymerase chain reaction-positive, scrub typhus patients, and 82 negative control serum samples from non-scrub typhus cases were used for evaluation of the recombinant TSA-based ELISA. We compared the performance of the mixed-TSA ELISA with immunofluorescence assay (IFA), which is considered the gold standard method for the serological diagnosis of scrub typhus. The results indicated that the sensitivity of IgM mixed-TSA ELISA (80.7%) was significantly higher than that of IgM IFA (68.8%). We demonstrated that the mixed-TSA ELISA had a high sensitivity and specificity and can be used for screening of scrub typhus patient in the early phase of the disease.

Molecular characterization and phylogenetic analysis of dengue viruses imported into Taiwan during 2011-2016.

A total of 1,596 laboratory-confirmed imported dengue cases were identified in Taiwan during 2011-2016. Most of the imported cases arrived from Southeast Asia as well as the Indian subcontinent, the Pacific region, Latin America, Australia and Africa. Phylogenetic analyses of the complete envelope protein gene sequences from 784 imported dengue virus (DENV) isolates were conducted, and the results suggest that the DENV-1 genotype I and DENV-2 Cosmopolitan genotype comprise the predominant serotype/genotype of DENV strains circulating in Southeast Asia. The DENV-1 genotype III, DENV-3 genotype III and DENV-4 genotype I and II strains were found to be newly emerging in several Southeast Asian countries. Our results also showed that geographical restrictions of DENV-1 genotype I, DENV-1 genotype III and DENV-2 Cosmopolitan genotype are becoming blurred, indicating the extensive introductions and continuous expansions of DENV strains between nations in Southeast Asia. In this study, we present the geographic distribution and dynamic transmission of DENV strains circulating in Southeast Asian countries. In addition, we demonstrated local dengue epidemics caused by several imported DENV strains in Taiwan during 2011-2016.

Snake venomics and antivenomics of Protobothrops mucrosquamatus and Viridovipera stejnegeri from Taiwan: keys to understand the variable immune response in horses.

The proteomes of the venoms of the snakes Viridovipera stejnegeri and Protobothrops mucrosquamatus from Taiwan were characterized by N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of in-gel generated tryptic peptides. Proteins belonging to the following toxin classes were identified: metalloproteinase, phospholipase A(2) (PLA(2)), serine proteinase, C-type lectin-like, CRISP, l-amino acid oxidase, disintegrin, and peptides (vasoactive and inhibitors of SVMPs). Nine horses were immunized with a mixture of these venoms. All horses developed a satisfactory immune response against lethality of the venom of V. stejnegeri, whereas only three horses reached the accepted neutralizing potency against the venom of P. mucrosquamatus. Antivenoms were prepared from pools of 'good responder' (GR) and 'poor responder' (PR) horses and compared by antivenomics and neutralization tests. A similar neutralizing response was observed between the GR and PR antivenoms against the venom of V. stejnegeri, whereas antivenom from PR had a lower neutralizing activity against effects of P. mucrosquamatus venom than antivenom from GR. The low potency of the plasma of some horses against this venom is a consequence of the low immunogenicity of the neurotoxic PLA(2) trimucrotoxin. Our results provide clues for innovating the immunization scheme to generate improved antivenoms.

Annexin II binds to capsid protein VP1 of enterovirus 71 and enhances viral infectivity.

Enterovirus type 71 (EV71) causes hand, foot, and mouth disease (HFMD), which is mostly self-limited but may be complicated with a severe to fatal neurological syndrome in some children. Understanding the molecular basis of virus-host interactions might help clarify the largely unknown neuropathogenic mechanisms of EV71. In this study, we showed that human annexin II (Anx2) protein could bind to the EV71 virion via the capsid protein VP1. Either pretreatment of EV71 with soluble recombinant Anx2 or pretreatment of host cells with an anti-Anx2 antibody could result in reduced viral attachment to the cell surface and a reduction of the subsequent virus yield in vitro. HepG2 cells, which do not express Anx2, remained permissive to EV71 infection, though the virus yield was lower than that for a cognate lineage expressing Anx2. Stable transfection of plasmids expressing Anx2 protein into HepG2 cells (HepG2-Anx2 cells) could enhance EV71 infectivity, with an increased virus yield, especially at a low infective dose, and the enhanced infectivity could be reversed by pretreating HepG2-Anx2 cells with an anti-Anx2 antibody. The Anx2-interacting domain was mapped by yeast two-hybrid analysis to VP1 amino acids 40 to 100, a region different from the known receptor binding domain on the surface of the picornavirus virion. Our data suggest that binding of EV71 to Anx2 on the cell surface can enhance viral entry and infectivity, especially at a low infective dose.

Pigment epithelium-derived factor (PEDF) promotes tumor cell death by inducing macrophage membrane tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

Pigment epithelium-derived factor (PEDF) is an intrinsic anti-angiogenic factor and a potential anti-tumor agent. The tumoricidal mechanism of PEDF, however, has not been fully elucidated. Here we report that PEDF induces the apoptosis of TC-1 and SK-Hep-1 tumor cells when they are cocultured with bone marrow-derived macrophages (BMDMs). This macrophage-mediated tumor killing is prevented by blockage of TNF-related apoptosis-inducing ligand (TRAIL) following treatment with the soluble TRAIL receptor. PEDF also increases the amount of membrane-bound TRAIL on cultured mouse BMDMs and on macrophages surrounding subcutaneous tumors. PEDF-induced tumor killing and TRAIL induction are abrogated by peroxisome proliferator-activated receptor γ (PPARγ) antagonists or small interfering RNAs targeting PPARγ. PEDF also induces PPARγ in BMDMs. Furthermore, the activity of the TRAIL promoter in human macrophages is increased by PEDF stimulation. Chromatin immunoprecipitation and DNA pull-down assays confirmed that endogenous PPARγ binds to a functional PPAR-response element (PPRE) in the TRAIL promoter, and mutation of this PPRE abolishes the binding of the PPARγ-RXRα heterodimer. Also, PPARγ-dependent transactivation and PPARγ-RXRα binding to this PPRE are prevented by PPARγ antagonists. Our results provide a novel mechanism for the tumoricidal activity of PEDF, which involves tumor cell killing via PPARγ-mediated TRAIL induction in macrophages.

Pigment epithelium-derived factor is an intrinsic antifibrosis factor targeting hepatic stellate cells.

The liver is the major site of pigment epithelium-derived factor (PEDF) synthesis. Recent evidence suggests a protective role of PEDF in liver cirrhosis. In the present study, immunohistochemical analyses revealed lower PEDF levels in liver tissues of patients with cirrhosis and in animals with chemically induced liver fibrosis. Delivery of the PEDF gene into liver cells produced local PEDF synthesis and ameliorated liver fibrosis in animals treated with either carbon tetrachloride or thioacetamide. In addition, suppression of peroxisome proliferator-activated receptor gamma expression, as well as nuclear translocation of nuclear factor-kappa B was found in hepatic stellate cells (HSCs) from fibrotic livers, and both changes were reversed by PEDF gene delivery. In culture-activated HSCs, PEDF, through the induction of peroxisome proliferator-activated receptor gamma, reduced the activity of nuclear factor-kappa B and prevented the nuclear localization of JunD. In conclusion, our observations that PEDF levels are reduced during liver cirrhosis and that PEDF gene delivery ameliorates cirrhosis suggest that PEDF is an intrinsic protector against liver cirrhosis. Direct inactivation of HSCs and the induction of apoptosis of activated HSCs may be two of the mechanisms by which PEDF suppresses liver cirrhosis.

Pigment epithelium-derived factor induces interleukin-10 expression in human macrophages by induction of PPAR gamma.

AIM: In search for the anti-inflammation mechanism of PEDF, we investigate whether pigment epithelium-derived factor (PEDF) induces the gene expression of interleukin (IL)-10 in human macrophages and determine the molecular basis of this induction. MAIN METHODS: Human macrophages derived from a monocytic cell line, THP-1, and peripheral monocytes were treated with PEDF. IL-10 expression was assessed by quantitative real-time PCR, enzyme-linked immunosorbent assay, semi-quantitative reverse transcriptase (RT)-PCR, and promoter-reporter assay. Activity of extracellular signal-regulated kinase 2 (ERK2) and p38 mitogen-activated protein kinase (MAPK) was assessed by immunoblotting using antibodies targeting phosphorylated kinases forms. Elk-1 and ATF-2 phosphorylation was determined as well. Pharmacological inhibitors were used to examine the involvement of ERK, p38 MAPK, and peroxisome proliferator-activated receptor gamma (PPARgamma) on the IL-10 expression induced by PEDF. KEY FINDINGS: PEDF increased the levels of IL-10 mRNA and protein in THP-1 cells and human macrophages derived from peripheral monocytes. Blockade of activity of ERK or p38 MAPK attenuated PEDF effects on induction of PPARgamma and IL-10. PEDF increased the transcriptional activity of IL-10 promoter. The effect was synergistically augmented by PPARgamma agonist, but attenuated by inhibitors of PPARgamma, ERK or p38 MAPK. These results showed that PEDF promotes IL-10 expression at transcriptional level, and that this is achieved through the ERK2/p38MAPK-dependent PPARgamma expression. SIGNIFICANCE: The anti-inflammatory property of PEDF may in part through the induction of IL-10 in macrophages. Our study supports the therapeutic potential of PEDF and PPARgamma agonists in inflammatory diseases.