kDa Gene Research Articles

Genetic Typing, Based on the 56-Kilodalton Type-Specific Antigen Gene, of Orientia tsutsugamushi Strains Isolated from Chiggers Collected from Wild-Caught Rodents in Taiwan

Orientia tsutsugamushi is the etiological agent of scrub typhus, a mite-borne, febrile illness that occurs in the Asia-Pacific region. We conducted strain characterization of O. tsutsugamushi isolates from chiggers obtained from rodents based the nucleotide sequence of the 56-kDa outer membrane protein gene. With the use of PCR, a total of 68 DNA sequences of 56-kDa antigen genes were amplified. Phylogenetic analysis revealed that there were at least six definable clusters among the 68 isolates: 37% Karp-related strains (25/68), 27% TA763 strains (18/68), 12% JG-related strains (8/68), 19% Kato-related strains (13/68), 4% divergent strains (3/68), and 1% representing a Gilliam prototype strain (1/68). Overall, the O. tsutsugamushi genotypes exhibited a high degree of diversity, similar to that seen in strains from the rest of the areas where scrub typhus is endemic. Moreover, the 56-kDa protein sequence similarity between O. tsutsugamushi isolates from mites and those from human patients (H. Y. Lu et al., Am. J. Trop. Med. Hyg. 83:658-663, 2010) were striking, thus highlighting potential risk factors for this emerging zoonotic disease.

Phylogenetic Analysis of the 56 kDa Protein Genes of Orientia tsutsugamushi in Southwest Area of Korea

This study was conducted to determine which genotypes were present in southwestern Korea. Nested polymerase chain reaction (PCR) and DNA sequence analysis targeting the Orientia tsutsugamushi-specific 56-kDa protein gene was performed with samples of blood and eschar. Of the 69 PCR-positive samples, 61 clustered with the Boryong previously isolated in Korea. CUH 4-6 had sequence homology of 100% with Kato, and CUH 4-3 had homology of 99.8% with Kato and formed the Kato cluster. CUH 4-57, CUH 4-31, CUH 4-142, and CUH 4-324 formed a Kawasaki cluster. CUH 4-271 had sequence homology of 100% with Jecheon and formed a Karp cluster. CUH 4-117 had homology of 99.8% with Neimeng-65, and Gilliam cluster. The most common genotype of O. tsutsugamushi in the southwestern part of Korea is the Boryong genotype. We also identified O. tsutsugamushi of the Kato, Neimeng-65 and Kawasaki genotypes, which had not been identified before in Korea.

Molecular epidemiology of Orientia tsutsugamushi in Cambodia and Central Vietnam reveals a broad region-wide genetic diversity

Scrub typhus is an acute infectious disease caused by an obligate intracellular bacterium Orientia tsutsugamushi following the bite of infected trombiculid mites of the genus Leptotrombidium. This zoonotic disease is a major cause of febrile illness in the Asia-Pacific region, with a large spectrum of clinical manifestations from unapparent or mild disease to fatal disease. O. tsutsugamushi is characterized by a very high genomic plasticity and a large number of antigenic variants amongst strains. The 56-kDa type specific antigen (TSA) gene, encoding the major antigenic protein, was used as reference to investigate the genetic relationships between the strains and to genotype O. tsutsugamushi isolates. The open reading frame of the 56-kDa TSA gene of 41 sequences (28 Cambodian and 13 Vietnamese strains) from patient samples were sequenced and used for genotyping. The 28 Cambodian isolates clustered into 5 major groups, including Karp (43.5%), JG-v (25%), Kato/TA716 (21.5%), TA763 (3.5%) and Gilliam (3.5%). Karp (77%), TA763 (15.5%) and JG-v (7.5%) strains were identified amongst the 13 Vietnamese isolates. This is the first countrywide genotyping description in Cambodia and in Central Vietnam. These results demonstrate the considerable diversity of genotypes in co-circulation in both countries. The genotyping result might raise awareness amongst Cambodian and Vietnamese clinicians of the high genetic diversity of circulating O. tsutsugamushi strains and provides unique and beneficial data for serological and molecular diagnosis of scrub typhus infections as well as raw materials for future studies and vaccine development.

Genetic variability of the 18 kDa/HP6 protective antigen in Taenia saginata and Taenia asiatica: Implications for vaccine development

Genomic characterization of the genes encoding the Taenia 18 kDa/HP6 protective antigens was carried out for Taenia saginata and T. asiatica using 42 taeniid isolates comprising 23 samples of T. saginata, 13 samples of T. asiatica and 6 samples of T. solium. The corresponding sequences from all taeniid isolates were PCR-amplified with specific primers and then sequenced. All the genes, and other described taeniid gene homologues, had the same genomic structure. Surprisingly, the T. saginata TSA18 gene showed nucleotide variability within the 23 samples analyzed. This resulted in two distinct genotypes with 96% DNA sequence similarity and deduced amino acid sequences with 21 substitutions, mainly located in the second exon which contains the fibronectin type III domain. In regards to T. asiatica, the 18 kDa gene (TASI18) was very similar to the T. saginata antigen homologues, both at the DNA and deduced amino acid sequence levels, and the TSOL18 gene was conserved among T. solium isolates as previously described. The implications of these findings on the future development of taeniid vaccines are discussed.

つつが虫病患者由来の Karp 型 <i>Orientia tsutsugamushi </i>56-kDa タンパク遺伝子シークエンスによる亜型分類

To determine the Karp-type Orientia tsutsugamushi subtype in northern Japan, i.e., Yamagata, Niigata, and Akita Prefectures, we analyzed the partial nucleotide sequence of the 56-kDa protein-encoding gene of 30 isolates from scrub typhus cases. Based on sequencing results, we classified isolates into two groups of 27 and 3 isolates. Nucleotide sequences of 27 isolates were homologous to the yeo-joo strain, classified as a JP-1 subtype. The three isolates were each homologous to a stain of CMM1, KNP1, or KNP2, classified as JP-2 subtypes. Phylogenetic tree analysis showed the 27 isolates forming a cluster with the yeo-joo strain and the three isolates with the CMM, KNP1, and KNP2 strains and therefore belonging to these subtypes. The Karp-type O. tsutsugamushi JP-2 subtype predominates in Japan, the JP-1 subtype probably the predominates in the area investigated. O. tsutsugamushi JP-1 subtype strains must therefore be isolated from subjects in this area and comprehensively studied.

The Wlds transgene reduces axon loss in a Charcot-Marie-Tooth disease 1A rat model and nicotinamide delays post-traumatic axonal degeneration

Charcot-Marie-Tooth disease (CMT) is the most common inherited neuropathy and a duplication of the peripheral myelin protein of 22 kDa (PMP22) gene causes the most frequent subform CMT1A. Clinical impairments are determined by the amount of axonal loss. Axons of the spontaneous mouse mutant Wallerian degeneration slow (Wlds) show markedly reduced degeneration following various types of injuries. Protection is conferred by a chimeric Wlds gene encoding an N-terminal part of ubiquitination factor Ube4b and full length nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1). Nmnat1 enzyme generates nicotinamide adenine dinucleotide (NAD) from nicotinamide mononucleotide. Here, in a Pmp22 transgenic animal model of Charcot-Marie-Tooth disease type 1A (CMT rat), the Wlds transgene reduced axonal loss and clinical impairments without altering demyelination. Furthermore, nicotinamide – substrate precursor of the Nmnat1 enzyme – transiently delayed posttraumatic axonal degeneration in an in vivo model of acute peripheral nerve injury, but to a lower extent than Wlds. In contrast, 8 weeks of nicotinamide treatment did not influence axonal loss or clinical manifestations in the CMT rat. Therefore, nicotinamide can partially substitute for the protective Wlds effect in acute traumatic, but not in chronic secondary axonal injury. Future studies are needed to develop axon protective therapy in CMT1A which may be combined with therapeutic strategies aimed at downregulation of toxic PMP22 overexpression.

Comparison of Conventional, Nested, and Real-Time Quantitative PCR for Diagnosis of Scrub Typhus

Orientia tsutsugamushi is the causative agent of scrub typhus. For the diagnosis of scrub typhus, we investigated the performances of conventional PCR (C-PCR), nested PCR (N-PCR), and real-time quantitative PCR (Q-PCR) targeting the O. tsutsugamushi-specific 47-kDa gene. To compare the detection sensitivities of the three techniques, we used two template systems that used plasmid DNA (plasmid detection sensitivity), including a partial region of the 47-kDa gene, and genomic DNA (genomic detection sensitivity) from a buffy coat sample of a single patient. The plasmid detection sensitivities of C-PCR, N-PCR, and Q-PCR were 5 × 10(4) copies/μl, 5 copies/μl, and 50 copies/μl, respectively. The results of C-PCR, N-PCR, and Q-PCR performed with undiluted genomic DNA were negative, positive, and positive, respectively. The genomic detection sensitivities of N-PCR and Q-PCR were 64-fold and 16-fold (crossing point [Cp], 37.7; 426 copies/μl), respectively. For relative quantification of O. tsutsugamushi bacteria per volume of whole blood, we performed real-time DNA PCR analysis of the human GAPDH gene, along with the O. tsutsugamushi 47-kDa gene. At a 16-fold dilution, the copy number and genomic equivalent (GE) of GAPDH were 1.1 × 10(5) copies/μl (Cp, 22.64) and 5.5 × 10(4) GEs/μl, respectively. Therefore, the relative concentration of O. tsutsugamushi at a 16-fold dilution was 0.0078 organism/one white blood cell (WBC) and 117 organisms/μl of whole blood, because the WBC count of the patient was 1.5 × 10(4) cells/μl of whole blood. The sensitivities of C-PCR, N-PCR, and Q-PCR performed with blood samples taken from patients within 4 weeks of onset of fever were 7.3% (95% confidence interval [CI], 1.6 to 19.9), 85.4% (95% CI, 70.8 to 94.4), and 82.9% (95% CI, 67.9 to 92.8), respectively. All evaluated assays were 100% specific for O. tsutsugamushi. In conclusion, given its combined sensitivity, specificity, and speed, Q-PCR is the preferred assay for the diagnosis of scrub typhus.

Isolation of a Novel Orientia Species ( O. chuto sp. nov.) from a Patient Infected in Dubai

In July 2006, an Australian tourist returning from Dubai, in the United Arab Emirates (UAE), developed acute scrub typhus. Her signs and symptoms included fever, myalgia, headache, rash, and eschar. Orientia tsutsugamushi serology demonstrated a 4-fold rise in antibody titers in paired serum collections (1:512 to 1:8,192), with the sera reacting strongest against the Gilliam strain antigen. An Orientia species was isolated by the in vitro culture of the patient's acute blood taken prior to antibiotic treatment. The gene sequencing of the 16S rRNA gene (rrs), partial 56-kDa gene, and the full open reading frame 47-kDa gene was performed, and comparisons of this new Orientia sp. isolate to previously characterized strains demonstrated significant sequence diversity. The closest homology to the rrs sequence of the new Orientia sp. isolate was with three strains of O. tsutsugamushi (Ikeda, Kato, and Karp), with a nucleotide sequence similarity of 98.5%. The closest homology to the 47-kDa gene sequence was with O. tsutsugamushi strain Gilliam, with a nucleotide similarity of 82.3%, while the closest homology to the 56-kDa gene sequence was with O. tsutsugamushi strain TA686, with a nucleotide similarity of 53.1%. The molecular divergence and geographically unique origin lead us to believe that this organism should be considered a novel species. Therefore, we have proposed the name "Orientia chuto," and the prototype strain of this species is strain Dubai, named after the location in which the patient was infected.

Antigenic Drift of Orientia tsutsugamushi in South Korea as Identified by the Sequence Analysis of a 56-kDa Protein-Encoding Gene

This study was performed to determine the nationwide antigenic diversity of Orientia tsutsugamushi in South Korea. Sequence analysis was performed around variable domains I and II of a 56-kDa protein-encoding gene. We used eschar to overcome the disadvantages of conventional serotyping. The serological passive hemagglutination assay (PHA) was assessed based on the genotyping results. We analyzed 153 isolates from scrub typhus patients in major endemic areas and found that Boryong was the major strain (68.6%). New strains were also identified: Taguchi (19.6%), Kanda/Kawasaki (9.2%), and UAP7 (1.3%). PHA yielded significantly fewer positive results among Kawasaki strains (P < 0.001), which are not included in the PHA antigen panel. In South Korea, Boryong was still the predominant strain, but the sequence analysis identified new changes in minor strains (30.1%). This antigenic drift had a negative effect on the PHA results. Periodic surveillance of the contemporary strains using sequence analysis is needed.

Graded Attenuation of TCR Signaling Elicits Distinct Autoimmune Diseases by Altering Thymic T Cell Selection and Regulatory T Cell Function

Mice with a mutation of the zeta-associated protein of 70 kDa gene (skg mutation) are genetically prone to develop autoimmune arthritis, depending on the environment. In a set of mice with the mutation, the amount of zeta-associated protein of 70 kDa protein as well as its tyrosine phosphorylation upon TCR stimulation decreased from +/+, skg/+, skg/skg, to skg/- mice in a stepwise manner. The reduction resulted in graded alterations of thymic positive and negative selection of self-reactive T cells and Foxp3(+) natural regulatory T cells (Tregs) and their respective functions. Consequently, skg/- mice spontaneously developed autoimmune arthritis even in a microbially clean environment, whereas skg/skg mice required stimulation through innate immunity for disease manifestation. After Treg depletion, organ-specific autoimmune diseases, especially autoimmune gastritis, predominantly developed in +/+, at a lesser incidence in skg/+, but not in skg/skg BALB/c mice, which suffered from other autoimmune diseases, especially autoimmune arthritis. In correlation with this change, gastritis-mediating TCR transgenic T cells were positively selected in +/+, less in skg/+, but not in skg/skg BALB/c mice. Similarly, on the genetic background of diabetes-prone NOD mice, diabetes spontaneously developed in +/+, at a lesser incidence in skg/+, but not in skg/skg mice, which instead succumbed to arthritis. Thus, the graded attenuation of TCR signaling alters the repertoire and the function of autoimmune T cells and natural Tregs in a progressive manner. It also changes the dependency of disease development on environmental stimuli. These findings collectively provide a model of how genetic anomaly of T cell signaling contributes to the development of autoimmune disease.

The antitumor efficacy of IL-24 mediated by E1A and E1B triple regulated oncolytic adenovirus

Background: IL-24 (interleukin-24) is a promising, multi-functional anti-cancer agent able to selectively induce tumor cell apoptosis while sparing normal cells. Additionally, IL-24 can enhance the immune response to tumors and suppress tumor angiogenesis. In this study, we introduced IL-24 into the oncolytic adenovirus, Ad·sp·E1A(∆24·E1B(∆55)·IL-24. in which E1A was engineered to target Rb (retinoblastoma) deficient or dysfunctional tumors. The survivin promoter (sp), was used to drive expression of IL-24, thereby allowing it to target most tumors. Finally, the 55 KDa gene of E1B was also deleted, thereby preventing replication in normal cells.Results: Ad·sp·E1A(∆24)·E1B(∆55)·IL-24 showed enhanced anti-tumor effects over the E1, singly regulated oncolytic adenovirus, ONYX-015, in in vitro experiments. Furthermore, Ad·sp·E1A(∆24)·E1B(∆55)·IL-24 could effectively inhibit the progression of NCI-H460 lung carcinoma xenografts in nude mice. Methods: The antitumor effect of Ad·sp·E1A(∆24)·E1B(∆55)·IL-24 was assessed by MTT assay and crystal violet staining in a panel of tumor cells. Cell staining and western blotting for caspase activation were used to assess apoptosis. We assessed the anti-tumor effects of Ad·sp·E1A(∆24)·E1B(∆55)·IL-24 in a xenograft model. Conclusion: This is the first study to use an E1A and E1B triple regulated oncolytic adenovirus vector carrying IL-24 to treat large tumors. We attained efficient antitumor effects both in vitro and in vivo, which provides an experimental foundation for clinical cancer therapy.

Detection of spotted fever group Rickettsia spp. from bird ticks in the U.K.

Migratory birds are known to play a role in the long-distance transportation of microorganisms. To investigate whether this is true for rickettsial agents, we undertook a study to characterize tick infestation in populations of the migratory passerine bird Riparia riparia (Passeriformes: Hirundinidae), the sand martin. A total of 194 birds were sampled and ticks removed from infested birds. The ticks were identified as female Ixodes lividus (Acari: Ixodidae) using standard morphological and molecular techniques. Tick DNA was assayed to detect Rickettsia spp. using polymerase chain reaction and DNA was sequenced for species identification. A single Rickettsia spp. was detected in 100% of the ticks and was designated Rickettsia sp. IXLI1. Partial sequences of 17-kDa and ompA genes showed greatest similarity to Rickettsia sp. TCM1, an aetiological agent of Japanese spotted fever-like illness, previously described in Thailand. Phylogenetic analysis showed that Rickettsia sp. IXLI1 fitted neatly into a group containing strains Rickettsia japonica, Rickettsia sp. strain Davousti and Rickettsia heilongjiangensis. In conclusion, this research shows that U.K. migratory passerine birds host ticks infected with Rickettsia species and contribute to the geographic distribution of spotted fever rickettsial agents.

Rickettsia hoogstraalii sp. nov., isolated from hard- and soft-bodied ticks

A novel spotted fever group Rickettsia was found in Haemaphysalis sulcata ticks collected from sheep and goats in Croatia in 2006. At the same time, a genetically identical organism was co-isolated with the embryonic cell line CCE3 obtained from the soft tick Carios capensis in Georgia, USA. In this study, further phenotypic and genotypic characteristics of the novel rickettsial strain present in H. sulcata ticks were investigated. Based on the cultivation of bacteria in mosquito and Vero cell cultures, the presence of rickettsiae in tick tissues and cell cultures [confirmed by transmission electron microscopy (TEM)] and the amplification and sequencing of five rickettsial genes, it was demonstrated that the novel Rickettsia strain fulfils the criteria to be classified as a novel species. The name Rickettsia hoogstraalii sp. nov. is proposed for the new strain. Rickettsia hoogstraalii sp. nov., an obligately intracellular bacterium, was grown in Vero cells and arthropod CCE3, ISE6 and C6/36 cell lines. The morphology of the cells of the novel species was typical of SFG rickettsiae. The small coccobacillary appearance of the bacteria was apparent with light microscopy. A Gram-negative bacterial cell wall and a cytoplasmic membrane separated by a narrow periplasmic space were visible by TEM. To date, Rickettsia hoogstraalii sp. nov. has been isolated from two species of ticks, H. sulcata and C. capensis. The novel species appears to be geographically widely distributed, having been detected in Croatia, Spain and Georgia, USA. Although no information is available regarding the possible pathogenicity of the novel species for vertebrate hosts, R. hoogstraalii sp. nov. has a cytopathic effect in Vero, CCE3 and ISE6 cells. Sequence analyses of the 16S rRNA, 17 kDa, gltA, ompA and ompB genes indicated that even though R. hoogstraalii sp. nov. was closely related to Rickettsia felis, it represents a separate species within the spotted fever group. The type strain of R. hoogstraalii sp. nov. is strain Croatica(T) (=DSM 22243(T)=UTMB 00003(T)).

Bacterial Diversity in &lt;I&gt;Amblyomma americanum&lt;/I&gt; (Acari: Ixodidae) With a Focus on Members of the Genus &lt;I&gt;Rickettsia&lt;/I&gt;

The lone star tick, Amblyomma americanum (Acari: Ixodidae), is commonly reported from people and animals throughout the eastern U.S. and is associated with transmission of a number of emerging diseases. To better define the microbial communities within lone star ticks, 16S rRNA gene based analysis using bacteria-wide primers, followed by sequencing of individual clones (n = 449) was used to identify the most common bacterial operational taxonomic units (OTUs) present within colony-reared and wild A. americanum. The colony-reared ticks contained primarily sequence affiliated with members of the genus Coxiella (89%; 81/91), common endosymbionts of ticks, and Brevibacterium (11%; 10/91). Similarly, analysis of clones from unfed wild lone star ticks revealed that 96.7% (89/92) of all the OTUs identified were affiliated with Coxiella-like endosymbionts, as compared with only 5.1-11.7% (5/98-9/77) of those identified from wild lone star ticks after feeding. In contrast, the proportion of OTUs identified as Rickettsia sp. in wild-caught ticks increased from 2.2% (2/92) before feeding to as high as 46.8% (36/77) after feeding, and all Rickettsia spp. sequences recovered were most similar to those described from the spotted fever group Rickettsia, specifically R. amblyommii and R. massiliae. Additional characterization of the Rickettsiales tick community by polymerase chain reaction, cloning, and sequencing of 17 kDa and gltA genes confirmed these initial findings and suggested that novel Rickettsia spp. are likely present in these ticks. These data provide insight into the overall, as well as the rickettsial community of wild lone star ticks and may ultimately aid in identification of novel pathogens transmitted by A. americanum.

Rickettsia helveticain Patient with Meningitis, Sweden, 2006

Pathogenicity of Rickettsia helvetica is relatively unknown. We isolated a spotted fever group rickettsial organism from a patient with subacute meningitis. Nucleotide sequences of the 16S rRNA, ompB, and 17kDa genes identified the isolate as R. helvetica. This organism may be associated with serious infections such as central nervous system disorders.

Dissemination of Spotted Fever Rickettsia Agents in Europe by Migrating Birds

Migratory birds are known to play a role as long-distance vectors for many microorganisms. To investigate whether this is true of rickettsial agents as well, we characterized tick infestation and gathered ticks from 13,260 migratory passerine birds in Sweden. A total of 1127 Ixodes spp. ticks were removed from these birds and the extracted DNA from 957 of them was available for analyses. The DNA was assayed for detection of Rickettsia spp. using real-time PCR, followed by DNA sequencing for species identification. Rickettsia spp. organisms were detected in 108 (11.3%) of the ticks. Rickettsia helvetica, a spotted fever rickettsia associated with human infections, was predominant among the PCR-positive samples. In 9 (0.8%) of the ticks, the partial sequences of 17kDa and ompB genes showed the greatest similarity to Rickettsia monacensis, an etiologic agent of Mediterranean spotted fever-like illness, previously described in southern Europe as well as to the Rickettsia sp.IrITA3 strain. For 15 (1.4%) of the ticks, the 17kDa, ompB, gltA and ompA genes showed the greatest similarity to Rickettsia sp. strain Davousti, Rickettsia japonica and Rickettsia heilongjiangensis, all closely phylogenetically related, the former previously found in Amblyomma tholloni ticks in Africa and previously not detected in Ixodes spp. ticks. The infestation prevalence of ticks infected with rickettsial organisms was four times higher among ground foraging birds than among other bird species, but the two groups were equally competent in transmitting Rickettsia species. The birds did not seem to serve as reservoir hosts for Rickettsia spp., but in one case it seems likely that the bird was rickettsiemic and that the ticks had acquired the bacteria from the blood of the bird. In conclusion, migratory passerine birds host epidemiologically important vector ticks and Rickettsia species and contribute to the geographic distribution of spotted fever rickettsial agents and their diseases.

Familial ALS with G298S Mutation in TARDBP: A Comparison of CSF Tau Protein Levels with those in Sporadic ALS

We report a 52-year-old Japanese man showing both upper and lower motor neuron signs with familial amyotrophic lateral sclerosis (ALS). Analysis of the TAR DNA-binding protein of 43 kDa (TDP-43) gene (TARDBP) revealed a glycine-to-serine substitution at position 298 (G298S). Cerebrospinal fluid (CSF) level of total tau protein (CSF-tau) of our patient was found to be highly elevated compared with those of sporadic ALS cases and controls. The elevated CSF-tau level might be related to the damage of neurons exhibiting a large number of TDP-43 inclusions in familial ALS with this mutation.

Characterisation of Orientia tsutsugamushi genotypes from wild rodents and chigger mites in Korea

Orientia tsutsugamushi is a causative agent of scrub typhus, and a Gram-negative obligate intracellular bacterium that is transmitted to humans by the bite of infected Leptotrombidium mites. In Korea, the incidence of scrub typhus has increased remarkably in recent years. A total of 6022 cases of scrub typhus were reported in 2007, and the incidence of scrub typhus infection was 13.2 cases per 100 000 persons. To develop a national strategy for the control and prevention of this disease that is based upon scientific data, we surveyed the infection rate and analysed the distribution of O. tsutsugamushi genotypes among wild rodents and engorged chigger mites in Korea. The specimens used in the present study were obtained from rodents because rodents have been recognised as a natural reservoir of O. tsutsugamushi and a host of chigger mites. At monthly intervals between August 2005 and July 2007, Sherman live traps were used to capture wild rodents living in four regions of Korea. Specimens were obtained from two areas in southern Korea, Chogye in Chollanam province and Ganjeon in Gyeongsangnam province, and from two areas in the middle region of Korea, Jangan and Songsan in Gyeonggi province. Chigger mites were collected from the wild rodents that had been captured. The 56-kDa genes of O. tsutsugamushi were collected from the blood or spleens of the wild rodents, and were detected from the pools of 30 chigger mites by nested PCR amplification, which was carried out as described by Furuya et al. [1]. In the present study, genotypes were determined by PCR-RFLP using DdeI. Analysis of the nucleotide sequence homology among the nested PCR products was performed using the CLUSTALX (http://clustal.org) and Tree Explorer (Tamura; http://evolgen.biol.metrou.ac.jp/TE/TE_man.html) programs in order to confirm the genotype in cases that could not be determined by RFLP. The rates of positivity among wild rodents from Chogye, Ganjeon, Jangan and Songsan, were 4.5% (12 ⁄266), 5.0% (10 ⁄201), 12.4% (25 ⁄202) and 5.0% (13 ⁄258), respectively, as shown in Table 1. To monitor the infection rate in mites, we calculated the MPR (minimum positive rate) using the equation: number of positive mites ⁄total number of mites examined. The MPRs were 0.6% (6 ⁄1071), 0.3% (12 ⁄4215), 0.9% (26 ⁄2893) and 1.3% (85 ⁄6752) in Chogye, Ganjeon, Jangan and Songsan respectively. The rates of positivity for O. tsutsugamushi were 6.5% (60 ⁄927) and 0.9% (129 ⁄14 931) in the wild rodents and chigger mites, respectively. In a previous study, Ree et al. [2] analysed the incidence of O. tsutsugamushi by IFA and PCR using 4315 individual mites collected from eight localities in Korea and reported that the infection rate was 1.5%, which was slightly higher than the result reported in the present study. From these results, we were able to determine that there is a high risk of scrub typhus infection in the natural environment of Korea. The Boryong genotype was the most common, at a rate of 76.7% in wild rodents and 82.9% in chigger mites, followed by the Gilliam (10%), Pajoo (8.3%) and Karp (5%) genotypes in the wild rodents and the Karp (11.6%), Pajoo (3.1%), Yoncheon (1.6%) and Kawasaki (0.8%) genotypes in the chigger mites. The Kato genotype was not detected in any of the specimens. The geographical distribution of the Boryong genotype was slightly higher in southern Korea than in mid-Korea, while the Gilliam, Karp and

Genetic characterisation of ompA, ompB and gltA genes from Candidatus Rickettsia rioja

A. Portillo, V. Ibarra, S. Santiba´n˜ez, L. Pe´rez-Marti´nez, J. R. Blanco and J. A. OteoA´ rea de Enfermedades Infecciosas, Hospital San Pedro-Centro de Investigacio´n Biome´dica de La Rioja(CIBIR), La Rioja, SpainRickettsiosis are emerging and re-emerging infec-tions distributed all over the world. In the lastyears ‘new’ Rickettsia species have been involvedas human pathogens [1–3]. Candidatus Rickettsiarioja is a ‘new’ spotted fever group Rickettsiainvolved in Dermacentor-borne necrosis erythemaand lymphadenopathy (DEBONEL) or tick-borne-lymphadenopathy (TIBOLA) cases from Spain[4,5]. In 2006, a fragment of the ompA gene fromEDTA-treated blood of two patients who devel-oped DEBONEL after being bitten by Dermacentormarginatus in La Rioja, was submitted to GenBank(GenBank accession no. EF028201). This Rickettsiaspecies has also been amplified from ticksremoved from patients with DEBONEL.The aim of this study was to characterise thenucleotide sequence of ompA, ompB, citrate sintase(gltA) and 17 KDa genes from Candidatus R. rioja.As part of a retrospective study, 15 DNA extractsused for ompA PCR that showed 98% similaritywith Rickettsia raoultii and with uncultured Rick-ettsia sp. RpA4, DnS14 and DnS28, were furtheranalysed for the presence of ompB, gltA and17 KDa. Specimens were obtained from twoblood samples and nine D. marginatus removedfrom DEBONEL patients and from four D. mar-ginatus removed from asymptomatic people.For our 15 specimens, when ompA nucleotidesequences were compared with GenBank data,>99% similarity was found with Candidatus Rick-ettsia rioja (Fig. 1). Unfortunately, all attempts toamplify ompB and gltA genes from the two bloodsamples failed. However, PCR amplicons forompB and gltA were obtained from 12⁄13D. marginatus (92.3%). Subsequent sequence anal-ysis of ompB PCR products showed highestsimilarity ( 99% identity with Candidatus R. rioja. For theompB and gltA amplicons obtained from 12D. marginatus, highest identities for ompB andgltA were found with partial genes from R. raoultii(<98.9% and <99.7%, respectively). These com-parisons suggest that Candidatus R. rioja can beconsidered a new Rickettsia species as soon as anestablished isolate is available.ACKNOWLEDGEMENTS

VEGI-armed oncolytic adenovirus inhibits tumor neovascularization and directly induces mitochondria-mediated cancer cell apoptosis

Vascular endothelial cell growth inhibitor (VEGI) is a member of the tumor necrosis factor superfamily and plays an important role in vascular homeostasis. In this study, to investigate the anticancer therapeutic potential of this gene, a secreted isoform of VEGI (VEGI-251) was inserted into a selectively replicating adenovirus with E1B 55 kDa gene deletion (ZD55) to construct ZD55-VEGI-251. We report here that secreted VEGI-251 produced from ZD55-VEGI-251-infected cancer cells potently inhibits endothelial cell proliferation, tube formation in vitro and angiogenesis of chick chorioallantoic membrane in vivo. Additionally, ZD55-VEGI-251 infection leads to a much more severe cytopathic effect than control viruses on several human cancer cell lines, including cervical cancer cell line HeLa, hepatoma cell line SMMC-7721 and colorectal cancer cell line SW620. Further study reveals that the increased cytotoxicity is a result of VEGI-251 autocrine-dependent, mitochondria-mediated apoptosis accompanied by caspase-9 activation, enhanced caspase-3 activation and PARP cleavage. Moreover, ZD55-VEGI-251-treatment of athymic nude mice bearing human cervical and colorectal tumor xenografts markedly suppressed tumor growth. Our findings indicate that the combined effect of antiangiogenesis and apoptosis-induction activity makes the VEGI-251-armed oncolytic adenovirus a promising therapeutic agent for cancer.