Emergence of Japanese encephalitis virus genotype V in the Republic of Korea

<p>Abstract</p> <p>Background</p> <p>Japanese encephalitis virus (JEV) genotype V reemerged in Asia (China) in 2009 after a 57-year hiatus from the continent, thereby emphasizing a need to increase regional surveillance efforts. Genotypic characterization was performed on 19 JEV-positive mosquito pools (18 pools of <it>Culex tritaeniorhynchus </it>and 1 pool of <it>Cx. bitaeniorhynchus</it>) from a total of 64 positive pools collected from geographically different locations throughout the Republic of Korea (ROK) during 2008 and 2010.</p> <p>Findings</p> <p>Two regions of the JEV genome were sequenced from 19 pools; the envelope gene and the nonstructural protein 5 (NS5)/3'-untranslated region (UTR). Eighteen pools of <it>Culex tritaeniorhynchus </it>and one pool of <it>Cx. bitaeniorhynchus </it>were positive for genotype I and genotype V, respectively. Sequence alignment of the complete E gene from <it>Cx. bitaeniorhynchus </it>showed high amino acid similarity (98.8%) to the Muar strain, characterized as the first report of genotype V, isolated from an encephalitis patient in Malaysia in 1952.</p> <p>Conclusion</p> <p>This study represents the first report of JEV genotype V in the ROK. The reemergence of genotype V in Asia (China and ROK) after more than a half-century and its discovery in <it>Cx. bitaeniorhynchus</it>, a mosquito species previously unknown to carry JEV in the ROK, emphasizes the need for enhanced JE surveillance to monitor the dynamics of JEV strains within the region. Future findings may have implications with regard to JEV vaccination/prevention strategies.</p

Metagenomic Approach to Characterizing Disease Epidemiology in a Disease-Endemic Environment in Northern Thailand

In this study, we used a metagenomic approach to analyze bacterial communities from diverse populations (humans, animals, and vectors) to investigate the role of these microorganisms as causative agents of disease in human and animal populations. Wild rodents and ectoparasites were collected from 2014 to 2018 in Nan province, Thailand where scrub typhus is highly endemic. Samples from undifferentiated febrile illness (UFI) patients were obtained from a local hospital. A total of 200 UFI patient samples were obtained and 309 rodents and 420 pools of ectoparasites were collected from rodents (n = 285) and domestic animals (n = 135). The bacterial 16S rRNA gene was amplified and sequenced with the Illumina. Real-time PCR and Sanger sequencing were used to confirm the next-generation sequencing (NGS) results and to characterize pathogen species. Several pathogens were detected by NGS in all populations studied and the most common pathogens identified included Bartonella spp., Rickettsia spp., Leptospira spp., and Orientia tsutsugamushi. Interestingly, Anaplasma spp. was detected in patient, rodent and tick populations, although they were not previously known to cause human disease from this region. Candidatus Neoehrlichia, Neorickettsia spp., Borrelia spp., and Ehrlichia spp. were detected in rodents and their associated ectoparasites. The same O. tsutsugamushi genotypes were shared among UFI patients, rodents, and chiggers in a single district indicating that the chiggers found on rodents were also likely responsible for transmitting to people. Serological testing using immunofluorescence assays in UFI samples showed high prevalence (IgM/IgG) of Rickettsia and Orientia pathogens, most notably among samples collected during September–November. Additionally, a higher number of seropositive samples belonged to patients in the working age population (20–60 years old). The results presented in this study demonstrate that the increased risk of human infection or exposure to chiggers and their associated pathogen (O. tsutsugamushi) resulted in part from two important factors; working age group and seasons for rice cultivation and harvesting. Evidence of pathogen exposure was shown to occur as there was seropositivity (IgG) in UFI patients for bartonellosis as well as for anaplasmosis. Using a metagenomic approach, this study demonstrated the circulation and transmission of several pathogens in the environment, some of which are known causative agents of illness in human populations

Amplicon-Based Next Generation Sequencing for Rapid Identification of Rickettsia and Ectoparasite Species from Entomological Surveillance in Thailand

Background: Next generation sequencing (NGS) technology has been used for a wide range of epidemiological and surveillance studies. Here, we used amplicon-based NGS to species identify Rickettsia and their arthropod hosts from entomological surveillance. Methods: During 2015–2016, we screened 1825 samples of rodents and ectoparasites collected from rodents and domestic mammals (dog, cat, and cattle) across Thailand for Rickettsia. The citrate synthase gene was amplified to identify Rickettsia to species, while the Cytochrome Oxidase subunit I (COI) and subunit II (COII) genes were used as target genes for ectoparasite identification. All target gene amplicons were pooled for library preparation and sequenced with Illumina MiSeq platform. Result: The highest percentage of Rickettsia DNA was observed in fleas collected from domestic animals (56%) predominantly dogs. Only a few samples of ticks from domestic animals, rodent fleas, and rodent tissue were positive for Rickettisia DNA. NGS based characterization of Rickettsia by host identified Rickettsia asembonensis as the most common bacteria in positive fleas collected from dogs (83.2%) while “Candidatus Rickettsia senegalensis” was detected in only 16.8% of Rickettsia positive dog fleas. Sequence analysis of COI and COII revealed that almost all fleas collected from dogs were Ctenocephalides felis orientis. Other Rickettsia species were detected by NGS including Rickettsia heilongjiangensis from two Haemaphysalis hystricis ticks, and Rickettsia typhi in two rodent tissue samples. Conclusion: This study demonstrates the utility of NGS for high-throughput sequencing in the species characterization/identification of bacteria and ectoparasite for entomological surveillance of rickettsiae. A high percentage of C. f. orientis are positive for R. asembonensis. In addition, our findings indicate there is a risk of tick-borne Spotted Fever Group rickettsiosis, and flea-borne murine typhus transmission in Tak and Phangnga provinces of Thailand

Genotyping of <i>Orientia tsutsugamushi</i> from individual mites by next-generation sequencing.

<p>Genotyping of <i>Orientia tsutsugamushi</i> from individual mites by next-generation sequencing.</p

Abundance and diversity of field-collected trombiculid mites on wild-caught rodents and small mammals.

<p>(A) Abundance of chigger(s) from three dominant small mammal species. (B) Diversity of chigger(s) collected from animal(s).</p

Chigger infestation rates and population diversity of mites recovered from small mammals, Thailand.

<p>Chigger infestation rates and population diversity of mites recovered from small mammals, Thailand.</p

Heterogeneity of <i>Orientia tsutsugamushi</i> genotypes in field-collected trombiculid mites from wild-caught small mammals in Thailand

<div><p>Trombiculid mites are the vectors of scrub typhus, with infected larval mites (chiggers) transmitting the causative agent, <i>Orientia tsutsugamushi</i>, during feeding. Co-existence of multiple <i>O</i>. <i>tsutsugamushi</i> strains within infected mites has previously been reported in naturally infected, laboratory-reared mite lines using molecular methods to characterize the 56-kDa type-specific antigen (TSA) gene. In the current study, more advanced next-generation sequencing technology was used to reveal the heterogeneity of O. <i>tsutsugamushi</i> genotypes in field-collected trombiculid mites from rodents and small mammals in scrub typhus-endemic areas of Thailand. Twenty-eight trombiculid mites collected from 10 small mammals were positive for <i>O</i>. <i>tsutsugamushi</i>, corresponding to a prevalence rate of 0.7% within the mite population. Twenty-four of the infected mites were <i>Leptotrombidium</i> spp., indicating that this genus is the main vector for <i>O</i>. <i>tsutsugamushi</i> transmission in Thailand. In addition, <i>O</i>. <i>tsutsugamushi</i> was detected in the mite genera <i>Ascoschoengastia</i>, <i>Blankaartia</i>, <i>Gahrliepia</i>, and <i>Lorillatum</i>. Of the 10 infested small animal hosts, six had 2–10 infected mites feeding at the time of collection. Deep sequencing was used to characterize mixed infections (two to three <i>O</i>. <i>tsutsugamushi</i> genotypes within an individual mite), and 5 of the 28 infected mites (17.9%) contained mixed infections. Additionally, 56-kDa TSA gene sequence analysis revealed identical bacterial genotypes among co-feeding mites with single or mixed infections. These results suggest that co-feeding transmission may occur during the feeding process, and could explain the occurrence of mixed infections in individual mites, as well as the recovery of multiple infected mites from the same host. This study also revealed highly diverse within-host <i>O</i>. <i>tsutsugamushi</i> genotypes. The occurrence of multiple <i>O</i>. <i>tsutsugamushi</i> genotypes within individual mites has important implications, and could provide a mechanism for pathogen evolution/diversification in the mite vector.</p></div

Prevalence of <i>Orientia tsutsugamushi</i> in rodents, small mammals, and associated trombiculid mites captured from June–August 2015 from five provinces in three regions of Thailand.

<p>Prevalence of <i>Orientia tsutsugamushi</i> in rodents, small mammals, and associated trombiculid mites captured from June–August 2015 from five provinces in three regions of Thailand.</p

<i>Orientia tsutsugamushi</i>-positive rate in mite populations collected from 10 animals, Thailand.

<p><i>Orientia tsutsugamushi</i>-positive rate in mite populations collected from 10 animals, Thailand.</p

Relationship between co-feeding mites and diversity of Orientia tsutsugamushi 56-kDa TSA gene sequence-based genotypes detected in mite populations.

<p>(A) Network graph demonstrates the potential co-feeding transmission of <i>O</i>. <i>tsutsugamushi</i> genotypes (yellow circle) among infected mites on the same host (indicated by colored lines). Only hosts with multiple infected mites were included in the analysis. Hosts infested with single <i>O</i>. <i>tsutsugamushi</i>-infected mite were indicated with grey line. The graph was created in the igraph R package. (B) Heatmap demonstrating a pair-wise distance matrix of the 56-kDa TSA gene sequences among 36 <i>O. tsutsugamushi</i> genotypes identified in 28 infected mites. The heatmap was generated using the Heatmap.2 function of the gplots R package.</p