Effects of root-colonizing fungi on pioneer Pinus thunbergii seedlings in primary successional volcanic mudflow on Kuchinoerabu Island, Japan.

Root-colonizing fungi, such as mycorrhizal fungi and dark septate endophyte fungi, are often found on pioneer plant species during early primary succession. However, little is known about which fungal species are responsible for the establishment of pioneer plants when these symbionts colonize simultaneously. We investigated the root-colonizing fungal communities of Pinus thunbergii that established prior to lichens, bryophytes, and short-lived herbaceous plants in a primary successional volcanic mudflow site on Kuchinoerabu Island, Japan. We collected a total of 54 current-year and 1- to 2-year-old seedlings. The colonization of root fungi was evaluated by direct observation of key structures (e.g., mantle, arbuscule, microsclerotia, and hyphae) and molecular analysis. Of the 34 current-year seedlings collected, only 12 individuals were colonized by ectomycorrhizal (ECM) fungi. By contrast, all 1- to 2-year-old seedlings were colonized by ECM fungi. Seedlings colonized by pine-specific ECM fungi, specifically Rhizopogon roseolus and Suillus granulatus, showed higher nitrogen and phosphorus contents in their needles compared to non-ECM seedlings. Arbuscular mycorrhizal fungi and dark septate endophyte fungi were found in only two and three individuals, respectively. The high density of mycophagous deer on Kuchinoerabu-jima may contribute to the favored dispersal of ECM fungi over other root-colonizing fungi. In conclusion, the seedling establishment of P. thunbergii at the volcanic mudflow may be largely supported by ECM fungi, with negligible effects of arbuscular mycorrhizal fungi and dark septate endophytes.

Zoonotic Infection with Oz Virus, a Novel Thogotovirus.

Oz virus is a novel thogotovirus isolated from ticks that causes lethal infection in mice. We conducted serosurveillance of Oz virus infection among humans and wild mammals in Japan using virus-neutralization tests and ELISAs. Results showed that Oz virus may be naturally infecting humans and other mammalian hosts.

Severe Fever with Thrombocytopenia Syndrome Virus RNA in Semen, Japan.

Severe fever with thrombocytopenia syndrome virus (SFTSV) can be transmitted between humans. We describe a case of severe fever with thrombocytopenia syndrome in which SFTSV RNA was detected in semen after its disappearance from serum. Our findings indicate possible sexual transmission of this emerging virus.

Effects of a herbicide on paddy predatory insects depend on their microhabitat use and an insecticide application.

Indirect effects of agrochemicals on organisms via biotic interactions are less studied than direct chemical toxicity despite their potential relevance in agricultural landscapes. In particular, the role of species traits in characterizing indirect effects of pesticides has been largely overlooked. Moreover, it is still unclear whether such indirect effects on organisms are prevalent even when the organisms are exposed to direct toxicity. We conducted a mesocosm experiment to examine indirect effects of a herbicide (pentoxazone) on aquatic predatory insects of rice paddies. Because the herbicide selectively controls photosynthetic organisms, we assumed that the effects of the herbicide on predatory insects would be indirect. We hypothesized that phytophilous predators such as some Odonata larvae, which cling to aquatic macrophytes, would be more subject to negative indirect effects of the herbicide through a decrease in abundance of aquatic macrophytes than benthic, nektonic, and neustonic predators. Also, we crossed-applied an insecticide (fipronil) with herbicide application to examine whether the indirect effects of the herbicide on the assembling predators act additively with direct adverse effects of the insecticide. The herbicide application did not decrease the abundance of phytoplankton constitutively, and there were no clear negative impacts of the herbicide on zooplankton and prey insects (detritivores and herbivores). However, the abundance of aquatic macrophytes was significantly decreased by the herbicide application. Although indirect effects of the herbicide were not so strong on most predators, their magnitude and sign differed markedly among predator species. In particular, the abundance of phytophilous predators was more likely to decrease than that of benthic, nektonic, and neustonic predators when the herbicide was applied. However, these indirect effects of the herbicide could not be detected when the insecticide was also applied, seemingly due to fipronil's high lethal toxicity. Our study highlights the importance of species traits such as microhabitat use, which characterize biotic interactions, for predicting indirect effects of agrochemicals. Given that indirect effects of the chemicals vary in response to species traits and direct toxicity of other chemicals, efforts to explain this variation are needed to predict the realistic risks of indirect effects of agrochemicals in nature.

Viral load and inflammatory cytokine dynamics associated with the prognosis of severe fever with thrombocytopenia syndrome virus infection: An autopsy case.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by a novel bunyavirus. The mechanism underlying disease progression remains unknown, and effective treatment strategy for SFTS is yet to be completely established, making its increasing incidence and subsequent mortality a great concern. Here, we present the autopsy case of a patient with rapidly progressed, fatal SFTS infection. Her viral titer and serum cytokines levels were measured daily and compared with the values of a survivor of the infection. Our findings elucidate the clinical features and pathophysiology of SFTS.

Community responses of aquatic insects in paddy mesocosms to repeated exposures of the neonicotinoids imidacloprid and dinotefuran.

Pesticides are one of major threats to wetland environments and their communities, and thus the information about ecological impact assessment of agro-chemicals on ecosystems is essential for future effective pesticides management. Here, effects of the yearly application of two neonicotinoids, imidacloprid and dinotefuran on aquatic insect communities of experimental rice fields were assessed during two years of monitoring. Both neonicotinoid-treated fields and controls were monitored biweekly throughout the 5-month experimental period until harvest (late October) in each year. Maximum concentrations of imidacloprid (157.5 µg/l in 2014 and 138.0 µg/l in 2015) and dinotefuran (10.54 µg/l in 2014 and 54.05 µg/l in 2015) in water were relatively similar in both years, but maximum residues of imidacloprid (245.45 µg/kg) and dinotefuran (419.5 µg/kg) in the sediment in the second-year were 18 and 175 times higher than in the first year, respectively, with great variability of concentrations among sampling dates. In addition, remaining soil residues of both neonicotinoids were approximately 1 µg/kg (ppb) at the start of the second-year. A total of 6265 individuals of 18 aquatic species belonging to 7 orders were collected. No differences in the number of species between controls and the two neonicotinoids-treated paddies were found between years. However, clear differences in community structures of aquatic insects among the imidacloprid- and dinotefuran-treated mesocosms, and controls and between years were shown by PRC analysis. In particular, imidacloprid likely decreased Crocothemisia servilia mariannae nymphs, Chironominae spp. larvae, and Aedes albopictus larvae, whereas dinotefuran tended to decrease Guignotus japonicus, Orthetrum albistylum speciosum nymphs, and Tubiificidae spp. In addition, long-living species of Coleoptera and Odonata were most sensitive to both neonicotinoids. Changes in composition of feeding functional groups (FFGs) of aquatic insects were more prominent in the first year and became subtler in the second year. One of the possibilities of this phenomenon may be functional redundancy in which species that had low sensitivity to imidacloprid and dinotefuran replaced the vacant niche caused by decreases of other species with high susceptibility within the same feeding functions, although further studies are needed to verify this explanation. Thus, feeding functional traits can be a good indicator for evaluation of changes in ecosystem processes under pesticides exposures. Consequently, the current study emphasized that more realistic prediction of community properties after the repeated application of agrochemicals in successive years should consider for 1) long-term population monitoring, 2) cumulative effects at least over the years, and 3) species' functional traits.

Comparative ecotoxicity of imidacloprid and dinotefuran to aquatic insects in rice mesocosms.

There are growing concerns about the impacts of neonicotinoid insecticides on ecosystems worldwide, and yet ecotoxicity of many of these chemicals at community or ecosystem levels have not been evaluated under realistic conditions. In this study, effects of two neonicotinoid insecticides, imidacloprid and dinotefuran, on aquatic insect assemblages were evaluated in experimental rice mesocosms. During the 5-month period of the rice-growing season, residual concentrations of imidacloprid were 5-10 times higher than those of dinotefuran in both soil and water. Imidacloprid treatment (10kg/ha) reduced significantly the populations of, Crocothemis servilia mariannae and Lyriothemis pachygastra nymphs, whereas those of Orthetrum albistylum speciosum increased slightly throughout the experimental period. However, Notonecta triguttata, which numbers were high from the start, later declined, indicating possible delayed chronic toxicity, while Guignotus japonicus disappeared. In contrast, dinotefuran (10kg/ha) did not decrease the populations of any species, but rather increased the abundance of some insects, particularly Chironominae spp. larvae and C. servilia mariannae nymphs, with the latter being 1.7x higher than those of controls. This was an indirect effect resulting from increased prey (e.g., chironomid larvae) and lack of competition with other dragonfly species. The susceptibilities of dragonfly nymphs to neonicotinoids, particularly imidacloprid, were consistent with those reported elsewhere. In general, imidacloprid had higher impacts on aquatic insects compared to dinotefuran.

A Single Amino Acid Substitution in the NS2A Protein of Japanese Encephalitis Virus Affects Virus Propagation In Vitro but Not In Vivo.

We identified a unique amino acid of NS2A113, phenylalanine, that affects the efficient propagation of two Japanese encephalitis virus strains, JaTH160 and JaOArS982, in neuroblastoma Neuro-2a cells but not in cell lines of extraneural origin. This amino acid did not affect viral loads in the brain or survival curves in mice. These findings suggest that virus propagation in vitro may not reflect the level of virus neuroinvasiveness in vivo.

The world first two cases of severe fever with thrombocytopenia syndrome: An epidemiological study in Nagasaki, Japan.

Severe fever with thrombocytopenia syndrome (SFTS) caused by the SFTS virus (SFTSV), a novel phlebovirus belonging to the family Bunyaviridae, was reported in China for the first time in 2009. We observed two cases where the SFTSV was isolated for the first time in Nagasaki, Japan, in 2005. Two males in their 60s, a farmer and a hunter, respectively, living in Nagasaki developed SFTS during the same period. The patients developed similar clinical symptoms and signs, such as fever, loss of consciousness, and multiple organ dysfunction. The farmer died and the hunter survived. A retrospective diagnosis of SFTS was made in 2013, and genetic analysis revealed that the patients were infected with different SFTSV strains. Retrospective analysis of cytokine production in non-fatal case revealed interleukin (IL)-6, IL-8 and interferon-γ level of acute phase was low and could be potential prognostic factors. As there are no epidemiological studies of positive rate of SFTSV antibody in people living in endemic areas in Japan, a field study was performed. Volunteers at high risk for tick bites, such as hunters, farmers, and soldiers, were recruited in 6 regions, including the areas where the SFTS cases occurred. Three hundred and twenty six volunteers in Nagasaki prefecture were examined and none of these tested positive for the SFTSV antibody. Our data indicates that the risk for SFTSV infection is not high in Nagasaki prefecture. Further collection of blood samples from endemic areas is warranted for the prevention of SFTSV infection.

Application of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for the detection of SFTSV-specific human IgG and IgM antibodies by indirect ELISA.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease that was first reported in China in 2011. It is caused by SFTS virus (SFTSV) which is a member of the Phlebovirus genus in the Bunyaviridae family. SFTSV has been classified as a BSL3 pathogen. There is a need to develop safe and affordable serodiagnostic methods for proper clinical management of infected patients. METHODS: The full length nucleocapsid (N) gene of SFTSV Yamaguchi strain was amplified by RT-PCR and cloned to an expression vector pQE30. The recombinant (r) SFTSV-N protein was expressed by using Escherichia coli (E. coli) expression system and purified under native conditions. rSFTSV-N protein based indirect IgG and IgM enzyme linked immunosorbent assay (ELISA) systems were established to detect specific human IgG and IgM antibodies, respectively. One hundred fifteen serum samples from clinically suspected-SFTS patients were used to evaluate the newly established systems and the results were compared with the total antibody detecting sandwich ELISA system. RESULTS: The native form of recombinant (r) SFTSV-N protein was expressed and purified. Application of the rSFTSV-N protein based indirect IgG ELISA to the 115 serum samples showed results that perfectly matched those of the total antibody sandwich ELISA with a sensitivity and specificity of 100 %. The rSFTSV-N protein based indirect IgM ELISA missed 8 positive samples that were detected by the total antibody sandwich ELISA. The sensitivity and specificity of rSFTSV-N-IgM capture ELISA were 90.59 and 100 %, respectively. CONCLUSIONS: The rSFTSV-N protein is highly immunoreactive and a good target for use as an assay antigen in laboratory diagnosis. Its preparation is simpler in comparison with that used for the total antibody sandwich system. Our rSFTSV-N protein-based IgG and IgM ELISA systems have the advantage of distinguishing two types of antibodies and require small volume of serum sample only. They are safe to use for diagnosis of SFTS virus infection and especially fit in large-scale epidemiological investigations.

Qualitative differences in brain-infiltrating T cells are associated with a fatal outcome in mice infected with Japanese encephalitis virus.

Japanese encephalitis (JE) is the most important form of viral encephalitis in Asia. The critical factors determining mortality and severity of JE virus (JEV) infection remain unclear. We identified brain-infiltrating T cells associated with a fatal outcome of JEV infection in mice. Dying mice were defined as those that lost more than 25 % of their body weight by day 13 and died by day 21, while surviving mice were defined as those that lost less than 10 % by day 13, based on the result of the survival time course study. Two groups of five mice that demonstrated brain virus titers of >1 × 10(6) pfu/g were randomly selected from the dying and surviving groups and used in the analyses. Cytokine patterns in brains were first examined, revealing a higher ratio of Th1-related cytokine genes in dying mice. The expression levels of CD3, CD8, CD25, and CD69 increased in JEV-infected mice relative to mock-infected mice. However, expression levels of these cell-surface markers did not differ between the two groups. T-cell receptor (TCR) usage and complementary determining region 3 (CDR3) sequences were analyzed in the brain-infiltrating T cells. T cells expressing VA8-1, VA10-1, and VB2-1 increased in both groups. However, the dominant T-cell clones as defined by CDR3 amino acid sequence differed between the two groups. The results indicate that the outcome of JEV infection, death or survival, was determined by qualitative differences in infiltrating T-cell clones with unique CDR3 amino acid sequences.

Different acute toxicity of fipronil baits on invasive Linepithema humile supercolonies and some non-target ground arthropods.

Fipronil is one of the most effective insecticides to control the invasive ant Linepithema humile, but its effectiveness has been assessed without considering the genetic differences among L. humile supercolonies. We hypothesized that the susceptibility of the ant to fipronil might differ among supercolonies. If so, dosage and concentration of fipronil may need to be adjusted for effective eradication of each supercolony. The relative sensitivities of four L. humile supercolonies established in Hyogo (Japan) to fipronil baits were examined based on their acute toxicity (48-h LC(50)). Toxicities of fipronil to seven ground arthropods, including four native ant species, one native isopoda, and two cockroaches were also determined and compared to that of L. humile supercolonies using species sensitivity distributions. Marked differences in susceptibility of fipronil were apparent among the supercolonies (P < 0.008), with the 'Japanese main supercolony' (271 µg L(-1)) being five to ten times more sensitive to fipronil than other colonies (1183-2782 µg L(-1)). Toxicities to non-target species (330-2327 µg L(-1)) were in the same range as that of L. humile, and SSDs between the two species groups were not significantly different (t = -1.389, P = 0.180), suggesting that fipronil's insecticidal activity is practically the same for L. humile as for non-target arthropods. Therefore, if the invasive ant is to be controlled using fipronil, this would also affect the local arthropod biodiversity. Only the 'Japanese main supercolony' can be controlled with appropriate bait dosages of fipronil that would have little impact on the other species.

Detection of east/central/south African genotype of chikungunya virus in Myanmar, 2010.

In 2010, chikungunya virus of the East Central South African genotype was isolated from 4 children in Myanmyar who had dengue-like symptoms. Phylogenetic analysis of the E1 gene revealed that the isolates were closely related to isolates from China, Thailand, and Malaysia that harbor the A226V mutation in this gene.

NS1' protein expression facilitates production of Japanese encephalitis virus in avian cells and embryonated chicken eggs.

Japanese encephalitis virus (JEV), which belongs to the genus Flavivirus of the family Flaviviridae, is a leading cause of meningo-encephalitis in Asian countries. The flavivirus non-structural protein 1 (NS1) plays a role in virus replication and in the elicitation of an immune response. The NS1' protein found among the members of the JEV subgroup is an extended form of NS1 and is generated by a -1 ribosomal frameshift. This protein is known to be involved in viral pathogenicity; however, its specific function is still unknown. Here, we describe an investigation of the molecular function of NS1' protein through the production of JEV NS1'-expressing and -non-expressing clones and their infection of avian and mammalian cells. Efficient NS1' protein expression was observed in avian cells and was found to facilitate JEV production in both avian cultured cells and embryonated chicken eggs. NS1' protein was observed to co-localize with NS5 protein and resulted in increased viral RNA levels in avian cells. These findings clearly indicate that NS1' enhances the production of JEV in avian cells and may facilitate the amplification/maintenance role of birds in the virus transmission cycle in nature.

Type-I interferon response affects an inoculation dose-independent mortality in mice following Japanese encephalitis virus infection.

BACKGROUND: The laboratory mouse model is commonly employed to study the pathogenesis of encephalitic flaviviruses such as Japanese encephalitis virus (JEV). However, it is known that some strains of these viruses do not elicit a typical mortality dose response curve from this organism after peripheral infection and the reason for it has not yet been fully understood. It is suggested that induction of more vigorous Type-I IFN (IFN-I) response might control early virus dissemination following increasing infectious challenge doses of the virus. Thus, the objective of this study was to examine this suggested role of IFN-I in the mortality of mice infected with various doses of JEV. METHODS: Inbred 129 mice and their IFNAR KO (A129) mice were subcutaneously inoculated with 100, 102, 104 or 106 pfu of JaOArS982 strain of JEV. Mice were weighed daily and observed for clinical signs. Virus titers in the brains and spleens of JEV-infected mice were determined by plaque forming assays. The upregulated mRNA levels of genes related to IFN-I response of mice were examined by real-time PCR. RESULTS: The mortality rates of 129 mice infected with JaOArS982 did not significantly increase despite the increase in inoculation dose and no significant difference of viral loads was observed between their brains. However, there was clear elevation of the mRNA levels of interferon regulatory factor (IRF)3, IRF7, IRF9, MDA5 and RIG-I at 24 hours post-infection depending on the inoculation dose. In A129 mice, length of survival days and the viral loads of spleen and brain were observed to be inoculation dose-dependent. CONCLUSIONS: From these results, it is suggested that early IFN-I response elicited by high inoculation doses of JEV provides an anti-viral effect during the early phase of infection. Accordingly, virus replication is counteracted by IFN-I response at each increasing inoculation dose resulting in the interference of impending severe disease course or fatal outcome; hence, this might explain the inoculation dose-independent mortality in mice caused by Japanese encephalitis virus.

pH-dependent virucidal effects of weak acids against pathogenic viruses.

BACKGROUND: Weak acids, such as acetic acid, show virucidal effects against viruses, and disinfectants are considered effective virucidal agents possibly because of their low pH, depending on the proton concentration. This study aimed to evaluate the efficacy of different weak acids (acetic, oxalic, and citric acids) and eligible vinegars under different pH conditions by comparing their inactivation efficacies against enveloped and non-enveloped viruses. METHODS: Acetic, oxalic, and citric acids were adjusted to pH values of 2, 4 and 6, respectively. They were also diluted from 1 M to 0.001 M with distilled water. Enveloped influenza A virus (FulV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and non-enveloped feline calicivirus (FCV) were treated with adjusted weak acids for up to 30 min. These viruses were also reacted with white distilled vinegar (WDV) and grain-flavored distilled vinegar (GV) for up to 30 min. Infectious viral titers after the reactions were expressed as plaque-forming units per mL. RESULTS: Acetic acid showed virucidal effects against FulV at pH 4, whereas citric and oxalic acids did not. Acetic and citric acids inactivated SARS-CoV-2 at pH 2, whereas oxalic acid did not. All acids showed virucidal effects against FVC at pH 2; however, not at pH 4. The virucidal effects of the serially diluted weak acids were also reflected in the pH-dependent results. WDV and GV significantly reduced FulV titers after 1 min. SARS-CoV-2 was also susceptible to the virucidal effects of WDV and GV; however, the incubation period was extended to 30 min. In contrast, WDV and GV did not significantly inactivate FCV. CONCLUSIONS: The inactivation efficacy of weak acids is different even under the same pH conditions, suggesting that the virucidal effect of weak acids is not simply determined by pH, but that additional factors may also influence these effects. Moreover, eligible vinegars, the main component of which is acetic acid, may be potential sanitizers for some enveloped viruses, such as FulV, in the domestic environment.

Can warming accelerate the decline of Odonata species in experimental paddies due to insecticide fipronil exposure?

Systemic insecticides are one of the causes of Odonata declines in paddy fields. Since rising temperatures associated with global warming can contribute to strengthen pesticide toxicity, insecticide exposures under increasing temperatures may accelerate the decline of Odonata species in the future. However, the combined effects of multiple stressors on Odonata diversity and abundance within ecosystems under various environmental conditions and species interactions are little known. Here, we evaluate the combined effects of the insecticide fipronil and warming on the abundance of Odonata nymphs in experimental paddies. We show that the stand-alone effect of the insecticide exposure caused a significant decrease in abundance of the Odonata community, while nymphs decreased synergistically in the combined treatments with temperature rise in paddy water. However, impacts of each stressor alone were different among species. This study provides experimental evidence that warming could accelerate a reduction in abundance of the Odonata community exposed to insecticides (synergistic effect), although the strength of that effect might vary with the community composition in targeted habitats, due mainly to different susceptibilities among species to each stressor. Community-based monitoring in actual fields is deemed necessary for a realistic evaluation of the combined effects of multiple stressors on biodiversity.

Development of an entirely cloned cDNA-based reverse genetics system for Tofla virus of orthonairovirus.

The genus Orthonairovirus includes highly pathogenic tick-borne viruses such as the Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV). A reverse genetics system is an indispensable tool for determining the viral factors related to pathogenicity. Tofla orthonairovirus (TFLV) is a recently identified virus isolated from ticks in Japan and our research has suggested that TFLV is a useful model for studying pathogenic orthonairoviruses. In this study, we successfully established a reverse genetics system for TFLV using T7 RNA polymerase. Recombinant TFLV was generated by transfecting cloned complementary DNAs encoding the TFLV genome into BSR T7/5 cells expressing T7 RNA polymerase. We were able to rescue infectious recombinant TFLV mutant (rTFLVmt) and wild-type TFLV (rTFLVpt) viruses, which exhibited indistinguishable growth kinetics in mammalian cells and pathogenicity in A129 mice compared with the authentic virus. Our approach provides a valuable method for establishing reverse genetics system for orthonairoviruses.

Clinical Factors Associated with SFTS Diagnosis and Severity in Cats.

Severe fever with thrombocytopenia syndrome (SFTS) is a potentially fatal tick-borne zoonosis caused by SFTS virus (SFTSV). In addition to tick bites, animal-to-human transmission of SFTSV has been reported, but little is known about feline SFTSV infection. In this study, we analyzed data on 187 cats with suspected SFTS to identify biomarkers for SFTS diagnosis and clinical outcome. Body weight, red and white blood cell and platelet counts, and serum aspartate aminotransferase and total bilirubin levels were useful for SFTS diagnosis, whereas alanine aminotransferase, aspartate aminotransferase and serum SFTSV RNA levels were associated with clinical outcome. We developed a scoring model to predict SFTSV infection. In addition, we performed a phylogenetic analysis to reveal the relationship between disease severity and viral strain. This study provides comprehensive information on feline SFTS and could contribute to the protection of cat owners, community members, and veterinarians from the risk of cat-transmitted SFTSV infection.

Development of simple and rapid assay to detect viral RNA of tick-borne encephalitis virus by reverse transcription-loop-mediated isothermal amplification.

BACKGROUND: Tick-borne encephalitis virus (TBEV) is a causative agent of acute central nervous system disease in humans. It has three subtypes, far eastern (FE), Siberian (Sib) and European (Eu) subtypes, which are distributed over a wide area of Europe and Asia. The objective of this study was to develop a simple and rapid assay for the detection of TBEV RNA by using reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) method that can differentiate the three subtypes of TBEV and can be used for clinical diagnosis and epidemiological study. METHODS: Primers for TBEV-specific and subtype-specific RT-LAMP assay were designed to target the consensus sequence in NS1 of all subtypes and the consensus sequence in the E gene of each subtype, respectiveluy. In vitro transcribed RNA of Oshima strain that belongs to FE subtype was serially diluted and used to examine the sensitivity of the assay. Cross-reactivity of subtype-specific RT-LAMP assay was tested by using the RNA of Oshima and Sofjin (FE), IR-99 (Sib) and Hochosterwitz (Eu) strains. RNA extracted from the mixtures of TBEV and ticks, and of TBEV and human blood, and the mouse tissues infected with TBEV, were evaluated in the assay. Positive amplification was observed by real-time monitoring of turbidity and by visual detection of color change. RESULTS: The sensitivity of TBEV-specific RT-LAMP assay was 102 copies of target RNA per reaction volume. FE-specific RT-LAMP assay amplified viral genes of Oshima and Sofjin strains but not of IR-99 and Hochosterwitz strains, and of Japanese encephalitis virus. RT-LAMP assay for Sib and for Eu specifically amplified viral genes of IR-99 and Hochosterwitz strains, respectively. We also showed that tick or human blood extract did not inhibit the amplification of viral gene during the assay. Furthermore, we confirmed that the TBEV RT-LAMP could detect virus RNA from peripheral and central nervous system tissues of laboratory mice infected with TBEV. CONCLUSION: TBEV RT-LAMP assay offers a sensitive, specific, rapid and easy-to-handle method for the detection of TBEV RNA in tick samples and this may be applied in the clinical samples collected from TBE-suspected patients.