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.

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.

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.

Undesirable dispersal via a river pathway of a single Argentine ant supercolony newly invading an inland urban area of Japan.

Invasive ants pose a risk to human well-being and social/ecosystem stability. Linepithema humile Mayr is among the most damaging invasive ants worldwide. Most L. humile populations invade ports/wharfs isolated from surrounding landscapes, but unfortunately, a new population was discovered in an inland urban area (Nara Prefecture) of Japan in 2021. In this study, first, the supercolony type of the Nara L. humile population was identified via a hostility test, and then its distribution pattern was characterized. In aggression tests between L. humile from Nara and four supercolonies (haplotypes LH1, LH2, LH3, LH4), this ant showed extremely strong hostility against all supercolonies exept LH2, which was detected only in Japan in its introduced range. In Nara, L. humile was abundant in and around the urban river. Simulations revealed that using this environment for movement/dispersal increased the annual dispersal ability by 14 times compared with that achieved via ground (125 m), as mentioned in the literature. Therefore, river channels can serve as major pathways of long-distance dispersal for L. humile invading inland urban areas. Since applying chemical strategies around rivers is problematic, preventing L. humile from moving to rivers from initial invasion sites is crucial.

Global warming intensifies the interference competition by a poleward-expanding invader on a native dragonfly species.

Rapid climate warming has boosted biological invasions and the distribution or expansion polewards of many species: this can cause serious impacts on local ecosystems within the invaded areas. Subsequently, native species may be exposed to threats of both interspecific competition with invaders and temperature rises. However, effects of warming on interspecific interactions, especially competition between invader and native species remains unclear. To better understand the combined threats of biological invasions and warming, the effect of temperature on competitive interactions between two dragonfly species, the expanding Trithemis aurora from Southeast Asia and the Japanese native Orthetrum albistylum speciosum were assessed based on their foraging capacity. Although the stand-alone effect of temperature on foraging intake of the native dragonfly was not apparent, its intake significantly decreased with increasing temperatures when the invader T. aurora was present. Such reductions in foraging might lead to displacement of the native species through competition for food resources. This suggests that impacts of invader species against native species are expected to be more severe when interspecific competition is exacerbated by temperature rises.

Development of a Novel Positive Pressure Protective Suit for a Biosafety Level 4 Laboratory in Japan.

Biosafety level 4 (BSL-4) laboratories are necessary to study microorganisms that are highly pathogenic to humans and have no prevention or therapeutic measures. Currently, most BSL-4 facilities have suit-type laboratories to conduct experiments on highly pathogenic microorganisms. In 2021, the first Japanese suit-type BSL-4 laboratory was constructed at Nagasaki University. Positive pressure protection suit (PPPS) is a primary barrier that protects and isolates laboratory workers from pathogens and the laboratory environment. Here, we developed a novel PPPS originally designed to be used in the Nagasaki BSL-4 laboratory. We modified several parts of a domestic chemical protective suit, including its front face shield, cuff, and air supply hose, for safe handling of microbiological agents. The improved suit, PS-790BSL4-AL, showed resistance to several chemicals, including quaternary ammonium disinfectant, and did not show any permeation against blood and phages. To validate the suit's integrity, we also established an airtight test that eliminated individual differences for quantitative testing. In conclusion, our developed suit performs sufficiently as a primary barrier and allows for the safe handling of pathogens in our new BSL-4 laboratory.

Acute toxicity of fipronil to an invasive ant, Lepisiota frauenfeldi.

Slow-acting fipronil is one of the best components for controlling invasive ants. However, its efficacy against invasive Lepisiota frauenfeldi, which recently invaded Japan, remains unclear. Here, its acute toxicity to Le. frauenfeldi was assessed, and its lethal concentrations were compared with those against other invasive ants (Linepithema humile and Solenopsis invicta). The LC10 and LC50 values of fipronil for Le. frauenfeldi were significantly lower than the previously reported values for Li. humile and/or S. invicta, and its LC90 value against Le. frauenfeldi was in the same range as that required for Li. humile extermination. Additionally, Le. frauenfeldi can be more sensitive to fipronil than non-target arthropods. Therefore, recent fipronil-based Li. humile and S. invicta eradication/control programs may be effective against Le. frauenfeldi as well. Moreover, applying fipronil at dosages appropriate for Le. frauenfeldi would lead to effective Le. frauenfeldi extermination/control with low damage to other native species/ants.

First report of the genera Paralobella and Blasconura (Collembola: Neanuridae: Neanurinae) from Japan with the description of three new species.

This study reports the first record of the genera Paralobella Cassagnau Deharveng, 1984 and Blasconura Cassagnau, 1983, three new species from Kuchinoerabu-jima and Miyako-jima of Japan, respectively. Paralobella kuchierabu sp. nov. can be distinguished from all known genus members by distinct morphological characteristics: yellow body color, mandible with 3 teeth, presence of chaeta O on head, and tubercle De of Th. IIIII with 5 (4+s) chaetae, tubercle Di of Th. I with 1 chaeta, tubercles De and Dl of Abd. V separated. Blasconura triangulum sp. nov. is characterized by a tubercle Fr fused to An, unguis without an inner tooth, and 2 very closely placed tubercles Di on head. Blasconura cordis sp. nov. is unique in the genus, with a tubercle An fused to Cl. Other characteristic features of the species are the absence of chaeta O on head, tubercle Oc with 3 chaetae, Ant. I with 7 chaetae, and tubercle De of Th. III with 5 (4+s) chaetae. A key to all species of the genus Paralobella and Blasconura is provided.

Comprehensive Surveillance of Virus Infection among Captive African Pygmy Hedgehogs in Japan.

African pygmy hedgehogs (Atelerix albiventris) are popular exotic pets in Japan, and their breeding numbers have recently increased. Although various diseases have been reported in hedgehogs, including skin, respiratory, neurological, and neoplastic diseases, most of the causes remain unidentified. In this study, we investigated herpesvirus, adenovirus, and coronavirus infections among 150 African pygmy hedgehogs in Japan and evaluated the correlations between virus infection and diseases. A novel herpesvirus named Atelerix albiventris herpesvirus 1 (AAHeV), and African pygmy hedgehog adenovirus 1 (AhAdV-1) were detected in 14 and 3 oral swab samples, respectively. AAHeV infection may be related to neurological clinical signs. Interestingly, no hedgehog with a neoplastic disorder tested positive for AAHeV. Further research is required to determine the pathogenicity and prevalence of the detected viruses.

Erratum to: "Efficacy of 265-nm ultraviolet light in inactivating infectious SARS-CoV-2" [Journal of Photochemistry and Photobiology].

[This corrects the article DOI: 10.1016/j.jpap.2021.100050.].

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.

Epidemiological study of Kabuto Mountain virus, a novel uukuvirus, in Japan.

Kabuto Mountain virus (KAMV), the new member of the genus Uukuvirus, was isolated from the tick Haemaphysalis flava in 2018 in Japan. To date, there is no information on KAMV infection in human and animals. Therefore, serological surveillance of the infection among humans and wild mammals was conducted by virus-neutralization (VN) test and indirect immunofluorescence assay (IFA). Sera of 24 humans, 59 monkeys, 171 wild boars, 233 Sika deer, 7 bears, and 27 nutria in Yamaguchi Prefecture were analyzed by VN test. The positive ratio of humans, monkeys, wild boars, and Sika deer were 20.8%, 3.4%, 33.9% and 4.7%, respectively. No positive samples were detected in bears and nutria. The correlation coefficients between VN test and IFA in human, monkey, wild boar, and Sika deer sera were 0.5745, 0.7198, 0.9967 and 0.9525, respectively. In addition, KAMV was detected in one pool of Haemaphysalis formosensis ticks in Wakayama Prefecture. These results indicated that KAMV or KAMV-like virus is circulating among many wildlife and ticks, and that this virus incidentally infects humans.

Seroepidemiological surveys of tick-borne encephalitis virus and novel tick-borne viruses in wild boar in Nagasaki, Japan.

In Japan, tick-borne viruses such as tick-borne encephalitis virus (TBEV) and severe fever with thrombocytopenia syndrome virus have been identified in humans, animals, and ticks. In addition, novel tick-borne viruses have been isolated from ticks in Japan. This study aimed to determine the seroprevalence of TBEV and novel viruses, particularly Tofla virus (TFLV), Kabuto Mountain virus (KAMV), and Muko virus (MUV) in wild boar in Nagasaki, Japan. Enzyme-linked immunosorbent assays and neutralization tests were performed to detect antibodies against each virus. Wild boar serum tested positive for antibodies against KAMV, TFLV, and TBEV, but not MUV. This study revealed the seroprevalence of newly identified tick-borne viruses and TBEV in animals residing in the Nagasaki area. The seroprevalence of these viruses in sentinel animals may inform policies aimed at preventing tick-borne virus disease outbreaks.

Dry-Heat Tolerance of Egg Sacs of Invasive Latrodectus Spiders (Araneae: Theridiidae) in Japan: Implications for Efficient Control/Extermination.

Strategic responses to invasive Latrodectus widow spiders are a global challenge due to the risks they pose to health and ecosystems. Chemical strategies involving the use of pyrethroids are effective against adult spiders, but as their populations rebound, chemical control becomes costly and unsustainable for eradication. A major obstacle is the inefficacy of insecticides against eggs, which are covered by a protective silk egg sac. Eradication of invasive spiders must focus on destroying progeny. Here, the responses of eggs in egg sacs of two invasive Latrodectus spiders in Japan (Latrodectus hasseltii (Thorell) and Latrodectus geometricus (C.L. Koch)) to short-term dry-heat exposure were examined. To test whether the dry-heat tolerance of the egg sacs of both spider species differed, lethal temperature (LT) was determined based on the hatching rate of eggs from egg sacs subjected to a range of temperatures. Hatching in both species failed completely when the egg sacs were exposed to temperatures of 55°C and above for 10 min, but the LT to reduce hatching by 90% (LT90) differed significantly between L. hasseltii (50. 9°C) and L. geometricus (52. 8°C). Our study highlights the efficacy of dry heat in suppressing hatching and thus shows the possibility for effective extermination of these noxious invasive pests. Further exploration and investigation of the effects of humidity and heat exposure time on egg sacs under field conditions are needed to guide Latrodectus spider control strategies.

Legacy of pre-eruption vegetation affects ground-dwelling arthropod communities after different types of volcanic disturbance.

Volcanic eruptions are one of the largest natural disturbances and are followed by the establishment of novel plant and animal communities in terrestrial ecosystems. However, the role of pre-eruption vegetation in the establishment of arthropod communities after volcanic disturbances is currently unknown. Here, we asked whether the legacy of pre-eruption vegetation mediates the community structure of ground-dwelling arthropods after volcanic disturbances. The 2015 eruption in Kuchinoerabu-jima Island, southwest Japan, caused two types of disturbances [a pyroclastic flow and a lahar (i.e., mudflow)] in three types of forests (broad-leaved, black pine, and cedar). We hypothesized that pre-eruption vegetation would influence the community structure of ground-dwelling arthropods after the disturbance, and we expected that these effects from vegetation would be more prevalent for the less severe disturbances. The total abundance of ground-dwelling arthropods decreased more in the lahar than the pyroclastic flow, and arthropod species composition showed a greater change after the lahar. These findings suggest that the lahar disturbance was more severe than the pyroclastic disturbance. Contrary to expectations, the difference in the arthropod species composition among the vegetation types was greatest after the lahar. After the pyroclastic flow, leaf litter remained to some degree with all the vegetation types. After the lahar disturbance, however, although the litter in the cedar forests remained, the litter disappeared completely from broad-leaved and black pine forests. The disappearance of litter from these two forest types after the lahar may be responsible for the greater difference in arthropod species composition among the vegetation types. This study shows that the legacy effects of pre-eruption vegetation on terrestrial arthropod communities after volcanic disturbance were different depending on the type of disturbance. Focusing on the role of pre-eruption biotic factors would contribute to a better understanding of the recovery processes of terrestrial ecosystems after large natural disturbances.

Severe Fever with Thrombocytopenia Syndrome in Cats and Its Prevalence among Veterinarian Staff Members in Nagasaki, Japan.

In this study, we investigated severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) infection in cats in Nagasaki, Japan. In total, 44 of 133 (33.1%) cats with suspected SFTS were confirmed to be infected with SFTSV. Phylogenetic analyses of SFTSV isolates from cats indicated that the main genotype in Nagasaki was J1 and that unique reassortant strains with J2 (S segment) and unclassified genotypes (M and L segments) were also present. There were no significant differences in virus growth in cell cultures or fatality in SFTSV-infected mice between the SFTSV strains that were isolated from recovered and fatal cat cases. Remarkably, SFTSV RNAs were detected in the swabs from cats, indicating that the body fluids contain SFTSV. To evaluate the risk of SFTSV infection when providing animal care, we further examined the seroprevalence of SFTSV infection in veterinarian staff members; 3 of 71 (4.2%) were seropositive for SFTSV-specific antibodies. Our results provide useful information on the possibility of using cats as sentinel animals and raised concerns of the zoonotic risk of catching SFTSV from animals.

The novel therapeutic target and inhibitory effects of PF-429242 against Zika virus infection.

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus of African origin that is transmitted by Aedes mosquitoes. ZIKV was historically limited to Africa and Asia, where mild cases were reported. However, ZIKV has recently been responsible for major global outbreaks associated with a wide range of neurological complications. Since no antiviral therapy exists for ZIKV, drug discovery research for ZIKV is crucial. Intracellular lipids regulated by sterol regulatory element-binding proteins (SREBPs) are important in flavivirus pathogenesis. PF-429242 has been reported to inhibit the activity of site-1 protease (S1P), which regulates the expression of SREBP target genes. Our primary objective in this study is to elucidate the mechanism of the antiviral activity of PF-429242 against the African genotype (ZIKVMR-766) and Asian genotypes (ZIKV H/PF 2013 and ZIKV PRVABC59) using several primate-derived cell lines. The virus titer was determined via a focus-forming assay; we used flow cytometry to quantify intracellular lipids in ZIKV-infected and mock-treated cells. The PF-429242 molecule effectively suppressed ZIKV infection in neuronal cell lines; T98G, U-87MG, SK-N-SH and primary monocytes cell, indicating that PF-429242 molecule can be used therapeutically. A strong reduction in ZIKV replication was observed at 12 µM and 30 µM in in neuronal cell lines and primary monocytes, respectively. Interestingly, the inhibitory effects of the PF-429242 molecule were observed when it was tested on various ZIKV-lineage infections. Lipid quantification reveals that ZIKV increases lipogenesis in infected cells, while the exogenous addition of cholesterol effectively blocks ZIKV replication. Furthermore, the supplementation of oleic acid increases the ZIKV titer. Fenofibrate, an inhibitor of lipid droplet formation, reduces the ZIKV titer. Collectively, our results demonstrate that the development of antiviral drugs against ZIKV could be based on key regulators of lipid metabolism. In addition, this study reveals that the mechanism of the PF-429242-mediated suppression among flavivirus infections is not entirely identical. Our results warrant further evaluation of PF-429242 as a prospective antiviral drug, given the multiple advantageous properties of this compound, such as its limited toxicity, neuroprotective properties, and broad spectrum of capabilities.