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Rabies virus (RABV) causes fatal neurological disease. Pre-exposure prophylaxis (PrEP) and post-exposure prophylaxis (PEP) using inactivated-virus vaccines are the most effective measures to prevent rabies. In Japan, HEP-Flury, the viral strain, used as a human rabies vaccine, has historically been propagated in primary fibroblast cells derived from chicken embryos. In the present study, to reduce the cost and labor of vaccine production, we sought to adapt the original HEP-Flury (HEP) to Vero cells. HEP was repeatedly passaged in Vero cells to generate ten- (HEP-10V) and thirty-passaged (HEP-30V) strains. Both HEP-10V and HEP-30V grew significantly better than HEP in Vero cells, with virulence and antigenicity similar to HEP. Comparison of the complete genomes with HEP revealed three non-synonymous mutations in HEP-10V and four additional non-synonymous mutations in HEP-30V. Comparison among 18 recombinant HEP strains constructed by reverse genetics and vesicular stomatitis viruses pseudotyped with RABV glycoproteins indicated that the substitution P(L115H) in the phosphoprotein and G(S15R) in the glycoprotein improved viral propagation in HEP-10V, while in HEP-30V, G(V164E), G(L183P), and G(A286V) in the glycoprotein enhanced entry into Vero cells. The obtained recombinant RABV strain, rHEP-PG4 strain, with these five substitutions, is a strong candidate for production of human rabies vaccine. [ABSTRACT FROM AUTHOR]

Two human patients with Macacine alphaherpesvirus 1 infection were identified in Japan in 2019. Both patients had worked at the same company, which had a macaque facility. The rhesus-genotype B virus genome was detected in cerebrospinal fluid samples from both patients. [ABSTRACT FROM AUTHOR]

Background: The annual rabies vaccination coverage in dogs among 47 prefectures in Japan has been reported to range from 42.3% to 92.4%, and the overall coverage has been steadily declining. Given the presence of unregistered dogs and the small number of stray dogs, the true vaccination coverage is likely to be even lower. Methods: We conducted a cross-sectional survey of dog owners to identify the owner characteristics associated with dog rabies vaccination. People in Japan who currently own dogs were recruited and answered a questionnaire consisting of four sections: (i) demographic characteristics, (ii) education history associated with medicine, (iii) factors related to veterinary services, and (iv) dog characteristics. Results: A total of 534 dog owners covering 629 dogs were surveyed. Vaccination within the prior 12 months was the major outcome (56.1%). The associated variables were (a) owner education level, (b) knowledge about mandatory vaccination, (c) having a family veterinary clinic, (d) frequency of visiting a veterinary clinic, and (e) having ever been advised to vaccinate their dog. Conclusions: Although causality cannot be implied, our findings indicate improving owners' knowledge about mandatory vaccination, facilitating attachment to a veterinary clinic, and veterinarians providing vaccination advice might increase the uptake of dog rabies vaccination. The finding in Japan did not deviate from Asian and African countries with rabies, and the sample estimate of annual vaccination coverage was lower than the reported estimate among registered dogs. [ABSTRACT FROM AUTHOR]

Because adult and juvenile eel gobies usually hide within the burrows of muddy substrates, their diversity and life history have not yet been fully elucidated. We investigated larval specimens of the eel gobies collected on Okinawa Island in southern Japan. The genus Trypauchenopsis was previously thought to consist of only one species, but our larval collection identified two species, Trypauchenopsis limicola and Trypauchenopsis intermedia, distinguished by their species‐specific melanophore arrangements and differences in their fin‐ray counts. Taenioides kentalleni were previously known from only two specimens worldwide. A third specimen of this species has now been added from the larval collection. In addition to the three species above, Taenioides gracilis and Caragobius urolepis were identified and the larval morphologies of the five species were described for the first time. All the larvae collected in the present study were at late postflexion stage. T. limicola, T. intermedia and T. gracilis were presumably collected in the estuaries and beaches when approaching their adult habitats at the end of pelagic life. They were 8.5–10.3 mm in standard length, and otolith analysis suggests that their pelagic larval durations are a little longer than 1 month (average 34–37 days). The larval occurrence suggested that the spawning season of T. limicola is May–December, when the water temperature is warmer than approximately 20°C. Our work reveals that studying the larval stage can provide new information on the taxonomy and life history of the elusive cryptobenthic fish. [ABSTRACT FROM AUTHOR]

To investigate the seroprevalence of severe fever with thrombocytopenia syndrome (SFTS) among wild and companion animals on Tsushima Island, Japan, SFTS virus (SFTSV)-specific ELISA and virus-neutralizing tests were conducted on 50 wild boars, 71 Sika deer, 84 dogs, 323 domestic cats, and 6 Tsushima leopard cats. In total, 1 wild boar (1.8%), 2 dogs (2.4%), 7 domestic cats (2.2%), and 1 Tsushima leopard cat (16.7%) were positive for anti-SFTSV antibodies. Among the 11 positive animals, 10 were collected after 2019, and all were found on the southern part of the island. SFTSV, thus far, seems to be circulating within a limited area of Tsushima Island. To protect humans and animals, including endangered Tsushima leopard cats, from SFTSV infection, countermeasures are needed to prevent the spread of SFTSV on Tsushima Island. [ABSTRACT FROM AUTHOR]

Severe fever with the thrombocytopenia syndrome virus (SFTSV) causes fatal disease in humans, cats, and cheetahs. In this study, the information on seven dogs with SFTS was summarized. All dogs showed anorexia, high fever, leukopenia, and thrombocytopenia, two dogs showed vomiting and loose stool, and five dogs had tick parasites. All dogs also had a history of outdoor activity. The SFTSV gene was detected in all dogs. Remarkably, three dogs (43%) died. SFTSV was isolated from six dogs and the complete genomes were determined. A significant increase in anti-SFTSV-IgG antibodies was observed in two dogs after recovery, and anti-SFTSV-IgM antibodies were detected in four dogs in the acute phase. Using an ELISA cut-off value of 0.410 to discriminate between SFTSV-negative and positive dogs, the detection of anti-SFTSV-IgM antibodies was useful for the diagnosis of dogs with acute-phase SFTS. Four out of the ninety-eight SFTSV-negative dogs possessed high anti-SFTSV IgG antibody titers, indicating that some dogs can recover from SFTSV infection. In conclusion, SFTSV is lethal in some dogs, but many dogs recover from SFTSV infection. [ABSTRACT FROM AUTHOR]

Seal parapoxvirus (SPPV) infection has been reported among pinnipeds in aquaria in Japan; however, its seroprevalence is unknown. Therefore, an enzyme-linked immunosorbent assay (ELISA) was developed for serological diagnosis of SPPV infection. The gene encoding the major envelope protein of SPPV was cloned into the eukaryotic expression vector pAcGFP1-N1, which encodes the green fluorescence protein (GFP), thereby producing a fusion protein (Env-GFP). Parental and cloned vector DNA was independently transfected into cultured seal cells for the expression of GFP and Env-GFP. The wells of an ELISA plate were coated with either GFP- or Env-GFP-transfected cell lysates. The light absorbance of each serum sample was adjusted by subtracting the absorbance of GFP-coated wells from that of Env-GFP-coated wells. Sera from two spotted seals (Phoca largha), six beluga whales (Delphinapterus leucas), three Pacific white-sided dolphins (Lagenorhynchus obliquidens), and ten bottlenose dolphins (Tursiops truncatus) from an aquarium in Japan were examined using the ELISA. Positive reactions were not observed, except in one preserved sample collected ten years ago from a naturally SPPV-infected spotted seal. The established ELISA could be useful in screening marine mammal sera for anti-SPPV antibodies. [ABSTRACT FROM AUTHOR]

Adenoviruses have been reported to infect a variety of birds. Here, we isolated a novel adenovirus from the liver of a dead owl chick (Bengal eagle owl; Bubo bengalensis) at a raptor-breeding facility in Japan and determined the complete genome sequence of the virus. We performed necropsies on the dead owl chicks and found that they had enlarged livers, pericardial edema, and focal necrosis of the liver tissue. Transmission electron microscopy of the liver tissue revealed a virus-like structure, appearing as paracrystalline arrays in the nucleus, and immunohistochemical staining with anti-adenovirus antibodies showed positive reactions in hepatocytes and other cells. Attempts to isolate the virus from homogenized liver tissue of a dead owl chick showed a cytopathic effect on chicken-derived cultured cells after multiple blind passages. Further, we determined the complete genome sequence of this virus and performed phylogenetic analysis, revealing that this adenovirus belongs to the genus Aviadenovirus, forming a cluster with fowl and turkey aviadenoviruses. The amino acid sequence divergence between the DNA polymerase of this virus and its closest known adenovirus relative is approximately 29%, implying that this virus can be assigned to a new species in the genus Aviadenovirus. Based on our data, this novel owl adenovirus is a likely cause of fatal infections in owls, which may threaten wild and captive owl populations. Further, this virus is unique among raptor adenoviruses in that it infects chicken-derived cultured cells, raising the importance of further investigations to evaluate interspecies transmission of this virus. [ABSTRACT FROM AUTHOR]

In 2014, an outbreak of Getah virus (GETV) infection occurred in Japan in a horse population that was inoculated with a vaccine against GETV. In this study, we investigated the seroprevalence of GETV infection among wild boars in Japan. Interestingly, the highest rate of anti-GETV-positive wild boars was observed in 2013, which gradually decreased during 2014-2016. The results suggested that GETV spread among wild boars around 2012, resulting in the 2014 outbreak. [ABSTRACT FROM AUTHOR]

Three species of Schismatogobius de Beaufort 1912, distinguished by their morphology and mitochondrial DNA sequences, were found in freshwater streams in the Ryukyu Archipelago, Japan. Although two species were previously known from Japan ( S. roxasi Herre 1936 and S. ampluvinculus Chen, Shao, and Fang 1995), the taxonomy needs to be revised. To identify these species, the holotype morphology of S. marmoratus (Peters 1868), S. bruynisi de Beaufort 1912, and S. roxasi, originally described from the Philippines and Indonesia, were examined and re-described here, because relatively little information about their diagnostic characters was provided in the original descriptions. The three Japanese species were identified as S. ampluvinculus, S. marmoratus, and a new species. They were distinguished from each other and from their congeners by the banding pattern of the body, markings on the pectoral fins, pigment patterns on the ventral surface of the head and pelvic fin, body depth at the pelvic-fin origin, pre-anal length, and pectoral-fin ray counts. Although the new species had been regarded as S. roxasi in previous publications, we show that it is actually not S. roxasi and that it also differs from all other nominal species of Schismatogobius. This is described as a new species, S. ninja. Additionally, this is the first record of S. marmoratus from Japan. [ABSTRACT FROM AUTHOR]

The genus Borrelia comprises arthropod-borne bacteria, which are infectious agents in vertebrates. They are mainly transmitted by ixodid or argasid ticks. In Hokkaido, Japan, Borrelia spp. were found in deer and Haemaphysalis ticks between 2011 and 2013; however, the study was limited to a particular area. Therefore, in the present study, we conducted large-scale surveillance of ticks and wild animals in the western part of the main island of Japan. We collected 6,407 host-seeking ticks from two regions and 1,598 larvae obtained from 32 engorged female ticks and examined them to elucidate transovarial transmission. In addition, we examined whole blood samples from 190 wild boars and 276 sika deer, as well as sera from 120 wild raccoons. We detected Borrelia spp. in Haemaphysalis flava, Haemaphysalis megaspinosa, Haemaphysalis kitaokai, Haemaphysalis longicornis, and Haemaphysalis formosensis. In addition, we isolated a strain from H. megaspinosa using Barbour-Stoenner-Kelly medium. The minimum infection rate of ticks was less than 5%. Transovarial transmission was observed in H. kitaokai. Phylogenetic analysis of the isolated strain and DNA fragments amplified from ticks identified at least four bacterial genotypes, which corresponded to the tick species detected. Bacteria were detected in 8.4%, 15%, and 0.8% of wild boars, sika deer, and raccoons, respectively. In this study, we found seasonal differences in the prevalence of bacterial genotypes in sika deer during the winter and summer. The tick activity season corresponds to the season with a high prevalence of animals. The present study suggests that a particular bacterial genotype detected in this study are defined by a particular tick species in which they are present. [ABSTRACT FROM AUTHOR]

Among the tick-borne orbiviruses (genus Orbivirus, family Reoviridae), 36 serotypes are currently classified within a single virus species, Great Island virus. In this study, we report the first characterization of a tick-borne orbivirus isolated from the tick Ixodes turdus in Japan, which we identified as a new member of the species Great Island virus. The virus isolate, designated Muko virus (MUV), replicated and induced cytopathic effects in BHK-21, Vero E6, and CCL-141 cells and caused high mortality in suckling mice after intracerebral inoculation. Full genome sequence analysis showed that MUV shared the greatest phylogenetic similarity with Tribeč virus in terms of the amino acid sequences of all viral proteins except for outer capsid protein 1 (OC1; VP4 of MUV). Analysis of genome segment 9 in MUV detected an uninterrupted open reading frame that overlaps with VP6 (Hel), which putatively encodes a molecular and functional equivalent of NS4 from Great Island virus. Our study provides new insights into the geographic distribution, genetic diversity, and evolutionary history of the members of the species Great Island virus. [ABSTRACT FROM AUTHOR]

Cattle do not generally appear to develop severe viremia when infected with Japanese encephalitis virus (JEV), and they can be infected without showing clinical signs. However, two cattle in Japan recently died from JEV infection. In this study, we investigated the presence of different species of mosquitoes and flavivirus in a cowshed in the southwest region of Japan. In this cowshed, the two most common species of mosquitoes collected were Culex tritaeniorhynchus (including Culex pseudovishnui) and Anopheles sinensis. We performed virus isolation from the collected mosquitoes and obtained two flaviviruses: JEV and a novel insect-specific flavivirus, tentatively designated Yamadai flavivirus (YDFV). Phylogenetic analysis revealed that all three JEV isolates belonged to JEV genotype I and were closely related to a JEV strain that was isolated from the brains of cattle exhibiting neurological symptoms in Japan. Genetic characterization of YDFV revealed that the full genome RNA (10,863 nucleotides) showed homology with the Culex-associated insect-specific flaviviruses Quang Binh virus (79 % identity) and Yunnan Culex flavivirus (78 % identity), indicating that YDFV is a novel insect-specific flavivirus. [ABSTRACT FROM AUTHOR]

Egg masses of Luciogobius ryukyuensis were found in spawning grounds around the lowest reach of the adult's habitat in the tidally influenced area of streams on Okinawa Island. The eggs were attached to the underside of stones and were cared for by a solitary male fish. The number of eggs within an egg mass was 191-1368. The eggs were elliptical, measuring 2.0-2.6 mm in length, and 0.6-0.8 mm in width. Early development of L. ryukyuensis was described from laboratory-reared specimens. Newly hatched larvae, measuring 2.8-3.3 mm in body length, had an open mouth, pigmented eyes, pectoral fin buds, an orange-colored yolk sac, and characteristic melanophores along the dorsal and ventral midlines of the body. The yolk was completely absorbed at days 2-3. Notochord flexion began at day 10 and was completed at day 15. The fish started settling on the bottom of the tank at day 34 (14.1 mm in body length) when the body surface started to be covered by intense pigmentation. The eggs and newly hatched larvae of L. ryukyuensis were of standard size of the genus and their morphologies closely resemble those of L. guttatus. [ABSTRACT FROM AUTHOR]

The article presents a study which performed sequence analysis and molecular characterization of parapoxvirus on nodular lesions in a Phoca Largha seal in Japan. The purpose was to clarify the genetic relationship between parapoxviruses in both pinnipeds and cetaceans in Japan and other countries. Deoxyribonucleic acid (DNA) was extracted from the lesions to detect orthopoxvirus and parapoxvirus DNA using the polymerase chain reaction (PCR) method. It showed how the partial nucleotide sequence of the envelope gene determined from the PCR product were closely related to 2 parapoxvirus strains from Alaskan spotted seals. Since virus-neutralizing (VN) antibody against canine distemper virus (CDV) was not detected, it concludes that the lesions were infections caused by pinniped parapoxvirus.

The article discusses results of a study that determines the full genome sequence of the equine isolate of Japanese encephalitis virus (JEV) strain. It notes that there has been only one reported case of equine Japanese encephalitis for the past 25 years. Results of the sequence analysis showed that the JEV genome is similar to that of the dominant JEV genotype circulating in Japan. The neurovirulence of genotype I was high as it was for genotype III. The study indicated the need and importance of continuous vaccination of horses against JEV in Japan.

The morphology of Eleotris acanthopoma collected from Moorea in French Polynesia is described. This is the first record of this species from French Polynesia, greatly expanding the known range, which was previously only considered to extend from southern Japan to New Caledonia. Nucleotide sequences of the mitochondrial ND5 gene of several Eleotris species and related genera indicate that E. acanthopoma from Moorea belongs to the same lineage as E. acanthopoma from Japan and the Philippines. Despite being separated by a distance of approximately 10,000 km, two of the specimens from Moorea and one from the Philippines had identical nucleotide sequences. Results of this study indicate that extensive dispersal occurs during the pelagic larval stage of this species. [ABSTRACT FROM AUTHOR]

Although raccoons (Procyon Iotor) are susceptible to influenza viruses, highly pathogenic avian influenza virus (H5N1) infection in these animals has not been reported. We performed a serosurvey of apparently healthy feral raccoons in Japan and found specific antibodies to subtype H5N1 viruses. Feral raccoons may pose a risk to farms and public health. [ABSTRACT FROM AUTHOR]

Drifting fish larvae were collected with a plankton net in the lower reaches of a freshwater area of the Teima Stream, Okinawa Island, Japan, during 24 hr periods each month from June 1998 to October 1999 (except July 1998). Newly hatched larvae of several gobioid and two pipefish species were collected, and their morphology was described. The larval occurrences suggested that most species spawn mainly from spring to fall, with some Rhinogobius species spawning in winter. Larvae of all fishes occurred predominantly during hours after dusk throughout the year. It is suggested that larvae of amphidromous fishes spawned in freshwater streams on Okinawa Island begin to drift soon after hatching at dusk and complete their exit from freshwater areas into the estuary and sea by midnight. [ABSTRACT FROM AUTHOR]

The article presents a study on the detection of antibodies in raccoons, raccoon dogs, and wild boars against Japanese Encephalitis Virus (JEV) in Japan. It states that JEV is transmitted to animal species including humans, horses and pigs through Culex tritaeniorhynchus and causes acute infection on the central nervous systems resulting to encephalitis. It adds that the results from the examined wild animals between males and females or between adults and juveniles showed no significant differences of JEV seroprevalence. It also adds that wild boars were more seropositive than raccoons for JEV antibody. The study concludes that JEV was more common in wild mammals which indicates that JEV infection in humans in Japan could still be possibly endemic.

Reproductive biology and early development of two species of sleepers, Eleotris acanthopoma Bleeker, 1853, and E. fusca (Forster, 1801), were investigated in streams on Okinawa Island in southern Japan. Gonadal examination and morphology of the genital papillae indicated that E. acanthopoma matured at a smaller body size (ca. 28 mm in standard length) than E. fusca (ca. 50 mm). Mature ovaries were composed of oocytes that could be categorized into two size classes. Larger females of both species had several hundred thousand developed oocytes in the larger size class and may spawn them at one or several consecutive spawning events. Egg masses of both species were found in habitats typically occupied by adults and were deposited, often sparsely, on the underside of objects. Form of the egg masses and morphology of eggs and newly hatched larvae of both species were almost identical. Eggs were a nearly spherical pyriform in shape, with the widest diameters measuring approximately 0.4 mm. Newly hatched larvae were very small (1.0-1.4 mm in notochord length) and undeveloped. The mouth opened and the eyes became pigmented 3 days after hatching, and all of their yolk was consumed 4 days after hatching. Reproductive strategies of both species were characterized by high fecundity through production of small eggs and small newly hatched larvae, with high fecundity likely to mitigate the presumed increased risk associated with widespread larval dispersal. [ABSTRACT FROM AUTHOR]

Jingmen tick virus (JMTV) and the related jingmenvirus-termed Alongshan virus are recognized as globally emerging human pathogenic tick-borne viruses. These viruses have been detected in various mammals and invertebrates, although their natural transmission cycles remain unknown. JMTV and a novel jingmenvirus, tentatively named Takachi virus (TAKV), have now been identified during a surveillance of tick-borne viruses in Japan. JMTV was shown to be distributed across extensive areas of Japan and has been detected repeatedly at the same collection sites over several years, suggesting viral circulation in natural transmission cycles in these areas. Interestingly, these jingmenviruses may exist in a host tick species-specific manner. Vertical transmission of the virus in host ticks in nature was also indicated by the presence of JMTV in unfed host-questing Amblyomma testudinarium larvae. Further epidemiological surveillance and etiological studies are necessary to assess the status and risk of jingmenvirus infection in Japan. [ABSTRACT FROM AUTHOR]

Severe fever with thrombocytopenia syndrome virus (SFTSV) is the causative agent of SFTS, an emerging tick-borne disease in East Asia, and is maintained in enzootic cycles involving ticks and a range of wild animal hosts. Direct transmission of SFTSV from cats and dogs to humans has been identified in Japan, suggesting that veterinarians and veterinary nurses involved in small-animal practice are at occupational risk of SFTSV infection. To characterize this risk, we performed a sero-epidemiological survey in small-animal-practice workers and healthy blood donors in Miyazaki prefecture, which is the prefecture with the highest per capita number of recorded cases of SFTS in Japan. Three small-animal-practice workers were identified as seropositive by ELISA, but one had a negative neutralization-test result and so was finally determined to be seronegative, giving a seropositive rate of 2.2% (2 of 90), which was significantly higher than that in healthy blood donors (0%, 0 of 1000; p < 0.05). The seroprevalence identified here in small-animal-practice workers was slightly higher than that previously reported in other high-risk workers engaged in agriculture and forestry in Japan. Thus, enhancement of small-animal-practice workers' awareness of biosafety at animal hospitals is necessary for control of SFTSV. [ABSTRACT FROM AUTHOR]

A woman in her fifties showed symptoms of fever, loss of appetite, vomiting, and general fatigue 2 days after she was bitten by a sick cat, which had later died, in Yamaguchi prefecture, western Japan, in June 2016. She subsequently died of multiorgan failure, and an autopsy was performed to determine the cause of death. However, the etiological pathogens were not quickly identified. The pathological features of the patient were retrospectively re-examined, and the pathology of the regional lymph node at the site of the cat bite was found to show necrotizing lymphadenitis with hemophagocytosis. The pathological features were noted to be similar to those of patients reported to have severe fever with thrombocytopenia syndrome (SFTS). Therefore, the lymph node section was retrospectively tested immunohistochemically, revealing the presence of the SFTS virus (SFTSV) antigen. The sick cat showed similar symptoms and laboratory findings similar to those shown in human SFTS cases. The patient had no history of tick bites, and did not have skin lesions suggestive of these. She had not undertaken any outdoor activities. It is highly possible that the patient was infected with SFTSV through the sick cat's bite. If a patient gets sick in an SFTS-endemic region after being bitten by a cat, SFTS should be considered in the differential diagnosis. [ABSTRACT FROM AUTHOR]

The article focuses on the seroprevalence of pseudorabies virus (PRV) in wild boars in Japan. PRV is known to be the causative agent for the epidemic disease Aujeszky's disease that causes high mortality in newborn piglets and abortion in pregnant sows. In the virus neutralization (VN) test performed by the authors, it was disclosed that 6 came out as positive for VN antibody out of the 173 serum samples. It concluded that as PRV continues to affect Japan, caution is needed due to possible transmission from wild boars to pigs.

Because serosurveys of Japanese encephalitis virus (JEV) among wild animals and pigs may not accurately reflect risk for humans in urban/residential areas, we examined seroprevalence among dogs and cats. We found that JEV-infected mosquitoes have spread throughout Japan and that dogs, but not cats, might be good sentinels for monitoring JEV infection in urban/residential areas. [ABSTRACT FROM AUTHOR]

The article presents a study on canine distemper virus (CDV) infection in Procyon lotor or wild raccoons in Japan. It says that CDV is the cause of lethal disease in the members of the order Carnivora in which raccoons are conceived to be reservoirs of CDV to wild and zoo animals. The study used 106 raccoon sera from prefectures of Hyogo and Osaka which were analyzed. The analysis showed that 32.1% of the raccoons had a virus-neutralizing (VN) antibody; however, it states that there is no significant difference in CDV seroprevalence with regards to places, gender and body weights. The study concludes that CDV is an epidemic within the feral raccoons in Japan and shows that it might have spread in the wild animals of the country.

Francisella tularensis (F. tularensis) is the etiological agent of the zoonotic disease tularemia. F. tularensis subspecies holarctica biovar japonica has rarely been isolated in Japan and is considered to have moderate virulence, although the biological properties of fresh isolates have not been analyzed in detail. Here, we analyzed the virulence of two strains of F. tularensis subspecies holarctica biovar japonica (NVF1 and KU-1) and their phenotypic stability during serial passages in Eugon chocolate agar (ECA) and Chamberlain's chemically defined medium (CDM) based agar (CDMA). C57BL/6 mice intradermally inoculated with 101 colony-forming units of NVF1 or KU-1 died within 9 days, with a median time to death of 7.5 and 7 days, respectively. Both NVF1 and KU-1 strains passaged on ECA 10 times had comparable virulence prior to passaging, whereas strains passaged on ECA 20 times and on CDMA 50 times were attenuated. Attenuated strains had decreased viability in 0.01% H2O2 and lower intracellular growth rates, suggesting both properties are important for F. tularensis virulence. Additionally, passage on ECA of the KU-1 strains altered lipopolysaccharide antigenicity and bacterial susceptibility to β-lactam antibiotics. Our data demonstrate F. tularensis strain virulence in Japan and contribute to understanding phenotypic differences between natural and laboratory environments. [ABSTRACT FROM AUTHOR]

In Japan, indigenous tick-borne phleboviruses (TBPVs) and their associated diseases first became evident in 2013 by reported human cases of severe fever with thrombocytopenia syndrome (SFTS). In this study, we report a novel member of the genus Phlebovirus designated as Kabuto Mountain virus (KAMV), which was isolated from the ixodid tick Haemaphysalis flava in Hyogo, Japan. A complete viral genome sequencing and phylogenetic analyses showed that KAMV is a novel member of TBPVs, which is closely related to the Uukuniemi and Kaisodi group viruses. However, unlike the Uukuniemi group viruses, the 165-nt intergenic region (IGR) in the KAMV S segment was highly C-rich in the genomic sense and not predicted to form a secondary structure, which are rather similar to those of the Kaisodi group viruses and most mosquito/sandfly-borne phleboviruses. Furthermore, the NSs protein of KAMV was highly divergent from those of other TBPVs. These results provided further insights into the genetic diversity and evolutionary relationships of TBPVs. KAMV could infect and replicate in some rodent and primate cell lines. We evaluated the infectivity and pathogenicity of KAMV in suckling mice, where we obtained a virulent strain after two passages via intracerebral inoculation. This is the first report showing the existence of a previously unrecognized TBPV in Japan, other than the SFTS virus. [ABSTRACT FROM AUTHOR]

During the course of tick-borne virus surveillance in Japan, three independent isolates of probably the same virus were obtained from three geographically distant populations of the hard tick Haemaphysalis flava . Genome analyses of the three isolates demonstrated that they were closely related but distinct strains of a novel virus, designated Tarumizu tick virus (TarTV), which has a genome of 12 double-stranded RNA segments. The development of the virus-induced cytopathic effects on BHK cells significantly varied according to virus strains. Ten out of 12 segments of TarTV appeared to encode putative orthologs or functional equivalents of viral proteins of Colorado tick fever virus (CTFV) and Eyach virus, suggesting that TarTV is the third member of the genus Coltivirus in the family Reoviridae . This was supported by the facts that the 5′- and 3′-terminal consensus sequences of coltivirus genomes were found also in TarTV genome, and segment 9 of TarTV had sequence and structural features that may mediate a stop codon read-through as observed in that of CTFV. However, segment 7 and 10 of TarTV had no significant sequence similarities to any other proteins of known coltiviruses. Electron microscopic analysis demonstrated that TarTV particle had a non-enveloped bilayer icosahedral structure, and viral inclusion bodies were formed in infected cells. TarTV could infect and replicate in several mammalian cell lines tested, but show no clinical symptoms in intracerebrally inoculated mice. Taken together, our findings provide new insights into genetic diversity and evolution of the genus Coltivirus . [ABSTRACT FROM AUTHOR]

A novel rhabdovirus was isolated from the serum of a healthy Japanese wild boar ( Sus scrofa leucomystax ) and identified using the rapid determination system for viral nucleic acid sequences (RDV), next-generation sequencing, and electron microscopy. The virus was tentatively named wild boar rhabdovirus 1 (WBRV1). Phylogenetic analysis of the entire genome sequence indicated that WBRV1 is closely related to Tupaia rhabdovirus (TRV), which was isolated from cultured cells of hepatocellular carcinoma tissue of tree shrew. TRV has not been assigned to any genus of Rhabdoviridae till date. Analysis of the L gene indicated that WBRV1 belongs to the genus Vesiculovirus. These observations suggest that both TRV and WBRV1 belong to a new genus of Rhabdoviridae. Next-generation genome sequencing of WBRV1 revealed 5 open reading frames of 1329, 765, 627, 1629, and 6336 bases in length. The WBRV1 gene sequences are similar to those of other rhabdoviruses. Epizootiological analysis of a population of wild boars in Wakayama prefecture in Japan indicated that 6.5% were positive for the WBRV1 gene and 52% were positive for WBRV1-neutralizing antibodies. Furthermore, such viral neutralizing antibodies were found in domestic pigs in another prefecture. WBRV1 was inoculated intranasally and intraperitoneally into SCID and BALB/c mice and viral RNA was detected in SCID mice, suggesting that WBRV1 can replicate in immunocompromised mice. These results indicate this novel virus is endemic in wild animals and livestock in Japan. [ABSTRACT FROM AUTHOR]

Abstract: In the spring of 2007, seven raccoon dogs and a weasel were captured near the city of Tanabe in Wakayama prefecture, Japan. The causative agent of the animals’ death 1–2days after capture was identified as canine distemper virus (CDV) by virus isolation, immunostaining with an anti-CDV polyclonal antibody, and a commercially available CDV antigen-detection kit. Sequence analysis of hemagglutinin genes indicated the isolated viruses belong to genotype Asia-1 and possess the substitution from tyrosine (Y) to histidine (H) at position 549 that is associated with the spread of CDV to non-canine hosts. A serosurvey for CDV was then conducted among wild animals in the region. The animals assayed consisted of 104 raccoons, 41 wild boars, 19 raccoon dogs, five Sika deer, two badgers, one weasel, one marten, one Siberian weasel and one fox. Virus-neutralization (VN) tests showed that, except for fox and weasel, all of the species assayed had VN antibodies to CDV. Interestingly, 11 of the 41 wild boars (27%) and two of the five Sika deer assayed possessed VN antibodies to CDV. These findings indicate that CDV infection was widespread among wild mammals during this epizootic. [Copyright &y& Elsevier]

As a part of risk analysis for consumption of meat from wild animals, the prevalence of Campylobacter spp. in wild deer and boar in Japan was investigated. C. hyointestinalis subsp. hyointestinalis (C. hyointestinalis) was isolated from 2.8% (7/253) of the wild deer and 22.1% (71/321) of the wild boar examined. All 23 wild deer isolates and 141 (72.7%) wild boar isolates carried both chcdt - I and chcdt-II genes. The remaining 53 (27.3%) wild boar isolates had only the chcdt - II gene. By whole-genome sequence analysis, we detected 38–40 virulence- and survival-associated genes (motility, chemotactic, adhesion, invasion, toxin, glycosylation, iron uptake, drug resistance, and stress response), which had been identified in C. jejuni and C. coli. In conclusion, our study highlights C. hyointestinalis as a possible cause of food-borne disease in humans and emphasizes the importance of food hygiene in the processing of wild meats for human consumption. • Campylobacter hyointestinalis was more prevalent in wild boar (22.1%) than in deer (2.8%). • Whole genome sequencing detected 40 pathogenicity-related genes in the strains. • C. jejuni and C. coli were not isolated from any wild animals. • C. hyointestinalis should be reconsidered as a risk when consuming wild game meats. [ABSTRACT FROM AUTHOR]

• Rabbit hepatitis E virus (HEV) circulates among feral rabbits in Japan. • Persistent HEV infection was observed in a rabbit experimentally infected with the HEV isolate JP-59. • Further studies are required to determine the zoonotic risk of rabbit HEV. Rabbit hepatitis E virus (HEV) has been detected among rabbits and recently isolated from immunocompromised patients, suggesting zoonotic transmission. In this study, HEV infection among feral rabbits (Oryctolagus cuniculus) was assessed by detection of anti-HEV antibodies and HEV RNA. The prevalence of anti-HEV antibodies in sera was of 33 % (20/60) and HEV RNA was detected from only one of fecal swabs (1.7 %, 1/58). Furthermore, one naïve rabbit was intravenously inoculated with the suspension of the HEV-positive fecal specimen, exhibiting persistent HEV shedding in feces, intermittent viremia, seroconversion to anti-HEV IgM and IgG, and high alanine aminotransferase (ALT) values, indicating persistent HEV infection. The isolate JP-59 had a length of 7,282 bp excluding a poly (A) tail and possessed the characteristic 93 bp-insertion in ORF1. Phylogenetic analysis indicated that JP-59 formed a cluster with other rabbit HEV isolates from rabbits and human origin. The JP-59 shared the nucleotide sequence identities less than 87 % with other rabbit HEVs, suggesting that a novel rabbit HEV strain was circulating in Japan. [ABSTRACT FROM AUTHOR]

• Nycteribia spp. are potential vectors of Bartonella among Miniopterus fuliginosus. • Bartonella prevalence in Nycteribia and Penicilidia bat flies is 31.7 % (89/281). • The bat flies harbored three species of Bartonella. • One is common to the host bats and the bat flies. • The others may be bat fly-specific Bartonella. We examined Bartonella prevalence in 281 bat flies collected from 114 eastern bent-wing bats (Miniopterus fuliginosus) in Japan and phylogenetically analyzed with other bat fly and bat strains. The bat flies were identified as Penicilidia jenynsii (PJ; n = 45), Nycteribia allotopa (NA; n = 157), and novel Nycteribia species (NS; n = 79). Bartonella DNAs were detected in 31.7 % (89/281) of bat flies by PCR targeting the citrate synthase (gltA) gene. The prevalence of Bartonella DNA among the bat flies was 47.1 % (74/157) in NA, 15.2 % (12/79) in NS, and 6.7 % (3/45) in PJ. Bartonella bacteria were also isolated from two NA and one NS. A phylogenetic analysis of the gltA sequences revealed that bat fly-associated strains were classified into three lineages and the same lineages of Bartonella were commonly detected from both Nycteribia bat flies and Miniopterus bats. These results suggest that Nycteribia bat flies are potential vectors for transmitting Bartonella among Miniopterus bats. [ABSTRACT FROM AUTHOR]

The sika deer (Cervus nippon) is one of the most common species of wildlife in Japan. This study aimed to reveal the prevalence of tick-borne protozoan parasites in wild sika deer living in western Japan. We used nested polymerase chain reaction (PCR) to detect the 18S rRNA gene of tick-borne apicomplexan parasites (Babesia , Theileria, and Hepatozoon spp.) from 276 blood and liver samples from sika deer captured in the Yamaguchi, Oita, Kagoshima, Okayama, Ehime, Kochi, and Tokushima Prefectures. In total, 259 samples (259/276; 93.8%) tested positive in the nested PCR screening. Gene sequencing revealed that 99.6% (258/259) of positive samples contained Theileria sp. (sika 1), while Theileria sp. (sika 2), another Theileria species, was detected in only 3 samples. We also found that one sample from a sika deer captured in Kagoshima contained the gene of an unidentified Babesia sp. related to Babesia sp. Kh-Hj42, which was previously collected from tick in western Siberia. In conclusion, we found a high prevalence of piroplasms in sika deer from western Japan, and DNA analysis revealed that Theileria sp. (sika 1) had the highest infection rate. [ABSTRACT FROM AUTHOR]

• SFTSV infection in cats was characterized by an acute onset and high mortality. • Cats infected with SFTSV showed fever, icterus, thrombocytopenia, and leukopenia frequently. • Outdoor cats were commonly at risk of SFTSV infection in the endemic area. • The almost cat SFTSV was genetically close to human SFTSV. • SFTS in cats is a public health issue, affecting pet owners and animal hospitals. Severe fever with thrombocytopenia syndrome (SFTS) is a recently discovered emerging infectious disease. A zoonotic disease with a high fatality rate in human beings, clinical information on SFTS virus (SFTSV) infection in animals is important. Since 2017, we have diagnosed 24 client-owned cats living in western Japan with SFTS, by genetic and serological testing. In this study, we characterized the clinical features of SFTS in cats and their associated risk factors, by evaluating the clinical parameters retrospectively. A phylogenetic analysis on SFTSV was also conducted. There were no obvious tendencies in age or sex, outdoor cats were commonly at risk of SFTSV infection. All infected cats showed acute onset of clinical signs including anorexia and lethargy, while 68.2% of the cats showed fever and 41.7% showed vomiting. The case fatality rate was 62.5%. Thrombocytopenia, leukopenia, and elevated serum total bilirubin, serum amyloid A, and creatinine phosphokinase concentration were the characteristic findings in the first clinical blood examination. Phylogenic analysis revealed that regional clustered viruses infect both humans and cats. For pet owners and animal hospitals, SFTS in small animals could be an important public health issue. [ABSTRACT FROM AUTHOR]

Migratory birds carry ticks harboring various pathogens, including the zoonotic Yezo virus. In Hokkaido, Japan, we collected ticks from migratory birds during 2020-2021. Eight of 385 pools, comprising 2,534 ticks, tested positive for Yezo virus RNA, suggesting Yezo virus might be spread through the flyways of migratory birds.

Francisella tularensis is an intracellular gram-negative bacterium known as the causative agent of tularemia, which can be transmitted to humans by direct contact with wild animals or by tick bites. Although F. tularensis is highly pathogenic, its recent prevalence in Japan is underreported due to the small number of reported cases. To clarify the current situation of F. tularensis in wild animals, we conducted surveillance on various species of wild animals in Yamaguchi prefecture. In this study, we screened 809 samples collected from 90 Japanese black bears, 105 Japanese monkeys, 168 sika deer, 205 wild boars, and 84 bats. For seroprevalence analysis, we tested 177 serum samples from 75 black bears and 102 monkeys using the microagglutination test. The results showed that serums from five black bears exhibited slight agglutination. Western blot was performed as a confirmatory test on these five samples, but no positive signals were detected. Additionally, molecular surveillance was conducted using DNA extracted from 464 whole blood and 168 tissues, targeting the gene encoding 23 KDa hypothetical protein by real-time PCR and outer membrane protein A gene by conventional PCR. No positive samples of F. tularensis were detected by either real-time or conventional PCR. Although we did not detect any F. tularensis-positive samples through serological and molecular analyses, continuous surveillance studies are necessary since sporadic human cases have been reported in Japan., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

In recent years, bedside ultrasound examinations have been used in many clinical departments and are called point-of-care ultrasound (POCUS). Regarding POCUS in the cardiac field, a protocol called focus (focused) cardiac ultrasound (FoCUS) has been developed in Europe and the United States, is being used clinically, and an educational syllabus has been created. According to them, FoCUS is defined as a point-of-care cardiac ultrasound examination using standardized limited sections and protocols. FoCUS is primarily intended to be performed by non-cardiologists, and in order to avoid making mistakes in judgment, it is important to be familiar with its limitations and it is necessary to understand pathological conditions that can only be diagnosed using conventional comprehensive echocardiography. The Japanese Society of Echocardiography has edited this clinical guideline because we believe that FoCUS should be used effectively and appropriately in Japan, and that appropriate education is essential to popularize FoCUS in Japan. Furthermore, lung POCUS has recently come into clinical use. Lung POCUS is useful for the diagnosis and follow-up of heart failure when used in conjunction with FoCUS, and is especially useful in primary care where chest X-rays are not available. The working group that created this manual agreed that it is desirable to educate patients about lung POCUS in conjunction with FoCUS, so we decided to include the basic techniques of lung POCUS and how to use them in this manuscript., (© 2024. Japanese Society of Echocardiography.)

During their blood-feeding process, ticks are known to transmit various viruses to vertebrates, including humans. Recent viral metagenomic analyses using next-generation sequencing (NGS) have revealed that blood-feeding arthropods like ticks harbor a large diversity of viruses. However, many of these viruses have not been isolated or cultured, and their basic characteristics remain unknown. This study aimed to present the identification of a difficult-to-culture virus in ticks using NGS and to understand its epidemic dynamics using molecular biology techniques. During routine tick-borne virus surveillance in Japan, an unknown flaviviral sequence was detected via virome analysis of host-questing ticks. Similar viral sequences have been detected in the sera of sika deer and wild boars in Japan, and this virus was tentatively named the Saruyama virus (SAYAV). Because SAYAV did not propagate in any cultured cells tested, single-round infectious virus particles (SRIP) were generated based on its structural protein gene sequence utilizing a yellow fever virus-based replicon system to understand its nationwide endemic status. Seroepidemiological studies using SRIP as antigens have demonstrated the presence of neutralizing antibodies against SAYAV in sika deer and wild boar captured at several locations in Japan, suggesting that SAYAV is endemic throughout Japan. Phylogenetic analyses have revealed that SAYAV forms a sister clade with the Orthoflavivirus genus, which includes important mosquito- and tick-borne pathogenic viruses. This shows that SAYAV evolved into a lineage independent of the known orthoflaviviruses. This study demonstrates a unique approach for understanding the epidemiology of uncultured viruses by combining viral metagenomics and pseudoinfectious viral particles., Competing Interests: Competing interests statement:The authors declare no competing interest.

Luciogobius griseus n. sp., belonging to the Luciogobius platycephalus complex, is described on the basis of 40 specimens from the Nansei Islands, southwestern Japan (subtropical area). The new species is generally found in intertidal gravel sediments subjected to freshwater runoff from springs on coastal lines or river mouths and is characterized by the following combination of characters: total second dorsal-fin rays 912 (modally 11); total anal-fin rays usually 1214 (modally 13); pectoral-fin rays 1215 (modally 13); vertebrae 17 or 18 + 23 or 24 = 4042 (18 + 23 = 41); uppermost 24 (23) rays on pectoral fin free; 812 pectoral-fin rays branched (uppermost free rays and sometimes lowermost ray unbranched); pectoral-fin membrane not strongly concave anteriorly (except for free rays); pelvic fins united, forming a disk; head relatively short, 13.920.8% of standard length (SL); relatively short pre-pelvic fin, length 14.422.1% of SL; relatively long pre-dorsal fin, length 68.972.9% of SL; relatively long pre-anal fin, length 63.567.7% of SL; relatively short pelvic fin, length 2.84.7% of SL; distance between posterior end of pelvic fin and anus relatively long, 32.036.4% of SL (aforementioned morphometrics each distinguishing L. griseus n. sp. from other species in the L. platycephalus complex); and fresh specimens with greenish dark brown or gray body. A key to the L. platycephalus complex is provided, together with limited descriptions and remarks on the other two members of the complex.

Getah virus (GETV), an arthropod-borne virus transmitted by mosquitoes, has been isolated from several animals. GETV infection in horses shows clinical signs such as fever, rash, and edema in the leg. Noma horses are one of the eight Japanese native horses. The present study aimed to clarify the occurrence of GETV infection in Noma horses. Serum samples collected from Noma horses were analyzed using a virus neutralization test and enzyme-linked immunosorbent assay and showed that the anti-GETV antibody titers in the samples collected in 2017 were significantly higher than those collected in 2012. We concluded that a seroconversion of anti-GETV antibodies was occurred in the Noma horse population around 2012, providing evidence of the GETV epidemic in Japan circa 2012.

In Japan, hepatitis E virus (HEV) causes hepatitis in humans through the consumption of raw or undercooked meat, including game meat. In the present study, nationwide surveillance of HEV infection among a total of 5,557 wild animals, including 15 species, was conducted in Japan. The prevalence of anti-HEV antibodies in wild boar was 12.4%, with higher positive rates in big boars (over 50 kg, 18.4%) than in small individuals (less than 30 kg, 5.3%). Furthermore, HEV RNA was more frequently detected in piglets than in older boars. Interestingly, the detection of HEV among wildlife by ELISA and RT-PCR suggested that HEV infection in Sika deer was a very rare event, and that there was no HEV infection among wild animals except for wild boar, Sika deer and Japanese monkeys. In conclusion, wild boar, especially piglets, are at high risk of HEV infection, while other wild animals showed less risk or no risk of HEV transmission.

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.

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.

The prevalence of Shiga toxin-producing Escherichia coli O157 (STEC O157) strains in wild deer and boar in Japan was investigated. STEC O157 strains were isolated from 1.9% (9/474) of the wild deer and 0.7% (3/426) of the wild boar examined. Pulsed-field gel electrophoresis (PFGE) analysis classified the wild deer and boar strains into five and three PFGE patterns, respectively. The PFGE pattern of one wild boar strain was similar to that of a cattle strain that had been isolated from a farm in the same area the wild boar was caught, suggesting that a STEC O157 strain may have been transmitted between wild boar and cattle. Clade analysis indicated that, although most of the strains were classified in clade 12, two strains were classified in clade 7. Whole-genome sequence (WGS) analysis indicated that all the strains carried mdfA, a drug resistance gene for macrolide antibiotics, and also pathogenicity-related genes similar to those in the Sakai strain. In conclusion, our study emphasized the importance of food hygiene in processing meat from Japanese wild animals for human consumption.

We investigated the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among dogs in the Tokyo area via enzyme-linked immunosorbent assay (ELISA) using the spike protein as the target antigen. Plasma samples from 494 household dogs and blood-donor dogs were tested from July 2020 to January 2021. Of these samples, three showed optical densities that were higher than the mean plus two standard deviations of the mean of the negative-control optical densities (ODs). Of these three samples, only the sample with the highest OD by ELISA was confirmed positive by virus neutralization testing. The positive dog presented no SARS-CoV-2-related symptoms. The positivity rate of SARS-CoV-2 infections among dogs in the Tokyo area was approximately 0.2%.