Replication of single viruses across the kingdoms, Fungi, Plantae, and Animalia.

It is extremely rare that a single virus crosses host barriers across multiple kingdoms. Based on phylogenetic and paleovirological analyses, it has previously been hypothesized that single members of the family Partitiviridae could cross multiple kingdoms. Partitiviridae accommodates members characterized by their simple bisegmented double-stranded RNA genome; asymptomatic infections of host organisms; the absence of an extracellular route for entry in nature; and collectively broad host range. Herein, we show the replicability of single fungal partitiviruses in three kingdoms of host organisms: Fungi, Plantae, and Animalia. Betapartitiviruses of the phytopathogenic fungusRosellinia necatrix could replicate in protoplasts of the carrot (Daucus carota), Nicotiana benthamiana and Nicotiana tabacum, in some cases reaching a level detectable by agarose gel electrophoresis. Moreover, betapartitiviruses showed more robust replication than the tested alphapartitiviruses. One of the fungal betapartitiviruses, RnPV18, could persistently and stably infect carrot plants regenerated from virion-transfected protoplasts. Both alpha- and betapartitiviruses, although with different host preference, could replicate in two insect cell lines derived from the fall armyworm Spodoptera frugiperda and the fruit fly Drosophila melanogaster. Our results indicate the replicability of single partitiviruses in members of three kingdoms and provide insights into virus adaptation, host jumping, and evolution.

Identification and epidemiological study of an uncultured flavivirus from ticks using viral metagenomics and pseudoinfectious viral particles.

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.

Development of an entirely plasmid-based reverse genetics system for 12-segmented double-stranded RNA viruses.

The family Reoviridae is a nonenveloped virus group with a double-stranded (ds) RNA genome comprising 9 to 12 segments. In the family Reoviridae, the genera Cardoreovirus, Phytoreovirus, Seadornavirus, Mycoreovirus, and Coltivirus contain virus species having 12-segmented dsRNA genomes. Reverse genetics systems used to generate recombinant infectious viruses are powerful tools for investigating viral gene function and for developing vaccines and therapeutic interventions. Generally, this methodology has been utilized for Reoviridae viruses such as Orthoreovirus, Orbivirus, Cypovirus, and Rotavirus, which have genomes with 10 or 11 segments, respectively. However, no reverse genetics system has been developed for Reoviridae viruses with a genome harboring 12 segments. Herein, we describe development of an entire plasmid-based reverse genetics system for Tarumizu tick virus (TarTV) (genus Coltivirus, family Reoviridae), which has a genome of 12 segments. Recombinant TarTVs were generated by transfection of 12 cloned complementary DNAs encoding the TarTV genome into baby hamster kidney cells expressing T7 RNA polymerase. Using this technology, we generated VP12 mutant viruses and demonstrated that VP12 is an N-glycosylated protein. We also generated a reporter virus expressing the HiBiT-tagged VP8 protein. This reverse genetics system will increase our understanding of not only the biology of the genus Coltivirus but also the replication machinery of the family Reoviridae.

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.

Histopathological Characterization of Cases of Spontaneous Fatal Feline Severe Fever with Thrombocytopenia Syndrome, Japan.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tickborne infectious disease caused by SFTS virus (SFTSV). We report 7 cases of spontaneous fatal SFTS in felines. Necropsies revealed characteristic lesions, including necrotizing lymphadenitis in 5 cases and necrotizing splenitis and SFTSV-positive blastic lymphocytes in all cases. We detected hemorrhagic lesions in the gastrointestinal tract in 6 cases and lungs in 3 cases, suggesting a more severe clinical course of SFTS in felids than in humans. We noted necrotic or ulcerative foci in the gastrointestinal tract in 3 cases, the lung in 2 cases, and the liver in 4 cases. We clarified that blastic lymphocytes are predominant targets of SFTSV and involved in induction of necrotic foci. We also found that thymic epithelial cells were additional targets of SFTSV. These results provide insights for diagnosing feline SFTS during pathological examination and demonstrate the similarity of feline and human SFTS cases.

Toyo virus, a novel member of the Kaisodi group in the genus Uukuvirus (family Phenuiviridae) found in Haemaphysalis formosensis ticks in Japan.

Ticks are important vector arthropods that transmit various pathogens to humans and other animals. Tick-borne viruses are of particular concern to public health as these are major agents of emerging and re-emerging infectious diseases. The Phenuiviridae family of tick-borne viruses is one of the most diverse groups and includes important human pathogenic viruses such as severe fever with thrombocytopenia syndrome virus. Phenuivirus-like sequences were detected during the surveillance of tick-borne viruses using RNA virome analysis from a pooled sample of Haemaphysalis formosensis ticks collected in Ehime, Japan. RT-PCR amplification and Sanger sequencing revealed the nearly complete viral genome sequence of all three segments. Comparisons of the viral amino acid sequences among phenuiviruses indicated that the detected virus shared 46%-70% sequence identity with known members of the Kaisodi group in the genus Uukuvirus. Furthermore, phylogenetic analysis of the viral proteins showed that the virus formed a cluster with the Kaisodi group viruses, suggesting that this was a novel virus, which was designated "Toyo virus" (TOYOV). Further investigation of TOYOV is needed, and it will contribute to understanding the natural history and the etiological importance of the Kaisodi group viruses.

Distribution of Japanese Encephalitis Virus, Japan and Southeast Asia, 2016-2018.

During 2016-2018, we conducted surveillance for Japanese encephalitis virus (JEV) in mosquitoes and pigs in Japan, Thailand, the Philippines, and Indonesia. Phylogenetic analyses demonstrated that our isolates (genotypes Ia, Ib, III, IV) were related to JEV isolates obtained from the same regions many years ago. Indigenous JEV strains persist in Asia.

The complete genomic sequence of Rhinolophus gammaherpesvirus 1 isolated from a greater horseshoe bat.

In a comprehensive research project on bat viruses, we successfully isolated a novel herpesvirus from the spleen of a greater horseshoe bat (Rhinolophus ferrumequinum) in Japan using a cell line established from the kidney of the same bat. This herpesvirus was a novel gammaherpesvirus (Rhinolophus gammaherpesvirus 1; RGHV-1), which belonged to the genus Percavirus. The whole RGHV-1 genome (147,790 bp) showed that 12 of the 84 genes predicted to contain open reading frames did not show any homology to those of other herpesviruses.

Detection and isolation of tick-borne bacteria (Anaplasma spp., Rickettsia spp., and Borrelia spp.) in Amblyomma varanense ticks on lizard (Varanus salvator).

Ticks are one of the arthropods that play an important role in the transmission of numerous pathogens to livestock and humans. We investigated the presence of tick-borne bacteria in 23 Amblyomma varanense that fed on a water monitor (Varanus salvator) in Indonesia. Anaplasmataceae and borreliae were detected by PCR in 17.4% and 95.7% of ticks, respectively. "Candidatus Rickettsia sepangensis", spotted fever group of Rickettsia, was detected in 21.7% of ticks. The water monitor is a common reptile that is widely encountered in city areas in Asian countries. Our results suggested that Am. varanense on water monitor in Indonesia harbored several kinds of bacteria.

Getah virus epizootic among wild boars in Japan around 2012.

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.

Bustos virus, a new member of the negevirus group isolated from a Mansonia mosquito in the Philippines.

We isolated two distinct viruses from mosquitoes collected in Bustos, Bulacan province, Philippines, in 2009. These viruses show rapid replication and strong cytopathic effects in mosquito C6/36 cells. Whole-genome analysis of these viruses demonstrated that both viruses belong to the negevirus group. One of the viruses, from Culex vishunui mosquitoes, is a new strain of Negev virus. The other virus, from a Mansonia sp. mosquito, is a new negevirus designated Bustos virus. Gene expression analysis of the Bustos virus revealed that infected cells contain viral subgenomic RNAs that probably include open reading frame (ORF) 2 or ORF3. Purified Bustos virus particles contained at least three proteins, and the major component (a probable major capsid protein) is encoded by ORF3. Bustos virus did not show infectivity in mammalian BHK-21 cells, suggesting that it is an insect-specific virus, like other known negeviruses.

Detection of novel ferret coronaviruses and evidence of recombination among ferret coronaviruses.

In an epidemiological study of ferret coronaviruses (FRCoVs), novel FRCoV strains (Saitama-1 and Aichi-1) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequence analysis of partial RNA-dependent RNA polymerase (RdRp) genes. Phylogenetic analysis indicated that these strains belonged to different clusters from other FRCoV strains. Next, the nucleotide sequence of the 3'-terminal region of Saitama-1 (8271 bases) strain was determined and compared with those of the other FRCoVs, indicating that the Saitama-1 strain differed from the previously reported MSU-1 and MSU-2 strains in the regions encoding spike (S) protein, nucleocapsid, and open reading frame 7b. Furthermore, the results of SimPlot analysis indicated that FRCoV (MSU-2 strain) emerged via a recombination event of S protein between the MSU-1 and Saitama-1 strains. This mechanism is similar to that responsible for the emergence of type II feline coronavirus. This information will be useful for understanding the pathogenesis of FRCoV in ferrets.

Isolation of Japanese encephalitis virus and a novel insect-specific flavivirus from mosquitoes collected in a cowshed in Japan.

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.

Genetic and biological characterization of Muko virus, a new distinct member of the species Great Island virus (genus Orbivirus, family Reoviridae), isolated from ixodid ticks in Japan.

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 Tribec 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.

Persistent natural infection of a Culex tritaeniorhynchus cell line with a novel Culex tritaeniorhynchus rhabdovirus strain.

Culex tritaeniorhynchus rhabdovirus (CTRV) is a mosquito virus that establishes persistent infection without any obvious cell death. Therefore, occult infection by CTRV can be present in mosquito cell lines. In this study, it is shown that NIID-CTR cells, which were derived from Cx. tritaeniorhynchus, are persistently infected with a novel strain of CTRV. Complete genome sequencing of the infecting strain revealed that it is genetically similar but distinct from the previously isolated CTRV strain, excluding the possibility of contamination. These findings raise the importance of further CTRV studies, such as screening of CTRV in other mosquito cell lines.

Analysis of Mosquito-Borne Flavivirus Superinfection in Culex tritaeniorhynchus (Diptera: Culicidae) Cells Persistently Infected with Culex Flavivirus (Flaviviridae).

Superinfection exclusion is generally defined as a phenomenon in which a pre-existing viral infection prevents a secondary viral infection; this has also been observed in infections with mosquito-borne viruses. In this study, we examined the superinfection exclusion of the vertebrate-infecting flaviviruses, Japanese encephalitis virus (JEV) and dengue virus (DENV), by stable and persistent infection with an insect-specific flavivirus, Culex flavivirus (CxFV), in a Culex tritaeniorhynchus Giles cell line (CTR cells). Our experimental system was designed based on the premise that wild Cx. tritaeniorhynchus mosquitoes naturally infected with CxFV are superinfected with JEV by feeding on JEV-infected animals. As a result, we found no evidence of the superinfection exclusion of both JEV and DENV by pre-existing CxFV infection at the cellular level. However, JEV superinfection induced severe cytopathic effects on persistently CxFV-infected CTR cells. These observations imply the possibility that JEV superinfection in CxFV-infected Cx. tritaeniorhynchus mosquitoes has an adverse effect on their fitness.

First isolation and characterization of a mosquito-borne orbivirus belonging to the species Umatilla virus in East Asia.

An orbivirus was isolated from a sample from the ornithophilic mosquito Culex sasai in Japan. The virus, designated Koyama Hill virus (KHV), replicated to high titer in a mosquito cell line and to a low titer in an avian cell line, but the release of progeny viruses was not observed in mammalian cell lines inoculated with KHV. Electron microscopic examination of KHV-infected mosquito cells showed approximately 70-nm virus particles and viral tubules typical of members of the genus Orbivirus, family Reoviridae. KHV efficiently replicated in Cx. sasai mosquitoes, suggesting a potential vector species for KHV transmission in nature. Full-length viral genome sequencing and phylogenetic analysis revealed that KHV is closely related to Umatilla virus (UMAV) and Stretch Lagoon orbivirus (SLOV). This suggests that KHV is a new member of the species Umatilla virus, an orbivirus species not previously observed in East Asia. The KHV genome segment encoding NS1 contains a notable sequence deletion and heterogeneity compared with a prototype UMAV, which may affect its growth properties and pathogenicity in host cells. These results provide new insights into the genetic diversity and geographic distribution of members of the species Umatilla virus.

Characterization and arbovirus susceptibility of cultured CERNI cells derived from sika deer (Cervus nippon).

Cervus nippon (sika deer) are widely distributed throughout eastern Asia. Deer possess a variety of antibodies against several zoonotic pathogens, indicating that they act as reservoir of zoonoses. In this study, we reported the characterization of cultured cells derived from sika deer and evaluated their susceptibility to arthropod-borne viruses to clarify their usefulness in virological studies. Cells derived from testicular tissue in Dulbecco's modified eagle medium with 16% fetal bovine serum started growing as primary cultured cells. The diploid cells consisted of 68 chromosomes, consistent with those of Japanese sika deer previously reported. The phylogenetic analysis showed the cells formed a robust clade with Japanese population of C. nippon, indicating that the cultured cells established in this study were originated from the Japanese sika deer. The cells immortalized by the simian virus 40 T-antigen were predominantly spindle-shaped cells exhibiting adhesive properties, and cultivated at 37°C and 5% CO2, which are common culture conditions for many mammalian cell lines. Western blotting analysis indicated that the cultured cells were multiple types of cells that coexist, including at least epithelial, fibroblast, and also Leydig cells. We confirmed that the cells have susceptibility to several arboviruses distributed in Japan: Getah virus, Japanese encephalitis virus, Oz virus, and severe fever with thrombocytopenia syndrome virus, but not to Tarumiz tick virus. From these results, the cells contribute to clarify the role of sika deer as a reservoir of zoonoses in nature and deer-associated experimental research at the cellular and molecular levels.

Xenorhabdus ishibashii sp. nov., isolated from the entomopathogenic nematode Steinernema aciari.

Gram-negative bacteria of the genus Xenorhabdus exhibit a mutualistic association with steinernematid entomopathogenic nematodes and a pathogenic relationship with insects. Here we describe two isolates of the entomopathogenic nematode Steinernema aciari collected from China and Japan. 16S rRNA gene sequence similarity and phylogenetic analysis indicated that the isolates obtained from S. aciari belonged to the genus Xenorhabdus. Multilocus sequence analysis based on five universal protein-coding gene sequences revealed that the isolates were closely related to Xenorhabdus ehlersii DSM 16337(T) and Xenorhabdus griffiniae ID10(T) but that they exhibited <97 % sequence similarity with these reference strains, which indicated that the isolates were distinct from previously described species. Based on these genetic differences and several differential phenotypic traits, we propose that the isolates represent a novel species of the genus Xenorhabdus, for which we propose the name Xenorhabdus ishibashii sp. nov. The type strain is GDh7(T) ( = DSM 22670(T)  = CGMCC 1.9166(T)).

Characterization of Dak Nong virus, an insect nidovirus isolated from Culex mosquitoes in Vietnam.

In this study, we isolated and characterized an insect nidovirus from the mosquito Culex tritaeniorhynchus Giles (Diptera: Culicidae) in Vietnam, as an additional member of the new family Mesoniviridae in the order Nidovirales. The virus, designated "Dak Nong virus (DKNV)," shared many characteristics with Cavally virus and Nam Dinh virus, which have also been discovered recently in mosquitoes, and these viruses should be considered members of a single virus species, Alphamesonivirus 1. DKNV grew in cultured mosquito cells but could not replicate in the cultured vertebrate cells tested. N-terminal sequencing of the DKNV structural proteins revealed two posttranslational cleavage sites in the spike glycoprotein precursor. DKNV is assumed to be a new member of the species Alphamesonivirus 1, and the current study provides further understanding of viruses belonging to the new family Mesoniviridae.