Differential role of NSs genes in the neurovirulence of two genogroups of Akabane virus causing postnatal encephalomyelitis.

Akabane virus (AKAV) is a member of the genus Orthobunyavirus, family Peribunyaviridae. In addition to AKAV strains that cause fetal Akabane disease, which is characterized by abortion in ruminants, some AKAV strains cause postnatal infection characterized by nonsuppurative encephalomyelitis in ruminants. Here, we focused on the NSs protein, a virulence factor for most viruses belonging to the genus Orthobunyavirus, and we hypothesized that this protein would act as a neurovirulence factor in AKAV strains causing postnatal encephalomyelitis. We generated AKAV strains that were unable to produce the NSs protein, derived from two different genogroups, genogroups I and II, and then examined the role of their NSs proteins by inoculating mice intracerebrally with these modified viruses. Our results revealed that the neurovirulence of genogroup II strains is dependent on the NSs protein, whereas that of genogroup I strains is independent of this protein. Notably, infection of primary cultured bovine cells with these viruses suggested that the NSs proteins of both genogroups suppress innate immune-related gene expression with equal efficiency. These results indicate differences in the determinants of virulence of orthobunyaviruses.

Complete genome sequences of epizootic hemorrhagic disease virus serotypes 5 and 6 isolated in Japan.

Here, we report the complete genome sequences of epizootic hemorrhagic disease (EHD) virus serotypes 5 (EHDV-5) and 6 (EHDV-6) isolated in the Yaeyama Islands of Okinawa Prefecture, Japan. The EHDV-5 strain, ON-11/E/16, which was isolated in 2016, is, to our knowledge, the second EHDV-5 strain to be isolated after the first was isolated in Australia in 1977. In each of the genome segments, ON-11/E/16 was most closely related to EHDV strains of different serotypes isolated in Australia and Japan. Our results support the idea that various serotypes of EHDV have been circulating while causing reassortment in the Asia-Pacific region. In all genome segments, the EHDV-6 strain, ON-3/E/14, which was isolated in 2014, was highly similar to EHDV-6 strain HG-1/E/15, which was detected in affected cattle during the EHD epidemic in Hyogo prefecture in 2015. Therefore, these two EHDV-6 strains, ON-3/E/14 and HG-1/E/15, may have the same origin. However, it is unclear whether EHDV-6 was transmitted directly between the locations where those strains were isolated/detected (approx. 1,500 km apart) or whether EHDV-6 strains of the same origin entered each location at different times. In addition, we cannot rule out the possibility that EHDV-6 infection has spread unnoticed through asymptomatic cattle in other areas of Japan. Therefore, further investigation into EHDV infection in cattle is necessary for a more detailed understanding of the ecology of EHDV in Japan.

Isolation of Culicoides- and Mosquito-Borne Orbiviruses in the Southwestern Islands of Japan Between 2014 and 2019.

The circulation of arboviruses in livestock ruminants has often gone unrecognized owing to the fact that a significant percentage of arboviruses probably induce subclinical infections and/or negligible symptoms in infected animals. To determine the current situation of arbovirus circulation in the Yaeyama Islands, attempts to isolate viruses from bovine blood samples collected between 2014 and 2019 have been made. In total, 308 blood samples were collected during the study period, and 43 of them induced cytopathic effects (CPEs) in cell cultures. The identification of the CPE agents was performed by reported RT-PCR assays and a high-throughput analysis with a next-generation sequencing platform. The obtained viruses consisted of an orthobunyavirus (Peaton virus), Culicoides-borne orbiviruses (bluetongue virus serotypes 12 and 16, epizootic hemorrhagic disease virus [EHDV] serotypes 5, 6, and 7, D'Aguilar virus, and Bunyip Creek virus), and potential mosquito-borne orbiviruses (Yunnan orbivirus, Guangxi orbivirus, and Yonaguni orbivirus). Most of the orbiviruses were recovered from washed blood cells with mosquito cell cultures, suggesting that this combination was more efficient than other combinations such as plasma/blood cells and hamster cell lines. This marked the first time that the isolation of EHDV serotypes 5 and 6 and three potential mosquito-borne orbiviruses was recorded in Japan, showing a greater variety of orbiviruses on the islands than previously known. Genetic analysis of the isolated orbiviruses suggested that the Yaeyama Islands and its neighboring regions were epidemiologically related. Some of the viruses, especially the potential mosquito-borne orbiviruses, were isolated during several consecutive years, indicating their establishment on the islands.

Isolation of epizootic hemorrhagic disease virus serotype 7 from cattle showing fever in Japan in 2016 and improvement of a reverse transcription-polymerase chain reaction assay to detect epizootic hemorrhagic disease virus.

Epizootic hemorrhagic disease (EHD) is an arthropod-borne disease of wild and domestic ruminants caused by the EHD virus (EHDV). To date, seven EHDV serotypes have been identified. In Japan, strain Ibaraki of EHDV serotype 2 has caused outbreaks of Ibaraki disease in cattle. In addition, EHDV serotype 7 (EHDV-7) has caused large-scale EHD epizootics. In mid-September 2016, eight cattle at a breeding farm in Fukuoka Prefecture, Japan developed fever. Since EHDV-7 was detected in sentinel cattle in western Japan in 2016, we suspected that the cause of this fever might be an EHDV-7 infection. In this study, we tested cattle for EHDV-7 and some other viruses. Consequently, EHDV was isolated from washed blood cells collected from three of the eight cattle, and genetic analysis of genome segment 2 revealed that this isolate was EHDV-7. Moreover, all affected cattle tested positive for anti-EHDV-7 neutralizing antibodies. Our results suggest that the fever was caused by EHDV-7 infection. In addition, we modified a conventional reverse transcription polymerase chain reaction assay for the specific detection of EHDV. This modified assay could detect various strains of EHDV isolated in Japan, Australia, and North America. Furthermore, the assay permitted the detection of EHDV-7 in blood cells collected from seven of the eight cattle. We believe that this modified assay will be a useful tool for the diagnosis of EHD.

Genomic analysis of putative novel serotypes of Tibet orbivirus isolated in Japan.

Tibet orbivirus (TIBOV) was initially isolated in Tibet in 2009 and subsequently in Guangdong, Hunan, and Yunnan, China. We document the first isolation of TIBOV outside of China: two TIBOV isolates from Culicoides collected in 2009 and 2010 in Kagoshima, Japan. Their complete genome sequences were also determined. Our results suggest that the two virus isolates are of novel serotypes, evident by variability within genome segment 2 encoding VP2. These new putative TIBOV serotypes will help with future virus surveillance and with the evaluation of its potential to cause disease in domestic ruminants.

Complete Genome Sequences of Two Akabane Virus Strains Causing Bovine Postnatal Encephalomyelitis in Japan.

Akabane virus (AKAV) (genus Orthobunyavirus, family Peribunyaviridae) is an arthropod-borne virus that causes congenital abnormalities in ruminants. Here, we report the complete genome sequences of two AKAV strains causing nonsuppurative encephalomyelitis in cattle by postnatal infection in Japan.

Identification and characterization of a novel orbivirus, Yonaguni orbivirus, isolated from cattle on the westernmost island of Japan.

A novel orbivirus (genus Orbivirus, family Reoviridae), designated Yonaguni orbivirus (YONOV), was isolated from bovine blood collected on a subtropical island of Japan in 2015. The YONOV genome (20,054 nucleotides in total) has a coding arrangement similar to those of mosquito-borne orbiviruses. YONOV has a close genetic relationship to mosquito-borne orbiviruses, especially to Mobuck virus (MBV), which was isolated in North America. However, YONOV and MBV share less than 74% nucleotide sequence identity in the major subcore protein (T2) coding sequence, which satisfies the criterion for species demarcation. It is still uncertain whether YONOV should be assigned to a novel species in the genus Orbivirus.

Surveillance of Culicoides biting midges in northern Honshu, Japan, during the period of Akabane virus spread.

A surveillance of Culicoides biting midges with light suction traps was conducted in the northern region of Honshu, main island of Japan, during the summers and autumns of 2009 and 2010. A total of 106 trap collections across 37 cattle farms were investigated for the structure and distribution of Culicoides species. Forty-thousand and one hundred forty-nine specimens of Culicoides biting midges were identified at the species level, and ≥19 species were included in the specimens. Culicoides oxystoma, which is a known major vector of Akabane virus (AKAV), appeared not to have expanded in northern Honshu during the surveillance. Of the potential AKAV vectors suggested by a previous laboratory experiment, C. tainanus and C. punctatus widely infested cowsheds across northern Honshu. The AKAV circulation was confirmed by serological surveillance of sentinel cattle in northern Honshu during the summer and autumn of 2010 and, consequently, >200 calves affected by the virus were identified as of spring 2011. Our surveillance demonstrated that C. tainanus and C. punctatus were widely spread and often dominated at cattle farms in/around the seroconverted regions, and our results thus suggest that these species played a critical role in the AKAV transmission in 2010. Because the distribution ranges of C. tainanus and C. punctatus cover almost all of mainland Japan, a potential risk of AKAV transmission might be expected even in areas outside the range of C. oxystoma.

Oral Susceptibility of Japanese Culicoides (Diptera: Ceratopogonidae) Species to Akabane Virus.

To test their virus susceptibility and capacity as transmission vectors, Japanese Culicoides species were artificially fed a mixture of Akabane virus (AKAV) and bovine blood, and virus recovery was attempted from infected midges 9-11 d post-exposure. Culicoides tainanus, C. punctatus, C. humeralis, C. jacobsoni, C. oxystoma, and C. asiana were found to be orally susceptible to AKAV. Virus titers in single infected midges of C. tainanus, C. oxystoma, C. punctatus, and C. jacobsoni ranged from 100.75 to 104.0 TCID50 (tissue culture infectious dose). The titers in the infected C. oxystoma were significantly higher than those in the other infected species. Viral RNA was detected from both midges testing positive and those testing negative for infectious virus particles, but the viral RNA copies in the infectious virus-negative midges were significantly lower than those in the infectious virus-positive midges. Lower viral amplification, limited dissemination or both caused by tissue barriers might occur in infected midges from which infectious viruses were undetectable. A fully disseminated infection was developed in orally infected C. oxystoma and C. tainanus. This finding indicates their capacity to transmit AKAV, assuming that salivary gland barriers have limited effects on viral entry to and replication in salivary gland tissue. This result also suggests that the other orally susceptible species are potentially competent for AKAV transmission and would be considered active vectors of its spread.

Congenital abnormalities in calves associated with Peaton virus infection in Japan.

Peaton virus (PEAV; family Peribunyaviridae, genus Orthobunyavirus) appears to be capable of producing congenital malformations in ruminants; however, its pathogenicity remains unknown given its relatively low incidence. We evaluated the relationship between congenital abnormalities of calves and PEAV infection by serologic, epidemiologic, pathologic, and virologic investigations using specimens from 31 malformed calves in the years 1996-2016 in Japan. Antibody testing was carried out for known teratogenic viruses, including Akabane, Aino, Chuzan, and bovine viral diarrhea viruses, in the precolostral sera of these abnormal calves, but all results were negative. However, all 31 malformed calves were positive for antibodies against PEAV. A PEAV-specific gene was amplified from central nervous system tissues from a stillborn calf delivered in April 2007, and its nucleotide sequence was identical with that of PEAV isolated from healthy sentinel cattle in September 2006. These findings indicate that PEAV can cause bovine congenital anomalies.

Full genome sequence of a Sathuvachari virus strain isolated in the southwestern-most archipelago of Japan.

Two virus strains, tentatively designated as ON-6/P/05 and ON-7/E/05, were isolated from blood samples of healthy cattle in the Yaeyama Islands, located in the southwestern-most region of Japan, in 2005. Ultrastructural observations of infected baby hamster (BHK-21) cells revealed that the viruses had features consistent with those of orbivirus. As with other orbiviruses, the viral genome consists of 10 double-stranded RNA segments. The full genome sequence of ON-6/P/05 was determined and shared high nucleotide and amino acid identities (90.07-98.22% nucleotide identity; 96.16-99.72% amino acid identity) with that of Sathuvachari virus (SVIV), a member of the species Sathuvachari virus of the genus Orbivirus, originally isolated from starlings collected in southern India in 1963. The sequence of segment two of ON-7/E/05 was identical to that of ON-6/P/05. The isolation of SVIV from cattle also indicated that the virus has a wider host range than previously thought. The potential pathogenicity of SVIV in domestic animals should be considered in future disease surveillance within its distribution range.

Epizootic Hemorrhagic Disease Virus Serotype 6 Infection in Cattle, Japan, 2015.

During October-December 2015, an epizootic hemorrhagic disease outbreak occurred in cattle in Japan. Forty-six animals displayed fever, anorexia, cessation of rumination, salivation, and dysphagia. Virologic, serologic, and pathologic investigations revealed the causative agent was epizootic hemorrhagic disease virus serotype 6. Further virus characterization is needed to determine virus pathogenicity.

Differential Distribution of Salmonella Serovars and Campylobacter spp. Isolates in Free-Living Crows and Broiler Chickens in Aomori, Japan.

Salmonella and Campylobacter cause foodborne enteritis mainly via the consumption of raw/undercooked contaminated poultry meat and products. Broiler flocks are primarily colonized with these bacteria; however, the underlying etiology remains unclear. The present study was conducted in order to obtain further information on the prevalence and genotypic distribution of Salmonella and Campylobacter in free-living crows and broiler flocks in a region for 2 years, thereby facilitating estimations of the potential risk of transmission of C. jejuni from crows to broiler flocks. Salmonella serovars Bredeney and Derby were isolated from 8 and 3 out of 123 captured crows, respectively, both of which are not common in broiler chickens. Campylobacter were isolated from all 89 crows tested and C. jejuni was prevalent (85 crows). Pulsed field gel electrophoresis showed broad diversity in the crow isolates of C. jejuni. However, 3 crow isolates and 2 broiler isolates showing similar banding patterns were assigned to different sequence types in multi-locus sequence typing. These results indicate that crows do not share Salmonella serovars with broilers, and harbor various genotypes of C. jejuni that differ from those of broiler flocks. Thus, our results indicate that crows are not a potential vector of these bacteria to broiler flocks in this region.

Characterization of genome segments 2, 3 and 6 of epizootic hemorrhagic disease virus strains isolated in Japan in 1985-2013: Identification of their serotypes and geographical genetic types.

We characterized genome segments 2, 3 and 6 (Seg-2, Seg-3 and Seg-6) of 11 Japanese strains of epizootic hemorrhagic disease (EHD) virus (EHDV) isolated in 1985-2013. The Japanese strains were divisible into two groups based on phylogenetic analyses of the nucleotide sequences of Seg-2 and Seg-6. In both of the phylogenetic trees based on Seg-2 and Seg-6, seven of the 11 Japanese strains were grouped together with EHDV-2 and EHDV-7 strains, and the other four Japanese strains were grouped with EHDV-1 strains. The phylogenetic analysis of Seg-2 among EHDV strains identified 10 of the 11 Japanese strains as EHDV-1, EHDV-2 or EHDV-7. The other Japanese strain, ON-4/B/98, isolated from an asymptomatic cow in 1998 was in the same group as the EHDV-2 and EHDV-7 strains in the phylogenetic trees based on Seg-2 and Seg-6, but the results suggested that the strain belongs to another serotype. We thus conducted a serum neutralization test to identify that serotype by using anti-EHDV-2 and anti-EHDV-7 rabbit sera. We observed that the ON-4/B/98 strain was not sufficiently neutralized by any of the antisera, which suggests that the strain could be assigned into a new serotype, tentatively named 'EHDV-10.' Sequences of Seg-3 were also determined, and all of the Japanese strains were grouped together with Australian strains, suggesting that the Japanese strains are a part of EHDV distributed in the Asia-Pacific region. The data obtained herein would be beneficial for the diagnosis and prevention of EHD in Japan and neighboring countries.

Congenital Malformations of Calves Infected with Shamonda Virus, Southern Japan.

In 2015 and 2016, we observed 15 malformed calves that were exposed to intrauterine infection with Shamonda virus, a Simbu serogroup orthobunyavirus, in Japan. Characteristic manifestations were arthrogryposis and gross lesions in the central nervous system. Our results indicate that this arbovirus should be considered a teratogenic virus in ruminants.

Resurgence of bovine ephemeral fever in mainland Japan in 2015 after a 23-year absence.

In September and October 2015, suspected cases of bovine ephemeral fever (BEF) were reported in the mainland region of Kagoshima Prefecture and on Tanegashima Island. The genome of the BEF virus (BEFV) was detected in the diseased cows and the cows that had recovered. The serum obtained from the affected cows contained high titers of BEFV-neutralizing antibody. In total, 18 affected cows were demonstrated to be infected with BEFV during the outbreak. Our findings showed evidence that BEF occurred in mainland Japan after a 23-year absence. Phylogenetic analysis based on the surface glycoprotein (G) gene revealed that BEFVs detected in the affected cows were genetically distinct from previous Japanese BEFVs, but were close to BEFVs circulating in Taiwan and mainland China in recent years. Amino acid substitution in the neutralizing epitope domains of the G protein was limited between the detected viruses and the vaccine strain (YHL isolate), and high titers of the neutralizing antibody against the YHL isolate were induced in the infected cattle during the disease occurrences. Therefore, current BEF vaccines probably elicit protective immunity against the BEFVs detected in 2015, although their effectiveness should be assessed. Since the BEFV vaccination rates are estimated to be low, a BEF outbreak should be considered a possibility in mainland Japan.

Molecular characterization of an Akabane virus isolate from West Java, Indonesia.

We isolated an arbovirus from bovine blood in Indonesia. The arbovirus was obtained from the plasma of a cow showing no clinical symptoms in West Java in February 2014, and was identified as Akabane virus (AKAV) by AKAV-specific RT-PCR and subsequent sequence analysis. Phylogenetic analysis based on partial S segment indicated the AKAV isolate, WJ-1SA/P/2014, was most closely related with two isolates from Israel and Turkey reported in 2001 and 2015, respectively, and that WJ-1SA/P/2014 isolate belongs to AKAV genogroup Ib. This is the first isolation of AKAV from Indonesia.

Monitoring for bovine arboviruses in the most southwestern islands in Japan between 1994 and 2014.

BACKGROUND: In Japan, epizootic arboviral infections have severely impacted the livestock industry for a long period. Akabane, Aino, Chuzan, bovine ephemeral fever and Ibaraki viruses have repeatedly caused epizootic abnormal births and febrile illness in the cattle population. In addition, Peaton, Sathuperi, Shamonda and D'Aguilar viruses and epizootic hemorrhagic virus serotype 7 have recently emerged in Japan and are also considered to be involved in abnormal births in cattle. The above-mentioned viruses are hypothesized to circulate in tropical and subtropical Asia year round and to be introduced to temperate East Asia by long-distance aerial dispersal of infected vectors. To watch for arbovirus incursion and assess the possibility of its early warning, monitoring for arboviruses was conducted in the Yaeyama Islands, located at the most southwestern area of Japan, between 1994 and 2014. RESULTS: Blood sampling was conducted once a year, in the autumn, in 40 to 60 healthy cattle from the Yaeyama Islands. Blood samples were tested for arboviruses. A total of 33 arboviruses including Akabane, Peaton, Chuzan, D' Aguilar, Bunyip Creek, Batai and epizootic hemorrhagic viruses were isolated from bovine blood samples. Serological surveillance for the bovine arboviruses associated with cattle diseases in young cattle (ages 6-12 months: had only been alive for one summer) clearly showed their frequent incursion into the Yaeyama Islands. In some cases, the arbovirus incursions could be detected in the Yaeyama Islands prior to their spread to mainland Japan. CONCLUSIONS: We showed that long-term surveillance in the Yaeyama Islands could estimate the activity of bovine arboviruses in neighboring regions and may provide a useful early warning for likely arbovirus infections in Japan. The findings in this study could contribute to the planning of prevention and control for bovine arbovirus infections in Japan and cooperative efforts among neighboring countries in East Asia.

Serological survey of caprine arthritis-encephalitis virus infection in Japan.

A serological survey of caprine arthritis-encephalitis virus (CAEV) infection was conducted from September 2006 to February 2007 in Japan. A total of 857 serum samples were collected from 113 herds in 28 prefectures and were analyzed for the presence of CAEV antibodies using agar gel immunodiffusion test. The seroprevalence of CAEV infection at the herd and animal levels was 15.0% (17/113) and 10.0% (86/857), respectively. Large farms with more than 10 goats and with animals for dairy and breeding purposes had higher seroprevalence (P<0.05). The results of this study provide useful information to consider effective control programs against CAEV infection in Japan.

Broad-range detection of arboviruses belonging to Simbu serogroup lineage 1 and specific detection of Akabane, Aino and Peaton viruses by newly developed multiple TaqMan assays.

TaqMan assays were developed for the broad-range detection of arboviruses belonging to Simbu serogroup lineage 1 in the genus Orthobunyavirus and also for the specific detection of three viruses in the lineage, Akabane, Aino and Peaton viruses (AKAV, AINOV and PEAV, respectively). A primer and probe set was designed for the broad-range detection of Simbu serogroup lineage 1 (Pan-Simbu1 set) mainly targeting AKAV, AINOV, PEAV, Sathuperi and Shamonda viruses (SATV and SHAV), and the forward and reverse primers of the Pan-Simbu1 set were also used for the specific detection of AKAV with another probe (AKAV-specific set). In addition, two more primer and probe sets were designed for AINOV- and PEAV-specific detection, respectively (AINOV- and PEAV-specific sets). All of the four primer and probe sets successfully detected targeted viruses, and thus broad-range and specific detection of all the targeted viruses can be achieved by using two multiplex assays and a single assay in a dual (two-color) assay format when another primer and probe set for a bovine ß-actin control is also used. The assays had an analytical sensitivity of 10 copies/tube for AKAV, at least 100 copies/tube for AINOV, 100 copies/tube for PEAV, one copy/tube for SATV and at least 10 copies/tube for SHAV, respectively. Diagnostic sensitivity of the assays was tested with field-collected bovine samples, and the results suggested that the sensitivity was higher than that of a conventional RT-PCR. These data indicate that the newly developed TaqMan assays will be useful tools for the diagnosis and screening of field-collected samples for infections of AKAV and several other arboviruses belonging to the Simbu serogroup lineage 1.