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.

Low Molecular-Weight Curdlan, (1→3)-ß-Glucan Suppresses TLR2-Induced RANKL-Dependent Bone Resorption.

Fungal ß-glucan is a potent immunological stimulator, and that it activates both the innate immune system and adaptive immunity. Curdlan is (1→3)-ß-glucan, a linear form of ß-glucan with a high molecular weight; it modulates the immune response. However, its role in bone tissue is controversial, and the effects of curdlan on bone tissues are unknown. Toll-like receptors (TLRs) play critical roles in innate immunity, and various ligands for TLRs are thought to regulate the host defense mechanisms against pathogens. TLR2 is known to form heterodimers with TLR6, and the TLR2-TLR6 heterodimer (TLR2/6) recognizes diacylated lipopeptides from Gram-positive bacteria. In the present study, we prepared low molecular-weight curdlan, (1→3)-ß-D-glucan, and examined its effects on bone resorption induced by TLR2/6 signaling. In co-cultures of bone marrow cells and osteoblasts, low molecular-weight curdlan suppressed the osteoclast formation induced by TLR2/6 ligand, and attenuated bone resorption in mouse calvarial organ cultures. Curdlan acted on mouse osteoblasts and suppressed the expression of receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL), a key molecule for osteoclastogenesis. Curdlan also acted on mouse bone marrow macrophages and suppressed RANKL-dependent osteoclast differentiation from osteoclast precursor cells. The present study indicates that low molecular-weight curdlan attenuated TLR2-induced inflammatory bone resorption. Curdlan, (1→3)-ß-glucan may be a natural agent with beneficial effects on bone health in humans.

Molecular identification of field-collected Culicoides larvae in the southern part of Japan.

Although Culicoides biting midges act as a vector of important human and domestic animal diseases, their ecology is poorly understood. The lack of proper identification systems of Culicoides larvae is one of the main obstacles to progress in research. Based on mitochondrial sequences of 19 Japanese Culicoides species, we designed a universal primer set to amplify the partial sequence of the mitochondrial cytochrome c oxidase I (cox 1). The polymerase chain reaction product amplified from extracted DNA of Culicoides larvae using the primer set was directly sequenced, and species identification based on the variation at cox1 was conducted. Using the molecular identification system, we sorted 243 specimens of field-collected larvae from the southern part of Japan into 10 species including Culicoides arakawae (Arakawa), Culicoides oxystoma Kieffer, and Culicoides brevitarsis Kieffer, which are regarded as vectors of important livestock animal diseases. Eight species of Culicoides larvae, including C. arakawae and C. oxystoma, were recovered from active paddy fields and an abandoned paddy field. The result suggests that paddy fields contribute to breeding a variety of Culicoides species and maintenance and spread of Culicoides-borne pathogens. In contrast, larvae of C. brevitarsis were collected from cattle dung in pastures. The molecular identification system described herein using nucleotide sequences successfully achieved larval identification and will be useful for a better understanding of larval habitats of Culicoides biting midges.

Genetic reassortment between Sathuperi and Shamonda viruses of the genus Orthobunyavirus in nature: implications for their genetic relationship to Schmallenberg virus.

The recent outbreak of malformations in ruminants in Northern Europe caused by Schmallenberg virus induced us to analyze the genetic properties of the related orthobunyaviruses and clarify their relationship. The sequencing of three genomic RNA segments of Sathuperi, Shamonda and Douglas viruses (SATV, SHAV and DOUV) revealed that the M RNA segment of SATV and DOUV had a high degree of sequence identity with that of Schmallenberg virus, but the S and L RNA segments closely matched those of SHAV. Phylogenetic analysis of the three genomic RNA segments indicated that Schmallenberg virus is a reassortant, with the M RNA segment from SATV and the S and L RNA segments from SHAV.

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.

Genetic engineering approach to develop next-generation reagents for endotoxin quantification.

The bacterial endotoxin test, which uses amebocyte lysate reagents of horseshoe crab origin, is a sensitive, reproducible and simple assay to measure endotoxin concentration. To develop sustainable raw materials for lysate reagents that do not require horseshoe crabs, three recombinant protease zymogens (factor C, derived from mammalian cells; factor B; and the proclotting enzyme derived from insect cells) were prepared using a genetic engineering technique. Recombinant cascade reagents (RCRs) were then prepared to reconstruct the reaction cascade in the amebocyte lysate reagent. The protease activity of the RCR containing recombinant factor C was much greater than that of recombinant factor C alone, indicating the efficiency of signal amplification in the cascade. Compared with the RCR containing the insect cell-derived factor C, those containing mammalian cell-derived factor C, which features different glycosylation patterns, were less susceptible to interference by the injectable drug components. The standard curve of the RCR containing mammalian cell-derived recombinant factor C had a steeper slope than the curves for those containing natural lysate reagents, suggesting a greater sensitivity to endotoxin. The present study supports the future production of recombinant reagents that do not require the use of natural resources.

Genetic characterization of Aino and Peaton virus field isolates reveals a genetic reassortment between these viruses in nature.

Sequence determination and phylogenetic analysis were conducted using the S, M and L RNA segments of the 10 Aino, 6 Peaton and 1 Sango virus (AINOV, PEAV and SANV) field isolates of the genus Orthobunyavirus in the family Bunyaviridae, respectively. The Japanese AINOV strains were genetically stable, but the sequence differences between the Japanese and Australian AINOV strains were considerably larger than those among the Japanese AINOV strains. A similar result was found in the genetic relationship among Japanese and Australian PEAVs, and SANV which was isolated in Nigeria and was thought as a synonym of PEAV, suggesting that geographic separation contributed significantly to the evolution of those viruses. The Australian AINOV strain B7974 is more closely related to the Australian PEAV strain CSIRO110 than to the Japanese AINOV strains in the S and L RNA segments, while the phylogenetic position of the M RNA segment of the B7974 strain was clustered with those of the Japanese AINOV strains. Our findings indicate that the B7974 strain is a reassortment with the M RNA segment derived from AINOV and the S and L RNA segments derived from an Australian PEAV.

Phylogenetic relationships of the G gene sequence of bovine ephemeral fever virus isolated in Japan, Taiwan and Australia.

The G gene encoding the neutralization antigen of bovine ephemeral fever virus (BEFV) was characterized in order to define the virus's molecular epidemiology in Japan and the genetic relationships among the Japanese, Taiwanese and Australian isolates. The nucleotide and amino acid sequences of the gene were highly conserved among the Japanese strains, regardless of the year of isolation, and were closely related to the Taiwanese strains. By phylogenetic analysis, the Japanese and Taiwanese strains were classified clearly into three chronological clusters: 1966, 1984-1989 and 1996-2004, indicating that the epidemics of bovine ephemeral fever may occur almost simultaneously in both countries by the same genotype. On the other hand, the Australian strains were distantly related to these East Asian strains and placed in the independent fourth cluster of the phylogenetic tree. It is suggested that three amino acid substitutions at residues 224, 271 and 499 in the neutralizing epitopes, of which two generate new glycosylation sequences, are responsible for antigenic variations of bovine ephemeral fever virus. The cross-neutralization test using the bovine ephemeral fever virus isolated in Japan demonstrated that the vaccine developed based on the oldest Japanese strain, YHL, appears to still be effective for controlling bovine ephemeral fever in Japan.

A limulus amoebocyte lysate activating activity (LAL activity) that lacks biological activities of endotoxin found in biological products.

Pyrogenic substances in influenza HA (IHA) vaccine have been controlled by the pyrogen test or the mouse body weight decreasing toxicity (BWD) test. We examined the possibility of replacing the animal tests with the endotoxin test Commercial IHA vaccines were found to show considerable levels of LAL activity ranging from 0.2 to 160 EU/ml. However, a batch of the vaccine having even 100 EU/ml of LAL activity showed neither pyrogenicity in rabbits nor tumor necrosis factor alpha (TNF-alpha) induction in RAW264.7 cells. The LAL activity of IHA vaccine was abolished by a monoclonal antibody that recognizes LPS-binding epitope of LAL factor C. The activity of IHA vaccine showed different physicochemical properties from those of LAL activity of endotoxin. LAL activity of endotoxin is known to be sensitive to polymyxin B treatment and was found to be resistant to polyoxyethylene 10 cetyl ether (Brij56) treatment. On the contrary, the LAL activity of IRA vaccine was shown to be resistant to polymyxin B but sensitive to Brij56 treatment. The difference in sensitivity of the two LAL activities to polymyxin B and Brij56 might suggest the possibility of their discriminative measurements.