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ABSTRACT Dogs are model animals that can be used to study the gut microbiome. Although the gut microbiome is assumed to be closely related to aging, information pertaining to this relationship in dogs is limited. Here, we examined the association between the canine gut microbiome and age via a bacterial 16S rRNA gene amplicon sequence analysis in a colony of 43 Japanese purebred Shiba Inu dogs. We found that microbial diversity tended to decrease with aging. A differential abundance analysis showed an association of a single specific microbe with aging. The age-related coabundance network analysis showed that two microbial network modules were positively and negatively associated with aging, respectively. These results suggest that the dog gut microbiome is likely to vary with aging. Gut microbiome analysis in a Shiba Inu purebred dog colony shows age-related transition of not only microbial diversity, a specific microbe, but also modules of a microbial coabundance network.

Influenza D virus (IDV) contains seven genome segments. The M and NS segments are regulated via splicing to express two proteins each (P42 and M1 from the M segment and NS1 and NS2 from the NS segment). Previously, we created an eight-segment IDV by separating the NS1 and NS2 genes into independent monocistronic segments. In this study, we designed another eight-segment IDV with two divided M segments, transcribing either P42 or M1 mRNA independently. Specifically, we constructed two plasmids for reverse genetics: one for viral RNA (vRNA) synthesis of the P42 segment with silent mutations at the splicing donor site and one for vRNA synthesis of the M1 segment with deletion of the intron in the M segment. We successfully created the virus via reverse genetics using these two segments and six other vRNA synthesis plasmids (PB2, PB1, P3, HEF, NP, and NS). Furthermore, we generated a nine-segment IDV including the P42, M1, NS1, and NS2 segments. These systems enable independent functional and interactive analyses of M and NS segment-encoded proteins for viral infectivity. Additionally, the eight- and nine-segment IDVs exhibited significantly reduced replication in cell culture compared with wild-type IDV, suggesting a strategy for attenuated live vaccine development.

Leukemia inhibitory factor (LIF) receptor, an interleukin 6 cytokine family signal transducer (Il6st, also known as Gp130) that is expressed in the uterine epithelium and stroma, has been recognized to play an essential role in embryo implantation. However, the molecular mechanism underlying Gp130-mediated LIF signaling in the uterine epithelium during embryo implantation has not been elucidated. In this study, we generated mice with uterine epithelium specific deletion of Gp130 ( Gp130 ecKO). Gp130 ecKO females were infertile due to the failure of embryo attachment and decidualization. Histomorphological observation revealed that the endometrial shape and embryo position from Gp130 ecKO were comparable to those of the control, and uterine epithelial cell proliferation, whose attenuation is essential for embryo implantation, was controlled in Gp130 ecKO. Comprehensive gene expression analysis using RNA-seq indicates that epithelial Gp130 regulates the expression of estrogen- and progesterone-responsive genes in conjunction with immune response during embryo implantation. We also found that an epithelial remodeling factor, snail family transcriptional repressor 1 (Snai1), was markedly reduced in the pre-implantation uterus from Gp130 ecKO. These results suggest that not only the suppression of uterine epithelial cell proliferation, but also Gp130-mediated epithelial remodeling is required for successful implantation in mice.

Virus purification by cesium chloride (CsCl) density gradient, which generally requires an expensive ultracentrifuge, is an essential technique in virology. Here, we optimized virus purification by CsCl density gradient using general centrifugation (40,000 × g , 2 h, 4 °C), which showed almost the same purification ability as conventional CsCl density gradient ultracentrifugation (100,000 × g , 1 h, 4 °C) using phages S13′ and φEF24C. Moreover, adenovirus strain JM1/1 was also successfully purified by this method. We suggest that general centrifugation can become a less costly alternative to ultracentrifugation for virus purification by CsCl densiy gradient and will thus encourage research in virology.

The raccoon dog ( Nyctereutes procyonoides ), classified in the order Carnivora within the family Canidae , is native to East Asia and widely distributed throughout Japan due to its adaptability to various environments. Despite the close relationship between raccoon dogs and other animals, viruses infecting raccoon dogs have not been thoroughly investigated in Japan. In this study, we performed metatranscriptomic analyses using fecal samples collected from latrines of wild raccoon dogs in two locations on mainland Japan. Nearly complete viral genomes were identified, including viruses belonging to the genus Kobuvirus (CaKoV), an unclassified canine sapelovirus within the subfamily Ensavirinae (CaSaV), the Genius Mamastrovirus (CaAstV), unclassified hepe-astro-like virus (bastrovirus-like) (Bast-like V), and an unclassified dicistrovirus (DiciV) within the family Dicistroviridae . Phylogenetic analyses revealed that raccoon dog CaKoV, CaSaV, and CaAstV are related to canine strains but form independent clusters specific to raccoon dogs, suggesting they have evolved within this host population. Bast-like V, detected for the first time in raccoon dogs, showed high sequence identity with viruses previously identified in Chinese shrews. The shared insectivorous nature of these hosts and in silico host range predictions suggest that Bast-like Vs may originate from arthropod viruses. Although DiciV is likely of dietary origin due to its arthropod hosts, the large number of sequence reads detected and the phylogenetic clustering of raccoon dog DiciVs with mammalian DiciVs indicate the need to assess their potential infectivity in mammals and the risk of spillover. These findings suggest that raccoon dogs harbor endemic viruses within the canine population and may act as potential vectors for viruses with unknown infectivity in mammals but with spillover risk.

The combined use of phage and antibiotics can show synergistic antimicrobial effects, so-called phage–antibiotic synergy (PAS). Here, we screened and examined PAS against Pseudomonas aeruginosa in vitro . Testing four different phages infecting P. aeruginosa , phage KPP22 classified within the family Myoviridae genus Pbunavirus showed PAS with the widest range of antibiotics, and showed PAS with anti- Pseudomonas drugs such as piperacillin and ceftazidime. Thus, evidence suggests that the combined use of phage and antibiotics is a promising therapeutic strategy against P. aeruginosa infections, with consideration needed regarding the optimal selection and adequate application timing of these phages and antibiotics.

Cattle are major reservoirs of the provisionally named influenza D virus, which is potentially involved in the bovine respiratory disease complex. Here, we conducted a serological survey for the influenza D virus in Japan, using archived bovine serum samples collected during 2010-2016 from several herds of apparently healthy cattle in various regions of the country. We found sero-positive cattle across all years and in all the prefectural regions tested, with a total positivity rate of 30.5%, although the positivity rates varied among regions (13.5-50.0%). There was no significant difference in positivity rates for Holstein and Japanese Black cattle. Positivity rates tended to increase with cattle age. The herds were clearly divided into two groups: those with a high positive rate and those with a low (or no) positive rate, indicating that horizontal transmission of the virus occurs readily within a herd. These data demonstrate that bovine influenza D viruses have been in circulation for at least 5 years countrywide, emphasizing its ubiquitous distribution in the cattle population of Japan.

Subclinical mastitis causes economic losses due to reduced milk yield and elevated somatic cell counts (SCCs), despite no visible clinical signs. A higher incidence of subclinical mastitis has been reported in cattle infected with bovine leukemia virus (BLV). Levamisole (LMS), known for its immunomodulatory properties, has been suggested as a potential alternative to antibiotics for mastitis treatment; however, its efficacy in BLV-infected cows, particularly in relation to proviral load (PVL), remains unclear. This study aimed to evaluate the therapeutic effect of LMS on subclinical mastitis and its impact on milk immune responses by classifying BLV-infected cows based on PVL. A total of 42 dairy cows with subclinical mastitis (48 quarters) were grouped as BLV-negative, low-PVL, or high-PVL using a PVL cut-off value of 17.8 copies/10 ng DNA, and were administered LMS orally. Changes in viable bacterial counts, SCCs, and milk leukocyte populations were compared. LMS administration significantly reduced the SCC and milk macrophage numbers, especially in BLV-negative and low-PVL cows. These results suggest that LMS may improve subclinical mastitis in certain BLV-infected cows and that PVL may serve as a useful indicator for treatment responsiveness. However, the limited effect in high-PVL cows and the small sample size have limitations, warranting further investigation.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis and is closely related to the human T cell leukemia virus. Since BLV infection mostly occurs via cell-to-cell transmission, BLV infectivity is generally measured by culturing BLV-infected cells with reporter cells that form syncytia upon BLV infection. However, this method is time-consuming and requires skill. To visualize the infectivity of BLV, we developed a new assay called the luminescence syncytium induction assay (LuSIA) that is based on a new reporter cell line designated CC81-BLU3G. CC81-BLU3G is stably transfected with pBLU3-EGFP, which contains the BLV long terminal repeat U3 region linked to the enhanced-green fluorescence protein (EGFP) gene. CC81-BLU3G expresses the EGFP in response to BLV Tax expression specifically, and forms fluorescing syncytia when transfected with an infectious BLV plasmid or when cultured with BLV-infected cells. Compared to the conventional assay, LuSIA was more specific and detected cattle samples with low proviral loads. The fluorescing syncytia was easily detected by eye and automated scanning and LuSIA counts correlated strongly with the proviral load of infected cattle (R 2  = 0.8942).

Vanitaracin A, an anti-hepatitis B virus polyketide, has been previously isolated from Talaromyces sp. In the present study, we searched for novel compounds in the culture broth obtained from a vanitaracin A-producing fungus under various conditions. Three novel compounds (vanitaracin C, vanitaraphilone A, and 2-hydroxy-4-(hydroxymethyl)-6-methylbenzaldehyde) were isolated, and their structures were determined using spectroscopic methods (1D/2D NMR and MS). In addition, the antiviral spectrum of vanitaracin A was examined by measuring its antiviral activities against rabies virus, Borna disease virus 1, and bovine leukemia virus. This compound exhibited antiviral activity against bovine leukemia virus, which is the causative agent of enzootic bovine leukosis. The anti-bovine leukemia virus effects of other compounds isolated from the vanitaracin A-producing fungus, namely, vanitaracins B and C, vanitaraphilone A, and 2-hydroxy-4-(hydroxymethyl)-6-methylbenzaldehyde, were also evaluated. Vanitaracin B, vanitaraphilone A and 2-hydroxy-4-(hydroxymethyl)-6-methylbenzaldehyde were also found to exhibit activity against bovine leukemia virus. These findings reveal the broad-spectrum antiviral activity of the vanitaracin scaffold and suggest several candidates for the development of anti-bovine leukemia virus drugs.