Oz virus (OZV), a member of the genus Thogotovirus in the family Orthomyxoviridae , is an emerging tick-borne virus reported in Japan. A fatal human case and seroepidemiological evidence of widespread exposure among wild animals and humans suggest its potential public health significance. However, no animal models suitable for pathogenic studies or evaluation of countermeasures are available for OZV. Here, we have established a lethal mouse model of OZV infection using cell-adapted virus and mice lacking type I interferon signaling (B6 Ifnar1 KO mice). OZV infection resulted in 100% mortality and was characterized by robust viral replication in the liver and spleen, severe hepatitis, and acute liver injury. Using this model, we also demonstrated that oral administration of T-705, an antiviral drug widely used against RNA viruses, as well as immunization with an inactivated whole virus particle vaccine, protected B6 Ifnar1 KO mice from lethal OZV infection by mitigating the acute hepatitis. The present study provides critical insights into OZV pathogenesis and establishes a practical in vivo platform for the development of countermeasures against OZV infection. ### Competing Interest Statement The authors have declared no competing interest. Japan Society for the Promotion of Science, 25K18810, 25K09431 Japan Agency for Medical Research and Development, JP223fa627005, JP24wm0225044

Reverse genetics (RG) systems are essential tools for basic virological studies and applied studies using engineered recombinant viruses in various research fields. While the circular polymerase extension reaction (CPER) has been widely applied to prepare a full-length infectious complementary DNA (cDNA) of positive-sense RNA viruses, its use for negative-sense RNA viruses (mononegaviruses) remains limited. Here, we report the first CPER-based RG system for rabies virus (RABV), a member of mononegaviruses. Infectious RABV was successfully rescued from cells transfected with helper plasmids and the CPER product, the assembled overlapping DNA fragments encoding the full-length viral genome cDNA and regulatory elements. Using this system, we generated wild-type, point-mutant, reporter-expressing, and chimeric RABVs, all of which retained their expected biological properties. Deep sequencing revealed that CPER-derived viruses occasionally harbor low-frequency mutations undetectable by Sanger sequencing, highlighting PCR-related artifacts as a limitation. In addition, CPER products with a pUC19 backbone could be directly applied for Escherichia coli transformation and cloning of RABV full-genome cDNA plasmids, offering a flexible, ligase-free cloning strategy for conventional RG. Our work establishes CPER as a versatile platform for engineering recombinant RABVs, facilitating rapid generation and genetic manipulation of RABV with potential applications for research on other mononegaviruses.IMPORTANCEReverse genetics systems allow researchers to generate recombinant viruses with precise genetic modifications, advancing studies of viral replication, pathogenicity, and vaccine development. However, constructing a full-length viral genome expressing plasmids is often time-consuming and technically demanding. To bypass the cloning process, a simple, cloning-free reverse genetics platform based on the circular polymerase extension reaction (CPER) has been applied for positive-sense RNA viruses. In this study, we applied the CPER-based reverse genetics system for rabies virus (RABV), a mononegavirus, enabling rapid and flexible generation of recombinant RABVs, including mutant, reporter-expressing, and chimeric clones. Our approach greatly facilitates genetic engineering of RABV and provides a versatile framework that can be extended to other mononegaviruses, thereby accelerating both basic and applied virology research.

Rotavirus C (RVC) causes acute gastroenteritis in neonatal piglets. Despite the clinical importance of RVC infection, the distribution and prevalence in pig populations in most African countries remains unknown. In this study, we identified RVC in Zambian pigs by metagenomic analysis. The full genome sequence of the RVC revealed two different VP4 sequences, implying that two different RVC strains (ZP18-77-c1 and ZP18-77-c2) were present in the same sample. Genetic analyses demonstrated that all segments of ZP18-77-c1 and ZP1877-c2 showed high nucleotide sequence identities (87.7-94.5%) to known porcine RVC strains, and ZP18-77c1 and ZP18-77-c2 strains were assigned to genotype constellations, G1-P[4]/P[14]-I13-R5-C5-M1-A7-N9-T10E5-H1. We further screened RVC genomes among pig feces collected in Zambia (n = 147) by RT-qPCR, and 78 samples (53.1%) were positive. This study demonstrated the first full genome sequence of African RVC strains with a relatively high prevalence of RVC infection in the pig populations in Zambia.

Rotavirus B (RVB) causes diarrhea in humans and pigs. Although various RVB strains were identified in humans and various animals globally, little is known about the epidemiology RVB infection in Africa. In this study, we attempted to examine the prevalence of RVB infection in pig populations in Zambia. Metagenomic analyses were conducted on pig feces collected in Zambia to detect double stranded RNA viruses, including RVB. To clarify the prevalence of RVB infection in pig populations in Zambia, 147 fecal samples were screened for the RVB detection by RT-qPCR. Full genome sequence of a detected RVB was determined by Sanger sequencing and genetically analyzed. The metagenomic analyses revealed that RVB sequence reads and contigs of RVB were detected from one fecal sample collected from pigs in Zambia. RT-qPCR screening detected RVB genomes in 36.7

Members of the genus Borrelia are arthropod-borne spirochetes that are human and animal pathogens. Vertebrate hosts, including wild animals, are pivotal to the circulation and maintenance of Borrelia spirochetes. However, information on Borrelia spirochetes in vertebrate hosts in Zambia is limited. Thus, we aimed to investigate the presence of Borrelia spirochetes in wild animals and cattle in Zambia. A total of 140 wild animals of four species and 488 cattle DNA samples from /near the Kafue National Park were collected for real-time PCR screening, followed by characterization using three different genes with positive samples. Five impalas and 20 cattle tested positive using real-time PCR, and sequence analysis revealed that the detected Borrelia were identified to be Borrelia theileri, a causative agent of bovine borreliosis. This is the first evidence of Borrelia theileri in African wildlife and cattle in Zambia. Our results suggest that clinical differentiation between bovine borreliosis and other bovine diseases endemic in Zambia is required for better treatment and control measures. As this study only included wild and domestic animals in the Kafue ecosystem, further investigations in other areas and with more wildlife and livestock species are needed to clarify a comprehensive epidemiological status of Borrelia theileri in Zambia.

Highly pathogenic avian influenza viruses (HPAIVs) with H5 and H7 hemagglutinin (HA) subtypes are derived from their low pathogenic counterparts following the acquisition of multiple basic amino acids in their HA cleavage site. It has been suggested that consecutive adenine residues and a stem-loop structure in the viral RNA region that encodes the cleavage site are essential for the acquisition of the polybasic cleavage site. By using a reporter assay to detect non-templated nucleotide insertions, we found that insertions more frequently occurred in the RNA region (29 nucleotide-length) encoding the cleavage site of an H5 HA gene that was predicted to have a stem-loop structure containing consecutive adenines than in a mutated corresponding RNA region that had a disrupted loop structure with fewer adenines. In virus particles generated by using reverse genetics, nucleotide insertions that created additional codons for basic amino acids were found in the RNA region encoding the cleavage site of an H5 HA gene but not in the mutated RNA region. We confirmed the presence of virus clones with the ability to replicate without trypsin in a plaque assay and to cause lethal infection in chicks. These results demonstrate that the stem-loop structure containing consecutive adenines in HA genes is a key molecular determinant for the emergence of H5 HPAIVs.

Human metapneumovirus (HMPV) has been a notable etiological agent of acute respiratory infection in humans, but it was not discovered until 2001, because HMPV replicates only in a limited number of cell lines and the cytopathic effect (CPE) is often mild. To promote the study of HMPV, several groups have generated green fluorescent protein (GFP)-expressing recombinant HMPV strains (HMPVGFP). However, the growing evidence has complicated the understanding of cell line specificity of HMPV, because it seems to vary notably among HMPV strains. In addition, unique A2b clade HMPV strains with a 180-nucleotide duplication in the G gene (HMPV A2b(180nt-dup) strains) have recently been detected. In this study, we re-evaluated and compared the cell line specificity of clinical isolates of HMPV strains, including the novel HMPV A2b(180nt-dup) strains, and six recombinant HMPVGFP strains, including the newly generated recombinant HMPV A2b(180nt-dup) strain, MG0256-EGFP. Our data demonstrate that VeroE6 and LLC-MK2 cells generally showed the highest infectivity with any clinical isolates and recombinant HMPVGFP strains. Other human-derived cell lines (BEAS-2B, A549, HEK293, MNT-1, and HeLa cells) showed certain levels of infectivity with HMPV, but these were significantly lower than those of VeroE6 and LLC-MK2 cells. Also, the infectivity in these suboptimal cell lines varied greatly among HMPV strains. The variations were not directly related to HMPV genotypes, cell lines used for isolation and propagation, specific genome mutations, or nucleotide duplications in the G gene. Thus, these variations in suboptimal cell lines are likely intrinsic to particular HMPV strains.

Highly pathogenic avian influenza viruses with H5 and H7 hemagglutinin (HA) subtypes evolve from low-pathogenic precursors through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been observed to occur naturally only in these HA subtypes, little is known about the genetic basis for the acquisition of the polybasic HA cleavage site. Here we show that consecutive adenine residues and a stem-loop structure, which are frequently found in the viral RNA region encoding amino acids around the cleavage site of low-pathogenic H5 and H7 viruses isolated from waterfowl reservoirs, are important for nucleotide insertions into this RNA region. A reporter assay to detect nontemplated nucleotide insertions and deep-sequencing analysis of viral RNAs revealed that an increased number of adenine residues and enlarged stem-loop structure in the RNA region accelerated the multiple adenine and/or guanine insertions required to create codons for basic amino acids. Interestingly, nucleotide insertions associated with the HA cleavage site motif were not observed principally in the viral RNA of other subtypes tested (H1, H2, H3, and H4). Our findings suggest that the RNA editing-like activity is the key mechanism for nucleotide insertions, providing a clue as to why the acquisition of the polybasic HA cleavage site is restricted to the particular HA subtypes.IMPORTANCE Influenza A viruses are divided into subtypes based on the antigenicity of the viral surface glycoproteins hemagglutinin (HA) and neuraminidase. Of the 16 HA subtypes (H1 to -16) maintained in waterfowl reservoirs of influenza A viruses, H5 and H7 viruses often become highly pathogenic through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been known since the 1980s, the genetic basis for nucleotide insertions has remained unclear. This study shows the potential role of the viral RNA secondary structure for nucleotide insertions and demonstrates a key mechanism explaining why the acquisition of the polybasic HA cleavage site is restricted to particular HA subtypes in nature. Our findings will contribute to better understanding of the ecology of influenza A viruses and will also be useful for the development of genetically modified vaccines against H5 and H7 influenza A viruses with increased stability.

We analyzed the pathogenicity of various serotypes of Listeria monocytogenes using a Balb/c mouse intravenous injection model. The survival rates of mice inoculated with strains NS1/2b (serotype 1/2b), NS3b (serotype 3b) and NS 4b (serotype 4b) were 60, 63.6 and 63.6%, respectively. Although the survival rates were similar, the bacterial growth in the liver of NS3b-infected mice was 144.5-fold higher than that in the liver of NS4b-infected mice. Histopathological analyses suggest that the NS4b strain replicated more in monocytes/macrophages, whereas the NS3b strain replicated more in hepatocytes. These results raise a possibility that the serotype 4b strains replicated more in monocytes/macrophages compared to the other serotype strains. To assess this, we isolated CD11b-positive cells from mouse livers infected with EGDe (serotype 1/2a), NS1/2b, NS3b, NS4b and the serotype 4b strains 51414 and F17 and counted the number of live bacteria in these cells. CD11b-positive cells from the NS4b-, 51414- and F17-infected mice possessed 24.4- to 42.7-fold higher numbers of live bacteria than those from mice infected with EGDe and NS3b strains. These results suggest that serotype 4b strains replicated more in monocytes/macrophages than the other serotypes, and this may be involved in the pathogenicity of serotype 4b strains, particularly in the dissemination of L. monocytogenes through the host body.

Equine herpesvirus type 4 (EHV-4) is one of the most important pathogens in horses. To clarify the key genes of the EHV-4 genome that cause abortion in female horses, we determined the whole genome sequences of a laboratory strain and 7 Japanese EHV-4 isolates that were isolated from 2 aborted fetuses and nasal swabs of 5 horses with respiratory disease. The full genome sequences and predicted amino acid sequences of each gene of these isolates were compared with of the reference EHV-4 strain NS80567 and Australian isolates that were reported in 2015. The EHV-4 isolates clustered in 2 groups which did not reflect their pathogenicity. A comparison of the predicted amino acid sequences of the genes did not reveal any genes that were associated with EHV-4-induced abortion.

We examined the effects of encapsulated lactic acid bacteria administrated orally to lactating cattle on the intestinal flora. A dose of 3 X 10¹¹ colony forming unit (cfu) of freeze-dried Lactobacillus coryniformis subsp. torquens (JCM1099) encapsulated in an enteric capsule capable of bypassing the rumen was administered for seven days. DNA was extracted from feces 0 and 24 hr after daily administration. Metagenomic analysis showed an increasing trend of the alpha diversity, an index of the species diversity. Furthermore, principal component analysis of intestinal flora revealed that cattle could be differentiated by JCM1099 capsule and suspension administration via principal components 1, 2, and 3. We conclude that administration of encapsulated JCM1099 can alter the intestinal bacterial flora of cattle.

Marine Mammal ScienceVolume 31, Issue 2 p. 818-827 Notes Brown adipose tissue expresses uncoupling protein 1 in newborn harbor seals (Phoca vitulina) Yuta Sakurai, Yuta Sakurai Department of Aquatic Bioscience and Industry, Graduate School of Bio-Industry, Tokyo University of Agriculture, Abashiri, 099-2493 Japan Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, JapanSearch for more papers by this authorYuko Okamatsu-Ogura, Yuko Okamatsu-Ogura Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, JapanSearch for more papers by this authorMasayuki Saito, Masayuki Saito Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, JapanSearch for more papers by this authorKazuhiro Kimura, Corresponding Author Kazuhiro Kimura Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, JapanCorresponding author (e-mail: k-kimura@vetmed.hokudai.ac.jp).Search for more papers by this authorRyo Nakao, Ryo Nakao Research Center for Zoonosis Control, Hokkaido University, Sapporo 001-0020, JapanSearch for more papers by this authorAiko Ohnuma, Aiko Ohnuma Research Center for Zoonosis Control, Hokkaido University, Sapporo 001-0020, JapanSearch for more papers by this authorMari Kobayashi, Mari Kobayashi Department of Aquatic Bioscience and Industry, Graduate School of Bio-Industry, Tokyo University of Agriculture, Abashiri, 099-2493 Japan NPO Marine Wildlife Center of Japan, Abashiri, 093-0042 JapanSearch for more papers by this author Yuta Sakurai, Yuta Sakurai Department of Aquatic Bioscience and Industry, Graduate School of Bio-Industry, Tokyo University of Agriculture, Abashiri, 099-2493 Japan Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, JapanSearch for more papers by this authorYuko Okamatsu-Ogura, Yuko Okamatsu-Ogura Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, JapanSearch for more papers by this authorMasayuki Saito, Masayuki Saito Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, JapanSearch for more papers by this authorKazuhiro Kimura, Corresponding Author Kazuhiro Kimura Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, JapanCorresponding author (e-mail: k-kimura@vetmed.hokudai.ac.jp).Search for more papers by this authorRyo Nakao, Ryo Nakao Research Center for Zoonosis Control, Hokkaido University, Sapporo 001-0020, JapanSearch for more papers by this authorAiko Ohnuma, Aiko Ohnuma Research Center for Zoonosis Control, Hokkaido University, Sapporo 001-0020, JapanSearch for more papers by this authorMari Kobayashi, Mari Kobayashi Department of Aquatic Bioscience and Industry, Graduate School of Bio-Industry, Tokyo University of Agriculture, Abashiri, 099-2493 Japan NPO Marine Wildlife Center of Japan, Abashiri, 093-0042 JapanSearch for more papers by this author First published: 07 December 2014 https://doi.org/10.1111/mms.12193Citations: 2Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume31, Issue2April 2015Pages 818-827 RelatedInformation

The role played by bats as a potential source of transmission of Leptospira spp. to humans is poorly understood, despite various pathogenic Leptospira spp. being identified in these mammals. Here, we investigated the prevalence and diversity of pathogenic Leptospira spp. that infect the straw-colored fruit bat (Eidolon helvum). We captured this bat species, which is widely distributed in Africa, in Zambia during 2008–2013. We detected the flagellin B gene (flaB) from pathogenic Leptospira spp. in kidney samples from 79 of 529 E. helvum (14.9%) bats. Phylogenetic analysis of 70 flaB fragments amplified from E. helvum samples and previously reported sequences, revealed that 12 of the fragments grouped with Leptospira borgpetersenii and Leptospira kirschneri; however, the remaining 58 flaB fragments appeared not to be associated with any reported species. Additionally, the 16S ribosomal RNA gene (rrs) amplified from 27 randomly chosen flaB-positive samples was compared with previously reported sequences, including bat-derived Leptospira spp. All 27 rrs fragments clustered into a pathogenic group. Eight fragments were located in unique branches, the other 19 fragments were closely related to Leptospira spp. detected in bats. These results show that rrs sequences in bats are genetically related to each other without regional variation, suggesting that Leptospira are evolutionarily well-adapted to bats and have uniquely evolved in the bat population. Our study indicates that pathogenic Leptospira spp. in E. helvum in Zambia have unique genotypes.

Ticks are one of the most important blood-sucking vectors for infectious microorganisms in humans and animals. When feeding they inject saliva, containing microbes, into the host to facilitate the uptake of blood. An understanding of the microbial populations within their salivary glands would provide a valuable insight when evaluating the vectorial capacity of ticks. Three tick species (Ixodes ovatus, I. persulcatus and Haemaphysalis flava) were collected in Shizuoka Prefecture of Japan between 2008 and 2011. Each tick was dissected and the salivary glands removed. Bacterial communities in each salivary gland were characterized by 16S amplicon pyrosequencing using a 454 GS-Junior Next Generation Sequencer. The Ribosomal Database Project (RDP) Classifier was used to classify sequence reads at the genus level. The composition of the microbial populations of each tick species were assessed by principal component analysis (PCA) using the Metagenomics RAST (MG-RAST) metagenomic analysis tool. Rickettsia-specific PCR was used for the characterization of rickettsial species. Almost full length of 16S rDNA was amplified in order to characterize unclassified bacterial sequences obtained in I. persulcatus female samples. The numbers of bacterial genera identified for the tick species were 71 (I. ovatus), 127 (I. persulcatus) and 59 (H. flava). Eighteen bacterial genera were commonly detected in all tick species. The predominant bacterial genus observed in all tick species was Coxiella. Spiroplasma was detected in Ixodes, and not in H. flava. PCA revealed that microbial populations in tick salivary glands were different between tick species, indicating that host specificities may play an important role in determining the microbial complement. Four female I. persulcatus samples contained a high abundance of several sequences belonging to Alphaproteobacteria symbionts. This study revealed the microbial populations within the salivary glands of three species of ticks, and the results will contribute to the knowledge and prediction of emerging tick-borne diseases.

The cytochrome P450 (CYP) 1–3 families are involved in xenobiotic metabolism, and are expressed primarily in the liver. Ostriches (Struthio camelus) are members of Palaeognathae with the earliest divergence from other bird lineages. An understanding of genes coding for ostrich xenobiotic metabolizing enzyme contributes to knowledge regarding the xenobiotic metabolisms of other Palaeognathae birds. We investigated CYP1–3 genes expressed in female ostrich liver using a next-generation sequencer. We detected 10 CYP genes: CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2W2, CYP2AC1, CYP2AC2, CYP2AF1, and CYP3A37. We compared the gene expression levels of CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2AF1, and CYP3A37 in ostrich liver and determined that CYP2G19 exhibited the highest expression level. The mRNA expression level of CYP2G19 was approximately 2–10 times higher than those of other CYP genes. The other CYP genes displayed similar expression levels. Our results suggest that CYP2G19, which has not been a focus of previous bird studies, has an important role in ostrich xenobiotic metabolism.

In order to survey arenaviruses in the Republic of Zambia, we captured 335 rodents from three cities between 2010 and 2011. Eighteen Luna virus (LUNV) and one lymphocytic choriomeningitis virus (LCMV)-related virus RNAs were detected by one-step RT-PCR from Mastomys natalensis and Mus minutoides, respectively. Four LUNV strains and one LCMV-related virus were isolated, and the whole genome nucleotide sequence was determined by pyrosequencing. Phylogenetic analyses revealed that the LUNV clade consists of two branches that are distinguished by geographical location and that the LCMV-related virus belongs to the LCMV clade, but diverges from the typical LCMVs. Comparison of nucleoprotein amino acid sequences indicated that the LCMV-related virus could be designated a novel arenavirus, which was tentatively named as the Lunk virus. Amino acid sequences of the GP, NP, Z and L proteins showed poor similarity among the three Zambian arenavirus strains, i.e. Luna, Lunk and Lujo virus.

To investigate arenavirus in Zambia, we characterized virus from the kidneys of 5 arenavirus RNA-positive rodents (Mastomys natalensis) among 263 captured. Full-genome sequences of the viruses suggested that they were new strains similar to Lassa virus-related arenaviruses. Analyzing samples from additional rodents and other species can elucidate epizootiologic aspects of arenaviruses.

The present study was designed to determine the effects of physiological stress on milk-somatic cell counts (SCC) and function of bovine peripheral blood leukocytes (PBL). Nine healthy lactating cows were used in the examination. Five cows were transported 100 km for 4 hr (transported group; TG), and 4 cows were penned (non-transported group; NTG). Blood and milk samples were collected at 0, 2, and 4 hr after loading, and at 2 hr, and 1, 2, 3, and 6 days after unloading. The following activities were measured: adhesion receptor (CD 18 and L-selectin) expression of neutrophils and monocytes, migration capacity and percentage of apoptotic cells of neutrophils, serum soluble L-selectin (sL-selectin), plasma cortisol, and SCC. A significant increase in plasma cortisol and milk SCC was observed in TG. Leukocytosis, derived from neutrophils was recorded in TG, and was indicated by apoptotic measurement as an increase of young cells from the marginal pool. Increased migration and decreased surface expression of both L-selectin and CD 18 in neutrophils were observed after transportation. Elevated serum sL-selectin was also noted as a result of transportation. The present study indicated that transport stress modulates peripheral blood neutrophil function, particularly enhancing migration capacity, and causes diapedesis across the mammary epithelium. Increased milk SCC in transported cattle might be due to these phenomena, and severe physiological stress may bring about an increase in SCC in milk.

Eight splenectomized calves were inoculated with Theileria orientalis sergenti (Tos)-infected tick gland homogenate (5 calves) or infected erythrocyte suspension (3 calves). Clinical characteristics were different in calves post-infection. Animals were divided into 3 groups on the basis of susceptibility as high, middle, and low. Increase in mRNA of IFN-gamma, IL-2, and TNF-alpha was observed in peripheral blood mononuclear cells at the peak of infection and was seen to be related with pyrexia and parasitemia. Expression of IL-1, IL-4, and inducible nitric oxide synthase was not observed. Decreased plasma nitrite/nitrate level was observed in the groups. The results of this study indicate that Th1 response is the predominant response in Tos infection, and this response is also related with their clinical characteristics.