Molecular characterization of an avian rotavirus a strain detected from a large-billed crow (Corvus macrorhynchos) in Japan.

Avian rotaviruses A (RVAs) are occasionally transmitted to animals other than the original hosts across species barriers. Information on RVAs carried by various bird species is important for identifying the origin of such interspecies transmission. In this study, to facilitate an understanding of the ecology of RVAs from wild birds, we characterized all of the genes of an RVA strain, JC-105, that was detected in a fecal sample of a large-billed crow (Corvus macrorhynchos) in Japan. All of the genes of this strain except for the VP4 and VP7 genes, which were classified as novel genotypes (P[56] and G40, respectively), were closely related to those of the avian-like RVA strain detected from a raccoon, indicating the possibility that crows had been involved in the transmission of avian RVAs to raccoons. Our findings highlight the need for further viral investigations in wild birds and mammals to understand the mechanisms of avian-to-mammal RVA transmission.

Coordinated regulation of phosphatidylinositol 4-phosphate and phosphatidylserine levels by Osh4p and Osh5p is an essential regulatory mechanism in autophagy.

Macroautophagy (hereafter autophagy) is an intracellular degradative pathway in budding yeast cells. Certain lipid types play essential roles in autophagy; yet the precise mechanisms regulating lipid composition during autophagy remain unknown. Here, we explored the role of the Osh family proteins in the modulating lipid composition during autophagy in budding yeast. Our results showed that osh1-osh7∆ deletions lead to autophagic dysfunction, with impaired GFP-Atg8 processing and the absence of autophagosomes and autophagic bodies in the cytosol and vacuole, respectively. Freeze-fracture electron microscopy (EM) revealed elevated phosphatidylinositol 4-phosphate (PtdIns(4)P) levels in cytoplasmic and luminal leaflets of autophagic bodies and vacuolar membranes in all deletion mutants. Phosphatidylserine (PtdSer) levels were significantly decreased in the autophagic bodies and vacuolar membranes in osh4∆ and osh5∆ mutants, whereas no significant changes were observed in other osh deletion mutants. Furthermore, we identified defects in autophagic processes in the osh4∆ and osh5∆ mutants, including rare autophagosome formation in the osh5∆ mutant and accumulation of autophagic bodies in the vacuole in the osh4∆ mutant, even in the absence of the proteinase inhibitor PMSF. These findings suggest that Osh4p and Osh5p play crucial roles in the transport of PtdSer to autophagic bodies and autophagosome membranes, respectively. The precise control of lipid composition in the membranes of autophagosomes and autophagic bodies by Osh4p and Osh5p represents an important regulatory mechanism in autophagy.

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.

Chronic Toxoplasma infection affects gene expression of drug-metabolizing enzymes in mouse liver.

Toxoplasma gondii is an intracellular protozoan parasite causing toxoplasmosis, an infectious disease affecting warm-blooded vertebrates worldwide. Many drug-metabolizing enzymes are located in the liver, a major organ of drug metabolism, and their function can be affected by pathogen infection.Using next-generation sequencing (RNA-seq) and quantitative polymerase chain reaction (qPCR), changes in the hepatic expressions of drug-metabolizing enzymes were analysed in mice chronically infected with T. gondii. The analysis found that, among drug-metabolizing enzymes, 22 genes were upregulated and 28 genes were downregulated (≥1.5-fold); of these 5 and 17 genes, respectively, were cytochromes P450 (Cyp or P450).Subsequent qPCR analysis showed that six P450 genes were upregulated significantly (≥1.5-fold, p < 0.05), namely, Cyp1b1, Cyp2c29, Cyp2c65, Cyp2d9, Cyp2d12, and Cyp3a59, whereas nine P450 genes were downregulated significantly (≥1.5-fold, p < 0.05), namely, Cyp2c38, Cyp2c39, Cyp2c44, Cyp2c69, Cyp2d40, Cyp2e1, Cyp3a11, Cyp3a41, and Cyp3a44.Moreover, metabolic assays in infected mouse liver using typical P450 substrates revealed that midazolam 1'-hydroxylation and testosterone 2-hydroxylation activities decreased significantly (≥1.5-fold, p < 0.05), whereas testosterone 16-hydroxylation activity increased significantly (≥1.5-fold, p < 0.05).Chronic Toxoplasma infection affects drug metabolism, at least partly, by altering the gene expressions of drug-metabolizing enzymes, including P450s.

Record of Cryptosporidium serpentis from Goniurosaurus kuroiwae sengokui (Reptilia, Squamata, Eublepharidae) in Tokashikijima, Okinawa Prefecture, Japan.

In reptiles, infection with Cryptosporidium parasite can be lethal, especially in species of family Eublepharidae such as Eublepharis macularius and Goniurosaurus splendes species. We investigated the presence of Cryptosporidium spp. in wild reptiles, especially Goniurosaurus spp., in five islands of Japan. Nested PCR was performed to detect Cryptosporidium spp. in 38 fecal samples of 9 reptile species from five islands of Japan, and 3 (3/8) positive samples of Goniurosaurus kuroiwae sengokui from Tokashikijima, Okinawa Prefecture were detected. A BLAST search of the detected gene sequences revealed that the all 3 samples were identified as Cryptosporidium serpentis, a reptile-specific Cryptosporidium parasite. This is the first record of Cryptosporidium from a wild Goniurosaurus species in Japan and worldwide.

Unique asymmetric distribution of phosphatidylserine and phosphatidylethanolamine in Toxoplasma gondii revealed by nanoscale analysis.

Toxoplasma gondii is a highly prevalent obligate apicomplexan parasite that is important in clinical and veterinary medicine. It is known that glycerophospholipids phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn), especially their expression levels and flip-flops between cytoplasmic and exoplasmic leaflets, in the membrane of T. gondii play important roles in efficient growth in host mammalian cells, but their distributions have still not been determined because of technical difficulties in studying intracellular lipid distribution at the nanometer level. In this study, we developed an electron microscopy method that enabled us to determine the distributions of PtdSer and PtdEtn in individual leaflets of cellular membranes by using quick-freeze freeze-fracture replica labeling. Our findings show that PtdSer and PtdEtn are asymmetrically distributed, with substantial amounts localized at the luminal leaflet of the inner membrane complex (IMC), which comprises flattened vesicles located just underneath the plasma membrane (see Figs. 2B and 7). We also found that PtdSer was absent in the cytoplasmic leaflet of the inner IMC membrane, but was present in considerable amounts in the cytoplasmic leaflet of the middle IMC membrane, suggesting a barrier-like mechanism preventing the diffusion of PtdSer in the cytoplasmic leaflets of the two membranes. In addition, the expression levels of both PtdSer and PtdEtn in the luminal leaflet of the IMC membrane in the highly virulent RH strain were higher than those in the less virulent PLK strain. We also found that the amount of glycolipid GM3, a lipid raft component, was higher in the RH strain than in the PLK strain. These results suggest a correlation between lipid raft maintenance, virulence, and the expression levels of PtdSer and PtdEtn in T. gondii.

Generation and characterization of a genetically modified live rabies vaccine strain with attenuating mutations in multiple viral proteins and evaluation of its potency in dogs.

Live rabies vaccines have advantageous features that can facilitate mass vaccination for dogs, the most important reservoirs/transmitters of rabies. However, some live vaccine strains have problems in their safety, namely, risks from the residual pathogenicity and the pathogenic reversion of live vaccine strains. The reverse genetics system of rabies virus provides a feasible option to improve the safety of a live vaccine strain by, for example, artificially introducing attenuating mutations into multiple viral proteins. It was previously demonstrated in separate studies that introduction of amino acid residues Leu at position 333 in the viral glycoprotein (G333), Ser at G194, and Leu/His at positions 273/394 in the nucleoprotein (N273/394) enhance the safety of a live vaccine strain. In this study, to test our hypothesis that combinational introduction of these residues would significantly increase the safety level of a vaccine strain, we generated a novel live vaccine candidate, ERA-NG2, that is attenuated by mutations at N273/394 and G194/333, and we examined its safety and immunogenicity in mice and dogs. ERA-NG2 did not cause any clinical signs in mice after intracerebral inoculation. After 10 passages in suckling mouse brains, ERA-NG2 retained all of the introduced mutations except the mutation at N394 and the highly attenuated phenotype. These findings indicate that the ERA-NG2 is highly and stably attenuated. After confirming that ERA-NG2 induced a virus-neutralizing antibody (VNA) response and protective immunity in mice, we immunized dogs intramuscularly with a single dose (105-7 focus-forming units) of ERA-NG2 and found that, at all of the tested doses, the strain induced a VNA response in dogs without inducing any clinical signs. These findings demonstrate that ERA-NG2 has a high level of safety and a substantial level of immunogenicity in dogs and thus is a promising live vaccine candidate that can facilitate vaccination in dogs.

Avian paramyxovirus serotype-1 isolation from migratory birds and environmental water in southern Japan: An epidemiological survey during the 2018/19-2021/2022 winter seasons.

Newcastle disease caused by highly pathogenic viruses of avian paramyxovirus serotype-1 (APMV-1) is a highly contagious poultry disease. Although a large-scale epidemic of Newcastle disease had occurred in Japan between the 1950s and the 2000s, there have been no outbreaks anywhere since 2010. In addition, there are no reports of epidemiological surveys of APMV-1 in wild birds in Japan in the last 10 years. We conducted the first epidemiological survey of APMV-1 in the Izumi plain, Kagoshima prefecture of southern Japan from the winter of 2018 to 2022. A total of 15 APMV-1 strains were isolated, and isolation rates from roosting water and duck fecal samples were 2.51% and 0.10%, respectively. These results indicate that the isolation method from environmental water may be useful for efficient surveillance of APMV-1 in wild birds. Furthermore, this is the first report on the success of APMV-1 isolation from environmental water samples. Genetic analysis of the Fusion (F) gene showed that all APMV-1 isolates were closely related to virus strains circulating among waterfowl in Far East Asian countries. All isolates have avirulent motifs in their cleavage site of F genes, all of which were presumed to be low pathogenic viruses in poultry. However, pathogenicity test using embryonated chicken eggs demonstrated that some isolates killed all chicken embryos regardless of viral doses inoculated (102 -106 50% egg infectious dose). These results indicated that APMV-1 strains, which are potentially pathogenic to chickens, are continuously brought into the Izumi plain by migrating wild birds.

Genetic characterization of bovine respiratory syncytial viruses in Japan between 2017 and 2019.

Bovine respiratory syncytial virus (BRSV) strains that were detected in Kagoshima prefecture and isolated in Hokkaido between 2017 and 2019, together with a BRSV vaccine strain, were subjected to full-genome sequencing. The BRSV strains identified in Japan were found to be genetically close to each other but distant from the vaccine strains. The deduced amino acids at positions 206 and 208 of the glycoprotein (G protein), which form one of the major epitopes of the recent Japanese BRSV strains, were different from those of the vaccine strains. Therefore, the recent Japanese BRSV strains might be antigenically different from the BRSV vaccine strains.

Molecular characterisation of a novel avian rotavirus A strain detected from a gull species (Larus sp.).

A recent study demonstrated the possibility that migratory birds are responsible for the global spread of avian rotavirus A (RVA). However, little is known about what types of RVAs are retained in migratory birds. In this study, to obtain information on RVA strains in migratory birds, we characterised an RVA strain, Ho374, that was detected in a faecal sample from a gull species (Larus sp.). Genetic analysis revealed that all 11 genes of this strain were classified as new genotypes (G28-P[39]-I21-R14-C14-M13-A24-N14-T16-E21-H16). This clearly indicates that the genetic diversity of avian RVAs is greater than previously recognised. Our findings highlight the need for investigations of RVA strains retained in migratory birds, including gulls.

Establishment of a reverse genetics system for rabies virus strain Komatsugawa.

The rabies virus strain Komatsugawa isolated from a dog in Tokyo in the 1940s retains biological properties as a field strain, providing an effective model for studying rabies pathogenesis. To facilitate molecular studies on the pathogenesis, this study aimed to establish a reverse genetics system for the Komatsugawa strain. By transfecting the full-length genome plasmid of this strain, infectious virus with artificially introduced genetic markers in its genome was rescued. The recombinant strain had biological properties similar to those of the original strain. These findings indicate that a reverse genetics system for the Komatsugawa strain has successfully been established.

The Amino Acid at Position 95 in the Matrix Protein of Rabies Virus Is Involved in Antiviral Stress Granule Formation in Infected Cells.

Stress granules (SGs) are dynamic structures that store cytosolic messenger ribonucleoproteins. SGs have recently been shown to serve as a platform for activating antiviral innate immunity; however, several pathogenic viruses suppress SG formation to evade innate immunity. In this study, we investigated the relationship between rabies virus (RABV) virulence and SG formation, using viral strains with different levels of virulence. We found that the virulent Nishigahara strain did not induce SG formation, but its avirulent offshoot, the Ni-CE strain, strongly induced SG formation. Furthermore, we demonstrated that the amino acid at position 95 in the RABV matrix protein (M95), a pathogenic determinant for the Nishigahara strain, plays a key role in inhibiting SG formation, followed by protein kinase R (PKR)-dependent phosphorylation of the α subunit of eukaryotic initiation factor 2α (eIF2α). M95 was also implicated in the accumulation of RIG-I, a viral RNA sensor protein, in SGs and in the subsequent acceleration of interferon induction. Taken together, our findings strongly suggest that M95-related inhibition of SG formation contributes to the pathogenesis of RABV by allowing the virus to evade the innate immune responses of the host. IMPORTANCE Rabies virus (RABV) is a neglected zoonotic pathogen that causes lethal infections in almost all mammalian hosts, including humans. Recently, RABV has been reported to induce intracellular formation of stress granules (SGs), also known as platforms that activate innate immune responses. However, the relationship between SG formation capacity and pathogenicity of RABV has remained unclear. In this study, by comparing two RABV strains with completely different levels of virulence, we found that the amino acid mutation from valine to alanine at position 95 of matrix protein (M95), which is known to be one of the amino acid mutations that determine the difference in virulence between the strains, plays a major role in SG formation. Importantly, M95 was involved in the accumulation of RIG-I in SGs and in promoting interferon induction. These findings are the first report of the effect of a single amino acid substitution associated with SGs on viral virulence.

Improved method for avian influenza virus isolation from environmental water samples.

Environmental water-targeted surveillance of migratory aquatic birds at overwintering sites is potentially one of the most effective approaches for understanding the ecology of avian influenza viruses (AIVs). In this study, we improved the method for AIV isolation from environmental water samples by making a minor modification to our previously reported process. We experimentally demonstrated that the AIV recovery efficiency of the modified method was 10-100-fold higher than that of the original method. This improved isolation method allowed us to isolate a considerably larger number of AIV isolates from environmental water samples collected at an overwintering site for tens of thousands of migratory aquatic birds in Japan during the 2018/2019 winter season, compared with those during previous winter seasons. Genetic and phylogenetic analyses revealed that AIVs of the same subtypes with multiple genetic constellations were circulating in a single overwintering site during a single winter season. These findings indicate that our improved isolation method contributes to enhance environmental water-targeted surveillance and to a better understanding of AIV ecology in migratory aquatic bird populations by monitoring ongoing AIV circulation.

Establishment of a reverse genetics system for avian rotavirus A strain PO-13.

Avian rotavirus A (RVA) is one of major enteric pathogens that cause diarrhoea in young avian individuals. Importantly, some of the avian RVA strains of G18P[17] genotype are naturally transmitted to and cause clinical diseases in mammalian species, indicating their potential risks to animal health. Although molecular information on the pathogenesis by avian RVA strains will be useful for estimating their risks, the absence of a reverse genetics (RG) system for these strains has hindered the elucidation of their pathogenic mechanisms. In this study, we aimed to establish an RG system for the avian G18P[17] prototype strain PO-13, which was isolated from a pigeon in Japan in 1983 and was experimentally shown to be pathogenic in suckling mice. Transfection with plasmids expressing 11 genomic RNA segments of the strain resulted in rescue of the infectious virus with an artificially introduced genetic marker on its genome, indicating that an RG system for the PO-13 strain was successfully established. The rescued recombinant strain rPO-13 had biological properties almost identical to those of its wild-type strain (wtPO-13). Notably, both rPO-13 and wtPO-13 induced diarrhoea in suckling mice with similar efficiencies. It was thus demonstrated that the RG system will be useful for elucidating the pathogenic mechanisms of the PO-13 strain at the molecular level. This is the first report of the establishment of an RG system for an avian RVA strain.

Prevalence and organ tropism of crane-associated adenovirus 1 in cranes overwintering on the Izumi plain, Japan.

Crane-associated adenovirus 1 (CrAdV-1) is a proposed novel virus in the genus Aviadenovirus, first detected in faecal samples from hooded cranes (a vulnerable crane species) on the Izumi plain, a major overwintering site for migratory cranes in Japan. CrAdV-1 was genetically characterized in that study; however, its virological characteristics remain largely unclear. To investigate the prevalence and organ tropism of CrAdV-1, we collected swab and organ samples from dead or debilitated cranes on the Izumi plain. CrAdV-1 gene was detected in 47% (45/95) of tested cranes, comprising mainly hooded cranes but also white-naped and sandhill cranes. These results indicate that CrAdV-1 shedding is widespread among cranes overwintering on the Izumi plain. Phylogenetic analyses revealed that the 68 nucleotide sequences determined from the positive swabs formed a single cluster, suggesting phylogenetic differences between CrAdV-1 and other aviadenoviruses. CrAdV-1 prevalence showed a significant linear increase with time through the overwintering period (November to February), especially among juveniles. These findings indicate that CrAdV-1 spreads mainly by transmission between juveniles progressively through the overwintering period. The CrAdV-1 gene-positive rate was significantly higher in cloacal swabs than conjunctival or tracheal swabs. Copy numbers for the partial CrAdV-1 gene sequence were markedly high in the colon samples from three of the four cranes investigated for organ tropism. We also detected relatively high copy numbers in the cerebrum, trachea, lung and heart, suggesting that CrAdV-1 mainly targets these four organs and transmitted via the faecal-oral route and airborne transmission. These results contribute to further understanding of the virological characteristics of CrAdV-1.

Protozoan and Rickettsial Pathogens in Ticks Collected from Infested Cattle from Turkey.

Diseases caused by tick-transmitted pathogens including bacteria, viruses, and protozoa are of veterinary and medical importance, especially in tropical and subtropical regions including Turkey. Hence, molecular surveillance of tick-borne diseases will improve the understanding of their distribution towards effective control. This study aimed to investigate the presence and perform molecular characterization of Babesia sp., Theileria sp., Anaplasma sp., Ehrlichia sp., and Rickettsia sp. in tick species collected from cattle in five provinces of Turkey. A total of 277 adult ticks (males and females) were collected. After microscopic identification, tick pools were generated according to tick species, host animal, and sampling sites prior to DNA extraction. Molecular identification of the tick species was conducted through PCR assays. Out of 90 DNA pools, 57.8% (52/90) were detected to harbor at least 1 pathogen. The most frequently-detected pathogens were Babesia bovis, with a minimum detection rate of 7.9%, followed by Ehrlichia sp. (7.2%), Theileria annulata (5.8%), Coxiella sp. (3.3%), Anaplasma marginale (2.5%), Rickettsia sp. (2.5%), and B. occultans (0.7%). Rickettsia sp. identified in this study include Candidatus Rickettsia barbariae, R. aeschlimannii, and Rickettsia sp. Chad. All sequences obtained from this study showed 99.05−100% nucleotide identity with those deposited in GenBank (query cover range: 89−100%). This is the first molecular detection of Rickettsia sp. Chad, a variant of Astrakhan fever rickettsia, in Turkey. Results from this survey provide a reference for the distribution of ticks and tick-borne pathogens in cattle and expand the knowledge of tick-borne diseases in Turkey.

Essential roles of phosphatidylinositol 4-phosphate phosphatases Sac1p and Sjl3p in yeast autophagosome formation.

Autophagy is regulated by phosphoinositides. We have previously shown that phosphatidylinositol 4-phosphate (PtdIns(4)P) is localized in the autophagosomal membrane. Additionally, in yeast cells, phosphatidylinositol 4-kinases Pik1p and Stt4p play important roles in the formation of the autophagosome and its fusion with the vacuole, respectively. In this study, we analyzed the primary role of PtdIns(4)P phosphatases in yeast autophagy. The PtdIns(4)P labeling densities in the membranes of the vacuoles, mitochondria, nucleus, endoplasmic reticulum, and plasma membrane dramatically increased in the phosphatase deletion mutants sac1∆ and sjl3∆, and the temperature-sensitive mutant sac1ts/sjl3∆ at the restrictive temperature. GFP-Atg8 processing assay indicated defective autophagy in the sac1∆ and sac1ts/sjl3∆ mutants. In contrast to the localization of PtdIns(4)P in the luminal leaflet of autophagosomal membranes in the wild-type yeast, PtdIns(4)P was localized in both the luminal and cytoplasmic leaflets of the autophagosomal membranes in the sac1∆ strain. In addition, the number of autophagic bodies in the vacuole significantly decreased in the sac1∆ strain, although autophagosomes were present in the cytoplasm. In the sac1ts/sjl3∆ strain, the number of autophagosomes in the cytoplasm dramatically decreased at the restrictive temperature. Considering that the numbers of autophagosomes and autophagic bodies in the sjl3∆ strain were comparable to those in the wild-type yeast, we found that the autophagosome could not be formed when PtdIns(4)P phosphatase activities of both Sac1p and Sjl3p were diminished. Together, these results indicate that the turnover of PtdIns(4)P by phosphatases is essential for autophagosome biogenesis.

Molecular Detection and Characterization of Babesia and Theileria in Cattle and Water Buffaloes from Southern Luzon, Philippines.

Babesia and Theileria are tick-borne protozoan parasites that can cause significant economic losses in the cattle industry. This study aimed to contribute to the limited epidemiological data on Theileria orientalis as well as Babesia bigemina and B. bovis in large ruminants in the Philippines. Blood samples of 412 cattle and 108 water buffalo collected from four provinces in Southern Luzon, Philippines, were initially tested for the 18S rRNA gene of piroplasms through nested PCR. Positive samples were further subjected to species-specific PCR. The 18s rRNA of piroplasms was detected in 123 (29.9%) cattle and three (2.8%) water buffaloes. Theileria orientalis was found to be the most common piroplasm in cattle with a detection rate of 17.5%, followed by Babesia bovis and B. bigemina. Co-infections were also observed. Two water buffaloes were found infected with B. bovis, while one was positive for B. bigemina. The phylogenetic tree for B. bovis showed clustering of the isolates in two clades together with isolates from other countries, and a third separate clade. Meanwhile, the T. orientalis isolates in this study were distributed in three clades together with reported isolates from other countries. This study confirms the presence of T. orientalis in the Philippines and reports the genetic diversity of B. bovis and T. orientalis.

Characterization of an avian rotavirus A strain isolated from a velvet scoter (Melanitta fusca): implication for the role of migratory birds in global spread of avian rotaviruses.

Avian G18P[17] rotaviruses with similar complete genome constellation, including strains that showed pathogenicity in mammals, have been detected worldwide. However, it remains unclear how these strains spread geographically. In this study, to investigate the role of migratory birds in the dispersion of avian rotaviruses, we analysed whole genetic characters of the rotavirus strain RK1 that was isolated from a migratory species of birds [velvet scoter (Melanitta fusca)] in Japan in 1989. Genetic analyses revealed that the genotype constellation of the RK1 strain, G18-P[17]-I4-R4-C4-M4-A21-N4-T4-E4-H4, was highly consistent with those of other G18P[17] strains detected in various parts of the world, supporting the possibility that the G18P[17] strains spread via migratory birds that move over a wide area. Furthermore, the RK1 strain induced diarrhoea in suckling mice after oral gastric inoculation, indicating that at least some of the rotaviruses that originated from migratory birds are infectious to and pathogenic in mammals. In conclusion, it was demonstrated that migratory birds may contribute to the global spread of avian rotaviruses that are pathogenic in mammalian species.

Characterization of Single-Chain Fv Fragments of Neutralizing Antibodies to Rabies Virus Glycoprotein.

Rabies has almost a 100% case-fatality rate and kills more than 59,000 people annually around the world. There is no established treatment for rabies. The rabies virus (RABV) expresses only the glycoprotein (RABVG) at the viral surface, and it is the target for the neutralizing antibodies. We previously established mouse monoclonal antibodies, 15-13 and 12-22, which showed neutralizing activity against the RABV, targeting the sequential and conformational epitopes on the RABVG, respectively. However, the molecular basis for the neutralizing activity of these antibodies is not yet fully understood. In this study, we evaluated the binding characteristics of the Fab fragments of the 15-13 and 12-22 antibodies. The recombinant RABVG protein, in prefusion form for the binding analysis, was prepared by the silkworm-baculovirus expression system. Biolayer interferometry (BLI) analysis indicated that the 15-13 Fab interacts with the RABVG, with a KD value at the nM level, and that the 12-22 Fab has a weaker binding affinity (KD ~ µM) with the RABVG compared to the 15-13 Fab. Furthermore, we determined the amino acid sequences of both the antibodies and the designed single-chain Fv fragments (scFvs) of the 15-13 and 12-22 antibodies as another potential biopharmaceutical for targeting rabies. The 15-13 and 12-22 scFvs were successfully prepared by the refolding method and were shown to interact with the RABVG at the nM level and the µM level of the KD, respectively. These binding characteristics were similar to that of each Fab. On the other hand, differential scanning fluorometry (DSF) revealed that the thermal stability of these scFvs decreases compared to their Fabs. While the improvement of the stability of scFvs will still be required, these results provide insights into the neutralizing activity and the potential therapeutic use of antibody fragments for RABV infection.