Primary and immortalized cell lines derived from the Amami rabbit (Pentalagus furnessi) and evolutionally conserved cell cycle control with CDK4 and Cyclin D1.

The Amami rabbit (Pentagulus furnessi) is a dark brown-furred rabbit classified as an endangered species and only found in the Amami Islands of Japan. They are often called living fossils because they retain primitive characteristics of ancient rabbits that lived approximately 1 million years ago, such as short feet and hind legs and small ears. Although the ancient rabbit has disappeared due to the competition with European rabbit (Oryctolagus cuniculus) in the most of the Asian area, Amami rabbit survived since Amami Islands has isolated from Japan and Taiwan. Although Amari rabbit is one of the protected animals, their population decreases each year due to human activities, such as deforestation and roadkill. In this study, we collected roadkill samples of Amami rabbits and established primary and immortalized fibroblast cell lines. Combined expression of human-derived mutant Cyclin-dependent kinase 4, Cyclin D1, and hTERT allowed us to immortalize fibroblasts successfully in three individuals of Amami rabbits. The immortalized fibroblasts dramatically extended the cell culture period, when it was compared with the cell culture period of wild type cells. Furthermore, the immortalized cells maintained their normal chromosomal pattern (2n = 46). Our results suggest that cellular senescence which mainly regulated by p16-RB signaling pathway is conserved in animal evolution at least from 1 million years ago.

Extended proliferation of chicken- and Okinawa rail-derived fibroblasts by expression of cell cycle regulators.

Although immortalized cultured cells are useful for various functional assays or transcriptome analysis, highly efficient and reproducible immortalization methods have not been developed in avian-derived cells. We introduced the simian virus 40 T antigen (SV40T) and human papillomavirus (HPV)-E6E7 to chick and Okinawa rail (endangered species)derived fibroblast. As a result, neither the SV40T nor E6E7 genes could induce avian cell immortality. Accordingly, we attempted to use a recently developed immortalization method, which involved the coexpression of mutant cyclin-dependent kinase 4 (CDK4), Cyclin D, and TERT (K4DT method) in these avian cells. Although the K4DT method could not efficiently induce the efficient immortalization in mass cell population, cellular division until the senescence was significantly extended by K4DT, we succeeded to obtain the immortalized avian cells (chick K4DT: one clone, Okinawa rail K4DT: three clones, Okinawa rail K4DT + telomerase RNA component: one clone) with K4DT expression. We conclude that K4DT expression is used to extend the cell division and immortalization of avian-derived cells.

Chick derived induced pluripotent stem cells by the poly-cistronic transposon with enhanced transcriptional activity.

Induced pluripotent stem (iPS) cell technology lead terminally differentiated cells into the pluripotent stem cells through the expression of defined reprogramming factors. Although, iPS cells have been established in a number of mammalian species, including mouse, human, and monkey, studies on iPS cells in avian species are still very limited. To establish chick iPS cells, six factors were used within the poly-cistronic reprogramming vector (PB-R6F), containing M3O (MyoD derived transactivation domain fused with Oct3/4), Sox2, Klf4, c-Myc, Lin28, and Nanog. The PB-R6F derived iPS cells were alkaline-phosphatase and SSEA-1 positive, which are markers of pluripotency. Elevated levels of endogenous Oct3/4 and Nanog genes were detected in the established iPS cells, suggesting the activation of the FGF signaling pathway is critical for the pluripotent status. Histological analysis of teratoma revealed that the established chick iPS cells have differentiation ability into three-germ-layer derived tissues. This is the first report of establishment of avian derived iPS cells with a single poly-cistronic transposon based expression system. The establishment of avian derived iPS cells could contribute to the genetic conservation and modification of avian species.

Expression of Six Proteins Causes Reprogramming of Porcine Fibroblasts Into Induced Pluripotent Stem Cells With Both Active X Chromosomes.

In this study, we created porcine-induced pluripotent stem (iPS) cells with the expression of six reprogramming factors (Oct3/4, Klf4, Sox2, c-Myc, Lin28, and Nanog). The resulting cells showed growth dependent on LIF (leukemia inhibitory factor) and expression of multiple stem cell markers. Furthermore, the iPS cells caused teratoma formation with three layers of differentiation and had both active X chromosomes (XaXa). Our iPS cells satisfied the both of important characteristics of stem cells: teratoma formation and activation of both X chromosomes. Injection of these iPS cells into morula stage embryos showed that these cells participate in the early stage of porcine embryogenesis. Furthermore, the RNA-Seq analysis detected that expression levels of endogenous pluripotent related genes, NANOG, SOX2, ZFP42, OCT3/4, ESRRB, and ERAS were much higher in iPS with six factors than that with four reprogramming factors. We can conclude that the expression of six reprogramming factors enables the creation of porcine iPS cells, which is partially close to naive iPS state. J. Cell. Biochem. 118: 537-553, 2017. © 2016 Wiley Periodicals, Inc.

Characterization of Highly Pathogenic Avian Influenza Virus A(H5N6), Japan, November 2016.

Highly pathogenic avian influenza viruses (HPAIVs) A(H5N6) were concurrently introduced into several distant regions of Japan in November 2016. These viruses were classified into the genetic clade 2.3.4.4c and were genetically closely related to H5N6 HPAIVs recently isolated in South Korea and China. In addition, these HPAIVs showed further antigenic drift.

Genetic variation of major histocompatibility complex genes in the endangered red-crowned crane.

Populations that have drastically decreased in the past often have low genetic variation, which may increase the risk of extinction. The genes of major histocompatibility complex (MHC) play an important role in the adaptive immune response of jawed vertebrates. Maintenance of adaptive genetic diversity such as that of MHC genes is important for wildlife conservation. Here, we determined genotypes of exon 3 of MHC class IA genes (MHCIA) and exon 2 of MHC class IIB genes (MHCIIB) to evaluate genetic variation of the endangered red-crowned crane population on Hokkaido Island, Japan, which experienced severe population decline in the past. We identified 16 and 6 alleles of MHCIA and MHCIIB, respectively, from 152 individuals. We found evidence of a positive selection at the antigen-binding sites in MHCIA exon 3 and MHCIIB exon 2. The phylogenetic analyses indicated evidence of trans-species polymorphism among the crane MHC genes. The genetic variability in both classes of MHC genes at the population level was low. No geographic structure was found based on the genetic diversity of microsatellite and MHC genes. Our study provides useful data for the optimal management of the red-crowned crane population in Hokkaido and can contribute to future studies on MHC genes of the continental populations of the red-crowned crane and other crane species.

Gene duplication and concerted evolution of mitochondrial DNA in crane species.

The gene duplication in mitochondrial DNA (mtDNA) has been reported in diverse bird taxa so far. Although many phylogenetic and population genetic analyses of cranes were carried out based on mtDNA diversity, whether mtDNA contains duplicated regions is unknown. To address the presence or absence of gene duplication in cranes and investigate the molecular evolutionary features of crane mtDNA, we analyzed the gene organization and the molecular phylogeny of mtDNA from 13 crane species. We found that the mtDNA in 13 crane species shared a tandem duplicated region, which consists of duplicated sequence sets including cytochrome b (Cytb), NADH6, control region (CR) and three genes of tRNA. The gene order in the duplicated region was identical among all the 13 crane species, and the nucleotide sequences found within each individual showed high similarities. In addition, phylogenetic trees based on homologous sequences of CR and Cytb indicated the possibility of concerted evolution among the duplicated genes. The results suggested that the duplication event occurred in the common ancestor of crane species or some older ancestors.

Characterization of H5N6 highly pathogenic avian influenza viruses isolated from wild and captive birds in the winter season of 2016-2017 in Northern Japan.

On 15 November 2016, a black swan that had died in a zoo in Akita prefecture, northern Japan, was strongly suspected to have highly pathogenic avian influenza (HPAI); an HPAI virus (HPAIV) belonging to the H5N6 subtype was isolated from specimens taken from the bird. After the initial report, 230 cases of HPAI caused by H5N6 viruses from wild birds, captive birds, and domestic poultry farms were reported throughout the country during the winter season. In the present study, 66 H5N6 HPAIVs isolated from northern Japan were further characterized. Phylogenetic analysis of the hemagglutinin gene showed that the H5N6 viruses isolated in northern Japan clustered into Group C of Clade 2.3.4.4 together with other isolates collected in Japan, Korea and Taiwan during the winter season of 2016-2017. The antigenicity of the Japanese H5N6 isolate differed slightly from that of HPAIVs isolated previously in Japan and China. The virus exhibited high pathogenicity and a high replication capacity in chickens, whereas virus growth was slightly lower in ducks compared with that of an H5N8 HPAIV isolate collected in Japan in 2014. Comprehensive analyses of Japanese isolates, including those from central, western, and southern Japan, as well as rapid publication of this information are essential for facilitating greater control of HPAIVs.

Low Genetic Variation of Red-Crowned Cranes on Hokkaido Island, Japan, Over the Hundred Years.

The red-crowned crane (Grus japonensis) is recognized internationally as an endangered species. Migratory populations breed in eastern Russia and northeastern China, whereas the resident population inhabits the island of Hokkaido, Japan. Although the population inhabiting Hokkaido had experienced a severe bottleneck by the end of the 19th century, the population size has recovered to about 1500 and continues to increase now thanks to conservation efforts. A previous study reported that no marked genetic differences were seen in the island population, and that the genetic variation of the whole population on Hokkaido was lower than that of the continental population. However, the precise genetic structure of the island population in the past or near present remains unclear. To better understand the spatiotemporal changes in the genetic structure of the island population, we performed mitochondrial DNA (mtDNA) analyses using stuffed specimens (years 1878-2001) and tissue or blood samples (years 1970-2014). We found three haplotypes in the island population, one of which was a novel mtDNA haplotype in 1997 and 2007 samples. In addition, there was no clear difference in the haplotype frequency through the time span. These results suggest that the low genetic variation of the island population persisted for the last hundred years. It is thus nearly impossible for the island population to recover its genetic variation in isolation. Conservation plans for this species should therefore include the promotion of genetic exchanges between the continental and island populations, such as through artificial introduction to Hokkaido.

Immortalized prairie vole-derived fibroblasts (VMF-K4DTs) can be transformed into pluripotent stem cells and provide a useful tool with which to determine optimal reprogramming conditions.

The cellular conditions required to establish induced pluripotent stem cells (iPSCs), such as the number of reprogramming factors and/or promoter selection, differ among species. The establishment of iPSCs derived from cells of previously unstudied species therefore requires the extensive optimization of programming conditions, including promoter selection and the optimal number of reprogramming factors, through a trial-and-error approach. While the four Yamanaka factors Oct3/4, Sox2, Klf4, and c-Myc are sufficient for iPSC establishment in mice, we reported previously that six reprogramming factors were necessary for the creation of iPSCs from primary prairie vole-derived cells. Further to this study, we now show detailed data describing the optimization protocol we developed in order to obtain iPSCs from immortalized prairie vole-derived fibroblasts. Immortalized cells can be very useful tools in the optimization of cellular reprogramming conditions, as cellular senescence is known to dramatically decrease the efficiency of iPSC establishment. The immortalized prairie vole cells used in this optimization were designated K4DT cells as they contained mutant forms of CDK4, cyclin D, and telomerase reverse transcriptase (TERT). We show that iPSCs derived from these immortalized cells exhibit the transcriptional silencing of exogenous reprogramming factors while maintaining pluripotent cell morphology. There were no observed differences between the iPSCs derived from primary and immortalized prairie vole fibroblasts. Our data suggest that cells that are immortalized with mutant CDK4, cyclin D, and TERT provide a useful tool for the determination of the optimal conditions for iPSC establishment.

Isolation and characterization of major histocompatibility complex class II B genes in cranes.

In this study, we isolated and characterized the major histocompatibility complex (MHC) class II B genes in cranes. Genomic sequences spanning exons 1 to 4 were amplified and determined in 13 crane species and three other species closely related to cranes. In all, 55 unique sequences were identified, and at least two polymorphic MHC class II B loci were found in most species. An analysis of sequence polymorphisms showed the signature of positive selection and recombination. A phylogenetic reconstruction based on exon 2 sequences indicated that trans-species polymorphism has persisted for at least 10 million years, whereas phylogenetic analyses of the sequences flanking exon 2 revealed a pattern of concerted evolution. These results suggest that both balancing selection and recombination play important roles in the crane MHC evolution.

Novel approach for detecting classical swine fever virus from swabs of wild boar cut tails using nested real-time PCR.

We devised a method to detect the classical swine fever virus (CSFV) in tail-wiped swabs from wild boars. The CSFV gene in swabs was detected with high sensitivity using nested real-time polymerase chain reaction (PCR), which is a combination of reverse transcription-PCR (RT-PCR) and real-time PCR. We compared CSFV gene detection from boar tissue using the conventional and our tail-wiped swab method. The tail-wiped swab method showed sensitivity and specificity of 100% (26/26) and 98.8% (172/174), respectively compared to the conventional method. Thus, the swab-based CSFV detection method was considered to have detection sensitivity comparable to that of conventional methods. Additionally, we conducted surveillance for CSFV in wild boars on Awaji Island. CSFV was detected in 10.7% (45/420) of samples.

Organoids containing neural-like cells derived from chicken iPSCs respond to poly:IC through the RLR family.

There is still much room for development in pluripotent stem cell research on avian species compared to human stem cell studies. Neural cells are useful for the evaluation of risk assessment of infectious diseases since several avian species die of encephalitis derived from infectious diseases. In this study, we attempted to develop induced pluripotent stem cells (iPSCs) technology for avian species by forming organoids containing neural-like cells. In our previous study, we established two types iPSCs from chicken somatic cells, the first is iPSCs with PB-R6F reprogramming vector and the second is iPSCs with PB-TAD-7F reprogramming vector. In this study, we first compared the nature of these two cell types using RNA-seq analysis. The total gene expression of iPSCs with PB-TAD-7F was closer to that of chicken ESCs than that of iPSCs with PB-R6F; therefore, we used iPSCs with PB-TAD-7F to form organoids containing neural-like cells. We successfully established organoids containing neural-like cells from iPSCs using PB-TAD-7F. Furthermore, our organoids responded to poly:IC through the RIG-I-like receptor (RLR) family. In this study, we developed iPSCs technology for avian species via organoid formation. In the future, organoids containing neural-like cells from avian iPSCs can develop as a new evaluation tool for infectious disease risk in avian species, including endangered avian species.

Cultured fibroblasts of the Okinawa rail present delayed innate immune response compared to that of chicken.

The Okinawa rail is endemic to Okinawa Island and is categorized as an endangered animal. In this study, we focused on innate immunity because it is the first line of host defense. In particular, signals recognizing foreign RNA (e.g., viruses) are important for host defense because they activate the host immune system. The retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) families (RIG-I, MDA5, and LGP2) are sensors that activate innate immunity. Therefore, we analyzed these functions in the Okinawa rail using genomic and cellular analyses of fibroblasts. Fibroblasts can be obtained from dead individuals, allowing these cells to be obtained from dead individuals, which is particularly useful for endangered species. The MDA5 gene of Okinawa rail was sequenced using the Sanger method following PCR amplification and extraction of the amplified sequence from agarose gel. Additionally, mRNA expression analysis of cultured fibroblasts exposed to poly I:C was done. The MDA5 gene was found to be a mutated nonfunctional gene in the Okinawa rail. The mRNA expression rates of inflammatory cytokine genes type I IFN, and Mx1 were slower in Okinawa rail than in chicken cultured fibroblasts. Similar to the mRNA expression results, cell number and live cell ratio also slowly decreased in the Okinawa rail compared with chicken cultured fibroblasts, indicating that the innate immune reaction differs between chicken and the Okinawa rail. To the best of our knowledge, this is the first experimental evaluation of the loss of function of the Okinawa rail innate immune genes. In conclusion, our results provide a basis for conservation strategies for the endangered Okinawa rail.

Sequencing methods for HA and NA genes of avian influenza viruses from wild bird feces using Oxford Nanopore sequencing.

We developed a method to determine the sequences of hemagglutinin (HA) and neuraminidase (NA) from RNA extracted directly from wild bird fecal samples, using Nanopore Flongle. We determined the nucleotide sequences and subtypes of HA and NA in 16 and 15 samples respectively, using Flongle. The results of HA and NA subtyping determined by the conventional method were consistent with their subtypes determined by our method, thereby the applicability of this method in the identification of HA and NA subtypes. In addition, the homology between the HA fragments in this and the Sanger methods ranged from 98.5 % to 100 %. Compared with conventional PCR with the Sanger method, this method can easily determine HA and NA subtypes and sequences directly from the fecal samples. It is easier to implement and has lower running costs (USD100$) than other NGS-based methods, making it a useful tool for avian influenza surveillance in wild birds.

Detection of H5N1 High Pathogenicity Avian Influenza Viruses in Four Raptors and Two Geese in Japan in the Fall of 2022.

In the fall of 2022, high pathogenicity avian influenza viruses (HPAIVs) were detected from raptors and geese in Japan, a month earlier than in past years, indicating a shift in detection patterns. In this study, we conducted a phylogenetic analysis on H5N1 HPAIVs detected from six wild birds during the 2022/2023 season to determine their genetic origins. Our findings revealed that these HPAIVs belong to the G2 group within clade 2.3.4.4b, with all isolates classified into three subgroups: G2b, G2d, and G2c. The genetic background of the G2b virus (a peregrine falcon-derived strain) and G2d viruses (two raptors and two geese-derived strains) were the same as those detected in Japan in the 2021/2022 season. Since no HPAI cases were reported in Japan during the summer of 2022, it is probable that migratory birds reintroduced the G2b and G2d viruses. Conversely, the G2c virus (a raptor-derived strain) was first recognized in Japan in the fall of 2022. This strain might share a common ancestor with HPAIVs from Asia and West Siberia observed in the 2021/2022 season. The early migration of waterfowl to Japan in the fall of 2022 could have facilitated the early invasion of HPAIVs.

The dynamics of the microbiome in Ixodidae are shaped by tick ontogeny and pathogens in Sarawak, Malaysian Borneo.

Tick-borne diseases have recently been considered a potential emerging public health threat in Malaysia; however, fundamental studies into tick-borne pathogens and microbiome appear limited. In this study, six tick species (Ixodes granulatus, Haemaphysalis hystricis, Haemaphysalis shimoga, Dermacentor compactus, Dermacentor steini and Dermacentor atrosignatus) collected from two primary forests and an oil palm plantation in Sarawak, Malaysian Borneo, were used for microbiome analysis targeting bacterial 16S rDNA using next-generation sequencing (NGS). In addition, bacterial species were further characterized in conventional PCRs to identify potential pathogens. Sequences generated from NGS were first filtered with the Decontam package in R before subsequent microbial diversity analyses. Alpha and beta analyses revealed that the genus Dermacentor had the highest microbial diversity, and H. shimoga significantly differed in microbial composition from other tick species. Alpha and beta diversities were also significantly different between developmental stages of H. shimoga. Furthermore, we observed that some bacterial groups were significantly more abundant in certain tick species and developmental stages of H. shimoga. We tested the relative abundances using pairwise linear discriminant analysis effect size (LEfSe), which also revealed significant microbial composition differences between Borrelia-positive and Borrelia-negative I. granulatus ticks. Finally, pathogenic and potentially pathogenic bacteria circulating in different tick species, such as Rickettsia heilongjiangensis, Ehrlichia sp., Anaplasma sp. and Bartonella spp. were characterized by PCR and sequencing. Moreover, Coxiella and Francisella-like potential symbionts were identified from H. shimoga and D. steini, respectively. More studies are required to unravel the factors associated with the variations observed in this study.

The rise of hyperabundant native generalists threatens both humans and nature.

In many disturbed terrestrial landscapes, a subset of native generalist vertebrates thrives. The population trends of these disturbance-tolerant species may be driven by multiple factors, including habitat preferences, foraging opportunities (including crop raiding or human refuse), lower mortality when their predators are persecuted (the 'human shield' effect) and reduced competition due to declines of disturbance-sensitive species. A pronounced elevation in the abundance of disturbance-tolerant wildlife can drive numerous cascading impacts on food webs, biodiversity, vegetation structure and people in coupled human-natural systems. There is also concern for increased risk of zoonotic disease transfer to humans and domestic animals from wildlife species with high pathogen loads as their abundance and proximity to humans increases. Here we use field data from 58 landscapes to document a supra-regional phenomenon of the hyperabundance and community dominance of Southeast Asian wild pigs and macaques. These two groups were chosen as prime candidates capable of reaching hyperabundance as they are edge adapted, with gregarious social structure, omnivorous diets, rapid reproduction and high tolerance to human proximity. Compared to intact interior forests, population densities in degraded forests were 148% and 87% higher for wild boar and macaques, respectively. In landscapes with >60% oil palm coverage, wild boar and pig-tailed macaque estimated abundances were 337% and 447% higher than landscapes with <1% oil palm coverage, respectively, suggesting marked demographic benefits accrued by crop raiding on calorie-rich food subsidies. There was extreme community dominance in forest landscapes with >20% oil palm cover where two pig and two macaque species accounted for >80% of independent camera trap detections, leaving <20% for the other 85 mammal species >1 kg considered. Establishing the population trends of pigs and macaques is imperative since they are linked to cascading impacts on the fauna and flora of local forest ecosystems, disease and human health, and economics (i.e., crop losses). The severity of potential negative cascading effects may motivate control efforts to achieve ecosystem integrity, human health and conservation objectives. Our review concludes that the rise of native generalists can be mediated by specific types of degradation, which influences the ecology and conservation of natural areas, creating both positive and detrimental impacts on intact ecosystems and human society.

Comparison of the Genetic Diversity of the Captive and Wild Populations of the Tsushima Leopard Cat Using a GRAS-Di Analysis.

The Tsushima leopard cat (Prionailurus bengalensis euptilurus) (TLC) is a regional population of the Amur leopard cat (P. bengalensis euptilurus) that lives only on the Tsushima Island in Japan and is threatened with extinction. Because the TLC population is small, genetic management is important. In this study, we obtained the draft genome of the TLC and identified single-nucleotide polymorphism (SNP) markers using a genotyping by random amplicon sequencing-direct (GRAS-Di) analysis. We genotyped 31 captive individuals and 50 wild individuals, of which 48 were from a previous study. The identified SNPs were used to clarify the genetic diversity and genetic structure of the wild and captive populations of the TLC. The size of the genome was estimated to be about 2.42 Gb. The number of SNP markers developed was 139, and although PID and probability of exclusion obtained using these SNP markers were not as high as those reported in the studies of other wild species, these SNP markers could be used to identify individuals and parentage. Moreover, the genetic diversity indices of the captive population were similar to those of the wild population. These SNP markers will be useful for understanding the ecology of the TLC and planning conservation strategies.

Draft genome sequence data of Indian rhinoceros, Rhinoceros unicornis.

The Indian rhinoceros (Rhinoceros unicornis) is a large herbivore found in northern India and southern Nepal. It is a critically endangered species, with an estimated population of approximately 3,600 in the wild. Genetic factors, such as the loss of genetic diversity and the accumulation of deleterious variations, are critical risk factors for the extinction of endangered species, such as the Indian rhinoceros. To support the conservation efforts of the Indian rhinoceros, we assembled its draft genome. The new genomic data will enable the study of functional genes associated with the ecological and physiological characteristics of Indian rhinoceros and help us establish more effective conservation measures. The muscles of an Indian rhinoceros that died from prostration at a zoo were collected, and the samples were stored at the National Institute for Environmental Studies (Tsukuba, Japan). Sequence data were obtained using an Illumina NovaSeq 6000 platform for short reads and an Oxford Nanopore Technologies PromethION for long reads. We generated approximately 235.2 Gbp of data. From these sequences, we assembled a 2,375,051,758 bp genome consisting of 7,615 contigs. The genome data are available from the National Center Biotechnology Information BioProject database under accession number BOSQ00000000.