Cocirculation of Genetically Distinct Highly Pathogenic Avian Influenza H5N5 and H5N1 Viruses in Crows, Hokkaido, Japan.

We isolated highly pathogenic avian influenza (HPAI) H5N5 and H5N1 viruses from crows in Hokkaido, Japan, during winter 2023-24. They shared genetic similarity with HPAI H5N5 viruses from northern Europe but differed from those in Asia. Continuous monitoring and rapid information sharing between countries are needed to prevent HPAI virus transmission.

Genetic diversity of H5N1 and H5N2 high pathogenicity avian influenza viruses isolated from poultry in Japan during the winter of 2022-2023.

High pathogenicity avian influenza viruses (HPAIVs) of the H5N1 and H5N2 subtypes were responsible for 84 HPAI outbreaks on poultry premises in Japan during October 2022-April 2023. The number of outbreaks during the winter of 2022-2023 is the largest ever reported in Japan. In this study, we performed phylogenetic analyses using the full genetic sequences of HPAIVs isolated in Japan during 2022-2023 and those obtained from a public database to identify their genetic origin. Based on the hemagglutinin genes, these HPAIVs were classified into the G2 group of clade 2.3.4.4b, whose ancestors were H5 HPAIVs that circulated in Europe in late 2020, and were then further divided into three subgroups (G2b, G2d, and G2c). Approximately one-third of these viruses were classified into the G2b and G2d groups, which also included H5N1 HPAIVs detected in Japan during 2021-2022. In contrast, the remaining two-thirds were classified into the G2c group, which originated from H5N1 HPAIVs isolated in Asian countries and Russia during the winter of 2021-2022. Unlike the G2b and G2d viruses, the G2c viruses were first detected in Japan in the fall of 2022. Importantly, G2c viruses caused the largest number of outbreaks throughout Japan over the longest period during the season. Phylogenetic analyses using eight segment genes revealed that G2b, G2d, and G2c viruses were divided into 2, 4, and 11 genotypes, respectively, because they have various internal genes closely related to those of avian influenza viruses detected in wild birds in recent years in Asia, Russia, and North America, respectively. These results suggest that HPAIVs were disseminated among migratory birds, which may have generated numerous reassortant viruses with various gene constellations, resulting in a considerable number of outbreaks during the winter of 2022-2023.

Isolation and complete genomic characterization of a Movar 33/63-like Japanese bovine herpesvirus 4 from a calf with respiratory disease.

Bovine herpesvirus 4 (BoHV-4) is an indigenous virus in cattle prevalent mainly in North and South American countries and European countries, but the genomic sequences and genetic characteristics of Japanese strains have not been reported. BoHV-4 is suspected, but not proven, to be associated with various diseases. In the present study, we isolated BoHV-4 from a 10-month-old Japanese Black calf with respiratory symptoms in Japan. To identify the genetic characteristics of the isolate named strain SG20, complete genome sequencing was performed using a combination of next-generation and Sanger sequencing technologies. The complete long unique coding region (LUR) of SG20 was found to comprise 108,819 nucleotides with 41.4% GC content and contain at least 78 open reading frames. It shares 83.4 to 99.3% overall nucleotide identity with six BoHV-4 strains available in the database. The deduced amino acid sequence alignment revealed that SG20 contains genotype 1-specific features of BoHV-4, such as amino acid substitutions and insertions within the glycoprotein B region. Phylogenetic analyzes based on the nucleotide sequences of ORF20 indicated that the virus belonged to genotype 1 (Movar 33/63-like group). The strain was also analyzed using the complete LUR and placed in the same clade as a strain recently isolated from China, but it was distinct from American and European BoHV-4 strains of genotype 1. Although further genomic and epidemiologic information is needed, our results help elucidate the molecular epidemiology of BoHV-4 and provide a foundation for future studies.

Genetics of H5N1 and H5N8 High-Pathogenicity Avian Influenza Viruses Isolated in Japan in Winter 2021-2022.

In winter 2021-2022, H5N1 and H5N8 high-pathogenicity avian influenza (HPAI) viruses (HPAIVs) caused serious outbreaks in Japan: 25 outbreaks of HPAI at poultry farms and 107 cases in wild birds or in the environment. Phylogenetic analyses divided H5 HPAIVs isolated in Japan in the winter of 2021-2022 into three groups-G2a, G2b, and G2d-which were disseminated at different locations and times. Full-genome sequencing analyses of these HPAIVs revealed a strong relationship of multiple genes between Japan and Siberia, suggesting that they arose from reassortment events with avian influenza viruses (AIVs) in Siberia. The results emphasize the complex of dissemination and reassortment events with the movement of migratory birds, and the importance of continual monitoring of AIVs in Japan and Siberia for early alerts to the intrusion of HPAIVs.

Experimental Infection of Chickens with H5N8 High Pathogenicity Avian Influenza Viruses Isolated in Japan in the Winter of 2020-2021.

During the winter of 2020-2021, numerous outbreaks of high pathogenicity avian influenza (HPAI) were caused by viruses of the subtype H5N8 in poultry over a wide region in Japan. The virus can be divided into five genotypes-E1, E2, E3, E5, and E7. The major genotype responsible for the outbreaks was E3, followed by E2. To investigate the cause of these outbreaks, we experimentally infected chickens with five representative strains of each genotype. We found that the 50% chicken infectious dose differed by up to 75 times among the five strains, and the titer of the E3 strains (102.75 50% egg infectious dose (EID50)) was the lowest, followed by that of the E2 strains (103.50 EID50). In viral transmission experiments, in addition to the E3 and E2 strains, the E5 strain was transmitted to naïve chickens with high efficiency (>80%), whereas the other strains had low efficiencies (<20%). We observed a clear difference in the virological characteristics among the five strains isolated in the same season. The higher infectivity of the E3 and E2 viruses in chickens may have caused the large number of HPAI outbreaks in Japan during this season.

Different Infectivity and Transmissibility of H5N8 and H5N1 High Pathogenicity Avian Influenza Viruses Isolated from Chickens in Japan in the 2021/2022 Season.

H5N8 and H5N1 high pathogenicity avian influenza viruses (HPAIVs) caused outbreaks in poultry farms in Japan from November 2021 to May 2022. Hemagglutinin genes of these viruses belong to clade 2.3.4.4B and can be divided phylogenetically into the following groups: 20A, 20E, and 21E. In this study, we compared the infectivity and transmissibility of HPAIVs from three groups of chickens. Representative strains from 20A, 20E, and 21E groups are A/chicken/Akita/7C/2021(H5N8)(Akita7C), A/chicken/Kagoshima/21A6T/2021(H5N1)(Kagoshima6T), and A/chicken/Iwate/21A7T/2022(H5N1)(Iwate7T), respectively. Fifty percent lethal dose of Akita7C in chickens (103.83 fifty percent egg infectious dose (EID50)) was up to seven times lower than those of Kagoshima6T and Iwate7T (104.50 and 104.68 EID50, respectively). Mean death times for Akita7C- and Kagoshima6T-infected chickens (3.45 and 3.30 days, respectively) were at least a day longer than that of Iwate7T (2.20 days). Viral titers of the trachea and cloaca of Iwate7T-infected chicken were the highest detected. The transmission rate of the Akita7C strain (100%) was markedly higher than those of the two strains (<50%). These data suggest that the infectivity and transmissibility of the Akita7C strain (H5N8) in chickens are higher than those of H5N1 viruses, providing fundamental information needed for formulating effective prevention and control strategies for HPAI outbreaks.

Whole-genome sequencing of live attenuated bovine adenovirus type 7 vaccine strain TS-GT suggests biomarkers for virulence attenuation.

Bovine adenovirus type 7 (BAdV-7) is one of the most important respiratory and enteric pathogens in the cattle industry. Although live attenuated vaccines are used to control the virus in Japan, limited information is available on the genomic regions that determine viral pathogenicity. We determined the complete genome sequence of the attenuated BAdV-7 strain TS-GT. The genome is 30,052 bp long and contains 45-bp inverted terminal repeats and 30 predicted genes. A genome sequence comparison showed that 99.9% of the TS-GT genome is identical to the prototypic and pathogenic BAdV-7 strain Fukuroi; however, the TS-GT genome contains a novel mutation and four indels. We describe here potential relationships between these genomic changes and the biological characteristics of BAdV-7.

Genetically Diverse Highly Pathogenic Avian Influenza A(H5N1/H5N8) Viruses among Wild Waterfowl and Domestic Poultry, Japan, 2021.

Genetic analyses of highly pathogenic avian influenza H5 subtype viruses isolated from the Izumi Plain, Japan, revealed cocirculation of 2 genetic groups of clade 2.3.4.4b viruses among migratory waterfowl. Our findings demonstrate that both continuous surveillance and timely information sharing of avian influenza viruses are valuable for rapid risk assessment.

Novel single nucleotide polymorphisms in the bovine leukemia virus genome are associated with proviral load and affect the expression profile of viral non-coding transcripts.

Bovine leukemia virus (BLV) infects bovine B-cells and causes malignant lymphoma, resulting in severe economic losses in the livestock industry. To control the spread of BLV, several studies have attempted to clarify the molecular mechanisms of BLV pathogenesis, but the details of the mechanism are still enigmatic. Currently, viral non-coding RNAs are attracting attention as a novel player for BLV pathogenesis because these transcripts can evade the host immune response and are persistently expressed in latent infection. One of the viral non-coding RNA, AS1, is encoded in the antisense strand of the BLV genome and consists of two isoforms, AS1-L and AS1-S. Although the function of the AS1 is still unknown, the AS1 RNA might also have some roles because it keeps expressing in tumor tissues. In the present study, we identified novel single nucleotide polymorphisms (SNPs) located in the AS1 coding region and indicated that individuals infected with BLV with minor SNPs showed low proviral load. To evaluate the effect of identified SNPs, we constructed infectious clones with these SNPs and found that their introduction affected the expression profile of AS1 RNA; the amount of AS1-L isoform increased compared with the wild type, although the total amount of AS1 RNA remained unchanged. Prediction analysis also suggested that the introduction of SNPs changed the secondary structure of AS1 RNA. These results explain part of the relationship between BLV expansion in vivo and the expression profile of AS1, although further analysis is required.

Complete Genome Sequence of Bovine Adenovirus Type 7 Strain Fukuroi, Isolated from a Cow with Respiratory Disease.

We determined the complete genome sequence of the bovine adenovirus type 7 prototype strain Fukuroi using next-generation sequencing technology. We found that the viral genome is 30,034 bp long and has the shortest inverted terminal repeats among known adenoviruses.

Establishment of a simplified inverse polymerase chain reaction method for diagnosis of enzootic bovine leukosis.

Enzootic bovine leukosis (EBL) is a malignant B-cell lymphoma of cattle caused by infection with bovine leukemia virus (BLV). It is defined by clonal and neoplastic expansion of BLV-infected B cells. Currently, multiple examinations are able to comprehensively diagnose this condition. Inverse polymerase chain reaction (PCR) is a useful method to determine retrovirus integration sites. Here, we established a simplified inverse PCR method, involving the evaluation of clonality and similarity of BLV integration sites, to clinically diagnose EBL, and we also assessed its reliability. We found that the novel BLV inverse PCR could detect clonal expansion of infected cells even if they constituted only 5% of the total number of cells, while not amplifying any fragments from BLV-uninfected cells, thus confirming its sufficient sensitivity and specificity for use in EBL diagnosis. Furthermore, 50 clinical cases of bovine leukemia were analyzed using BLV inverse PCR and other PCR-based methods, wherein our method most efficiently determined virus-dependent bovine leukemia, including unidentified clinical cases observed in a previous report. Following further clinical investigations to enhance its reliability, the proposed BLV inverse PCR method has the potential to be applied to EBL diagnosis.

Sequence and unique phylogeny of G genes of bovine respiratory syncytial viruses circulating in Japan.

Bovine respiratory syncytial virus (BRSV) is an etiologic agent of bovine respiratory disease. The rapid evolutionary rate of BRSV contributes to genetic and antigenic heterogeneity of field strains and causes occasional vaccine failure. We conducted molecular epidemiologic characterization of BRSV circulating in Japan to obtain genetic information for vaccine-based disease control. Phylogenetic analysis of G and F gene sequences revealed that all of the isolated Japanese BRSV strains clustered in the same genetic subgroup, which was distinct from the 9 known groups. We assigned the Japanese group to subgenotype X. The Japanese isolates formed 2 temporal clusters: isolates from 2003 to 2005 clustered in lineage A; isolates from 2017 to 2019 formed lineage B. The alignment of the deduced amino acid sequences of the G gene revealed that the central hydrophobic region responsible for viral antigenicity is conserved in all of the isolates; unique amino acid mutations were found mainly in mucin-like regions. Our results suggest that BRSV has evolved uniquely in Japan to form the new subgenotype X; the antigenic homogeneity of the viruses within this group is inferred.

Phylogenetic and antigenic analysis of bovine parainfluenza virus type 3 isolated in Japan between 2002 and 2019.

Bovine parainfluenza virus type 3 (BPIV3) is one of the most important viral respiratory pathogens of cattle. In addition to the classical BPIV3 genotype A (BPIV3a), new genetic groups, genotype B (BPIV3b) and C (BPIV3c), have been identified and isolated in certain parts of the world. The present study aimed to investigate the genetic and antigenic characteristics of BPIV3 circulating in Japan. Seventy-three BPIV3 field strains were isolated from nasal samples of cattle between 2002 and 2019. Phylogenetic analysis of the phosphoprotein and hemagglutinin-neuraminidase genes showed that the isolates clustered into two genotypes, BPIV3a (49 %) and BPIV3c (51 %). The BPIV3a strains had more wide genetic variation than the rest of the genotypes. Additionally, new variants were obtained and designated them tentatively as subgroup 4 of the BPIV3a. The first Japanese BPIV3c was isolated in 2012, but here the BPIV3c NM2 strain was isolated from a sample collected four years earlier than the previous report. The antigenicity of ten BPIV3 strains including all three genotypes was assessed with a viral cross-neutralization test. Anti-sera against BPIV3a and BPIV3b cross-reacted well with both homologous and heterologous viruses. On the other hand, anti-sera against BPIV3c had reduced cross-reactivity to the heterologous viruses. Overall, our findings showed that genetically and antigenically divergent BPIV3 is prevalent in cattle in Japan. These results could provide a reference for molecular epidemiological characterization of BPIV3 and vaccine development.

Establishment of Novel Cells Stably Secreting Various Human IL-18 Recombinant Proteins.

BACKGROUND: The immunotherapies against cancer, autoinmmune diseases or infection are remarkable development. These days programmed cell death (PD)-1 antibody-induced immune checkpoint blockade or chimeric antigen receptor-T cells (CAR-T) have been shown to have eminent therapeutic effects on tumor development. We have focused on adoptive transfer with human gamma delta T cells for novel immunotherapies. Additionally, IL-18 is one of the cytokines that enhances cytokine secretion and cytotoxicity of human gamma delta T cells. METHOD: Thus, we established novel cell lines stably expressing and secreting various types of human recombinant IL-18 proteins to their culture supernatants using episomal vector. We also differentiated primary cultured human gamma delta T cells from peripheral blood mononuclear leukocytes to validate biological activity of the IL-18 proteins using measuring IFN-γ by ELISA. RESULTS AND CONCLUSION: Finally, we demonstrated that the supernatant could activate human gamma delta T cells using monitoring interferon gamma in culture medium.

Establishment of Novel Reporter Cells Stably Maintaining Transcription Factor-driven Human Secreted Alkaline Phosphatase Expression.

BACKGROUND: Transcriptional regulation is a very important and pivotal function in myriad biological responses. Thus, methods to determine transcriptional activity are required in not only basic medical research but also in drug discovery. We established novel reporter constructs using human secreted embryonic alkaline phosphatase (SEAP) and Epstein-Barr virus nuclear antigen (EBNA) 1, which can maintain constructs synchronized to host cell replication. METHODS: We established nuclear factor-kappa B (NFkB) or interferon regulatory factor (IRF) driven SEAP expression constructs and then, introduced them into culture cells. RESULTS: The cells maintain reporter constructs for a long period in the culture and produce SEAP into culture supernatant in response to each specific ligand such as lipopolysaccharide (LPS) and interferon- beta. Measuring SEAP with chemiluminescence makes it possible to get high standard dynamic range applying to high-throughput screening in drug discovery in both 96 and 384 well format. We can also use it to determine transcriptional activity in the cells transfected with expression plasmid or treated with various toll-like receptor (TLR) ligands in a concentration-dependent manner and time-dependent manner. Finally, we demonstrated drug screening using a number of natural products library. CONCLUSION: We for the first time established the two novel reporter cells and validated their quality and accuracy enough to carry out drug screening.

Effect of IL-18 on the Expansion and Phenotype of Human Natural Killer Cells: Application to Cancer Immunotherapy.

When pathogenic stresses are recognized by innate immune cells, inflammasomes are assembled and caspase-1 is activated, resulting in the conversion of pro-IL-18 into mature IL-18. Because natural killer (NK) cells express IL-18 receptors, IL-18 may play roles in immune functions of NK cells. In the present study, we examined the effect of IL-18 on NK cells derived from lung cancer patients and healthy adult volunteers. When peripheral blood NK cells were stimulated with IL-2, the cells formed clusters beginning on day 5-6 and proliferated thereafter, in which the number of NK cells increased by 10-fold in 10 days. When IL-18 was added, cell clusters were observed as early as on day 4 and NK cells proliferated vigorously. On day 10, the expansion rate was 56-fold on average, showing that IL-18 promoted the expansion of NK cells. It was also notable that IL-18 enhanced the expression of CD80, CD86, HLA-DR and HLA-DQ on NK cells, suggesting that IL-18 conferred NK cells an APC-like phenotype. When cellular cytotoxicity was determined, APC-like NK cells efficiently killed tumor cells and anti-tumor activity was augmented by the addition of tumor antigen-specific mAbs. In addition, IFN-γ was produced by APC-like NK cells in response to tumor cells, and the cytokine production was further enhanced by mAbs. Taken together, IL-18 not only promoted the expansion of NK cells, but also changed the phenotype of NK cells. IL-2/IL-18-induced NK cells might, therefore, serve as a bridge between innate immunity and adaptive immunity and be useful for cancer immunotherapy.

Expansion of human γδ T cells for adoptive immunotherapy using a bisphosphonate prodrug.

Cancer immunotherapy with human γδ T cells expressing Vγ2Vδ2 T cell receptor (also termed Vγ9Vδ2) has shown promise because of their ability to recognize and kill most types of tumors in a major histocombatibility complex (MHC) -unrestricted fashion that is independent of the number of tumor mutations. In clinical trials, adoptive transfer of Vγ2Vδ2 T cells has been shown to be safe and does not require preconditioning. In this report, we describe a method for preparing highly enriched human Vγ2Vδ2 T cells using the bisphosphonate prodrug, tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1-bisphosphonate (PTA). PTA stimulated the expansion of Vγ2Vδ2 cells to purities up to 99%. These levels were consistently higher than those observed after expansion with zoledronic acid, the most commonly used stimulator for clinical trials. Cell numbers also averaged more than those obtained with zoledronic acid and the expanded Vγ2Vδ2 cells exhibited high cytotoxicity against tumor cells. The high purity of Vγ2Vδ2 cells expanded by PTA increased engraftment success in immunodeficient NOG mice. Even low levels of contaminating αß T cells resulted in some mice with circulating human αß T cells rather than Vγ2Vδ2 cells. Vγ2Vδ2 cells from engrafted NOG mice upregulated CD25 and secreted tumor necrosis factor-α and interferon-γ in response to PTA-treated tumor cells. Thus, PTA expands Vγ2Vδ2 T cells to higher purity than zoledronic acid. The high purities allow the successful engraftment of immunodeficient mice without further purification and may speed up the development of allogeneic Vγ2Vδ2 T cell therapies derived from HLA-matched normal donors for patients with poor autologous Vγ2Vδ2 T cell responses.

Live Cell Labeling with Terpyridine Derivative Proligands to Measure Cytotoxicity Mediated by Immune Cells.

Immunotherapy using immune checkpoint inhibitors and CAR-T cells has revolutionized treatment for patients with malignant tumors. However, measuring tumor cell cytotoxicity mediated by immune effector cells in clinical laboratories has been difficult due to the requirement for radioactive substances. In this study, a series of novel terpyridine derivative proligands were synthesized, and a non-radioactive cellular cytotoxicity assay using the newly synthesized compounds was developed for use in preclinical and clinical studies for cancer immunotherapy. Once internalized into target cells, the compounds are hydrolyzed by esterases, resulting in the intracellular accumulation of the negatively charged terpyridine derivatives. When the labeled target cells are recognized and killed by immune effector cells, the integrity of the cell membrane is disrupted, and the terpyridine derivatives are released. Upon combining the culture supernatant with europium (Eu3+ ), the cytotoxicity of immune effector cells for the target cells can be quantitatively determined by measuring the intensity of the Eu3+ /ligand-derived time-resolved fluorescence. Thus, the assay developed in this study would facilitate the development of novel cancer immunotherapies.

Auto-amplification system for prostaglandin F2α in bovine corpus luteum.

The bovine corpus luteum (CL) is hypothesized to utilize a local auto-amplification system for prostaglandin (PG) F2α production. The objective of the present study was to determine if such a PGF2α auto-amplification system exists in the bovine CL, and if so, which factors regulate it. PGF2α significantly stimulated intra-luteal PGF2α production in all luteal phases, but did not affect PGE2 production. The stimulatory effect of exogenous PGF2α on CL PGF2α production was lower at the early luteal phase. Indomethacin, an inhibitor of prostaglandin-endoperoxide synthase (PTGS), significantly suppressed the PGF2α-stimulated PGF2α production by luteal tissue, indicating that the PGF2α in the medium was of luteal origin. Consistent with these secreted-PGF2α profiles, PGF2α receptor (PTGFR) protein expression was higher during the mid and late luteal phases than at early and developing luteal phases. Treatment of cultured bovine luteal cells obtained from the mid-luteal phase with PGF2α (1 µM) significantly increased the expressions of PTGS2, PGF synthase (PGFS), and carbonyl reductase1 (CBR1) at 24 hr post-treatment. Together, these results suggest the presence of a local auto-amplification system for PGF2α mediated by PTGS2, PGFS, and CBR1 in the bovine CL, which may play an important role in luteolysis.