The bovine leukemia virus-derived long non-coding RNA AS1-S binds to bovine hnRNPM and alters the interaction between hnRNPM and host mRNAs.

Viruses utilize several strategies to cause latent infection and evade host immune responses. Long non-coding RNA (lncRNA), a class of non-protein-encoding RNA that regulates various cellular functions by interacting with RNA-binding proteins, plays important roles for viral latency in several viruses, such as herpesviruses and retroviruses, due to its lack of antigenicity. Bovine leukemia virus (BLV), which belongs to the family Retroviridae, encodes the BLV-derived lncRNA AS1-S, which is a major transcript expressed in latently infected cells. We herein identified bovine heterogeneous nuclear ribonucleoprotein M (hnRNPM), an RNA-binding protein located in the nucleus, as the binding partner of AS1-S using an RNA-protein pull-down assay. The pull-down assay using recombinant hnRNPM mutants showed that RNA recognition motifs (RRMs) 1 and 2, located in the N-terminal region of bovine hnRNPM, were responsible for the binding to AS1-S. Furthermore, RNA immunoprecipitation (RIP) assay results showed that the expression of AS1-S increased the number of mRNAs that co-immunoprecipitated with bovine hnRNPM in MDBK cells. These results suggested that AS1-S could alter the interaction between hnRNPM and host mRNAs, potentially interfering with cellular functions during the initial phase of mRNA maturation in the nucleus. Since most of the identified mRNAs that exhibited increased binding to hnRNPM were correlated with the KEGG term "Pathways in cancer," AS1-S might affect the proliferation and expansion of BLV-infected cells and contribute to tumor progression. IMPORTANCE BLV infects bovine B cells and causes malignant lymphoma, a disease that greatly affects the livestock industry. Due to its low incidence and long latent period, the molecular mechanisms underlying the progression of lymphoma remain enigmatic. Several non-coding RNAs (ncRNAs), such as miRNA and lncRNA, have recently been discovered in the BLV genome, and the relationship between BLV pathogenesis and these ncRNAs is attracting attention. However, most of the molecular functions of these transcripts remain unidentified. To the best of our knowledge, this is the first report describing a molecular function for the BLV-derived lncRNA AS1-S. The findings reported herein reveal a novel mechanism underlying BLV pathogenesis that could provide important insights for not only BLV research but also comparative studies of retroviruses.

Upregulation of host genes during disease progression in bovine leukemia virus infection is independent of overexpression of viral transcriptional regulators in vitro.

Bovine leukemia virus (BLV) is a member of the genus Deltaretrovirus within the family Retroviridae that infects bovine B cells, causing persistent lymphocytosis and enzootic bovine leukosis (EBL) in a small fraction of infected cattle. As changes in the transcriptome of infected cells are important for BLV disease progression, comprehensive analysis of gene expression in different disease states is required. In this study, we performed an RNA-seq analysis using samples from non-EBL cattle with and without BLV infection. Subsequently, a transcriptome analysis was conducted in combination with previously obtained RNA-seq data from EBL cattle. We found several differentially expressed genes (DEGs) between the three groups. After screening and confirmation of target DEGs using real-time reverse transcription polymerase chain reaction, we found that 12 target genes were significantly upregulated in EBL cattle compared to BLV-infected cattle without lymphoma. In addition, the expression levels of B4GALT6, ZBTB32, EPB4L1, RUNX1T1, HLTF, MKI67, and TOP2A were significantly and positively correlated with the proviral load in BLV-infected cattle. Overexpression experiments revealed that these changes were independent of BLV tax or BLV AS1-S expression in vitro. Our study provides additional information on host gene expression during BLV infection and EBL development, which may be helpful for understanding the complexity of transcriptome profiles during disease progression.

Current research and future directions for realizing the ideal One-Health approach: A summary of key-informant interviews in Japan and a literature review.

The COVID-19 pandemic has highlighted the importance of the One Health (OH) approach, which considers the health of humans, animals, and the environment in preventing future pandemics. A wide range of sustainable interdisciplinary collaborations are required to truly fulfill the purpose of the OH approach. It is well-recognized, however, that such collaborations are challenging. In this study, we undertook key-informant interviews with a panel of stakeholders from Japan to identify their perceived needs and challenges related to OH research. This panel included scientists, government officials, journalists, and industry stakeholders. By combining a thematic analysis of these interviews and a literature review, we summarized two key themes pertinent to the effective implementation of OH research: types of required research and systems to support that research. As a technological issue, interviewees suggested the importance of research and development of methodologies that can promote the integration and collaboration of research fields that are currently fragmented. An example of such a methodology would allow researchers to obtain high-resolution metadata (e.g. ecological and wildlife data) with high throughput and then maximize the use of the obtained metadata in research, such as in environmental DNA analysis, database construction, or the use of computational algorithms to find novel viral genomes. In terms of systems surrounding OH research, some interviewees stressed the importance of creating a sustainable research system, such as one that has continuous budget support and allows researchers to pursue their academic careers and interests. These perceptions and challenges held by Japanese stakeholders may be common to others around the world. We hope this review will encourage more researchers and others to work together to create a resilient society against future pandemics.

Endemic infections of bovine viral diarrhea virus genotypes 1b and 2a isolated from cattle in Japan between 2014 and 2020.

Bovine viral diarrhea virus (BVDV) is a causative agent of bovine viral diarrhea. In Japan, a previous study reported that subgenotype 1b viruses were predominant until 2014. Because there is little information regarding the recent epidemiological status of BVDV circulating in Japan, we performed genetic characterization of 909 BVDV isolates obtained between 2014 and 2020. We found that 657 and 252 isolates were classified as BVDV-1 and BVDV-2, respectively, and that they were further subdivided into 1a (35 isolates, 3.9%), 1b (588, 64.7%), 1c (34, 3.7%), and 2a (252, 27.7%). Phylogenetic analysis using entire E2 coding sequence revealed that a major domestic cluster in Japan among BVDV-1b and 2a viruses were unchanged from a previous study conducted from 2006 to 2014. These results provide updated information concerning the epidemic strain of BVDV in Japan, which would be helpful for appropriate vaccine selection.

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.

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.

Development of an in situ hybridization assay using an AS1 probe for detection of bovine leukemia virus in BLV-induced lymphoma tissues.

Enzootic bovine leukosis (EBL) is a malignant B cell lymphoma caused by infection with bovine leukemia virus (BLV). Histopathological examination is commonly used for diagnosis of the disease, but observation of lymphoma alone does not confirm EBL because cattle may be affected by sporadic forms of lymphoma that are not associated with BLV. Detection of BLV in tumor cells can be definitive evidence of EBL, but currently, there is no technique available for such a purpose. In this study, we focused on a viral non-coding RNA, AS1, and developed a novel in situ hybridization assay for the detection of BLV from formalin-fixed paraffin-embedded (FFPE) tissues. RNA-seq analysis revealed that all examined B lymphocytes derived from clinical EBL abundantly expressed AS1 RNA, indicating a possible target for detection. The in situ hybridization assay using an AS1 probe clearly detected AS1 RNA in fetal lamb kidney cells persistently infected with BLV. The utility of this assay in clinical samples was assessed using three EBL-derived lymph node specimens and one BLV-negative specimen, and AS1 RNA was detected specifically in the EBL-derived tissues. These results suggest that AS1 RNA is a useful target for the detection of BLV from FFPE specimens of tumor tissues. This technique is expected to become a powerful tool for EBL diagnosis.

Production of immunogenic recombinant L1 protein of bovine papillomavirus type 9 causing teat papillomatosis.

Bovine papillomavirus type 9 (BPV9) is a causative agent of severe teat papillomatosis. Considering the lack of efficient BPV culture methods, recombinant proteins such as virus-like particles developed through genetic engineering may serve as a useful tool for developing effective vaccines against BPV infection. In this study, we successfully produced immunogenic particles composed of recombinant L1 protein of BPV9 (rBPV9-L1), using a baculovirus expression system. rBPV9-L1-immunized mice produced BPV9-specific IgG, which did not cross-react with BPV type 6, which is another causative agent of teat papillomatosis. Hence, immunogenic rBPV9-L1 is potentially applicable as a vaccine candidate for teat papillomatosis.

The chemokines CCL2 and CXCL10 produced by bovine endometrial epithelial cells induce migration of bovine B lymphocytes, contributing to transuterine transmission of BLV infection.

Bovine leukemia virus (BLV) causes a lymphoproliferative disease in cattle and is transmitted horizontally and vertically via infected lymphocytes. Although transplacental infection is considered the predominant route of vertical transmission of BLV, the molecular mechanisms of this process remain to be elucidated. Notably, how BLV passes through the blood-placental barrier remains unclear, given that BLV is transmitted primarily by cell-to-cell contact. One hypothesis is that B cell migration to the placenta may be induced by certain endometrium-expressed chemokines. To test this hypothesis, we performed an in vitro cell migration assay using bovine B cell lines and endometrial epithelial cells. Cell migration assays showed that two bovine B cell lines, BL2M3 and BL3.1 cells, were attracted to the supernatant of bovine endometrial epithelial cells (BEnEpCs). Quantitative real-time RT-PCR showed that expression levels of mRNAs encoding the chemokines CCL2 and CXCL10 were higher in BEnEpCs than in MDBK cells. Additionally, an inhibition assay using immune serum against CCL2 and CXCL10 showed suppression of migration of bovine B cell lines. A syncytium assay showed that cells expressing BLV envelope (Env) protein fused with BEnEpCs. Here we found that bovine B cells are attracted by chemokines produced in the endometrium and that cells expressing BLV Env protein fused with endometrium epithelial cells. These results explain part of the molecular mechanism of transplacental transmission during BLV infection, although further analysis will be required. Advances in these areas are expected to contribute to controlling the spread of BLV.

Identification of Novel Linear Epitopes Located in the Infectious Bronchitis Virus Spike S2 Region.

We identified novel linear epitopes on the infectious bronchitis virus (IBV) spike S2 region. The conformational structure of the IBV spike protein was predicted from a homologous protein, human coronavirus NL63 spike. Although the obtained structure was incomplete, most of the IBV spike protein structure was predicted; the N-terminus of the S1 region could not be predicted due to its variability. In the model, the region located in the proximity of the fusion peptide appeared to be well conserved, and we evaluated the antigenicity of these domains, which are involved in the membrane fusion machinery. Western blotting revealed that IBV TM86 spike residues 686-723 were antigenic. Epitope mapping analysis using synthesized peptides revealed that IBV TM86 spike 669-685 (SNFSTGAFNISLLLTPP), 686-697 (SNPRGRSFIEDL), and 692-703 (SFIEDLLFTSVE) residues were major linear epitopes; two identified epitopes (686-697 and 692-703) were covered by the fusion peptide, and the other epitope (669-685) was adjacent to the fusion peptide. Although the identified epitopes are identically located as the neutralizing epitope in severe acute respiratory syndrome coronavirus, the recombinant protein that includes those epitopes could not elicit neutralizing antibodies against IBV. This is the first report describing IBV spike S2 epitopes located in the proximity of the fusion peptide, and it is suggested that the spike fusion machinery of IBV may differ from that of severe acute respiratory syndrome coronavirus, or, alternatively, IBV may have another mechanism to penetrate the cell membrane.

Turkey herpesvirus with an insertion in the UL3-4 region displays an appropriate balance between growth activity and antibody-eliciting capacity and is suitable for the establishment of a recombinant vaccine.

We constructed turkey herpesvirus (HVT) vector vaccines in which the VP2 gene of infectious bursal disease virus (IBDV) was inserted into the HVT genome in the following regions: UL3-4, UL22-23, UL45-46, and US10-SORF3. We then evaluated the relationship between the gene insertion site and the capacity of the virus to elicit antibodies. rHVT/IBD (US10) showed good growth activity in vitro, with growth comparable to that of the parent HVT. On the other hand, rHVT/IBD (UL3-4), rHVT/IBD (UL22-23), and rHVT/IBD (UL45-46) exhibited decreased growth activity in chicken embryo fibroblast (CEF) cells compared to the parent HVT. However, the rHVT/IBD (US10) elicited lower levels of virus-neutralizing (VN) antibodies compared to the other constructs. rHVT/IBD (UL3-4) and rHVT/IBD (UL45-46) appeared to be similar in their ability to elicit VN antibodies. Based on the results of in vitro and in vivo assays, rHVT/IBD (UL3-4) was selected for further testing. In a challenge assay, rHVT/IBD (UL3-4) protected chickens from challenge with virulent Marek's disease virus serotype 1 and IBDV. In conclusion, the site of gene insertion may have a strong effect on the growth of the vector virus in vitro and its antibody-eliciting capacity. Insertions in the UL3-4 region permitted a balance between growth activity and VN-antibody-eliciting capacity, and this region might therefore be an appropriate insertion site for IBDV VP2.

Fiber knob domain lacking the shaft sequence but fused to a coiled coil is a candidate subunit vaccine against egg-drop syndrome.

Egg-drop syndrome (EDS) virus is an avian adenovirus that causes a sudden drop in egg production and in the quality of the eggs when it infects chickens, leading to substantial economic losses in the poultry industry. Inactivated EDS vaccines produced in embryonated duck eggs or cell culture systems are available for the prophylaxis of EDS. However, recombinant subunit vaccines that are efficacious and inexpensive are a desirable alternative. In this study, we engineered chimeric fusion proteins in which the trimeric fiber knob domain lacking the triple ß-spiral motif in the fiber shaft region was genetically fused to trimeric coiled coils, such as those of the engineered form of the GCN4 leucine zipper peptide or chicken cartilage matrix protein (CMP). The fusion proteins were expressed predominantly as soluble trimeric proteins in Escherichia coli at levels of 15-80mg/L of bacterial culture. The single immunization of chickens with the purified fusion proteins, at a dose equivalent to 10µg of the knob moiety, elicited serum antibodies with high hemagglutination inhibition (HI) activities, similar to those induced by an inactivated EDS vaccine. A dose-response analysis indicated that a single immunization with as little as 1µg of the knob moiety of the CMP-knob fusion protein was as effective as the inactivated vaccine in inducing antibodies with HI activity. The immunization of laying hens had no apparent adverse effects on egg production and effectively prevented clinical symptoms of EDS when the chickens were challenged with pathogenic EDS virus. This study demonstrates that the knob domain lacking the shaft sequence but fused to a trimeric coiled coil is a promising candidate subunit vaccine for the prophylaxis of EDS in chickens.

Decreased neutralizing antigenicity in IBV S1 protein expressed from mammalian cells.

We evaluated the antigenicity of recombinant infectious bronchitis virus (IBV) S1 protein expressed in mammalian cells. Recombinant S1 was expressed as a secreted protein fused with a trimerization motif peptide, then purified using Ni Sepharose. The purified protein was analyzed by Western blotting, mixed with oil adjuvant, and administered to 29-day-old specific-pathogen-free chickens. Six weeks after immunization, anti-IBV neutralizing titer and anti-S1 ELISA titer were determined; immunized chickens then were inoculated with IBV via the trachea and ciliary activity was observed. Results showed that the recombinant S1 protein was highly glycosylated, and the neutralizing antigenicity of recombinant S1 protein was lower than that of inactivated virus. However, anti-S1 ELISA indicated that the recombinant S1 protein induced antibodies against S1. These results suggest that the recombinant S1 may retain non-neutralizing epitopes but have unnatural glycosylation pattern and conformation, resulting in lacking neutralizing conformational epitopes. In conclusion, the neutralizing antigenicity of recombinant S1 protein expressed from mammalian cells was decreased, and was not sufficient to induce neutralizing antibodies.

The haemagglutination activity of equine herpesvirus type 1 glycoprotein C.

Equine herpesvirus type 1 (EHV-1) has haemagglutination (HA) activity toward equine red blood cells (RBCs), but the identity of its haemagglutinin is unknown. To identify the haemagglutinin of EHV-1, the major glycoproteins of EHV-1 were expressed in 293T cells, and the cells or cell lysates were mixed with equine RBCs. The results showed that only EHV-1 glycoprotein C (gC)-producing cells adsorbed equine RBCs, and that the lysate of EHV-1 gC-expressing cells agglutinated equine RBCs. EHV-1 lacking gC did not show HA activity. HA activity was inhibited by monoclonal antibodies (MAbs) specific for gC, but not by antibodies directed against other glycoproteins. In addition, HA activity was not inhibited by the addition of heparin. These results indicate that EHV-1 gC can bind equine RBCs irrespective of heparin, in contrast to other herpesvirus gC proteins.

Characterization of glycoproteins in equine herpesvirus-1.

In this study, we attempted to express twelve glycoproteins of equine herpesvirus-1 (EHV-1) in 293T cells and to characterize these using monoclonal antibodies (MAbs) and horse sera against EHV-1. Expression of glycoprotein B (gB), gC, gD, gG, gI and gp2 was recognized by immunoblot analysis using horse sera, but that of gE, gH, gK, gL, gM and gN was not. Four MAbs recognized gB, four recognized gC and one recognized gp2. Two MAbs against gB cross-reacted with EHV-4. Interestingly, coexpression of gE and gI and gM and gN enhanced their antigenicity. Furthermore, immunoblot analysis of gp2 showed that different molecular masses of gp2 were recognized by the MAb against gp2 and horse sera against EHV-1. In this study, it was demonstrated that at least six glycoproteins were immunogenic to horses, and coexpression of gE and gI and gM and gN was important for enhancement of antigenicity.

Identification of a major immunogenic region of equine herpesvirus-1 glycoprotein E and its application to enzyme-linked immunosorbent assay.

A major immunogenic region of equine herpesvirus (EHV)-1 glycoprotein E (gE) was identified. Firstly, the various fragments of EHV-1 gE were expressed as fusion proteins with glutathione S-transferase (GST) in Escherichia coli and their antigenicities were compared by immunoblot analysis using sera from horses experimentally infected with EHV-1. Thirty-three amino acids of gE (a.a. 169-201) specifically and sensitively reacted with the antibodies induced by EHV-1 but not EHV-4 infection. The corresponding region of EHV-4 gE (a.a. 169-199) did not react with antibodies to EHV-1, indicating that this region is specific for each virus. In addition, when the antigenicities of three 20-mer synthetic peptides of EHV-1 gE, gE1(169-188), gE1(176-195) and gE1(182-201) were compared by enzyme-linked immunosorbent assay (ELISA), gE1(169-188) was found to contain a major B-cell epitope. ELISA using two synthetic peptides, gE1(169-188) and gG4(319-330), previously identified as the major EHV-4-specific epitope in gG, was developed and could specifically detect antibodies to EHV-1 and EHV-4, respectively. In Japan, the EHV-1 deleted in gE gene (EHV-1 ΔgE) virus is expected to be introduced in the field as a new modified live vaccine. This ELISA did not react with antibodies induced by inoculation with EHV-1 ΔgE, indicating that it is a useful method to differentiate between EHV-1 infection and EHV-1 ΔgE inoculation. In conclusion, the ELISA described herein, using synthetic peptides, is a simple method to distinguish between EHV-1 and EHV-4 infections and will be suitable as a vaccine marker after introduction of EHV-1 ΔgE into field horses.

Establishment of canine and feline cells expressing canine signaling lymphocyte activation molecule for canine distemper virus study.

Signaling lymphocyte activation molecule (SLAM) is one of the receptors for canine distemper virus (CDV). In this study, canine and feline cells expressing canine SLAM, designated A-72/cSLAM and CRFK/cSLAM, were established for the in vitro study of canine distemper. Recent CDV isolates, KDK-1 and 246, which belong to genotypes Asia/H1 and Asia/H2, respectively, rapidly grew and produced distinct syncytia in both the SLAM-expressing cells. The virus-neutralizing (VN) test was successfully performed using these cells, and the results indicated that sera from dogs experimentally infected with KDK-1 had higher VN titers for homologous strain KDK-1 than for heterologous strain 246 and the vaccine Onderstepoort. These newly established cells expressing canine SLAM would help virological and serological analyses of canine distemper.

Further Development of an Equine Cell Line that can be Propagated over 100 Times.

Cell lines originating from horses are necessary for isolation and propagation of equine herpesviruses (EHV). Although we established an equine-derived cell line, FHK-Tcl3, propagation ceased after fewer than 40 passages. In this study, FHK-Tcl3 cell propagation continued beyond 40 passages, achieving over 100 passages. FHK-Tcl3 cells were then cloned by limiting dilution at the 100th passage. Cloned cells were termed FHK-Tcl3.1. FHK-Tcl3.1 cells grew well and were propagated every 3 to 4 days by splitting 1:5. In addition, EHV-1, -2 and -4 showed a clear cytopathic effect (CPE) in FHK-Tcl3.1 cells, and this CPE was very similar to those seen in parental FHK-Tcl3 and primary fetal horse kidney cells. FHK-Tcl3.1 cells continue to propagate and the current passage record is over 100 times after cloning. Therefore, this cell appears to have been immortalized. FHK-Tcl3.1 cells have potential for growth and diagnosis of various equine viruses, including equine herpesviruses.

Establishment of a novel equine cell line for isolation and propagation of equine herpesviruses.

In the present study, an equine-derived cell line was established by transfecting primary fetal horse kidney (FHK) cells with expression plasmid encoding simian virus 40 (SV40) large T antigen and then cloning them by limiting dilution. The cloned cell line, named FHK-Tcl3, grew well and could be propagated over 30 times by splitting them 1:3. Equine herpesvirus (EHV)-1 and EHV-4 replicated well in FHK-Tcl3. EHV-2 and EHV-4 were isolated from samples collected from horses in the field using FHK-Tcl3, and EHV-3 also propagated in FHK-Tcl3. These results indicated that this novel cell line, FHK-Tcl3, can be used for isolation and propagation of equine herpesviruses.