Genetically modified rabies virus vector-based bovine ephemeral fever virus vaccine induces protective immune responses against BEFV and RABV in mice.

Bovine ephemeral fever (BEF), caused by the bovine ephemeral fever virus (BEFV), is associated with an acute febrile infection in cattle and widespread in tropical and subtropical areas, leading to great economic losses to cattle and milk industry. However, no efficacious BEF vaccine is currently available in China. Herein, we generated a recombinant rabies virus (RABV) expressing BEFV glycoprotein (LBNSE-BG), utilizing a reverse genetics system based on the recombinant rabies virus strain LBNSE. It was found that mice immunized with LBNSE-BG produced robust neutralizing antibodies against both BEFV and RABV, and developed complete protection from lethal RABV challenge. Further studies showed that LBNSE-BG activated more dendritic cells (DCs), B cells and T cells in immunized mice than the parent virus LBNSE. Collectively, these findings demonstrate that the recombinant LBNSE-BG described here has the potential to be developed as a cost-effective and efficacious bivalent vaccine for cattle use in endemic areas of BEF and rabies.

The immunogenicity of the secretory GΔTM protein of bovine ephemeral fever virus stably expressed by mammalian cells.

Bovine ephemeral fever virus (BEFV) causes an acute febrile disease in cattle and water buffalo. The disease has an impact on dairy and beef production in tropical and subtropical countries. Vaccination is used for disease prevention and control. In this study, we developed a recombinant lentivirus to produce mammalian stable cells expressing histidine-tagged BEFV G protein with a deleted transmembrane domain (GΔTM) as a secretory protein. In addition, guinea pigs were immunised with the purified GΔTM protein and booster immunised at a 3-week interval. The mammalian stable cells were able to continuously produce GΔTM protein for a minimum of 25 passages. All of the mammalian stable cells expressing GΔTM protein could react specifically with a BEFV convalescent bovine serum. Serum samples from the immunised guinea pigs could react strongly and specifically with the purified GΔTM protein. Moreover, post-immunised guinea pig sera contained antibodies that could neutralise BEFV. These results indicate that the G protein without a transmembrane domain can be used as a subunit vaccine for the prevention and control of BEFV. The availability of the mammalian stable cells, which constitutively express GΔTM protein, could facilitate the potential use of the secretory protein for BEFV diagnosis and vaccine development.

MiR-3470b promotes bovine ephemeral fever virus replication via directly targeting mitochondrial antiviral signaling protein (MAVS) in baby hamster Syrian kidney cells.

BACKGROUND: Bovine ephemeral fever virus (BEFV), the causative agent of bovine ephemeral fever, is an economically important pathogen of cattle and water buffalo. MicroRNAs (miRNAs) are endogenous 21-23 nt small non-coding RNA molecules that binding to a multiple of target mRNAs and functioning in the regulation of viral replication including the miRNA-mediated antiviral defense. However, the reciprocal interaction between bovine ephemeral fever virus replication and host miRNAs still remain poorly understood. The aim of our study herein was to investigate the exact function of miR-3470b and its molecular mechanisms during BEFV infection. RESULTS: In this study, we found a set of microRNAs induced by BEFV infection using small RNA deep sequencing, and further identified BEFV infection could significantly up-regulate the miR-3470b expression in Baby Hamster Syrian Kidney cells (BHK-21) after 24 h and 48 h post-infection (pi) compared to normal BHK-21 cells without BEFV infection. Additionally, the target association between miR-3470b and mitochondrial antiviral signaling protein (MAVS) was predicted by target gene prediction tools and further validated using a dual-luciferase reporter assay, and the expression of MAVS mRNA and protein levels was negatively associated with miR-3470b levels. Furthermore, the miR-3470b mimic transfection significantly contributed to increase the BEFV N mRNA, G protein level and viral titer, respectively, whereas the miR-3470b inhibitor had the opposite effect on BEFV replication. Moreover, the overexpression of MAVS or silencing of miR-3470b by its inhibitors suppressed BEFV replication, and knockdown of MAVS by small interfering RNA also promoted the replication of BEFV. CONCLUSIONS: Our findings is the first to reveal that miR-3470b as a novel host factor regulates BEFV replication via directly targeting the MAVS gene in BHK-21 cells and may provide a potential strategy for developing effective antiviral therapy.

Characterization of a recombinant Newcastle disease virus expressing the glycoprotein of bovine ephemeral fever virus.

Bovine ephemeral fever (BEF) is caused by the arthropod-borne bovine ephemeral fever virus (BEFV), which is a member of the family Rhabdoviridae and the genus Ephemerovirus. BEFV causes an acute febrile infection in cattle and water buffalo. In this study, a recombinant Newcastle disease virus (NDV) expressing the glycoprotein (G) of BEFV (rL-BEFV-G) was constructed, and its biological characteristics in vitro and in vivo, pathogenicity, and immune response in mice and cattle were evaluated. BEFV G enabled NDV to spread from cell to cell. rL-BEFV-G remained nonvirulent in poultry and mice compared with vector LaSota virus. rL-BEFV-G triggered a high titer of neutralizing antibodies against BEFV in mice and cattle. These results suggest that rL-BEFV-G might be a suitable candidate vaccine against BEF.

Kinetics of selected plasma cytokines during innate-adaptive immune response transition in adult cattle infected with the bovine ephemeral fever virus.

While virus neutralizing antibodies are known to be variably protective against bovine ephemeral fever (BEF) virus (BEFV) infections, the cytokine events that mediate the nascent adaptive immune response have not been defined in cattle. This study determined the plasma kinetics of IL-2, IFN-γ, IL-6, and IL-10 during the period of innate-immune response transition and evaluated the relationship between the virus neutralizing antibody response and viraemia in BEFV-infected cattle. Plasma from four virus-infected and uninfected negative control animals was tested by cytokine-specific immunoenzymatic assays, viraemia monitored by qRT-PCR, and virus neutralizing antibody titres determined using a standard protocol. Unlike the negative controls, plasma IL-6 and IL-10 were increased in all the virus-infected animals starting several days prior to initiation of viraemia. In one animal, plasma IL-2 and IFN-γ were consistently higher than in the other three virus-infected animals and the negative control mean. The animal with the strongest IL-2 and IFN-γ responses had the shortest viraemia while the heifer with the lowest IL-2/IFN-γ indices demonstrated the longest viraemia. Evidently, increase in plasma IL-6 and IL-10 precedes seroconversion during BEFV infections in cattle suggesting the two cytokines may influence immunological events that pave way to B-cell activation and seroconversion. While there is remarkable variability in IL-2 and IFN-γ expression amongst BEFV-infected animals, increased plasma levels of the two cytokines appear to be associated with a shorter viraemia. Ongoing studies will help define the precise role of T cells in anti-BEFV adaptive immune responses.

Experimental infection with bovine ephemeral fever virus and analysis of its antibody response cattle.

Bovine ephemeral fever (BEF) is an arthropod-borne viral disease that occurs throughout mainland China. LS11 obtained in the 2011 BEF epidemic was a wild strain, and its virulence and antibody response have never been studied in China. Therefore, the issues were investigated in this work. Experimental cattle were intravenously infected with different doses of BEF virus, and some non-infected cattle were simultaneously monitored. Blood and serum samples were collected from all animals over the course of our study. Infected cattle were challenged for a second time with BEF virus to determine protective period of the antibodies. BEF virus was detected in blood samples from infected cattle, but not in monitored cattle. The neutralizing antibodies (nAbs) against BEFV were easier to be detected and persisted for longer periods in cattle infected with higher doses of BEFV than in those infected with lower doses. When the titer of nAbs was equal to 5 or 6, re-infected cattle still could mount a challenge against BEFV. However, after 3 or 6months, when nAbs were no longer apparent, re-infected cattle displayed typical symptoms of BEF. Our findings indicated that vaccination should be performed once the titer of nAb decreased to 5 or 6.

Kinetics of pro-inflammatory cytokines, interleukin-10, and virus neutralising antibodies during acute ephemeral fever virus infections in Brahman cattle.

While fever and inflammation are hallmark features of bovine ephemeral fever (BEF), the cytokine networks that underlie the acute phase of the disease have not been empirically defined in cattle. This study characterised the plasma kinetics of proinflammatory cytokines (IL-1ß, IL-6, TNF-α) and IL-10 during acute BEF and elucidated on the relationship between the onset of the virus neutralizing antibody response and resolution of viraemia in natural BEF virus (BEFV) infections in cattle. Plasma from three BEFV-infected and three uninfected cattle was tested for the study cytokines by a cELISA, viraemia monitored by qRT-PCR, and virus neutralizing antibody titres determined using a standard protocol. Unlike the negative controls, plasma concentrations of IL-1ß, TNF-α, IL-6, and IL-10 were consistently increased in the three virus-infected animals. Two of the infected heifers were recumbent and pyrexic on the first day of monitoring and increased cytokine production was already in progress by the time viraemia was detected in all the three infected animals. In all the virus-infected heifers, IL-1ß was the most strongly expressed cytokine, IL-6 and IL-10 manifested intermediate plasma concentrations while TNF-α was the least expressed and demonstrated bi-phasic peaks three and five days after the onset of pyrexia. In two of the BEFV-infected heifers, viraemia resolved on the day of seroconversion while in the other infected animal, viral RNA was detectable up to three days after seroconversion. The present data document variable increase in plasma IL-1ß, IL-6, TNF-α, and IL-10 during natural BEFV infections and the fact that upregulation of all but TNF-α precedes seroconversion. In addition to virus neutralising antibodies, it is likely that cytokine-mediated cellular mechanisms may be required for resolution of viraemia in BEF. Considering the anti-inflammatory properties of IL-10, its upregulation may potentially antagonise the fever response in BEFV-infected cattle.

Relationships of bovine ephemeral fever epizootics to population immunity and virus variation.

Bovine ephemeral fever is an arthropod-borne bovine viral disease caused by infection with bovine ephemeral fever virus which belongs to genus Ephemerovirus within the family Rhabdoviridae. In this study, serological data and virological information about the disease and the virus, spanning from 2001 to 2013, were employed to analyze the relationships of bovine ephemeral fever epizootics to population immunity and virus variation. National and regional surveillance data indicated that 2 of the 3 major epizootics and 87% regional outbreaks were associated with lower neutralizing antibody titers and immunity coverage, reflecting the importance of population immunity for the control of bovine ephemeral fever. Phylogenetic analysis and sequence comparison demonstrated that Taiwanese bovine ephemeral fever viruses were >96.0% and >97.6% similar to the East Asian isolates in nucleotide and amino acid sequences, respectively. These analyses supported that the Taiwanese viruses shared the same gene pool with the strains of the other East Asian countries, mainly Japan.

The protective effectiveness of an inactivated bovine ephemeral fever virus vaccine.

Bovine ephemeral fever (BEF) is an important viral disease of cattle. Despite the extensive use of inactivated vaccines for the prevention of BEF, a controlled study of their field effectiveness has never been performed. We conducted a large field effectiveness study of a BEF inactivated vaccine, during a large BEF outbreak. Neutralizing antibody titers measured in 385 heifers and calves 1 month after 2(nd) vaccination averaged 1:91.8 (CI95%=76.6-110). The effectiveness study enrolled 2780 cows in nine herds. In two herds cows vaccinated twice, 1 year before the outbreak and once 2-3 months before outbreak onset were compared with non-vaccinated cows. Average vaccine effectiveness of three vaccine doses compared to no vaccination was 47% (CI95%=34-57) in these herds. In two other herds cows vaccinated twice 1 year before the outbreak and twice again 2-3 months before outbreak were compared with cows vaccinated only twice 2-3 months prior to the outbreak. Average vaccine effectiveness of four doses compared to two doses was 49% (CI95%=25-65) in these herds. In five herds cows vaccinated twice 2-3 months before outbreak onset were compared with non-vaccinated cows. This vaccination schedule was shown to be non-effective (average effectiveness=2%, CI95%=-14-17). Milk production analysis on one of the effected herds, in which 56% vaccine effectiveness and an absolute reduction of 27% in morbidity were documented, revealed a net milk production loss of 175.9kg/sick cow (CI95%=127.9-223.9) and an average gain of 37kg for each vaccinated cow (CI95%=-3.6-77.7). This study indicates that despite the fact that two vaccine doses of the tested inactivated vaccine elicited high titers of neutralizing antibodies, partial protection was induced only when at least 3 doses were administrated before natural challenge.

Evolution of bovine ephemeral fever virus in the Australian episystem.

Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus that causes a debilitating disease of cattle in Africa, Asia, and Australia; however, its global geodynamics are poorly understood. An evolutionary analysis of G gene (envelope glycoprotein) ectodomain sequences of 97 BEFV isolates collected from Australia during 1956 to 2012 revealed that all have a single common ancestor and are phylogenetically distinct from BEFV sampled in other geographical regions. The age of the Australian clade is estimated to be between 56 and 65 years, suggesting that BEFV has entered the continent on few occasions since it was first reported in 1936 and that the 1955-1956 epizootic was the source of all currently circulating viruses. Notably, the Australian clade has evolved as a single genetic lineage across the continent and at a high evolutionary rate of ∼10(-3) nucleotide substitutions/site/year. Screening of 66 isolates using monoclonal antibodies indicated that neutralizing antigenic sites G1, G2, and G4 have been relatively stable, although variations in site G3a/b defined four antigenic subtypes. A shift in an epitope at site G3a, which occurred in the mid-1970s, was strongly associated with a K218R substitution. Similarly, a shift at site G3b was associated primarily with substitutions at residues 215, 220, and 223, which map to the tip of the spike on the prefusion form of the G protein. Finally, we propose that positive selection on residue 215 was due to cross-reacting neutralizing antibody to Kimberley virus (KIMV). This is the first study of the evolution of BEFV in Australia, showing that the virus has entered the continent only once during the past 50 to 60 years, it is evolving at a relatively constant rate as a single genetic lineage, and although the virus is relatively stable antigenically, mutations have resulted in four antigenic subtypes. Furthermore, the study shows that the evolution of BEFV in Australia appears to be driven, at least in part, by cross-reactive antibodies to KIMV which has a similar distribution and ecology but has not been associated with disease. As BEFV and KIMV are each known to be present in Africa and Asia, this interaction may occur on a broader geographic scale.

Safety, immunogenicity and duration of immunity elicited by an inactivated bovine ephemeral fever vaccine.

Bovine ephemeral fever (BEF) is an economically important viral vector-borne cattle disease. Several live-attenuated, inactivated and recombinant vaccines have been tested, demonstrating varying efficacy. However, to the best of our knowledge, duration of immunity conferred by an inactivated vaccine has never been reported. In the last decade, Israel has faced an increasing number of BEF outbreaks. The need for an effective vaccine compatible with strains circulating in the Middle East region led to the development of a MONTANIDE™ ISA 206 VG (water-in-oil-in-water), inactivated vaccine based on a local strain. We tested the safety, immunogenicity and duration of immunity conferred by this vaccine. The induced neutralizing antibody (NA) response was followed for 493 days in 40 cows vaccinated by different protocols. The vaccine did not cause adverse reactions or a decrease in milk production. All cows [except 2 (6.7%) which did not respond to vaccination] showed a significant rise in NA titer of up to 1:256 following the second, third or fourth booster vaccination. Neutralizing antibody levels declined gradually to 1:16 up to 120 days post vaccination. This decline continued in cows vaccinated only twice, whereas cows vaccinated 3 or 4 times showed stable titers of approximately 1:16 for up to 267 days post vaccination. At least three vaccinations with the inactivated BEF vaccine were needed to confer long-lasting immunity. These results may have significant implications for the choice of vaccination protocol with inactivated BEF vaccines. Complementary challenge data should however be added to the above results in order to determine what is the minimal NA response conferring protection from clinical disease.

Antigenic characterization of bovine ephemeral fever rhabdovirus G and GNS glycoproteins expressed from recombinant baculoviruses.

Recombinant baculoviruses expressing the BEFV envelope glycoprotein G and non-structural glycoprotein G(NS) were constructed. The G protein expressed in insect cells was located on the cell surface and induced spontaneous cell fusion at mildly acidic pH. The expressed G protein reacted with MAbs to continuous and conformational neutralization sites (G1, G2, G3b and G4), but not to conformational site G3a. The expressed G(NS) protein was also located on the cell surface but did not exhibit fusogenic activity. The G(NS) protein reacted with polyclonal antiserum produced from vaccinia-virus-expressed recombinant G(NS) but did not react with G protein antibodies. A His(6)-tagged, soluble form of the G protein was expressed and purified by Ni(2+)-NTA chromatography. The purified G protein reacted with BEFV-neutralizing MAbs to all continuous and conformational antigenic sites. The highly protective characteristics of the native BEFV G protein suggest that the secreted, baculovirus-expressed product may be a useful vaccine antigen.

[Expression, purification and antigenic characterization of the Epitope-G1 gene of bovine ephemeral fever virus in Escherichia coli].

The epitope-G1 gene, cloned from the pMD-G plasmid including G protein gene of bovine ephemeral fever virus (BEFV), was subcloned into expression vector pGEX-4T-1 to construct pGEX-G1 recombinant plasmid successfully. The pGEX-G1 was transformed into E. coli BL21(DE3) to be induced with IPTG. The optimal expression conditions for G1 gene were obtained, which included reaction temperature 16 degrees C, induction time 18h and IPTG concentration 0.1 mmol/L. The soluble target protein was purified with Glutathione Sepharose TM(4B) and the purity reached 80%. The inclusion body washed with 2% deoxycholic acid sodium salt and dissolved with 0.5% N-lauroyl sarcosine sodium was recovered by the way of dialysis, then the protein was purified with Glutathione Sepharose TM(4B) and its purity was above 85%. The protein purified had nicer reaction activity by analysis of Western blot. The target protein was used as coating antigen to detect the sera against BEFV by an indirect ELISA. The 12 positive sera to BEFV were detected and the average of OD490 was 1.813 +/- 0.231, while the average of OD490 from 12 negative sera was 0.359 +/- 0.032, and the distinction was very remarkable (P < 0.01). All the rabbits inoculated with the target protein had produced high titer of antibodies, which indicated that the target protein had immunological activity. The average of OD49, detecting the 8 positive sera to rabies virus (RV) with the target protein purified was 0.324 +/- 0.031 which closed the datum obtained from the negative sera to BEFV, and it showed no cross-reaction between the sera to RV and BEFV. All the results above indicated that the target protein expressed had nicer biological activity and specificity, so the protein could be used as coating antigen to develop ELISA Kit for diagnosing BEF.

DNA sequence analysis of glycoprotein G gene of bovine ephemeral fever virus and development of a double oil emulsion vaccine against bovine ephemeral fever.

The surface glycoprotein G is considered as the major neutralizing and protective antigen of bovine ephemeral fever virus (BEFV). Comparison of the deduced amino acid sequence of G protein of BEFV isolates during the period 1984-2004 outbreaks in Taiwan showed amino acid substitutions in the neutralizing epitopes. All the isolates differ markedly in the neutralizing epitope at the same amino acid positions compared to the currently available killed vaccine strain (Tn73). Tn88128 strain isolated in 1999 showed the maximum variability of 12 amino acids, 5 amino acid in the neutralization epitope and 7 apart from, respectively. Combinations of both Tn88128 (1999) and commercially available vaccine strain (Tn73) were developed and its safety was evaluated in mice, guinea pigs, calves, and pregnant cows. None of the animals showed any adverse effect or clinical signs. Calves were immunized with commercial vaccine (Tn73) and, combined vaccine (Tn73 and Tn88128), respectively, with adjuvants such as Al-gel and water-in-oil-in-water (w/o/w) oil and PBS alone and challenged with Tn88128 strains. Except PBS administered animals, all the vaccinated animals showed protective immune response. However, animals immunized with combined vaccine plus w/o/w adjuvant elicited stronger neutralization antibodies and long lasting immunity compared to other vaccines.

Bovine ephemeral fever in Taiwan.

Bovine ephemeral fever (BEF) is a vector-borne disease of cattle, spanning tropical and subtropical zones of Asia, Australia, and Africa, caused by Ephemerovirus of the Rhabdoviridae. Taiwan has had 3 BEF epizootics, occurring in 1989, 1996, and 1999, since the vaccination regimen was initiated in 1984, given once a year in the spring with a single-dose formaldehyde-inactivated vaccine using the 1983 isolate as the seed virus. This study evaluated the 1999 population immunity against BEF virus in Taiwanese dairy cows with a neutralization test and whether the recent BEF virus isolates have mutated significantly from the vaccine virus. In March 1999, before vaccination, 94% of the animals studied were already seropositive, suggestive of an endemic or persistent infection from the previous year. By June 1999, when 51% of herds had been vaccinated, the antibody level rose, and by September 1999, the serum-neutralizing antibody (SNA) level fell to a minimum, preceding the outbreak of BEF in October 1999, during which the antibody levels of vaccinated cows continued to decline while those of unvaccinated cows rose sharply. The results suggest that, in 1999, vaccine-induced immunity was partially protective against BEE Because the current single-dose vaccination regimen resulted in minimal population immunity by September, a booster vaccination given in late summer may be advisable for future disease control. Analysis of the glycoprotein gene of Taiwanese isolates between 1983 and 1999 showed a 97.4-99.6% homology, with an alteration of 4 amino acids in antigenic sites G1, G3b, and G3c. Phylogenetic analysis of Taiwanese isolates revealed at least 2 distinct clusters: the 1983-1989 isolates and the 1996-1999 isolates. Both were distinct from 2 Japanese strains and the Australian BB7721 strain. Thus, at least 2 distinct BEF viruses, which had diverged before 1983, existed in Taiwanese dairy cows.

Vaccinia virus-expressed bovine ephemeral fever virus G but not G(NS) glycoprotein induces neutralizing antibodies and protects against experimental infection.

Two related glycoproteins (G and G(NS)) encoded in the bovine ephemeral fever virus (BEFV) genome were expressed from recombinant vaccinia viruses (rVV). Both proteins were detected in lysates of rVV-infected cells by labelling with D-[6-3H]glucosamine or by immuno-blotting. The recombinant G protein (mol. mass 79 kDa) appeared slightly smaller than the native G protein but reacted with monoclonal antibodies directed against all defined neutralizing antigenic sites (G1, G2, G3a, G3b and G4). The recombinant G(NS) protein (mol. mass 90kDa) was identical in size to the native G(NS) protein and failed to react by immuno-fluorescence with anti-G protein monoclonal or poly-clonal antibodies. Antisera raised in rabbits against rVV-G or rVV-G(NS) both reacted strongly by immuno-fluorescence and immuno-electron microscopy with BEFV-infected cells. The G protein was localized intracellularly in the endoplasmic reticulum/Golgi complex and at the cell surface associated with budding and mature virus particles. The G(NS) protein also localized intracellularly in the endoplasmic reticulum/Golgi complex; however, at the cell surface it was associated with amorphous structures and not with budding or mature virions. Rabbits vaccinated with rVV-G developed high levels of antibodies which neutralized BEFV grown in either mammalian or insect cells. Cattle vaccinated with rVV-G also produced neutralizing antibodies and were protected against experimental BEFV infection. In contrast, rVV-G(NS) vaccinated rabbits and cattle failed to produce neutralizing antibodies and, after challenge, BEFV was isolated from two-thirds of the vaccinated cattle.

Field trials of ephemeral fever vaccines.

Various bovine ephemeral fever (BEF) vaccines were tested between 1982 and 1984 in 24 Queensland herds; neutralising antibody responses were monitored and six of the herds were closely observed following natural challenge with ephemeral fever. A vaccine regime of two consecutive vaccinations with attenuated virus combined with the adjuvant Quil A provided excellent protection against BEF for at least 12 months, whereas one vaccination with the Quil A vaccine or two vaccinations with vaccine containing the adjuvant aluminium hydroxide gel did not provide significant protection. Antibody responses were highest for the vaccine incorporating Quil A when it was given as two consecutive injections. There was an apparent relationship between neutralising antibody response and the level of protection.

Epidemiologic analysis of spatial clustering of bovine ephemeral fever outbreaks. II. Principal component analysis.

The principal component analysis (PCA) was applied to analyze a correlation matrix of three variables on epidemic data of bovine ephemeral fever (BEF) outbreaks. These original data were summarized from the official outbreak report of Fukuoka Prefecture. The first and the second principal components of the PCA were interpreted as the infectious potency due to BEF virus and the prevention against BEF virus infection, respectively. The BEF outbreak areas were able to be classified epidemically into 4 groups by using the two principal components. The valuable epidemiological insights can be reasonably obtained from an application of the PCA. The results provided an important information for a further BEF vaccination campaign in the western part of Japan.

An outbreak of bovine ephemeral fever in Nagasaki Prefecture in 1988.

In October, 1988, bovine ephemeral fever (BEF) occurred in Nagasaki Prefecture and throughout Kyushu island, with the exception Miyazaki Prefecture. The first outbreak occurred in Hirado-shi on October 17. The total number of diseased cattle was 24 in 24 farms in Nagasaki Prefecture. The clinical findings were mainly sudden fever, anorexia, and instability in standing. The serum neutralizing antibodies against BEF virus (BEFV) rose in all infected cattle. Twelve strains of the virus were isolated in HmLu-1 cell cultures made directly from the heparinized blood of 17 infected cattle. The buffy coat was mainly collected from the samples and washed three times with phosphate buffered saline. These isolates were all neutralized by an antiserum against BEFV (Yamaguchi strain). With the aid of an electron microscope, a representative of isolates named Hirado-9 with a length of 150 nm was seen in the sample of infected HmLu-1 cell cultures. Both Hirado-9 and Yamaguchi strains reacted with antisera. The outbreak of BEF in 1988 was the first since 1971 in Nagasaki Prefecture. The result proved that BEFV can be easily isolated in HmLu-1 cell culture from the washed blood cells of infected cattle.