INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Ibaraki disease (IBAD) is an arthropod-borne viral disease of cattle characterized by fever, anorexia, and deglutitive disorder (15, 16). Since the first recognition of the disease in Japan in 1959, epizootic occurrences of the disease have been reported in Japan, Korea, and Taiwan (1, 11). The causative agent of IBAD is the Ibaraki virus (IBAV), which belongs to the epizootic hemorrhagic disease virus (EHDV) serogroup in the genus Orbivirus in the family Reoviridae. Ten different serotypes of EHDV are known to exist worldwide (6). IBAV has been demonstrated to be closely related serologically to but distinct from the Alberta strain of EHDV serotype 2 (EHDV-2) (2, 18).

The virion contains 10 double-stranded (ds) genome segments. Each of the segments encodes various structural and nonstructural viral proteins (9, 10, 13). One of these, segment 3 of the RNA genome (RNA3), encodes a serogroup-specific antigen, VP3. Recently, researchers demonstrated that reverse transcription (RT)-PCR with primers based on the sequence of RNA3 was a useful tool for the detection and differentiation of the EHDV serogroup (8, 12).

IBAD occurred on an epidemic scale in the late summer to autumn of 1982 and 1987 in the western parts of Japan. After the last outbreak in 1987, the next epidemic of the disease occurred from August to November 1997 in the same area. In the latter outbreak, numerous abortions and stillbirths were reported among cattle, in addition to the typical symptoms of IBAD. The viruses were isolated from the blood of affected and apparently healthy animals and from aborted fetuses and Culicoides biting midges. This paper describes the identification of the suspected causal agent and the genetic comparison with previous isolates of IBAV and EHDV by PCR-restriction fragment length polymorphism (RFLP) analysis.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Viruses and cells. The Ibaraki-2 (16) and Y87061 strains of IBAV were isolated from the blood of infected cows in 1959 and 1987, respectively. The following EHDV strains were used in this experiment: New Jersey (serotype 1), Alberta (serotype 2), CSIRO439 (serotype 2), CSIRO157 (serotype 7), CSIRO753 (serotype 8), CSIRO775 (serotype 9), and DPP 59 (serotype 10). All viruses were propagated on hamster lung (HmLu-1) cells and baby hamster kidney (BHK-21) cells. The cells were grown in Eagle's minimum essential medium (MEM; Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 0.295% tryptose phosphate broth (Difco Laboratories, Detroit, Mich.), 0.15% NaHCO₃, 2 mM L-glutamine, and 10% calf serum. The viruses were inoculated onto a cell monolayer that had been washed three times with Earle's solution and were cultured with serum-free MEM. Infectious culture fluid was harvested when the cells showed a complete cytopathic effect (CPE).

Virus isolation. The heparinized blood samples were collected from cattle with typical symptoms of IBAD and from cattle raised in the same cowshed with the affected animals. Blood samples were also taken from dams which had a miscarriage or a stillbirth. These blood samples were separated into plasma and erythrocytes, and the erythrocytes were washed three times with phosphate-buffered saline to eliminate the antibodies. The internal organs of the fetuses, the placenta, and Culicoides biting midges were homogenized with MEM. All samples were stored at 80°C until use. The cells grown in test tubes were washed three times with Earle's solution before inoculation with samples. After inoculation with 0.1 ml of the samples, the cells were incubated with 0.4 ml of MEM supplemented with antibiotics at 37°C for 7 days. The cultures were passaged in the same manner until a CPE was observed.

Production of hyperimmune serum. Hyperimmune sera against viruses were raised in rabbits. Fluid from cultures of the virus on BHK-21 cells was centrifuged at 5,600 × g for 20 min at 4°C and was concentrated by precipitation with 50% saturated ammonium sulfate. Purified virus was obtained at the interface of the discontinuous gradient of 20 and 50% (wt/vol) sucrose in phosphate-buffered saline after centrifugation at 100,700 × g for 2 h at 4°C (SW 28.1 rotor; Beckman Coulter Inc., Fullerton, Calif.). The rabbits were immunized once intradermally with a mixture of purified virus and Freund's complete adjuvant (Dia-iatron Co., Ltd., Tokyo, Japan) and, 3 weeks later, subcutaneously with a mixture of purified virus and Freund's incomplete adjuvant (Dia-iatron Co., Ltd.). The serum was collected 10 days after the booster immunization.

Serum neutralization test. Antiserum was serially diluted twofold with serum-free MEM in a flat-bottom 96-well microplate (Sumitomo Bakelite Co., Tokyo, Japan). One hundred 50% tissue culture infective doses of virus were added to each dilution. The mixtures were incubated at 37°C for 1 h, and then HmLu-1 cells suspended in serum-free medium (GIT; Wako Pure Chemical Industries, Ltd., Osaka, Japan) were added to each well. After incubation at 37°C for 7 days in a humidified 5% CO₂ atmosphere, the antibody titers were expressed as a reciprocal of the highest dilution of sera that completely inhibited the CPE.

Preparation of viral dsRNA. The viral dsRNA was extracted from infected BHK-21 cells by the method described by Siaz-Ruiz and Kaper (17). Briefly, infected BHK-21 cells were homogenized in TE buffer (2 mM Tris-HCl, 1 mM EDTA [pH 8.0]). After centrifugation, the supernatant was disrupted in 1% sodium dodecyl sulfate-0.4 M NaCl. The nucleic acid was precipitated by phenol extraction and ethanol precipitation. The pellet was resuspended in 1 mM EDTA solution (pH 5.0), and then an equal volume of 4 M LiCl solution was added and the mixture was kept at 4°C for 8 h. After removal of the precipitant by centrifugation, an equal volume of 8 M LiCl solution was added to precipitate the viral dsRNA, and the mixture was kept at 4°C for 8 h. The final pellet was reconstituted in TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]). The viral dsRNA was separated on a 0.9% agarose gel (FMC Bioproducts, Rockland, Maine) for 60 min at 100 V. The gel was stained with ethidium bromide and was visualized under UV light and photographed.

RNA extraction and RT-PCR. Viral RNA was extracted from virus culture fluid with a High Pure Viral RNA kit in accordance with the manufacturer's instruction (Roche Diagnostics-Boehringer Mannheim, Mannheim, Germany). The primers (L3-1 [5'-CCCAGATGTTCAATAGCGAACCTAATC-3'] and L3-2 [5'-TAACATTTCGTTA TAGCAATAGTAGTT-3']) were synthesized on the basis of the sequence of RNA3 of EHDV described elsewhere (12). RT-PCR was performed with a TaKaRa RNA PCR kit (with RTase from avian myeloblastosis virus), version 2.1 (TaKaRa Shuzo Co., Ltd., Shiga, Japan), with some modifications. Briefly, the cDNA was synthesized at 42°C for 30 min after preincubation with both primers at 94°C for 4 min and then on ice. For PCR amplification, the PCR mixture (5 mM MgCl₂, 1× PCR buffer, 2.5 U of Taq polymerase) was added to the RT reaction mixture. PCR was carried out with 28 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and elongation at 72°C for 90 s. The PCR products were separated on a 1.5% agarose gel at 100 V for 40 min. After staining with ethidium bromide, the gel was visualized under UV light and photographed.

RFLP analysis of PCR products. The complete nucleotide sequence of the RNA3 of an Australian isolate of EHDV-2 and the partial nucleotide sequence of IBAV have been reported elsewhere (7). The predicted PCR products of EHDV-2 and IBAV have been shown to be cut at five and at least two sites, respectively, with restriction enzyme Sau3AI, and those of IBAV has been shown to be cut at at least one site with restriction enzyme HaeIII. The PCR products were digested with HaeIII and Sau3AI (TaKaRa Shuzo Co., Ltd.), followed by ethanol precipitation. All reactions were performed at 37°C for 60 min. Restriction fragments were separated on a 2.0% agarose gel, and the gel was observed as described above.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Virus isolation. IBAD occurred from August through November 1997 in nine prefectures in the western part of Japan. During that outbreak, 242 cattle showed clinical symptoms and were diagnosed with IBAD on the basis of serological examination to confirm the seroconversion to IBAV. During the same period, several hundred cases of abortions and stillbirths occurred among cattle in the same prefectures. A total of 114 virus isolates were obtained from blood samples from both affected and apparently healthy cattle and from the samples from the fetuses and Culicoides biting midges. Fifty-three, 3, and 11 isolates were isolated from erythrocytes, plasma, and whole blood, respectively, of cattle exhibiting typical IBAD symptoms and cattle raised in the same cowshed with affected animals. These 67 isolates are referred to as IBAD relatives. Thirty-five and two isolates were from the aborted fetuses and placentas, respectively. Seven isolates were obtained from the blood of dams that had a miscarriage or stillbirth and from the blood of cattle raised in the same cowshed with the affected animals. These 44 isolates are referred to as abortion or stillbirth relatives. Three virus isolates were obtained from Culicoides biting midges caught during the epidemic period of the disease.

Neutralization test. An isolate, designated KS(H-22)P/97, was chosen from among the isolates of IBAD relatives, and its serological relationship to IBAV and EHDV was investigated via the cross-neutralization test. As shown in Table 1, antisera to the Ibaraki-2 and Y87061 strains of IBAV equally neutralized the homologous strain at titers up to 64 to 128 as well as the CSIRO439 strain of EHDV. The Alberta strain of EHDV was neutralized by antisera to Ibaraki-2 and Y87061 at titers up to 8 and 16, respectively. KS(H-22)P/97 was neutralized by these sera up to a titer of 8. On the other hand, antiserum to KS(H-22)P/97 neutralized the homologous strain at a titer of 64, whereas the neutralization titers of the serum against other strains of IBAV and EHDV were less than 16. 

Genomic dsRNA profiles of viruses. The genomic dsRNA profile of KS(H-22)P/97 was determined by agarose gel electrophoresis and was compared with those of strains Ibaraki-2 and Y87061 of IBAV and the CSIRO439 strain of EHDV (Fig. 1). The migration patterns of the genome segments of strain KS(H-22)P/97 were identical to those of the other three strains of the virus. Segments 7 and 8 of the viruses seemed to migrate together.

View larger version (65K): [in this window] [in a new window]   FIG. 1.   Agarose gel electrophoresis of viral dsRNA. The dsRNA extracted from infected BHK-21 cells was electrophoresed through a 0.9% agarose gel and was stained with ethidium bromide. Lanes: 1, mock-infected cells; 2, IBAV Ibaraki-2; 3, IBAV Y87061; 4, KS(H-22)P/97; 5, EHDV-2 CSIRO439; M, pHY marker (TaKaRa Shuzo Co., Ltd.). The numbers 1 to 10 on the right indicate RNA segments 1 to 10, respectively.

RT-PCR. To confirm the specificity of RT-PCR with the synthesized primer pair, PCR was done with cDNA transcribed from dsRNA extracted from IBAV and a different serotype of EHDV. The specific PCR product, with an expected size of 659 bp, was amplified from the Australian EHDV isolates, i.e., CSIRO439, CSIRO157, CSIRO753, CSIRO775, and DPP 59, as well as the Ibaraki-2 and Y87061 strains of IBAV (Fig. 2A). No specific band was amplified for the North American EHDV isolates, the New Jersey and Alberta strains (Fig. 2A). The specific band was amplified from IBAD relatives, abortion or stillbirth relatives, and isolates from Culicoides biting midges in 1997. Figure 2B shows the results of RT-PCR for representative isolates from different prefectures, Hyogo (HG), Okayama (OY), Fukuoka (FO), Saga (SG), Nagasaki (NS), Kumamoto (KM), Oita (OI), Miyazaki (MZ), Kagoshima (KS), and Okinawa (ON).

View larger version (28K): [in this window] [in a new window]   FIG. 2.   PCR amplification of IBAV and EHDV. (A) Lanes: 1, IBAV Ibaraki-2; 2, EHDV-1 New Jersey; 3, EHDV-2 Alberta; 4, EHDV-2 CSIRO439; 5, EHDV-7 CSIRO157; 6, EHDV-8 CSIRO753; 7, EHDV-9 CSIRO775; 8, EHDV-10 DPP 59; M, pHY marker. (B) Lanes: 1, IBAV Ibaraki-2; 2, IBAV Y87061; 3, isolate from HG; 4, isolate from OY; 5, isolate from FO; 6, isolate from SG; 7, isolate from NS; 8, isolate from KM; 9, isolate from OI; 10, isolate from MZ; 11, isolate from KS; 12, isolate from ON; M, pHY marker.

RFLP analysis of PCR products. The identities of the specific PCR products were analyzed by RFLP analysis. The PCR products were digested with either the HaeIII or the Sau3AI restriction enzyme. Analysis of the RFLP patterns obtained with HaeIII grouped the viruses into two types of Australian EHDV and IBAV strains. All 114 isolates recovered in 1997 had the same RFLP pattern as those of IBAV (Fig. 3A). The RFLP patterns obtained with Sau3AI were rather complicated (Fig. 4B). Three Australian EHDV isolates, CSIRO439, CSIRO157, and CSIRO775, had the same RFLP pattern as the Ibaraki-2 strain of IBAV. Australian EHDV strains CSIRO753 and DPP 59 and IBAV strain Y87061 each had different RFLP patterns. However, all 114 isolates recovered in 1997 had identical RFLP patterns, and the patterns were distinct from those of EHDV and formerly recovered IBAV isolates (Fig. 3B).

View larger version (33K): [in this window] [in a new window]   FIG. 3.   RFLP patterns of PCR products amplified from field isolates in 1997. PCR products were digested with HaeIII (A) and Sau3AI (B). Lanes: 1, IBAV Ibaraki-2; 2, IBAV Y87061; 3, isolate from HG; 4, isolate from OY; 5, isolate from FO; 6, isolate from SG; 7, isolate from NS; 8, isolate from KM; 9, isolate from OI; 10, isolate from MZ; 11, isolate from KS; 12, isolate from ON; M, pHY marker.

View larger version (34K): [in this window] [in a new window]   FIG. 4.   RFLP patterns of PCR products amplified from IBAV and EHDV. PCR products were digested with HaeIII (A) and Sau3AI (B). Lanes: 1, IBAV Ibaraki-2; 2, EHDV-2 CSIRO439; 3, EHDV-7 CSIRO157; 4, EHDV-8 CSIRO753; 5, EHDV-9 CSIRO775; 6, EHDV-10 DPP 59; M, pHY marker.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

During the epidemic of IBAD in 1997, affected cattle showed typical symptoms of the disease, i.e., fever, anorexia, and disturbance of deglutition. Sixty of the animals died of aspiration pneumonia, dehydration, and emaciation resulting from difficulty with swallowing. In such animals, degeneration of striated muscular tissue was observed in the esophagus, larynx, pharynx, tongue, and skeletal muscles. These clinical and pathological findings are identical to those that occur with IBAD (15). Although subclinical or inapparent infection with IBAV has frequently occurred, abortion and stillbirth of cows had not been reported in previous outbreaks. During the outbreak in 1997, numerous cases of abortions and stillbirths among cattle were observed together with IBAD. A total 114 virus isolates were obtained from the blood of affected animals and cattle raised in the area where the epidemic occurred and from fetuses and Culicoides biting midges that were suspected as being the vectors. The results of RFLP analysis with HaeIII and Sau3AI with these viruses revealed that all of the isolates were identical and had the same origin. Furthermore, the virus seemed to cause not only typical symptoms of IBAD but also abortions and stillbirths in cows.

The results of a neutralization test indicated that strain KS(H-22)P/97 was closely related to IBAV and EHDV-2. However, the isolate was distinct from strains of both virus groups. Strain CSIRO439 was most closely related to the prototype Ibaraki-2 and Y87061 strains of IBAV, as described previously (5), while strain KS(H-22)P/97 was distinct from the Ibaraki-2 and Y87061 strains of IBAV and the CSIRO439 strain of EHDV-2. The antigenic relationship between KS(H-22)P/97 and the North American EHDV-2 strains is not clear. The genomic dsRNA profile revealed that strain KS(H-22)P/97 belongs to the same genotype that includes the Ibaraki-2, Y87061, and CSIRO439 strains.

The sequence of RNA3 of EHDV has been demonstrated to be highly conserved, with more than 90% homology among cognate genes of the same EHDV topotype (3, 7, 19). The primer pair based on the sequence of the RNA3 gene of EHDV allowed successive detection of Australian EHDV and IBAV strains as well as isolates recovered in 1997. McColl and Gould (12) revealed that the specific PCR product of IBAV was not obtained at the high-stringency annealing temperature at 65°C but that it was obtained when the temperature was reduced to 37°C. In this experiment, the specific products were obtained from both EHDV and IBAV at an annealing temperature of 60°C. The serogroup-specific primer used in this study, however, did not amplify the genes of the North American EHDV strains. Although the nucleotide sequences of RNA3 of the Australian EHDV and North American EHDV strains had 79% homologies (7, 19), the nucleotide sequences of the North American EHDV strains were substituted at the 3' end of the primer annealing site. The failure of specific amplification in North American EHDV strains might be caused by this substitution. The RT-PCR could not directly distinguish IBAV from Australian EHDV strains, contrary to the results described by McColl and Gould (12).

RFLP analysis of PCR products allowed grouping of the viruses. Analysis of the RFLP pattern obtained by digestion with HaeIII grouped viruses into distinct topotypes of Australian EHDV and IBAV (Japanese EHDV) strains. The isolates recovered in 1997 were grouped into IBAV. Although the RFLP pattern obtained by digestion with Sau3AI indicated that the genomic variation existed in a topotype among Australian EHDV and IBAV strains, further information including determination of the nucleotide sequence would be needed to reveal the phylogenic association. The RFLP pattern obtained by digestion with Sau3AI suggested that the isolates recovered in 1997 were unique strains of IBAV. The serological surveillance data indicated that many arboviruses, including EHDV and IBAV, are present in eastern and southeastern Asia (4, 14). In Japan, import controls on animals and preventive efforts with vaccines have been used. The live attenuated vaccine derived from the Ibaraki-2 strain has been demonstrated to be effective and safe. National surveillance and intensive monitoring of yearlings as sentinel cattle have been in place for a number of years, and until 1997, IBAD had not been observed and no animals in the sentinel groups had seroconverted since 1987. In addition to this history, the fact that the outbreak of IBAD and reproductive problems had occurred in southern parts of Japan in 1997 suggests that the virus was probably introduced into Japan by infected Culicoides biting midges carried on the wind from places where climate conditions were suitable.