Identification and PCR-restriction fragment length polymorphism analysis of a variant of the Ibaraki virus from naturally infected cattle and aborted fetuses in Japan

Abstract

One hundred fourteen field isolates of the Ibaraki virus (IBAV), a member of the epizootic hemorrhagic disease virus serotype 2 (EHDV-2), were isolated from blood samples of affected and apparently healthy cattle and Culicoides biting midges and from blood samples of dams and internal organs of aborted fetuses during an outbreak of Ibaraki disease in the southern part of Japan in 1997. In this outbreak, 242 cattle showed typical symptoms of the disease, and several hundred dams had miscarriages or stillbirths. The viruses that induced typical Ibaraki disease and reproductive problems among cattle were identical and were antigenically closely related to but distinct from previous isolates of IBAV and EHDV-2. The virus was considered to be a putative agent of this outbreak. Reverse transcription-PCR based on segment 3 of the RNA genome of EHDV-2 and restriction fragment length polymorphism analysis of the PCR products were conducted to compare the genomes of the viruses. The results suggested that the virus isolated in 1997 was a variant of IBAV and might be exotic.

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.

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.

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.

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.