| Project/Area Number |
14360110
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
General fisheries
|
|---|
| Research Institution | HIROSHIMA UNIVERSITY |
|---|
Principal Investigator |
NAKAI Toshihiro Hiroshima University, Graduate School of Biosphere Science, Professor, 大学院・生物圏科学研究科, 教授 (60164117)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
MISE Kazuyuki Kyoto University, Graduate School of Agriculture, Associate Professor, 大学院・農学研究科, 助教授 (90209776)
|
|---|
| Project Period (FY) |
2002 – 2003
|
| Project Status |
Completed (Fiscal Year 2003)
|
| Budget Amount *help |
¥15,000,000 (Direct Cost: ¥15,000,000)
Fiscal Year 2003: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2002: ¥12,300,000 (Direct Cost: ¥12,300,000)
|
|---|
| Keywords | betanodavirus / viral nervous necrosis / in vitro infectious RNA transcription system / viral coat protein gene / RNA-dependent RNA polymerase gene / reassortant virus / host specificity / SJNNV / リアソータント / 血清型 / トランスフェクション |
|---|
| Research Abstract |
Betanodaviruses, the causal agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNAs as the genomes. Betanodaviruses have marked host specificity although the primary structures of the viral RNAs and encoded proteins are similar among betanodaviruses. However, no mechanism underlying the host specificity has yet been reported. We first constructed a cDNA-mediated infectious RNA transcription system for striped jack nervous necrosis virus (SJNNV) and sevenband grouper nervous necrosis virus (SGNNV). To construct full-length cDNA clones of the SJNNV genomic RNA1 and RNA2, the complete 5'-and 3'-noncoding sequences of both segments were determined using the RACE method. Based on the terminal sequences obtained, we performed RT-PCR and constructed plasmid clones containing full-length cDNA copies of both RNAs, positioned downstream of a T7 RNA polymerase promoter. These plasmids were cleaved at a unique restriction site just downstream of the 3' terminus of each
… More
SJNNV sequence and transcribed in vitro into RNA with a cap structure analog. The transcript mixture was transfected into fish cell line E-11. Using indirect immunofluorescence with anti-SJNNV serum, fluorescing cells were observed specifically in these transfected cells, and this culture supernatant exhibited pathogenicity to striped jack larvae. This is the first report of betanodavirus cDNA clones from which infectious genomic RNAs can be transcribed. Then, we tested two reassortants between SJNNV and SGNNV for the infectivity in originated host fish. When striped jack and sevenband grouper larvae were bath-challenged with the reassortant virus comprising SJNNV RNA1 and SGNNV RNA2, sevenband grouper was exclusively killed similar to inoculation with SGNNV Conversely, inoculations with the reassortant virus comprising SGNNV RNA1 and SJNNV RNA2 killed striped jack but did not affect sevenband grouper. Immunofluorescent microscopic studies using anti-SJNNV polyclonal antibodies revealed that both the reassortants multiplied at the brains, spinal cords, and retinas of infected fish similar to infection with parental virus inoculations. These results indicate that viral RNA2 and/or encoded coat protein control host specificity in SJNNV and SGNNV. Less
|
