| Hypoviruses are a group of unencapsidated, cytoplasmically replicating RNA viruses which can reduce virulence of Cryphonectria parasitica and alter host biological processes, resulting in suppressed pigmentation, reduced asexual production, and loss of female fertility. The discovery of the hypovirus presented a new avenue for biological control of chestnut blight. Hypoviruses lack an extracellular route of infection, they are transmitted from infected to non-infected fungal strains through hyphal anastomosis, but the transmission is limited by vegetative incompatibility (vic).The effects of virus strains on the virus transmission in C. parasitica were observed in this study. In laboratory assays by replicating vegetative incompatibility (vic) genotypes in independent fungal isolates, we quantified the transmission of three CHV1 viruses between isolates heteroallelic at one or two vic loci, and demonstrated that different CHV1 virus isolates determined their transmission abilities. The Chinese local hypovirus CHV1-CN280 showed higher transmission efficiency than the other two sequenced hypoviruses CHV1-EP713 and CHV1-Euro7 in this study.The results suggested that using the hypoviruses with higher transmission abilities could increase the effciencies of the biological control of chestnut blight.By constructing cDNA library and RT-PCR technology, the whole genome sequence CHV1-CN280 was determined. The virus showed extensive identities with other three sequenced hypoviruses:CHV1-EP713, CHV1-Euro7 and CHV1-Euro7, with an average of 61% and 94% identities at nuceotide level and 59% and 95% identities at amino acids level respectively. Compared with the other three sequenced hypovirus, the 3'end of CHV1-CN280 was relatively conservative, but the 5'end of CHV1-CN280 was very different from the other sequenced hypoviruses. The junctions of ORFA and ORFB were UAAUG in CHV1-EP713,CHV1-Euro7,CHV1-EP721 and CHV2-NB58, but in CHV1-CN280, it was AUGUAUAA:the UAA was the stop codon of ORFA, and the AUG was the initiation codon of ORFB. A pair of primers was designed to determine junctions of ORFA and ORFB in other 12 CHV1 hypoviruses. The results showed that The junction mode AUGUAUAA was very common as well as UAAUG. Another pair of primers was designed according to the conserved sequences in the 5'-UTR and ORFA region of CHV1 genome and the reverse transcription PCR (RT-PCR) method was developed for the population genetics analysis of CHV1. A total of 12 CHV1 hypoviruses was partially sequenced, combining the partially sequences of other CHV1 hypoviruses determined before, comparison analysis was performed. The results showed that all 36 CHV1 hypoviruses could be grouped into two groupes and eight subtypes. Considering the great differences between the two groupes and the enormous differences that CHV1-CN280 genomic sequence share with other three sequenced hypoviruses, it is suggested that the CHV1 hypoviruses similar to CHV1-CN280 should be classified into a new subspecies.The full-lenghth cDNA clone of the CHV1-CN280 was constructed by accurately joining viral cDNA fragments according to the whole genome sequence with aid of restriction enzymes. By transfection, CHV1-CN280 was successfully introduced into virus-free fungal host strains CN280(v-) and BB-1-5. All of the transfected strains showed similar phenotype to CN280 and the regenerated dsRNA showed similar transmission abilities to CHV1-CN280 which was much higher than CHV1-EP713, conforming the high transmission ability was the property of the virus.The availability of infectious cDNA clone of CHV1-CN280 makes it possible to dissect the molecular basis of a viral genome that controls the horizontal transmission ability and to understand other aspects of interaction between host and virus.
|
PDF Full Text Request