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Journal of Virology, February 2007, p. 1813-1820, Vol. 81, No. 4
0022-538X/07/$08.00+0     doi:10.1128/JVI.01625-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Genome Sequence, Full-Length Infectious cDNA Clone, and Mapping of Viral Double-Stranded RNA Accumulation Determinant of Hypovirus CHV1-EP721{triangledown}

Haiyan Lin,1 Xiuwan Lan,1 Hong Liao,1 Todd B. Parsley,2,{dagger} Donald L. Nuss,2 and Baoshan Chen1,3*

Guangxi Key Laboratory of Subtropical Bioresource Conservation and Utilization,1 Key Laboratory of Ministry of Education of China for Microbial and Plant Genetic Engineering, Guangxi University, 100 Daxiu Road, Nanning, Guangxi 530004, People's Republic of China,3 Center for Biosystems Research, 5115 Plant Sciences Building, University of Maryland Biotechnology Institute, College Park, Maryland 207422

Received 29 July 2006/ Accepted 16 November 2006

Cryphonectria parasitica strain EP721 is infected with a strain of hypovirus CHV1, CHV1-EP721, and exhibits typical hypovirulence-associated traits such as reduced pigmentation and reduced asexual sporulation. However, the accumulation of the viral double-stranded RNA (dsRNA) in this hypovirus-infected C. parasitica strain is atypically low. We now report the complete nucleotide sequence and construction of a full-length infectious cDNA clone for hypovirus CHV1-EP721. The genome sequence of CHV1-EP721 was determined to be 12,724 bp in length and to share extensive homology with two other hypovirus strains, CHV1-Euro7 and CHV1-EP713, with an average of 99% and 90% identities at the nucleotide level and 99% and 92% identities at the amino acid level, respectively. CHV1-EP721 was successfully introduced into virus-free fungal host strain EP721(-v) by transfection with transcripts derived from a full-length viral cDNA. The transfected strain had a phenotype indistinguishable from that of EP721, and the accumulation of CHV1-EP721 dsRNA in the transfectant was lower than those transfected by CHV1-Euro7 and CHV1-EP713 transcripts. Through the construction of chimeric viruses by domain swapping using infectious cDNA clones of CHV1-EP721, CHV1-EP713, and CHV1-Euro7 hypoviruses, the determinant for the low level of viral dsRNA accumulation in CHV1-EP721 was mapped to the second of two CHV1-EP721 open reading frames (ORFs), ORF B. Further refined swapping of domains within ORF B identified a 2.5-kb coding region between p48 and the polymerase domain of CHV1-EP721 as being responsible for the low viral dsRNA accumulation. Evidence is also provided that low rates of hypovirus transmission through conidial spores correlates with low viral dsRNA accumulation.

* Corresponding author. Mailing address: Guangxi Key Laboratory of Subtropical Bioresource Conservation and Utilization, Guangxi University, 100 Daxiu Road, Nanning, Guangxi 530004, People's Republic of China. Phone: 86-771-3239566. Fax: 86-771-3237873. E-mail: chenbs2008{at}163.com.

{triangledown} Published ahead of print on 29 November 2006.

{dagger} Present address: ImQuest Bioscience, 7340 Executive Way, Suite R, Frederick, MD 21704.

Journal of Virology, February 2007, p. 1813-1820, Vol. 81, No. 4
0022-538X/07/$08.00+0     doi:10.1128/JVI.01625-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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