Cloning of complete genome sets of six dsRNA viruses using an improved cloning method for large dsRNA genes.
Cloning full-length large (>3 kb) dsRNA genome segments from small amounts of dsRNA has thus far remained problematic. Here, a single-primer amplification sequence-independent dsRNA cloning procedure was perfected for large genes and tailored for routine use to clone complete genome sets or individual genes. Nine complete viral genome sets were amplified by PCR, namely those of two human rotaviruses, two African horsesickness viruses (AHSV), two equine encephalosis viruses (EEV), one bluetongue virus (BTV), one reovirus and bacteriophage Phi12. Of these amplified genomes, six complete genome sets were cloned for viruses with genes ranging in size from 0.8 to 6.8 kb. Rotavirus dsRNA was extracted directly from stool samples. Co-expressed EEV VP3 and VP7 assembled into core-like particles that have typical orbivirus capsomeres. This work presents the first EEV sequence data and establishes that EEV genes have the same conserved termini (5' GUU and UAC 3') and coding assignment as AHSV and BTV. To clone complete genome sets, one-tube reactions were developed for oligo-ligation, cDNA synthesis and PCR amplification. The method is simple and efficient compared to other methods. Complete genomes can be cloned from as little as 1 ng dsRNA and a considerably reduced number of PCR cycles (22-30 cycles compared to 30-35 of other methods). This progress with cloning large dsRNA genes is important for recombinant vaccine development and determination of the role of terminal sequences for replication and gene expression.
Biochemistry Division, Onderstepoort Veterinary Institute, Onderstepoort, 0110 South Africa1.
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Potgieter
MRC Diarrhoeal Pathogens Research Unit, Medunsa 0204, Pretoria, South Africa2.
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Steele
Biochemistry Division, Onderstepoort Veterinary Institute, Onderstepoort, 0110 South Africa1.
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van Dijk
D000356:African Horse Sickness Virus; D003001:Cloning, Molecular / Q000379:methods*; D017910:Cystoviridae; D018076:DNA, Complementary / Q000032:analysis / Q000096:biosynthesis; D021141:Nucleic Acid Amplification Techniques; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000235:genetics*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics*; D012087:Reoviridae; D020079:Terminal Repeat Sequences
D018076:DNA, Complementary; D012330:RNA, Double-Stranded; D012367:RNA, Viral