| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Animal: RNA Viruses |
Biochemistry Division, Onderstepoort Veterinary Institute, Onderstepoort, 0110 South Africa1
MRC Diarrhoeal Pathogens Research Unit, Medunsa 0204, Pretoria, South Africa2
Author for correspondence: Albie van Dijk. Fax +61 2 6246 4173. e-mail albievandijk{at}hotmail.com
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 
12. 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.
This article has been cited by other articles:
|
|
 |
|
  A. C. Potgieter, N. A. Page, J. Liebenberg, I. M. Wright, O. Landt, and A. A. van Dijk Improved strategies for sequence-independent amplification and sequencing of viral double-stranded RNA genomes J. Gen. Virol., June 1, 2009; 90(6): 1423 - 1432. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Matthijnssens, C. A. Potgieter, M. Ciarlet, V. Parreno, V. Martella, K. Banyai, L. Garaicoechea, E. A. Palombo, L. Novo, M. Zeller, et al. Are Human P[14] Rotavirus Strains the Result of Interspecies Transmissions from Sheep or Other Ungulates That Belong to the Mammalian Order Artiodactyla? J. Virol., April 1, 2009; 83(7): 2917 - 2929. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Jiang, S. Ji, Q. Tang, X. Cui, H. Yang, B. Kan, and S. Gao Molecular characterization of a novel adult diarrhoea rotavirus strain J19 isolated in China and its significance for the evolution and origin of group B rotaviruses J. Gen. Virol., October 1, 2008; 89(10): 2622 - 2629. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Cao, M. Barro, and Y. Hoshino Porcine Rotavirus Bearing an Aberrant Gene Stemming from an Intergenic Recombination of the NSP2 and NSP5 Genes Is Defective and Interfering J. Virol., June 15, 2008; 82(12): 6073 - 6077. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Wirblich, B. Bhattacharya, and P. Roy Nonstructural Protein 3 of Bluetongue Virus Assists Virus Release by Recruiting ESCRT-I Protein Tsg101 J. Virol., January 1, 2006; 80(1): 460 - 473. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. C. Potgieter, M. Cloete, P. J. Pretorius, and A. A. van Dijk A first full outer capsid protein sequence data-set in the Orbivirus genus (family Reoviridae): cloning, sequencing, expression and analysis of a complete set of full-length outer capsid VP2 genes of the nine African horsesickness virus serotypes J. Gen. Virol., May 1, 2003; 84(5): 1317 - 1326. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
