02495naa a2200169 a 450000100080000000500110000800800410001910000180006024500900007826000090016850000730017752019190025070000180216970000220218770000200220977300960222914686112009-08-21       2005    bl ---       0--  u    #d1 aGASPAR, J. O.  aA primer pair allows amplification of part of the 3'-terminal of carlaviruses genome.  c2005  aEdição dos Resumos da XVI National Meeting of Virology, nov. 2005.  aThe genus Carlavirus belongs to the family Flexiviridae 9Adams et al. Arch. Virol. 149:1045, 2004) and has Carnation latent virus (CLV) as the type member. Badge et al. (Eur. J. Plant Pathol. 102:305, 1996) described a primer (named Carla-Uni), which in association with a oligo d (T21) primer, permits the amplification of a ~ 120 nt fragment lovated at the 11 kDa gene and the polyA tract. This PCR amplification has been used as a diagnostic test for carlaviruses. In order to get a longer PCR product, we compared published sequences of the coat protein gene and found that the amino acids sequence GLGVPTE is conserved in almost al the carlavirus sequenced. This allowed us to design a degenerate primer to that region which, when used with a oligo d(T21) primer, produced in a RT-PCR reaction a fragment og ~930 pb from three carlaviruses tested. This new sense primer has the degenerated sequence 5' - GGBYTNGGBGTNCCNACNGA-3', where B= C or G or T, Y = C or T, N = A or C or G or T. The primer pair was used to amplify sequences from Potato virus S, Cole lantent virus (both transmitted by aphids) and Cowpea mild mottle virus (transmitted by whitefly), all producing a fragment of ~930pb. To verify that the DNA fragments generated by PCR were of viral origin, that from Cowpea mild mottle virus was cloned e sequenced. The amplified fragment (936 pb) comprised the coat protein gene, the 11K gene and a short untraslated sequence before de poly(A) tract. The partial sequencing of the CP gene showed the highest identify (78,3% nt and 93,5% aa) with the isolate H of Cowpea mild mottle virus (AF024628). Our designed primer have the advantage that about 930 nt from the 3' terminus, comprising part of the CP gene, the 11K gene and the terminal untranslated region can be amplified for sequencing, allowing the detection of carlaviruses and identification of unknown viruses as mebers of this virus genus.1 aBELINTANI, P.1 aALMEIDA, A. M. R.1 aKITAJIMA, E. W.  tVirus Reviews & Research, Rio de Janeirogv. 10, p. 140, Nov. 2005. Supplement, ref. P-191.