Identification of Brazilian flaviviruses by a simplified reverse transcription-polymerase chain reaction method using Flavivirus universal primers.
We report a simplified reverse transcription-polymerase chain reaction (RT-PCR) method for identification of Brazilian flaviviruses based on the patterns of electrophoretic separation of the amplicons. The RT-PCR was done on the culture fluids of Aedes albopictus C6/36 cells infected with Brazilian flaviviruses, without previous extraction of viral RNA, using Flavivirus universal primers that anneal to highly conserved sequences within the nonstructural protein 5 and 3'- non translated region of the virus genome. Genomes of 13 Brazilian Flavivirus isolates were amplified. It was not possible to amplify the genome of Bussuquara virus. Analysis of the RT-PCR products gave reproducible results and three distinct amplicon patterns were observed. Cacipacoré (800-850 basepairs [bp]) and yellow fever viruses (600 bp) yielded a single amplicon; dengue virus types 1 and 2 (650 and 550 bp), dengue virus type 4 (550 and 450 bp), Iguape (650-600 bp and 750-700 bp), St. Louis encephalitis (700 and 650-600 bp), and Rocio viruses (600 and 500-550 bp) yielded two amplicons; and Ilheus virus yielded five amplicons, two larger than 1,000 bp, one 650-700 bp, one 550-600 bp, and one 450-500 bp. The analysis of amplicon DNA sequences of six viruses showed homology with the 3'- nontranslated region of Flavivirus genome. The use of the Flavivirus universal primers in this simple RT-PCR technique is suitable as a screening test for the genus Flavivirus, with the exception of Bussuquara virus, in Brazilian isolates in tissue culture fluid.
Figueiredo
L T
LT
Unidade Multidisciplinar de Pesquisa em Virologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, SP, Brazil.
Batista
W C
WC
Kashima
S
S
Nassar
E S
ES
D000330:Aedes; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000032:analysis* / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D018067:Flaviviridae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016679:Genome, Viral; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral