Direct sequencing of genomic cDNA fragments amplified by the polymerase chain reaction for molecular epidemiology of dengue-2 viruses

Abstract

A nucleotide fragment encoding amino acids 29 to 94 in the E-protein of 28 dengue-2 isolates of diverse geographic and host origins was examined by direct sequencing of a polymerase chain reaction (PCR)-amplified product, and compared to six previously published sequences. Nucleotide divergence ranged from 0 to 19.8% corresponding to a maximum of 9% divergence in the amino acid sequence. Taking a divergence of 6% between the nucleotide sequence as a cut off for genotype classification, six groups have been established. Southeast Asian and the Jamaican 1983 genotypes show a high rate of similarity (> 95.2%). Our results suggest that virus of this group is now circulating as the dominant topotype in Brazil (1990) and in French Guyana (1986-1991). African strains fall into two groups, one endemic group (1970-1990) and one epidemic group (1986-1987). The three other groups correspond to viruses from Sri Lanka (1982) and the Seychelles (1977), from Puerto Rico (1973) and from Tahiti (1975). Our approach appears to be valuable characterizing dengue isolates, easily and rapidly.

Molecular epidemiology of type 1 and 2 dengue viruses in Brazil from 1988 to 2001

“…Subsequently, in 1986, the first outbreak of greater proportions caused by DEN-1 occurred in the met-and Alagoas classified them into the Asian/ American-Asian genotype (18). Similar results were also obtained by nucleotide sequence analysis of another E protein gene segment (nucleotides 85 to 282) of three DEN-2 samples isolated in Brazil in 1990 (7). Nucleotide sequence analysis of the NS5/ 3'NC junction area of two DEN-1 samples isolated in 1990 and 1994 from Rio de Janeiro and São Paulo States, respectively, classiropolitan area of Rio de Janeiro and then spread towards the urban areas in the Northeast and Midwest regions of Brazil.…”

“…Unlike other RNA viruses, some segments of the dengue virus genome have a high degree of stability where fixed mutations are common. Partial sequencing of some genomic regions has been successfully employed to determine the genetic variation of dengue viruses and to characterize genotypes within serotypes (6)(7)(8)(9)(10)(11).…”

“…About 80% of notified dengue cases in the Americas occurred in Brazil, and in the last years more than 1 million cases have been reported in all five Brazilian geographic regions (17). In spite of the importance of dengue as a serious public health problem in Brazil, only a few dengue viruses isolated from endemic regions have been analyzed with respect to genomic variability (6,7,9,16,(18)(19)(20).…”

Geographic distribution and evolution of yellow fever viruses based on direct sequencing of genomic cDNA fragments

“…In this study, we have examined a large panel of YF virus isolates including some from older (Ghana 1927, Zaire 1959, Ethiopia 1961, Senegal 1927and 1965 and recent (Burkina Faso 1983, Nigeria 1986, Mali 1987, Cameroon 1990) epidemics, by direct sequencing of a gene fragment obtained from genomic RNA subjected to reverse transcriptase (RT) and PCR. This methodology was valuable for epidemiological analysis and classification of dengue 2 viruses (Deubel et al, 1993) and was therefore developed to support epidemiological investigations of YF. We have examined the primary nucleotide sequence of an E protein gene fragment of 21 African YF virus isolates, including the two original isolates obtained from humans in Ghana (Asibi) and in Senegal (FV) in 1927.…”

“…pseudoscutellaris AP61 cells. For viral RNA preparation, viruses were passaged once in AP61 cells and viral RNA was phenol-extracted from the cytoplasmic lysate of infected cells and ethanolprecipitated as previously described (Deubel et al, 1993). First strand cDNA was primed and DNA was subsequently amplified using 20-mer oligonucleotide primers chosen to frame an E gene region.…”