Sequence Diversity of the Capsid Gene and the Nonstructural Gene NS2B of Dengue-3 Virus in Vivo

Abstract

Previously, we studied the envelope (E) gene of dengue virus and reported that dengue-3 virus is present as a quasispecies. To investigate the extent of intrahost sequence variation of other dengue viral genes, we examined in this study the capsid (C) gene and the nonstructural gene, NS2B, derived directly from plasma dengue viruses from 18 confirmed dengue-3 patients. Using reverse transcription-PCR, multiple clones of a 360-nucleotide region covering the C gene and of a 404-nucleotide region covering the NS2B gene from each patient were completely sequenced and analyzed. Our findings of the intrahost sequence variation of the C and the NS2B genes (mean pairwise p-distance: 0.12 to 1.02%, and 0.16 to 1.20%, respectively) demonstrate the quasispecies structure of dengue virus in vivo. A linear relationship was found between the extent of sequence variation of the C and NS2B proteins, suggesting that intrahost sequence variation of dengue-3 virus is likely to reflect genetic drift. The extent of intrahost sequence variation observed is in the same range as that of acute human immunodeficiency virus or hepatitis C virus infection, indicating that the random mutation frequency of dengue virus is similar to that of other RNA viruses in vivo. Consistent with a previous report of the E gene, the observations of genome-defective clones in both the C and the NS2B genes (3.9 and 5.0% of the clones, respectively) suggest a higher frequency of defective viruses in vivo. These findings would add to our understanding of the evolution of dengue-3 virus.

Study of Sequence Variation of Dengue Type 3 Virus in Naturally Infected Mosquitoes and Human Hosts: Implications for Transmission and Evolution

“…3A and C). For the C gene, ten clones from each patient were analyzed as reported previously (26). The mean diversity ranged from 0.13 to 0.41%, and the overall mean diversity was 0.23%, which, in agreement with the results observed for the E gene, was higher than that in the mosquitoes (Table 1).…”

“…We next examined another gene, the C gene, through RT-PCR of a 360-nucleotide region covering the entire C gene ( Fig. 1) (26). Among the 21 clones examined, there were six nucleotide substitutions out of 6,678 nucleotides sequenced, corresponding to a mean diversity of 0.09% (Table 1).…”

Inter- and Intra-Host Evolution of Dengue Viruses and the Inference to the Pathogenesis

“…The involvement of DI particles has been wellstudied in many RNA viruses, such as LCMV and alphavirus, which leads to virus persistence and increased cytopathology and inflammation. Our study, consistent with previous studies (Wang, Lin et al 2002;Wang, Sung et al 2002), also suggested the DI particles exist in human plasma with dengue viral infection. Combinations of DI particles with standard virus in the viral quasispecies leading to curing of the infection or increasing virus replication remain to be studied.…”

Molecular study of the quasispecies evolution of a typical pigeon paramyxovirus type 1 after serial passages in pigeons by contact

“…Therefore, truncated HN proteins might be expressed at the surface of the cells and incorporated into newly formed virus particles, possibly with consequences for the binding abilities and the infectivity of the virus (Huang et al , 2004). However, the question arises whether these aberrant and defective genomic RNAs, detected inside the infected cells, are actually packaged into the newly formed virus particles, excreted and transmitted to susceptible hosts, as shown in other models (Cabot et al , 2000;Kissi et al , 1999;Wang et al , 2003). This question could be assessed by studying the virus heterogeneity after virus purification from both organs and cloacal and tracheal swabs.…”