Dengue virus structural differences that correlate with pathogenesis

Abstract

The understanding of dengue virus pathogenesis has been hampered by the lack of in vitro and in vivo models of disease. The study of viral factors involved in the production of severe dengue, dengue hemorrhagic fever (DHF), versus the more common dengue fever (DF), have been limited to indirect clinical and epidemiologic associations. In an effort to identify viral determinants of DHF, we have developed a method for comparing dengue type 2 genomes (reverse transcriptase PCR in six fragments) directly from patient plasma. Samples for comparison were selected from two previously described dengue type 2 genotypes which had been shown to be the cause of DF or DHF. When full genome sequences of 11 dengue viruses were analyzed, several structural differences were seen consistently between those associated with DF only and those with the potential to cause DHF: a total of six encoded amino acid charge differences were seen in the prM, E, NS4b, and NS5 genes, while sequence differences observed within the 5' nontranslated region (NTR) and 3' NTR were predicted to change RNA secondary structures. We hypothesize that the primary determinants of DHF reside in (i) amino acid 390 of the E protein, which purportedly alters virion binding to host cells; (ii) in the downstream loop (nucleotides 68 to 80) of the 5' NTR, which may be involved in translation initiation; and (iii) in the upstream 300 nucleotides of the 3' NTR, which may regulate viral replication via the formation of replicative intermediates. The significance of four amino acid differences in the nonstructural proteins NS4b and NS5, a presumed transport protein and the viral RNA polymerase, respectively, remains unknown. This new approach to the study of dengue virus genome differences should better reflect the true composition of viral RNA populations in the natural host and permit their association with pathogenesis.

FIG. 1

Schematic of the DEN-2 RNA genome showing (from top to bottom) the genome organization, the RT-PCR strategy (overlapping PCR fragments are shown as blocks), and the sequencing strategy. Arrows pointing to the right indicate regions sequenced 3′→5′ on the minus-sense strand; arrows pointing to the left indicate regions sequenced 3′→5′ on the plus-sense strand. UTR, untranslated region.

FIG. 2

Phylogenetic tree generated by maximum parsimony analysis of nucleotide sequences from the entire genome of 11 DEN-2 viruses, sequences of previously published strains and representatives of DEN-1, -3, and -4. Viruses are listed by strain number (Table 1). Branch lengths (proportional to the bar, which equals 1,000) represent the number of nucleotide substitutions between the viruses over the 10,724 nt of the genome used for comparison. Bootstrap values are shown in italics above the branches.

FIG. 3

Predicted RNA secondary structures formed by the complete 5′ NTR of two genotypes of DEN-2: sample 0131, representing the American genotype (a), and sample K0010, representing the Southeast Asian genotype (b). Nucleotide differences between the genotypes (positions 69 and 77) are underlined. B, 5′-terminal bulge; L, 3′-terminal loop.

FIG. 4

Predicted RNA secondary structures formed by the complete 3′ NTR of two genotypes of DEN-2: sample 0131, representing the American genotype (a), and sample K0010, representing the Southeast Asian genotype (b).