A role of viral variation in determining the host response

The role of dengue virus variation in determining host responses and clinical outcome to infection is largely unknown due to the paucity of data on the viral genome structure. Previous studies have concentrated on structural genes, often from an epidemiological perspective. With the development of high-throughput capillary sequencing for viral genomes, investigation of their functional aspects becomes possible, but only with large commitments of time and money.

We are developing a rapid and cost-effective mechanism for dengue resequenc-ing, utilizing an array platform approach that has already been developed to track the genetic diversity of the SARS CoV (Wong et al 2004), using high-density custom microarrays manufactured by Nimblegen Systems. For the pilot array, we first created reference consensus sequences to the four serotypes of dengue, derived from all available dengue genomes in GenBank. Resequencing probes were synthesized in situ onto the array using our previous bioinformatics approaches. While the probes were able to distinguish between the four serotypes of dengue, the resultant sequence was not complete, giving sequencing errors primarily in regions where there were multiple polymorphisms within the same resequencing probe. To overcome this problem, we generated a second consensus sequence derived from viruses isolated in Southeast Asia and conducted allelic association analysis of single nucleotide polymorphisms which occur in close proximity using a novel bioinformatics approach. This new approach is now able to sequence over 95% of the genome (Fig. 1), with further improvement possible with a development of the bioinformatics interpretation of the hybridization pattern. With this new tool, rapid and cost effective whole genome sequencing is becoming a reality, allowing the investigation of viral variation in host—pathogen interaction to become routine.

To enable the community of dengue researchers to make use of this new data flow we have developed the DengueInfo website and database (www.dengueinfo.org) that is specifically focused on the genomic aspect of dengue biology. It will serve as a repository for full length and near full length dengue genome sequences. It also unifies the sometimes poorly standardised GenBank annotations. The website offers a powerful query interface that allows one to find sequences that match very flexible search criteria. To help researchers investigate any link between genome and severity we also store, where available, some clinical information such as disease outcome.

In a preliminary investigation, we compared the genomic sequence of the prototype strain New Guinea C (NGC) dengue 2 virus, with a clinical isolate TSV01 (GenBank AY037116) and identified nine variations leading to non-conserved amino acid substitutions (Fig. 2). We then investigated possible consequences of these genomic variations on the host response using our HepG2 in vitro model and microarray readout (described above). SAM analysis revealed 88 genes that were

Serogroup DENV-1

DENV-2

DENV-3 DENV-4 Background

FIG. 1. Dengue resequencing by microarray hybridization. The figure shows the result of a hybridization to a dengue 1 PCR amplification to a chip containing probes to all four sero-groups (partitioned into sections, as labelled to the left). In this hybridization, accurate calls were made to 98.3% of the whole genome.

TABLE 4 Expression profile differences between NGC and TSV01 in HepG2 cells

Genes found to be

Biological process

Genes in pathway

significant

Pathway P value

Interferon-mediated immunity

41

9

8.36E-13

Immunity and defence

786

18

2.78E-08

differentially expressed between these strains, predominantly because NGC initiated a relatively small host response (Table 4). Interestingly, the most statistically important difference was in interferon mediated immunity (Table 4).

We predict that there are likely to be naturally occurring variations in the dengue genome, most likely in the non structural genes, that manipulate the host response. Future work will look at this variation and also investigate specific variations using a site directed approach.

Conclusions

The work presented here points towards the beginning of a deeper understanding of the dynamics of this human—virus interaction.

FIG. 1. Dengue resequencing by microarray hybridization. The figure shows the result of a hybridization to a dengue 1 PCR amplification to a chip containing probes to all four sero-groups (partitioned into sections, as labelled to the left). In this hybridization, accurate calls were made to 98.3% of the whole genome.

Variations Virus
FIG. 2. Protein variation as a result of genomic sequence difference between dengue 2 strains NGC and TSV01.

We would like to fully acknowledge our partners in the Singapore EDEN study, in particular the study leader Adrian Ong from Tan Tock Seng Hospital, the study nurse Diana Tan Bee Har and Lee Ching Ng, Tim Hart and Bijon Kumarsil from the Environmental Health Institute and Ooi Eng Eong from the Defense Science Organization. In addition at GIS we would like to thank Frans Verhoef, for Bioinformatic support and LEONG Wan Yee, Aw Poh Kim Pauline and Khoo Chen Ai, for technical assistance. We would also like to thank the participants in this study for their willingness to be involved. This work is in part funded through a grant from the Singapore Tissue Network and partly from a grant from NITD. At GIS the work is funded through A*STAR.

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