Molecular and viral research on dengue hemorrhagic fever

为了阐明与登革出血热发展相关的各种病毒和宿主因素，我们进行了一项为期三年的研究项目。我们在孟加拉国、菲律宾和越南登革热流行期间分离了一些登革病毒株，并测定了病毒株的核苷酸序列。并与常规数据库中的序列进行了比较。我们目睹了21世纪初菲律宾登革病毒2型的基因型转变，从地方株到世界株。而在孟加拉国和越南，登革热病毒在当地进化，并在这些国家引起周期性流行病。在与登革出血热和登革热病毒的比较中，我们没有发现两组之间有任何显著差异。在流式细胞术分析中，我们清楚地证明了登革病毒可以感染外周B细胞和NK细胞，这表明登革病毒在人体中的靶细胞并不总是特定的细胞群，而是病毒可以根据其对每个病毒群的亲和力选择靶细胞。后来，通过使用不同的细胞系从一名患者中分离出两种不同的病毒株，隔离一株病毒在蚊细胞中分离到，并在细胞中生长良好，而在人B细胞中生长不好。另一种病毒是用人单核细胞培养细胞系K562分离的，在B细胞系中生长良好。这两种病毒E蛋白的氨基酸发生了改变，提示了细胞嗜性的决定因素。登革病毒种群缺口很可能在决定登革病毒在人类感染中的致病机制方面具有一定的作用。进一步澄清这种登革病毒种群缺口的重要性对于了解确定人类登革病毒感染严重程度的机制是必要的。

In order to elucidate various virus and host factors that are relevant to the development of dengue hemorrhagic fever, we conducted a three-year research project. We isolated a number of dengue virus strains during dengue epidemic in Bangladesh, the Philippines, and Vietnam and determined nucleotide sequences of the virus strains. The data was compared with each other as well as the sequences obtained in conventional data base. We witnessed the genotype shift of dengue virus 2 in the Philippines during the early 2000s from local strain to cosmopolitan strain. While in Bangladesh and Vietnam, dengue viruses evolved locally and caused periodic epidemics in the countries. In the comparisons made with viruses from dengue hemorrhagic fever and dengue fever, we could not detect any significant differences between the two groups. In the flow cytometry analysis, we clearly demonstrated that dengue virus can infect peripheral B cells and NK cells suggesting the target of dengue virus in humans is not always on a particular cell group but the virus can choose the target cells according to its affinity to each virus group.This idea was further strengthened later by the fact that two distinct virus strains were isolated from one patient by using different cell lines for isolation. One virus stain was isolated by mosquito cells and grow well in the cells but not human B cell line. While the other virus was isolated by K562, a culture cell line form human monocyte, grow well in B cell lines. Those two viruses possessed an amino acid alternation in E protein, suggesting a determinant of cell tropism. It is very likely that dengue virus population gap may have some effect to determine dengue virus pathogenesis in human infection. Further clarification of the importance of this dengue virus population gap is necessary to understand the mechanism to determine the severity in dengue virus infections in humans.