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行政院國家科學委員會補助專題研究計畫成果報告
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※ 登革病毒與 B 細胞及嗜中性細胞的互動 ※
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計畫類別：個別型計畫
計畫編號：NSC90-2320-B-006-074-執行期間：20010801～20020731
計畫主持人：陳舜華
執行單位：國立成功大學微生物學科（所）
參與人員: 林育雯
一、中文摘要 流行病學調查報告指出，台灣已有 四個血清型登革病毒同時存在，由此警 訊顯示登革流行的頻率將大增，而且出 現出血/休克等致死症狀的機會將提 高。事實上，近三年以來，登革在南台 灣持續發燒，每年都有感染流行，尤其 民國八十七年的流行，有數例嚴重出血 /休克症狀群，並造成一名患者死亡。 登革目前在台灣已衍然形成一顆不定 時炸彈，不知何時將爆發大流行。民國 八十九年我們提計劃時已大聲急呼，今 年不幸(民國九十一年)在高雄地區數 千人的大流行，更驗証我們的說法。無 奈我們對病毒致病機轉仍不完全了 解，目前僅能以症狀支持療法維持重症 病人生命，無法針對病毒妥善治療，更 遑論有效預防病毒造成的傷害。 登革病毒的致病機制轉眾說紛 芸，因此民國八十七年感染流行期間， 我們採集許多病人檢體，藉由分析檢 體，重新探討致病機轉。我們發現人類 血液中Ｂ細胞及嗜中性白血球在病徵 上扮演相當重要角色。這二種細胞具有 病毒進入所需的受體，極有可能被毒感 染活化後，引起病人的病態現象。但以 前並無太多相關研究，而從病人檢體 中，因採樣時間通常太晚，我們無法獲 得這些完整訊息。所以在這一計劃中， 我們提議以非常敏感方法來探討，病毒 是否能感染及活化 B 細胞及嗜中性白 血球，引起一連串的病態反應。研究所 得的結果將有助於我們解答登革病毒 感染造成出血/休克的致病機轉，並助 於臨床上發展有效的治療方法及安全 的疫苗。目前這計劃有關 B 細胞研究結 果所寫成論文，已被 J our nal of Vir ology 接受刊登。 關鍵詞：登革病毒、Ｂ細胞、嗜中性白 血球 AbstractDengue virus (DV) infection causes symptoms in humans range from mild fever (DF) to fatal hemorrhage fever (DHF) and shock syndrome (DSS). Severe DHF/DSS occurs mostly in patients secondarily infected by a different serotype. Recent
epidemiological surveys showed Taiwan has all 4 serotypes, so there is increasing potential of endemic outbreak and developing fatal DHF/DSS. Indeed,
2 dengue outbreak has been reported in southern Taiwan for five consecutive years. One big outbreak was in 1988. Hundreds and thousands people have been infected in this year outbreak. Several patients developed DHF/DSS and death in these 2 major outbreaks. Although dengue infection poses a serious threat to public health of Taiwan, unfortunately, there is no specific
antiviral treatment or prophylaxis vaccine because the lack of understanding its pathogenesis.
By analyzing the clinical samples collected from patients during outbreak, our research group found the immune mechanisms mediated by B
Lymphocytes (B cells) and neutrophils play an important role in pathological changes of dengue patients. These 2 type cells bear Fc receptors, so they are potential targets for DV. However, whether virus can infect and activate these cells in the early stage of infection to trigger pathological processes
observed in dengue patients is poorly understood. It is very difficult to obtain such information from clinical samples due to the time constraint in collecting samples and complexity. This grant is, therefore, proposed to investigate the interaction between DV and B cells as well neutrophils. The results obtained from this study will help us to design effective antiviral therapy and safe
vaccine to prevent fatal dengue infection.
The manuscr ipt of B cell study pr oposed in this pr ojected has been accepted for publication in J our nal of Vir ology.
Keywords：Dengue virus, B cells, and neutrophils
二、緣由與目的
Infection with any of the four
serotype DV(-1, -2, -3, and -4),
mosquito-born flaviviruses, can cause self-limiting DF or severe DHF and DSS. The incidence of fatal DHF cases has increased sharply in Asia over the last two decades, making it a leading cause of morbidity. Several mechanisms have been proposed to explain the
pathogenesis of DV infection. A long-standing hypothesis,
antibody-dependent enhancement (ADE), proposes that preexisting
non-neutralizing antibodies enhance DV infection of monocytes via the Fc
receptor. More recently,
immunopathogenesis theories suggest that the ADE results in increased T-cell activation and cytokine production, which subsequently activate complement to damage endothelial cells. We have found that, besides monocytes and T cells, B cells also contribute to
pathogenesis by producing high titers of anti-platelet and anti-endothelial cell autoantibodies, particularly, in DHF/DSS patients, which could induce
coagulopathy and vasculopathy, two major pathologies of DHF/DSS.
Throughout DV infection, virus and cytokines are detected in patient blood, and peripheral blood mononuclear cells (PBMC) are found to be one of the most common recovery sites of virus. In PBMC, virus is detected frequently in the adherent monocytes. In addition, a study has previously established that only B cells and monocytes in human PBMC support virus replication and that monocytes produce more viruses than B cells. Based on these observations, and particularly based on ADE, monocytes are thought to be the major cell target for DV. However, two clinical
observations of infected patients with evident syndromes revealed that viral antigen appeared only on B cells and identified that B cells, not monocytes,
3 are infected with the virus. Whether DV replicates actively in B cells is still an open question. Because B cells and monocytes have Fc receptors, they are both potential targets for
antibody-enhanced infection. B cells could also secrete cytokines such as interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α), which are found to be elevated specifically in DHF/DSS patients and affect endothelial cells. Additionally, increased levels of IL-6 has been shown to correlate with a
deficiency in coagulation factor XII and with elevated levels of anti-platelet and anti-endothelial autoantibodies as well as fibrinolytic component, such as
tissue-type plasminogen activator in dengue patients. In this report, we characterized DV replication,
antibody-enhanced virus infection, and cytokine responses in human B cells, and compared them to the corresponding response of monocytes.
三、結果與討論
Active DV r eplication in human pr imar y B cells. The presence of
replication template (negative-strand RNA intermediate), viral antigens
including core and nonstructural proteins, and increasing amount of viruses over time of post-infection indicated that DV actively replicated in B cells.
Surprisingly, we found that not only were the ranges of virus produced by infected B cells and monocytes overlapping but also the mean values were not statistically different (P > 0.6) between these two cell types at any time point in 8 donors we tested.
Antibody enhanced DV2 r eplication in pr imar y B cells and monocytes. Dengue immune serum
slightly increased viral replication by an average of 2.2-fold in both infected B
cells and monocytes (P > 0.05). RT-PCR assays detected a 1.3-fold increase of the negative-strand RNA genome in infected B cells with DV3 immune serum compared to that with normal serum further confirming that ADE increases virus replication (Fig. 1B). Flow cytometric analyses of NS1 expression were performed on infected B cells from 3 donors. We found that, in the presence of DV3 immune serum, not only was the percentage of cells positive for viral NS1 protein increased
significantly (P < 0.05) but also the mean fluorescence intensity of NS1 expression in infected B cells was augmented about 2.8-fold [78 + 32 (control serum) vs 221 + 92 (immune serum)].
Cytokine r esponses in DV-infected B cells and monocytes. Both B cells
and monocytes can produce IL-6 and TNF-α, so the cytokine responses of these two cell types after infection were examined. DV2 infection significantly increased the level of these two
cytokines in both B cells and monocytes (P < 0.05) 48 h after infection. Similar to the virus yield, the ranges these two cytokines produced by infected B cells and monocytes overlapped. Although the average amount of these two
cytokines secreted by infected B cells was ess than that by infected monocytes, this difference did not attain statistical significance (P = 0.37). Antibody also slightly enhanced cytokine production in infected B cells and monocytes.
Conclusion: DV is able to replicate
in B cells and the levels of virus replication, antibody-enhanced virus replication, and cytokine responses observed in B cells were not statistically different from those in monocytes. These results suggest that B cells may amplify and spread virus during infection. B cells could also secret cytokines and
4 autoantibodies to trigger pathologic
responses during infection. Therefore, B cells play an important role in DV pathogenesis.
四計劃成果自評
Although this project got extremely poor score and low budget funding (NT$
531, 300) from NSC, we still carried it through for its significance in dengue pathogenesis. The detailed
characterization of DV replication and cytokine production in virus-infected human B cells finally got approval of reviewers and was accepted for publication in the prestigious
