登革病毒感染的登革出血熱/登革休克症候群:動物模式

行政院衛生署 90 年度委託研究計畫

成果年報(期終報告)

(自 90 年 8 月 至 91 年 7 月止)

計畫名稱：

登革病毒感染的登革出血熱/登革休克症候群:動物模式

計畫編號：DOH 91 -TD-11 (NSC91-3112-P006-005Y)

研究起訖：

90/8/1 – 91/7/31 (延期至 91/10/31)

申請機構：國立成功大學

主 持 人： 黎煥耀

職 稱： 教授

聯絡電話： 06-2353535 x 5643

電子郵件： hylei@mail.ncku.edu.tw

聯 絡 人： 黎煥耀

傳 真： 06-2097825

填表日期： 92.1.30

註:請依契約書第四條之規定時程繳交，一式二份

目 錄

頁 碼

封面

目錄

一、年度預期目標及所要完成工作項目

( 1 )

二、半年度(期中)已執行完成之工作項目及初步成果

( 2 )

三、半年度(期中)執行研究中所遭遇之問題與困難

( 4 )

四、半年度(期中)經費使用狀況

( 5 )

共

( 5 )頁

頁數限制：2 頁 一、預定完成工作項目及實際執行情形

預定完成工作項目 實際執行情形

Improve the mouse infection model to mimic human dengue hemorrhagic fever and dengue shock syndrome

100%

a. Increase the virus load in vivo to enhance the development of DHF/DSS.

100%

b. Secondary dengue infection. 100%

c. Additional inoculation of dengue virus.

頁數限制：5 頁 二、半年﹙期中﹚初步成果

We have learned that:

a. There is only transient viremia detected by RT-PCR at day 1-3 after intravenous injection of 1 x 108 PFU of dengue 2 virus. Dengue virus less than 1 x 108 PFU is not effective.

b. The CD69 early activation marker detected by flow cytometry was observed maximum at day 1 on leukocytes.

c. The mitogen-stimulated T cell proliferation was impaired after dengue infection in mice.

d. The dengue virus-infected mice will develop paralysis, hepatic and brain inflammation at 14 days post infection. The dengue virus was re-isolated at this late stage.

e. The strain susceptibility to dengue virus-induced paralysis is different. A/J (H-2a) strain is most sensitive while BALB/c (H-2d) or B6 (H-2 b) strains are resistant.

f. Anti-platelet and anti-endothelial cell auto-antibodies were generated after dengue virus infection, and these antibody can cause lyse platelet or endothelial cells in the presence of complement.

We investigated whether dengue virus can induce more severe course. It was known that antigen can induced biphasic (1 h and 24 h) hypersensitivity on antigen-sensitized mice. The unique early-type hypersensitivity (ETH) is elicited within 1 h post antigen challenge and has the characteristics of increased

vasopermeability, which is different from the 24-h cell-mediated hypersensitivity. The type 2 Dengue virus was cultured in C6/36 cells, and concentrated by

ultracentrifugation. The semi-purified dengue virus had titer equivalent to 5 x108 ~ 1 x 109 PFU/ mg protein. We used this semi-purified protein to sensitize and challenge the mice. The dengue antigen induced both 1-h and 24-h hypersensitivity. Both hypersensitivity are dengue antigen-specific, no ear swelling was detected on naive mice. Furthermore, dengue antigen could also induce both types of hypersensitivity on dengue virus-infected mice.

The intravenous injection of antigen on antigen-sensitized mice manifests anaphylactic symptoms such as hemorrhage, hypotension, hypothermia,

hemoconcentration, thrombocytopenia, and anaphylactic death. The

hemoconcentration and thrombocytopenia were manifested as early as 2 min after intravenous injection of dengue antigen (100 microgram equivalent to 5 x 107 PFU) on dengue antigen/CFA primed mice. The hemoconcentration sustained for 1 h while the thrombocytopenia maintained till 48 h. The plasma proteins traced by Evans blue were leaked into tissue. Some of the mice will die within half an hour due to the loss of effective blood volume. The dengue protein-induced hemoconcentration was dose-dependent, the minimum amount of dengue protein to induce

Dengue virus causes mouse mortality when the virus is given intracerebrally. We further found that dengue virus also caused encephalitis when it was

administrated intravenously. The detection of virus on various organs was performed by RT-PCR. Organs such as liver and brain, but not spleen or lung, had dengue positive strain of RNA, indicating that mouse cells are permissible for dengue virus by peripheral infection. The mouse primary hepatocyte or brain cells infected with Dengue 2 virus showed cytopathic. When the dengue antigen-sensitized mice were challenged with these Dengue 2 infected-cells intraperitoneally, the

hemoconcentration and thrombocytopenia was manifested. This suggests that dengue virus-infected cells can also present the dengue antigen to cause the anaphylactic response in dengue antigen-sensitized mice. Furthermore, the mice were infected with type 2 Dengue virus, two months later, they were challenged with the dengue antigen intravenously. The thrombocytopenia was manifested at 10 min after challenge. Based on the above data, we conclude that dengue virus can infect the mouse, stimulate the dengue antigen-specific ETH. When the mouse is re-stimulated with the dengue antigens, the anaphylactic response such as hemoconcentration and thrombocytopenia is manifested.

During this process, the in vivo target of dengue virus is not clearly demonstrated. Although monocytes (especially immature dendritic cells),

lymphocytes, endothelial cells, and hepatocytes can be infected by dengue virus in vitro, how are their in vivo infectious status are not clear yet. In our mouse study, the dengue genome can only be detected in blood by RT-PCR at day 2 post dengue virus injection. But we can not detect dengue genome in any organs such as spleen, lymph node, liver, lung, heart, thymus, brain, kidney after 3 days post injection.

Surprisingly, the dengue genome was re-detected on spinal cord, lung in paralyzed mice at 14 days after infection. It is not clear at this moment where the dengue virus hides. Is there a second viremia at the later time in addition to the early viremia? We will try to determine where the dengue virus replicates, where the dengue virus antigens express on tissues or organs. Either virus isolation, detection of viral genome by RT-PCR, or detection of dengue virus-induced antigen by

immunohistochemical staining will be used. The virus load will be quantitated when it is required. We have developed many different monoclonal antibodies from dengue virus-infected mice that can bind dengue virus associated antigens. To develop more efficient mouse infection model, the conditions for inoculation of dengue virus will be refined.

頁數限制：1 頁 三、半年﹙期中﹚研究中所遭遇之問題與困難，並請自評是否符合進度。 □進度超前 x 符合進度 □落後 ( )月 ◎所遭遇之問題與困難 （篇幅不足，請自行複製） 第 4 頁

四、半年(期中)經費使用狀況 項 目 本年度核定金額 消 耗 狀 況 人事費 445550 386,850(86.8%) 業務費 (含管理費) 394450 0(0%) 合計 840,000 386,850(46%) （篇幅不足，請自行複製） 第 5 頁