Bovine herpesvirus type 4 (BHV-4)是一種γ型皰診病毒(gamma-herpesvirus)，牛隻經常發現有此病毒感染。當BHV-4感染宿主細胞之後，其基因依表現的先後順序可分為三期：immediate early gene ( IE ), early gene ( E ), late gene ( L ) 等三個時期。IE基因的產物在病毒進入細胞後最早表現，並能調控E及L基因的表現，我們實驗室發現BHV-4上的IE-1 基因區域可進行alternative transcription，此段基因除了產生IE-1 mRNA外，亦可產生另一基因產物E-1 mRNA。然而，目前對於這些產物的功用仍不清楚。
　　在以往BHV-4主要是利用牛腎臟上皮細胞(MDBK)做為感染的細胞，但我們實驗室發現BHV-4在感染牛頸動脈內皮細胞(BAEC)所生成病毒產量較在MDBK感染產生病毒的量增加100 ~ 1000倍，顯示BAEC對於BHV-4感染的感受性較高。我們亦發現到BHV-4的IE-2基因產物在BAEC中能夠活化IE-1 promoter region的表現，而MDBK中則無此活化的現象。利用IE-1 promoter region截短的方式，我們已找出IE-1 promoter region在內皮細胞受到IE-2活化調控的區域，位在E-1基因上游 –483 至 -248間的位置。為了瞭解BHV-4在這二種細胞中表現差異的原因，本論文之重心擬在探討BHV-4感染不同細胞後的基因表現及調控的情形。
　　為了瞭解IE-2活化IE-1 promoter region的現象是否只有在受到BHV-4感染的BAEC才會發生而不在受感染之MDBK中發生，首先我們將含有IE-1、IE-2 之promoter的質體分別送入此二種細胞中，而後再感染病毒觀察此二種 promoter被病毒感染後活化的情形。在BAEC感染BHV-4後可看到明顯的IE-1 promoter region表現量增加的情形， 而IE-2 promoter的表現則不受病毒感染的調控。然而在MDBK中以BHV-4感染後IE-1及IE-2 promoter表現量都沒有受病毒感染而活化的現象。我們進一步將截短的IE-1 promoter送入BAEC中，再以病毒感染，發現病毒活化IE-1 promoter region的區域，也是在 E-1基因上游–483 ~ -314之間，與之前利用IE-2產物活化的區域相當接近，因此我們推論BHV-4感染內皮細胞後，可以藉由IE-2的生成，對IE-1 promoter region進行活化。可能是這種調控作用使IE-1 gene region做不同的表現，因而影響病毒在內皮細胞產量。
　　為了釐清IE-2在不同細胞對於IE-1 promoter region作用後，是否會影響基因的表現型態，因此我們另行構築了一個含有完整基因區域的質體，其中同時包含了IE-1與E-1的promoter和結構基因的區域，我們將其命名為pIE1F，將此質體送入細胞後，由所表現RNA經由RT-PCR的方式來探討IE-1、 E-1基因在不同細胞中表現的情形。我們將pIE1F以暫時性轉染的方式分別送入內皮細胞的BAEC、HMEC-1與上皮細胞的MDBK中，觀察到在pIE1F單獨存在細胞中之時，只能夠表現出IE-1的mRNA。若在pIE1F轉染同時外再加入能夠表現IE-2的pCRIE2做共同暫時性轉染時後，可於內皮細胞中觀察到能夠產生出E-1的mRNA來，而在MDBK中，不管是否有IE-2存在都不會有E-1 mRNA之表現。我們的實驗顯示出，雖然BHV-4能夠成功的感染不同的細胞，但是在感染細胞後，由細胞內病毒的基因表現經由病毒基因及細胞基因產物的共同作用，受到了調節，以致於影響病毒力價的產生，這種相互影響的作用似乎隨著細胞的種類不同而有所差異。這可能是造成BHV-4病毒在不同細胞中產生病毒量不同之主要原因。

　　Bovine herpesvirus type 4 (BHV-4), a ubiquitous virus in cattle, belongs to the gammaherpesvirinae. There are three phases of herpesvirus gene expression in a cascade-like manner and the genes of the virus were classified as immediate early (IE), early (E) and late (L) genes accordingly. Herpesvirus IE gene products are the earliest gene regulators expressed and are essential for the induction of E and L genes. In our pervious studies, we found that BHV-4 IE-1 gene had underwent an alternative transcription to produce an early gene product, E-1 RNA.
　　The most common used cells for BHV-4 propagation is Madin Darby bovine kidney cells (MDBK). However, recently we had demonstrated that bovine arterial endothelial cells (BAEC) are more susceptible to BHV-4 infection than MDBK. Our previous studies have found that BHV-4 IE-1 promoter expression in BAEC can be activated by IE-2 gene product, but not in MDBK, and the response region is around –483 to –248 upstream of E-1 gene. The aim of this study is to analyze the gene expression of IE and its regulation in different cells infected with BHV-4.
　　Our results showed that BHV-4 infection enhanced the expression activity of IE-1 promoter region, but the infection did not change the expression of IE-2 promoter in BAEC. However, neither IE-1 promoter region nor IE-2 promoter in MDBK could be activated by BHV-4 infection. Furthermore, we used a series of progressively deleted fragment of IE-1 promoter region to locate the regulatory region. We found the regulatory region was between –483 to –314 upstream of E-1 mRNA. The regulatory region activated by virus infection was very close to the region regulated by IE-2 gene product. The results confirm that BHV-4 IE-1 promoter region can be regulated by IE-2 in endothelial cells, not in epithelial cells.
　　In order to further understand IE-1 gene expression patterns and regulation by IE-2 product in different cell types, we have constructed a vector containing full length of BHV-4 IE-1 gene, pIE1F, that consisted of IE-1 promoter and structure gene. The gene was transfected into endothelial and epithelial cells separately. The gene expression patterns of IE-1 and E-1 were analyzed by RT-PCR. The data indicated IE-2 that cotransfection could trigger E-1 expression only in endothelial cells, not in MDBK. The results also showed that the pattern of BHV-4 gene expression was regulated by IE-2 gene product and the regulation is cell-specific. The different regulation mechanisms of viral gene may contribute to the difference of viral susceptibility of different host cell types.

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