Expression of Koi Herpesvirus in Peripheral Blood Leucocyte Cells Isolated from Latently Infected Koi

錦鯉疱疹病毒 (koi herpesvirus, KHV)，目前被分類於鯉魚疱疹病毒第三型 (Cyprinid herpesvirus 3, CyHV-3)，為具封套雙股DNA病毒，其核酸大小為295kbp。會造成錦鯉 (Cyprinus carpio koi)與鯉魚 (Cyprinus carpio carpio) 80-100%的死亡率，造成經濟上極大損失；病毒最佳感染溫度為18-28℃。當細胞培養溫度上升至30℃時，病毒會開始停止複製且基因停止轉錄。而在臨床上，當溫度低於18℃或高於28℃時，受感染魚隻臨床症狀會消失並造成潛伏感染；而這些帶原魚可能會在季節變化時，將體內潛伏感染之KHV傳播給健康魚隻，而造成大規模的疾病爆發。而本實驗室以原位雜交 (in situ hybridization) 可在發病魚的巨噬細胞核與質中偵測到病毒核酸；且在2008年翁的實驗中，以KHV病魚乳劑感染健康錦鯉的巨噬細胞，透過Real-time PCR技術，可發現病毒核酸隨時間增加而上升。故初步認為KHV可在巨噬細胞中複製。 因此本實驗主要研究目的為偵測KHV是否會在帶原魚隻血球細胞中潛伏，並利用溫度變化以及ConA刺激，偵測是否表現或合成完整病毒顆粒；根據實驗結果顯示，無論健康魚或帶原魚在23℃下進行攻毒後7-13天，血液中會有病毒早期與晚期的mRNA表現，而在32℃下則僅有早期mRNA表現，顯示在不適當的感染溫度下病毒在周邊血液白血球中無法完成完整的複製；另外可以發現健康魚在23℃第一次接觸病毒時，不會產生中和力價，且魚隻在攻毒後10天開始出現死亡；而32℃攻毒，魚隻不會出現死亡的現象但也沒有中和力價的出現；另外帶原魚在不同溫度下接觸病毒時，會產生中和力價使魚隻可以耐過感染，顯示帶原魚可以有效耐過病毒的第二次感染；另外以TO與KF-1細胞株進行KHV攻毒，可以發現對KHV不具感受性之細胞僅會表現早期的mRNA，而不會出現晚期mRNA之表現。感染KHV後的TKF則會表現早期與晚期mRNA。Koi herpesvirus (KHV), designated Cyprinid herpesvirus 3 (CyHV-3), is the etiological agent of an emerging and mortal disease in koi (Cyprinus carpio koi) and common carp (Cyprinus carpio carpio). The genome comprises a linear double-stranded DNA of 295 kbp. The disease is seasonal; afflict fish when the water temperature is 18 to 28°C. Virus propagation and virus gene transcription are turned off when cell are moved to a nonpermissive temperature of 30°C. And CyHV-3 can persist asymptomatically for long periods in the fish if the temperature out of optimal temperature or latency sustained, bursts of new infection occur after exposure to permissive temperatures and stress. Our previous data showed that thymidine kinase gene of KHV was detected in macrophages of infected koi by in situ hybridization and Weng found out that KHV TK gene copy number increased in the head kidney leukocyte derived from koi when detected by real-time PCR. In our study, experimental result showed out, the mRNA of Orf4 (early transcription gene) and Orf2 (late transcription gene) can be detected both in KHV infected naive and carrier carp at 7-13 days postinfection (d.p.i.) when temperature at 23°C. But at 32°C only Orf4 could be detected, demonstrated the virus is unable to complete the replication in peripheral blood leucocytes (PBL) under nonpermissive temperature. In naive carp immersed with KHV the peak of the mortality at 10 d.pi. and serum neutralization (SN) titer can’t be detected within this duration at 23°C. Howerer at 32°C, the naive carp immersed with KHV showed normal appearance without death. The carrier carp exposed to KHV in both permissive and nonpermissive temperature appeared no death and higher SN titer. Moreover only Orf4 could be detected in TO and KF-1 that appeared not susceptibile to KHV, and both Orf4 and Orf2 can be detected in KHV infected TKF