TW201812005A

TW201812005A - Novel fish pathogenic virus

Info

Publication number: TW201812005A
Application number: TW106127062A
Authority: TW
Inventors: 馮張客; 新吳佳; 路克葛萊斯; 葛魯艾德戴; 瓦納斯佛格斯; 德胡克莉亞凡; 馬丁戴斯
Original assignee: 荷蘭商英特威國際公司
Priority date: 2016-08-11
Filing date: 2017-08-10
Publication date: 2018-04-01
Also published as: CN109890409A; TWI765902B; WO2018029301A1; SG11201900912PA

Abstract

The present invention pertains to a novel fish pathogenic virus causing disease in fish, tentatively called Lates Calcarifer Herpes Virus (LCHV), to cell cultures comprising said virus, DNA fragments and corresponding proteins of the said virus, to vaccines on the basis of said virus, DNA and/or protein and to antibodies reactive with said virus and diagnostic test kits for the detection of said virus.

Description

Novel fish pathogen virus

本發明係關於一種會在魚類中引起疾病之新穎魚類病原病毒；包含該病毒之細胞培養物、該病毒之DNA片段及相應蛋白質；基於該病毒、DNA及/或蛋白質之疫苗，及與該病毒反應之抗體，及用於偵測該病毒之診斷測試套組。The present invention relates to a novel fish pathogenic virus which causes disease in fish; a cell culture comprising the virus, a DNA fragment of the virus and a corresponding protein; a vaccine based on the virus, DNA and/or protein, and the virus The antibody to the reaction, and the diagnostic test kit for detecting the virus.

在過去幾十年裏，全球魚類之消費量出現大幅增加。此同等考慮了對諸如鮭魚、大菱鮃、大比目魚及鱈魚的冷水魚類及諸如亞洲海鱸(Asian sea bass) (尖吻鱸)、吳郭魚、虱目魚、鰤魚、紅魽、石斑魚及軍曹魚的熱帶魚類之消耗。因此，養魚場的數目及大小出現增漲，以滿足持續增加之市場需求。如自例如畜牧學已知，大量緊密生活在一起之動物易受各類疾病之影響，甚至受在大規模商業養殖時期之前鮮為人知或幾乎見不到或甚至未知之疾病的影響。此在魚類養殖方面亦係同樣之情況。在2015年，報導了尤其是在越南(Vietnam)及新加坡(Singapore)之養魚場中爆發之亞洲海鱸(尖吻鱸)之一種新疾病。觀測到魚類經歷了類似鱗片脫落疾病之症狀。鱗片脫落疾病係由最近經分離且描述於WO2014/191445中之虹彩病毒科(Iridoviridae)之病毒引起。然而，此等病例使用鱗片脫落疾病病毒DNA特異性PCR引子測試對鱗片脫落疾病呈陰性。當在野外(例如在養魚場中，由此不在受控制之實驗室環境中)發現時常常與此新疾病相關之主要臨床徵象(並非所有此等症狀一定會在每一條患病之魚中出現)可描述為如下。存在發病百分比高之臨床徵象之急性發作，很多魚類受影響且自臨床徵象發作起4至7日內死亡率高達60% (通常30%至70%)。此等臨床徵象可描述為如下：受影響魚類具有全身性皮膚病灶，變得無生氣，且顯示顯著食慾缺乏。隨著疾病進展，皮膚病灶變得愈加嚴重，導致皮膚變暗，伴有淡白色斑點及鰭及尾部之糜爛，使魚類呈現幽靈狀外觀。眼睛變得腫脹且輕微混濁。經常注意到之該疾病之內部徵象為脾及腎增大。腎變得脆弱且可輕易分離。常常可觀測到肝有些蒼白。鰓隨著疾病進展而變得蒼白。本發明之目標本發明之一目標係提供此疾病之病原體。此使得能夠提供以對抗該疾病為目標之偵測方法及疫苗。In the past few decades, global fish consumption has increased significantly. This is equally considered for cold-water fish such as squid, turbot, halibut and squid, and such as Asian sea bass (barramundi), squid, milkfish, squid, red squid, grouper And the consumption of tropical fish by cobia. As a result, the number and size of fish farms have increased to meet the growing market demand. As is known, for example, from animal husbandry, a large number of closely living animals are susceptible to a variety of diseases, even those that are little known or barely visible or even unknown prior to the period of large-scale commercial farming. This is also the case in fish farming. In 2015, a new disease of the Asian sea otter (Barramundi), which broke out in fish farms in Vietnam and Singapore, was reported. Fish were observed to experience symptoms similar to scale-off diseases. Scale-eating disease is caused by a virus that has recently been isolated and described in the Iridoviridae family of WO 2014/191445. However, these cases were negative for scale shedding disease using the DNA-specific PCR primer test for scale-slipping disease virus. The main clinical signs often associated with this new disease when found in the wild (eg in fish farms, thus not in a controlled laboratory environment) (not all such symptoms must occur in every diseased fish) ) can be described as follows. There is an acute onset of clinical signs with a high percentage of onset, many fish are affected and mortality is as high as 60% (usually 30% to 70%) within 4 to 7 days from the onset of clinical signs. These clinical signs can be described as follows: The affected fish have systemic skin lesions that become lifeless and show a significant loss of appetite. As the disease progresses, the skin lesions become more severe, causing the skin to darken, with pale white spots and smashing of the fins and tails, giving the fish a ghostly appearance. The eyes become swollen and slightly turbid. The internal signs of the disease are often noted as enlarged spleen and kidney. The kidneys become fragile and can be easily separated. It is often observed that the liver is somewhat pale.变得 becomes pale as the disease progresses. OBJECT OF THE INVENTION One object of the present invention is to provide a pathogen for this disease. This makes it possible to provide detection methods and vaccines aimed at combating the disease.

已發現如此處上文所描述之疾病之病原體係疱疹病毒及異疱疹病毒科(Alloherpesviridae)之成員。其係屬於雙股DNA病毒之二十面體病毒。該病毒與異疱疹病毒科-對魚類或兩棲動物具有致病性之疱疹病毒科-之病毒具有一定水準(雖然較低)之相似性。異疱疹病毒科之病毒係經包膜的，具有二十面體及球形至多形性之幾何結構。其直徑大約150-200 nm。基因組係線性且非分段的，長度在約100 kbp至250 kbp之間。本發明之病毒具有約130 kbp之基因組。圖1顯示表明已知異疱疹病毒科與新發現病毒之間的關係之系統樹。在此樹中，新病毒係指代為尖吻鱸疱疹病毒(LCHV)。此樹係使用第5版MEGA程式且使用標準設置來製作。(MEGA5：使用最大可能、進化距離及最大簡約法之分子進化遺傳分析(Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods). Koichiro Tamura，Daniel Peterson，Nicholas Peterson，Glen Stecher，Masatoshi Nei及Sudhir Kumar. Mol. Biol. Evol. 28(10): 2731-2739. 2011 doi:10.1093/molbev/msr121 2011年5月4日提前獲得出版)。異疱疹病毒科，更特定而言在魚類中發現之異疱疹病毒科概述於Hanson, L等人之綜述論文(Viruses 3: 2160-2191, 2011)中。根據本發明之疱疹病毒發現於亞洲海鱸(尖吻鱸)中。至今尚未有疱疹病毒被描述為對亞洲海鱸致病。亦無法排除該病毒對其他(亞)熱帶魚類具有致病性。在已知異疱疹病毒科中，鯰魚疱疹病毒第1型(Ictalurid herpesvirus 1；IHV1)係已知最近之家族成員。此病毒對河鯰具有致病性。然而，在核苷酸水準方面，根據本發明之病毒與鯰魚疱疹病毒第1型之間的總序列一致性遠低於60%。與相關性更小之鰻魚疱疹病毒(Anguilid Herpesvirus；AHV)及錦鯉疱疹病毒(Koi Herpesvirus；KHV)之序列一致性更低。表1顯示在新疱疹病毒中鑑別出之若干基因與IHV1中之同源基因之間的對比。該表顯示所鑑別出之最重要ORF(左手側欄)，其限制條件為具有小於300個鹼基對之經鑑別ORF不納入序列編號中(但僅僅出於此表之目的而忽略)，在DNA上之位置(針對該病毒之一個代表性實例，其寄存在Institut Pasteur，參見如下)，IHV1中之相應ORF，且已在該表中指示了在此等已知ORF下在胺基酸水準方面之序列一致性水準。該病毒之代表性實例已以寄存號碼CNCM I - 5118 (尖吻鱸疱疹病毒)寄存於微生物培養國家保藏中心(Collection Nationale de Cultures de Microorganisms；CNCM)，巴斯德研究院，25 Rue du Docteur Roux, F-75724 Paris Cedex 15，法國。表 1 新穎之疱疹病毒與IHV1之間的對比表 1 之說明 ORF：LCHV中之開放閱讀框架(數目) 框架：基因組上之閱讀框架(1、2、3，負數指相反股) 長度：鹼基對中LCHV ORF之長度 AA長度：BLAST搜索中所命中之胺基酸之長度 IHV Id.：LCHV ORF與相應IHV ORF之間的胺基酸一致性百分比 IHV-1：鯰魚疱疹病毒第1型同系物：基於與已知蛋白質之同源性之由LHCV ORF編碼之蛋白質的假定功能定義當前病毒之野生型形式係呈複製 勝任型形式之病毒，如可自患病魚類，尤其亞洲海鱸中分離出，且能夠在自其分離出呈野生型形式之病毒之魚類物種的健康魚類中誘發同一疾病。在經不活化或減毒之後，根據定義野生型病毒能夠以其野生型形式誘發疾病。經分離病毒 係如下病毒：自在自然界中的患病主體中通常與該病毒相關之組織中脫離，且在不存在其他病毒及細菌之情況下轉移至諸如培養皿、燒瓶或生物反應器之容器。經分離病毒之一個實例係存在於生物反應器中之特定細胞株之細胞培養物中的病毒。經分離 DNA 片段係自其如存在於相應天然出現之複製勝任型生物中之天然(完整)DNA中取出之DNA片段。此類DNA片段可按原樣存在於穩定化流體中，或可以重組方式轉移至另一生物之DNA中。在各情況下，在本發明之意義上，DNA片段仍係分離的。經分離蛋白質 係自其天然環境中獲得之蛋白質，亦即獲自其與相應天然出現之複製勝任型生物之天然關聯物之蛋白質。疫苗係可安全投與受試者動物且能夠針對病原微生物而在該動物內誘導保護性免疫，亦即誘導有成效的防治性治療之醫藥組合物。在此意義上，有成效的防治性治療係輔助預防或改善該病原體之感染、或由該感染引起、產生於治療後病原性病原體攻擊之病症的治療，尤其在此類攻擊之後減小其在主體中之負載，且視情況輔助預防或改善產生於治療後病原體感染之一或多個臨床表現。開放閱讀框架 (ORF)係具有編碼蛋白質或肽之潛能之閱讀框架之部分。ORF係不含有終止密碼子之密碼子之繼續延伸。Members of the pathogenic system herpesvirus and alloherpesviridae of the disease as described herein above have been found. It belongs to the icosahedral virus of the double-stranded DNA virus. The virus has a certain (although lower) similarity to the herpesvirus family - the herpesvirus family that is pathogenic to fish or amphibians. The virus of the herpesvirus family is enveloped and has an icosahedral and spherical to polymorphic geometry. It is approximately 150-200 nm in diameter. The genome is linear and non-segmented and is between about 100 kbp and 250 kbp in length. The virus of the present invention has a genome of about 130 kbp. Figure 1 shows a phylogenetic tree showing the relationship between the known herpesvirus family and the newly discovered virus. In this tree, the new virus is referred to as the herpes labia virus (LCHV). This tree is created using the 5th version of the MEGA program and using standard settings. (MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods). Koichiro Tamura, Daniel Peterson, Nicholas Peterson, Glen Stecher, Masatoshi Nei And Sudhir Kumar. Mol. Biol. Evol. 28(10): 2731-2739. 2011 doi:10.1093/molbev/msr121 was published on May 4, 2011). The herpesvirus family, and more particularly the herpesvirus family found in fish, is outlined in a review paper by Hanson, L et al. (Viruses 3: 2160-2191, 2011). The herpes virus according to the present invention is found in the Asian sea otter (Barramundi). So far, no herpes virus has been described as causing disease in Asian jellyfish. It is also impossible to rule out that the virus is pathogenic to other (sub)tropical fish. In the known herpesvirus family, the herpes labyrinvirus type 1 (Ictalurid herpesvirus 1; IHV1) is known to be a recent family member. This virus is pathogenic to river otters. However, at nucleotide levels, the overall sequence identity between the virus according to the invention and the herpes simplex virus type 1 is well below 60%. The sequence consistency of the lesser related Anguilid Herpesvirus (AHV) and Koi Herpesvirus (KHV) is lower. Table 1 shows the comparison between several genes identified in the herpes simplex virus and homologous genes in IHV1. The table shows the most important ORF identified (left hand sidebar) with the restriction that the identified ORF with less than 300 base pairs is not included in the sequence number (but ignored for the purposes of this table only), The position on the DNA (for a representative example of the virus, deposited in Institut Pasteur, see below), the corresponding ORF in IHV1, and the amino acid level at these known ORFs has been indicated in the table The sequence consistency level of the aspect. A representative example of this virus has been deposited with the deposit number CNCM I - 5118 ( Barramundi herpesvirus) at the Collection Nationale de Cultures de Microorganisms (CNCM), Pasteur Institute, 25 Rue du Docteur Roux , F-75724 Paris Cedex 15, France. Table 1 Comparison between novel herpesvirus and IHV1 Description of Table 1 ORF: open reading frame in LCHV (number) Framework: reading frame on the genome (1, 2, 3, negative refers to the opposite strand) Length: length of the LCHV ORF in the base pair AA Length: BLAST search Length of amino acid hit IHV Id.: Percent amino acid identity between LCHV ORF and corresponding IHV ORF IHV-1: Herpesvirus type 1 homologue: based on homology to known proteins The putative function of the protein encoded by the LHCV ORF defines that the wild-type form of the current virus is a replication -competitive form of the virus, such as can be isolated from diseased fish, especially from Asian jellyfish, and can be isolated from wild-type The same disease is induced in healthy fish species of the fish species of the virus. After inactivation or attenuation, the wild type virus is capable of inducing disease in its wild-type form by definition. The isolated virus is a virus that is normally detached from a tissue associated with the virus in a subject in nature and transferred to a container such as a petri dish, flask or bioreactor in the absence of other viruses and bacteria. An example of an isolated virus is a virus present in a cell culture of a particular cell strain in a bioreactor. The isolated DNA fragment is a DNA fragment which is removed from its native (intact) DNA as found in the corresponding naturally occurring replication competent organism. Such DNA fragments may be present in the stabilizing fluid as such or may be recombinantly transferred to the DNA of another organism. In each case, the DNA fragments are still isolated in the sense of the present invention. An isolated protein is a protein obtained from its natural environment, i.e., a protein obtained from its natural association with a corresponding naturally occurring replication competent organism. Vaccines are pharmaceutical compositions that can be safely administered to a subject animal and that are capable of inducing protective immunity against the pathogenic microorganism, i.e., inducing effective therapeutic treatment. In this sense, a productive prophylactic treatment is a treatment that assists in preventing or ameliorating the infection of the pathogen, or a condition caused by the infection, resulting from a pathogenic pathogen attack after treatment, especially after such an attack. The load in the subject, and optionally assists in preventing or ameliorating one or more clinical manifestations of the pathogen infection resulting from the treatment. The open reading frame (ORF) is part of a reading frame that encodes the potential of a protein or peptide. The ORF is a continuation of the codon that does not contain a stop codon.

在第一實施例中，新穎病毒之特徵為一種經分離之疱疹病毒，其係異疱疹病毒科之成員，且其野生型形式在亞洲海鱸中會引起疾病，該疾病之特徵在於以下徵象(在實驗室環境中在病毒之腹膜內攻擊之後)：臨床徵象在攻擊之後大約3日發作，全身性皮膚病灶(大量地，通常超過病例之50%)導致皮膚變暗伴隨有蒼白斑點及鰭糜爛，無生氣伴隨有觀測到之游動平衡喪失(「觀測到之」意謂其可見到但並非在所有情況下，僅出現在極其無生氣魚類中)，(幾乎完全)缺乏食慾，鰓蓋呼吸速率增加，及在攻擊之後約2週發生死亡。在另一實施例中，該病毒具有對應於具有根據SEQ ID NO: 1之序列之DNA的DNA。實際上此意謂在此DNA之全長上之一致性水準超過70%，較佳地超過71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%，超過99%或甚至高達100%。應注意，目前咸信在位置80,000周圍(在130k個鹼基對中)病毒具有內部重複。基於與其他疱疹病毒之同源性，由此可能該病毒可按四基因組異構體形式存在(如Mahiet等人在Structural variability of the Herpes Simplex Virus 1 genome In Vitro and In Vivo ; Journal of Virology，2012年8月，第86卷，第16號，第8592-8601頁中所解釋)。有可能新穎病毒除在bp 80,000周圍之重複之外額外具有一或多個內部重複。然而，此尚未確立。SEQ ID NO: 1對應於新穎病毒之可能基因組異構體中之一者。在該病毒之另一實施例中，在其DNA中，至少95%之開放閱讀框架(尤其是包含至少300個鹼基對之ORF)，例如至少96%、97%、98%、99%或100%，與具有根據SEQ ID NO: 1之序列之DNA的相應開放閱讀框架具有至少80%的序列一致性，例如至少81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或甚至高達100%一致性。應注意，出於界定本發明之目的，用於測定一致性水準之適合程式係NCBI鹼基局部比對檢索工具(Basic Local Alignment Search Tool)之核苷酸blast程式(blastn)，其使用「比對兩個或更多個序列」選項及標準設置(http://blast.ncbi.nlm.nih.gov/Blast.cgi)。在另一實施例中，作為異疱疹病毒科之成員的經分離之疱疹病毒具有以寄存號碼CNCM I - 5118 寄存在法國、巴黎、巴斯德研究院微生物培養國家保藏中心(Collection Nationale de Cultures de Microorganisms ；CNCM之病毒的鑑別特徵中之至少一者。此意謂該病毒可經鑑別為根據本發明之新穎之異疱疹病毒，亦即尖吻鱸疱疹病毒。在另一實施例中，鑑別特徵係選自由以下組成之群：1)病毒具有在其全長上與根據SEQ ID NO: 1之序列至少70%(或至少71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%)SEQ ID NO: 一致之DNA序列，具有上述關於內部重複之限制條件；或2)呈其野生型形式之病毒在亞洲海鱸中會引起疾病，該疾病之特徵為以下徵象：臨床徵象在攻擊之後大約3日發作，全身性皮膚病灶(大量地，通常超過病例之50%)導致皮膚變暗伴隨有蒼白斑點及鰭糜爛，無生氣伴隨有觀測到之游動平衡喪失(「觀測到之」意謂其可見到但並非在所有情況下，僅出現在極其無生氣魚類中)，(幾乎完全)缺乏食慾，鰓蓋呼吸速率增加，及在攻擊之後約2週發生死亡，或3)病毒包含主包膜蛋白(MEP)基因，該MEP基因與如SEQ ID NO: 2中所示之核苷酸序列的一致性水準為至少80%；或4)病毒包含dUTP酶基因，該dUTP酶基因與如SEQ ID NO: 4中所示之核苷酸序列的一致性水準為至少80%；或病毒包含末端酶基因，該末端酶基因與如SEQ ID NO: 6中所示之核苷酸序列的一致性水準為至少80%；或病毒包含聚合酶基因，該聚合酶基因與如SEQ ID NO: 8中所示之核苷酸序列的一致性水準為至少80%。在新穎之疱疹病毒之另一實施例中，其中該病毒作為異疱疹病毒科之成員且其野生型形式在亞洲海鱸中會引起疾病，該病毒之特徵在於該病毒具有對應於具有根據SEQ ID NO: 1之序列之DNA的DNA。此意謂該病毒具有在其全長上與根據SEQ ID NO: 1之DNA至少70%一致之DNA (具有關於內部重複之以上限制條件)。一致性水準可更高，例如71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或甚至100%。在新穎之疱疹病毒之再一實施例中，其中該病毒作為異疱疹病毒科之成員且其野生型形式在亞洲海鱸中會引起疾病，該病毒之特徵在於具有如下DNA：其中至少95%之開放閱讀框架(尤其包含至少300個鹼基對之ORF)，例如至少96%、97%、98%、99%或100%，與具有根據SEQ ID NO: 1之序列之DNA的相應開放閱讀框架具有至少80%的序列一致性，例如至少81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或甚至高達100%一致性。在其他實施例中，新穎病毒可基於其主包膜蛋白之編碼DNA序列(ORF28)及其dUTP酶之編碼DNA序列(ORF1)而區別於異疱疹病毒科之已知成員。結果表明，該病毒之主包膜蛋白與即使異疱疹病毒科之其他物種中最接近物種之MEP的序列一致性水準僅為30%。dUTP酶與異疱疹病毒科之其他物種之最接近dUTP酶的序列一致性水準僅為45%。編碼MEP及dUTP酶之DNA序列之典型實例分別顯示於SEQ ID NO: 2及SEQ ID NO: 4中。其各別胺基酸序列，亦即由根據SEQ ID NO: 2及SEQ ID NO: 4之DNA片段編碼之蛋白(術語「由……編碼」不排除其他DNA產生同一蛋白質，或換言之：「由特定DNA序列編碼」意謂蛋白質可基於特定序列合成，但亦可能藉由使用另一序列合成)，顯示於SEQ ID NO: 3及SEQ ID NO: 5中。應理解，對於本文涵蓋之特定蛋白，在病原體之個別代表之間可存在天然變異。在例如主包膜蛋白序列中，的確存在引起微小變化之基因變異。對於dUTP酶而言亦如此。首先，存在所謂「第二及第三鹼基中之擺動」，其說明核苷酸可能出現變化而在其編碼之胺基酸序列中仍不被注意：例如三聯體TTA、TTG、TCA、TCT、TCG及TCC均編碼白胺酸。此外，可在胺基酸序列中發現根據本發明之新病毒的代表之間的微小變異。此等變異可由總序列中之一或多個胺基酸差異或由該序列中之一或多個胺基酸之缺失、取代、插入、反轉或添加來反映。基本上不改變生物及免疫活性之胺基酸取代已例如由Neurath等人描述於「蛋白質(The Proteins)」 Academic Press New York (1979)中。在進化中頻繁出現之相關胺基酸或替代物之間的胺基酸替代尤其係Ser/Ala、Ser/Gly、Asp/Gly、Asp/Asn、Ile/Val(參見Dayhof, M.D., Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington D.C., 1978，第5卷，增刊3)。其他胺基酸取代包括Asp/Glu、Thr/Ser、Ala/Gly、Ala/Thr、Ser/Asn、Ala/Val、Thr/Phe、Ala/Pro、Lys/Arg、Leu/Ile、Leu/Val及Ala/Glu。基於此資訊，Lipman及Pearson開發了一種用於快速且靈敏之蛋白質對比(Science 227, 1435-1441, 1985)以及確定同源蛋白質之間的功能性相似性之方法。本發明之例示性實施例之此類胺基酸取代以及具有缺失及/或插入之變異屬於本發明之範疇內。此解釋了為何例如MEP及dUTP酶當自根據本發明之病毒之不同代表分離時可能具有顯著低於100%之同源性水準，然而仍代表根據本發明之病毒之MEP或dUTP酶。通常，為根據本發明之MEP或dUTP酶之蛋白質分別與SEQ ID NO: 3及SEQ ID NO: 5之胺基酸序列具有至少70%之序列一致性，由此與此等序列具有70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或甚至100%之序列一致性。因此，特定言之，與此等蛋白質(亦即MEP及dUTP酶)相關之實施例(A至G)如下： A：一種包含主包膜蛋白(MEP)基因之經分離之疱疹病毒，其特徵在於該病毒係異疱疹病毒科之成員，該病毒在亞洲海鱸中會引起疾病，且該MEP基因之核苷酸序列與如SEQ ID NO: 2中所示之核苷酸序列的一致性水準為至少80% (例如81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%)。 B：一種包含dUTP酶基因之經分離之疱疹病毒，其特徵在於該病毒係異疱疹病毒科之成員，該病毒在亞洲海鱸中會引起疾病，且該dUTP酶基因之核苷酸序列與如SEQ ID NO: 4中所示之核苷酸序列的一致性水準為至少80% (例如81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%)。 C：一種具有MEP基因及dUTP酶基因之經分離之疱疹病毒，其特徵在於該MEP基因之核苷酸序列與如SEQ ID NO: 2中所示之核苷酸序列的一致性水準為至少80%，且該dUTP酶基因之核苷酸序列與如SEQ ID NO: 4中所示之核苷酸序列的一致性水準為至少80% (例如81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%)。 D：一種包含主包膜蛋白(MEP)基因之經分離之疱疹病毒，其特徵在於該病毒係異疱疹病毒科之成員，該病毒在亞洲海鱸中會引起疾病，且該MEP基因在PCR反應中與如SEQ ID NO: 21及SEQ ID NO: 22中所示之引子組反應而得到277 +/- 10個鹼基對之PCR產物。 E：一種包含dUTP酶基因之經分離之疱疹病毒，其特徵在於該病毒係異疱疹病毒科之成員，該病毒在亞洲海鱸中會引起疾病，且該dUTP酶基因在PCR反應中與如SEQ ID NO: 23及SEQ ID NO: 24中所示之引子組反應而得到346 +/- 10個鹼基對之PCR產物。 F：一種包含MEP基因及dUTP酶基因之經分離之疱疹病毒，其特徵在於該MEP基因在PCR反應中與如SEQ ID NO: 21及SEQ ID NO: 22中所示之引子組反應而得到277 +/- 10個鹼基對之PCR產物，且該dUTP酶基因在PCR反應中與如SEQ ID NO: 23及SEQ ID NO: 24中所示之引子組反應而得到346 +/- 10個鹼基對之PCR產物。 G：一種經分離之疱疹病毒，其特徵在於MEP基因之核苷酸序列與如SEQ ID NO: 2中所示之核苷酸序列的一致性水準為至少80% (或至少81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%)，且dUTP酶基因之核苷酸序列與如SEQ ID NO: 4中所示之核苷酸序列的一致性水準為至少80% (或至少81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%)，且其特徵在於病毒DNA在PCR反應中與如SEQ ID NO: 21及SEQ ID NO: 22中所示之引子組反應而得到277 +/- 10個鹼基對之PCR產物，且在PCR反應中與如SEQ ID NO: 23及SEQ ID NO: 24中所示之引子組反應而得到346 +/- 10個鹼基對之PCR產物實施例D至G利用使用針對根據本發明之病毒的主包膜蛋白基因序列或dUTP酶基因序列之引子組的PCR測試。選擇其序列描繪於SEQ ID NO: 21至22及SEQ ID NO: 23至24中之兩個不同之引子組。使用與病毒之主包膜蛋白基因反應的第一引子組(SEQ ID NO: 21至22)之PCR測試包膜使用兩個引子LCHV MEP FW及LCHV MEP REV (參見實例部分中之表2b)。使用第二引子組(SEQ ID NO: 23至24)之PCR測試與病毒之dUTP酶基因反應且使用兩個引子LCHV dUTP FW及LCHV dUTP REV(參見實例部分中之表2c)。更詳細地描述於實例部分中之該等測試係標準PCR測試。若對第一引子組之PCR產物之分析揭示了約277個鹼基對之PCR產物或若對第二引子組之PCR產物之分析揭示了約346個鹼基對之PCR產物，且病毒係異疱疹病毒科之成員且在亞洲海鱸中會引起疾病，則此明確地說明經分析病毒係根據本發明之病毒。出於本發明之目的，約277個鹼基對之PCR產物係長度在277 + 10個鹼基對與277 - 10個鹼基對之間的PCR產物。約346個鹼基對之PCR產物係長度在346 + 10個鹼基對與346 - 10個鹼基對之間的PCR產物。與特異於新穎病毒之其他蛋白質，亦即末端酶及聚合酶相關之其他實施例(H至K)尤其如下： H：一種包含末端酶基因之經分離之疱疹病毒，其特徵在於該病毒係異疱疹病毒科之成員，該病毒在亞洲海鱸中會引起疾病，且該末端酶基因在PCR反應中與如SEQ ID NO: 25及SEQ ID NO: 26中所示之引子組反應而得到585 +/- 10個鹼基對之PCR產物。 I：一種包含聚合酶基因之經分離之疱疹病毒，其特徵在於該病毒係異疱疹病毒科之成員，該病毒在亞洲海鱸中會引起疾病，且該聚合酶基因在PCR反應中與如SEQ ID NO: 27及SEQ ID NO: 28中所示之引子組反應而得到314 +/- 10個鹼基對之PCR產物。 J：一種包含末端酶基因及聚合酶基因之經分離之疱疹病毒，其特徵在於該末端酶基因在PCR反應中與如SEQ ID NO: 25及SEQ ID NO: 26中所示之引子組反應而得到585 +/- 10個鹼基對之PCR產物，且該聚合酶基因在PCR反應中與如SEQ ID NO: 27及SEQ ID NO: 28中所示之引子組反應而得到314 +/- 10個鹼基對之PCR產物。 K：一種經分離之疱疹病毒，其特徵在於末端酶基因之核苷酸序列與如SEQ ID NO: 6中所示之核苷酸序列的一致性水準為至少80% (或至少81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%)，且聚合酶基因之核苷酸序列與如SEQ ID NO: 8中所示之核苷酸序列的一致性水準為至少80% (或至少81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%)，且其特徵在於病毒DNA在PCR反應中與如SEQ ID NO: 25及SEQ ID NO: 26中所示之引子組反應而得到585 +/- 10個鹼基對之PCR產物，且在PCR反應中與如SEQ ID NO: 27及SEQ ID NO: 28中所示之引子組反應而得到314 +/- 10個鹼基對之PCR產物。實施例H至K利用使用針對根據本發明之病毒的末端酶基因序列或聚合酶基因序列之引子組的PCR測試。選擇其序列描繪於SEQ ID NO: 25至26及SEQ ID NO: 27至28中之兩個不同之引子組。使用與病毒之末端酶基因反應之第一引子組(SEQ ID NO: 25至26)之PCR測試使用兩個引子LCHV TER FW及LCHV TER REV (參見實例部分中之表2d)。使用第二引子組(SEQ ID NO: 27至28)之PCR測試與病毒之聚合酶基因且使用兩個引子LCHV POL FW及LCHV POL REV (參見實例部分中之表2e)。更詳細地描述於實例部分中之該等測試係標準PCR測試。若對第一引子組之PCR產物之分析揭示了約585個鹼基對之PCR產物或若對第二引子組之PCR產物之分析揭示了約314個鹼基對之PCR產物，且病毒係異疱疹病毒科之成員且在亞洲海鱸中會引起疾病，則此明確地說明經分析病毒係根據本發明之病毒。基於上述新穎病毒之主包膜蛋白及dUTP酶的DNA編碼序列，亦提供本發明之以下實施例L至O： L：一種包含編碼主包膜蛋白之基因之(經分離)DNA片段，其特徵在於該基因與如SEQ ID NO: 2中所示之MEP基因之核苷酸序列的一致性水準為至少80% (或至少81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%)；及M：一種由此DNA片段編碼之(經分離)主包膜蛋白。 N：一種包含編碼dUTP酶之基因之(經分離)DNA片段，其特徵在於該基因與如SEQ ID NO: 4中所示之dUTP酶基因之核苷酸序列的一致性水準為至少80% (或至少81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%)；及O：一種由此DNA片段編碼之(經分離) dUTP酶。目前咸信若干其他基因亦適用於鑑別根據本發明之新穎病毒。此等基因中之一者係對應於ORF224 (SEQ ID NO: 10)之基因，其編碼膜(糖基)蛋白(SEQ ID NO: 11)。另一基因係與鯰魚疱疹病毒第1型(IHV1) TK (胸苷激酶)基因同源之基因。在IHV-1中此基因對應於ORF5 (Hanson等人, Virology. 1994年8月1日; 202(2):659-64)。新穎病毒具有在胺基酸水準方面與IHV-1之ORF 5具有33%之一致性之保守域「去氧核苷激酶」。用於鑑別新穎病毒之另一基因(SEQ ID NO: 12)係包含ORF206、編碼主衣殼蛋白(SEQ ID NO: 13)之基因。根據本發明之再一實施例關於任何具有長度為至少100個核苷酸之開放閱讀框架之(經分離)DNA片段，其中該DNA與具有根據SEQ ID NO: 1之序列的DNA之開放閱讀框架具有至少80% (或至少81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%)的序列一致性。儘管已發現30至40個核苷酸之長度足以區分根據本發明之病毒之DNA與公知之任何病毒之DNA，但實際相關長度，尤其針對基於相應蛋白質之相應次單位疫苗，係至少100個核苷酸(或至少150、200、250或甚至至少300個核苷酸)，以便對應於具有對應於病毒蛋白質之相關免疫原性抗原決定基的相關且突出之3D一致性之蛋白質。因此在另一實施例中，本發明亦關於由此類DNA片段編碼之(經分離)蛋白質。在一個實施例中，本發明亦關於包含呈複製勝任型形式的根據本發明之新穎病毒之細胞培養物(亦即在人工培養容器中限定數目細胞類型的人工培養物，亦稱作細胞株；亦描述細胞在其天然環境之外的受控條件下生長之製程)。若干魚類細胞株潛在地可用於支持根據本發明之病毒之複製。可用於生長根據本發明之病毒之細胞株的一個實例係亞洲海鱸之腦細胞之細胞株。用於分離此細胞株之方法已尤其是由Hasoon等人描述於In Vitro Cell. Dev. Biol. - Animal 47:16-25 (2011)中。潛在地可用於支持該病毒複製之細胞株的另一實例由Chi等人: 「Persistent infection of betanodavirus in a novel cell line derived from the brain tissue of barramundi Lates calcarifer」, Chi SC, Wu YC, Cheng TM, Dis Aquat Organ. 2005年6月; 65(2):91-8. PMID: 16060261揭示。亦已確立，使用常用方法之海鱸鰭原生細胞培養物可用於支持該病毒之複製。可用於生長該病毒之其他細胞株係來自皮膚、腦、心臟，亦即病毒可能在其中複製之器官之細胞。在又一實施例中，本發明關於用於對抗魚類疱疹病毒疾病之疫苗，其中該疫苗包含根據本發明之疱疹病毒或如上述之免疫原性蛋白質及醫藥學上可接受之載劑。此類載劑可簡單如水，只要其適合使材料以臨床上相關量投與而不會引起不可接受之副作用。典型載劑係油與水之乳液、不可溶佐劑(通常鋁或其他鹽或大免疫刺激聚合分子)於水中之懸浮液，或可溶佐劑(諸如皂苷、PAMP、卡波莫(carbopol)或其他免疫刺激分子)之溶液。通常，載劑包含此項技術中通常已知的穩定劑及防腐劑。在另一實施例中，疫苗包含根據本發明之疱疹病毒呈活減毒(亦即複製勝任但不再能夠誘導如最初野生型病原體所誘導之全套症狀)或不活化形式。已使用不同技術設計單純疱疹病毒之實驗性疫苗。疫苗係由肽、(重組)病毒蛋白質、病毒蛋白質之混合物、完整及部分(fractionated)之經殺死病毒(參見例如Yasumoto, S.等人於Fish Pathology 41: 141-145 (2006)中，其描述包含捕集在脂質體隔室內之不活化完整錦鯉疱疹病毒之疫苗)、複製缺陷型病毒及減毒之複製勝任型病毒(如由Koelle, D.M.及L. Corey. 2003: Recent Progress in Herpes Simplex Virus Immunobiology and Vaccine Research. Clin Microbiol Rev. 16(1): 96-113所概述)組成。各方法具有特定優點及缺點，其已由Stanberry於2000年論述(Stanberry, L. R., A. L. Cunningham, A. Mindel, L. L. Scott, S. L. Spruance, F. Y. Aoki及C. J. Lacey. 2000: Prospects for control of herpes simplex virus disease through immunization. Clin. Infect. Dis. 30:549-566.)。已知在細胞培養物中毒性疱疹病毒株之活體外連續繼代產生減毒後代，或換言之，產生引發保護性免疫反應而不會引起疾病之臨床症狀之非毒性複製勝任病毒株。舉例而言，馬利克(Marek's)疾病病毒(MDV)之減毒藉由以下方式達成：使用一種稱為Rispens或CVI988之保護性疫苗，藉由使毒性病毒在活體外連續繼代直至所得分離物變成無毒的(Rispens BH, Vloten H, Mastenbroek N, Maas HJ, Schat KA. 1972: Control of Marek's disease in the Netherlands. 1. Isolation of an avirulent Marek's disease virus (strain CVI988) and its use in laboratory vaccination trials. Avian Dis. 16:108-125)。相對應地，據描述，經常在路徑內涉及DNA複製及轉錄調節之複基因涉及MDV之從頭減毒，且向未來MD及由此相應疱疹病毒疫苗之合理設計提供目標。亦已描述了魚類疱疹病毒之藉由連續繼代之減毒(尤其是參見Noga, E.J.等人, Can. J. Fish. Aquat. Sci. 38: 925-929, 1981)。如通常已知，病毒之減毒可為自發的或可藉由藥物誘導(突變或其他自然條件諸如UV光；參見例如Mutation Research 768, 2016, 53-67及J . gen . Virol , 1985,66 , 2271-2277)。減毒背後之根本基因機制常常得不到充分理解，但遺傳變化(突變、缺失等)及/或其在病毒基因組中之積聚係疱疹病毒株之減毒之基礎。突變涉及多重病毒機制，包括病毒之複製能力、擴散等。活體內病毒複製及感染性所必需之某些分子路徑對於複製培養物而言可能非必需，且涉及此類路徑之彼等基因可能在長期培養期間更易於改變基因。若基因組中之此類逢機突變及缺失經表徵，且隨著新一代定序技術之發展，則對較大病毒諸如疱疹病毒之整個基因組進行定序變得相對簡單，此使得能夠合理設計減毒。根據關於疱疹病毒減毒之文獻，複基因已成為其中基因功能障礙導致病毒發生功能性減毒之可能目標。涉及病毒複製之基因(可能已在活減毒單純疱疹疫苗中經歷突變)之詳細概述由Roizman及Knipe於2001年給出(Roizman, B.及D. M. Knipe: Herpes simplex viruses and their replication，第2399-2459頁。於D. M. Knipe, P. M. Howley及D. E. Griffin (編), Fields virology，第4版，第2卷. Lippincott, Philadelphia, Pa中)。另外，此類突變可用於建立使用不連續複製病毒之疫苗方法。突變病毒在提供轉錄中所需的非突變基因之經基因工程改造之細胞株中生長。舉例而言，當編碼gH之晚期基因UL22缺失之單純疱疹病毒感染非補足細胞時，後代病毒粒子可離開細胞但無法感染次級細胞(Koelle及Corey, 2003，如上文所引用)。以下係疱疹病毒基因之列表，其中功能障礙或缺失已被描述為導致錦鯉疱疹病毒-一種異疱疹病毒-或其他疱疹病毒發生功能性減毒。此等基因係用於使根據本發明之疱疹病毒減毒之目標基因： 1) 錦鯉疱疹病毒之胸苷激酶(TK)基因。TK基因亦已描述於河鯰疱疹病毒(CCV)中，所述CCV為一種與描述於本發明中之病毒相對密切相關之病毒(Hanson LA, Kousoulas KG及RL Thune. 1994: Channel catfish herpesvirus (CCV) encodes a functional thymidine kinase gene: elucidation of a point mutation that confers resistance to Ara-T. Virology 202(2):659-64)。 2) 錦鯉疱疹病毒之d-UTP酶基因。 3) 編碼錦鯉疱疹病毒之ORF57之基因，如以Genbank寄存號N° NC_009127提供，其中ORF57開始及終止密碼子位於99382及100803位置(Boutier M, Ronsmans M, Ouyang P, Fournier G, Reschner A等人 (2015): Rational Development of an Attenuated Recombinant Cyprinid Herpesvirus 3 Vaccine Using Prokaryotic Mutagenesis and In Vivo Bioluminescent Imaging. PLoS Pathog 11(2): e1004690)。 4) 如在牛疱疹病毒、馬疱疹病毒及假性狂犬病病毒(假性狂犬病(Aujeszky's disease))中發現之gD (EHV en BHV)/gp50 (PRV)基因。此基因編碼可產生中和抗體之糖蛋白。本發明亦關於一種使用如上文所描述之疫苗預防性地治療動物(亦即治療動物以預防治療後相應野生型病原體感染)之方法，其包含向動物系統地投與疫苗。系統地投與疫苗意指投與疫苗使得其到達身體之循環系統(包含心血管及淋巴系統)，由此整體地而非特定位置(諸如胃腸道)地影響身體。系統性投與可例如藉由投與抗原至肌肉組織中(肌內)、真皮中(皮內)、皮膚下面(皮下)、黏膜下面(黏膜下)、靜脈中(靜脈內)、體腔中(腹膜內)等來進行。本發明亦體現在與根據本發明之病毒反應之抗體或抗血清，及用於偵測與根據本發明之病毒或與其抗原物質反應之抗體之診斷測試套組，其中該測試套組包含根據本發明之病毒或其抗原物質。本發明亦體現在用於偵測根據本發明之疱疹病毒或其抗原物質之診斷測試套組，其中該測試套組包含與根據本發明之病毒或與其抗原物質反應之抗體或如上文所描述之PCR引子組。現將使用以下實例進一步闡釋本發明。實例實例 1 ： 尖吻鱸 疱疹病毒之發現 用於分離感染原之血清及組織樣品之收集 在新加坡之亞洲海鱸(尖吻鱸 )養魚場中觀測到患病魚類。患病魚類呈現出在養殖者看來相似於鱗片脫落疾病之臨床症狀(Gibson-Kueh等人, J Fish Dis. 2012年1月;35(1):19-27. doi: 10.1111/j.1365-2761.2011.01319.x. PMID: 22103767；de Groof等人, PLoS Pathog. 2015年8月 7日;11(8):e1005074. doi: 10.1371/journal.ppat.1005074. PMID: 26252390)。然而，當更仔細地研究疾病症狀時，似乎患病魚類顯示更急性之感染(3至10日而非大於15日)，伴隨有更高發病率。皮膚病灶不太嚴重，但相比於鱗片脫落疾病更具全身性。皮膚病灶與鱗片脫落疾病病灶的不同之處在於整個魚體更暗且更無光澤，帶有蒼白黏液之斑點。觀測到一定程度之鱗片缺失，但其不顯著，且並非主要臨床徵象。此不同於由鱗片脫落疾病病毒所引起之鱗片缺失，由鱗片脫落疾病病毒引起之鱗片缺失具有局部化的斑狀病灶，其更深入地受壞死影響，伴隨有嚴重的鱗片缺失。鱗片脫落疾病病毒亦通常引起更長期的爆發。基於觀測到之差異及鱗片脫落疾病病毒PCR得到陰性結果之事實，基於對受影響養魚場之臨床觀測，疑似存在一種不同的病毒感染原(infectious viral agent)。因此決定進行針對病毒分離之跟蹤研究，且發現了一種新病毒。除鱗片缺失之外，對患病魚類之其他觀測結果係無生氣、嚴重食慾缺乏、混濁/腫脹眼睛之急性發作及高死亡率(高達受影響魚類之30%至70%)。從受影響的魚類取出樣品(血清、腎臟、脾臟)。將所彙集之血清樣品儲存在-70℃下直至進一步分析。將所彙集之腎臟樣品保持在+4℃下直至次日均質化。用於分離感染原之組織樣品之組織均質化 藉由使用均質化棒在SVDB (標準疫苗稀釋液緩衝液= PBS)中手動研磨來均質化腎臟樣品，且樣品最終在SVDB中以1:9 (w/v)稀釋且隨後在健他黴素中預處理1小時。在+4℃下將均質化樣品以5,500 rpm離心10分鐘，且隨後收集無細胞上清液。在接種單層之前一天，在T75燒瓶中將海鱸腦(Seabass brain；SBB)細胞以2 × 10⁴ 個細胞/平方公分接種在EMEM + 10% FBS +健他黴素(gentamycin)+雙性黴素中。對此實驗，細胞在亦添加有HEPES及碳酸氫鈉之培養基中生長，且細胞經優化為在不含CO₂ 之此等條件下生長。次日，用添加有0.1 mL或0.3 mL未經稀釋之無細胞上清液之新鮮培養基來更換培養基(15 mL)將燒瓶在28℃下培育。在3日之後在添加有未經稀釋之無細胞上清液(0.1 mL及0.3 mL)之燒瓶中觀測CPE，且在第5日採集(第1代)。將假定感染原命名為V511。根據上文方案，將CPE引發物(CPE-causing agent)在SBB細胞上繼代額外3次。將CPE引發物命名為V511/SBB_4P。對受影響魚類之細胞及血清中之 CPE 引發物的培養物上清液進行病毒偵測 使用由De Vries等人(2011) PLoS ONE 6(1):e16118所描述之VIDISCA-454技術分析受影響魚類之血清樣品及CPE引發物之第4代採集物V511/SBB_4P之培養物上清液。在兩種類型之樣品中，獲得疑似來源於新穎之魚類病原體之序列。使用此等序列來衍生PCR引子以用於習知PCR及定量PCR (參見表2a至表2e)。新序列之鹼基局部比對檢索揭示培養物上清液中CPE引發物及血清中疑似感染原顯示某種與異疱疹病毒科病毒的同源性程度。因此將此新病毒稱作尖吻鱸疱疹病毒(LCHV)。全基因組定序 如所描述(de Vries M等人 PLoS One. 2011;6(1):e16118. doi:10.1371/ journal.pone.0016118)，將病毒培養物上清液樣品以10,000 × g離心10分鐘且用TurboDNase (Thermofisher)處理，之後藉由Boom提取方法(Boom R等人J Clin Microbiol. 1990;28(3):495-503)提取核酸。使用dsDNA片段化酶(New England Biolabs)剪切樣品。使用AMPure XP珠粒(agencourt AMPure XP PCR，Beckman Coulter)以1:1.8 (樣品:珠粒)比率純化經剪切樣品以移除酶。在純化之後，用DNA聚合酶I大(克列諾(Klenow))片段(New England Biolabs)對樣品進行末端修復。用AMPure XP珠粒(agencourt AMPure XP PCR，Beckman Coulter)以1:1.8 (樣品:珠粒)比率純化經末端修復之樣品以移除酶，之後藉由使用克列諾片段(3'→5'外切核酸酶(Exo))(New England Biolabs)對樣品進行A尾端化。使用AMPure XP珠粒(agencourt AMPure XP PCR，Beckman Coulter)以1:1.8 (樣品:珠粒)比率純化樣品以移除聚合酶。藉由使用T4連接酶(Thermofisher)將來自NEBNext Multiplex Oligos for Illumina (New England Biolabs)之泡狀連接子(Bubble adaptors)連接至A尾端化樣品。藉由使用AMPure XP珠粒(agencourt AMPure XP PCR，Beckman Coulter)進行大小選擇，首先以1:0.5 (樣品:珠粒)比率進行以確保移除大部分大小大於400 bp之片段，隨後向上清液添加另外AMPure XP珠粒(agencourt AMPure XP PCR，Beckman Coulter)以得到1:0.85 (樣品:珠粒)之最終比率，來結合200 bp至400 bp之間的DNA片段且移除小於200 bp之片段。在大小選擇之後，藉由使用來自NEBNext Multiplex Oligos for Illumina (New England Biolabs)之USER酶打開泡狀連接子。然後，使用來自NEBNext Multiplex Oligos for Illumina (New England Biolabs)之連接子特定引子及Q5 hotstart mastermix (New England Biolabs)進行28次循環的PCR；30秒98℃，以及10秒98℃及75秒65℃、隨後5分鐘65℃之循環。在PCR之後，藉由使用AMPure XP珠粒(agencourt AMPure XP PCR，Beckman Coulter)以1:0.5 (樣品:珠粒)比率對樣品進行大小選擇，以移除大小大於400 bp之片段，且向上清液添加另外AMPure XP珠粒(agencourt AMPure XP PCR，Beckman Coulter)以得到1:0.85 (樣品:珠粒)之最終比率，來結合200 bp至400 bp之間的DNA片段且移除小於200 bp之片段。隨後，經由Qubit dsDNA HS分析套組(Thermofisher)量測DNA之濃度，使用高靈敏性DNA分析套組在生物分析儀上檢查大小。將DNA稀釋至2.49ng/μl之濃度。藉由使用MiSeq (Illumina)使用成對末端定序及v2套組(Illumina)對DNA進行定序。系統發育分析 初始系統發育分析係基於在疾病爆發之樣品中所發現之尖吻鱸疱疹病毒的208 bp DNA片段(SEQ ID NO: 14)。此片段顯示在經轉譯核苷酸層次具有與鯰魚疱疹病毒第1型NP_041153.2及其他魚類病毒之ORF62之同源性。使用BLAST鹼基局部比對檢索工具(http://blast.ncbi.nlm.nih.gov/ Blast.cgi)及多序列比對工具ClustalW產生核苷酸及蛋白質序列比對。使用MEGA5軟體使用鄰近歸併法來建立系統樹，其中在空隙或插入之情況下有部分缺失(MEGA5：Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods (使用最大可能、進化距離及最大簡約法之分子進化遺傳分析). Koichiro Tamura, Daniel Peterson, Nicholas Peterson, Glen Stecher, Masatoshi Nei及Sudhir Kumar. Mol. Biol. Evol. 2011 28(10): 2731-2739. 2011 doi:10.1093/molbev/msr121)。此系統發育分析之結果呈現於圖1中，其基於LCHV之208 bp DNA片段與IHV1 ORF62之同源性分析描繪LCHV之系統樹。系統發育分析確認LCHV係異疱疹病毒科中之新發現病毒。實例 2 ：使用 PCR 、 qPCR 分析偵測尖吻鱸疱疹病毒 引子設計 對疾病爆發之樣品中發現之尖吻鱸疱疹病毒的208 bp DNA片段設計PCR引子。此片段顯示在經轉譯核苷酸水準方面與鯰魚疱疹病毒第1型NP_041153.2之ORF62之同源性。亦對MEP、dUTP酶、末端酶及聚合酶基因設計引子。表2a：對尖吻鱸疱疹病毒之208 bp DNA片段設計之引子表2b：對尖吻鱸疱疹病毒之MEP設計之引子表2c：對尖吻鱸疱疹病毒之dUTP酶設計之引子表2d：對尖吻鱸疱疹病毒之末端酶設計之引子表2e：對尖吻鱸疱疹病毒之聚合酶設計之引子 PCR 及凝膠電泳 使用Veriti 96孔熱循環器(Applied Biosystems)進行習知PCR。製備含有1× Supertaq緩衝液、0.02 U/µl Supertaq酶、0.2 mM去氧核糖核苷三磷酸(dNTP)、1 µM正向引子及1 µM反向引子之主混合物。對於各樣品，在48 µl PCR混合物中添加2.0 µl DNA模板，使用2 µL無菌水作為陰性對照。將PCR程式設計為以95℃下之60秒初始化開始，隨後分別在95℃、基於引子之Tm的引子組特定退火溫度及72℃下進行各自持續30秒的變性、退火及延伸，重複40次。程式以在72℃下10分鐘之最終延伸結束。在115伏特下用1.5%瓊脂糖凝膠中之1×溴化乙錠及1× TAE緩衝液負載樣品，持續60分鐘。qPCR 分析定量聚合酶鏈反應使用BioRad CFX 96系統進行。qPCR實驗使用探針快速主混合物(Probe Fast Master Mix)以及序列特定探針進行。各反應存在含有1×探針快速qPCR主混合物(KAPA)、200 nM正向引子、200 nM反向引子及200 nM探針之18 µl主混合物。引子對2用於qPCR分析，最佳化Tm為60.7℃。探針DNA序列係CGCGGGATGACCTCTTCTCG (SEQ ID NO: 31)且所使用標記物係5' 6FAM及3' TAMRA。向18 µl主混合物中添加2 µl DNA模板。各反應一式兩份地進行，且在嵌入CFX系統之前使所有盤以2,200 × g旋轉4分鐘。使用以95.0℃下3分鐘開始、隨後進行95.0℃下3秒及60.7℃下30秒之40次重複之程式，出現擴增。標準線 使用含有具有尖吻鱸疱疹病毒一致性序列構築體(藉由GenScript合成[合成如上所述之208 bp片段且在質體載體中選殖])之pUC57載體之稀釋系列作為陽性對照，效能與精確性之指示物及樣品定量之指示物。載體以每qPCR反應1.0 × 10¹ 個複本/2微升至1.0 × 10⁹ 個複本/2微升範圍溶解且稀釋在水中。稀釋系列包括在所有qPCR實驗中且儲存在-20℃下。樣品中尖吻鱸疱疹病毒DNA之定量藉由支持軟體(CFX-Manager 3.1版)進行，該軟體使用獲自稀釋系列之資料來建立標準線。進行之所有qPCR實驗均顯示98.0%與100.2%之間的效能及0.997與0.999之間的精確性，強調了經設計用於定量LCHV DNA之qPCR方法之穩定性。實例 3 ： 新穎之感染原在亞洲海鱸中之實驗性感染 V511 / SBB _ 4P 在亞洲海鱸 ( 尖吻鱸 ) 中之實驗性感染 在此實驗中，用V511以腹膜內及同居方式感染魚，以研究是否V511係在野外爆發疾病之原因。繼感染之後在不同時間點取樣不同器官以確定感染之過程。表3：處理組及槽配置。藉由腹膜內(IP)注射0.3 mL於SBB細胞株V511/SBB_4P上生長之未經稀釋V511無細胞上清液(CFS)來攻擊具25條亞洲海鱸之一組。CSF之病毒滴度係3.2 × 10⁶ TCID₅₀ /mL (參見以下滴定方案)。具25條魚之第二組在由網分離之同一槽中同居(表3)。網允許水在兩個槽半部之間自由移動且允許兩個處理組之魚之間進行密切接近但不直接接觸。在實驗開始時，魚之平均重量為18公克。在攻擊之前將魚餓約24小時以確保胃腸道排空，從而降低受傷風險。在攻擊之前藉由根據標準程序用Aqui-S鎮靜來將魚麻醉。將在IP攻擊組中之魚網住、給服鎮靜劑且對骨盆鰭之底端與頂端之間的中線進行IP注射。攻擊之後，將魚置放於指定槽中用於恢復及觀測。將未感染的同居魚引入由網分離之相鄰槽分區中。記錄死亡率及臨床徵象。在攻擊後第4日、第7日、第11日、第14日及第18日，在同居組中隨機取樣3條魚且將其進行屍體解剖以用於採集魚組織而用於進一步測試。取樣包括脾臟、心臟、腦、血清、肝臟、腸、鰓、皮膚及腎臟，以便更好理解在經由自然途徑感染之後新穎感染原之感染過程。在攻擊後第4日、第7日、第11日及第14日，在IP感染組中隨機取樣3條魚且將其進行屍體解剖以用於採集腎臟組織而用於進一步測試。在第17日，自3條以實驗方式感染之魚中取樣脾臟、心臟、腦、血清、肝臟、腸、鰓、皮膚及腎臟。至攻擊後第18日，所有魚已經取樣或已死於感染(參見表6)。樣品收集概述於表4及表5中。表4：以實驗方式IP感染之組中魚組織之取樣. 表5：同居組中魚組織之取樣. 表6中呈現之結果顯示IP攻擊途徑及同居攻擊途徑皆能夠產生與在指標性亞洲海鱸養魚場中觀測到之臨床徵象相似之臨床徵象。觀測到之大體臨床徵象係無生氣、食慾缺乏、皮膚、鰭及眼睛病灶，亦在指標性養魚場之受影響養殖魚類中相似地觀測到了該等臨床徵象。表6：在IP攻擊處理組及同居攻擊處理組中觀測到之臨床徵象此顯示在受控實驗室環境(其中與諸如養魚場之野外環境相比，無其他病原體存在且存在較少應激子)中，在(IP)攻擊之後典型症狀係1)臨床徵象在攻擊之後約3天發作，2)全身性皮膚病灶可能導致皮膚變暗伴隨有蒼白斑點及鰭糜爛，3)因極度無生氣導致之游動平衡喪失，4)幾乎完全缺乏食慾，5)鰓蓋呼吸速率增加及6)在攻擊之後約2週發生死亡。可在一些魚中觀測到腫脹及混濁眼睛。死亡率及%累積死亡率記錄顯示於表7中。表7：每日死亡率及%累積死亡率記錄*。 *不包括針對組織收集而經取樣之魚。 V511/SBB_4P病毒上清液自經IP攻擊之魚傳播至未經處理之同居魚。兩組經歷相似臨床症狀。該等臨床症狀亦相似於在指標性養魚場觀測到之臨床症狀(初始爆發)。相比於同居組，IP感染組經受更急性且更嚴重的疾病，疾病徵象在感染後3日內出現。相比之下，同居攻擊之魚在感染後第7日顯示初始臨床徵象。使用衍生自在野外疾病研究期間收集之組織樣品之感染物質及活體外後續繼代，吾人能夠經由IP感染途徑及同居感染途徑二者複製在爆發期間觀測到之臨床症狀。亦展示疾病自IP感染之魚傳播至在同一水空間中之未經處理的同居魚，證實了此病原體之感染性質。實例 4 ： 在感染實驗 ( 實例 3 ) 期間收集之組織樣品之樣品製備、組織均質化、 DNA 分離 器官樣品之均質化 使用Precellys 24均質儀將在上述實驗中收集之魚器官樣品(表4、表5)均質化。使用存在6,500 rpm下20秒與10秒間隔之兩個循環的程式在磷酸鹽緩衝鹽水(PBS)中製備10%器官均質物。在一個循環中完成心臟樣品、脾臟樣品、腎臟樣品、腦樣品、腸樣品及肝臟樣品之均質化，皮膚樣品及鰓樣品均質化兩次。在均質化期間所有樣品均保存在冰上且儲存在-80℃下。DNA 提取使用MagNA Pure 96系統及MagNA Pure 96 DNA及病毒NA套組進行DNA提取。針對提取，將250 µl MagNA Pure 96外部溶解緩衝液添加至200 µl樣品中。使用預安置之外部溶解方案將DNA分離且在50 µl Milli-Q水中溶離。將DNA儲存於-20℃下直至進一步使用。實例 5 ： 病毒之培養及病毒之滴定 海鱸腦 ( SBB ) 細胞株之建立及培養 ：細胞株SBB最初在Intervet Norbio Singapore Pte Ltd (MSD AH之分部)衍生自亞洲海鱸腦細胞之經胰蛋白酶處理之懸浮液。用於衍生海鱸腦細胞之程序已由Hasoon等人在In Vitro Cell. Dev. Biol. - Animal 47: 16-25 (2011)中及由Chi等人Dis Aquat Organ. 2005年6月;65(2):91-8描述。 SBB培養基由補充有2 mM L-麩醯胺酸及110 mg/L丙酮酸鈉之899 ml E-MEM、100 ml FCS (10%)及(視情況選用之) 1 mL新黴素多黏菌素(Neomycin Polymyxin)抗生素溶液1000×儲備液構成。細胞在28℃及5% CO₂ 下常規地生長。在開始培養之前將培養基保存在4℃下。使用一安瓿之冷凍SBB儲備物來開始培養。藉由使安瓿在28℃水中升溫來使來自液氮之細胞快速解凍。將細胞懸浮液添加至試管中且用9倍體積之培養基緩慢稀釋。隨後，對細胞進行計數。將懸浮液分配至適當培養瓶或滾瓶中且在28℃及5% CO₂ 下培育。燒瓶或滾瓶中之接種密度約為3 × 10⁴ 個細胞/平方公分。在6至24小時之後或細胞完全附著之後，更新培養基以移除殘餘DMSO (冷凍培養基由90%培養基及10% DMSO構成)。將細胞進一步培育3至7天或直至達至匯合。對於滾瓶，需要0.2至0.5 rpm之滾速。滾瓶可具有480、960及1750 cm² 之不同表面積。一旦達至匯合，使細胞繼代。繼代可以3.0 × 10⁴ 個細胞/平方公分之初始接種密度每3至4天進行一次。替代地，當以1.0 × 10⁴ 個細胞/平方公分之密度塗覆時繼代可每7天發生一次。將用於細胞繼代之試劑(培養基、PBS、胰蛋白酶/EDTA)預溫熱至28℃。丟棄培養基且使用適當體積之PBS(對於T25燒瓶為3 mL)洗滌匯合單層一次。隨後丟棄PBS，且在28℃下將細胞在補充有1% (vol/vol)之2.5%胰蛋白酶溶液及1% (vol/vol)之2% EDTA溶液的相同體積之PBS中培育15分鐘。在脫離之後，添加相同體積之新鮮培養基且將細胞再懸浮並計數。以適合於培養瓶或滾瓶之培養體積以所需細胞密度設置新燒瓶。對於冷凍細胞，在程序之前，將培養基及2×濃縮之冷凍培養基(80% (vol/vol)培養基加20% (vol/vol) DMSO)保存在4℃下。如上所述處理匯合的細胞培養物直至且包括胰蛋白酶處理。將細胞再懸浮、計數、進一步再懸浮於適合量之培養基中，且在渦流懸浮液的同時逐滴添加相等體積之2×冷凍培養基。用每安瓿5.25 × 10⁶ 個細胞填充用於液氮儲存之安瓿以啟動T175或用2.25 × 10⁶ 個細胞填充以啟動T75。使用尖吻鱸疱疹病毒接種 SBB 細胞在設置接種實驗之前，使自液氮儲存培養之細胞繼代至少一次。在以3.0 × 10⁴ 個細胞/平方公分接種在組織培養瓶中之前，使細胞繼代且培養24小時。接種體包括來自病毒之先前繼代之新鮮或冷凍-解凍培養物未經稀釋之上清液。將培養基自燒瓶移除。隨後在28℃下將燒瓶接種至少60分鐘。當使用培養基中之LCHV病毒之先前繼代的採集物接種細胞時，較佳使用每細胞0.001-0.01 TCID₅₀ 之MOI。在移除接種體之後(在60分鐘之後，但此非絕對要求)，添加新鮮培養基且培養細胞直至使用倒置顯微鏡觀測到完全CPE (通常在2至4天之後)。藉由收集以800 × g旋轉5分鐘以移除殘渣的培養物上清液採集病毒。替代地，可藉由過濾來使上清液澄清。澄清的上清液用於後續繼代或PCR/DNA/EM分析或在-70℃下冷凍。可使用採集物之(定量) PCR分析及/或滴定來確認病毒之複製。使用DNA定序技術以及EM確認病毒之身分。使用MagNA Pure 96系統及MagNA Pure 96 DNA及病毒NA套組(實例4)將用於(定量) PCR之DNA自組織培養基分離。在 SBB 細胞上滴定病毒 如上所述培養SBB細胞。在測試之前一天，在培養基(EMEM + 10% FCS + L-Glu + NaPyr)中製備含有6.0 × 10⁴ 個細胞/毫升之SBB細胞懸浮液。使用100 µL此細胞懸浮液接種微量滴定盤之96孔。將盤在28℃及5% CO₂ 下培育24小時。在此培育期之後單層為約50%匯合。在測試當天，藉由以下製備各病毒樣品之10倍連續稀釋液直至達至10^- ⁷ ：將0.5 mL樣品轉移至含有4.5 mL冷(0-20℃)滴定培養基(具有減少之FCS之培養基；50% EMEM + L-Glu + NaPyr + 50%培養基)之試管，混合且轉移0.5 mL於含有4.5 mL滴定培養基之下一個試管中，隨後小心混合，轉移等。第1行及第12行及第A列及第H列充當陰性對照且用100微升/孔新鮮滴定培養基接種。在第B列至第G列(10個孔/稀釋液)，微量滴定盤用100微升/孔病毒稀釋液(10^- ² 、10^- ³ 、10^- ⁴ 、10^- ⁵ 、10^- ⁶ 、10^- ⁷ )接種。在操作期間，病毒稀釋液之溫度保持在0℃與20℃之間。將盤在28℃及5% CO₂ 下培育6天。6日病毒培育期之後，使用倒置顯微鏡對盤篩檢LCHV特異性CPE。CPE之特徵在於為細胞圍聚，隨後細胞脫離/溶解(圖2)。將顯示LCHV特異性CPE之各孔計分為陽性。根據由Reed及Muench, am. J. Epidemiol. (1938) 27(3): 493-497所描述之方法及計算來確定TCID₅₀ 。自滴定分析中之陽性孔分離之DNA樣品的qPCR分析確認了病毒之存在及複製。結果以3.0 × 10⁴ 個細胞/平方公分接種組織培養瓶且培養24小時。24小時之後，在胰蛋白酶處理之後對一個燒瓶中之細胞進行計數以確定燒瓶中存在之細胞的實際數目。自隨後接種之其他燒瓶中移除培養基。將由來自培養基中之LCHV病毒之先前繼代(病毒之繼代數目在4至8之間)的未稀釋培養物上清液構成之每細胞0.001 TCID₅₀ 的接種體塗覆至單層上且培育60分鐘。移除接種體且添加新鮮培養基至細胞培養瓶中。培養細胞直至使用倒置顯微鏡觀測到完全CPE。藉由收集以800 × g旋轉5分鐘以移除殘渣的培養物上清液採集病毒。樣品取自(1)用於感染單層之未稀釋接種體，(2)使用新鮮培養基替代接種體之後1小時採集之燒瓶的培養物上清液，(3)呈50% CPE之單層及(4)呈100% CPE之單層。對樣品(1)、(3)及(4)進行滴定，且對樣品(1)、(2)、(3)及(4)進行qPCR分析。結果展示於表8中。使用Olympus CKX41倒置顯微鏡以40倍放大率捕捉圖像。此等圖像展示於圖2中。圖2A顯示培養瓶中呈90%匯合之SBB細胞之形態學(p18)，且圖2B顯示呈50% CPE之SBB細胞之形態學。表8：在SBB細胞上之LCHV生長之詳細結果。實例 6 ： 電子顯微法 電子顯微法 將具有純碳膜之400網孔之銅網格在空氣中曝露於輝光放電20秒以使膜表面為親水性的。將病毒樣品以10 µl之體積置放於塗佈碳之網格上且將其培育約2分鐘。使用濾紙印乾過量樣品且將10 µl水置放於網格上且再次藉由印吸立即移除。隨後將10 µl 1%乙酸鈾醯置放於網格上用於染色。30秒之後，藉由印吸移除過量染色劑且在於電子顯微鏡中查看之前將樣本乾燥若干分鐘。在80 kV下操作之JEOL 1011透射電子顯微鏡中觀測樣本。使用SIS Veleta 2kx2k照相機記錄圖像。使用JEOL 1011透射電子顯微鏡捕捉LCHV培養物樣品之圖像以確認經鑑別之病毒係疱疹病毒且排除任何其他病毒之存在。使用第5代LCHV以0.01之MOI接種SBB (p9)細胞。在採集之後於-80℃下將病毒儲存培養基中且獲得10⁴ ^. ⁴³ TCID₅₀ /mL之效價。製備1 µl樣品用於電子顯微法，其中兩個圖像顯示於圖3中。在圖A中，可觀測到具有約100 nm直徑之兩個黑點，與普通疱疹病毒之衣殼之平均直徑(115-130 nm)相符。圖B顯示放大圖，其中可清晰地觀測到此粒子之二十面體輪廓線。在樣品中未發現包膜病毒。圖3顯示使用電子顯微鏡捕捉之LCHV病毒。在圖3A中可辨識兩種疱疹病毒(黑點)。比例尺係500 nm。圖3B顯示其中一個點之放大圖，清晰地顯示出二十面體輪廓線。上文呈現之實例描述來自患病魚類之新穎病原體之偵測及分離。可在使用經分離病原體以實驗方式感染健康魚類之後重現相同疾病症狀。自以實驗方式感染之魚類中分離之感染原與最初分離之病原體相同。此證明上文所描述之疾病症狀僅可歸因於所發現之病原體，即尖吻鱸疱疹病毒。實例 7 ： 原生細胞培養物海鱸鰭細胞 建立了來自海鱸鰭細胞(SBF)之原生細胞培養物。在培養中，將細胞培養了至少5代。如下建立培養物。將魚麻醉。修整尾側鰭(尾部)且將其在PBS +健他黴素0.3% +恩氟沙星(enrofloxacin) 0.002%+雙性黴素0.5%中洗滌三次。使用解剖刀片將鰭切成組織片段。將片段轉移至25 cm2組織培養瓶中，所述組織培養瓶含有補充有20% FCS及健他黴素0.3% +恩氟沙星0.002% +雙性黴素0.5%之L15培養基。將燒瓶在28℃下在不含CO₂ 之含濕氣培育箱中培育。基於殘渣之存在及pH，視需要改變培養基(L15)。視細胞密度而定，使用PBS中之0.125%胰蛋白酶藉由胰蛋白酶處理，直至細胞脫離且以1:1-3之間的低比率分裂來使細胞繼代。在初始繼代期間，在補充有20% FCS之L15培養基中培養細胞。在後續繼代中，將FCS百分比減小至10%。尖吻鱸疱疹病毒之接種使用如實例5中所描述之程序進行。感染後第4天觀測到100% CPE。實例 8 ： 額外新穎之病毒株 自不同於指標性養魚場之養魚場獲得病魚之樣品，其中觀測到尖吻鱸疱疹病毒感染之臨床症狀。然而，使用描述於實例2中之引子組2 (SEQ ID NO: 17與SEQ ID NO: 18)之PCR分析得到陰性PCR結果。作為替代方案，使用基於聚合酶(ORF21)及末端酶(ORF 37)序列設計之引子組用於PCR，其中ORF具有與其他異疱疹病毒科 相比相對高水準之胺基酸保守。用於PCR之引子呈現於表2d及表2e中。此等PCR得到陽性結果。對末端酶PCR片段進行定序，且顯示出與呈現於SEQ ID NO: 1中之尖吻鱸疱疹病毒之序列具有97%一致性。SEQ ID NO: 29呈現如SEQ ID NO: 1中所呈現之LCHV之末端酶PCR的PCR產物。SEQ ID NO: 30呈現自不同於指標性養魚場之養魚場獲得的LCHV之末端酶PCR之PCR產物。得出結論，疾病之爆發係由另一LCHV株引起。In a first embodiment, the novel virus is characterized by an isolated herpesvirus which is a member of the Herpesviridae family and whose wild-type form causes disease in Asian jellyfish, which is characterized by the following signs ( In the laboratory environment after intraperitoneal challenge of the virus): Clinical signs appear about 3 days after the attack, and systemic skin lesions (in large numbers, usually more than 50% of the cases) cause darkening of the skin accompanied by pale spots and fin erosion , lifeless with the observed loss of swimming balance ("observed" means that it is visible but not in all cases, only in extremely lifeless fish), (almost completely) lack of appetite, cover the breath The rate increases and death occurs about 2 weeks after the attack. In another embodiment, the virus has DNA corresponding to DNA having the sequence according to SEQ ID NO: 1. In practice, this means that the consistency level over the entire length of the DNA exceeds 70%, preferably more than 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, more than 99% or even as high as 100%. It should be noted that the current virus has an internal repeat around the 80,000 position (in 130k base pairs). Based on homology to other herpesviruses, it is possible that the virus can exist as a four-genomic isoform (eg Mahiet et al.Structural Variability Of The Herpes Simplex Virus 1 Genome In Vitro And In Vivo ; Journal of Virology, August 2012, Vol. 86, No. 16, pp. 8592-8601). It is possible that the novel virus additionally has one or more internal iterations in addition to the repetition around bp 80,000. However, this has not been established. SEQ ID NO: 1 corresponds to one of the possible genomic isoforms of the novel virus. In another embodiment of the virus, at least 95% of the open reading frames (especially comprising at least 300 base pairs of ORFs) in its DNA, eg, at least 96%, 97%, 98%, 99% or 100% having a sequence identity of at least 80% with a corresponding open reading frame of DNA having the sequence according to SEQ ID NO: 1, for example at least 81%, 82%, 83%, 84%, 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even up to 100% identity. It should be noted that for the purpose of defining the present invention, a suitable program for determining the level of consistency is the nucleotide blast program (blastn) of the NCBI Base Local Alignment Search Tool, which uses "ratio" For two or more sequences" options and standard settings (http://blast.ncbi.nlm.nih.gov/Blast.cgi). In another embodiment, the isolated herpes virus that is a member of the Herpesviridae has a registry numberCNCM I - 5118 At least one of the identification features of the virus collected by the Collection Nationale de Cultures de Microorganisms (CNCM) in France, Paris, and the Pasteur Institute. This means that the virus can be identified as novel according to the present invention. Herpes simplex virus, ie, herpes labia virus. In another embodiment, the identifying characteristic is selected from the group consisting of: 1) the virus has at least 70% of its sequence and its sequence according to SEQ ID NO: (or at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%) , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) SEQ ID NO: Consistent DNA A sequence having the above-mentioned restriction on internal duplication; or 2) a virus in its wild-type form causes disease in Asian jellyfish, which is characterized by the following signs: clinical signs appear about 3 days after the attack, systemic Skin lesions (in large numbers, usually more than 50% of cases) cause darkening of the skin accompanied by pale spots and fin erosion, with no activity accompanied by observed movements Loss of weight ("observed" means that it is visible but not in all cases, only in extremely lifeless fish), (almost completely) lack of appetite, increased respiratory rate, and about 2 weeks after the attack Death occurs, or 3) the virus comprises a major envelope protein (MEP) gene having a consensus level of at least 80% with the nucleotide sequence as set forth in SEQ ID NO: 2; or 4) the virus comprises dUTP An enzyme gene having a consensus level of at least 80% with a nucleotide sequence as set forth in SEQ ID NO: 4; or a virus comprising a terminal enzyme gene, such as SEQ ID NO: 6 The nucleotide sequence shown has a level of identity of at least 80%; or the virus comprises a polymerase gene having a consensus level of at least 80% with the nucleotide sequence set forth in SEQ ID NO: 8. . In another embodiment of the novel herpesvirus, wherein the virus is a member of the Herpesviridae family and its wild-type form causes disease in Asian jellyfish, the virus is characterized in that the virus has a corresponding SEQ ID NO: DNA of the DNA of the sequence of 1. This means that the virus has DNA that is at least 70% identical to its DNA according to SEQ ID NO: 1 over its entire length (with the above restrictions on internal repeats). The level of consistency can be higher, such as 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%. In a further embodiment of the novel herpesvirus, wherein the virus is a member of the Herpesviridae family and its wild-type form causes disease in Asian jellyfish, the virus is characterized by having the following DNA: at least 95% thereof An open reading frame (particularly comprising at least 300 base pairs of ORFs), for example at least 96%, 97%, 98%, 99% or 100%, corresponding to an open reading frame having DNA according to the sequence of SEQ ID NO: Having at least 80% sequence identity, such as at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even up to 100% consistency. In other embodiments, the novel virus can be distinguished from a known member of the Herpesviridae based on the coding DNA sequence of its major envelope protein (ORF28) and its dUTP enzyme coding DNA sequence (ORF1). The results showed that the sequence identity of the major envelope protein of the virus and the MEP of the closest species in other species of the Herpesviridae was only 30%. The sequence homology of the dUTP enzyme closest to the dUTP enzyme of the other species of the Herpesviridae is only 45%. Typical examples of DNA sequences encoding MEP and dUTP enzymes are shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively. The respective amino acid sequence, that is, the protein encoded by the DNA fragment according to SEQ ID NO: 2 and SEQ ID NO: 4 (the term "encoded by" does not exclude other DNA from producing the same protein, or in other words: "by "Specific DNA sequence encoding" means that the protein can be synthesized based on a particular sequence, but may also be synthesized by using another sequence, as shown in SEQ ID NO: 3 and SEQ ID NO: 5. It will be appreciated that for a particular protein encompassed herein, there may be a natural variation between the individual representatives of the pathogen. In, for example, the major envelope protein sequence, there is indeed a genetic variation that causes minor changes. The same is true for the dUTP enzyme. First, there is a so-called "wobble in the second and third bases", which indicates that the nucleotide may change and is still not noticed in the amino acid sequence it encodes: for example, triplet TTA, TTG, TCA, TCT TCG and TCC both encode leucine. Furthermore, minor variations between the representatives of the novel viruses according to the invention can be found in the amino acid sequence. Such variations may be reflected by one or more amino acid differences in the total sequence or by the deletion, substitution, insertion, inversion or addition of one or more amino acids in the sequence. Amino acid substitutions which do not substantially alter biological and immunological activity have been described, for example, by Neurath et al. in "The Proteins" Academic Press New York (1979). Amino acid substitutions between related amino acids or alternatives that frequently occur in evolution are especially Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, MD, Atlas of protein). Sequence and structure, Nat. Biomed. Res. Found., Washington DC, 1978, Vol. 5, Supplement 3). Other amino acid substitutions include Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Thr/Phe, Ala/Pro, Lys/Arg, Leu/Ile, Leu/Val, and Ala/Glu. Based on this information, Lipman and Pearson developed a method for rapid and sensitive protein comparison (Science 227, 1435-1441, 1985) and for determining functional similarity between homologous proteins. Such amino acid substitutions and variations with deletions and/or insertions of exemplary embodiments of the invention are within the scope of the invention. This explains why, for example, MEP and dUTP enzymes may have a homology level significantly lower than 100% when isolated from different representatives of the virus according to the invention, yet still represent the MEP or dUTP enzyme of the virus according to the invention. Typically, the protein of the MEP or dUTP enzyme according to the invention has at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 5, respectively, thereby having 70% of the sequence, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% Sequence identity of 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%. Thus, in particular, the examples (A to G) associated with these proteins (i.e., MEP and dUTP enzymes) are as follows: A: an isolated herpesvirus comprising a major envelope protein (MEP) gene, characterized In the virus is a member of the Herpesviridae family, which causes disease in Asian jellyfish, and the nucleotide sequence of the MEP gene is in agreement with the nucleotide sequence as shown in SEQ ID NO: 2. At least 80% (eg 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%) , 96%, 97%, 98%, 99% or 100%). B: an isolated herpesvirus comprising a dUTP enzyme gene, characterized in that the virus is a member of the herpesvirus family, the virus causes disease in the Asian jellyfish, and the nucleotide sequence of the dUTP enzyme gene is The nucleotide sequence shown in SEQ ID NO: 4 has a level of identity of at least 80% (eg, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%). C: an isolated herpesvirus having a MEP gene and a dUTP enzyme gene, characterized in that the nucleotide sequence of the MEP gene and the nucleotide sequence as shown in SEQ ID NO: 2 have a level of at least 80 %, and the nucleotide sequence of the dUTP enzyme gene is at least 80% identical to the nucleotide sequence as shown in SEQ ID NO: 4 (eg, 81%, 82%, 83%, 84%, 85) %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%). D: an isolated herpesvirus comprising a major envelope protein (MEP) gene, characterized in that the virus is a member of the herpesvirus family, the virus causes disease in the Asian jellyfish, and the MEP gene is in a PCR reaction The PCR product was obtained by reacting with the primer set as shown in SEQ ID NO: 21 and SEQ ID NO: 22 to obtain 277 +/- 10 base pairs. E: an isolated herpesvirus comprising a dUTP enzyme gene, characterized in that the virus is a member of the herpesvirus family, the virus causes disease in the Asian jellyfish, and the dUTP enzyme gene is in the PCR reaction with ID NO: 23 and the primer set shown in SEQ ID NO: 24 were reacted to give a PCR product of 346 +/- 10 base pairs. F: an isolated herpesvirus comprising a MEP gene and a dUTP enzyme gene, characterized in that the MEP gene is reacted with a primer set as shown in SEQ ID NO: 21 and SEQ ID NO: 22 in a PCR reaction to obtain 277 +/- 10 base pair PCR product, and the dUTP enzyme gene reacts with the primer set as shown in SEQ ID NO: 23 and SEQ ID NO: 24 in a PCR reaction to give 346 +/- 10 bases Paired PCR products. G: an isolated herpesvirus characterized by a nucleotide sequence of the MEP gene having a level of identity with a nucleotide sequence as set forth in SEQ ID NO: 2 of at least 80% (or at least 81%, 82%) , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100%), and the nucleotide sequence of the dUTP enzyme gene and the nucleotide sequence as shown in SEQ ID NO: 4 are at least 80% (or at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% And characterized in that the viral DNA is reacted with a primer set as shown in SEQ ID NO: 21 and SEQ ID NO: 22 in a PCR reaction to obtain a PCR product of 277 +/- 10 base pairs, and in PCR PCR products in the reaction which are reacted with the primer set as shown in SEQ ID NO: 23 and SEQ ID NO: 24 to obtain 346 +/- 10 base pairs. Examples D to G utilize the use of the virus according to the present invention. PCR test of the primer set of the major envelope membrane protein gene sequence or the dUTP enzyme gene sequence. The primer set is selected to be depicted in two different primer sets of SEQ ID NOS: 21 to 22 and SEQ ID NOS: 23 to 24. The PCR test envelope using the first primer set (SEQ ID NOS: 21 to 22) reactive with the major envelope protein gene of the virus used two primers LCHV MEP FW and LCHV MEP REV (see Table 2b in the Examples section). The PCR test using the second primer set (SEQ ID NOS: 23 to 24) was reacted with the viral dUTP enzyme gene and two primers LCHV dUTP FW and LCHV dUTP REV were used (see Table 2c in the Examples section). These test series standard PCR tests are described in more detail in the Examples section. Analysis of the PCR product of the first primer set revealed that about 277 base pair PCR products or analysis of the PCR product of the second primer group revealed a PCR product of about 346 base pairs, and the virus is different. A member of the herpesvirus family and causing disease in Asian jellyfish, this clearly indicates that the analyzed virus is a virus according to the invention. For the purposes of the present invention, a PCR product of about 277 base pairs is a PCR product of between 277 + 10 base pairs and 277 - 10 base pairs in length. A PCR product of approximately 346 base pairs is a PCR product between 346 + 10 base pairs and 346 - 10 base pairs in length. Other examples (H to K) associated with other proteins specific for novel viruses, namely terminal enzymes and polymerases, are particularly as follows: H: an isolated herpesvirus comprising a terminal enzyme gene, characterized in that the virus is different A member of the herpesvirus family, which causes disease in Asian jellyfish, and the terminal enzyme gene is reacted with a primer set as shown in SEQ ID NO: 25 and SEQ ID NO: 26 in a PCR reaction to obtain 585 + /- 10 base pair PCR product. I: an isolated herpesvirus comprising a polymerase gene, characterized in that the virus is a member of the herpesvirus family, the virus causes disease in the Asian jellyfish, and the polymerase gene is in a PCR reaction with a SEQ ID NO: ID NO: 27 and the primer set shown in SEQ ID NO: 28 were reacted to give a PCR product of 314 +/- 10 base pairs. J: an isolated herpesvirus comprising a terminal enzyme gene and a polymerase gene, characterized in that the terminal enzyme gene is reacted in a PCR reaction with a primer set as shown in SEQ ID NO: 25 and SEQ ID NO: A 585 +/- 10 base pair PCR product was obtained, and the polymerase gene was reacted in a PCR reaction with the primer set as shown in SEQ ID NO: 27 and SEQ ID NO: 28 to obtain 314 +/- 10 Base pair PCR product. K: an isolated herpesvirus characterized in that the nucleotide sequence of the terminal enzyme gene and the nucleotide sequence as shown in SEQ ID NO: 6 are at least 80% (or at least 81%, 82) %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%), and the nucleotide sequence of the polymerase gene has a level of identity with the nucleotide sequence as shown in SEQ ID NO: 8 of at least 80% (or at least 81%, 82%, 83%) , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 %), and characterized in that the viral DNA is reacted with a primer set as shown in SEQ ID NO: 25 and SEQ ID NO: 26 in a PCR reaction to obtain a PCR product of 585 +/- 10 base pairs, and The PCR reaction was reacted with the primer set as shown in SEQ ID NO: 27 and SEQ ID NO: 28 to obtain a 314 +/- 10 base pair PCR product. Examples H to K utilize a PCR test using a primer set for a terminal enzyme gene sequence or a polymerase gene sequence of a virus according to the present invention. The primer set is selected to be depicted in two different primer sets of SEQ ID NOS: 25 to 26 and SEQ ID NOS: 27 to 28. The PCR test using the first primer set (SEQ ID NOS: 25 to 26) reactive with the terminal enzyme gene of the virus used two primers LCHV TER FW and LCHV TER REV (see Table 2d in the Examples section). The polymerase gene of the virus was tested using the PCR of the second primer set (SEQ ID NO: 27 to 28) and two primers LCHV POL FW and LCHV POL REV were used (see Table 2e in the Examples section). These test series standard PCR tests are described in more detail in the Examples section. Analysis of the PCR product of the first primer set revealed that about 585 base pair PCR products or analysis of the PCR product of the second primer group revealed a PCR product of about 314 base pairs, and the virus is different. A member of the herpesvirus family and causing disease in Asian jellyfish, this clearly indicates that the analyzed virus is a virus according to the invention. Based on the DNA coding sequences of the major envelope proteins and dUTP enzymes of the novel viruses described above, the following examples L to O of the present invention are also provided: L: an (isolated) DNA fragment comprising a gene encoding a major envelope protein, characterized The level of identity of the gene to the nucleotide sequence of the MEP gene as set forth in SEQ ID NO: 2 is at least 80% (or at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%); and M: a DNA Fragment-encoded (isolated) major envelope protein. N: an (isolated) DNA fragment comprising a gene encoding a dUTP enzyme, characterized in that the gene has a level of identity with the nucleotide sequence of the dUTP enzyme gene as shown in SEQ ID NO: 4 of at least 80% ( Or at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%); and O: a (isolated) dUTP enzyme encoded by this DNA fragment. It is currently known that several other genes are also suitable for identifying novel viruses according to the present invention. One of these genes corresponds to the gene of ORF224 (SEQ ID NO: 10), which encodes a membrane (glycosyl) protein (SEQ ID NO: 11). Another gene is a gene homologous to the salmon herpesvirus type 1 (IHV1) TK (thymidine kinase) gene. This gene corresponds to ORF5 in IHV-1 (Hanson et al., Virology. August 1, 1994; 202(2): 659-64). The novel virus has a conserved domain "deoxynucleoside kinase" which is 33% identical to the ORF 5 of IHV-1 in terms of amino acid level. Another gene (SEQ ID NO: 12) for identifying a novel virus comprises ORF206, a gene encoding a major capsid protein (SEQ ID NO: 13). According to still another embodiment of the present invention, there is directed to an (isolated) DNA fragment having an open reading frame of at least 100 nucleotides in length, wherein the DNA and the open reading frame having DNA according to the sequence of SEQ ID NO: Having at least 80% (or at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95) Sequence identity of %, 96%, 97%, 98%, 99% or 100%). Although it has been found that a length of 30 to 40 nucleotides is sufficient to distinguish the DNA of the virus according to the invention from the DNA of any known virus, the actual relevant length, in particular for the corresponding subunit vaccine based on the corresponding protein, is at least 100 cores. Glycoside (or at least 150, 200, 250 or even at least 300 nucleotides) to correspond to a protein having a relevant and prominent 3D identity corresponding to the relevant immunogenic epitope of the viral protein. Thus in another embodiment, the invention also relates to (isolated) proteins encoded by such DNA fragments. In one embodiment, the invention also relates to a cell culture comprising a novel virus according to the invention in a replication competent form (ie, an artificial culture of a defined number of cell types in an artificial culture vessel, also referred to as a cell strain; Processes for the growth of cells under controlled conditions outside their natural environment are also described). Several fish cell lines are potentially useful for supporting replication of the virus according to the invention. An example of a cell strain which can be used to grow a virus according to the present invention is a cell strain of brain cells of Asian jellyfish. Methods for isolating such cell lines have been described, inter alia, by Hasoon et al. in In Vitro Cell. Dev. Biol. - Animal 47: 16-25 (2011). Another example of a cell line potentially useful for supporting replication of the virus is by Chi et al.: "Persistent infection of betanodavirus in a novel cell line derived from the brain tissue of barramundi Lates calcarifer", Chi SC, Wu YC, ChengTM, Dis Aquat Organ. June 2005; 65(2): 91-8. PMID: 16060261 Revealed. It has also been established that sea scorpion native cell cultures using conventional methods can be used to support replication of the virus. Other cell lines that can be used to grow the virus are from the skin, brain, heart, that is, the cells of the organ in which the virus may replicate. In still another embodiment, the invention relates to a vaccine for combating a herpes simplex virus disease, wherein the vaccine comprises a herpesvirus according to the invention or an immunogenic protein as described above and a pharmaceutically acceptable carrier. Such carriers can be as simple as water, as long as they are suitable for administration of the material in clinically relevant amounts without causing unacceptable side effects. Typical carriers are emulsions of oil and water, insoluble adjuvants (usually aluminum or other salts or large immunostimulatory polymeric molecules) in water, or soluble adjuvants (such as saponins, PAMP, carbopol). Or a solution of other immunostimulatory molecules). Typically, the carrier comprises stabilizers and preservatives generally known in the art. In another embodiment, the vaccine comprises a live attenuated (i.e., replication competent but no longer capable of inducing a full set of symptoms as induced by the original wild-type pathogen) or an inactivated form of the herpesvirus according to the present invention. Experimental vaccines for herpes simplex virus have been designed using different techniques. Vaccines are killed by peptides, (recombinant) viral proteins, mixtures of viral proteins, intact and fractionated (see, for example, Yasumoto, S. et al., Fish Pathology 41: 141-145 (2006), Description of a replication-compact virus containing a non-activated intact koi herpes virus captured in a liposome compartment), a replication-defective virus, and attenuated replication-competitive virus (eg by Koelle, DM and L. Corey. 2003: Recent Progress in Herpes Simplex Virus Immunobiology and Vaccine Research. Clin Microbiol Rev. 16(1): 96-113) Composition. Each method has specific advantages and disadvantages that have been discussed by Stanberry in 2000 (Stanberry, LR, AL Cunningham, A. Mindel, LL Scott, SL Spruance, FY Aoki and CJ Lacey. 2000: Prospects for control of herpes simplex virus disease Through immunization. Clin. Infect. Dis. 30: 549-566.). It is known that in vitro exogenous passage of a virulent herpesvirus strain in a cell culture produces attenuated progeny, or in other words, a non-toxic replication competent virus strain that elicits a protective immune response without causing clinical symptoms of the disease. For example, the attenuation of Marek's disease virus (MDV) is achieved by using a protective vaccine called Rispens or CVI988, which allows the virulence virus to be continuously subcultured in vitro until the resulting isolate Becomes non-toxic (Rispens BH, Vloten H, Mastenbroek N, Maas HJ, Schat KA. 1972: Control of Marek's disease in the Netherlands. 1. Isolation of an avirulent Marek's disease virus (strain CVI988) and its use in laboratory vaccination trials. Avian Dis. 16:108-125). Correspondingly, it is described that complex genes that are frequently involved in DNA replication and transcriptional regulation in the pathway are involved in de novo de novo of MDV and provide a goal for the rational design of future MD and thus corresponding herpes virus vaccines. The fish herpes virus has also been described as being attenuated by successive passages (see especially Noga, E. J. et al., Can. J. Fish. Aquat. Sci. 38: 925-929, 1981). As is generally known, attenuating viruses can be spontaneous or can be induced by drugs (mutations or other natural conditions such as UV light; see for exampleMutation Research 768, 2016, 53-67 andJ . Gen . Virol , 1985,66 , 2271-2277). The underlying genetic mechanisms behind attenuation are often not fully understood, but genetic changes (mutations, deletions, etc.) and/or their accumulation in the viral genome are the basis of attenuated herpes virus strains. Mutations involve multiple viral mechanisms, including viral replication, spread, and the like. Certain molecular pathways necessary for viral replication and infectivity in vivo may not be necessary for replication cultures, and genes involved in such pathways may be more susceptible to alteration of genes during long term culture. If such mutations and deletions in the genome are characterized, and with the development of a new generation of sequencing techniques, it is relatively simple to sequence the entire genome of a larger virus such as herpes virus, which enables rational design reduction. poison. According to the literature on herpes virus attenuation, complex genes have become a possible target for functional dysfunction of the virus. A detailed overview of the genes involved in viral replication (which may have undergone mutations in live attenuated herpes simplex vaccines) was given by Roizman and Knipe in 2001 (Roizman, B. and DM Knipe: Herpes simplex viruses and their replication, 2399- 2459. In DM Knipe, PM Howley and DE Griffin (ed.), Fields virology, 4th edition, volume 2. Lippincott, Philadelphia, Pa). In addition, such mutations can be used to establish vaccine methods using discrete replication viruses. The mutant virus is grown in a genetically engineered cell line that provides a non-mutated gene required for transcription. For example, when herpes simplex virus encoding the late gene UL22 deletion of gH infects non-complementing cells, progeny virions can leave the cell but cannot infect secondary cells (Koelle and Corey, 2003, cited above). The following is a list of herpes virus genes in which dysfunction or deletion has been described as causing functional attenuation of the Koi herpes virus - a herpes simplex virus - or other herpes virus. These genes are the target genes for attenuating the herpesvirus according to the present invention: 1) the thymidine kinase (TK) gene of the Koi herpes virus. The TK gene has also been described in the herpes simplex virus (CCV), a virus that is relatively closely related to the virus described in the present invention (Hanson LA, Kousoulas KG and RL Thune. 1994: Channel catfish herpesvirus (CCV) Encodes a functional thymidine kinase gene: elucidation of a point mutation that confers resistance to Ara-T. Virology 202(2): 659-64). 2) The d-UTP enzyme gene of the Koi herpes virus. 3) The gene encoding ORF57 of Koi herpes virus, as provided by Genbank accession number N° NC_009127, where the start and stop codons of ORF57 are located at 99382 and 100803 (Boutier M, Ronsmans M, Ouyang P, Fournier G, Reschner A, etc.) (2015): Rational Development of an Attenuated Recombinant Cyprinid Herpesvirus 3 Vaccine Using Prokaryotic Mutagenesis and In Vivo Bioluminescent Imaging. PLoS Pathog 11(2): e1004690). 4) gD (EHV en BHV)/gp50 (PRV) gene found in bovine herpesvirus, equine herpesvirus and pseudorabies virus (Aujeszky's disease). This gene encodes a glycoprotein that produces a neutralizing antibody. The invention also relates to a method of prophylactically treating an animal (i.e., treating an animal to prevent infection by a corresponding wild-type pathogen after treatment) using a vaccine as described above, comprising systematically administering a vaccine to the animal. Systematic administration of a vaccine means that the vaccine is administered such that it reaches the body's circulatory system (including the cardiovascular and lymphatic systems), thereby affecting the body as a whole rather than a specific location, such as the gastrointestinal tract. Systemic administration can be by, for example, administering antigen to muscle tissue (intramuscular), dermis (intradermal), subcutaneous (subcutaneous), submucosal (submucosal), intravenous (intravenous), body cavity ( Intraperitoneal) and the like. The invention also relates to an antibody or antiserum reactive with a virus according to the invention, and a diagnostic test kit for detecting an antibody reactive with the virus according to the invention or with an antigenic substance thereof, wherein the test kit comprises Invented virus or antigenic substance thereof. The invention also resides in a diagnostic test kit for detecting a herpesvirus or antigenic substance thereof according to the invention, wherein the test kit comprises an antibody reactive with a virus according to the invention or with an antigenic substance thereof or as described above PCR primer set. The invention will now be further elucidated using the following examples. InstanceInstance 1 : Barramundi Herpes virus discovery Collection of serum and tissue samples used to isolate infectious agents Asian sea otters in Singapore (Barramundi Diseased fish are observed in fish farms. Diseased fish exhibit clinical symptoms similar to those of scale-shed disease in breeders (Gibson-Kueh et al., J Fish Dis. January 2012; 35(1): 19-27. doi: 10.1111/j.1365 -2761.2011.01319.x. PMID: 22103767; de Groof et al., PLoS Pathog. August 7, 2015; 11(8): e1005074. doi: 10.1371/journal.ppat.1005074. PMID: 26252390). However, when the disease symptoms were studied more closely, it appeared that the diseased fish showed a more acute infection (3 to 10 days instead of more than 15 days) with a higher incidence. Skin lesions are less severe, but are more systemic than flank exfoliation. The difference between skin lesions and scale-off disease lesions is that the entire fish body is darker and more dull, with spots of pale mucus. A certain degree of scale loss was observed, but it was not significant and was not the main clinical sign. This is different from the scale loss caused by the scale-shediasis virus, which is caused by the loss of scales caused by the scale-slipping disease virus, which has a localized plaque lesion, which is more deeply affected by necrosis, accompanied by severe scale loss. Scale-eating disease viruses also often cause longer-term outbreaks. Based on the observed differences and the fact that the scale-off disease virus PCR has a negative result, based on the clinical observation of the affected fish farm, it is suspected that there is a different viral infectious agent. It was therefore decided to conduct a follow-up study on virus isolation and discovered a new virus. In addition to the loss of scales, other observations of diseased fish were lifeless, severely loss of appetite, acute onset of turbid/swollen eyes, and high mortality (up to 30% to 70% of affected fish). Samples (serum, kidney, spleen) were taken from the affected fish. The pooled serum samples were stored at -70 °C until further analysis. The pooled kidney samples were kept at +4 °C until homogenization the next day.Tissue homogenization for isolating tissue samples of infectious agents The kidney samples were homogenized by manual grinding in a SVDB (standard vaccine diluent buffer = PBS) using a homogenized rod, and the samples were finally diluted 1:9 (w/v) in the SVDB and subsequently in the gytromycin Pretreatment in 1 hour. The homogenized sample was centrifuged at 5,500 rpm for 10 minutes at +4 °C, and then the cell-free supernatant was collected. One day before the inoculation of the monolayer, the Seabass brain (SBB) cells were 2 × 10 in a T75 flask.⁴ Cells/square centimeter were inoculated in EMEM + 10% FBS + gentamycin + amphotericin. In this experiment, cells were grown in medium supplemented with HEPES and sodium bicarbonate, and the cells were optimized to contain no CO.₂ It grows under these conditions. The next day, the flask was incubated at 28 ° C with fresh medium (0.1 mL) supplemented with 0.1 mL or 0.3 mL of undiluted cell-free supernatant. CPE was observed in flasks to which undiluted cell-free supernatants (0.1 mL and 0.3 mL) were added after 3 days, and collected on day 5 (passage 1). The hypothetical infection was named V511. According to the above protocol, the CPE-causing agent was subcultured 3 times on SBB cells. The CPE initiator was named V511/SBB_4P.In the cells and serum of affected fish CPE Primer culture supernatant for virus detection Analysis of culture supernatants of affected fish and serum culture of the fourth generation of CPE initiator V511/SBB_4P using the VIDISCA-454 technique described by De Vries et al. (2011) PLoS ONE 6(1):e16118 . In both types of samples, sequences suspected of being derived from novel fish pathogens were obtained. These sequences were used to derive PCR primers for use in conventional PCR and quantitative PCR (see Tables 2a through 2e). Local base alignment searches of the new sequences revealed that the CPE initiator in the culture supernatant and the suspected infectious agent in the serum showed some degree of homology to the herpesvirus. This new virus is therefore called the herpes labia virus (LCHV).Whole genome sequencing As described (de Vries M et al. PLoS One. 2011; 6(1): e16118. doi: 10.1371/ journal.pone.0016118), the virus culture supernatant samples were centrifuged at 10,000 x g for 10 minutes with TurboDNase (Thermofisher) treatment, followed by extraction of nucleic acids by the Boom extraction method (Boom R et al. J Clin Microbiol. 1990; 28(3): 495-503). Samples were cut using dsDNA fragmentase (New England Biolabs). The sheared samples were purified using a AMPure XP bead (agencourt AMPure XP PCR, Beckman Coulter) at a 1:1.8 (sample:bead) ratio to remove the enzyme. After purification, the samples were end-repaired with DNA polymerase I large (Klenow) fragment (New England Biolabs). The end-repaired sample was purified using AMPure XP beads (agencourt AMPure XP PCR, Beckman Coulter) at a ratio of 1:1.8 (sample:bead) to remove the enzyme, followed by the use of Klenow fragment (3'→5' The exonuclease (Exo) (New England Biolabs) was subjected to A tailing of the sample. The sample was purified using a AMPure XP bead (agencourt AMPure XP PCR, Beckman Coulter) at a 1:1.8 (sample:bead) ratio to remove the polymerase. Bubble adaptors from NEB Next Multiplex Oligos for Illumina (New England Biolabs) were ligated to the A-tailed sample by using T4 ligase (Thermofisher). Size selection by using AMPure XP beads (agencourt AMPure XP PCR, Beckman Coulter) was first performed at a 1:0.5 (sample:bead) ratio to ensure removal of most fragments larger than 400 bp in size, followed by supernatant Additional AMPure XP beads (agencourt AMPure XP PCR, Beckman Coulter) were added to obtain a final ratio of 1:0.85 (sample: beads) to bind DNA fragments between 200 bp and 400 bp and remove fragments less than 200 bp . After size selection, the vesicular linker was opened by using the USER enzyme from NEBNext Multiplex Oligos for Illumina (New England Biolabs). Then, 28 cycles of PCR were performed using a linker-specific primer from NEBNext Multiplex Oligos for Illumina (New England Biolabs) and Q5 hotstart mastermix (New England Biolabs); 30 seconds 98 ° C, and 10 seconds 98 ° C and 75 seconds 65 ° C Then, a cycle of 65 ° C for 5 minutes. After PCR, samples were size-selected by using AMPure XP beads (agencourt AMPure XP PCR, Beckman Coulter) at a ratio of 1:0.5 (sample: beads) to remove fragments larger than 400 bp in size and up-cleared Additional AMPure XP beads (agencourt AMPure XP PCR, Beckman Coulter) were added to obtain a final ratio of 1:0.85 (sample: beads) to bind DNA fragments between 200 bp and 400 bp and remove less than 200 bp. Fragment. Subsequently, the concentration of DNA was measured via a Qubit dsDNA HS assay kit (Thermofisher) and the size was checked on a bioanalyzer using a highly sensitive DNA assay kit. The DNA was diluted to a concentration of 2.49 ng/μl. The DNA was sequenced using a paired end sequencing and a v2 kit (Illumina) using MiSeq (Illumina).Phylogenetic analysis The initial phylogenetic analysis was based on a 208 bp DNA fragment of scorpion herpesvirus (SEQ ID NO: 14) found in samples of disease outbreaks. This fragment shows homology to the ORF 62 of the salmon herpesvirus type 1 NP_041153.2 and other fish viruses at the translated nucleotide level. Nucleotide and protein sequence alignments were generated using the BLAST base local alignment search tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the multiple sequence alignment tool ClustalW. The MEGA5 software uses a proximity merging method to create a phylogenetic tree with partial deletions in the case of voids or insertions (MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods (using maximum likelihood, evolution distance, and maximum). Molecular Evolutionary Genetic Analysis of the Simple Method). Koichiro Tamura, Daniel Peterson, Nicholas Peterson, Glen Stecher, Masatoshi Nei and Sudhir Kumar. Mol. Biol. Evol. 2011 28(10): 2731-2739. 2011 doi:10.1093/molbev/ Msr121). The results of this phylogenetic analysis are presented in Figure 1, which depicts the phylogenetic tree of LCHV based on homology analysis of the 208 bp DNA fragment of LCHV and IHV1 ORF62. Phylogenetic analysis confirmed the newly discovered virus in the LCHV lineage of the herpesvirus family.Instance 2 : use PCR , qPCR Analysis and detection of herpes simplex virus Primer design A PCR primer was designed for the 208 bp DNA fragment of the herpes labia virus found in the sample of the disease outbreak. This fragment shows homology to the ORF62 of carp herpesvirus type 1 NP_041153.2 in terms of translated nucleotide levels. Primers were also designed for MEP, dUTP enzymes, terminal enzymes, and polymerase genes. Table 2a: Introduction to the design of a 208 bp DNA fragment of the herpes labia virus Table 2b: Introduction to the MEP design of the herpes labia virus Table 2c: Introduction to the design of the dUTP enzyme for the herpes zoster virus Table 2d: Introduction to the end-enzyme design of the herpes zoster virus Table 2e: Introduction to polymerase design for herpes labby virus PCR Gel electrophoresis Conventional PCR was performed using a Veriti 96-well thermal cycler (Applied Biosystems). A master mix containing 1 x Supertaq buffer, 0.02 U/μl Supertaq enzyme, 0.2 mM deoxyribonucleoside triphosphate (dNTP), 1 μM forward primer, and 1 μM reverse primer was prepared. For each sample, 2.0 μl of DNA template was added to the 48 μl PCR mixture and 2 μL of sterile water was used as a negative control. The PCR program was designed to start with 60 seconds of initialization at 95 ° C, followed by denaturation, annealing and extension for 30 seconds each at 95 ° C, primer-based Tm-based primer set specific annealing temperature and 72 ° C. . The program ends with a final extension of 10 minutes at 72 °C. Samples were loaded with 1 x ethidium bromide and 1 x TAE buffer in a 1.5% agarose gel at 115 volts for 60 minutes.qPCR analysis Quantitative polymerase chain reaction was performed using the BioRad CFX 96 system. qPCR experiments were performed using a Probe Fast Master Mix and sequence specific probes. There was an 18 μl master mix containing 1× probe fast qPCR master mix (KAPA), 200 nM forward primer, 200 nM reverse primer and 200 nM probe for each reaction. Primer pair 2 was used for qPCR analysis with an optimized Tm of 60.7 °C. The probe DNA sequence was CGCGGGATGACCTCTTCTCG (SEQ ID NO: 31) and the markers used were 5' 6FAM and 3' TAMRA. Add 2 μl of DNA template to the 18 μl master mix. Each reaction was performed in duplicate and all disks were spun at 2,200 x g for 4 minutes prior to insertion into the CFX system. Amplification occurred using a procedure starting at 35.0 minutes at 35.0 °C followed by 40 repetitions at 95.0 °C for 3 seconds and 60.7 °C for 30 seconds.Standard line A dilution series containing the pUC57 vector with the herpes simplex virus hereditary sequence construct (synthesized by GenScript [synthesis of the 208 bp fragment as described above and colonized in the plastid vector]) as a positive control, potency and precision Indicators for the indication of sex and the quantification of samples. Vector with 1.0 × 10 per qPCR reaction¹ Replica/2 microliters to 1.0 × 10⁹ The replicates are in the range of 2 microliters and are diluted in water. The dilution series was included in all qPCR experiments and stored at -20 °C. The quantification of the herpes zoster virus DNA in the sample was performed by the support software (CFX-Manager version 3.1), which used the data from the dilution series to establish a standard line. All qPCR experiments performed showed an efficiency between 98.0% and 100.2% and an accuracy between 0.997 and 0.999, highlighting the stability of the qPCR method designed to quantify LCHV DNA.Instance 3 : Innovative infection of experimental infections in Asian sea otters V511 / SBB _ 4P Sea otter in Asia ( Barramundi ) Experimental infection In this experiment, fish were infected intraperitoneally and cohabiting with V511 to investigate whether V511 is a cause of disease in the wild. Different organs were sampled at different time points following infection to determine the course of infection. Table 3: Processing group and slot configuration. One group of 25 Asian jellyfish was challenged by intraperitoneal (IP) injection of 0.3 mL of undiluted V511 cell-free supernatant (CFS) grown on SBB cell line V511/SBB_4P. CSF virus titer is 3.2 × 10⁶ TCID₅₀ /mL (see the titration scheme below). The second group of 25 fish cohabited in the same trough separated by the net (Table 3). The mesh allows water to move freely between the two trough halves and allows close proximity but no direct contact between the fish of the two treatment groups. At the beginning of the experiment, the average weight of the fish was 18 grams. Hungry fish for about 24 hours before the attack to ensure gastrointestinal emptying, thereby reducing the risk of injury. Fish were anesthetized prior to challenge by sedation with Aqui-S according to standard procedures. The fish nets in the IP attack group were given a sedative and IP injections were made to the midline between the pelvic fins and the apex. After the attack, place the fish in the designated tank for recovery and observation. Uninfected cohabiting fish are introduced into adjacent trough partitions separated by a net. Mortality and clinical signs were recorded. On the 4th, 7th, 11th, 14th and 18th day after the challenge, 3 fish were randomly sampled in the cohabitation group and subjected to autopsy for fish tissue collection for further testing. Sampling includes the spleen, heart, brain, serum, liver, intestines, sputum, skin, and kidneys to better understand the infection process of the novel infectious agent after infection via the natural route. On days 4, 7, 11 and 14 post-challenge, 3 fish were randomly sampled in the IP-infected group and subjected to autopsy for collection of kidney tissue for further testing. On the 17th, spleen, heart, brain, serum, liver, intestine, sputum, skin and kidney were sampled from three experimentally infected fish. By the 18th day after the attack, all fish had been sampled or had died of infection (see Table 6). Sample collection is summarized in Tables 4 and 5. Table 4: Sampling of fish tissue in the experimentally IP-infected group. Table 5: Sampling of fish tissue in the cohabitation group. The results presented in Table 6 show that both the IP attack pathway and the cohabitation attack pathway are capable of producing clinical signs similar to those observed in the index Asian fish pond fish farm. The observed clinical signs were lifeless, loss of appetite, skin, fins, and eye lesions, and these clinical signs were similarly observed in affected farmed fish in indicator fish farms. Table 6: Clinical signs observed in the IP attack processing group and the cohabitation attack processing group This shows that in a controlled laboratory environment (where no other pathogens exist and fewer stressors are present than in a wild environment such as a fish farm), typical symptoms after the (IP) attack are 1) clinical signs after the attack About 3 days of episodes, 2) systemic skin lesions may cause skin darkening accompanied by pale spots and fin erosion, 3) loss of swimming balance due to extreme lifelessness, 4) almost complete lack of appetite, 5) sputum respiration rate Increase and 6) death occurred approximately 2 weeks after the attack. Swelling and opaque eyes can be observed in some fish. Mortality and % cumulative mortality records are shown in Table 7. Table 7: Daily Mortality and % Cumulative Mortality Record*. * Does not include fish sampled for tissue collection. The V511/SBB_4P virus supernatant was spread from the fish attacked by IP to the untreated cohabitation fish. Both groups experienced similar clinical symptoms. These clinical symptoms are also similar to the clinical symptoms (initial outbreaks) observed in indicator fish farms. Compared with the cohabitation group, the IP-infected group experienced more acute and more serious diseases, and the signs of the disease appeared within 3 days after infection. In contrast, cohabitation-stricken fish showed initial clinical signs on the 7th day after infection. Using infectious agents derived from tissue samples collected during field disease studies and subsequent passages in vitro, we were able to replicate the clinical symptoms observed during the outbreak via both the IP infection pathway and the co-infection pathway. The disease was also shown to spread from IP-infected fish to untreated cohabiting fish in the same water space, confirming the infectious nature of this pathogen.Instance 4 : In the infection experiment ( Instance 3 ) Sample preparation, tissue homogenization, collection of tissue samples collected during the period DNA Separation Homogenization of organ samples Fish organ samples (Table 4, Table 5) collected in the above experiments were homogenized using a Precellys 24 homogenizer. A 10% organ homogenate was prepared in phosphate buffered saline (PBS) using a program with two cycles of 20 seconds and 10 seconds at 6,500 rpm. The homogenization of the heart sample, the spleen sample, the kidney sample, the brain sample, the intestinal sample, and the liver sample was completed in one cycle, and the skin sample and the sputum sample were homogenized twice. All samples were kept on ice during homogenization and stored at -80 °C.DNA extract DNA extraction was performed using the MagNA Pure 96 system and the MagNA Pure 96 DNA and viral NA kits. For extraction, add 250 μl of MagNA Pure 96 External Lysis Buffer to the 200 μl sample. DNA was isolated using a pre-positioned external lysis protocol and dissolved in 50 μl of Milli-Q water. The DNA was stored at -20 ° C until further use.Instance 5 : Virus culture and virus titration Sea bream ( SBB ) Establishment and cultivation of cell lines : The cell line SBB was originally derived from a trypsinized suspension of Asian sea lice brain cells at Intervet Norbio Singapore Pte Ltd (part of MSD AH). The procedure for deriving sea blast cells has been performed by Hasoon et al. in In Vitro Cell. Dev. Biol. - Animal 47: 16-25 (2011) and by Chi et al. Dis Aquat Organ. June 2005; 2): Description of 91-8. SBB medium consisting of 899 ml E-MEM supplemented with 2 mM L-glutamic acid and 110 mg/L sodium pyruvate, 100 ml FCS (10%) and (optionally) 1 mL neomycin bacterium Neomycin Polymyxin antibiotic solution 1000× stock solution. Cells at 28 ° C and 5% CO₂ It grows conventionally. The medium was stored at 4 ° C before starting the culture. Start the culture using an ampoules of frozen SBB stock. The liquid nitrogen-containing cells were rapidly thawed by warming the ampoule in water at 28 °C. The cell suspension was added to the tube and slowly diluted with 9 volumes of medium. Subsequently, the cells were counted. Dispense the suspension into a suitable flask or roller bottle at 28 ° C and 5% CO₂ Under cultivation. Inoculation density in flasks or roller bottles is approximately 3 × 10⁴ Cells per square centimeter. After 6 to 24 hours or after the cells were completely attached, the medium was renewed to remove residual DMSO (freezing medium consisting of 90% medium and 10% DMSO). The cells are further incubated for 3 to 7 days or until confluence is reached. For roller bottles, a roll speed of 0.2 to 0.5 rpm is required. Roller bottles can be 480, 960 and 1750 cm² Different surface areas. Once the confluence is reached, the cells are subcultured. Subsequent can be 3.0 × 10⁴ The initial seeding density of cells per square centimeter is performed every 3 to 4 days. Alternatively, when at 1.0 × 10⁴ Subcultures can be generated every 7 days when the density of cells/square centimeter is applied. The reagents for cell passage (medium, PBS, trypsin/EDTA) were pre-warmed to 28 °C. The medium was discarded and the confluent monolayer was washed once with an appropriate volume of PBS (3 mL for T25 flask). The PBS was then discarded and the cells were incubated for 15 minutes at 28 °C in the same volume of PBS supplemented with 1% (vol/vol) 2.5% trypsin solution and 1% (vol/vol) 2% EDTA solution. After detachment, the same volume of fresh medium was added and the cells were resuspended and counted. The new flask is set at the desired cell density in a culture volume suitable for the culture flask or roller bottle. For frozen cells, medium and 2X concentrated freezing medium (80% (vol/vol) medium plus 20% (vol/vol) DMSO) were stored at 4 °C prior to the procedure. Confluent cell cultures are treated as described above and include trypsin treatment. The cells were resuspended, counted, further resuspended in a suitable amount of medium, and an equal volume of 2x frozen medium was added dropwise while the vortex suspension was added. Use 5.25 × 10 per ampere⁶ Cells filled with ampoules for liquid nitrogen storage to start T175 or use 2.25 × 10⁶ Cells are filled to start T75.Inoculation with herpes zoster virus SBB cell The cells cultured from the liquid nitrogen were subcultured at least once before the inoculation experiment was set. At 3.0 × 10⁴ The cells were subcultured and cultured for 24 hours before inoculation in cells in a tissue culture flask. The inoculum included a fresh or frozen-thawed culture from the previous passage of the virus without diluting the supernatant. The medium was removed from the flask. The flask was then inoculated at 28 ° C for at least 60 minutes. When cells are inoculated with previous passages of LCHV virus in the medium, it is preferred to use 0.001-0.01 TCID per cell.₅₀ MOI. After removal of the inoculum (after 60 minutes, but this is not absolutely required), fresh medium was added and the cells were cultured until complete CPE was observed using an inverted microscope (usually after 2 to 4 days). The virus was collected by collecting the culture supernatant which was spun at 800 × g for 5 minutes to remove the residue. Alternatively, the supernatant can be clarified by filtration. The clarified supernatant was used for subsequent passage or PCR/DNA/EM analysis or frozen at -70 °C. The replication of the virus can be confirmed using (quantitative) PCR analysis of the collection and/or titration. The identity of the virus was confirmed using DNA sequencing technology and EM. DNA for (quantitative) PCR was isolated from tissue culture media using the MagNA Pure 96 system and the MagNA Pure 96 DNA and Viral NA kit (Example 4).in SBB Titration of virus on cells SBB cells were cultured as described above. One day before the test, the preparation in the medium (EMEM + 10% FCS + L-Glu + NaPyr) contained 6.0 × 10⁴ Cell/ml of SBB cell suspension. 96 wells of the microtiter plate were inoculated with 100 μL of this cell suspension. Will be at 28 ° C and 5% CO₂ Cultivate for 24 hours. The monolayer was about 50% confluent after this incubation period. On the day of the test, a 10-fold serial dilution of each virus sample was prepared by up to 10^- ⁷ : Transfer 0.5 mL sample to a tube containing 4.5 mL of cold (0-20 ° C) titration medium (medium with reduced FCS; 50% EMEM + L-Glu + NaPyr + 50% medium), mix and transfer 0.5 mL to Contain a 4.5 mL titration medium under a test tube, then carefully mix, transfer, etc. Rows 1 and 12 and columns A and H served as negative controls and were inoculated with 100 microliters/well of fresh titration medium. In column B to column G (10 wells/diluent), microtiter plate with 100 μl/well virus dilution (10^- ² , 10^- ³ , 10^- ⁴ , 10^- ⁵ , 10^- ⁶ , 10^- ⁷ ) Inoculation. During the operation, the temperature of the virus dilution was maintained between 0 ° C and 20 ° C. Will be at 28 ° C and 5% CO₂ Cultivate for 6 days. After the 6-day virus incubation period, the plate was screened for LCHV-specific CPE using an inverted microscope. CPE is characterized by cell encapsulation followed by cell detachment/dissolution (Figure 2). Each well showing LCHV-specific CPE was scored as positive. The TCID is determined according to the method and calculation described by Reed and Muench, am. J. Epidemiol. (1938) 27(3): 493-497.₅₀ . qPCR analysis of DNA samples isolated from positive wells in the titration analysis confirmed the presence and replication of the virus.result At 3.0 × 10⁴ The cells were inoculated into tissue culture flasks and cultured for 24 hours. After 24 hours, the cells in one flask were counted after trypsin treatment to determine the actual number of cells present in the flask. The medium was removed from the other flasks that were subsequently inoculated. 0.001 TCID per cell consisting of undiluted culture supernatant from previous passages of LCHV virus in the culture medium (the number of passages of the virus between 4 and 8)₅₀ The inoculum was applied to a single layer and incubated for 60 minutes. The inoculum was removed and fresh medium was added to the cell culture flask. The cells were cultured until complete CPE was observed using an inverted microscope. The virus was collected by collecting the culture supernatant which was spun at 800 × g for 5 minutes to remove the residue. The sample was taken from (1) an undiluted inoculum for infecting a monolayer, (2) a culture supernatant of a flask collected 1 hour after replacing the inoculum with fresh medium, (3) a monolayer of 50% CPE and (4) A single layer of 100% CPE. Samples (1), (3), and (4) were titrated, and samples (1), (2), (3), and (4) were subjected to qPCR analysis. The results are shown in Table 8. Images were captured at 40x magnification using an Olympus CKX41 inverted microscope. These images are shown in Figure 2. Figure 2A shows the morphology of SBB cells at 90% confluence in culture flasks (p18), and Figure 2B shows the morphology of SBB cells at 50% CPE. Table 8: Detailed results of LCHV growth on SBB cells. Instance 6 : Electron microscopy Electron microscopy A 400 mesh grid of pure carbon film was exposed to glow discharge in air for 20 seconds to render the film surface hydrophilic. The virus samples were placed on a grid of coated carbon in a volume of 10 μl and incubated for about 2 minutes. Excess samples were printed using filter paper and 10 μl of water was placed on the grid and removed again by blotting. 10 μl of 1% uranyl acetate was then placed on the grid for staining. After 30 seconds, the excess stain was removed by blotting and the sample was dried for several minutes before being viewed in an electron microscope. Samples were observed in a JEOL 1011 transmission electron microscope operating at 80 kV. Images were recorded using a SIS Veleta 2kx2k camera. Images of LCHV culture samples were captured using a JEOL 1011 transmission electron microscope to confirm the identified viral herpesvirus and to exclude the presence of any other virus. SBB (p9) cells were seeded with a 5th generation LCHV at an MOI of 0.01. The virus was stored in medium at -80 ° C after collection and obtained 10⁴ ^. ⁴³ TCID₅₀ /mL titer. A 1 μl sample was prepared for electron microscopy, two of which are shown in Figure 3. In Figure A, two black spots with a diameter of about 100 nm were observed, consistent with the average diameter of the capsid of the common herpesvirus (115-130 nm). Figure B shows an enlarged view in which the icosahedral contour of the particle is clearly observed. No enveloped virus was found in the sample. Figure 3 shows the LCHV virus captured using an electron microscope. Two herpes viruses (black spots) can be identified in Figure 3A. The scale is 500 nm. Figure 3B shows an enlarged view of one of the points, clearly showing the icosahedral outline. The examples presented above describe the detection and isolation of novel pathogens from diseased fish. The same disease symptoms can be reproduced after experimentally infecting healthy fish using isolated pathogens. The infectious agent isolated from the experimentally infected fish is the same as the originally isolated pathogen. This demonstrates that the symptoms of the disease described above are attributable only to the pathogen found, namely the herpes labia virus.Instance 7 : Protocell culture A native cell culture from sea frog fin cells (SBF) was established. In culture, the cells are cultured for at least 5 passages. Cultures were established as follows. Anesthetize the fish. The caudal fin (tail) was trimmed and washed three times in PBS + gytromycin 0.3% + enrofloxacin 0.002% + amphotericin 0.5%. The fins were cut into tissue fragments using a scalpel blade. The fragment was transferred to a 25 cm2 tissue culture flask containing L15 medium supplemented with 20% FCS and gentamicin 0.3% + enfloxacin 0.002% + amphotericin 0.5%. The flask was at 28 ° C without CO₂ It is cultivated in a moisture-containing incubator. The medium (L15) is changed as needed based on the presence of the residue and the pH. Depending on the cell density, the cells were passaged by trypsin treatment using 0.125% trypsin in PBS until the cells were detached and split at a low ratio between 1:1-3. During the initial passage, cells were cultured in L15 medium supplemented with 20% FCS. In subsequent generations, the FCS percentage was reduced to 10%. Inoculation of the herpes zoster virus was carried out using the procedure described in Example 5. 100% CPE was observed on the 4th day after infection.Instance 8 : Additional novel virus strain A sample of sick fish was obtained from a fish farm different from the indicator fish farm, in which clinical symptoms of herpes zoster virus infection were observed. However, PCR analysis using primer set 2 (SEQ ID NO: 17 and SEQ ID NO: 18) described in Example 2 gave negative PCR results. As an alternative, a primer set designed based on the polymerase (ORF21) and terminal enzyme (ORF 37) sequences was used for PCR, where the ORF hasHerpesvirus It is more conservative than a relatively high level of amino acid. The primers for PCR are presented in Table 2d and Table 2e. These PCRs gave positive results. The terminal enzyme PCR fragment was sequenced and showed 97% identity to the sequence of the herpes labia virus presented in SEQ ID NO: 1. SEQ ID NO: 29 presents the PCR product of the end enzyme PCR of LCHV as presented in SEQ ID NO: 1. SEQ ID NO: 30 presents a PCR product of the end enzyme PCR of LCHV obtained from a fish farm different from the indicator fish farm. It was concluded that the outbreak of the disease was caused by another LCHV strain.

圖1顯示表明已知異疱疹病毒科與新發現病毒之間的關係之系統樹。圖2顯示CPE之特徵為細胞圍聚，隨後細胞脫離/溶解，圖2A顯示培養瓶中呈90%匯合之SBB細胞之形態學，圖2B顯示呈50% CPE之SBB細胞之形態學。圖3顯示使用電子顯微鏡捕捉之LCHV病毒，在圖3A中可辨識兩種疱疹病毒(黑點)。比例尺係500 nm。圖3B顯示其中一個點之放大圖，清晰地顯示出二十面體輪廓線。Figure 1 shows a phylogenetic tree showing the relationship between the known herpesvirus family and the newly discovered virus. Figure 2 shows that CPE is characterized by cell encapsulation followed by cell detachment/dissolution, Figure 2A shows the morphology of SBB cells at 90% confluence in culture flasks, and Figure 2B shows morphology of SBB cells at 50% CPE. Figure 3 shows the LCHV virus captured using an electron microscope, and two herpes viruses (black spots) can be identified in Figure 3A. The scale is 500 nm. Figure 3B shows an enlarged view of one of the points, clearly showing the icosahedral outline.

FR法國；微生物培養國家保藏中心(Collection Nationale de Cultures de Micro-organismes；CNCM)；2016/07/28；CNCM I-5118FR France; Collection Nationale de Cultures de Micro-organismes (CNCM); 2016/07/28; CNCM I-5118

Claims

An isolated herpesvirus that is a member of the family Alloherpesviridae and whose wild-type form causes disease in Asian sea bass, which is characterized by the following signs: clinical signs are attacking After about 3 days of onset, systemic skin lesions caused darkening of the skin accompanied by pale spots and fin erosion, with no life accompanied by observed loss of swimming balance, lack of appetite, increased respiratory rate of the lid, and occurred about 2 weeks after the attack. death.

The isolated herpesvirus of claim 1, wherein the virus has a DNA that is at least 70% identical in length to DNA having a sequence according to SEQ ID NO: 1.

The herpesvirus according to claim 2, wherein at least 95% of the open reading frame in the DNA of the virus has at least 80% sequence identity to the corresponding open reading frame of the DNA having the sequence according to SEQ ID NO: 1. .

An isolated herpesvirus, a member of the Herpesviridae family, having at least one Collection Nationale de Cultures deposited with the deposit number CNCM I - 5118 at the Institute of Microbial Culture in France, Paris, Pasteur Institute De Microorganisms; CNCM) Identification of viruses.

The isolated herpesvirus of claim 4, wherein the identifying characteristic is selected from the group consisting of: the virus having a full length DNA sequence that is at least 70% identical to the sequence according to SEQ ID NO: 1; wild type of the virus The form causes disease in Asian sea otters, which is characterized by the following signs: clinical signs appear about 3 days after the attack, systemic skin lesions, leading to darkening of the skin accompanied by pale spots and fin erosion, with no life accompanied by observations Loss of swimming balance, lack of appetite, increased respiratory rate, and death about 2 weeks after challenge; the virus contains the Major Envelope Protein (MEP) gene, as in SEQ ID NO: 2 The nucleotide sequence shown has a level of identity of at least 80%; the virus comprises a dUTP enzyme gene having a level of identity of at least 80% with the nucleotide sequence as set forth in SEQ ID NO: 4; The terminal enzyme (Terminase) gene has a level of identity with the nucleotide sequence as set forth in SEQ ID NO: 6 of at least 80%; the virus comprises a polymerase gene, and the core as shown in SEQ ID NO: Glycosidic acid sequence Consistency level is at least 80%.

An isolated herpesvirus which is a member of the Herpesviridae family and whose wild-type form causes disease in Asian jellyfish, wherein the virus has a full length and at least 70% of the DNA having the sequence according to SEQ ID NO: 1. Consistent DNA.

An isolated herpesvirus that is a member of the Herpesviridae family and whose wild-type form causes disease in Asian jellyfish, wherein the virus has the following DNA: at least 95% of which are open reading frames and have an SEQ ID The corresponding open reading frame of the DNA of the sequence of NO: 1 has at least 80% sequence identity.

An isolated herpesvirus comprising a Major Envelope Protein (MEP) gene, wherein: a) the virus is a member of the herpesvirus family, b) the virus causes disease in Asian jellyfish, c) The nucleotide sequence of the MEP gene is at least 80% identical to the nucleotide sequence as set forth in SEQ ID NO: 2.

An isolated herpesvirus comprising a dUTP enzyme gene, wherein: a) the virus is a member of the herpesvirus family, b) the virus causes disease in Asian jellyfish, c) the nucleotide sequence of the dUTP enzyme gene The level of identity to the nucleotide sequence as set forth in SEQ ID NO: 4 is at least 80%.

The herpesvirus according to claim 8 or 9, wherein the virus comprises the main envelope protein gene and the dUTP enzyme gene, wherein the nucleotide sequence of the MEP gene and the core as shown in SEQ ID NO: 2 The level of identity of the nucleotide sequence is at least 80%, and the nucleotide sequence of the dUTP enzyme gene is at least 80% identical to the nucleotide sequence as shown in SEQ ID NO: 4.

An isolated herpesvirus comprising a Major Envelope Protein (MEP) gene, wherein: a) the virus is a member of the herpesvirus family, b) the virus causes disease in Asian jellyfish, c) The MEP gene was reacted in a PCR reaction with a primer set as shown in SEQ ID NO: 21 and SEQ ID NO: 22 to give a PCR product of 277 +/- 10 base pairs.

An isolated herpesvirus comprising a dUTP enzyme gene, wherein: a) the virus is a member of the herpesvirus family, b) the virus causes disease in Asian jellyfish, c) the dUTP enzyme gene is in a PCR reaction The primer set as shown in SEQ ID NO: 23 and SEQ ID NO: 24 was reacted to give a PCR product of 346 +/- 10 base pairs.

An isolated herpesvirus comprising a terminal enzyme gene, wherein: a) the virus is a member of the herpesvirus family, b) the virus causes disease in Asian jellyfish, c) the terminal enzyme gene is in a PCR reaction The primer set as shown in SEQ ID NO: 25 and SEQ ID NO: 26 was reacted to give a 585 +/- 10 base pair PCR product.

An isolated herpesvirus comprising a polymerase gene, wherein: a) the virus is a member of the herpesvirus family, b) the virus causes disease in Asian jellyfish, c) the polymerase gene is in a PCR reaction The primer set as shown in SEQ ID NO: 27 and SEQ ID NO: 28 was reacted to give a PCR product of 314 +/- 10 base pairs.

A DNA fragment comprising a gene encoding a major envelope protein, wherein the gene has a level of identity with a nucleotide sequence of the MEP gene as set forth in SEQ ID NO: 2 of at least 80%.

A major envelope protein wherein the MEP has an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 3.

A DNA fragment comprising a gene encoding a dUTP enzyme, wherein the gene has a level of identity with a nucleotide sequence of the dUTP enzyme gene as set forth in SEQ ID NO: 4 of at least 80%.

A dUTP enzyme wherein the dUTP enzyme has an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 5.

A DNA fragment having an open reading frame of at least 100 nucleotides in length, wherein the DNA has at least 80% sequence identity to an open reading frame having DNA according to the sequence of SEQ ID NO: 1.

A protein encoded by a DNA fragment as claimed in claim 19.

A cell culture comprising a replication competent virus, wherein the culture comprises the herpesvirus of any one of claims 1 to 14.

A vaccine for combating a herpes virus disease in a fish, wherein the vaccine comprises the herpesvirus of any one of claims 1 to 14, or the protein of any one of claims 16, 18 and 20, and a pharmaceutically Acceptable carrier.

The vaccine of claim 22, wherein the vaccine comprises the herpesvirus of any one of claims 1 to 14, wherein the herpesvirus is in a live attenuated or inactivated form.

The use of a herpesvirus according to any one of claims 1 to 14 or a protein according to any one of claims 16, 18 and 20 for the manufacture of a vaccine for the prophylactic treatment of an animal.

An antibody or antiserum which is reacted with a virus according to any one of claims 1 to 14.

A diagnostic test kit for detecting an antibody that reacts with the virus of any one of claims 1 to 14 or an antigenic substance thereof, wherein the test kit comprises the virus of any one of claims 1 to 14. Or its antigenic substance.

A diagnostic test kit for detecting a herpesvirus or an antigenic substance thereof according to any one of claims 1 to 14, wherein the test kit comprises the virus according to any one of claims 1 to 14 or An antibody that reacts with an antigenic substance, or a PCR primer set as defined in any one of claims 11 to 14.