Cyprinid herpesvirus 3 (CyHV‐3) was identified by Bretzinger, Fischer‐Scherl, Oumouma, Hoffmann, and Truyen (1999) and Hedrick et al. (2000) as the aetiological agent of a viral disease termed KHVD, which can cause mortality as high as 80%–100% in common carp Cyprinus carpio (Reviewed in Gotesman, Kattlun, Bergmann, & El‐Matbouli, 2013). CyHV‐3 is a double‐stranded DNA virus consisting of a 295 kB genome encoding164 putative open reading frames (ORFs), and mass spectrometry analysis of viral particles has identified 40 proteins packaged in a mature virion including 22 structural, 3 capsid, 2 tegument and 13 envelope proteins (Michel, Leroy, et al., 2010). Furthermore, the immunogenic and vaccine potentials of several epitopes of CyHV‐3 have been investigated, including Orf12 (Kattlun, Menanteau‐Ledouble, & El‐Matbouli, 2014) which is readily recognized by the immune system of carp and Orf81 for which conflicting evidence exists (Kattlun et al., 2016; Zhou et al., 2014), although little such research has been conducted in goldfish (Carassius auratus ). In previous reports, our group elucidated pathogen–host interactions in CyHV‐3‐infected C. auratus through the use of monoclonal antibody‐linked pulldown assay followed by electro‐spray ionization mass spectrometry (ESI‐MS) as described in Gotesman, Menanteau‐Ledouble, and El‐Matbouli (2016). C. auratus is a non‐symptomatic carrier of CyHV‐3, and previous studies demonstrated that in C. auratus , several host defence proteins interact with CyHV‐3 (Bergmann et al., 2010; Gotesman, Abd‐Elfattah, Kattlun, Soliman, & El‐Matbouli, 2014). Interestingly, several of these proteins were not found to interact in the common carp, the susceptible carp host for CyHV‐3 (Gotesman, Soliman, & El‐Matbouli, 2013). A recent study by Torrent et al. (2016) has demonstrated that the IgMs of asymptotic CyHV‐3 surviving carp recognize an epitope derived from the amino‐terminal of the glycoprotein coded by the ORF149 of CyHV‐3 (Orf149). Monoclonal antibodies (mAbs) were generated by immunizing mice with purified CyHV‐3 particles (Cabon et al., 2017), and these mAbs were used to detect the virus in common carp brain cells by enzyme‐linked immunosorbent assay (Bergmann et al., 2017).

Pulldown assays use antibodies to capture a “bait” protein in an affinity resin, and in the present study, we applied a pulldown assay to investigate the proteins interacting with the Orf149 epitope: Orf149 mAbs were linked to N‐hydroxysuccinimide (NHS)‐activated agarose columns (Gotesman et al., 2016) to capture and identify host proteins interacting with CyHV‐3. Kidney samples of C. auratus previously infected by intraperitoneal injection with 200 µl of CyHV‐3 virus at a concentration of 10⁴ TCID₅₀ (Kattlun et al., 2014, 2016) were lysed using a Tissue Lyser (Qiagen). The sample were resuspended in non‐denaturing buffer (Gotesman et al., 2014; Gotesman, Soliman, et al., 2013) and passed through the columns by gravity filtration to expose the extracted host proteins to the agarose‐linked mAbs. The columns were rinsed 8 times with phosphate‐buffered saline (PBS) to ensure that unbound extracts were washed away as measured by spectrophotometry₂₈₀ (OD = 0) and the bound proteins were eluted from the column using glycine (pH 3) into microcentrifuge tubes containing neutralizing Tris base (pH 8). The entire eluted fraction was analysed by liquid chromatography tandem mass spectrometry LC‐MS/MS analysis (performed at the VetMedUni VetCore facilities) to elucidate host proteins that putatively interact with Orf149.

The majority of the proteins identified (Table 1A) were identical to proteins previously identified in C. carpio (Gotesman, Soliman, et al., 2013) as well as using a different epitope of CyHV3 species (Gotesman et al., 2014), including cytoskeletal, elongation factors and enzymatic proteins (Table 1A). The cytoskeletal protein actin (Sandquist, Kita, & Bement, 2011), which was detected in both CyHV‐3‐positive and CyHV‐3‐negative samples, serves as a track for both conventional and unconventional myosins (Moen, Johnsrud, Thomas, & Titus, 2011) and plays a role in intracellular translocation and cell remodelling (Gotesman, Hosein, & Gavin, 2010, 2011).

Another protein identified was the eukaryotic elongation factor 1 alpha (eEF1A) which has a diverse set of functions in the cell including interactions with the cytoskeleton (reviewed in Sasikumar, Perez, & Kinzy, 2012). Interestingly, certain RNA viruses interact with eF1A directly to aid in viral replication (Sasikumar et al., 2012) and this could explain the recovery of cytoskeletal protein actin by the pulldown assay. Because eEF1A’s activity is hijacked for viral propagation, it is plausible that an antibody targeting the glycoprotein Orf149 that interacts with the cell membrane of the host protein would also detect this cytoskeletal protein. This explanation is further supported by the fact that eEF1A was also pulled down by this assay.

Myeloid protein 1 is the final member of previously identified proteins. It is no surprise that a haemoglobin protein was recovered in the CyHV‐3‐positive samples (Table 1A) because CyHV‐3 is detectable in various regions of the circulatory system (Reviewed by Michel, Fournier, Lieffrig, Costes, & Vanderplasschen, 2010) including the hematopoietic tissue in the spleen (Lee et al., 2016).

The results from this trial also suggested that the mAb was able to capture the same metalloendopeptidase (metalloendopeptidase 042326) in both the infected and non‐infected samples (Table 1B). More importantly, a unique, mitochondrial cytochrome c protein (mitochondrial cytochrome C X4Z1X5) was also detected in the CyHV‐3‐positive samples. Intriguingly, this protein had not been previously implicated in CyHV‐3 infections (Table 1C). In our previous study, we identified interactions of CyHV‐3 with mitochondrial enzymes involved in ATP synthesis (Gotesman et al., 2014; Gotesman, Soliman, et al., 2013), and in this study, another mitochondrial protein was shown to interact with CyHV‐3. Cytochrome c‐like metalloendopeptidase is known to coordinate with metal ions for correct functions and has implications in immune function and disease (Bond & Jiang, 1997), and it is interesting to speculate what role the mitochondria plays in CyHV‐3 infection. Whether CyHV‐3 alters the activity of the mitochondrial machinery to produce higher amounts of ATP (Murata et al., 2000) or modulates apoptosis factors (Cotter & Blaho, 2009) to either increase viral replication (Aubert, Pomeranz, & Blaho, 2007; Zhou & Roizman, 2000) or the release of mature viruses via apoptosis (Zhang, Tang, & Xu, 2014), respectively, remains unclear. Interestingly, some viruses such as spring viremia of carp virus (SVCV) are known to modulate ROS (reactive oxidative species) production (Liu et al., 2017; Shao et al., 2016). Antimycin A (a small molecule inhibitor of cellular respiration) is known to inhibit the mitochondrial complex III, reducing ROS production in SVCV‐infected cells and inhibiting the transcription of SVCV glycoprotein and viral replication (Zhao et al., 2018). Alternatively, CyHV‐3 may curtail the production or release of ROS by the mitochondria to reduce the cell's natural viral defence mechanism (Gonzalez‐Dosal, Horan, & Paludan, 2012; Gonzalez‐Dosal et al., 2011). Such viral strategies have been previously reported, for example, among the important viral diseases that affect domesticated poultry, the fusogenically activated F and HN glycoproteins of Newcastle disease perturb mitochondrial fusion/fission haemostasis (Ren et al., 2019). The fact that CyHV‐3 can putatively interact with this aforementioned enzyme and other mitochondrial components raises interesting questions regarding how this virus modulates the natural host immune response and mitochondria for increased viability.

Pulldown assays have demonstrated good specificity in the past, and, because of the extensive cleaning steps, it is unlikely that our assay would have detected proteins that did not interact with the Orf149 protein. Indeed, using a different bait protein resulted in a different set of purified proteins. This confirmed that interactions between bait and prey proteins were critical in the purification process. In future research, it would be interesting to further investigate the interactions of CyHV‐3 with host proteins in different species, for example using other immunoprecipitation methods such as co‐immunoprecipitation.

The aquamedicine field is rapidly adapting unconventional approaches for the detection, characterization and treatment of emergent threats to marine and aquaculture industries (Reviewed by Gotesman, Menanteau‐Ledouble, Saleh, Bergmann, & El‐Matbouli, 2018). Interactions with host cells are one of the most critical aspects of viral infections; therefore, such studies can greatly improve our understanding of the disease. Moreover, such studies could potentially suggest new therapeutic possibilities.

Bretzinger，Fischer-Scherl，Oumouma，Hoffmann和Truyen（1999）和Hedrick等人鉴定出了塞浦路斯疱疹病毒3（CyHV-3）。（2000年）是被称为KHVD的病毒性疾病的病因，它可以导致鲤鱼Cyprinus carpio的死亡率高达80％–100％（在Gotesman，Kattlun，Bergmann和El-Matbouli进行综述，2013年）。CyHV-3是一种双链DNA病毒，由295 kB基因组组成，编码164个推定的开放阅读框（ORF），并且对病毒颗粒的质谱分析已鉴定出40种包装在成熟病毒体中的蛋白质，包括22种结构，3个衣壳，2个铁皮和13种包膜蛋白（Michel，Leroy等，2010）。此外，已经研究了CyHV-3几个表位的免疫原性和疫苗潜力，包括Orf12（Kattlun，Menanteau-Ledouble和El-Matbouli，2014年），鲤鱼和Orf81的免疫系统很容易识别它们，这些证据相互矛盾。尽管金鱼（Carassius auratus）的研究很少（Kattlun et al。，2016 ; Zhou et al。，2014）。在以往的报告中，我们阐明组中CyHV-3感染的病原体-宿主相互作用鲫鱼通过使用单克隆抗体连接的下拉测定的随后电喷雾电离质谱（ESI-MS）在Gotesman，Menanteau-描述Ledouble和El‐Matbouli（2016）。鲫鱼是证明CyHV-3，和以前的研究的非对症载体，在鲫鱼，几个宿主防御蛋白相互作用与CyHV-3（Bergmann等人，。2010 ; Gotesman，阿卜杜勒Elfattah，Kattlun，索利曼，＆El‐Matbouli，2014）。有趣的是，在普通鲤鱼（CyHV-3的易感鲤鱼宿主）中未发现其中的几种蛋白相互作用（Gotesman，Soliman和El-Matbouli，2013年）。Torrent等人的最新研究。（2016年）证明了存活的渐近性CyHV-3鲤鱼的IgM识别出一个表位，该表位来自CyHV-3（ORf149）的ORF149编码的糖蛋白的氨基末端。单克隆抗体（mAb）是通过用纯化的CyHV-3颗粒免疫小鼠而产生的（Cabon等，2017），这些mAb通过酶联免疫吸附法用于检测鲤鱼脑细胞中的病毒（Bergmann等。 ，2017）。

下拉检测使用抗体捕获亲和树脂中的“诱饵”蛋白，在本研究中，我们应用了下拉检测来研究与Orf149表位相互作用的蛋白：Orf149 mAb与N-羟基琥珀酰亚胺（NHS）激活连接琼脂糖柱（Gotesman et al。，2016）捕获和鉴定与CyHV-3相互作用的宿主蛋白。的肾样品鲫鱼先前通过腹膜内注射用200μlCyHV-3病毒的以10:1的浓度感染⁴ TCID ₅₀（Kattlun等人，2014，2016），使用组织Lyser（Qiagen）中裂解。将样品重悬于非变性缓冲液中（Gotesman et al。，2014; Gotesman，Soliman，et al。，2013），并通过重力过滤穿过色谱柱，使提取的宿主蛋白暴露于琼脂糖连接的mAb。将色谱柱用磷酸盐缓冲盐水（PBS）冲洗8次，以确保通过分光光度法₂₈₀（OD = 0）测量将未结合的提取物洗去，并使用甘氨酸（pH 3）将结合的蛋白从色谱柱中洗脱到微量离心管中含有中和的Tris碱（pH 8）。通过液相色谱串联质谱LC-MS / MS分析（在VetMedUni VetCore设施中进行）分析了整个洗脱部分，以阐明可能与Orf149相互作用的宿主蛋白。

鉴定出的大多数蛋白质（表1A）与以前在鲤鱼中鉴定出的蛋白质相同（Gotesman，Soliman等，2013），并且使用了不同的CyHV3抗原决定簇（Gotesman等，2014），包括细胞骨架，延伸因子和酶蛋白（表1A）。在CyHV-3阳性和CyHV-3阴性样品中均检测到的细胞骨架蛋白肌动蛋白（Sandquist，Kita，＆Bement，2011），可作为常规和非常规肌球蛋白（Moen，Johnsrud，Thomas， ＆提图斯，2011），并起着细胞内转运和细胞重塑（Gotesman，Hosein，与加文，一个角色2010，2011）。

鉴定出的另一种蛋白质是真核延伸因子1α（eEF1A），其在细胞中具有多种功能，包括与细胞骨架的相互作用（综述于Sasikumar，Perez和Kinzy，2012）。有趣的是，某些RNA病毒直接与eF1A相互作用，以帮助病毒复制（Sasikumar等，2012），这可以通过下拉法解释细胞骨架蛋白肌动蛋白的恢复。由于eEF1A的活性被劫持用于病毒繁殖，因此，与宿主蛋白的细胞膜相互作用的靶向糖蛋白Orf149的抗体也可能检测到该细胞骨架蛋白。eEF1A也被该测定法下拉的事实进一步支持了该解释。

髓样蛋白质1是先前鉴定的蛋白质的最终成员。毫不奇怪的是，在CyHV-3阳性样品中回收了血红蛋白蛋白（表1A），因为在循环系统的各个区域都可以检测到CyHV-3（Michel，Fournier，Lieffrig，Costes和Vanderplasschen综述，2010年））包括脾中的造血组织（Lee et al。，2016）。

该试验的结果还表明，单克隆抗体能够在感染和未感染的样品中捕获相同的金属内肽酶（金属内肽酶042326）（表1B）。更重要的是，在CyHV-3阳性样品中也检测到独特的线粒体细胞色素c蛋白（线粒体细胞色素C X4Z1X5）。有趣的是，该蛋白先前并未涉及CyHV-3感染（表1C）。在我们之前的研究中，我们确定了CyHV-3与参与ATP合成的线粒体酶的相互作用（Gotesman等人，2014 ; Gotesman，Soliman等人，2013），并且在这项研究中，另一种线粒体蛋白被证明与CyHV-3相互作用。已知细胞色素c样金属内肽酶可与金属离子协同发挥功能，并影响免疫功能和疾病（Bond＆Jiang，1997年），推测线粒体在CyHV-3感染中起什么作用很有趣。CyHV-3是否改变线粒体机制以产生更高量的ATP（Murata等，2000）或调节细胞凋亡因子（Cotter＆Blaho，2009）以增加病毒复制（Aubert，Pomeranz，＆Blaho，2007） ; Zhou＆Roizman，2000）或通过凋亡释放成熟病毒（Zhang，Tang，＆Xu，2014）分别不清楚。有趣的是，已知一些病毒，例如鲤鱼病毒的春季病毒血症（SVCV）可以调节ROS（反应性氧化物种）的产生（Liu等人，2017年; Shao等人，2016年）。抗霉素A（一种细胞呼吸抑制的小分子抑制剂）可以抑制线粒体复合物III，减少感染SVCV的细胞中ROS的产生，并抑制SVCV糖蛋白的转录和病毒复制（Zhao等人，2018）。另外，CyHV-3可能会抑制线粒体产生或释放ROS，从而降低细胞的天然病毒防御机制（Gonzalez-Dosal，Horan和Paludan，2012； Gonzalez-Dosal等，2011）。）。这样的病毒策略先前已有报道，例如，在影响家禽的重要病毒疾病中，新城疫的融合激活的F和HN糖蛋白扰乱了线粒体融合/裂变止血作用（Ren等，2019）。CyHV-3可能与上述酶和其他线粒体成分相互作用的事实引起了有关该病毒如何调节天然宿主免疫反应和线粒体以提高生存力的有趣问题。

下拉测定法在过去已显示出良好的特异性，并且由于广泛的清洁步骤，我们的测定法不太可能检测到与Orf149蛋白不相互作用的蛋白质。实际上，使用不同的诱饵蛋白会产生不同的纯化蛋白集。这证实了诱饵和猎物蛋白之间的相互作用在纯化过程中至关重要。在未来的研究中，进一步研究CyHV-3与不同物种中宿主蛋白的相互作用将是有趣的，例如使用其他免疫沉淀方法（例如共免疫沉淀）。

水产医学领域正在迅速适应非常规方法，以检测，表征和处理对海洋和水产养殖业的紧急威胁（Gotesman，Menanteau‐Ledouble，Saleh，Bergmann和El‐Matbouli综述，2018年）。与宿主细胞的相互作用是病毒感染最关键的方面之一。因此，这样的研究可以大大提高我们对这种疾病的了解。而且，这样的研究可能潜在地提出新的治疗可能性。