马疱疹病毒1型主要毒力基因分析及TK基因缺失载体的构建

为了解新疆马疱疹病毒1型（EHV-1）主要毒力基因遗传进化情况并构建TK基因缺失株，本研究以EHV-1 XJ2015株DNA为模板，对其主要毒力基因TK、gI和gE全长进行克隆、测序及生物信息学分析，并扩增TK基因左右重组臂TKL和TKR，构建质粒pUC-TKLR，将扩增后的增强绿色荧光蛋白（EGFP，含有CMV+polyA）插入pUC-TKLR质粒，构建TK基因缺失打靶质粒。TK、gI和gE基因同源性分析结果显示，XJ2015株与国外EHV-1分离株TK、gI和gE基因同源性均较高，分别为99.8%~100.0%、99.6%~100.0%和99.9%~100.0%；与EHV-3分离株同源性均最低，分别为72.9%、59.4%和62.1%；遗传进化分析显示，3个基因均与国外EHV-1同属于一个遗传进化分支，与EHV-9和EHV-4进化关系较近，但与EHV-3进化关系较远，表明XJ2015毒株与国外EHV-1毒株TK、gI、gE基因核苷酸上差异不明显，没有明显的地域性特征，功能基因保守且进化缓慢，同源基因功能相同或相近；经PCR扩增、酶切、测序及转染鉴定，本试验成功构建了用于TK基因缺失的打靶质粒pUC-TKLR-EGFP。通过对EHV-1主要毒力基因的分析及TK基因缺失打靶载体的构建，为新疆地区马鼻肺炎流行病学调查分析、TK基因缺失株的构建提供理论依据。     

Cyprinid herpesvirus 3 as a potential biological control agent for carp (Cyprinus carpio) in Australia: susceptibility of non‐target species

Carp (Cyprinus carpio L.) is a pest species in Australian waterways, and cyprinid herpesvirus 3 (CyHV‐3) is being considered as a potential biological control (biocontrol) agent. An important consideration for any such agent is its target specificity. In this study, the susceptibility to CyHV‐3 of a range of non‐target species (NTS) was tested. The NTS were as follows: 13 native Australian, and one introduced, fish species; a lamprey species; a crustacean; two native amphibian species (tadpole and mature stages); two native reptilian species; chickens; and laboratory mice. Animals were exposed to 100–1000 times the approximate minimum amount of CyHV‐3 required to cause disease in carp by intraperitoneal and/or bath challenge, and then examined clinically each day over the course of 28 days post‐challenge. There were no clinical signs, mortalities or histological evidence consistent with a viral infection in a wide taxonomic range of NTS. Furthermore, there was no molecular evidence of infection with CyHV‐3, and, in particular, all RT‐PCRs for viral mRNA were negative. As a consequence, the results encourage further investigation of CyHV‐3 as a potential biocontrol agent that is specific for carp.     

Can water disinfection prevent the transmission of infectious koi herpesvirus to naïve carp? – a case report

Hygienic measures such as disinfection are important tools for the maintenance of fish health in aquaculture. While little information is available on the disinfection of water intended for fish containment, Huwa‐San^®, a disinfectant used in food and water industries, was used for daily treatment at concentrations of approximately 60 ppm over a total period of 3 months (experiment 1) with a 3‐week treatment‐free interval after 2 months (experiment 2). During this period, koi herpesvirus (KHV) was added to the water of two aquaria, one used as a normal contact control, the other one receiving daily water disinfectant treatments that prevented KHV infection of carp. In the second experiment, Huwa‐San^® treatment was interrupted and KHV infection was prevalent. However, when naïve fish were introduced to the same aquarium after re‐application of disinfectant, KHV could not be detected in those naïve fish. Whilst KHV could not be detected in samples where disinfectant had been applied, it was present in samples of naïve fish cohabiting with infection contact control animals which had undergone no disinfectant treatment over experiments 1 and 2. The results presented here show that water treatment with a disinfectant may prevent transmission of infectious KHV to naïve carp cohabited with infected carp.     

Koi herpesvirus and carp edema virus threaten common carp aquaculture in Croatia

Common carp (Cyprinus carpio) is a very important fish species for warm-water aquaculture in Croatia. All Croatian carp farms are subjected to a surveillance programme for the presence of koi herpesvirus (KHV), causing a deadly disease called koi herpesvirus disease (KHVD). However, there is no surveillance for other viral pathogens of importance like carp edema virus (CEV), a causative agent of koi sleepy disease (KSD). During regular testing within the KHVD surveillance programme, we tested samples for CEV simultaneously. The screening indicated possible outbreaks of KHVD and KSD. During 2016, KHVD broke out in an isolated area and soon thereafter a KHV eradication programme was successfully performed. However, during 2018 and 2019, two additional mortality events occurred in lakes in the southern part of Croatia during the spring. Samples from both events tested positive for CEV. An epidemiological investigation confirmed the introduction of infected carps from an infected farm to one of the lakes. To prevent the spreading of CEV into open waters, it is of utmost importance to introduce CEV testing before fish movement or to perform regular testing of all carp farms in the country to determine CEV prevalence for the purpose of implementation of control measures.     

Virucidal effects of various agents—including protease—against koi herpesvirus and viral haemorrhagic septicaemia virus

In a search for alternative, environmentally friendly and effective disinfecting agents, a commercially available protease—Neutrase^®—was tested in this work for inactivation of koi herpesvirus (KHV) and of viral haemorrhagic septicaemia virus (VHSV). For comparison, the stability of these viral pathogens in similar configurations at various pH values and concentrations of peracetic acid or quicklime, typically used for disinfection, was tested. Therefore, virus suspensions were incubated with various concentrations of different agents for 24 hr and the titre of the remaining infectious particles was determined by virus titration. Furthermore, the treatment of both viruses, with the agents at concentrations that were previously appointed as effective, was also examined in the presence of solid material (quartz sand). All procedures investigated in this study, including the protease treatment, were able to reduce the titre of KHV and VHSV below the detection limit of the titration. Although further studies are necessary, this is the first report of the application of a protease for the inactivation of the selected fish pathogens, demonstrating the great potential of the latter for disinfection.     

Antiviral activity of exopolysaccharides from Arthrospira platensis against koi herpesvirus

Although koi herpesvirus (KHV) has a history of causing severe economic losses in common carp and koi farms, there are still no treatments available on the market. Thus, the aim of this study was to test exopolysaccharides (EPS) for its antiviral activity against KHV, by monitoring inhibition and cytotoxic effects in common carp brain cells. These substances can be easily extracted from extracellular algae supernatant and were identified as groups of sulphated polysaccharides. In order to reach this aim, Arthrospira platensis, which is well known for its antiviral activity of intra‐ and extracellular compounds towards mammalian herpesviruses, was investigated as standard organism and compared to commercial antiviral drug, ganciclovir, which inhibits the viral DNA polymerization. The antiviral activity of polysaccharides of A. platensis against KHV was confirmed in vitro using qualitative assessment of KHV life cycle genes, and it was found by RT‐PCR that EPS, applied at a concentration of >18 μg mL^?1 and a multiplicity of infection (MOI) of 0.45 of KHV, suppressed the viral replication in common carp brain (CCB) cells even after 22 days post‐infection, entirely. Further, this study presents first data indicating an enormous potential using polysaccharides as an additive for aquacultures to lower or hinder the spread of the KHV and koi herpesvirus disease (KHVD) in future.     

黑龙江地区鲤疱疹病毒3型的流行病学调查及其主要囊膜蛋白的抗原表位预测

2015-2016年对来自黑龙江地区46个养殖场送检的鲤鱼进行锦鲤疱疹病毒(Koi herpes virus,KHV)的分离鉴定,并应用DNAStar等软件对检出的阳性样本的主要囊膜蛋白(Major envelop protein,MEP)基因进行生物信息学分析。结果显示,KHV的检出率约为6.5%,3株阳性样本具有相同的MEP基因序列,称之为KHV-HLJ1516。MEP基因开放阅读框为771 bp,编码256个氨基酸,相对分子质量为28 280.58,等电点为8.40。对KHV-HLJ1516的MEP基因序列同源性分析表明,其与KHV-GZ11分离株仅有1个碱基不同,但并未导致氨基酸发生改变,碱基相似性为99%;而与HZ419分离株相比,KHV-HLJ1516的MEP基因有3个碱基发生突变,相应的氨基酸由Asn变为IIe。聚类分析结果显示,KHV-HLJ1516与KHV-GZ11处于同一分支,而HZ419位于另一分支。B细胞抗原表位预测结果显示,KHV-HLJ1516的MEP的抗原表位主要位于4~11,24~40,42~63,82~110,209~218,238~249氨基酸区段。     

表达鸡传染性法氏囊病病毒VP2基因的重组火鸡疱疹病毒的构建及生物学特性分析

鸡传染性法氏囊病（IBD）是一种严重危害养禽业的高度致死性和免疫抑制性传染病。为研制IBD重组火鸡疱疹病毒（HVT）活载体疫苗,本研究构建了表达鸡传染性法氏囊病病毒（IBDV）保护性抗原VP2基因的重组HVT并对其体外生物学特性进行了分析。通过RT-PCR扩增IBDV超强毒株VP2基因并克隆入pCI载体,获得重组真核表达质粒pCI-VP2。用限制性内切酶将携带CMV启动子的VP2基因表达框架切下,连接于入门质粒pENTR,构建获得重组入门质粒pENTR-VP2。将pENTR-VP2与HVT重组黏粒H3-Kan/ccdB进行LR重组反应,构建重组表达黏粒H3-VP2。用H3-VP2与其他4个相互重叠并覆盖HVT全基因组的黏粒共同转染鸡胚成纤维细胞（CEF）,拯救获得重组病毒rHVT-VP2。将重组病毒在CEF中连续传至20代后用PCR、间接免疫荧光试验和免疫印迹试验进行检测,并绘制重组病毒体外生长曲线,分析其体外复制特性。结果表明,重组病毒rHVT-VP2能够稳定表达VP2蛋白,rHVT-VP2在CEF中的复制能力与亲本病毒无明显差异。重组病毒rHVT-VP2免疫鸡后能够诱导产生IBDV中和抗体,并对IBDV强毒株攻击引起的死亡提供90%免疫保护。重组病毒rHVT-VP2的构建为研制IBD重组HVT活载体疫苗奠定了基础,对IBD的防控具有重要意义。