基于原位LAMP技术的牡蛎疱疹病毒(OsHV-1)易感宿主调查

Survey of Ostreid herpesvirus 1 (OsHV-1) susceptible hosts based on in situ LAMP technique

中文关键词: 牡蛎疱疹病毒  原位LAMP  易感性  流行病学调查

英文关键词: Ostreid herpesvirus 1  In situ LAMP  Susceptibility  Epidemiological study

中文摘要:

      牡蛎疱疹病毒(Ostreid herpesvirus 1, OsHV-1)给世界双壳贝类养殖业造成了严重的经济损失。10余种双壳贝类陆续被认定为易感宿主，仍有其他几种贝类仅有PCR核酸阳性数据，因确诊证据不足导致其易感性未得到充分评估。原位环介导等温核酸扩增(LAMP)检测技术相对传统原位杂交技术具有灵敏度高、方便快捷、可作为病原微生物感染证据的优点。为了在OsHV-1流行病学调查过程中实现病毒感染的快速检测和确诊，根据已报道的OsHV-1特异性LAMP检测引物，设计内引物，优化反应条件，建立了OsHV-1的原位LAMP检测方法。基于该方法对2019年以来采集的长牡蛎(Crassostrea gigas)、福建牡蛎(Crassostrea angulata)、栉孔扇贝(Chlamys farreri)、虾夷扇贝(Mizuhopecten yessoensis)、毛蚶(Scapharca subcrenata)和菲律宾蛤仔(Ruditapes philippinarum)样本进行检测。结果显示，毛蚶样本的OsHV-1原位LAMP检测结果呈阳性；其他几种贝类部分样本的实时定量PCR (qPCR)检测呈阳性，但原位LAMP检测呈阴性。对毛蚶样本的原位LAMP检测结果分析发现，病毒杂交信号主要分布在外套膜和肝胰腺等器官的结缔组织，推测感染的细胞为成纤维细胞和血淋巴细胞；在闭壳肌和斧足肌肉组织的肌细胞细胞核中也发现较多杂交信号。鳃丝内和周边偶现阳性信号，推测来自渗出的血淋巴细胞。基于原位LAMP技术的OsHV-1检测结果显示，毛蚶是OsHV-1的一种易感宿主，毛蚶结缔组织、肌肉组织和血淋巴细胞对该病毒有强亲嗜性。

英文摘要:

      Ostreid herpesvirus 1 (OsHV-1) has caused a serious economic loss to the global bivalve aquaculture industry. Given its wide host range and the frequent emergence of mutated strains, OsHV-1 infection threatens mollusk production. Unlike the common vertebrate herpesviruses that generally exhibit high host specificity, more than ten bivalve species have been identified as potentially susceptible to OsHV-1 infection, including members of Ostreoida, Pterioida, Arcoida, and Veneroida. A variety of OsHV-1 detection methods have been developed, such as PCR, in situ hybridization, in situ PCR, histopathology, transmission electron microscopy, ring-mediated isothermal nucleic acid amplification (LAMP), and recombinase polymerase amplification (RPA). The sensitivity and specificity of transmission electron microscopy detection are low, and specific complementary detection methods such as PCR are needed to confirm the occurrence of OsHV-1. In situ hybridization, although highly specific, has the disadvantages of low sensitivity, complicated procedures, and high investment of effort. PCR methods are still the most widely used for epidemiological investigation of OsHV-1. However, PCR cannot alone confirm infection, which has led to the emergence of unconfirmed cases of OsHV-1 infection in many species and regions. According to the World Organization for Animal Health´s (OIE) Aquatic Animal Manual, positive nucleic acid-specific detection combined with histopathology and transmission electron microscopy is required for the confirmed diagnosis of OsHV-1 infection. Other detection methods, such as LAMP and RPA, which use nucleic acid amplification alone to detect OsHV-1, can not confirm infection as the presence of nucleic acids is not equivalent to viral infection. As a result, the susceptibility of several bivalve species to OsHV-1 infection has not been evaluated. Instead, several obstacles remain with regards to the development of epidemiological surveillance programs and the implementation of quarantine, prevention, and control measures for OsHV-1 infection. Compared with traditional in situ hybridization and in situ PCR assays, in situ LAMP has the advantages of low nucleic acid amplification reaction temperature, less damage to tissue, constant temperature amplification that does not require special experimental equipment, and more convenient and efficient experimental design. In this study, we selected a set of LAMP primers designed for specific detection of OsHV-1, and a pair of loop primers were designed to improve the specificity and stability of the LAMP reaction on slides. An optimized in situ LAMP method for OsHV-1 detection was developed, which provides a rapid diagnostic method with high specificity and sensitivity to identify hosts that are potentially susceptible to OsHV-1 infection, and to characterize the distribution pattern and tissue affinity of the virus in new hosts. The optimized protocol and quantitative PCR (qPCR) were then used to detect OsHV-1 infection in bivalve samples (Crassostrea gigas, C. angulata, Chlamys farreri, Mizuhopecten yessoensis, Ruditapes philippinarum, and Scapharca subcrenata) collected from 2019 to 2021. OsHV-1 hybridization signals were observed in S. subcrenata samples alone, although positive qPCR results were obtained in more species. Further investigation of pathological characteristics and associated viral hybridization signals indicated that OsHV-1 infection always occurred in fibroblasts and hemocytes in the connective tissues of the mantle and hepatopancreas, nucleus of muscle cells in the foot, and adductor muscle. Signals were also occasionally observed in infiltrated hemocytes between and within the gill filaments. OsHV-1 hybridization signals were observed within the hemocytes infiltrating several organs of the furcula visceral cluster. The hemocytes of S. subcrenata seem to be particularly susceptible to OsHV-1 infection. Histopathological lesions and viral hybridization signals in the hepatopancreas organ were consistently observed in individuals with clinical signs, and we recommend the hepatopancreas as the preferred target organ for histopathological and in situ LAMP assays for OsHV-1 infection in S. subcrenata. In situ LAMP-based detection indicated that S. subcrenata was a susceptible host for OsHV-1, and the connective tissue, muscle tissue, and hemolymph cells had a strong affinity for the virus. At present, the susceptibility of many cultured and wild shellfish to OsHV-1 infection is still unknown. Since the emergence of mollusk mortalities associated with OsHV-1 infection occurred in China in the 1990s, its host range has expanded and changed along with the environment, which has complicated its prevention and control. In recent years, the scale of artificial breeding and cultivation of triploid C. gigas has expanded in China, and larval mortalities are frequently associated with OsHV-1 infection. The in situ LAMP detection method for OsHV-1 infection developed in this study proved to be convenient and fast and could be a valuable tool for the rapid detection and confirmation of OsHV-1 infection.