Coexistence in Field Samples of Two Variants of the Infectious Salmon Anemia Virus: A Putative Shift to Pathogenicity

Genetic reassortment plays an important role in the evolution of several segmented RNA viruses and in the epidemiology of their associated diseases. In particular, orthomyxoviruses show rapid fluctuation in the proportion of viral variants coexisting in an infected individual, especially under strong selective pressure. This is particularly relevant in salmon production carried out under confined and stressful conditions where one of the most feared pathogenic agents is the Infectious Salmon Anemia Virus, an orthomyxovirus family member whose biological behavior is only recently beginning to be understood. Pathogenicity of the virus has been mainly associated with deletions of the HPR region in coding segment 6 and the presence or absence of a specific insertion in a key region in coding segment 5. In this study we report, for the first time in Chile, the coexistence of two variants in fully asymptomatic fish. Of five samples analyzed, two were identified as the non-pathogenic variant, HPR0, and two as the highly pathogenic HPR7b variant, though with no clinical signs detectable in the fish. Interestingly, one of the samples unequivocally carried both variants, again without any clinical signs. Considering that in none of the samples the typical insertion in coding segment 5 was detected, it is our impression that this may represent a shift from the non-pathogenic HPR0 variant towards the highly infective HPR7b variant. If this were the case, the transition may be triggered first by deleting the corresponding sequence of the HPR region of segment 6, followed by the putative insertion in segment 5 to generate a virulent strain.     

The Putative Polymerase Sequence of Infectious Salmon Anemia Virus Suggests a New Genus within the Orthomyxoviridae

The infectious salmon anemia virus (ISAV) is an orthomyxovirus-like virus infecting teleosts. The disease caused by this virus has had major economic consequences for the Atlantic salmon farming industry in Norway, Canada, and Scotland. In this work, we report the cloning and sequencing of an ISAV-specific cDNA comprising 2,245 bp with an open reading frame coding for a predicted protein with a calculated molecular weight of 80.5 kDa. The putative protein sequence shows the core polymerase motifs characteristic of all viral RNA-dependent RNA polymerases. Comparison of the conserved motifs with the corresponding regions of other segmented negative-stranded RNA viruses shows a closer relationship with members of the Orthomyxoviridae than with viruses in other families. The putative ISAV polymerase protein (PB1) has a length of 708 amino acids, a charge of +22 at neutral pH, and a pI of 9.9, which are consistent with the properties of the PB1 proteins of other members of the family. Calculations of the distances between the different PB1 proteins indicate that the ISAV is distantly related to the other members of the family but more closely related to the influenza viruses than to the Thogoto viruses. Based on these and previously published results, we propose that the ISAV comprises a new, fifth genus in the Orthomyxoviridae.     

Electron Microscopy of Equine Infectious Anemia Virus

Equine infectious anemia (EIA) virus was observed in thin sections of infected cultured horse leukocytes by electron microscopy. The virus particles had a spherical shape and were between 80 and 120 nm in diameter. Most of them contained an electron-dense nucleoid 40 to 60 nm in diameter. They were observed to form by a process of budding from the plasma membrane and appeared to have thin surface projections. The particles described were not detected in uninfected cultured cells, and their appearance could be prevented by adding EIA immune serum to the inoculum. The implications of these findings in the classification of EIA virus are discussed.     

拼接信号序列单碱基变异提高马传染性贫血病毒mRNA拼接效率

分别以马传染性贫血(马传贫)驴强毒(D—A EIAV)RNA和马传贫驴白细胞弱毒疫苗(DLA EIAV)RNA为模板,利用RT—PCR的方法,克隆到马传贫强、弱毒株基因组外显子2及其下游的核苷酸序列。然后将报告基因CAT插入到EIAV内含子2env阅读框架中,构成CAT拼接报告系统。同时在强毒株重组表达质粒的基础上,将其外显子-3上游拼接受体位点的核苷酸序列CAG突变为弱毒株相应位置的核苷酸序列TAG,得到强毒单核苷酸突变株重组表达质粒。用构建的3个重组表达质粒DNA转染驴血白细胞,ELISA检测转染细胞CAT浓度。结果表明：EIAV强毒株重组表达质粒中CAT蛋白表达量最高,EIAV强毒株重组表达质粒次之,EIAV强毒突变株重组表达质粒最低。由于CAT基因被插入于各重组质粒中的EIAV内含子-2里,EIAV外显子-2、3之间的拼接可导致该基因的删除,因而其拼接效率低于EIAVmRNA外显子-2、3之间的拼接效率。实验数据表明,EIAV SA2拼接信号序列单碱基变异提高了SD2-SA2拼接效率;D—AEIAV SA2-SD2拼接效率比DLA EIAV相应位点拼接效率高。     

Structure of Equine Infectious Anemia Virus Matrix Protein

The Gag polyprotein is key to the budding of retroviruses from host cells and is cleaved upon virion maturation, the N-terminal membrane-binding domain forming the matrix protein (MA). The 2.8-A resolution crystal structure of MA of equine infectious anemia virus (EIAV), a lentivirus, reveals that, despite showing no sequence similarity, more than half of the molecule can be superimposed on the MAs of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV). However, unlike the structures formed by HIV-1 and SIV MAs, the oligomerization state observed is not trimeric. We discuss the potential of this molecule for membrane binding in the light of conformational differences between EIAV MA and HIV or SIV MA.     

Development of a Reverse Genetic System for Infectious Salmon Anemia Virus: Rescue of Recombinant Fluorescent Virus by Using Salmon Internal Transcribed Spacer Region 1 as a Novel Promoter

Infectious salmon anemia (ISA) is a serious disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), belonging to the genus Isavirus, family Orthomyxoviridae. There is an urgent need to understand the virulence factors and pathogenic mechanisms of ISAV and to develop new vaccine approaches. Using a recombinant molecular biology approach, we report the development of a plasmid-based reverse genetic system for ISAV, which includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). Salmon cells cotransfected with pSS-URG-based vectors expressing the eight viral RNA segments and four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex allowed the generation of infectious recombinant ISAV (rISAV). We generated three recombinant viruses, wild-type rISAV^901_09 and rISAVr^S6-NotI-HPR containing a NotI restriction site and rISAV^S6/EGFP-HPR harboring the open reading frame of enhanced green fluorescent protein (EGFP), both within the highly polymorphic region (HPR) of segment 6. All rescued viruses showed replication activity and cytopathic effect in Atlantic salmon kidney-infected cells. The fluorescent recombinant viruses also showed a characteristic cytopathic effect in salmon cells, and the viruses replicated to a titer of 6.5 × 10⁵ PFU/ml, similar to that of the wild-type virus. This novel reverse genetics system offers a powerful tool to study the molecular biology of ISAV and to develop a new generation of ISAV vaccines to prevent and mitigate ISAV infection, which has had a profound effect on the salmon industry.     

Electrostatic Architecture of the Infectious Salmon Anemia Virus (ISAV) Core Fusion Protein Illustrates a Carboxyl-Carboxylate pH Sensor

Segment 5, ORF 1 of the infectious salmon anemia virus (ISAV) genome, encodes for the ISAV F protein, which is responsible for viral-host endosomal membrane fusion during a productive ISAV infection. The entry machinery of ISAV is composed of a complex of the ISAV F and ISAV hemagglutinin esterase (HE) proteins in an unknown stoichiometry prior to receptor engagement by ISAV HE. Following binding of the receptor to ISAV HE, dissociation of the ISAV F protein from HE, and subsequent endocytosis, the ISAV F protein resolves into a fusion-competent oligomeric state. Here, we present a 2.1 Å crystal structure of the fusion core of the ISAV F protein determined at low pH. This structure has allowed us to unambiguously demonstrate that the ISAV entry machinery exhibits typical class I viral fusion protein architecture. Furthermore, we have determined stabilizing factors that accommodate the pH-dependent mode of ISAV transmission, and our structure has allowed the identification of a central coil that is conserved across numerous and varied post-fusion viral glycoprotein structures. We then discuss a mechanistic model of ISAV fusion that parallels the paramyxoviral class I fusion strategy wherein attachment and fusion are relegated to separate proteins in a similar fashion to ISAV fusion.     

Localised Infection of Atlantic Salmon Epithelial Cells by HPR0 Infectious Salmon Anaemia Virus

Infectious salmon anaemia (ISA) is an important, systemic viral disease of farmed Atlantic salmon, Salmo salar L. Endothelial cells are the main target cells for highly virulent HPR-deleted ISA virus (ISAV) types. Here we examine the pathogenesis of non-virulent ISAV HPR0 infections, presenting evidence of an epithelial tropism for this virus type, including actual infection and replication in the epithelial cells. Whereas all HPR0 RT-qPCR positive gills prepared for cryosection tested positive by immunohistochemistry (IHC) and immunofluorescent labelling, only 21% of HPR0 RT-qPCR positive formalin-fixed paraffin-embedded gills were IHC positive, suggesting different methodological sensitivities. Only specific epithelial cell staining was observed and no staining was observed in endothelial cells of positive gills. Furthermore, using an ISAV segment 7 RT-PCR assay, we demonstrated splicing of HPR0, suggesting initial activation of the replication machinery in the epithelial gill cells. Immunological responses were investigated by the expression of interferon-related genes (e.g. Mx and γIP) and by ELISA for presence of anti-ISAV antibodies on samples taken sequentially over several months during an episode of transient HPR0 infection. All fish revealed a variable, but increased expression of the immunological markers in comparison to normal healthy fish. Taken together, we conclude that HPR0 causes a localized epithelial infection of Atlantic salmon.     

High-Efficiency Rescue of Equine Infectious Anemia Virus from a CMV-Driven Infectious Clone

<正>Dear Editor,Equine infectious anemia virus (EIAV) belongs to the macrophage-tropic lentiviruses family and infects mainly equines, including horses, donkeys and mules. EIAV shares many similar characteristics in its viral biology and hostvirus immune regulation with other lentiviruses, such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), and has been accepted as a     

感染性马传染性贫血病毒嵌合克隆的构建

在已有的全长感染性克隆pFD3的基础上,构建了新的低拷贝的全长克隆pLGFD3-8。按照疫苗制备过程中env基因的变化情况,采用基因替换和定点突变的方法,构建了一系列具有马传染性贫血病毒(EIAV)强毒株env基因及其主要突变特征的嵌合克隆。利用这些克隆转染FDD细胞,并用逆转录酶活性检测和PCR方法确定其感染性。结果发现,在FDD细胞中传代3次后,可在细胞培养物中检测到逆转录酶活性和原病毒DNA的存在,在电镜下可以观察到典型的EIAV病毒颗粒。这一结果为进一步研究马传染性贫血病毒致病的分子机制和免疫保护机理奠定了良好的基础。     

Biological Characterization of Rev Variation in Equine Infectious Anemia Virus

Sequence analysis identified significant variation in the second exon of equine infectious anemia virus (EIAV) rev. Functional analysis indicated that limited amino acid variation in Rev significantly altered the export activity of the protein but did not affect Rev-dependent alternative splicing. EIAV Rev can mediate export through two independent cis-acting Rev-responsive elements (RREs), and differences among Rev variants were more pronounced when both RREs were present. Variation in Rev may be an important mechanism for regulation of virus replication in vivo and may contribute to changes in clinical disease.     

马传染性贫血病毒基因组部分转录图谱

以马传贫驴白细胞弱毒和驴强毒分别接种培养细胞和试验动物,分别在体外和体内条件,对病毒的各种转录产物进行克隆和序列分析。通过与病毒基因组的序列进行比较,用实验的方法绘制出了两株病毒在相应试验条件件下的转录图谱,确定了各拼接产物的外显子构成以及拼接供体和拼接受体位点的具体位置。发现两种病毒在外显子－3的上游拼接受体位点（SA2）的位置与已报道的EIAV毒株均不相同,且二者的拼接信号不一致。     

马传染性贫血病毒N-连接糖基化回复突变的感染性克隆构建

根据马传贫强毒株EIAV-L和疫苗株EIAV-FDD表面蛋白gp90的N-连接糖基化的变化规律,采用PCR定点突变的方法,对全长感染性克隆pLGFD3-8上的N-连接糖基化的差异区域进行改造后,构建成含有3个N-连接糖基化位点突变的感染性克隆pLGNl91N236N246.将其转染驴胎皮肤细胞(FDD),通过用逆转录酶活性、间接免疫荧光和RT-PCR方法检测而确定其感染性.结果表明,在FDD细胞中盲传三代后,在细胞培养物中可检测到逆转录酶活性,RT-PCR和间接免疫荧光检测均呈阳性,电镜下见到典型的EIAV颗粒.这一结果可能对N-连接糖基化在我国马传贫弱毒疫苗致弱机理的作用研究而奠定良好的基础.     

马传染性贫血病毒非编码区LTR嵌合克隆的生物学特性

将已构建的马传染性贫血病毒LTR强弱毒嵌合克隆衍生毒vLGFD9-12体内接种健康试验马,在150d观察期内,各组试验动物体症均未见异常。血液学分析发现,vLGFD9-12嵌合克隆衍生病毒与亲本弱毒疫苗株的白细胞与血红蛋白含量总体上没有明显的规律性的变化。在动物外周血中均检测到一定的病毒RNA拷贝数,但拷贝数较低。二者在诱导EIAV特异性淋巴细胞增殖功能和特异性细胞毒性杀伤反应中,亦具有相似的变化趋势和效应。本项研究为进一步确定我国马传贫弱毒疫苗株毒力致弱及免疫保护的分子机制奠定了重要的分子生物学基础。     

马传染性贫血病毒非编码区LTR嵌合克隆的生物学特性

将已构建的马传染性贫血病毒LTR强弱毒嵌合克隆衍生毒vLGFD9-12体内接种健康试验马,在150d观察期内,各组试验动物体症均未见异常.血液学分析发现,vLGFD9-12嵌合克隆衍生病毒与亲本弱毒疫苗株的白细胞与血红蛋白含量总体上没有明显的规律性的变化.在动物外周血中均检测到一定的病毒RNA拷贝数,但拷贝数较低.二者在诱导EIAV特异性淋巴细胞增殖功能和特异性细胞毒性杀伤反应中,亦具有相似的变化趋势和效应.本项研究为进一步确定我国马传贫弱毒疫苗株毒力致弱及免疫保护的分子机制奠定了重要的分子生物学基础.     

马传贫病毒主要基因变异研究进展

马传贫病毒是反转录病毒科慢病毒属的成员之一,它在体内要通过反转录酶的作用合成DNA,整合到宿主的基因组中进行复制,由于反转录酶没有校正功能,使病毒在体内复制过程中错误拷贝RNA基因组,导致高频率的遗传变异。近年国内外学者对EIAV的研究发现变异的基因主要集中在env、gag、LTR和S2等区域,这些区域基因的变异与病毒的毒力、病毒在体内的复制水平及免疫原性密切相关。由于EIAV是慢病毒中最简单的病毒,它具有独特的发病进程和明显的病程分界,使其成为研究包括HIV内其它慢病毒基因变异与临床症状之间相互关系的理想动物模型。因此对EIAV基因变异情况的研究具有重要意义。     

Epithelial Cadherin Determines Resistance to Infectious Pancreatic Necrosis Virus in Atlantic Salmon

Infectious pancreatic necrosis virus (IPNV) is the cause of one of the most prevalent diseases in farmed Atlantic salmon (Salmo salar). A quantitative trait locus (QTL) has been found to be responsible for most of the genetic variation in resistance to the virus. Here we describe how a linkage disequilibrium-based test for deducing the QTL allele was developed, and how it was used to produce IPN-resistant salmon, leading to a 75% decrease in the number of IPN outbreaks in the salmon farming industry. Furthermore, we describe how whole-genome sequencing of individuals with deduced QTL genotypes was used to map the QTL down to a region containing an epithelial cadherin (cdh1) gene. In a coimmunoprecipitation assay, the Cdh1 protein was found to bind to IPNV virions, strongly indicating that the protein is part of the machinery used by the virus for internalization. Immunofluorescence revealed that the virus colocalizes with IPNV in the endosomes of homozygous susceptible individuals but not in the endosomes of homozygous resistant individuals. A putative causal single nucleotide polymorphism was found within the full-length cdh1 gene, in phase with the QTL in all observed haplotypes except one; the absence of a single, all-explaining DNA polymorphism indicates that an additional causative polymorphism may contribute to the observed QTL genotype patterns. Cdh1 has earlier been shown to be necessary for the internalization of certain bacteria and fungi, but this is the first time the protein is implicated in internalization of a virus.     

Disease Induction by Virus Derived from Molecular Clones of Equine Infectious Anemia Virus

Equine infectious anemia virus (EIAV), a macrophage-tropic lentivirus, causes persistent infections of horses. A number of biologic features, including the rapid development of acute disease, the episodic nature of chronic disease, the propensity for viral genetic variation, and the ability for many infected animals to eventually control virus replication, render EIAV a potentially useful model system for the testing of antiretroviral therapies and vaccine strategies. The utility of the EIAV system has been hampered by the lack of proviral clones that encode promptly pathogenic viral stocks. In this report, we describe the generation and characterization of two infectious molecular clones capable of causing acute clinical syndromes similar to those seen in natural infections. Virus derived from clone p19/wenv17 caused severe debilitating disease at 5 to 7 days postinfection; initial febrile episodes were fatal in two of three infected animals. Virus derived from a second clone, p19/wenv16, caused somewhat milder primary febrile episodes by 10 to 12 days postinfection in two of two infected animals. Virus derived from both clones caused persistent infections such that some animals exhibited chronic equine infectious anemia, characterized by multiple disease episodes. The two virulent clones differ in envelope and rev sequences.