Immunity induced by recombinant attenuated IHNV (infectious hematopoietic necrosis virus)‐GN438A expresses VP2 gene‐encoded IPNV (infectious pancreatic necrosis virus) against both pathogens in rainbow trout

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are important pathogens in rainbow trout farming worldwide. Their co‐infection is also common, which causes great economic loss in juvenile salmon species. Development of a universal virus vaccine providing broadly cross‐protective immunity will be of great importance. In this study, we generated two recombinant (r) virus (rIHNV‐N438A‐ΔNV‐EGFP and rIHNV‐N438A‐ΔNV‐VP2) replacing the NV gene of the backbone of rIHNV at the single point mutation at residue 438 with an efficient green fluorescent protein (EGFP) reporter gene and antigenic VP2 gene of IPNV. Meanwhile, we tested their efficacy against the wild‐type (wt) IHNV HLJ‐09 virus and IPNV serotype Sp virus challenge. The relative per cent survival rates of two recombinant viruses against (wt) IHNV HLJ‐09 virus challenge were 84.6% and 81.5%, respectively. Simultaneously, the relative per cent survival rate of rIHNV‐N438A‐ΔNV‐VP2 against IPNV serotype Sp virus challenge was 88.9%. It showed the two recombinant viruses had high protection rates and induced a high level of antibodies against IHNV or IPNV. Taken together, these results suggest the VP2 gene of IPNV can act as candidate gene for vaccine and attenuated multivalent live vaccines and molecular marker vaccines have potential application for viral vaccine.     

Oral DNA vaccination of rainbow trout, Oncorhynchus mykiss (Walbaum), against infectious haematopoietic necrosis virus using PLGA [Poly(D,L-Lactic-Co-Glycolic Acid)] nanoparticles

A DNA vaccine against infectious haematopoietic necrosis virus (IHNV) is effective at protecting rainbow trout, Oncorhynchus mykiss, against disease, but intramuscular injection is required and makes the vaccine impractical for use in the freshwater rainbow trout farming industry. Poly (D,L-lactic-co-glycolic acid) (PLGA) is a U.S. Food and Drug Administration (FDA) approved polymer that can be used to deliver DNA vaccines. We evaluated the in vivo absorption of PLGA nanoparticles containing coumarin-6 when added to a fish food pellet. We demonstrated that rainbow trout will eat PLGA nanoparticle coated feed and that these nanoparticles can be detected in the epithelial cells of the lower intestine within 96 h after feeding. We also detected low levels of gene expression and anti-IHNV neutralizing antibodies when fish were fed or intubated with PLGA nanoparticles containing IHNV G gene plasmid. A virus challenge evaluation suggested a slight increase in survival at 6 weeks post-vaccination in fish that received a high dose of the oral vaccine, but there was no difference when additional fish were challenged at 10 weeks post-vaccination. The results of this study suggest that it is possible to induce an immune response using an orally delivered DNA vaccine, but the current system needs improvement.     

传染性造血器官坏死病核酸疫苗的构建及其在虹鳟接种部位的消长规律

本研究将传染性造血器官坏死病病毒(infectious hematopoietic necrosis virus,IHNV)分离株SD-12糖蛋白(glycoprotein,G)基因克隆进商业化载体pc DNA3.1(+),构建了IHNV G的表达载体,即传染性造血器官坏死病(infectious hematopoietic necrosis,IHN)核酸疫苗,命名为p IHNsd-G。采用背鳍基部肌肉注射的方式,以2μg/尾的剂量免疫虹鳟(Oncorhynchus mykiss)鱼苗(5.0±0.5)g。于免疫后第4天及第7天,利用real-time PCR技术检测免疫虹鳟头肾及接种部位肌肉组织Mx-1基因表达情况;于免疫后第21天,以100倍半数组织培养感染剂量(tissue culture infective dose,TCID50)采取腹腔注射的方式进行攻毒实验,计算核酸疫苗相对保护率(relative percent survival,RPS);于免疫后第60天及150天检测免疫虹鳟血清IHNV中和抗体效价;最后,以p IHNsd-G的启动子序列和氨苄青霉素抗性基因序列为目标基因,利用PCR方法监测p IHNsd-G在免疫虹鳟接种部位的动态分布情况。结果显示:Mx-1基因在头肾和接种部位肌肉中均显著上调表达,并且在接种部位肌肉组织中明显高于同一时间点的头肾组织;攻毒实验中p IHNsd-G对虹鳟的相对保护率高达94.4%;而在免疫后第60天,所有免疫虹鳟血清中均存在中和抗体,其最高效价高达320,在免疫后第150天,最高抗体效价为80,自此,说明已成功获得有效的IHN核酸疫苗。p IHNsd-G在虹鳟接种部位的PCR监测结果显示:在免疫后的第1天即可在注射部位的肌肉中检测到全部p IHNsd-G目标片段,在第84天时已经无法从注射部位肌肉中扩增出全长氨苄青霉素抗性基因,而所有目标基因在第150天时均消失不见。本研究在成功构建IHN核酸疫苗并系统地验证了其有效性的基础上,开展了该疫苗在接种部位的动态分析研究,为IHN核酸疫苗的研发和安全性评价研究提供了基础数据。     

First evidence of infectious hematopoietic necrosis virus (IHNV) in the Netherlands

In spring 2008, infectious hematopoietic necrosis virus (IHNV) was detected for the first time in the Netherlands. The virus was isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), from a put‐and‐take fishery with angling ponds. IHNV is the causative agent of a serious fish disease, infectious hematopoietic necrosis (IHN). From 2008 to 2011, we diagnosed eight IHNV infections in rainbow trout originating from six put‐and‐take fisheries (symptomatic and asymptomatic fish), and four IHNV infections from three rainbow trout farms (of which two were co‐infected by infectious pancreatic necrosis virus, IPNV), at water temperatures between 5 and 15 °C. At least one farm delivered trout to four of these eight IHNV‐positive farms. Mortalities related to IHNV were mostly <40%, but increased to nearly 100% in case of IHNV and IPNV co‐infection. Subsequent phylogenetic analysis revealed that these 12 isolates clustered into two different monophyletic groups within the European IHNV genogroup E. One of these two groups indicates a virus‐introduction event by a German trout import, whereas the second group indicates that IHNV was already (several years) in the Netherlands before its discovery in 2008.     

Inactivation of infectious pancreatic necrosis virus for vaccine use

Abstract. Formalin and β-propiolactone (BPL) were compared for efficacy in inactivating infectious pancreatic necrosis virus (IPNV) for vaccine use. Incubation with a 1:200 dilution of formalin at 20°C for four days or longer, or a 1:200 dilution of BPL at 4°C for six days or longer, completely inactivated the virus infectivity. However, whereas treatment with formalin caused only a slight reduction in anti-genicity as titrated by in vitro tests, treatment with BPL destroyed over 50% of the antigenicity of the virus. Virus inactivated with formalin also proved highly effective at inducing high titres of neutralizing antibody in trout.     

Appearance of Infectious Hematopoietic Necrosis Virus in Rainbow Trout,Oncorhynchus mykiss,During Seawater Adaptation

About 7% mortality occurred in rainbow trout, Oncorhynchus mykiss, during seawater adaptation at a marine farm in the South Sea of Korea during the winter of 2014. Most diseased fish showed petechial hemorrhaging of gills and internal fat with enlarged spleen. Although no parasites or bacteria were isolated from the diseased fish, all tissue filtrates produced cytopathic effects (CPEs) in fathead minnow and Chinook salmon embryo‐214 cells. The cell culture supernatant showing CPE contained specific 1527‐bp fragment for the infectious hematopoietic necrosis virus (IHNV) glycoprotein gene by polymerase chain reaction. Their nucleotide sequences shared 98.1–98.2% identities with IHNV RtUi02 isolated from rainbow trout in Korea. This isolate (RtGoH14) was closely related to Korean IHNV isolates of genogroup JRt rather than to those of North American and European genogroups. These results suggest that this IHNV isolate might have been introduced to rainbow trout farm (land‐based culture system) in Korea. This is the first report of IHNV infection in rainbow trout during seawater adaptation in Korea.     

Infectious haematopoietic necrosis virus from salmonids cultured in Korea

Abstract. Four isolates of infectious haematopoietic necrosis virus (IHNV) were obtained from rainbow trout, Oncorhynchus mykiss (Walbaum), and masu salmon, Oncorhynchus masou (Walbaum), fry during epizootics at hatcheries in Korea. The four isolates of IHNV were compared with three from salmonids in the USA (SRCV, OSV and RB-76) by analysis of virion proteins in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and neutralization tests, with two monoclonal antibodies raised against SRCV (MAb SRCV/A4) and RB-76 (MAb RB/B5). Based on the antigenicity and the size of the structural proteins, one Korean isolate from masu salmon (SCS) is similar to RB-76 and is an electropherotype 1. The other three isolates from rainbow trout (PRT, YRT and MRT) were identical to each other in the mobilities of their virion proteins in SDS-PAGE, and although their nucleocapsid (N) proteins comigrated with that of the RB-76 isolate, they differed from RB-76 in the smaller matrix 2 (M2) protein they contained. In addition, the three Korean isolates (PRT, YRT and MRT) could be divided into two groups by reactivity with MAb RB/B5. While the YRT isolate was neutralized by this MAb, the PRT and MRT isolates were not, suggesting that there are at least two neutralizing antigenic variants of IHNV in Korea.     

QTL for IHNV resistance and growth identified in a rainbow (Oncorhynchus mykiss) × Yellowstone cutthroat (Oncorhynchus clarki bouvieri) trout cross

Infectious hematopoietic necrosis virus (IHNV) is a major constraint to rainbow trout culture. Yellowstone cutthroat trout (Oncorhynchus clarki bouvieri) have greater resistance to this virus than do rainbow trout (O. mykiss), but the genetic mechanism of this resistance is not understood. We conducted a genome scan using a backcross of cutthroat trout into a rainbow trout background to estimate the number and locations of quantitative trait loci (QTL) associated with IHNV resistance and growth in trout. IHNV resistance was considered in terms of both survival (binary trait) and days to death (quantitative trait). The genetic map was scanned using interval mapping via two different approaches: one model considered survival alone and a second two-part model combined both survival and days to death. Three QTL were significantly (P ≤ 0.05) associated with virus resistance genome-wide, explaining 32.5% of the phenotypic variation. Cutthroat alleles at two of these QTL resulted in increased resistance to the pathogen, as expected. No growth QTL were detected in this cross. We suggest that these traits are genetically independent.     

传染性造血器官坏死病毒微型基因组表达干扰素对病毒的抑制效应

为构建传染性造血器官坏死病毒(IHNV HLJ-09)微型基因组并表达虹鳟IFN,采用RT-PCR扩增IHNV HLJ-09株的N、P、L、G和NV蛋白基因并亚克隆入真核表达载体pCI中,构建辅助质粒pCI-N、pCI-P、pCI-L、pCI-G和pCI-NV;将扩增获得的IHNV基因组两末端序列、增强型绿色荧光蛋白(EGFP)报告基因、虹鳟I型干扰素(IFN)基因克隆到真核表达载体pCI中构建出表达EGFP的IHNV微型基因组pCI-LFGT和表达IFN的IHNV微型基因组pCI-LFIT;将pCI-LFIT质粒转染已接种IHNV HLJ-09毒株的EPC细胞,实时荧光定量PCR法测定细胞中IHNV G基因RNA。结果显示:构建的微型基因组不论与辅助病毒还是与5个辅助质粒共转染,外源基因均能正确表达;pCI-LFIT质粒转染已接种病毒的EPC细胞组与对照组相比其中的病毒核酸显著减少。