Virulence of a chimeric recombinant infectious haematopoietic necrosis virus expressing the spring viraemia of carp virus glycoprotein in salmonid and cyprinid fish

Infectious haematopoietic necrosis virus (IHNV) and spring viraemia of carp virus (SVCV) are both rhabdoviruses of fish, listed as notifiable disease agents by the World Organization for Animal Health. Recombinant rhabdoviruses with heterologous gene substitutions have been engineered to study genetic determinants and assess the potential of these recombinant viruses for vaccine development. A recombinant IHNV (rIHNV), containing the full‐length genome of a European IHNV strain, was modified by deleting the glycoprotein (G) gene and replacing it with a European SVCV G‐gene to make the rIHNV‐Gsvcv. The chimeric rIHNV‐Gsvcv level of virulence in rainbow trout, common carp and koi was assessed, and its ability to induce a protective immune response in surviving koi against wild‐type SVCV infection was tested. The rIHNV‐Gsvcv infection of trout led to high mortality, ranging from 78% to 92.5%, after immersion. In contrast, no deaths occurred in juvenile common carp after infection with rIHNV‐Gsvcv by either immersion or intraperitoneal (IP) injection. Similarly, koi infected with rIHNV‐Gsvcv via IP injection had little to no mortality (≤9%). Koi that survived initial infection with a high dose of recombinant virus rIHNV‐Gsvcv were protected against a virulent SVCV challenge resulting in a high relative per cent survival of 82.5%.     

First detection and isolation of infectious haematopoietic necrosis virus from farmed rainbow trout in Nyeri County,Kenya

Infectious haematopoietic necrosis virus (IHNV) is the causative agent of infectious haematopoietic necrosis, a disease of salmonid responsible for great economic losses. The disease occurs in most parts of the world where rainbow trout is reared but has not been previously reported in Kenya. In this study, rainbow trout fry and growers from two farms in Nyeri County were screened for IHNV. Whole fry (n = 4 from each farm) and kidney samples from growers (n = 15 and n = 6 from the two farms, respectively) were collected and preserved for cell culture examination or PCR analysis. Screening of samples was done by PCR followed by sequencing of the glycoprotein gene of the virus. Demonstration of the virus was done by propagation in EPC cells followed by the indirect fluorescence antibody test (IFAT). The results revealed the presence of IHNV at low prevalence of 0.1 and 0.4 for the two farms. The virus was confirmed both by IFAT and by partial sequencing of the G gene. Phylogenetic analysis revealed that the Kenyan isolates were identical to those of the J genogroup found mostly in Asia. The findings have implications for biosecurity measures and import regulations for the Kenyan rainbow trout industry.     

一株传染性造血器官坏死病病毒的致病性研究

为了对分离于山东某虹鳟养殖场的一株传染性造血器官坏死病毒株(IHNV-Sn1203)进行致病性检测与研究,将该IHNV-Sn1203毒株进行虹鳟鱼苗人工回接感染实验。结果显示,8d内人工感染实验鱼累计死亡率高达100%。收集大批濒死的病鱼样本,制备病理组织切片;利用鲤上皮细胞(EPC)进行细胞感染实验、病毒电镜观察、空斑实验、病毒滴度检测和聚类分析。病理组织切片显示,该病毒可造成虹鳟造血器官广泛性坏死;细胞感染实验结果显示,接种24 h后EPC细胞出现葡萄串状典型细胞病变(cytopathic effect,CPE),72 h后大部分细胞崩解脱落形成网状孔洞;电镜下清晰可见弹状病毒粒子大量存在于细胞质内,其在EPC细胞上的滴度为108.36TCID50/mL,并能形成2~4 mm空斑。对病毒核蛋白氨基酸序列的聚类分析结果显示,该病毒与标准毒株RB-1和WRAC的同源性分别为97%和93%,与国内报道的zyx株具有最高的同源性(99%)。研究表明,IHNV-Sn1203毒株能够在鱼体及敏感细胞中稳定繁殖,产生典型病变,具有较高的病毒滴度,对虹鳟鱼苗有很高的感染性和致死性。     

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.     

Inhibition of infectious haematopoietic necrosis virus in cell cultures with peptide-conjugated morpholino oligomers

Delivery of phosphorodiamidate morpholino oligomers (PMO) into fish cells in vitro and tissues in vivo was examined. Uptake was evaluated by fluorescence microscopy and flow cytometry after treating cultured cells or live rainbow trout with 3' fluorescein-tagged PMO. Arginine-rich peptide conjugated to the 5' end of the PMO markedly enhanced cellular uptake in culture by 8- to 20-fold compared with non-peptide-conjugated PMO as determined by flow cytometry. Enhanced uptake of PMO conjugated to peptide was also observed in tissues of fish treated by immersion. The efficacy of PMO as inhibitors of infectious haematopoietic necrosis virus (IHNV) replication was determined in vitro. Peptide-conjugated PMOs targeting sequences within the IHNV genomic RNA (negative polarity) or antigenomic RNA (positive polarity) significantly inhibited replication in a dose-dependent and sequence-specific manner. A PMO complementary to sequence near the 5' end of IHNV genomic RNA was the most effective, diminishing titre by 97%, as measured by plaque assay and Western blot. These data demonstrate that replication of a negative-stranded non-segmented RNA virus can be inhibited by antisense compounds that target positive polarity viral RNA, or by a compound that targets negative polarity viral RNA.     

A single-step multiplex PCR for simultaneous detection of white spot syndrome virus and infectious hypodermal and haematopoietic necrosis virus in penaeid shrimp

White spot syndrome virus (WSSV) and infectious hypodermal and haematopoietic necrosis virus (IHHNV) are the major viral pathogens of penaeid shrimp worldwide (Lightner & Redman 1998). Litopenaeus vannamei was introduced into China from the Americas, and quickly became widely cultured. Following its introduction, both IHHNV and WSSV have become important pathogens of cultured penaeid shrimp and have had a huge impact on the culture industry in China in recent years.     

An isolate and sequence database of infectious haematopoietic necrosis virus (IHNV)

In the field of fish diseases, the amount of relevant information available is enormous. Internet‐based databases are an excellent tool for keeping track of the available knowledge in the field. Fishpathogens.eu was launched in June 2009 with the aim of collecting, storing and sorting data on fish pathogens. The first pathogen to be included was the rhabdovirus, viral haemorrhagic septicaemia virus (VHSV). Here, we present an extension of the database to also include infectious haematopoietic necrosis virus (IHNV). The database is developed, maintained and managed by the European Community Reference Laboratory for Fish Diseases and collaborators. It is available at http://www.fishpathogens.eu/ihnv .     

Pre-exposure to infectious hypodermal and haematopoietic necrosis virus or to inactivated white spot syndrome virus (WSSV) confers protection against WSSV in Penaeus vannamei (Boone) post-larvae

Larvae and post-larvae of Penaeus vannamei (Boone) were submitted to primary challenge with infectious hypodermal and haematopoietic necrosis virus (IHHNV) or formalin-inactivated white spot syndrome virus (WSSV). Survival rate and viral load were evaluated after secondary per os challenge with WSSV at post-larval stage 45 (PL45). Only shrimp treated with inactivated WSSV at PL35 or with IHHNV infection at nauplius 5, zoea 1 and PL22 were alive (4.7% and 4%, respectively) at 10 days post-infection (p.i.). Moreover, at 9 days p.i. there was 100% mortality in all remaining treatments, while there was 94% mortality in shrimp treated with inactivated WSSV at PL35 and 95% mortality in shrimp previously treated with IHHNV at N5, Z1 and PL22. Based on viral genome copy quantification by real-time PCR, surviving shrimp previously challenged with IHHNV at PL22 contained the lowest load of WSSV (0-1x10(3) copies microg-1 of DNA). In addition, surviving shrimp previously exposed to inactivated WSSV at PL35 also contained few WSSV (0-2x10(3) copies microg-1 of DNA). Consequently, pre-exposure to either IHHNV or inactivated WSSV resulted in slower WSSV replication and delayed mortality. This evidence suggests a protective role of IHHNV as an interfering virus, while protection obtained by inactivated WSSV might result from non-specific antiviral immune response.     

抗传染性胰腺坏死病毒VP3蛋白单克隆抗体的制备

利用纯化后的传染性胰腺坏死病毒(IPNV VP3)重组蛋白免疫BALB/c小鼠,通过细胞融合技术,采用间接ELISA和有限稀释法筛选杂交瘤细胞,利用染色体鉴定、蛋白印迹和免疫荧光等方法对单克隆抗体进行鉴定,共得到2株能稳定分泌特异性抗体的阳性细胞株,分别命名为2F1、4A7,亚类鉴定2株单抗均为IgG1亚类。ELISA检测其腹水效价,蛋白印迹检测表明获得的2株单抗均能特异性识别IPNV VP3蛋白;间接免疫荧光鉴定表明2株单抗均与IPNV发生反应;间接ELISA检测结果表明2株单抗均不与HSV、SVCV、HRV等病毒反应,与IPNV具有较强的特异性反应。     

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.     

Atlantic salmon,Salmo salar L. are broadly susceptible to isolates representing the North American genogroups of infectious hematopoietic necrosis virus

Beginning in 1992, three epidemic waves of infectious hematopoietic necrosis, often with high mortality, occurred in farmed Atlantic salmon Salmo salar L. on the west coast of North America. We compared the virulence of eleven strains of infectious hematopoietic necrosis virus (IHNV), representing the U, M and L genogroups, in experimental challenges of juvenile Atlantic salmon in freshwater. All strains caused mortality and there was wide variation within genogroups: cumulative mortality for five U‐group strains ranged from 20 to 100%, four M‐group strains ranged 30‐63% and two L‐group strains varied from 41 to 81%. Thus, unlike Pacific salmonids, there was no apparent correlation of virulence in a particular host species with virus genogroup. The mortality patterns indicated two different phenotypes in terms of kinetics of disease progression and final per cent mortality, with nine strains having moderate virulence and two strains (from the U and L genogroups) having high virulence. These phenotypes were investigated by histopathology and immunohistochemistry to describe the variation in the course of IHNV disease in Atlantic salmon. The results from this study demonstrate that IHNV may become a major threat to farmed Atlantic salmon in other regions of the world where the virus has been, or may be, introduced.     

Molecular tracing confirms that infection with infectious pancreatic necrosis virus follows the smolt from hatchery to grow‐out farm

Infectious pancreatic necrosis (IPN) is an important restraint to production of salmonids in aquaculture globally. In order to implement efficacious mitigation strategies for control of this disease, it is important to understand infection routes under current production systems. IPN virus has been shown to be transmitted vertically in Rainbow trout, from broodstock to fingerlings in hatcheries, and there is circumstantial evidence suggesting that vertical transmission can also occur in Atlantic salmon, in addition to horizontal transmission between grow‐out fish in farms. In this study, we show that the smolt carries infection with IPN from hatchery to the marine farm. We do this by comparing sequences from fish groups taken both in hatcheries and on corresponding marine grow‐out farms. We use statistical analysis to prove that sequences obtained from the same fish group in both hatchery and marine farm are more similar than sequences obtained from random fish groups on hatcheries and marine farms.     

Competition of infectious hypodermal and haematopoietic necrosis virus (IHHNV) with white spot syndrome virus (WSSV) for binding to shrimp cellular membrane

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV) are two widespread shrimp viruses. The interference of IHHNV on WSSV was the first reported case of viral interference that involved crustacean viruses and has been subsequently confirmed. However, the mechanisms underlying the induction of WSSV resistance through IHHNV infection are practically unknown. In this study, the interference mechanisms between IHHNV and WSSV were studied using a competitive ELISA. The binding of WSSV and IHHNV to cellular membrane of Litopenaeus vannamei was examined. The results suggested that there existed a mutual competition between IHHNV and WSSV for binding to receptors present on cellular membrane of L. vannamei and that the inhibitory effects of WSSV towards IHHNV were more distinct than those of IHHNV towards WSSV.     

用逆转录多聚酶链式反应从凡纳对虾中检出传染性皮下组织和造血器官坏死病毒

传染性皮下组织和造血器官坏死病毒 (ＩＨＨＮＶ) ,是一种细小病毒 ,它能感染所有起源于中胚层和外胚层的对虾组织细胞[1] 。在许多养殖对虾的国家都有ＩＨＨＮＶ ,特别是中美洲国家和地区[2 ] 的对虾养殖业深受其害。随着各国间对虾贸易的急剧增长 ,ＩＨＨＮＶ地理分布范围日益广泛 ,迄今为止 ,已扩散到中国台湾、新加坡、马来西亚、泰国、印度尼西亚、澳大利亚、菲律宾、厄瓜多尔、秘鲁等国家和地区[2 -4] 。红额角对虾 (Ｐｅｎａｅｕｓｓｔｙｌｉｒｏｓｔｒｉｓ)、斑节对虾 (Ｐ .ｍｏｎｏｄｏｎ)、短沟对虾 (Ｐ .ｓｅｍｉｓｕｌｃａｔｕ…     

Detection and molecular characterization of infectious spleen and kidney necrosis virus from major ornamental fish breeding states in Peninsular Malaysia

‘Gold standard’ OIE reference PCR assay was utilized to detect the presence of infectious spleen and kidney necrosis virus (ISKNV) in freshwater ornamental fish from Malaysia. From total of 210 ornamental fish samples representing 14 species, ISKNV was detected in 36 samples representing 5 fish species. All positive cases did not show any clinical signs of ISKNV. Three restriction enzymes analyses showed that the fish were infected by identical strains of the same virus species within Megalocytivirus genus. Major capsid protein (MCP) genes of 10 ISKNV strains were sequenced and compared with 9 other reference nucleotide sequences acquired from GenBank. Sequence analysis of MCP gene showed that all strains detected in this study were closely related to the reference ISKNV with nucleotide sequence identity that was ranging from 99.8% to 100%. In addition, phylogenetic analysis of MCP gene revealed that viruses from genus Megalocytivirus can be divided into three genotypes: genotype 1 include reference ISKNV and all other strains that were detected in this study, genotype 2 include viruses closely related to red sea bream iridovirus (RSIV), and genotype 3 include viruses closely related turbot reddish body iridovirus (TRBIV).