Infectious Hematopoietic Necrosis Virus Isolates Research Articles

Characterization and pathogenicity analysis of a newly isolated strain of infectious hematopoietic necrosis virus

Rainbow trout is one of the fastest-growing aquaculture species and infectious hematopoietic necrosis virus (IHNV) is endemic throughout almost all rainbow trout farms in China nowadays. In this study, IHNV GS21 was identified as the causative pathogen, which resulted in massive mortality of rainbow trout occurring in northwest China. GS21 isolate was propagated in Chinook salmon embryonic cell line (CHSE-214) and induced apparent cytopathic effects (CPE) at 3 days post-infection (dpi). Phylogenetic analysis revealed that GS21 isolate was clustered with other reported Chinese isolates within the J genogroup. Moreover, the complete cDNA sequence of GS21 isolate was obtained and it possesses more than 98 % of ANI values and 89 % of DDH values with other Chinese IHNV isolates. The detailed sequence analysis of G gene revealed the distinct amino acid substitutions of G230, G252, G270, and I277 in GS21 isolate. Furthermore, the artificially infected rainbow trout exhibited similar clinical disease symptoms as natural infection did. The cumulative mortality infected by GS21 isolate of 104 PFU/mL reached 93 % at approximately 13.5 °C. Additionally, viral loads in tissues increased first and declined then as well as the expression of immune-associated genes. Collectively, our results characterized a novel IHNV GS21 isolate that can lead to massive mortality in juvenile rainbow trout and provided a basis to define the pathogenic characteristics and evolutionary relationship of IHNV and host immune response against IHNV infection.

Genetics Reveal Long-Distance Virus Transmission Links in Pacific Salmon.

Simple SummaryThe transmission of viruses between host populations is essential for viruses to persist on the landscape. Therefore, the identification of specific transmission links can provide insights into how a virus moves from source to recipient (sink) populations, allowing for the development of strategies to interrupt transmission routes and control viral disease. For the fish pathogen infectious hematopoietic necrosis virus (IHNV), this study identifies three transmission links associated with the emergence of IHNV in coastal Washington steelhead trout populations between 2007 and 2011. The links were identified by the genetic typing of virus isolates obtained from coastal fish and potential source fish from the Columbia River Basin. Three exact genotype matches were found, indicating at least three introductions of virus from Columbia fish to coastal fish during years of the emergence event. Likely sources were juvenile fish in the Columbia region experiencing disease, and the first detected recipient populations in all cases were adult fish returning to coastal hatcheries. Variation in timing and distance for these three transmission links will provide Pacific Northwest fish health managers with a better understanding of IHNV transmission routes from Columbia region fish to coastal steelhead trout.In the coastal region of Washington State, a major pathogen emergence event occurred between 2007 and 2011 in which steelhead trout (Oncorhynchus mykiss) experienced a high incidence of infection and disease outbreaks due to the rhabdovirus infectious hematopoietic necrosis virus (IHNV). Genetic typing showed that the introduced viruses were in the steelhead-specific MD subgroup of IHNV and indicated the most likely source was a virus from the nearby Columbia River Basin. In the current study, full-length viral glycoprotein (G) gene sequences were determined for 55 IHNV isolates from both coastal and Columbia fish populations to identify specific source populations and infer mechanisms of transmission to coastal steelhead. We identified three transmission links based on exact fullG genotype matches between Columbia and coastal fish. In all cases, the likely source population was infected juvenile fish, and sink populations were adult fish returning to coastal rivers to spawn. The time intervals between detection in source and sink populations varied from 6 months to nearly 4 years, suggesting different transmission pathways. Surprisingly, distances between source and sink populations varied between 140 and 1000 km. These results confirm repeated introductions of virus from Columbia River Basin fish as the cause of emergence of MD virus on the Washington coast from 2007 to 2011.

Rapid Diagnostic Test to Detect and Discriminate Infectious Hematopoietic Necrosis Virus (IHNV) Genogroups U and M to Aid Management of Pacific Northwest Salmonid Populations.

Simple SummaryInfectious hematopoietic necrosis virus (IHNV) can cause severe disease and mortality in salmon and trout. Two major groups of the virus are found in the North American Columbia River Basin and Washington Coast, designated U and M. The U and M groups pose different risks depending on the fish host species. Here, we report the development of a new diagnostic test that can both detect the presence of IHNV and rapidly discriminate between the U and M groups. The new assay proved sensitive and specific when applied to free ranging, returning adult Pacific salmon. Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonids in North America, Europe, and Asia that is phylogenetically classified into five major virus genogroups (U, M, L, E, and J). The geographic range of the U and M genogroup isolates overlap in the North American Columbia River Basin and Washington Coast region, where these genogroups pose different risks depending on the species of Pacific salmon (Oncorhynchus spp.). For certain management decisions, there is a need to both test for IHNV presence and rapidly determine the genogroup. Herein, we report the development and validation of a U/M multiplex reverse transcription, real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) protein gene. The new U/M RT-rPCR is a rapid, sensitive, and repeatable assay capable of specifically discriminating between North American U and M genogroup IHNV isolates. However, one M genogroup isolate obtained from commercially cultured Idaho rainbow trout (O. mykiss) showed reduced sensitivity with the RT-rPCR test, suggesting caution may be warranted before applying RT-rPCR as the sole surveillance test in areas associated with the Idaho trout industry. The new U/M assay had high diagnostic sensitivity (DSe > 94%) and specificity (DSp > 97%) in free-ranging adult Pacific salmon, when assessed relative to cell culture, the widely accepted reference standard, as well as the previously validated universal N RT-rPCR test. The high diagnostic performance of the new U/M assay indicates the test is suitable for surveillance, diagnosis, and confirmation of IHNV in Pacific salmon from the Pacific Northwest regions where the U and M genogroups overlap.

Comparative Susceptibilities of Selected California Chinook Salmon and Steelhead Populations to Isolates of L Genogroup Infectious Hematopoietic Necrosis Virus (IHNV).

Simple SummaryChinook salmon in California conservation hatcheries have suffered disease outbreaks and significant mortality due to the fish virus infectious hematopoietic necrosis virus (IHNV) since at least the 1940s. Although steelhead trout in California are also occasionally infected with the same virus they do not typically experience disease. In this study the susceptibility of California Chinook salmon and steelhead trout was defined in controlled experiments that exposed groups of juvenile fish to California IHNV strains (L genogroup). The results confirmed high mortality among Chinook salmon but very low mortality of steelhead trout, despite identical virus exposures. Tests of varying conditions found increasing mortality of Chinook salmon with increasing virus dose, but reduced mortality at higher temperatures or with increased age or size of fish. Among Chinook salmon that survived virus exposure the persistence of virus was detected in one fish 8 months after virus exposure. These findings demonstrate that L genogroup IHNV in California has host specificity for Chinook salmon, and provide an understanding of factors that contribute to disease epidemics in California hatchery Chinook salmon.Salmonid species demonstrate varied susceptibility to the viral pathogen infectious hematopoietic necrosis virus (IHNV). In California conservation hatcheries, juvenile Chinook salmon (Oncorhynchus tshawytscha) have experienced disease outbreaks due to L genogroup IHNV since the 1940s, while indigenous steelhead (anadromous O. mykiss) appear relatively resistant. To characterize factors contributing to the losses of California salmonid fish due to IHNV, three populations of Chinook salmon and two populations of steelhead native to California watersheds were compared in controlled waterborne challenges with California L genogroup IHNV isolates at viral doses of 104–106 pfu mL−1. Chinook salmon fry were moderately to highly susceptible (CPM = 47–87%) when exposed to subgroup LI and LII IHNV. Susceptibility to mortality decreased with increasing age and also with a higher temperature. Mortality for steelhead fry exposed to two IHNV isolates was low (CPM = 1.3–33%). There was little intraspecies variation in susceptibility among populations of Chinook salmon and no differences in virulence between viruses strains. Viral persistence was demonstrated by the isolation of low levels of infectious IHNV from the skin of two juvenile Chinook salmon at 215 d post exposure. The persistence of the virus among Chinook salmon used for stocking into Lake Oroville may be an explanation for the severe epidemics of IHN at the Feather River hatchery in 1998–2002.

Differential virulence of infectious hematopoietic necrosis virus (IHNV) isolated from salmonid fish in Gangwon Province, Korea

The present study investigated the virulence and expression of innate immunity genes in isolates of infectious hematopoietic necrosis virus (IHNV) in Gangwon province, South Korea, by challenging rainbow trout, Atlantic salmon, and coho salmon. Eight IHNV isolates were used to infect RTG-2 cells for viral replication using plaque assays. Three isolates with the highest replication rates, the RtPc0314g and RtPc0314c isolates of the JRt-Shizuoka type and the RtPc0816g isolate of the JRt-Nagano type, were experimentally infected into the fish. In rainbow trout, both RtPc0314c and RtPc0314g isolates showed 100% cumulative mortality while the RtPc0816g isolate showed 60% cumulative mortality for 14 days. In contrast, all three isolates showed <60% cumulative mortality in Atlantic salmon and coho salmon. The expression of G genes in the kidney was higher than that in the spleen-infected fish, with the highest expression observed in the kidneys of rainbow trout. The relative expression levels of innate immunity genes were higher in rainbow trout than in Atlantic salmon and coho salmon. The expression level of immunoglobulin M increased until day 7, and the expression of type I interferon was higher in the spleen than in other tissues. The expression of Mx-1 was higher in the kidney and liver than other tissues. These results indicate that IHNV isolates from Gangwon province show host-specific virulence in rainbow trout and that their virulence and replication were higher in JRt-Shizuoka type than in JRt-Nagano type isolates.

First isolation and pathogenicity analysis of a genogroup U strain of infectious hematopoietic necrosis virus from rainbow trout in China.

Infectious hematopoietic necrosis virus (IHNV) is a major fish viral pathogen causing acute clinical disease and death in a variety of salmonids. IHNV isolates have been classified into five major genogroups according to the phylogenetic analysis of partial G gene fragments or the complete G gene sequence: U, M, E, L and J. Genogroup U strains have been reported in North America and Japan prior to 1982, and genogroup J is the only genogroup that has been reported in China. Here, one of IHNV strain (BjLL) was isolated from a local farm in China and were characterized in this study. The homogenate tissues of infected fry induced IHNV-positive cytopathic effects in epithelioma papulosum cyprinid (EPC) cells that were confirmed by RT-PCR and sequencing. The complete genome sequence of BjLL comprised 11,129 nucleotides, which had been submitted to GenBank (accession no. MF509592). By the sequence comparison and phylogenetic analysis for the G gene sequence of BjLL with 51 reference sequences in GenBank, we confirmed that this Chinese isolate belonged to genogroup U. Furthermore, virus exposure experiments with juvenile rainbow trout were conducted to assess the virulence and pathogenicity of BjLL. Compared with GS-2014 of genogroup J, BjLL was an obviously less virulent strain that could result in lower mortality. Besides, typical clinical symptoms and pathological damages could be seen in fish following infection of BjLL. The present study is the first report of genogroup U IHNV infection in China and will provide essential information for future studies on pathogenesis of IHNV BjLL and development of efficient control strategies.

Japanese amberjack Seriola quinqueradiata and red sea bream Pagrus major susceptibility to infectious hematopoietic necrosis virus (IHNV) isolate.

Infectious hematopoietic necrosis virus (IHNV) is known as a causative agent of heavy mortalities in farmed rainbow trout. However, there is limited information on its virulence for marine fish species. In this study, Japanese amberjack Seriola quinqueradiata and red sea bream Pagrus major were experimentally infected by intraperitoneal (IP) injection and immersion, with an IHNV isolate from rainbow trout in Japan, to evaluate the virulence of the virus for these fish species. The cumulative mortality for immersed rainbow trout was 15%. IHNV was isolated from all dead fish and 50% of the sequentially sampled rainbow trout. When Japanese amberjack were challenged by IP injection and immersion, the resulting cumulative mortality was 70% and 0%, respectively. The virus was isolated from all dead fish and 1 out of 3 Japanese amberjack sampled at 9 d post exposure. However, no mortality was observed in either of the red sea bream groups challenged with IHNV. IHNV was not isolated from any of the surviving red sea bream, or from any of the sequentially sampled fish. The viral titer on Japanese amberjack-derived YTF cells was in the same log range as that on FHM and RTH-149 cells, but the titers on the red sea bream cell lines SBK and GBRS were lower than the other cell lines, and were significantly different from the FHM and RTH-149 cell lines. These results suggest that Japanese amberjack has a low susceptibility to IHNV, and red sea bream has no or little susceptibility to the virus.

Survival of viral haemorrhagic septicaemia virus and infectious haematopoietic necrosis virus in the environment and dried on stainless steel.

Viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV) are important viral pathogens posing a serious threat to salmonid fish. Survival of two isolates of IHNV and one of VHSV was assessed at temperatures ranging from 4 to 25°C: (a) after drying on stainless steel, (b) in cell culture medium, (c) in filtered river water, (d) in unfiltered river water, and (e) survival, adsorption and desorption in river sediment and five typical soil types. The viruses survived 1 hr to > 84 days depending on the conditions. Survival was inversely related to temperature and organic and inorganic content. Both viruses remained infectious after being dried on stainless steel for several weeks highlighting the risk of mechanical transmission and persistence in a dry environment. Both adsorbed to the soils from the river water inoculum, with titres between 5.56x104 and 2.58x108 TCID50/ml after 1 hr. Clay soils adsorbed the least virus but had the greatest decrease in the river water inoculum (undetectable in ≤ 1 hr), and there was no desorption. Virus desorbed from the other soils into the surrounding water at different rates dependant on soil type (longest desorption was from chalk loam and sandy soil—detected at 28 days). When desorption was no longer detectable, virus persisted, adsorbed to the soil and remained infectious (the longest adsorption was detected in clay loam for ≥ 49 days, but all the viruses adsorbed to soils were likely to have survived longer than that detected, based on their rate of decay). The long survival of the viruses, particularly at cooler temperatures, highlights the risk of survival in the environment and waterborne spread. The data presented here are highly relevant for assessing risk of pathogen introduction via fomites (stainless steel) and for deciding on best control measures in the context of disease outbreaks.

Susceptibility of pike Esox lucius to VHSV and IHNV and potential transmission to rainbow trout Oncorhynchus mykiss.

Determining the origin of recurrent outbreaks of fish diseases occurring on fish farms is essential for disease prevention and control measures. In this study, we investigated the potential reservoir role of wild fish species living near salmonid farms which were regularly found to be positive for viral hemorrhagic septicemia virus (VHSV). In addition to VHSV, infectious hematopoietic necrosis virus (IHNV) was also isolated from several pike Esox lucius samples collected from a pond near the salmonid farms of interest. All isolates of VHSV and IHNV analyzed had 100% identical partial glycoprotein gene sequences. VHSV pike strain OO128-25 belonged to the Ia genotype and shared 99.1 to 99.5% nucleotide identity with strains recently isolated from the farms. IHNV pike strain OO121-8, European genotype, appeared to be different from strains from France characterized since the first isolation in 1987. Isolates representative of both viral species were highly virulent in rainbow trout Oncorhynchus mykiss. OO128-25 induced 65% mortality in pike fingerlings, whereas only weak mortality was observed with OO121-8, despite characteristic symptoms in infected fish. High levels of specific antibodies to VHSV and IHNV were detected in adult pike in the absence of clinical signs. Infection of rainbow trout in contact with experimentally VHSV- or IHNV-infected pike fingerlings indicates possible horizontal transmission. These results suggest that pike could act as a reservoir for VHSV and IHNV in the wild, providing additional evidence to explain viral persistence and resurgence in certain areas.

Genome and Phylogenetic Analysis of Infectious Hematopoietic Necrosis Virus Strain SNU1 Isolated in Korea.

Infectious hematopoietic necrosis virus (IHNV), one of the most important pathogenic fish viruses, affects trout fisheries and causes considerable economic losses. Currently, in Korea, more studies on IHNV infection are being reported. However, relatively less data is available on Korean isolates than on those from other countries. Few studies have focused on gene sequence analyses of IHNV glycoprotein (G) gene and almost none have focused on other gene fragments. Therefore, considering the dearth of adequate phylogenetic and genomic studies on Korean IHNV strains because of the lack of data, our study aimed to provide sufficient relevant data by sequencing the complete genome of the IHNV strain SNU1, which was recently isolated from a Korean rainbow trout farm. Moreover, we focused on expanding the perspectives on the phylogenesis of IHNV isolates from Korea and other Asian countries. IHNV was isolated from pooled hematopoietic tissue samples using Epithelioma papulosum cyprinid (EPC) cells, and phylogenetic analysis and genome study were conducted using complete G, N, and nonvirion (NV) gene sequences. Our main achievements were the development of a phylogenetic analytical method based on the NV gene and complete genome sequence analysis of the IHNV strain SNU1, which was compared with other Asian isolate sequences.

Phylogeography and evolution of infectious hematopoietic necrosis virus in China

Infectious hematopoietic necrosis virus (IHNV) is a well-known rhabdoviral pathogen of salmonid fish. In this study, a comprehensive analysis of 40 IHNV viruses isolated from thirteen fish farms in nine geographically dispersed Chinese provinces during 2012 to 2017 is presented. Identity of nucleotide and amino acid sequences among all the complete glycoprotein (G) genes from Chinese isolates was 98.0–100% and 96.7–100%, respectively. Coalescent phylogenetic analyses revealed that all the Chinese IHN virus characterized in this study were in a monophyletic clade that had a most recent common ancestor with the J Nagano (JN) subgroup within the J genogroup of IHNV. Within the Chinese IHNV clade isolates obtained over successive years from the same salmon fish farm clustered in strongly supported subclades, suggesting maintenance and diversification of virus over time within individual farms. There was also evidence for regional virus transmission within provinces, and some cases of longer distance transmission between distant provinces, such as Gansu and Yunnan. The data demonstrated that IHNV has evolved into a new subgroup in salmon farm environments in China, and IHNV isolates are undergoing molecular evolution within fish farms. We suggest that Chinese IHNV comprises a separate JC subgroup within the J genogroup of IHNV.

Phylogeny of the infectious hematopoietic necrosis virus in European aquaculture.

Infectious hematopoietic necrosis (IHN)—a highly lethal infectious salmonid disease—has caused substantial economic losses in the European production of rainbow trout (Oncorhynchus mykiss) since the late 1980s. The causal agent of IHN is the IHN virus (IHNV) introduced from overseas. However, until today, its phylogeographic spread in Europe remains poorly understood. We therefore sought to elucidate this unresolved topic by using the largest ever compiled dataset of European IHNV isolates (E isolates) (193 GenBank E isolates and 100 isolates from this study) for the complete glycoprotein (G) gene sequence. Our results clearly revealed that the active trout trade has left its traces in the E phylogeny. For example, the spread by trade of IHNV-infected trout was apparently the cause for the exposure of the E lineage to different local scenarios of selection and genetic drift, and therefore has led to the split of this lineage into various subordinated lineages. Accordingly, we also found evidence for E isolates being mixed Europe-wide by cross-border introduction events. Moreover, there were indications that this propagation of the E lineage within Europe corresponded with an extensive and rapid spread event, already during or shortly after its formation. Finally, in accordance with the high substitution rate of IHNV determined by previous studies, our dataset indicates that the mean period of occurrence of a single E haplotype is typically not longer than one calendar year.

Bivalent DNA vaccine induces significant immune responses against infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus in rainbow trout

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are important pathogens of salmon and trout. An active bivalent DNA vaccine was constructed with the glycoprotein gene of Chinese IHNV isolate Sn1203 and VP2–VP3 gene of Chinese IPNV isolate ChRtm213. Rainbow trout (5 g) were vaccinated by intramuscular injection with 1.0 µg of the bivalent DNA vaccine and then challenged with an intraperitoneal injection of IHNV, IPNV, or both, at 30 and 60 days post-vaccination (d.p.v.). High protection rates against IHNV were observed, with 6% and 10% cumulative mortality, respectively, compared with 90–94% in the mock-vaccinated groups. IPNV loads (531-fold and 135-fold, respectively) were significantly reduced in the anterior kidneys of the vaccinated trout. Significant protection against co-infection with IHNV and IPNV was observed, with cumulative mortality rates of 6.67% and 3.33%, respectively, compared with 50.0% and 43.3%, respectively, in the mock-vaccinated groups. No detectable infective IHNV or IPNV was recovered from vaccinated trout co-infected with IHNV and IPNV. The bivalent DNA vaccine increased the expression of Mx-1 and IFN-γ at 4, 7, and 15 d.p.v, and IgM at 21 d.p.v., and induced high titres (≥160) of IHNV and IPNV neutralizing antibodies at 30 and 60 d.p.v.

A effective DNA vaccine against diverse genotype J infectious hematopoietic necrosis virus strains prevalent in China

Infectious hematopoietic necrosis virus (IHNV) is the most important pathogen threatening the aquaculture of salmonid fish in China. In this study, a DNA vaccine, designated pIHNch-G, was constructed with the glycoprotein (G) gene of a Chinese IHNV isolate SD-12 (also called Sn1203) of genotype J. The minimal dose of vaccine required, the expression of the Mx-1 gene in the muscle (vaccine delivery site) and anterior kidney, and the titers of the neutralizing antibodies produced were used to evaluate the vaccine efficacy. To assess the potential utility of the vaccine in controlling IHNV throughout China, the cross protective efficacy of the vaccine was determined by challenging fish with a broad range of IHNV strains from different geographic locations in China. A single 100ng dose of the vaccine conferred almost full protection to rainbow trout fry (3g) against waterborne or intraperitoneal injection challenge with IHNV strain SD-12 as early as 4days post-vaccination (d.p.v.), and significant protection was still observed at 180d.p.v. Intragenogroup challenges showed that the DNA vaccine provided similar protection to the fish against all the Chinese IHNV isolates tested, suggesting that the vaccine can be widely used in China. Mx-1 gene expression was significantly upregulated in the muscle tissue (vaccine delivery site) and anterior kidney in the vaccinated rainbow trout at both 4 and 7d.p.v. Similar levels of neutralizing antibodies were determined with each of the Chinese IHNV strains at 60 and 180d.p.v. This DNA vaccine should play an important role in the control of IHN in China.

An efficient and simple method to increase the level of displayed protein on the yeast cell surface

BackgroundThe development of oral vaccines using yeast surface display technology is an area of intensive study in vaccine development, but the protein level displayed on yeast surfaces is not currently high enough to obtain a robust immune response. MethodsTo address this issue, we established an efficient and simple method of increasing the level of displayed protein on the yeast cell surface. We used the single chain variable fragment (scFv) of an antibody against the infectious hematopoietic necrosis virus isolate Sn1203 as a target display protein. The yeast-derived scFv was first displayed on the yeast surface by galactose induction, and then Escherichia coli-derived scFv was also displayed on the same yeast via an artificial anchoring condition to increase the total scFv level on the yeast surface. ResultsThe levels of yeast- and E. coli-derived scFv displayed on the yeast cell surface were analyzed by flow cytometry, western blotting, and fluorescent microscopy. The flow cytometry results indicated that when the cells were suspended in phosphate-buffered saline with 1mmol/L glutathione, 0.2mmol/L oxidized glutathione, and 5% dimethyl sulfoxide at 4°C for 6h, the E. coli-derived scFv protein was stably anchored to the yeast cell surface. The mean fluorescence intensity in these experiments, which is an indirect quantitative representation of the surface scFv expression, was three times higher in the treated cells than that in control cells. The western blotting results show two specific protein bands, the smaller of which was identified as the E. coli-derived scFv that was displayed on the yeast cell surface. Cell immunofluorescence is a more direct way to detect differentially produced proteins that are displayed on the yeast cell surface. The fluorescence microscopy results show that both fluorescence corresponding to the yeast-derived scFv and fluorescence corresponding to the E. coli-derived scFv can exist on the cell surface of same yeast cell. This confirms that the E. coli-derived scFv protein was successfully displayed on the yeast cell surface. ConclusionsThis method provides a rapid, simple, and high-efficiency strategy to increase the level of displayed protein on the yeast cell surface. Application of this technique may allow the yeast surface display system to be used to generate potential oral vaccines.

Diagnostic efficacy of molecular assays for the viral haemorrhagic septicaemia virus isolates from the Czech Republic

The diagnostic properties of the one-step real-time reverse-transcription polymerase chain reaction assay for viral haemorrhagic septicaemia virus detection were compared to methods currently in use in the Czech Republic, namely, virus isolation using the cell culture and conventional reverse-transcription polymerase chain reaction followed by the nested polymerase chain reaction. The assays were tested on a panel of 25 archived viral haemorrhagic septicaemia isolates and 8 archived infectious haematopoietic necrosis isolates obtained from monitoring and/or outbreaks of the diseases among farmed salmonids in the Czech Republic. The ability to detect the presence of the virus in the tissues of fish was tested on additional 32 field samples collected from the rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta) and brook trout (Salvelinus fontinalis). The real-time assay showed the highest analytic sensitivity by detecting the presence of viral nucleic acid in samples with 10-7 dilution, whereas the sensitivity of the conventional polymerase chain reaction peaked at 10-5. Diagnostic specificity of both molecular assays was confirmed by absence of cross-reactivity with the infectious haematopoietic necrosis virus isolates. This, along with consistent results in the detection of the virus in the fish tissues, confirms that the one-step real-time reverse-transcription polymerase chain reaction is currently an optimal stand-alone diagnostic method for the detection of the viral haemorrhagic septicaemia virus.

Infectious hematopoietic necrosis virus (IHNV) outbreak in farmed rainbow trout in Iran: Viral isolation, pathological findings, molecular confirmation, and genetic analysis

Infectious hematopoietic necrosis virus (IHNV) is the etiological agent of a contagious disease (IHN) mainly in salmonid fish. In the present study, we isolated and identified IHNV in trout fry from Iranian trout farms with unexplained high mortality in 2016. The affected fry showed cumulative mortality of 90% with the gross pathological signs including exophthalmia and hemorrhage of the eye, skin darkening, abdominal distension, ulceration of the snout, and the visceral pallor and yellowish fluid in the intestine. Histopathological examination revealed marked necrosis in the anterior kidney, liver and spleen with the intracytoplasmic inclusion bodies in the liver sections. Also, intranuclear inclusion body and marginated chromatin were observable in the hematopoietic cells of the kidney. The homogenates tissues of infected fry induced IHNV-positive cytopathic effects (CPE) in EPC cells and confirmed by RT-PCR reactions and sequencing. Phylogenetic analysis revealed the Iranian IHNV isolates belonged to the European (E) genogroup with 100% identity to some Italian isolates. This is the first report of IHNV infection in farmed trout fry in Iran describing the viral isolation, clinical symptoms, histopathological findings, molecular confirmation, and genetic analysis suggestion of the specific country of origin.

Molecular Evolution and Phylogeography of Co-circulating IHNV and VHSV in Italy.

Infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV) are the most important viral pathogens impacting rainbow trout farming. These viruses are persistent in Italy, where they are responsible for severe disease outbreaks (epizootics) that affect the profitability of the trout industry. Despite the importance of IHNV and VHSV, little is known about their evolution at a local scale, although this is likely to be important for virus eradication and control. To address this issue we performed a detailed molecular evolutionary and epidemiological analysis of IHNV and VHSV in trout farms from northern Italy. Full-length glycoprotein gene sequences of a selection of VHSV (n = 108) and IHNV (n = 89) strains were obtained. This revealed that Italian VHSV strains belong to sublineages Ia1 and Ia2 of genotype Ia and are distributed into 7 genetic clusters. In contrast, all Italian IHNV isolates fell within genogroup E, for which only a single genetic cluster was identified. More striking was that IHNV has evolved more rapidly than VHSV (mean rates of 11 and 7.3 × 10−4 nucleotide substitutions per site, per year, respectively), indicating that these viruses exhibit fundamentally different evolutionary dynamics. The time to the most recent common ancestor of both IHNV and VHSV was consistent with the first reports of these pathogens in Italy. By combining sequence data with epidemiological information it was possible to identify different patterns of virus spread among trout farms, in which adjacent facilities can be infected by either genetically similar or different viruses, and farms located in different water catchments can be infected by identical strains. Overall, these findings highlight the importance of combining molecular and epidemiological information to identify the determinants of IHN and VHS spread, and to provide data that is central to future surveillance strategies and possibly control.

Geography and host species shape the evolutionary dynamics of U genogroup infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is a negative-sense RNA virus that infects wild and cultured salmonids throughout the Pacific Coastal United States and Canada, from California to Alaska. Although infection of adult fish is usually asymptomatic, juvenile infections can result in high mortality events that impact salmon hatchery programs and commercial aquaculture. We used epidemiological case data and genetic sequence data from a 303 nt portion of the viral glycoprotein gene to study the evolutionary dynamics of U genogroup IHNV in the Pacific Northwestern United States from 1971 to 2013. We identified 114 unique genotypes among 1,219 U genogroup IHNV isolates representing 619 virus detection events. We found evidence for two previously unidentified, broad subgroups within the U genogroup, which we designated 'UC' and 'UP'. Epidemiologic records indicated that UP viruses were detected more frequently in sockeye salmon (Oncorhynchus nerka) and in coastal waters of Washington and Oregon, whereas UC viruses were detected primarily in Chinook salmon (Oncorhynchus tshawytscha) and steelhead trout (Oncorhynchus mykiss) in the Columbia River Basin, which is a large, complex watershed extending throughout much of interior Washington, Oregon, and Idaho. These findings were supported by phylogenetic analysis and by FST. Ancestral state reconstruction indicated that early UC viruses in the Columbia River Basin initially infected sockeye salmon but then emerged via host shifts into Chinook salmon and steelhead trout sometime during the 1980s. We postulate that the development of these subgroups within U genogroup was driven by selection pressure for viral adaptation to Chinook salmon and steelhead trout within the Columbia River Basin.

High throughput screening of recombinant antibodies against infectious hematopoietic necrosis virus from a combinatorial antibody library

Infectious hematopoietic necrosis virus (IHNV) is a significant rhabdoviral pathogen of salmonid fish. In this study, a single chain variable fragment (scFv) antibody library derived from rainbow trout (Oncorhynchus mykiss) and a glycoprotein fragment (named G4, 540 nt, 180 aa) of IHNV-Sn1203 isolate were co-expressed by a bacterial display technology. The library was subjected to three rounds of screening by flow cytometry (FCM) to select IHNV specific antibodies. Seven antibody clones with different mean fluorescence intensities (MFI) were obtained by picking colonies at random. The antibody clone with the highest MFI was expressed and purified. The purified IHNV-specific scFv antibody was used successfully in Western blotting, enzyme linked immunosorbent assay and an immunofluorescence antibody test. This method provides a high throughput means to screen an antibody library by flow cytometry and isolate an antibody that can be used as a potential universal reagent for the detection and confirmation IHNV strains that are prevalent throughout China. Statement of relevanceOutbreaks of infectious hematopoietic necrosis caused severe economic losses to salmon and trout aquaculture in China every year. In this study, a panel of recombinant antibodies against Chinese IHNV isolates was obtained. The isolated antibody was proven can be used as a universal diagnosis regent for IHNV prevalent in China. The study provides a novel method for rapid development of antibodies to emerging diseases.