Necrosis Virus In Rainbow Trout Research Articles

Development of a recombinant adenovirus-vectored vaccine against both infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss)

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are typical pathogens of rainbow trout Oncorhynchus mykiss, and the concurrent infection of the two viruses is very common among modern trout hatcheries, which has caused huge economic losses to the rainbow trout farming industry. To prevent and control the spread of IHNV and IPNV in juvenile trout simultaneously, in this study a bivalent recombinant adenovirus vaccine with IHNV Glycoprotein (G) and IPNV VP2 genes was developed. After immunizing juvenile trout with this bivalent vaccine via the immersion route, the expression levels of IHNV G and IPNV VP2 and the representative immune genes in vaccinated and control rainbow trout were tested to evaluate the correlation of immune responses with the expression of viral genes. The neutralizing antibody level induced by this bivalent vaccine as well as the protection efficacy of the vaccine against IHNV and IPNV was also evaluated. The results showed that IHNV G and IPNV VP2 were successfully expressed in juvenile trout, and all the innate and adaptive immune genes were up-regulated. This indicated that the level of the innate and adaptive immune responses were significantly increased, which might be induced by the high expression of the two viral proteins. Compared with the controls, high levels of neutralizing antibodies against IHNV and IPNV were induced in the vaccinated trout. Besides, the bivalent recombinant adenovirus vaccine showed high protection rate against IHNV, with the relative percent survival (RPS) of 81.25%, as well as against IPNV, with the RPS of 78.95%. Taken together, our findings clearly demonstrated that replication-defective adenovirus can be developed as a qualified vector for fish vaccines and IHNV G and IPNV VP2 were two suitable antigenic genes that could induce effective immune protection against these two pathogens. This study provided new insights into developing bivalent vectored vaccines and controlling the spread of IHNV and IPNV simultaneously in juvenile trout.

Long-Term Protection Elicited by an Inactivated Vaccine Supplemented with a Water-Based Adjuvant against Infectious Hematopoietic Necrosis Virus in Rainbow Trout (Oncorhynchus mykiss).

Previous inactivated vaccines against infectious hematopoietic necrosis (IHN) usually had a strong early immune protective effect but failed to provide long-term protection in rainbow trout (Oncorhynchus mykiss). To find a method for stabilizing the desired protective effect of IHN vaccines, we assessed the immune enhancement effect of four adjuvants on formaldehyde inactivated vaccine for IHN at 60 days postvaccination (dpv). The efficacy of a two-dose vaccination with the candidate adjuvant-formaldehyde inactivated vaccine for IHN was evaluated in terms of early protection and long-term protection (30 to 285 dpv). Neutralizing antibody titers were also measured at each time point. The Montanide GEL 02 PR (Gel 02) adjuvant significantly enhanced the immune protection provided by the IHN inactivated vaccine, whereas the immune-boosting effect of the other tested adjuvants lacked statistical significance. Both tested Gel 02-adjuvanted IHN inactivated vaccine dosages had a strong immune protection effect within 2 months postvaccination, with a relative percent of survival (RPS) of 89.01% to 100%, and the higher dosage provided complete protection at 204 dpv and a RPS of 60.79% on 285 dpv by reducing viral titers in rainbow trout. The neutralizing antibodies were observed only in vaccinated fish on 30 and 60 dpv. Through compatibility with an appropriate adjuvant, the highly immune protective effect of an IHN inactivated vaccine was prolonged from 60 dpv to at least 284 dpv; this novel adjuvant-IHN inactivated vaccine has promise as a commercial vaccine that provides the best available and longest duration of protection against IHN to rainbow trout. IMPORTANCE Infectious hematopoietic necrosis virus (IHNV) is one of the most serious pathogens threatening the global salmon and trout industry. However, there is currently only one commercialized infectious hematopoietic necrosis (IHN) vaccine, and it is inadequate for solving the global IHN problem. In this study, a promising adjuvanted inactivated vaccine with long-term protection was developed and comprehensively studied. We confirmed the presence of a late antiviral response stage in vaccinated rainbow trout that lacked detectable neutralizing antibodies, which are commonly recognized to be responsible for long-term specific protection in mammals. These findings further our understanding of unique features of fish immune systems and could lead to improved prevention and control of fish diseases.

An inactivated vaccine against infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss)

Infectious pancreatic necrosis virus (IPNV), belonging to the genus Aquabirnavirus within the family Birnaviridae, causes huge economic loss to the global salmonid industry every year. Recently, outbreaks of disease caused by genogroup I IPNV were found in many rainbow trout (Oncorhynchus mykiss) farms worldwide. An inactivated vaccine was prepared using a genogroup I IPNV isolate with an optimized procedure as incubation with β-propanolactone (BPL) at the final concentration of 0.5% at room temperature for 48 h. The inactivated vaccine was used to immunize rainbow trout, and the protection efficiency was evaluated by viral loads determination, immune-related genes quantification, and neutralizing antibody tests. The viral loads in immunized rainbow trout were significantly decreased and the strongest antiviral effect was observed on 30 days post-immunization (d.p.i). The expression of innate immune-related genes IFN-1, and Mx-1 genes were significantly up-regulated on 3, 7, and 15 d.p.i (p < 0.05), and adaptive immune-related genes CD4, CD8, and IgM genes were significantly up-regulated on 15 and 30 d.p.i (p < 0.05). Neutralizing antibodies were firstly detected on 30 d.p.i and the highest titer was observed on 45 d.p.i, which began to decrease on 60 d.p.i, but was still significantly higher than that in negative control fish. The results indicated that the vaccine prepared in this study could stimulate the non-specific and specific immune response and provide significant immune protection to the vaccinated rainbow trout.

Effects of Stocking Density on Stress Response and Susceptibility to Infectious Hematopoietic Necrosis Virus in Rainbow Trout.

The goals of this study were to examine the effect of stocking density on the stress response and disease susceptibility in juvenile rainbow trout (Oncorhynchus mykiss). Fish were sorted into one of 2 stocking densities (high density "HD", 20-40 kg/m³) or (low density, "LD", 4-8 kg/m³) and 3 stress indices (cortisol levels in serum and water, and neutrophil: lymphocyte (N:L) ratios from blood smears) were measured at multiple time points over 21 d. Serum cortisol was significantly increased at 1 h in LD samples and at 14 d in HD samples. Water cortisol concentrations were significantly higher in LD tanks as compared with HD tanks on day 14. N:L ratios were significantly higher in HD tanks on day 14 as compared with LD tanks and with baseline. The effect of stocking density on mortality after exposure to infectious hematopoietic necrosis virus (IHNV) was compared between fish held in HD or LD conditions, with or without prior acclimation to the different density conditions. No significant differences in survival were found between HD and LD treatments or between acclimated and nonacclimated treatments. Cumulative results indicate that 1) 1 to 4 gram rainbow trout did not generally demonstrate significant differences in stress indices at the density conditions tested over a 21-d period, 2) independent differences were found in 3 stress indices at day 14 after sorting into LD and HD holding conditions; and 3) LD and HD stocking densities did not have a significant effect on mortality due to IHNV.

Development of a live vector vaccine against infectious pancreatic necrosis virus in rainbow trout

Infectious pancreatic necrosis virus (IPNV) causes severe viral disease in salmonids, with major economic losses in the global rainbow trout aquaculture industry following from a high mortality rate. In the present study, we aimed to develop a safe and efficient vaccine to protect rainbow trout against IPNV infection. Administered via the immersion route, a live vector vaccine containing the coding region of the IPNV VP2 induced protective immune responses in rainbow trout. For best protection, juvenile rainbow trout should be immunized with a 1:100 dilution of the titer with 1 × 1010.0 ml−1 TCID50 of the live vector vaccine, via 10-min immersion. Expression of the IPNV VP2 gene was confirmed in spleens of vaccinated rainbow trout, and reached the highest level at 3 days post-vaccination (dpv) and thereafter gradually decreased between 3 and 15 dpv. The expression of TLR-3, TLR-7 and TLR-8 was upregulated after vaccination, and the highest expression levels of IFN-1, Mx-1, Mx-3, Vig-1 and Vig-2 were detected at 3 dpv. Four markers of the adaptive immune response (CD4, CD8, IgM and IgT) were continuously increased in the spleens of vaccinated fish as compared with controls, between 3 and 15 dpv. The cumulative percentage mortality differed significantly between the vaccinated fish and the controls (empty-plasmid-vaccinated). The results of the IPNV challenge showed that the live vector vaccine had a high protection rate against IPNV (relative percent survival value of 88.24), and the vaccinated rainbow trout displayed high levels of serum antibodies against IPNV infection. Taken together, our results demonstrate that this is a promising live vector vaccine that could be used to protect rainbow trout against IPNV infection.

Development of a live vector vaccine against infectious hematopoietic necrosis virus in rainbow trout

Infectious hematopoietic necrosis virus (IHNV) leads to serious disease and economic losses in the salmonid aquaculture industry. The present study aimed to develop an effective and efficient vaccine to protect rainbow trout (Oncorhynchus mykiss) against IHNV infection. Administered via the immersion route, a live vector vaccine containing the regions of the IHNV glycoprotein (G) induced immune responses in rainbow trout. Use of the immersion route induced more-efficient mucosal immunity than intramuscular injection vaccination. IHNV G gene expression was detected in the spleens of rainbow trout at 3, 7 and 15 days post-vaccination (dpv). The G gene expression continuously decreased between 3 and 15 dpv. In addition, the expression of TLR-3, TLR-7 and TLR-8 was upregulated after vaccination, and the highest expression levels of IFN-1, Mx-1, Mx-3, Vig-1 and Vig-2 were observed at 3 dpv. Four markers of the adaptive immune response (CD4, CD8, IgM and IgT) gradually increased. When experimental fish were challenged with IHNV by immersion, significant differences in cumulative percentage mortality were observed in the vaccinated fish and the unvaccinated (empty-plasmid-vaccinated) fish. The relative survival rate was 92% and 6% in the vaccinated group and empty-plasmid group, respectively. Serum antibody levels gradually increased in the vaccinated fish, unlike in the unvaccinated fish, after 7 dpv. Our results suggest there was a significant increase in fish immune responses and resistance to infection with IHNV following administration of the live vector vaccine. Therefore, this live vector vaccine is a promising vaccine that may be utilized to protect rainbow trout against IHNV.

Single-step genomic evaluation improves accuracy of breeding value predictions for resistance to infectious pancreatic necrosis virus in rainbow trout

The aim of this study was to compare the accuracy of breeding values (EBVs) predicted using the traditional pedigree based Best Linear Unbiased Prediction (PBLUP) and the single-step genomic Best Linear Unbiased Prediction (ssGBLUP) for resistance against infectious pancreatic necrosis virus (IPNV) in rainbow trout. A total of 2278 animals were challenged against IPNV and 768 individuals were genotyped using a 57 K single nucleotide polymorphism array for rainbow trout. Accuracies for both methods were assessed using five-fold cross-validation. The heritabilities were higher for PBLUP compared to ssGBLUP. The ssGBLUP accuracies outperformed PBLUP in 7 and 11% for days to death and binary survival, respectively. The ssGBLUP could be an alternative approach to improve the accuracy of breeding values for resistance against infectious pancreatic necrosis virus in rainbow trout, using information from genotyped and non-genotyped animals.

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.

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

AbstractAbout 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.

Association between MHC II beta chain gene polymorphisms and resistance to infectious haematopoietic necrosis virus in rainbow trout (Oncorhynchus mykiss, Walbaum, 1792)

Infectious haematopoietic necrosis virus (IHNV) is a serious pathogenic threat to rainbow trout (Oncorhynchus mykiss) and other salmonids. Major histocompatibility complex (MHC) polymorphisms have been closely linked to disease susceptibility. In this study, polymerase chain reaction-single-strand conformation polymorphism, cloning and sequencing were used to examine MHC class II β chain gene (DAB) polymorphisms and analyse their association with IHNV in rainbow trout. The DAB exon 2 was amplified from 104 resistant and 91 susceptible fish. Forty-five different alleles were identified, 30 of which were novel. Individuals possessed between one and four alleles, indicating that the genome harbours at least two closely related DAB exon 2 loci. The percentages of amino acid mutations in the peptide binding region (PBR) and non-PBR were 66.67% and 45.28% respectively. The rate of non-synonymous substitutions occurred at a significantly higher frequency than synonymous substitutions, suggesting the existence of balancing selection for maintenance of polymorphisms at the DAB exon 2 locus. The alleles Onmy-DAB*0201 (P < 0.001), Onmy-DAB*0904 (P < 0.001) and Onmy-DAB*1102 (P < 0.01) were associated with susceptibility to IHNV, while Onmy-DAB*0103, Onmy-DAB*0601 and Onmy-DAB*1402 were associated with resistance (P < 0.001). These alleles could be used as molecular markers for IHNV resistance breeding in rainbow trout.

Food pellets as an effective delivery method for a DNA vaccine against infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss, Walbaum)

A DNA vaccine based on the VP2 gene of infectious pancreatic necrosis virus (IPNV) was incorporated into feed to evaluate the effectiveness of this oral delivery method in rainbow trout. Lyophilized alginate–plasmid complexes were added to feed dissolved in water and the mixture was then lyophilized again. We compared rainbow trout that were fed for 3 consecutive days with vaccine pellets with fish that received the empty plasmid or a commercial pellet. VP2 gene expression could be detected in tissues of different organs in the rainbow trout that received the pcDNA-VP2 coated feed (kidney, spleen, gut and gill) throughout the 15 day time-course of the experiments. This pcDNA-VP2 vaccine clearly induced an innate and specific immune-response, significantly up-regulating IFN-1, IFN-γ, Mx-1, IL8, IL12, IgM and IgT expression. Strong protection, with relative survival rates of 78%–85.9% were recorded in the vaccinated trout, which produced detectable levels of anti-IPNV neutralizing antibodies during 90 days at least. Indeed, IPNV replication was significantly down-regulated in the vaccinated fish 45 days pi.

Occurrence and prevalence of infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss) cultured in cages in the Black Sea

Rainbow trout (Oncorhynchus mykiss) cultured in cage systems in the South Eastern Black Sea were surveyed for the type, occurrence and prevalence of infectious pancreatic necrosis virus (IPNV). Two nearby farms (designated as Farm A and Farm B) were visited monthly in 2007 and 2008. At each farm, 385 fish were selected randomly from five cages. Another farm with infected trout from a hatchery also was monitored for IPNV from the transfer to harvest. IPNV was found to be prevalent in both farms surveyed. In Farm A, IPNV was present throughout the growing period, from January to May, and all five randomly sampled cages tested positive for IPNV in March and April of 2007. In Farm B, IPNV was present only in February and March in 2007, and in 2008, IPNV was observed in January (two cages) and February (one cages) at low levels. Interestingly, IPNV was absent 2 weeks after transfer to the sea at 17.5°C. The same strain of IPNV, genotype III that was isolated from the same stock of fish at the hatchery, reoccurred when water temperatures dropped to 12°C in December in the Black Sea. Transferring fish to the sea at high water temperatures could lessen the negative impacts of IPNV on growth of rainbow trout in brackish water.

Differential virulence mechanisms of infectious hematopoietic necrosis virus in rainbow trout (Oncorhynchus mykiss) include host entry and virus replication kinetics

Host specificity is a phenomenon exhibited by all viruses. For the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV), differential specificity of virus strains from the U and M genogroups has been established both in the field and in experimental challenges. In rainbow trout (Oncorhynchus mykiss), M IHNV strains are consistently more prevalent and more virulent than U IHNV. The basis of the differential ability of these two IHNV genogroups to cause disease in rainbow trout was investigated in live infection challenges with representative U and M IHNV strains. When IHNV was delivered by intraperitoneal injection, the mortality caused by U IHNV increased, indicating that the low virulence of U IHNV is partly due to inefficiency in entering the trout host. Analyses of in vivo replication showed that U IHNV consistently had lower prevalence and lower viral load than M IHNV during the course of infection. In analyses of the host immune response, M IHNV-infected fish consistently had higher and longer expression of innate immune-related genes such as Mx-1. This suggests that the higher virulence of M IHNV is not due to suppression of the immune response in rainbow trout. Taken together, the results support a kinetics hypothesis wherein faster replication enables M IHNV to rapidly achieve a threshold level of virus necessary to override the strong host innate immune response.

Detection and quantification of infectious hematopoietic necrosis virus in rainbow trout ( Oncorhynchus mykiss) by SYBR Green real-time reverse transcriptase-polymerase chain reaction

A real-time reverse transcriptase-polymerase chain reaction assay using the fluorogenic dye SYBR Green I was developed for the detection and quantification of infectious hematopoietic necrosis virus (IHNV) infecting rainbow trout ( Oncorhynchus mykiss). Using primers designed for the IHNV nucleocapsid (N) and surface glycoprotein (G) genes, virus was detected in liver, kidney, spleen, adipose tissue, and pectoral fin samples from fish challenged in the laboratory via either injection or immersion and in fish collected from the field. The N- and G-gene amplicons provided melting curves with a single peak at 85.5 and 86.5 °C, respectively. Among different tissues tested, overall the N-gene was expressed in greater abundance than the G-gene in both laboratory-challenged and field samples. Kidney, liver, and spleen tissues had higher copies of the N- and G-genes compared to adipose tissue and pectoral fin. In samples from IHNV immersion challenge fish, the virus could be detected in the pectoral fin as early as 1 day post-challenge, and the viral load appears to decline by 6 days post-challenge. To evaluate the usefulness of non-invasive tissue sampling for IHNV detection, pectoral fin samples were collected from fish that were either apparently healthy or showing clinical signs of IHNV infection from commercial operations. Among the apparently healthy fish, using SYBR Green real-time RT-PCR the N-gene was detected in 2 out of 24 (8.3%), while the G-gene was detected in 8 of 24 (33%) fish. Among the fish showing clinical signs of IHNV infection, the N-gene was detected in 15 out of 36 (42%), while the G-gene was detected in 24 of 36 (67%) fish tested. Using a viral plaque assay, virus was detected in 4 of 24 (17%) apparently healthy fish and 33 of 36 (92%) fish showing clinical sign of IHNV infection. The higher level of IHNV detection by plaque assay compared to real-time RT-PCR might be due to the presence of more than one isolate in the field samples, and the inability to detect all the IHNV isolates using the current set of primers used in real-time RT-PCR. In conclusion, we developed a real-time RT-PCR assay for the detection and quantification of IHNV by SYBR Green real-time RT-PCR. This study demonstrates the potential of using fin clip as a non-invasive tissue source for detecting IHNV and possibly other viruses infecting salmonids in commercial aquaculture and in the field.

Identification of Additional Quantitative Trait Loci (QTL) Responsible for Susceptibility to Infectious Pancreatic Necrosis Virus in Rainbow Trout

Previous typing of microsatellite markers in a BC1F1 backcross rainbow trout Oncorhynchus mykiss revealed two quantitative trait loci (QTL) with a significant impact on suscep- tibility to infectious pancreatic necrosis. In the present study, additional genetic markers were mapped using the same strain in order to increase QTL resolution. A total of 199 individuals were screened at 226 marker loci (106 microsatellites, 116 amplified fragment length polymorphism markers, classical major histocompatibility complex molecules (MHC class I, nonclassical MHC class I, MHC class II) and tyrosinase) and the QTL were determined by single-point and interval mappings. Seven QTL were distinguished in the linkage groups RT-11, 12, 17, 23, 26, 29 and 31, in addition to the two already known in RT-3 and 22. The major QTL were those in RT-3, 12 and 22; whereas the classical MHC class I locus Onmy-UBA and the MHC class II locus Onmy-DAB were not associated with any of the QTL, a nonclassical MHC class I region with Onmy-UCA, Onmy-UDA, and Onmy-UEA mapped to the major QTL on RT-3.

A single major chromosomal region controls natural killer cell-like activity in rainbow trout.

We report the identification of a single major chromosomal region controlling natural killer (NK) cell-like activity in rainbow trout (Oncorhynchus mykiss). A genetic map based on 484 AFLP and 39 microsatellite genotypes from 106 doubled haploid fish was constructed. These fish were produced by androgenesis from a hybrid of two clonal lines divergent in NK-like activity. NK-like activities for 75 of the doubled haploids were quantified by an in vitro chromium release assay utilizing (51)Cr-labeled YAC-1 target cells. Composite interval mapping revealed a single major quantitative trait locus (QTL) associated with NK-like activity in this rainbow trout model. Genetic mapping revealed this QTL to also be unlinked to: fragmented MHC class I and MHC class II regions, the leukocyte receptor cluster, the natural killer cell enhancement factor ( NKEF) gene, the RAG-1 gene, and two QTL associated with resistance to infectious pancreatic necrosis virus in rainbow trout. Collectively, these results extend the utility of rainbow trout as an immunological model and are consistent with the idea that a single chromosomal region homologous to the natural killer cell complex (NKC) located on syntenic portions of mouse chromosome (Chr) 6, human Chr 12, and rat Chr 4 may exist in a lower vertebrate model.

Studies on pathogenesis following single and double infection with viral hemorrhagic septicemia virus and infectious hematopoietic necrosis virus in rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) were bath challenged with viral hemorrhagic septicemia (VHS) virus or infectious hematopoietic necrosis (IHN) virus or with both viruses simultaneously. The viral distribution and development of histologic lesions were examined using immunohistochemistry, while virus titer in kidney was determined by viral titration in cell culture. Single infections with VHS virus and IHN virus showed similar distributions of virus in internal organs. The early identification of virus in gill epithelium, 1 and 2 days postinfection (PI) for VHS virus and IHN virus, respectively, indicates that this organ is the point of entry for both viruses. The detection of VHS virus at 1 day PI and 3 days PI for IHN virus is indicative of kidney and spleen being the target organs for these viruses. A simultaneous infection of VHS virus and IHN virus resulted in both viruses establishing an infection. Further double infection did not result in a statistically significant lower titer of both viruses in kidney but a more restricted distribution of IHN virus in internal organs compared with the single infected group. The most striking finding is that, for IHN virus, virus was not detected in the brain in situ in the double-infected group. This study provides support for the conclusion that simultaneous infection with two piscine rhabdoviruses in a susceptible host results in some degree of interaction at the cell level, leading to a reduced systemic distribution of IHN virus.

Multiplication of Infectious Hematopoietic Necrosis Virus in Rainbow Trout following Immersion Infection: Whole-Body Assay and Immunohistochemistry

Abstract The sites of replication of infectious hematopoietic necrosis virus (IHNV) in infected tissues were detected in fingerling rainbow trout Oncorhynchus mykiss by in situ histologic techniques following immersion infection. Virus antigens in tissues were detected by a neutralizing mouse monoclonal antibody and a one-step anti-mouse biotin-streptavidin conjugated to horseradish peroxidase. The efficiency of infection and virulence of the virus determined by mortality rates showed high virulence of the selected IHNV isolates, and viral replication in individual fish showed that virus content of the fish increased rapidly from the second day to the seventh day postinfection. The earliest viral lesions following infection were detected in the epidermis of the pectoral fins, opercula, and ventral surface of the body. Virus lesions became evident in kidneys on the third day. By the fifth day, when there was a significant increase in virus titer, foci of viral replication were detected in gill tissue and i...

Multiplication of Infectious Hematopoietic Necrosis Virus in Rainbow Trout following Immersion Infection: Organ Assay and Electron Microscopy

Abstract Sequential spread of infectious hematopoietic necrosis virus (IHNV) to tissues of rainbow trout Oncorhynchus mykiss was examined following immersion infection with two different isolates of IHNV, a pathogenic strain and a nonpathogenic strain from rainbow trout. Virus strain 193–110 was highly pathogenic to 1-month-old rainbow trout and caused 100% mortality within 13 d, whereas strain RB-76 was much less virulent, causing 50% mortality by the 19th day. Virus titers of 1-month-old fingerling fish dying soon after infection were significantly higher than titers of those dying later. Assays of dissected tissues showed that gills of infected 2-month-old fingerlings contained virus as early as 16 and 20 h postinfection, with definite replication occurring at 48 h. The early presence of the virus in the gills followed shortly by appearance of the virus in the kidneys and spleen indicated that the virus spreads rapidly to the target organs. Virus was detected in many other organs at lower levels on the...

Detection and transmission of infectious hematopoietic necrosis virus in rainbow trout.

Detection and transmission of Infectious Hematopoietic Necrosis Virus in rainbow trout (Salmo gairdneri) was studied at a commercial trout hatchery. Transmission of virus was demonstrated via water, feed and contaminated eggs. If eggs from carrier females were incubated several weeks in virus-free water, the resulting fry did not become infected. However, if fry subsequently became infected they were lifetime carriers. Infectious virus was readily detectable in most tissues of moribund fish; in carriers it was detected in sex products of spawning fish, and in samples from the intestine of post-spawning fish, but not in samples from blood, feces, kidney, or liver. The carrier rate was not significantly different between sexes. It was concluded that adult carriers are the reservoir of infection and that transmission occurs primarily when carriers shed virus and expose susceptable fish or eggs.