Necrosis Virus (necrosis + virus)

Distribution by Scientific Domains


Selected Abstracts


Synergistic effects of esfenvalerate and infectious hematopoietic necrosis virus on juvenile chinook salmon mortality

ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 7 2005
Mark A. Clifford
Abstract Sublethal concentrations of pollutants may compromise fish, resulting in increased susceptibility to endemic pathogens. To test this hypothesis, juvenile chinook salmon (Oncorhynchus tshawytscha) were exposed to sublethal levels of esfenvalerate or chlorpyrifos either alone or concurrently with infectious hematopoietic necrosis virus (IHNV). Three trials were performed with fish exposed to concentrations of IHNV between 0.8 102 and 2.7 106 plaque-forming units/ml and to 5.0 ,g/L of chlorpyrifos or 0.1 ,g/L of esfenvalerate. The presence and concentration of IHNV in dead fish were assayed by virus isolation and plaque assay techniques, respectively. Among groups exposed to both esfenvalerate and IHNV, 83% experienced highly significant (p < 0.001) mortality, ranging from 20 to 90% at 3 d post-virus exposure, and cumulatively died from 2.4 to 7.7 d sooner than fish exposed to IHNV alone. This trend was not seen in any other treatment group. Virus assays of dead fish indicate a lethal synergism of esfenvalerate and IHNV. Chlorpyrifos had no observed effect on total mortality or IHNV susceptibility. The present results suggest that accepted levels of pollutants may be seemingly nonlethal to fish but, in fact, be acting synergistically with endemic pathogens to compromise survivorship of wild fish populations through immunologic or physiologic disruption. [source]


Characterization of susceptibility and carrier status of burbot, Lota lota (L.), to IHNV, IPNV, Flavobacterium psychrophilum, Aeromonas salmonicida and Renibacterium salmoninarum

JOURNAL OF FISH DISEASES, Issue 7 2010
M P Polinski
Abstract In this study, susceptibility and potential carrier status of burbot, Lota lota, were assessed for five important fish pathogens. Burbot demonstrated susceptibility and elevated mortality following challenge with infectious haematopoietic necrosis virus (IHNV) by immersion and to Aeromonas salmonicida by intraperitoneal (i.p.) injection. IHNV persisted in fish for at least 28 days, whereas A. salmonicida was not re-isolated beyond 17 days post-challenge. In contrast, burbot appeared refractory to Flavobacterium psychrophilum following intramuscular (i.m.) injection and to infectious pancreatic necrosis virus (IPNV) by immersion. However, i.p injection of IPNV resulted in re-isolation of virus from fish for the duration of the 28 day challenge. Renibacterium salmoninarum appeared to induce an asymptomatic carrier state in burbot following i.p. injection, but overt manifestation of disease was not apparent. Viable bacteria persisted in fish for at least 41 days, and bacterial DNA isolated by diagnostic polymerase chain reaction was detected from burbot kidney tissue 90 days after initial exposure. This study is the first to investigate susceptibility of burbot to selected fish pathogens, and this information will aid in efforts to culture and manage this species. [source]


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

JOURNAL OF FISH DISEASES, Issue 6 2010
S P Jonstrup
Abstract 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. [source]


Tissue tropism of nervous necrosis virus (NNV) in Atlantic cod, Gadus morhua L., after intraperitoneal challenge with a virus isolate from diseased Atlantic halibut, Hippoglossus hippoglossus (L.)

JOURNAL OF FISH DISEASES, Issue 8 2009
K Korsnes
Abstract Atlantic cod, Gadus morhua, averaging 100 g, were experimentally challenged by intraperitoneal injection of nervous necrosis virus (NNV) originating from Atlantic halibut. Cod tissues, including blood, gill, pectoral fin, barbel, ventricle, atrium, spleen, liver, lateral line (including muscle tissue), eye (retina) and brain, were sampled at day 25 and 130 and investigated by real-time RT-PCR for the presence of NNV. Relative quantifications at day 130 were calculated using the 2,,,Ct method. Immunosuppression by injection of prednisolone-acetate was introduced for a 30-day period, and tissue sampled at day 180 and relative quantification estimated. No mortality or clinical signs of disease were observed in the challenged group. The challenge resulted in detection of NNV in blood, spleen, kidney, liver, heart atrium and heart ventricle at day 25, and by the end of the experiment NNV showed a clear increase in brain and retina, suggesting these to be the primary tissues for viral replication. There was no increase in the relative amount of NNV in blood, atrium, ventricle, spleen, liver and kidney. Corticosteroid implants resulted in a weak increase in virus RNA in spleen, kidney, liver and brain. These findings suggest that Atlantic cod is susceptible to infection with NNV from halibut. The observed tissue tropism patterns suggest an initial viraemic phase, followed by neurotrophy. Head-kidney is the best tissue identified for possible NNV detection by non-lethal biopsy, but detection was not possible in all injected fish. [source]


Genetic analysis of aquabirnaviruses isolated from wild fish reveals occurrence of natural reassortment of infectious pancreatic necrosis virus

JOURNAL OF FISH DISEASES, Issue 7 2009
I Romero-Brey
Abstract In this study, we report the sequencing of the whole genome [including the 5, and 3, non-coding regions (NCR) of both segments A and B] of seven birnavirus strains isolated from wild fish from the Flemish Cap (FC) fishery at Newfoundland, Canada. From analysis and comparison of the sequences, most of the FC isolates clustered with the North American reference strains West Buxton (WB), Dry Mill and Jasper. One strain was included in the same genotype as the European strain Ab. In addition, at least in one case cohabitation of both type strains in an individual fish was demonstrated. These results clearly suggest the acquisition of the viruses from two different sources. The prevalence of the American type is easily explained by the close proximity of this fishing bank to the American coast whereas, although surprising, the presence of the European type strain could be because of migration of fish from European waters. In one strain, segment A and B sequences were typed differently (WB and Ab, respectively). These findings indicate natural reassortment between two strains of aquabirnaviruses in a host. [source]


Inactivated infectious haematopoietic necrosis virus (IHNV) vaccines

JOURNAL OF FISH DISEASES, Issue 10 2008
E Anderson
Abstract The inactivation dynamics of infectious haematopoietic necrosis virus (IHNV) by ,-propiolactone (BPL), binary ethylenimine (BEI), formaldehyde or heat and the antigenic and immunogenic properties of the inactivated vaccines were evaluated. Chemical treatment of IHNV with 2.7 mm BPL, 1.5 mm BEI or 50 mm formaldehyde abolished virus infectivity within 48 h whereas heat treatment at 50 or 100 C rendered the virus innocuous within 30 min. The inactivated IHNV vaccines were recognized by rainbow trout, Oncorhynchus mykiss, IHNV-specific antibodies and were differentially recognized by antigenic site I or antigenic site II IHNV glycoprotein-specific neutralizing monoclonal antibodies. The BPL inactivated whole virus vaccine was highly efficacious in vaccinated rainbow trout challenged by waterborne exposure to IHNV 7, 28, 42 or 56 days (15 C) after immunization. The formaldehyde inactivated whole virus vaccine was efficacious 7 or 11 days after vaccination of rainbow trout but performed inconsistently when tested at later time points. The other vaccines tested were not efficacious. [source]


Strong genetic influence on IPN vaccination-and-challenge trials in Atlantic salmon, Salmo salar L.

JOURNAL OF FISH DISEASES, Issue 8 2008
A Ramstad
Abstract Two series of experimental challenge trials were performed for evaluation of multivalent oil-adjuvanted vaccines with and without an infectious pancreatic necrosis virus (IPNV) antigen component. In both the trial series, Atlantic salmon were hatched, reared, vaccinated and subjected to temperature and light manipulation to induce smoltification. When ready for sea the fish were transported to the VESO Vikan experimental laboratory for bath or cohabitant challenge with IPNV. In the first series, four vaccination and bath challenge trials involving 2-year classes of experimental fish were conducted. In the second series, three groups of eyed eggs of Atlantic salmon allegedly differing in their innate resistance to IPNV were used (Storset, Strand, Wetten, Kjglum & Ramstad 2007). Hatching, rearing and smoltification were synchronized for each group, and fish from each genetic group were randomly allocated IPN vaccine, reference vaccine or saline before being placed into parallel tanks for bath or cohabitant challenge. In the first series of trials, IPN-specific mortality commenced on day 10,12 after bath challenge. Replicates showed similar results. In trials 1 and 2 belonging to the same experimental fish year class, the average cumulative control mortality reached 60.6% and 79.5%, respectively, whereas in trials 3 and 4 belonging to the following year class the control mortality was consistently below 50%. In the second series of trials, the experimental fish originating from allegedly IPN susceptible parents consistently showed the highest cumulative mortality among the unvaccinated controls (>75%) whereas smolts derived from allegedly IPNV resistant parents showed only 26,35% control mortality. The IPN-vaccinated fish experienced significantly improved survival vs. the fish immunized with reference vaccine, with RPS values above 75% in the IPN susceptible strain. In the IPN resistant strain, the protection outcomes were variable and in part non-significant. The outcome of both the trial series suggests that control mortalities above 50% are necessary to reliably demonstrate specific protection with IPN vaccines. [source]


Distribution of infectious pancreatic necrosis virus (IPNV) in wild marine fish from Scottish waters with respect to clinically infected aquaculture sites producing Atlantic salmon, Salmo salar L.

JOURNAL OF FISH DISEASES, Issue 3 2008
I S Wallace
Abstract This study represents the first large-scale investigation of IPNV in Scottish wild marine fish. Kidney samples were taken from 30 627 fish comprising 37 species and 45 isolations were made from nine different species, illustrating these as reservoirs of IPNV in Scottish waters. The estimated prevalence of IPNV in the Scottish marine environment was low at 0.15% (90% confidence intervals, (CI) of 0.11,0.19%). This was significantly greater in fish caught less than 5.0 km from IPN-positive fish farms in Shetland, at 0.58% (90% CI of 0.45,0.77%). This prevalence persisted and did not significantly decrease over the 16-month period of study. The estimated prevalence of IPNV for each positive species was less than 1% with the statistically non-significant exceptions of flounder, Platichthys flesus (L.), at 12.5% (90% CI of 0.64,47.06%) and saithe, Pollachius virens (L.), at 1.11% (90% CI of 0.49,2.19%). The 45 isolates were titrated and all but two were below the detection limit of the test (<55 PFU g,1). Titres of 3.8 102 PFU g,1 and 2.8 101 PFU g,1 were calculated from common dab, Limanda limanda (L.), and saithe, respectively. This study provides evidence that clinical outbreaks of IPN in farmed Atlantic salmon may cause a localized small increase in the prevalence of IPNV in wild marine fish. [source]


Field validation of experimental challenge models for IPN vaccines

JOURNAL OF FISH DISEASES, Issue 12 2007
A Ramstad
Abstract Atlantic salmon S1/2 pre-smolts from the VESO Vikan hatchery were assigned to study groups, i.p. immunized with commercially available, multivalent oil-adjuvanted vaccines with (Norvax Compact 6 , NC-6) or without (Norvax Compact 4 , NC-4) recombinant infectious pancreatic necrosis virus (IPNV) antigen. A control group received saline solution. When ready for sea, the fish were transported to the VESO Vikan experimental laboratory, where two identical tanks were stocked with 75 fish per group before being transferred to 10 C sea water and exposed by bath to first passage IPNV grown in CHSE-214 cells. The third tank containing 40 fish from each group was challenged by the introduction of 116 fish that had received an i.p injection of IPNV-challenge material. The remaining vaccinated fish were transported to the VESO Vikan marine field trial site and placed in two identical pens, each containing approximately 53 000 fish from the NC-6 group and 9000 fish from the NC-4 group. In the experimental bath challenge trial, the cumulative mortality was 75% and 78% in the control groups, and the relative percentage survival (RPS) of the NC-6-immunized fish vs. the reference vaccine groups was 60% and 82%, respectively. In the cohabitation challenge, the control mortality reached 74% and the IPNV-specific vaccine RPS was 72%. In both models, the reference vaccine lacking IPNV antigen gave a moderate but statistically significant non-specific protection. In the field, a natural outbreak of infectious pancreatic necrosis (IPN) occurred after 7 weeks lasting for approximately 3.5 months before problems due to winter ulcers became dominating. During this outbreak, mortality in the NC-4 groups were 33.5% and 31.6%, respectively, whereas mortality in the NC-6 groups were 6.9% and 5.3%, respectively, amounting to 81% IPNV-specific protection. In conclusion, the IPN protection estimates obtained by experimental challenges were consistent between tanks, and were confirmed by the field results. [source]


Isolation of a cyprinid herpesvirus 2 from goldfish, Carassius auratus (L.), in the UK

JOURNAL OF FISH DISEASES, Issue 11 2007
K R Jeffery
Abstract Haematopoietic necrosis virus [cyprinid herpesvirus 2 (CyHV-2)] was isolated during disease outbreaks in goldfish, Carassius auratus, at an ornamental fish retail site in southern England in 2004. Signs of disease included lethargy and inappetence and were first seen after water temperatures increased from 14,15 to 19,21 C. External gross pathology included pale patches on the gills and skin and internally the spleen was enlarged, often with distinctive white nodules. The most prominent histopathological changes observed were necrotic lesions in the spleen and kidney and focal patches of necrosis in the gill lamellae. Necrotic cells often contained nuclei with marginated chromatin and pale intranuclear inclusions. Ultrastructural examination of the spleen tissue revealed typical herpesvirus-like particles measuring 100 nm in diameter. The virus was isolated from extracts of gill tissue in KF-1 cells at 20 C and oligonucleotide primer sets were designed based on conserved gene sequences and used to amplify viral DNA by polymerase chain reaction (PCR). The PCR assays were then used to detect the virus in DNA extracted from tissues sampled during earlier disease investigations at the retail site owner's holding facility in 2002 and 2003 and stored at ,70 C since then. Polymerase gene-specific PCR amplification products obtained from tissue samples and from the virus isolated in cell culture shared 100% nucleotide sequence identity with the published sequence for CyHV-2. [source]


Analysis of the incidence of infectious pancreatic necrosis mortality in pedigreed Atlantic salmon, Salmo salar L., populations

JOURNAL OF FISH DISEASES, Issue 11 2006
D R Guy
Abstract A total of 77 124 Atlantic salmon post-smolts, representing 197 full-sib families produced by 149 males and 197 females, experienced a field challenge from infectious pancreatic necrosis virus (IPNV), following transfer to three separate seawater sites. The first IPN mortality was observed 45 days after transfer, and the duration of the epidemic varied between 37 and 92 days among sites. Mortalities were traced to their parental families by PIT (Passive Integrated Transpondes) tag records and DNA genotyping. Full-sib family mean incidence of mortality was calculated for each family on each site. Heritabilities were estimated based on the heterogeneity of chi-square using incidence within half-sib families and the variance in incidence among full-sib families, both on the observed and underlying liability scale. The observed correlation among families across sites was used to estimate genetic correlations. The overall mortality rate was 10.8%, with only small differences between sites, ranging from 10.3% to 11.9%. Heritabilities on the liability scale were found to be moderate to strong, and ranged between 0.24 and 0.81, with a pooled estimate of 0.43, greater than is typically associated with disease traits. Genetic correlations among sites were all substantial, between 0.71 and 0.78, and indicated that a substantial component of the genetic variation displayed within sites was common to all. The results show that field challenges can yield very good genetic information on family differences in resistance, especially when replicated over sites, which may then be developed for use in selection for breeding strains of Atlantic salmon with greater resistance to IPN. [source]


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

JOURNAL OF FISH DISEASES, Issue 10 2006
J Melena
Abstract 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 (0,1 103 copies ,g,1 of DNA). In addition, surviving shrimp previously exposed to inactivated WSSV at PL35 also contained few WSSV (0,2 103 copies ,g,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. [source]


Detection of infectious haematopoietic necrosis virus and infectious salmon anaemia virus by molecular padlock amplification

JOURNAL OF FISH DISEASES, Issue 4 2006
P J Millard
Abstract A new method for the molecular detection of the fish pathogens, infectious haematopoietic necrosis virus (IHNV) and infectious salmon anaemia virus (ISAV), is described. By employing molecular padlock probe (MPP) technology combined with rolling circle amplification (RCA) and hyperbranching (Hbr), it is possible to detect RNA target sequence from these viruses at levels comparable with those detected by the polymerase chain reaction (PCR), but without prior reverse transcription. The use of MPP technology combined with RCA and Hbr for the detection of IHNV and ISAV in fish exhibited selectivity comparable with that of PCR while potentially reducing the time and cost required for analysis. The method described was used to detect as few as 104 DNA oligonucleotide targets and was sequence-specific at the single base level. Viral RNA could be detected directly, either alone or in the presence of non-viral RNA from fish tissue. This technology is applicable for detecting a variety of microbes, in addition to IHNV and ISAV, and is ideal for further integration into a biosensor platform for on-site diagnosis of pathogen infection in fish. [source]


Aquatic birnavirus induces apoptosis through activated caspase-8 and -3 in a zebrafish cell line

JOURNAL OF FISH DISEASES, Issue 3 2005
J-R Hong
Abstract In this study, the possible influence of temperature on infectious pancreatic necrosis virus (IPNV)-induced apoptosis in a zebrafish liver epithelium (ZLE) cell line was investigated. At a lower temperature (18 C), there was expression of viral proteins VP2 and VP3 at 4 h post-infection (p.i.). At this time no expression was found in the high temperature group at 28 C. The cell survival ratio was 52 and 18% at 24 and 48 h p.i., respectively, during IPNV infection at 18 C. In addition, we assayed for apoptosis in IPNV-infected cells with terminal deoxynucleotidyl transferase (TdT)-mediated end labelling (TUNEL) of DNA at different dosages of virus. We found a ratio of apoptotic cells of 8 and 25% at 12 and 18 h p.i., respectively, in the multiplicity of infection (MOI) 1 group. The MOI 10 group had 20 and 45% apoptotic cells at 12 and 18 h, respectively. Furthermore, at 18 C IPNV activated the caspase-8 and 3 from 1.5 to 2 times at 12 and 18 h p.i., respectively. Taken together, these findings suggest that successful virus replication occurs at the low temperature (18 C) compared with the non-permissive temperature of 28 C. Thus, IPNV replication is capable of activating caspase-8 and -3 and inducing host apoptosis. [source]


Aquabirnaviruses isolated from marine organisms form a distinct genogroup from other aquabirnaviruses

JOURNAL OF FISH DISEASES, Issue 11 2004
C X Zhang
Abstract A phylogenetic tree of aquabirnaviruses, including marine birnaviruses (MABV) and infectious pancreatic necrosis virus (IPNV), was developed based on the nucleotide sequences and deduced amino acid sequences of the polyprotein and VP5 genes of genomic segment A. In the polyprotein of MABV strains, the amino acid sequences were very similar, with identities of 98.3,99.7%. Twenty-one unique amino acid residues were found in the deduced amino acid sequences of the polyprotein gene of MABV strains. The phylogenetic tree based on the nucleotide sequence of genomic segment A and polyprotein sequences showed that 31 aquabirnavirus strains were clustered into seven genogroups. All MABV strains isolated in Japan and Korea were clustered into one genogroup which was distinct from other aquabirnaviruses. The seventh genogroup containing all MABV strains showed amino acid sequence similarities of 80.7,90.6% with other genogroups. In VP5, four unique residues were found in MABV strains when compared with IPNV strains. The MABV strains exhibited amino acid sequence similarities of 63.9,86.4% with IPNV strains. The amino acid sequences of VP5 were conserved among MABV strains, but differed from those of IPNV strains. The MABV strains isolated from different host species and different geographical areas were very similar to each other, suggesting that the MABV are distinct from the other genogroups. [source]


Histopathological studies on viral nervous necrosis of sevenband grouper, Epinephelus septemfasciatus Thunberg, at the grow-out stage

JOURNAL OF FISH DISEASES, Issue 7 2004
S Tanaka
Abstract Viral nervous necrosis caused by sevenband grouper nervous necrosis virus (SGNNV) has occurred in grow-out stages (0,3 years old) of sevenband grouper, Epinephelus septemfasciatus, since the 1980s. In the present study, based on histopathological features of the central nervous system (CNS) in naturally diseased fish, pernasal infection experiments using grow-out fish were performed and pernasal infection was established as a putative invasion route of SGNNV. The definite SGNNV-targeted cells were determined by histopathological studies including indirect fluorescent antibody test and electron microscopy. Nerve cells in the olfactory lobe were most extensively necrotized with vacuolation followed by infiltration of microglia and macrophages. Purkinje cells and Golgi cells were extensively infected in the cerebellum. Megalocells and small nerve cell nuclei were also infected in the preoptic area, thalamus, medulla oblongata and spinal cord. Only a few small nerve cells were infected in the olfactory bulb and optic tectum. The retina of some diseased fish displayed vacuolated bipolar cells of the inner nuclear layer and in the ganglion cell layer. These SGNNV-infected nerve cells displayed viroplasmic inclusions containing virions, vacuoles and myelin-like structures. Based on observed histopathological changes, the lesion of the CNS was characterized by encephalitis but not encephalopathy. [source]


Establishment and characterization of two new cell lines derived from flounder, Paralichthys olivaceus (Temminck & Schlegel)

JOURNAL OF FISH DISEASES, Issue 11-12 2003
M S Kang
Abstract Two new cell cultures from flounder, Paralichthys olivaceus (Temminck & Schlegel), flounder fin (FFN) cells from fin tissue and flounder spleen (FSP) cells from spleen tissue, were established and characterized. The cells multiplied well in Eagle's minimum essential medium, supplemented with 10% foetal bovine serum, and have been subcultured more than 100 times, becoming continuous cell lines. Modal diploid chromosome number of FFN and FSP cells was 64 and 62, respectively. Polymerase chain reaction products were obtained from FFN and FSP cells with primer sets of microsatellite markers of flounder. Optimal growth temperature was 20 C and consisted of epithelioid cells. FFN and FSP cells showed cytopathic effects after inoculation of infectious pancreatic necrosis virus, marine birnavirus, chum salmon virus, infectious haematopoietic necrosis virus, spring viraemia of carp virus and hirame rhabdovirus. Thus these new cell lines may be useful for studying a wide range of fish viruses. [source]


Comparison of the efficiency and sensitivity of virus isolation and molecular methods for routine diagnosis of infectious haematopoietic necrosis virus and infectious pancreatic necrosis virus

JOURNAL OF FISH DISEASES, Issue 2 2002
-Maganja, D Barli
Infectious haematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are widely distributed fish pathogens in Europe. A reverse transcriptase,polymerase chain reaction (RT,PCR) assay was developed for the detection of both viruses as an alternative method to virus assay in cell culture. Oligonucleotide primers corresponding to highly conserved regions of glycoprotein G-gene sequences were used for IHNV. For the detection of IPNV the VP2-coding region was selected for RT,PCR amplification. Products of the expected size were amplified from total ribonucleic acid (RNA) extracts of infected cells. The optimized RT,PCR methods successfully detected viral RNA from ovarian and seminal fluids and other organs. To enhance the sensitivity and specificity of RT,PCR, a semi-nested PCR assay was tested using additional specific inner primers for reamplification of products obtained by RT,PCR. Because of the possibility of template carry-over contamination, a closed one step RT,PCR method was tested. This technically simplified approach was then combined with the PCR,enzyme linked immunosorbent assay (ELISA) method for the detection of amplification products and verification using specific biotinylated probes. The test provides an additional tool for the detection of IHNV and IPNV which is rapidly and easily performed and is highly sensitive, especially for the detection of IHNV in fish samples coinfected with IPNV. The PCR,ELISA method for the detection of RT,PCR products enables the screening of large numbers of samples and offers the possibility for automatisation of diagnostic work. [source]


Real-time PCR for the detection and quantitative analysis of IHNV in salmonids

JOURNAL OF FISH DISEASES, Issue 6 2001
K Overturf
The rapid identification and quantification of virus in diseased fish is a goal both conservationists and commercial aquaculturists have struggled to attain. Recently a technique for the detection of viral mRNA particles that uses fluorescent tagging and amplification has been developed. Utilizing primers and fluorescent labelled probes generated for the specific identification of the nucleocapsid (N) and glycoprotein (G) genes of infectious haematopoietic necrosis virus (IHNV), and an instrument that measures cyclic emittance of fluorescence, the presence or absence of virus can be easily and rapidly confirmed. This method is not only useful in confirming viral presence but is effective in measuring the relative or absolute quantity of virus present within the sample. This allows for the determination of the health status of a carrier fish by measuring the quantity of viral genomes or transcribed viral genes present. Because this method is based on sequence detection, instead of virus isolation in cell culture, it is also effective in determining the presence of pathogenic organisms from water, fish feeds, or other potential reservoirs of infection. [source]


Characterization of grouper nervous necrosis virus (GNNV)

JOURNAL OF FISH DISEASES, Issue 1 2001
S C Chi
Grouper nervous necrosis virus (GNNV) was isolated from moribund grouper larvae, Epinephelus sp., using a fish cell line GF-1. The present study describes the biochemical and biophysical properties of GNNV and the expression of GNNV in diseased grouper larvae. Viral protein was detectable in most of the GNNV-infected GF-1 cells by the fluorescent antibody technique (FAT) after 12 h post-infection (p.i.), although no cytopathic effect (CPE) appeared at that time. Clear CPE developed on the third day, and complete disintegration of the monolayer occurred over the subsequent two days. The infectivity of GNNV can be blocked following treatment at 60 C for 1 h. GNNV was sensitive to pH 3 and pH 10,12 with a 4 log10 drop in infectivity. Purified GNNV was analysed by SDS,PAGE, and then stained with periodic acid silver. The positive staining indicated that its two capsid proteins were glycoproteins. Genomic RNAs of GNNV were extracted from purified virions and analysed. The molecular weights of genomic RNAs were 1.02 106 and 0.50 106 Da. The T2 region of the coat protein gene of GNNV was amplified by polymerase chain reaction (PCR), and the multiple alignment of the T2 sequence of two GNNV isolates with four genotypes of fish nodaviruses revealed that these two isolates (GNNV9410 and GNNV9508) belong to the red-spotted grouper nervous necrosis virus (RGNNV) genotype. The tissue distribution of GNNV in naturally infected grouper larvae was investigated by in situ hybridization using a dig-labelled probe, which showed that GNNV was not only detected in the brain and retina, but also in the gill, skeletal muscle, liver, pyloric gland, intestine and blood cells in the heart. [source]


Development of a sensitive diagnostic assay for fish nervous necrosis virus based on RT-PCR plus nested PCR

JOURNAL OF FISH DISEASES, Issue 5 2000
L Dalla Valle
A polymerase chain reaction (PCR)-based assay to detect nervous necrosis virus (NNV) in fish was developed by using two sets of primers designed on a highly conserved region of the coat protein gene encoded by RNA2 of NNV. The first pair of primers amplified a fragment of 605 bp by one-step reverse-transcription (RT)-PCR, while the second pair amplified an internal segment of 255 bp by nested PCR. Addition of nested PCR increased the assay sensitivity 100-fold when carried out in a separate tube (two-step assay) and 10-fold when performed in the same tube (one-step assay). The sensitivity of the two-step assay was 104 times higher than that of virus cultivation. Nested PCR served also to confirm the specificity of the first amplification, as verified also by Southern hybridization analysis and direct sequencing. In species known to be susceptible to infection, such as European sea bass, Dicentrarchus labrax, and gilthead seabream, Sparus aurata, NNV was often detectable in brain tissue by RT-PCR alone but only by the two-step assay in blood, sperm, ovarian tissue or larvae. The same was true for sperm and ovarian tissue of shi drum, Umbrina cirrosa. NNV was also detected in the brains of Japanese red seabream, Pagrus major and brown meagre, Sciaena umbra, suggesting that these species can also be infected. No NNV was detected in samples of Artemia salina nauplii and rotifers obtained from a fish farm with an NNV outbreak. The inclusion of nested PCR in the assay appears to be necessary to screen out NNV-positive broodfish by blood sampling and testing of their larval progeny. [source]


Biology of the European large raspberry aphid (Amphorophora idaei): its role in virus transmission and resistance breakdown in red raspberry

AGRICULTURAL AND FOREST ENTOMOLOGY, Issue 1 2009
Lindsay S. McMenemy
Abstract 1,The European large raspberry aphid Amphorophora idaei Brner is the most important vector of viral diseases afflicting commercially grown red raspberry (Rubus idaeus L.) in Northern Europe, with European raspberry production amounting to 416 000 tonnes per annum. This review synthesizes existing knowledge on its biology and interactions with other organisms, including its host plant and the viral pathogens it vectors. 2,Information about trophic interactions with other insect herbivores and natural enemies is reviewed. Vine weevils Otiorhynchus sulcatus compromise aphid resistance in some raspberry cultivars, increasing A. idaei abundance by 80%. Parasitoids show mixed success in parasitizing A. idaei, although Aphidius ervi attack rates more than doubled when A. idaei fed on a partially susceptible raspberry cultivar, compared with a resistant variety. These findings are discussed in the context of potential biological control as part of an integrated pest and disease management framework. 3,Amphorophora idaei transmits four known viruses: Black raspberry necrosis virus, Raspberry leaf mottle virus, Raspberry leaf spot virus and Rubus yellow net virus, with A. idaei taking as little as 2 min to transmit some viruses. 4,Existing control strategies, including resistant cultivars, insecticides and eradication of disease from parent plants, are described. In particular, strong selection pressures have resulted in A. idaei overcoming genetic resistance in many raspberry cultivars and most insecticides are now ineffective. 5,Future directions for the sustained control of A. idaei are suggested, taking into consideration the possible effects of climate change and also changes in agronomic practices in U.K. agriculture. [source]


The competence of four thrips species to transmit and replicate four tospoviruses

PLANT PATHOLOGY, Issue 2 2004
T. Nagata
The tospoviruses Tomato spotted wilt virus (TSWV), Tomato chlorotic spot virus (TCSV), Groundnut ringspot virus (GRSV) and Chrysanthemum stem necrosis virus (CSNV) are well-known pathogens on tomato in Brazil. The thrips species Frankliniella occidentalis, F. schultzei, Thrips tabaci and T. palmi were studied for their competence to transmit these tospoviruses. Frankliniella occidentalis transmitted all four tospoviruses with different efficiencies. Frankliniella schultzei transmitted TCSV, GRSV and CSNV. Although F. schultzei has been reported as a vector of TSWV, the F. schultzei population in the present study did not transmit the TSWV isolate used. A population of T. tabaci known to transmit Iris yellow spot virus (onion isolate) did not transmit any of the studied tospoviruses, and nor did T. palmi. Replication of these tospoviruses could be demonstrated by ELISA, not only in the thrips species that could transmit them, but also in those that could not. The results strongly suggest that competence to transmit is regulated not only by the initial amount of virus acquired and replication, but also by possible barriers to virus circulation inside the thrip's body. [source]


A dual infection by infectious cuticular epithelial necrosis virus and a Chlamydia -like organism in cultured Litopenaeus vannamei (Boone) in Ecuador

AQUACULTURE RESEARCH, Issue 11 2001
R Jimenez
During 1996, microscopic examinations of post larvae and juveniles of moribund Litopenaeus vannamei showed multifocal necrosis in the cuticular epithelial tissues. In addition to these severe degenerative alterations in the epithelial cells typical of infectious cuticular epithelial necrosis virus (ICENV), columnar cells of the epithelium displayed small round intracytoplasmic inclusions in the necrotic tissue. Examination by electron microscopy of affected tissues demonstrated prokaryotic organisms in the cytoplasm of epithelial cells delineated by a distinct cytoplasmic vesicle; the prokaryotic organisms were morphologically similar to the genus Chlamydia. The necrotic tissue also showed the presence of particles of ICENV; the double infection by two different organisms in cuticular epithelial cells has not been reported previously. Two distinct stages in the intracellular development of a Chlamydia -like organism were recognized: (1) pleomorphic elementary bodies (EBs) that were spherical to oval were often observed in the process of division or in forming a common chain of three cells, the cells were surrounded by a rigid cell envelope and the presence of a cap or plaque hexagonally arrayed; (2) the reticular bodies (RBs) were forms often in the process of division. These cells had an electron-dense cytoplasm and contained a loose network of nuclear fibrils and a more fragile cell envelope. Regardless of the development stages of the Chlamydia -like organism within the cytoplasmic vesicles, ICENV particles were observed, either dispersed or in clusters, surrounded or inside the vesicles. The potential adverse impact of this dual infection on shrimp culture should be considered, especially in high-density operations. [source]