Infectious Pancreatic Necrosis Virus Carrier Research Articles

Restricted replication of viral hemorrhagic septicemia virus (VHSV) in a birnavirus-carrier cell culture.

Viral hemorrhagic septicemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV) are economically important pathogens of the salmonid aquaculture industry. In previous work we demonstrated that a cell line persistently infected with IPNV (EPCIPNV) exhibited antiviral activity against superinfection with the heterologous virus VHSV. This work extends our study by analyzing the replication of VHSV in the IPNV-persistently infected cells. At early and late stages of infection VHSV RNA synthesis, as well as VHSV-induced syncytia formation, were examined in EPCIPNV cultures. During the course of VHSV infection the accumulation of VHSV RNA is inhibited in EPCIPNV cells. Typical VHSV-induced membrane fusion at the late stages of infection is also absent in the IPNV carrier cultures. VHSV binding and fusion to EPCIPNV cells did not appear to be impaired, but a potent inhibitory effect on VHSV RNA synthesis is exerted at early times of infection in the IPNV carrier culture. In conclusion, the EPCIPNV cells are considered to be a useful system to study viral interference as well to analyze the mechanisms underlying the phenomenon of superinfection exclusion.

Immune responses to oral pcDNA-VP2 vaccine in relation to infectious pancreatic necrosis virus carrier state in rainbow trout Oncorhynchus mykiss

The VP2 gene of infectious pancreatic necrosis virus, encoded in an expression plasmid and encapsulated in alginate microspheres, was used for oral DNA vaccination of fish to better understand the carrier state and the action of the vaccine. The efficacy of the vaccine was evaluated by measuring the prevention of virus persistence in the vaccinated fish that survived after waterborne virus challenge. A real-time RT-qPCR analysis revealed lower levels of IPNV-VP4 transcripts in rainbow trout survivors among vaccinated and challenged fish compared with the control virus group at 45 days post-infection. The infective virus was recovered from asymptomatic virus control fish, but not from the vaccinated survivor fish, suggesting an active role of the vaccine in the control of IPNV infection. Moreover, the levels of IPNV and immune-related gene expression were quantified in fish showing clinical infection as well as in asymptomatic rainbow trout survivors. The vaccine mimicked the action of the virus, although stronger expression of immune-related genes, except for IFN-1 and IL12, was detected in survivors from the virus control (carrier) group than in those from the vaccinated group. The transcriptional levels of the examined genes also showed significant differences in the virus control fish at 10 and 45 days post-challenge.

Assessment of the Risk of White Sturgeon to become Infected and Potential Carriers of Infectious Pancreatic Necrosis Virus

Little scientific information is available to assess whether White Sturgeon Acipenser transmontanus can become infected and potential carriers of infectious pancreatic necrosis virus (IPNV). To assess this risk, monitoring results of adult and progeny White Sturgeon were examined from waters historically stocked with salmonid fish known to be IPNV carriers. From 1999 through 2004 White Sturgeon from a total of 30 separate families whose parentage came from waters historically stocked with IPNV carrier fish were tested. Duplicate groups of 25 juvenile Snake River White Sturgeon were waterborne exposed to 1.0×10(4) 50% tissue culture infective dose (TCID50)/mL of water for 1 h and an additional group was injected intraperitoneally with 1.0×10(5) TCID50/fish. A negative control group was handled similarly but was not exposed to the virus. No morbidity was detected in any of the treatment groups or the negative control. At 34, 40, 47, and 54 d postexposure to IPNV, virus reisolation was attempted on five fish from each group, and an additional five fish from each group were examined for histological changes consistent with an IPNV infection. At 34 and 40 d postinjection with IPNV, 20% (one of five) of the fish tested positive for the virus per sample interval; however, fish from groups that were waterborne-exposed to IPNV were all negative. At 47 and 54 d after exposure or injection with IPNV an additional five fish from each group were tested at each sample interval and all results were negative. Histological analysis of target tissue obtained from five fish per group at 34 and 54 d postinfection also failed to detect any consistent change associated with an IPNV infection. These results suggest that the risk of White Sturgeon to become infected and develop into potential carriers of IPNV is negligible.

Antiviral activity produced by an IPNV-carrier EPC cell culture confers resistance to VHSV infection

Infectious pancreatic necrosis virus (IPNV), a fish birnavirus, can establish a persistent infection on epithelioma papulosum cyprinid (EPC) cells producing a carrier state where a small fraction of IPNV-infected cells is maintained in the culture after continuous subculture. The EPC(IPNV) cells are resistant to challenge with IPNV as well as to challenge with viral hemorrhagic septicemia virus (VHSV), a rhabdovirus. In this work, the antiviral effect of the IPNV carrier culture conditioned medium (EPC(IPNV)-CM) was tested and analyzed in detail. EPC cells treated with the carrier culture supernatant become protected against VHSV challenge. Size-fractionation by filtration and acid and heat treatment showed that the IPNV persistently infected cells release an acid-resistant soluble factor in the molecular weight fraction bellow 50 kDa. The capacity of the EPC(IPNV)-CM to induce cytokine genes in EPC cells was also determined by real-time RT-PCR. We found that there is a positive correlation between up-regulation of mx gene expression in EPC cells treated with EPC(IPNV)-CM and protection against VHSV challenge. Our findings indicate that the control of IPNV multiplication in the carrier culture as well as the interference with rhabdovirus replication are connected to the production and release of an antiviral (interferon-like) factor to the medium.

Experimental IPN virus infection of Atlantic salmon parr; recurrence of IPN and effects on secondary bacterial infections in post-smolts

Infectious pancreatic necrosis (IPN) in Atlantic salmon ( Salmo salar L.) is a considerable problem in the Norwegian and Scottish fish farming industry. The disease is a main contributor to the post-smolt mortalities often observed in the first weeks after transfer to seawater. Asymptomatic carriers of infectious pancreatic necrosis virus (IPNV) are common, but the implications this has for the fish are largely unknown. In an experiment which lasted for 10 months, we aimed at investigating if vaccination, smoltification and seawater transfer may affect an IPNV carrier condition established in freshwater, and the possibility of reappearance of IPN in post-smolts. Vice versa, we wanted to see if this IPNV carrier condition could affect the protective effects of vaccines against bacterial pathogens, and the outcome of secondary bacterial infections. No changes in the IPNV carrier condition were registered during the period in freshwater, however a reactivation of the carrier condition occurred after transfer to seawater and 24% of the unvaccinated fish died due to IPN. The vaccine offered some unspecific protection against recurrence, and significantly fewer fish, 7%, died in the vaccinated group. In the challenge experiments with Aeromonas salmonicida and Vibrio salmonicida 15 weeks and 28 weeks after vaccination, respectively, the vaccine offered the same good protection against the bacterial pathogens in both IPNV carriers and in non-carriers. In the unvaccinated groups the time point of challenge with A. salmonicida concurred with the onset of IPN mortality in the IPNV carrier group, and significantly more fish in this group died compared to the control fish. The V. salmonicida challenge was performed several weeks after the IPN mortality had stopped. Mortality levels reached over 90% in both unvaccinated groups, but the fish started to die earlier and mortalities were significantly higher at day 8 till 12 after challenge in the IPNV carrier group, indicating that the fish could still be affected several weeks after the recurrence of IPNV. An asymptomatic carrier condition did not seem to have negative impact on the Atlantic salmon during the freshwater stage. However, we have demonstrated that an IPNV carrier state implies a risk of recurrence resulting in IPN mortality after seawater transfer. An oil-adjuvanted vaccine without IPNV antigens offered unspecific and significant protection against recurrence, and at the same time the specific protective effects against the bacterial pathogens were not impaired in the carrier fish.

A comparison between non‐destructive and destructive testing of Atlantic salmon, Salmo salar L., broodfish for IPNV – destructive testing is still the best at time of maturation

Two populations of Atlantic salmon broodstock, previously identified as infectious pancreatic necrosis virus (IPNV) carriers, were screened for IPNV at the time of stripping. Four hundred and ten broodfish were individually sampled of which 91 were detected as IPNV positive by virus culture of sonicated kidney homogenates combined with gonadal fluid, but none tested positive by the blood leucocyte assay. Thirty fish identified as IPNV carriers prior to maturation by the blood leucocyte assay were used in a separate study to compare non-destructive vs. destructive testing methods at stripping. IPNV was not detected using the blood leucocyte method at the time of stripping. RT-PCR and real-time PCR assays failed to detect IPNV from 13 blood samples, the virus was not isolated from milt (0/14) or sonicated ovarian fluid cell pellets (0/16) and only three fish tested positive by the standard culture of kidney homogenates. A third study of Atlantic salmon broodfish compared the IPNV isolation rates prior to maturation with the isolation rates at spawning during 1999-2001. In each year the percentage of IPNV-positive broodfish was significantly lower than in the pre-broodstock sample. While in pre-broodfish samples IPNV was detected by the blood leucocyte assay, no culture isolations or PCR positives were detected from non-destructive samples of the same individual broodfish at stripping. A consistent finding was that even for the kidney assay, the percentage of IPNV-positive fish in carrier populations was higher in pre-broodstock than in broodfish at stripping. These results indicate that destructive kidney sampling is still the most sensitive method for detecting IPNV carrier Atlantic salmon broodfish and that a change in IPNV carrier-status occurs during the maturation period.

In infectious pancreatic necrosis virus carrier Atlantic salmon, Salmo salar L., post‐smolts, almost all kidney macrophages ex vivo contain a low level of non‐replicating virus

The level of infection by infectious pancreatic necrosis virus (IPNV) of kidney macrophages from 12 asymptomatic carrier Atlantic salmon post-smolts was studied. Kidney leucocytes were fractionated on 34/51% Percoll gradients, allowed to adhere to plastic wells overnight, washed to remove non-adherent cells and cultured for up to 7 days with or without renewal of medium on day 3. On day 1, supernatants were harvested, macrophages were counted, lysed and IPNV in the supernatants and lysates was titred in chinook salmon embryo (CHSE-214) cells. The multiplicity of infection ranged between 1:2.2 and 1:7.4 (virus:macrophages). On day 3, the titres of IPNV in macrophage lysates decreased and in wells where the medium was renewed on day 3, IPNV was no longer detectable on day 7. In the supernatants, one fish was positive for IPNV on day 1, four fish on day 3 but none were detectably positive on day 7. In parallel wells in which the medium was not renewed, on day 7 IPNV was detected in macrophage lysates of three fish and the supernatants were also IPNV positive in two of these fish. This suggests that virus might be shed from infected macrophages and then reinfect other macrophages. When macrophages were serially diluted in wells and cultured for 24 h, IPNV could be cultured from macrophage lysates of wells containing between two and 70 macrophages. These results indicate that a very high proportion of the adherent kidney macrophages must be infected with very few non-replicating virions.

IPNV carrier Atlantic salmon growers do not express Mx mRNA and poly I:C-induced Mx response does not cure the carrier state

Injection of infectious pancreatic necrosis virus (IPNV) in post-smolt Atlantic salmon induced a rapid and persistent expression of Mx mRNA from day 1 to at least day 11 when Mx:β actin ratios were still at peak values of about 1.0. In contrast, an Atlantic salmon grower population, shown to be carriers of IPNV by culture of the virus from plastic adherent kidney leucocytes, showed no evidence of the expression of Mx transcripts. Nevertheless, IPNV-carrier growers showed a typical Mx response following injection of poly I:C, beginning on day 1, peaking on day 3 (mean Mx:β actin ratio 0.82) and disappearing by day 7. Notwithstanding such treatment, IPNV continued to persist in growers as the virus could still be isolated 14 days after poly I:C injection.

Infectious pancreatic necrosis virus infection in Atlantic salmon Salmo salar post-smolts affects the outcome of secondary infections with infectious salmon anaemia virus or Vibrio salmonicida.

Infectious pancreatic necrosis (IPN) virus (IPNV) infection in Atlantic salmon Salmo salar L. post-smolts and its influence on the outcome of secondary infections with infectious salmon anaemia (ISA) virus (ISAV) or Vibrio salmonicida were studied. The infections with ISAV or V salmonicida were performed both in a period of acute IPN and in the following IPNV carrier stage, 3 and 6 to 8 wk after experimental IPNV challenge, respectively. An IPNV carrier condition at low virus titre did not influence the mortality rates after secondary infections. Neither the ISAV infection nor the V. salmonicida infection in experimentally induced IPNV carriers resulted in mortalities different from those observed after challenge of IPNV-free fish. At higher IPNV titres in Atlantic salmon with acute IPN, the outcome of secondary infections was quite different from that observed in IPNV-free fish and in IPNV carriers. In 2 different experiments significantly more fish died when fish with acute IPN were infected with V salmonicida than when fish were infected with V salmonicida alone. Mortality also started earlier in the double-infected group than in the group challenged with V. salmonicida alone, 3 to 4 and 8 d after V salmonicida infection, respectively. Similar results were observed independent of whether mortalities due to IPN alone were registered in the experiments. When Atlantic salmon with acute IPN were infected with ISAV, significantly fewer fish died than when fish were infected with ISAV alone. The ongoing IPNV infection seemed to provide some protection against development of ISA.

Susceptibility of Arctic Char to Experimental Challenge with Infectious Hematopoietic Necrosis Virus (IHNV) and Infectious Pancreatic Necrosis Virus (IPNV).

Arctic char Salvelinus alpinus were exposed to the fish pathogens infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) to estimate susceptibility of the species to infection. Fish were immersion challenged at 46, 74, and 95 d posthatch for 5 h in viral concentration of 103 or 105 PFU(plaque-forming units)/mL and were monitored for 21 d postchallenge. Fish that died were assayed individually for virus-associated mortality, and survivors were assayed individually to estimate prevalence of virus carrier status. Arctic char succumbed to IHNV infection with population losses as high as 41%, and prevalence of IHNV carriers in surviving fish was 24% or less with virus titers in carrier fish ranging from 2.5 × 102 PFU/g to greater than 1.3 × 106 PFU/g. Infections with IPNV resulted in losses of 20% or less, and prevalence of IPNV carriers in surviving fish was as high as 82% with virus titers in carrier fish ranging from 2.5 × 102 PFU/g to greater than 5.0 × 106 PFU/g. Logistic regression models were developed for the mortality and infection data for each virus. For IHNV, the odds of dying or becoming infected were significantly greater for the fish from the 74-d age-group than for the fish from the 46-d or 95-d age-groups. Fish exposed to 105 PFU of IHNV/mL had significantly greater odds of dying or becoming infected than fish exposed to 103 PFU/mL. For IPNV, the odds of dying were significantly greater for the 74-d age-group compared with the other two age-groups. The 74-d fish were also significantly more likely to become infected than the 95-d-old fish. Fish exposed to 105 PFU of IPNV/mL had significantly greater odds of dying or becoming infected than fish exposed to 103 PFU of IPNV /mL.

Immunization of adult brook trout, Salvelinus fontinalis (Mitchill), fails to prevent the infectious pancreatic necrosis virus (IPNV) carrier state

Abstract. Adult brook trout, Salvelinus fontinalis (Mitchill), mounted a strong humoral immune response after injection with inactivated infectious pancreatic necrosis virus (IPNV) in Freund's complete adjuvant (FCA). However, this immunization did not prevent the fish from becoming IPNV carriers. After an injection challenge with virulent IPNV, the immunized and control fish (FCA or water) shed virus in the faeces and reproductive products and had IPNV‐infected leucocytes and visceral organs. Initially, from 1 to 3 weeks post‐challenge (wpc), immunized fish had a lower prevalence of infection and virus titres in the plasma, and fewer infected leucocytes than the control fish. Immunization did not prevent the eventual infection of the leucocytes; over 75% of the immunized and control fish had leucocyte‐associated viraemia from 6 to 15 wpc. When the organs were tested at 15 wpc, the immunized fish showed fewer infected organs per fish, and a lower prevalence of infection and virus titres in individual organs than the control fish, but these differences were not significant. Immunized male and female fish shed IPNV in the reproductive products, suggesting that immunization of adult fish would not prevent vertical transmission of IPNV to progeny.

Prevalence and pathogenicity of infectious pancreatic necrosis virus (IPNV) associated with feral lake trout, Salvelinus namaycush (Walbaum)

Abstract. A study was undertaken in 1987 to determine the prevalence of infectious panercatic necrosis virus (1PNV) infection in the lake trout population of Cornwall Lake. Alberta, Canada, and its pathogenicity to cultivable salmonid fish. Virological examination indicated that 44.4% of the adult lake trout in the lake, which is situated in a remote northern region of Alberta, were infected with the virus, mainly in the pyloric caeca and intestine. Virus was not detected in kidney, leucocytes, liver or gonads. In experimental immersion infection of brook trout fry, the virus caused a cumulative mortality of up to 74% in 30 days, beginning at 10 days post‐infection. Pyloric caeca, intestine and to some extent gills were found to be early sites of viral replication. The virus was less pathogenic to rainbow trout causing a cumulative mortality of 10% and the survivors were IPNV carriers for at least 3 months. The virus did not cause mortality in young lake trout, the natural host, but the infected fish carried the virus during the experimental period of 30 days.

Studies on the carrier state of infectious pancreatic necrosis virus infections in rainbow trout, Salmo gairdneri Richardson

Abstract. A rainbow trout population of infectious pancreatic necrosis virus carriers was studied over a one‐year period using both homogenization and co‐cultivation for virus isolation. The percentage of virus‐yielding fish was high between March and June, but declined during the latter part of the year. This was diametrically opposite to the trend in the serum antibody levels indicating that the marked humoral immune response resulted in a very significant reduction in the virus titres. The highest isolation rate was obtained from the kidneys after co‐cultivation underlining the very high sensitivity of this method for virus detection. Virus was occasionally isolated from the faeces indicating that this may well be a possible avenue for horizontal transmission of the virus. No virus was ever detected in gonadal tissue; the mechanism of vertical transmission of the virus is still very poorly understood.

Experimental induction of the carrier state in yearling brook trout: A model challenge protocol for IPNV immunization

Brook trout fry ( Salvelinus fontinalis) were not protected from infectious pancreatic necrosis virus (IPNV) challenge by immersion vaccination with inactivated, purified virus at concentrations of 10 7 to 10 9 pfu/ml. Mortalities in vaccinated groups were higher than for the unvaccinated control group and appeared to be dose-dependent. A challenge protocol for adult brook trout was developed for future vaccine trials. A single intraperitoneal injection of virulent, purified virus was sufficient to make long-lasting carriers of 16 month-old trout. Fish underwent a transient viremia, identified by virus isolation from plasma and leucocytes. Feces were the most reliable samples for identification of IPNV carriers by non-sacrificial testing. Many fish in the remaining infected group were still carriers 12 and 27 weeks post-infection.

Replication of infectious pancreatic necrosis virus in trout leucocytes and detection of the carrier state

Abstract. The exact cellular site of replication of infectious pancreatic necrosis virus (IPNV) in carrier fish is unknown. In order to determine if IPNV replicates in trout leucocytes, we purified leucocytes from normal (non‐carrier) trout and separated the cells into an adherent and a non‐adherent population. IPNV replicated in less than 0‐01 % of the adherent leucocytes with a yield of about 400 p.f.u./cell. IPNV also became associated with less than 0.07% of the non‐adherent leucocytes; either IPNV did not replicate in these cells or the yield was, at best, only a few p.f.u./cell. Trout persistently infected with IPNV (carrier fish) were tested for the presence of IPNV in leucocytes by co‐cultivating with a sensitive fish cell line; this same population of trout was also tested for IPNV by organ sampling using standard methods. Ninety‐eight per cent of the trout were positive for IPNV by organ sampling, but only 75 % yielded IPNV from leucocytes. Thus a blood sample from a living fish can be used to detect the presence of IPNV.