Inoculation of Infectious Pancreatic Necrosis Virus Serotype Sp did not cause Pancreas Disease in Atlantic Salmon (Salmo salar L.)

Atlantic salmon were selected from a fish farm with no previous record of pancreas disease (PD) or infectious pancreatic necrosis virus (IPNV) infection. Groups of fish were inoculated with either IPNV (strain Sp) from cell culture, organ material from fish with PD or control material as phosphate-buffered saline (PBS). Virological, histological and immunohistochemical examinations were carried out throughout the experiment. None of the fish died or showed clinical symptoms of PD. Histological examination revealed no pathological changes, and immunohistochemical studies were negative. Virus was isolated only sporadically from the group inoculated with organ material, whereas it was isolated consistently from the group inoculated with virus propagated in cell culture, as well as from in-contact control fish after the first week. In a latent carrier test, changes were entirely lacking in the first mentioned group, and were only slight in the last mentioned group. The data suggest that PD is not a transmissible disease, and that IPNV isolated from a PD outbreak does not play any part in the etiology of this disease.     

Eastern equine encephalomyelitis virus in experimentally infected bats.

Colonial bats (Myotis supp. and Eptesicus sp.) were infected with eastern equine encephalomyelitis virus by subcutaneous inoculation or by the bite of infected mosquitoes. Bats were maintained in an environment simulating conditions encountered in hibernacula or in summer maternal colonies. Virus was detected in the blood of hibernating bats at irregular intervals over a 42-day observation period; viremia perhaps was influenced by the amount of disturbance (arousal) involved in the blood sampling process. Target organs included brown fat, spleen, lung, kidneys, pancreas, and liver. Neutralizing antibody was not detected in sera collected from these bats between days 4 and 42 post-inoculation. In nonhibernating bats, virus was recovered from mammary glands, brown fat, pancreas, lungs, kidneys, and liver, in addition to blood. Attempts to infect bats orally or to transmit virus to suckling mice by the bite of viremic bats were unsuccessful. Virus was transmitted from viremic chickens to E. fuscus by the bite of Culiseta melanura and Aedes aegypti.     

Evaluation of a water concentration method for the detection of infectious pancreatic necrosis virus in a fish hatchery

A virus-adsorption-elution (viradel) method using electropositive microporous filters (Virosorb 1-MDS) was used for the concentration and détéction of infectious pancreatic necrosis virus (IPNV) at a hatchery with a past history of IPNV infection. Samples of fish tissue and unconcentrated water were also examined for the presence of IPNV. Virus was isolated from three of the nine concentrated water samples taken from various locations at the hatchery. Virus was also isolated from pooled fish tissues, but not from unconcentrated water samples. Representative numbers of viruses isolated from water and fish tissues were examined for virus neutralization in the presence of anti-IPNV amiserum; resistance to acid, ether, and heat; and replication in the presence of 5-bromo-2'-deoxyuridine (BUDR). When tested by dot-immunobinding assay using a panel of monoclonal antibodies (MABs), the viral isolates were found to belong to the West Buxton serotype of IPNV.     

Rotavirus Infection Induces Transient Pancreatic Involution and Hyperglycemia in Weanling Mice

Rotavirus is a ubiquitous double-stranded RNA virus responsible for most cases of infantile gastroenteritis. It infects pancreatic islets in vitro and is implicated as a trigger of autoimmune destruction of islet beta cells leading to type 1 diabetes, but pancreatic pathology secondary to rotavirus infection in vivo has not been documented. To address this issue, we inoculated 3 week-old C57Bl/6 mice at weaning with rhesus rotavirus, which is closely related to human rotaviruses and known to infect mouse islets in vitro. Virus was quantified in tissues by culture-isolation and enzyme-linked immunosorbent assay. A requirement for viral double stranded RNA was investigated in toll-like receptor 3 (TLR3)-deficient mice. Cell proliferation and apoptosis, and insulin expression, were analyzed by immunohistochemistry. Following rotavirus inoculation by gavage, two phases of mild, transient hyperglycemia were observed beginning after 2 and 8 days. In the first phase, widespread apoptosis of pancreatic cells was associated with a decrease in pancreas mass and insulin production, without detectable virus in the pancreas. These effects were mimicked by injection of the double-stranded RNA mimic, polyinosinic-polycytidylic acid, and were TLR3-dependent. By the second phase, the pancreas had regenerated but islets were smaller than normal and viral antigen was then detected in the pancreas for several days. These findings directly demonstrate pathogenic effects of rotavirus infection on the pancreas in vivo, mediated initially by the interaction of rotavirus double-stranded RNA with TLR3.     

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.     

Establishment and characterization of the Epithelioma Papulosum Cyprini (EPC) cell line persistently infected with Infectious Pancreatic Necrosis Virus (IPNV), an aquabirnavirus

Infectious pancreatic necrosis virus (IPNV), a type species of aquabirnaviruses in the family Birnaviridae, is an etiological agent of infectious pancreatic necrosis and has been isolated from epizootics of cultured salmonids. In the present study, an epithelioma papulosum cyprini (EPC) cell line persistently infected with IPNV (PI-EPC) was experimentally established by subculturing EPC cells surviving IPNV infection, and was characterized. PI-EPC cells were morphologically indistinguishable from EPC, but continued to grow and yield IPNV. PI-EPC cells showed no cytopathic effect due to IPNV inoculation, and susceptibility of PI-EPC cells against heterologous viruses was not different from that of EPC cells. Only one cell of 10^3.5 PI-EPC cells produced IPNV at approximately 10^0.5 50% tissue culture infectious dose (TCID50)/cell/day, which was approximately 1,000 times lower than that of normal EPC cells. PI-EPC cells that did not yield IPNV (N-PI-EPC) were screened. The IPNV genome was detected from both PI-EPC and N-PI-EPC cells, and the IPNV VP2 structural protein was detected from both cell lines, but no other IPNV proteins were observed by Western blot analysis with anti-IPNV serum. Thus, multiplication of IPNV in PI-EPC cells was regulated by some host cell factors, except interferon.     

Antiviral function of tilapia hepcidin 1–5 and its modulation of immune-related gene expressions against infectious pancreatic necrosis virus (IPNV) in Chinook salmon embryo (CHSE)-214 cells

Antimicrobial peptides, small cysteine-rich molecules, play vital roles in host defense mechanisms against pathogen infection. Recently, tilapia hepcidin (TH)1–5, was characterized, and its antimicrobial functions against several pathogens were reported. Herein, we investigated the antiviral functions of TH1-5 against infectious pancreatic necrosis virus (IPNV) in Chinook salmon embryo (CHSE)-214 cells. The presence of TH1-5 enhanced the survival of CHSE-214 cells infected with IPNV. Additionally, the number of plaques formed by the cytopathic effect of IPNV in CHSE-214 cells decreased when IPNV was preincubated with TH1-5. This observation demonstrates the antiviral function of TH1-5. Real-time PCR studies showed the modulation of interleukin, annexin, and other viral-responsive gene expressions by TH1-5. When TH1-5 and IPNV were used to co-treat CHSE-214 cells, then cells were re-challenged with IPNV at 24 h, the cells did not survive the IPNV infection. This shows that in the absence of TH1-5, viral re-challenge killed CHSE-214 cells. In conclusion TH1-5 protected CHSE-214 cells against IPNV by direct antimicrobial and immunomodulatory functions.     

Viral susceptibility of newly established cell lines from the Hawaiian monk seal Monachus schauinslandi

Ten of 11 cell lines, recently established from the snout (MS-SN), periorbital soft tissue (MS-EY), liver (MS-LV), kidney (MS-KD), lung (MS-LG), spleen (MS-SP), heart (MS-HT), thyroid (MS-TY), brain (MS-BR) and urinary bladder (MS-UB) of a juvenile Hawaiian monk seal Monachus schauinslandi, were evaluated in vitro for their susceptibility to 5 mammalian viruses: herpes simplex virus type 1 (HSV-1), vesicular stomatitis virus (VSV), reovirus type 3 (Reo-3), poliovirus type 1 (Polio-1) and vaccinia virus (Vac); 5 fish viruses: channel catfish herpesvirus (CCV), infectious hematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), fish rhabdovirus carpio (RC) and viral hemorrhagic septicemia virus (VHSV); and 2 marine mammal morbilliviruses: phocine distemper virus (PDV) and dolphin distemper virus (DMV). Four well-established continuous cell-lines of nonhuman primate (Vero) and fish (EPC, CHSE-214 and BB) origin served as controls to standardize the virus infectivity assays. Virus yields were quantified as 50% tissue culture infectious dose (TCID50) ml(-1) on Day 7 post-inoculation. Results of the viral challenge assays revealed that the monk seal cell lines shared a similar pattern of susceptibility to the mammalian viruses. Despite their different tissue origins, all monk seal cells were sensitive to HSV-1, Vac, VSV and Reo-3, but were refractory to Polio-1. A characteristic viral-induced cytopathic effect was noted with VSV and Reo-3, and distinct plaques were observed for HSV-1 and Vac. Monk seal cell lines were also susceptible to PDV and DMV, 2 morbilliviruses isolated from seals and dolphins, respectively. By contrast, these cell lines were generally resistant to VHSV, IHNV and IPNV, with varying susceptibility to RC and CCV. The wide range of viral susceptibility of these monk seal cell lines suggests their potential value in studying viruses of monk seals and other marine mammals.     

Spontaneous exocrine pancreas hypoplasia in specific pathogen-free C3HeB/FeJ and 101/H mouse pups causes steatorrhea and runting

Under specific pathogen-free conditions, 1.3% to 1.8% of litters born in our inbred 101/H and C3HeB/FeJ mouse colonies had pups with steatorrhea and runting. Clinically affected male and female pups were first identified when they were from 14 to 25 d old. Unaffected littermates were healthy and were weaned successfully. Postmortem findings in 8 clinically affected mice included a small, poorly differentiated exocrine pancreas comprising cytokeratin-negative duct-like structures but lacking recognizable acinar cells with their normal carboxypeptidase B-positive zymogen granules. Endocrine pancreas islets were unremarkable and contained insulin-positive beta cells and glucagon-positive alpha cells. There was mild inflammation of the hindgut but no evidence of intestinal pathogens or marked inflammation or necrosis of pancreas, either alone or as part of a multisystemic inflammatory condition. Sera from pups in 4 affected litters did not contain antibodies to reovirus 3, mouse coronavirus, rotavirus, or mouse adenovirus 2. Furthermore, 4 sets of parental mice and sentinel mice from the facility were negative for 13 viruses, bacteria, and parasites. C3HeB/FeJ and 101/H inbred strains may be genetically predisposed because the steatorrhea and runting was absent in 13 other mouse strains and subspecies bred in the specific pathogen-free facility. This condition resembles exocrine pancreas hypoplasia, but the inheritance is complex. A wider implication is that runting coupled with steatorrhea are phenotypic criteria to suspect pancreatic disease that could be used in the context of a mouse N-ethyl-N-nitrosourea-mutagenesis program to identify potential mutants with defects in pancreas development.     

Detection of infectious salmon anaemia virus (ISAV) by in situ hybridisation

An in situ hybridisation method was developed to detect infectious salmon anaemia virus (ISAV) in fixed tissues from Atlantic salmon Salmo salar L. Three DNA probes detected ISAV in heart, liver, kidney, spleen, caeca, and mid-gut from infected farmed Atlantic salmon obtained from a natural outbreak of ISA. The strongest signals were obtained using Probe S8, from Segment 8 of ISAV. Hybridisation was most prominent in the endothelial cells of heart tissue. The probes reacted specifically with ISAV; no hybridisation was evident in uninfected tissues from Atlantic salmon. Importantly, the probes did not cross react with the pathogens IHNV (haematopoietic necrosis virus), IPNV (infectious pancreatic necrosis virus), SPDV (salmon pancreas disease virus) and VHSV (viral haemorrhagic septicemia virus).     

Isolation in immunodeficient mice of Sarcocystis neurona from opossum (Didelphis virginiana) faeces, and its differentiation from Sarcocystis falcatula

Sarcocystis neurona was isolated in nude mice and gamma-interferon knockout mice fed sporocysts from faeces of naturally infected opossums (Didelphis virginiana). Mice fed sporocysts became lethargic and developed encephalitis. Protozoa were first found in the brain starting 21 days post-inoculation. Sarcocystis neurona was recovered in cell culture from the homogenate of liver, spleen and brain of a nude mouse 11 days after feeding sporocysts. The protozoa in mouse brain and in cell culture multiplied by schizogony and mature schizonts often had a residual body. Sarcocystis falcatula, which has an avian-opossum cycle, was not infective to nude or knockout mice. Protozoa were not found in tissues of nude mice or knockout mice after subcutaneous injection with culture-derived S. falcatula merozoites and sporocysts from the faeces of opossums presumed to contain only S. falcatula. Results demonstrate that S. neurona is distinct from S. falcatula, and that opossums are hosts for both species.     

Experimental Sendai virus infection in laboratory rats. I. Virus replication and immune response

Sixty 5 to 8 week old Sprague-Dawley (Crl:CD(SD)BR) rats were inoculated intranasally with 2000 egg infectious doses of egg-propagated Sendai virus. Virus was recovered from the upper respiratory tract and lungs on days 1 through 8 post-inoculation (PI). Serum antibody responses were measured for 12 rats over a 9 month period PI. Antibody was first detected at 7 days, peaked at 21 days, and was detected in 5 of the 12 rats at 9 months. A cell-mediated response, as measured by lymphocyte blastogenesis, also was detected at 7 days and peaked at 21 days, but was not detected at 6 months PI. Lung and serum interferon (IFN) was first detected at 3 hours and peaked at 6 hours, but was not detected by 160 hours. Lung IFN levels were 4 to 10 times those in the serum. These studies indicate that pathogenesis of Sendai virus infection in the rat is similar to that reported in the mouse, but that there are differences in the kinetics of both viral replication and morphologic changes, as described in the companion paper.     

Infectious pancreatic necrosis virus VP5 is dispensable for virulence and persistence

Infectious pancreatic necrosis virus (IPNV) is the causative agent of infectious pancreatic necrosis (IPN) disease in salmonid fish. Recent studies have revealed variation in virulence between isolates of the Sp serotype, associated with certain residues of the structural protein VP2. The isolates are also highly heterogenic in the coding region of the nonstructural VP5 protein. To study the involvement of this protein in the pathogenesis of disease, we generated three recombinant VP5 mutant viruses using reverse genetics. The "wild-type" recombinant NVI15 (rNVI15) virus is virulent, having a premature stop codon at nucleotide position 427, putatively encoding a truncated 12-kDa VP5 protein, whereas rNVI15-15K virus encodes a 15-kDa protein. Recombinant rNVI15-deltaVP5 virus contains a mutation in the initiation codon of the VP5 gene that ablates the expression of VP5. Atlantic salmon postsmolts were challenged to study the virulence characteristics of the recovered viruses in vivo. The role of VP5 in persistent infection was investigated by challenging Atlantic salmon fry with the recovered viruses, as well as with the low-virulence field strain Sp103 and a naturally occurring VP5-deficient mutant of Sp103. The results show that VP5 is not required for viral replication in vivo, and its absence does not alter the virulence characteristics of the virus or the establishment of persistent IPNV infection.     

Experimental Transmission of Infectious Pancreatic Necrosis Virus from the Blue Mussel,Mytilus edulis,to Cohabitating Atlantic Salmon (Salmo salar) Smolts

Integrated multitrophic aquaculture (IMTA) reduces the environmental impacts of commercial aquaculture systems by combining the cultivation of fed species with extractive species. Shellfish play a critical role in IMTA systems by filter-feeding particulate-bound organic nutrients. As bioaccumulating organisms, shellfish may also increase disease risk on farms by serving as reservoirs for important finfish pathogens such as infectious pancreatic necrosis virus (IPNV). The ability of the blue mussel (Mytilus edulis) to bioaccumulate and transmit IPNV to naive Atlantic salmon (Salmo salar) smolts was investigated. To determine the ability of mussels to filter and accumulate viable IPNV, mussels were held in water containing log 4.6 50% tissue culture infective dose(s) (TCID₅₀) of the West Buxton strain of IPNV ml⁻¹. Viable IPNV was detected in the digestive glands (DGs) of IPNV-exposed mussels as early as 2 h postexposure. The viral load in mussel DG tissue significantly increased with time and reached log 5.35 ± 0.25 TCID₅₀ g of DG tissue⁻¹ after 120 h of exposure. IPNV titers never reached levels that were significantly greater than that in the water. Viable IPNV was detected in mussel feces out to 7 days postdepuration, and the virus persisted in DG tissues for at least 18 days of depuration. To determine whether IPNV can be transmitted from mussels to Atlantic salmon, IPNV-exposed mussels were cohabitated with naive Atlantic salmon smolts. Transmission of IPNV did occur from mussels to smolts at a low frequency. The results demonstrate that a nonenveloped virus, such as IPNV, can accumulate in mussels and be transferred to naive fish.     

Molecular characterization of birnaviruses isolated from wild marine fishes at the Flemish Cap (Newfoundland)

Several isolates of aquatic birnaviruses were recovered from different species of wild fish caught in the Flemish Cap, a Newfoundland fishery close to the Atlantic coast of Canada. The nucleotide sequence of a region of the NS gene was identical among the isolates and was most similar to the Dry Mills and West Buxton reference strains of infectious pancreatic necrosis virus (IPNV). Phylogenetic analysis of the sequence of a region of the VP2 gene demonstrated that the isolates were most closely aligned with the American strains of IPNV serotype A1. Electron microscopy of virus structures clarified and concentrated from cultures of infected chinook salmon embryo (CHSE-214) cells revealed a majority of typical IPNV-like icosahedral particles, as well as a low proportion of type I tubules having a diameter of approximately 55 nm and a variable length of up to 2 microm. The tubules could be propagated in cell cultures, but always in the presence of low proportions of icosahedral particles. Cloning of selected isolates by serial dilution yielded preparations with a high proportion of the tubular structures with a density in CsCl gradients of approximately 1.30 g cm(-3). Polyacrylamide gel electrophoresis revealed the material in the band was composed of the IPNV pVP2 and VP2 proteins.     

Study of the viral interference between infectious pancreatic necrosis virus (IPNV) and infectious haematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss)

The resistance of rainbow trout (Oncorhynchus mykiss) to an infectious haematopoietic necrosis virus (IHNV) challenge following a preceding non-lethal infection with infectious pancreatic necrosis virus (IPNV) was investigated through experimental dual infections. Trout initially infected with IPNV were inoculated 14 days later with IHNV. Single infections of trout with 1 of the 2 viruses or with cell culture supernatant were also carried out and constituted control groups. No mortality was noted in fish after a single infection with IPNV. This virus had no influence on the head kidney leucocyte phagocytic activity and plasma haemolytic complement activity. IHNV induced a high mortality (72%) and reduced the macrophage phagocytic activity and complement haemolytic activity. It also induced a late production of anti-IHNV antibodies which occurred after clearance of the virus in the fish. In trout co-infected with both viruses, a mortality rate of 2% occurred and the immune parameters were similar to those observed in the fish infected with IPNV only, demonstrating that in co-infected trout IPNV inhibits the effects of IHNV. The studied parameters did not allow us to define the mechanism of interference occurring between these 2 viruses, but some hypothesis are put forward to explain the interference between the 2 viruses.     

Enhancement of mouse pancreatic regeneration and HIT-T15 cell proliferation with rat pancreatic extract

In this study, the effects of rat pancreatic extract (RPE) on regeneration of impaired mouse pancreas and proliferation of beta-cell line (HIT-T15) were investigated. RPE from the regenerating pancreas (2 days after 60% pancreatectomy) was treated to cure streptozotocin (STZ) induced diabetes in BALB/c mice. RPE-treated BALB/c mice for 21 consecutive days became euglycemic by day 30 and remained normoglycemic during a 150 day follow-up. Saline treated mice remained hyperglycemic sustained uncontrolled hyperglycemia. Islet neogenesis was observed in RPE-treated mice and confirmed by use of immunocytochemistry. Morphometric analysis of pancreas in reverted RPE-treated mice showed a new population of small islets compared with saline controls and an increased islet number. When HIT-T15 cells were treated with RPE, HIT-T15 cell proliferation and insulin secretion increased with increases in the RPE concentration. These results imply that RPE have the regeneration factors and help in the cure of diabetes.     

Fish viruses serologic comparison of the golden shiner and infectious pancreatic necrosis viruses

High titer neutralizing antiserum to golden shiner virus (GSV) was produced in a rabbit. The GSV antiserum had significant neutralization indices in tests with the original strain of GSV and with another more recent isolate. Neutralization indices were not significant when two strains of infectious pancreatic necrosis virus (IPNV) were reacted with GSV antiserum, and neither isolate of GSV showed significant reactivity in neutralization tests with polyvalent IPNV antiserum. Our results indicate that IPNV and GSV are serologically unrelated.