Experimental infection of Atlantic salmon Salmo salar pre-smolts by i.p. injection with new Irish and Norwegian salmonid alphavirus (SAV) isolates: a comparative study.

Atlantic salmon Salmo salar L. pre-smolts were experimentally infected with 2 different isolates of salmonid alphavirus (SAV): a Subtype 1 isolate from Ireland and a Subtype 3 isolate from Norway. Sequential samples of tissue and blood were collected during a period of 20 wk post injection and subjected to virus isolation from kidney tissue and serum, detection of viral nucleic acid in heart tissue and serum by real-time RT-PCR, detection of specific antibodies by virus neutralisation assay, and histopathological examination. Successful reproduction of pancreas disease (PD) was obtained by intraperitoneal (i.p.) injection of both isolates. No mortality was observed post infection in either group, but typical PD histopathological lesions in heart and pancreas tissue were observed with both isolates. The prevalence and severity of lesions in the pancreas, heart, skeletal muscle and brain were similar in both groups with only subtle differences recorded. Re-isolation of virus from kidney tissue was performed at 7 and 14 d post infection (d p.i.) only and was positive for both test groups at both sampling points. Isolation of virus from sera from both groups was positive at 4 to 14 d p.i., but was negative at later sampling points when antibody production had begun. Virus may be detected only during the acute phase using both methods. Specific neutralising antibodies could be detected for both test groups from Day 21 p.i. until the end of the experiment at 140 d p.i. Peak antibody titres were seen 70 d p.i. Using real-time RT-PCR, pancreas disease virus (PDV)-specific RNA was detected frequently in serum samples up to 14 d p.i. and occasionally thereafter. In contrast, viral RNA could still be detected in the heart tissue of fish from both groups for at least 140 d p.i.

Development and evaluation of a one-step real-time reverse transcription polymerase chain reaction assay for the detection of salmonid alphaviruses in serum and tissues.

We designed 4 primer pairs to amplify conserved regions of the E1 or nsP4 genes of salmonid alphavirus (SAV) and evaluated their performance in optimized 1-step SYBR green real-time RT-PCR (RRT-PCR) assays. A single primer pair, amplifying a 227 bp segment of E1 was then chosen for further study. This RRT-PCR was shown to be highly repeatable and reproducible over a wide range of RNA dilutions, with a linear relationship between cycle threshold (Ct) value and RNA concentration over a 10(7) dilution range. The limit of detection was calculated to be < or = 1.5 TCID50 ml(-1). When applied to sera previously screened by virus isolation for SAV viraemia, the RRT-PCR correctly identified all 13 culture-positive samples, as well as finding an additional 28 sera positive. Relative semi-quantitation of sera showed a very highly significant relationship between copy number and TCID50 (p < 0.001, R2 = 0.9563). Following experimental infection of salmon, heart samples were consistently positive until 21 d post infection (dpi), with (weak) positive signals still detectable in 50% of fish 70 dpi.

New subtype of salmonid alphavirus (SAV), Togaviridae, from Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss in Norway.

In Europe, 2 closely related alphaviruses (Togaviridae) are regarded as the causative agents of sleeping disease (SD) and salmon pancreas disease (SPD): SD virus (SDV) has been isolated from rainbow trout Oncorhynchus mykiss in France and the UK, while SPD virus (SPDV) has been isolated from salmon Salmo salar in Ireland and the UK. Farmed salmonids in western Norway also suffer from a disease called pancreas disease (PD), and this disease is also believed to be caused by an alphavirus. However, this virus has not yet been characterised at the molecular level. We have cultured a Norwegian salmonid alphavirus from moribund fishes diagnosed with cardiac myopathy syndrome (CMS) and fishes diagnosed with PD. The virus has also been found in salmon suffering from haemorrhagic smolt syndrome in the fresh water phase. The genomic organisation of the Norwegian salmonid alphavirus is identical to that in SPDV and SDV, and the nucleotide sequence similarity to the other 2 alphaviruses is 91.6 and 92.9%, respectively. Based on the pathological changes, host species and the nucleotide sequence, we suggest naming this virus Norwegian salmonid alphavirus (NSAV). Together with SPDV and SDV it constitutes a third subtype of salmonid alphavirus (SAV) species within the genus Alphavirus, family Togaviridae.

Update on viral vaccines for fish.

A substantial amount of research has been done on fish viruses affecting species in aquaculture. This review will focus on the salmonid industry, as this is the most industrialised part of fish farming where vaccines are extensively used. In spite of the amount of research performed, both in commercial companies and in academic organisations, few viral vaccines are licensed. As of today, all fish virus vaccines for sale are based upon inactivated virus or recombinant proteins. No live attenuated or DNA vaccines are currently licensed, but one DNA vaccine against IHN is being tested in controlled field trials in Canada. Vaccines against infectious pancreatic necrosis (IPN) have been sold for many years in Norway and are now also available in Chile. Most of the research on these vaccines has been performed by pharmaceutical companies, and not much information is available as scientific publications. It has also been difficult to establish reproducible IPN challenge models suitable for vaccine testing and this probably explains the lack of scientific publications. Quite the reverse is the case for the fish rhabdoviruses viral hemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV). The challenge models are reproducible, and both inactivated virus and DNA vaccines offer excellent protection. Recombinant subunit vaccines have so far shown unsatisfactory effect. Little information has been published regarding vaccine development against pancreas disease (PD) and infectious salmon anaemia (ISA). PD and ISA vaccines have been tested at the laboratory scale with good results, and two commercial ISA vaccines are currently available in Canada. Regarding nodaviruses, a few publications have shown effect of recombinant subunit formulations. However, nodaviruses cause disease early in the lifecycle of marine fish, and injection of these formulations into fish of a few grams is so far difficult on a commercial scale.

Immunization with viral antigens: infectious pancreatic necrosis.

Clinical IPN has traditionally been observed in brook trout (Salvelinus fontinalis) and rainbow trout (Oncorhynchus mykiss). However, during the past 10 years outbreaks of IPN have been reported frequently in farmed Atlantic salmon (Salmo salar L.) in Norway. Acute IPN with high mortality is observed in fry at start feeding and in smolt in the first six months after transfer to seawater. Today IPN is the most important infectious disease in Norway in farmed fish, giving economic losses of about 60 million USD yearly. A prophylactic strategy against this disease is strongly needed. Different strategies for developing IPN vaccines have been tested since the virus was first isolated in 1960. Vaccination with live virus has not been successful and is probably not an acceptable strategy for environmental risk reasons. Vaccination with inactivated virus has been tested in rainbow trout given by the oral route, by immersion and injection. Protection against challenge was obtained only by injection. IPN vaccines based on inactivated virus may be effective but are expensive. A subunit vaccine produced by fermentation is a more realistic strategy for fish vaccine production if the actual protective epitopes can be identified. Protective IPNV epitopes may include both B- and T-cell epitopes, but only B-cell epitopes have been examined so far. Epitope mapping with neutralising monoclonal antibodies indicates that the internal variable region of VP2 (aa 200-350) folds into an immunodominant structure including both serotype specific and conserved neutralisation epitopes that can renaturate spontaneously from E. coli-expressed recombinant VP2 (rVP2). Analysis with an IPNV group-A specific neutralising monoclonal antibody indicates that immunisation with recombinant protein containing the segment (aa 86-210) might induce protection against all IPNV serotypes. Subunit vaccines based on E. coli-expressed IPNV proteins have been tested in rainbow trout and Atlantic salmon. Vaccination by immersion in rainbow trout fry with bacterial lysate from E. coli expressing the IPNV Sp strain gene segment A induced protection against challenge with the IPNV Buhl strain. By injection of Atlantic salmon parr with partly purified E. coli-expressed rVP2 (N1 strain), increased resistance against IPN infection was demonstrated by challenge. In field trials it is shown that vaccination of pre-smolt with rVP2 included in a commercial oil/glucan adjuvanted multivalent bacterial vaccine gives protection against IPN in natural outbreaks, compared to fish vaccinated with the same vaccine without the IPNV component.

Mapping of neutralization epitopes on infectious pancreatic necrosis viruses.

We have characterized and mapped variable and conserved neutralization epitopes of serogroup A strains of aquatic birnaviruses. Epitope mapping using monoclonal antibodies (MAbs) and Escherichia coli-expressed deletion fragments of VP2 of the N1 strain of infectious pancreatic necrosis virus (IPNV) demonstrated that two variable epitopes, H8 and B9, depend on the variable region between amino acid 204-330. A conserved neutralization epitope, F2, was shown to depend on the same region as epitopes H8 and B9 but was additionally dependent on amino acids between 153-203. The neutralization epitopes H8, B9 and F2 were also shown to overlap by a competitive binding assay. One conserved neutralization epitope, AS-1, was not exposed on any of the recombinant VP2 deletion fragments and was therefore not possible to map. However, the MAbs AS1 and F2 were partly competitive indicating that these epitopes are overlapping. All neutralization epitopes were independent of a conserved non-neutralization epitope, E4. Our results demonstrate that the central third of VP2 contains several partly overlapping neutralization epitopes, both variable and conserved among serogroup A strains of IPNV.

Sequence of the large double-stranded RNA segment of the N1 strain of infectious pancreatic necrosis virus: a comparison with other Birnaviridae.

The cDNA sequence of the large dsRNA segment (segment A) of the N1 strain of infectious pancreatic necrosis virus (IPNV) has been determined. The nucleotide and deduced amino acid sequences were compared to the sequences of segment A of the Jasper strain of IPNV and to the sequences of segments A and B (5' and 3' flanking regions) of the 002-73 strain of infectious bursal disease virus (IBDV). The comparison demonstrated that the precursor protein of the major structural polypeptide, pVP2, is highly conserved at the N and C termini, whereas the amino acid sequence of an internal segment shows greater diversity between the strains. This internal segment probably carries the serotype-specific epitopes of birnaviruses. An alternative open reading frame (ORF) (444 bp) partly overlapping with the large ORF (2916 bp) of segment A was found to be conserved among the IPNV strains and is probably also present in the 002-73 strain of IBDV. This small ORF may encode a novel birnavirus polypeptide with an Mr of 17K. SDS-PAGE of radiolabelled purified IPNV particles revealed a band corresponding to the possible novel 17K polypeptide. Short terminal inverted repeats are found in segment A of the N1 and Jasper strains of IPNV and in segment B of the 002-73 strain of IBDV. Segment A of IPNV and segment B of IBDV also contain adjacent inverted repeats at their 5'-terminal flanking regions.

Antibody-mediated enhancement of BK virus infection in human monocytes and a human macrophage-like cell line.

We have monitored BK virus (BKV) antigen expression and multiplication in human monocytes and in a human macrophage (M luminal diameter)-like cell line (U937) in the presence or absence of dilution series of human or rabbit anti-BKV antisera. After infection with BKV alone, restricted expression (structural antigens and T-antigen) and multiplication was recorded in monocytes from some donors, while in U937 cells and monocytes from other donors, no signs of viral activity were detected. Monocyte cultures established from the same donor at different times demonstrated antigen expression/multiplication on two occasions but not on the third. A pronounced enhancement of BKV expression/multiplication in human monocytes and multiplication in U937 cells was seen with some dilutions of all antisera (human and rabbit) used. The pattern of enhancement and the dilution resulting in maximum viral activity was constant and seemed to be determined by the serum, but the exact level of enhancement for a given serum differed considerably in monocytes from different donors and seemed to be determined by the cells. In the latter respect, monocytes taken from the same donor some weeks apart showed variations at the same level, as did cells from different donors. PMA (phorbol-12-myristate-13-acetate) stimulation of monocytes and U937 cells resulted in stronger antibody enhancement in terms of infectivity, without affecting the number of monocytes showing antigen expression. No expression/multiplication of BKV was detected in the murine M luminal diameter-like cell line P338 DI.

Characterization of BK virus-specific antibodies in human sera by Western immunoblotting: use of a zwitterionic detergent for restoring the antibody-binding capacity of electroblotted proteins.

After initial problems related to denaturation of antigenic epitopes, we developed a Western immunoblotting method for the characterization of antibodies reacting with BK virus (BKV) structural polypeptides. When a zwitterionic detergent, Empigen BB, was added to the running buffer during electroblotting, the antibody-binding capacity of electrophoretically separated BKV polypeptides was partially restored. Antibodies reacting with different BKV antigens were detected and visualized by biotinylated anti-species-specific antibodies, peroxidase-conjugated streptavidine, and diaminobenzidine staining. Human sera containing anti-BKV antibodies reacted with VP1, but a serum containing antinuclear antibodies also reacted with VP4, -5 and -6 (histones). Serum from a rabbit inoculated with purified BKV reacted with VP1, and also with VP4, indicating that BKV inoculation may imply production of antibodies against histones.

Characterization of a new serotype of infectious pancreatic necrosis virus isolated from Atlantic salmon.

Virus particles isolated from hatchery reared fish with infectious pancreatic necrosis (IPN) were neutralized by homologous immune sera but not by immune sera raised against IPN virus serotype 1, 2, and 3. This virus isolate, called the N1 strain, was detected in one year old Atlantic salmon (Salmo salar) during an outbreak with histopathological lesions of IPN and slightly increased mortality. The polypeptide pattern of N1 virus differed markedly from that of the three classical IPN virus serotypes. Double stranded RNA isolated from the N1 virus particles, co-migrated during agarose gel electrophoresis with nucleic acid isolated from the IPN virus Jasper and Ab strains. Nucleic acid hybridizations using low stringency washing conditions and a synthetic DNA oligonucleotide probe (representing the 3' end of the A segment of the Jasper strain) gave positive results with the IPN virus Jasper, Ab, Sp, and N1 strains. The results presented in this paper show that the N1 isolate differs immunologically and biochemically from the IPN virus serotypes 1, 2, and 3 and may represent a new serotype of IPNV.

Neutralization test for BK virus: plaque reduction detected by immunoperoxidase staining.

We developed an immunoperoxidase staining test to detect structural antigens of BK virus (BKV) in Vero cell cultures. This test was used to examine the neutralizing activity of human and immunized animal sera. It was shown that sera positive for BKV antibodies measured by hemagglutination inhibition test and enzyme-linked immunosorbent assay (ELISA) were able to prevent expression of BKV structural antigens in cell cultures. The correlation between titers in the hemagglutination inhibition test, levels of BKV IgG measured by ELISA, and the titers assayed by the immunoperoxidase neutralization test was high. We suggest that this type of test may be used instead of conventional neutralization tests for other viruses with slowly developing cytopathogenic effects.

IgM antibodies in sera from patients with chronic active hepatitis reacting with the measles virus matrix protein and nucleoprotein.

The antigenic specificity of measles virus IgM antibodies in sera from patients with chronic active hepatitis not caused by hepatitis B virus has been examined. An immunosorbent column containing antihuman IgM covalently bound to Sepharose was used to pick up IgM from the sera. Radiolabelled measles virus antigens were then allowed to react with the IgM antibodies. The immune complexes were eluted and analysed by sodium dodecyl sulfate [SDS]-polyacrylamide gel electrophoresis. Four sera from patients with hepatitis B surface antigen [HBsAg]-negative chronic active hepatitis with high measles virus haemagglutination inhibition [HI] and complement fixation [CF] antibody titres and positive enzyme-linked immunosorbent assay [ELISA] for measles-virus-specific IgM were examined. The results were compared with those obtained using sera from patients with an acute measles virus infection and from healthy controls. In both patient groups, IgM antibodies with specificity against the matrix protein represented the major portion of the measles virus IgM. IgM antibodies against the measles virus nucleoprotein and probably against host-cell-derived actin were also present. The patient sera contained only traces of IgM antibodies with specificity against the measles virus haemagglutinin or fusion protein. No specific IgM antibodies were found in sera from healthy controls.

Measles virus-specific IgM antibodies in sera from patients with chronic active hepatitis.

The occurrence of measles virus-specific IgM antibodies in sera from patients with chronic active hepatitis not caused by Hepatitis B virus was examined by a specific enzyme-linked immunosorbent assay (ELISA). Using whole serum, specific IgM antibodies were detected in 12 of 23 sera from patients with hepatitis B surface antigen (HBsAg)-negative chronic active hepatitis. In nine of these sera the finding of specific IgM antibodies was verified by separation of IgM by density gradient centrifugation and examination of the fractions by ELISA. Most of the sera from the patients with measles virus-specific IgM antibodies had an elevated level of specific IgG antibodies compared to the level of IgG found in control sera. The significance of these findings in view of a possible persistent measles virus antigen production in patients with chronic active hepatitis is discussed.

Characterization of measles virus-specific antibodies in sera from patients with chronic active hepatitis.

Measles virus-specific antibodies in sera from patients with HBsAg-negative chronic active hepatitis and raised antibody titres against measles virus, have been examined by crossed immunoelectrophoresis. The immunoprecipitates were further analysed by SDS-polyacrylamide gel electrophoresis. Five measles virus-specific precipitation lines were demonstrated using measles virus-infected cells solubilized with Triton X-100. The three major precipitation lines were analysed by SDS-PAGE and contained the virus polypeptides: nucleoprotein, NP (MW approximately 60 000); haemoagglutinin, H (MW approximately 80 000) and fusion protein F1 (MW approximately 40 000). Considerably higher amounts of antibodies against these three virus polypeptides were demonstrated in the patient sera than in sera from healthy controls. By SDS-PAGE analysis of radiolabelled immune complexes adsorbed to Sepharose-protein A, antibodies against five measles virus polypeptides: NP, H, F1, P protein (MW approximately 70 000) and matrix protein, M (MW approximately 37 000) were demonstrated in the patient sera.

Measles virus specific precipitins in sera from patients with chronic active hepatitis.

Measles virus specific antibodies in sera from 29 patients with chronic active hepatitis have been examined using standard serological tests and immunoelectrophoretic techniques. Significantly raised CF antibody titres against measles virus antigen were demonstrated in 50% of the sera. The major immunoprecipitate obtained by immunoelectrophoresis was shown to contain aggregates of measles virus nucleocapsids. Antibodies against the haemagglutinin antigen of the virus envelope were also raised and were demonstrated by line immunoelectrophoresis.

Influence of hydrocortisone on granulocyte function and glucose metabolism.

Examination has been made of the influence of hydrocortisone on the in vitro phagocytosis and intracellular killing of Staphylococcus aureus by human neutrophils and the production of lactate and CO2 during phagocytosis of latex particles. In high concentrations, 0.5-2 mg per ml, hydrocortisone caused a significant reduction in the phagocytosis and the production of lactate. Neither the bactericidal activity nor the production of 14CO2 from (U-14C) glucose in phagocytizing leukocytes was influenced by these hydrocortisone concentrations.

Influence of antibodies and thermolabile serum factors on the bactericidal activity of human neutrophil granulocytes.

The influence of serum natibodies and thermolabile serum factors on the intracellular killing of Staphylococcus aureus by neutrophil granulocytes has been examined using a method which facilitates a precise in vitro evaluation of the phagocytic and bactericidal activities of human polymorphonuclear leucocytes. The bactericidal activity of the granulocytes was significantly less pronounced in the presence of serum absorbed with Staph. aureus or inactivated at 56 degrees C for 30 minutes than in the presence of untreated serum. Specific antibodies seemed to stimulate the intracellular killing of bacteria more than thermolabile serum factors.

Influence of IgG, F(ab')2 and IgM on the phagocytic and bactericidal activities of human neutrophil granulocytes.

The phagocytosis and intracellular killing of Staphylococcus aureus by humans granulocytes in the presence of immunoglobulin preparations have been examined. Isolated IgG from pooled human serum induced phagocytosis and intracellular killing of bacteria. F(ab')2 fragments had no significant effect, indicating that the Fc piece of the IgG molecule is of importance not only for the phagocytosis but also for the intracellular killing of bacteria. Isolated IgM stimulated the phagocytosis to a minor extent, with no enhancement of the bactericidal activity.