Detection of rainbow trout antibodies against viral haemorrhagic septicaemia virus (VHSV) by neutralisation test is highly dependent on the virus isolate used

Three serological tests, enzyme linked immunosorbent assay (ELISA), 50% plaque neutralisation test (50%PNT) and Western blotting (WB), were used to detect antibodies against viral haemorrhagic septicaemia virus (VHSV) in 50 rainbow trout broodstock from a rainbow trout farm endemically infected with VHS but with no clinical signs of infection. When the sera were examined by 50%PNT using the VHSV reference isolate DK-F1 or the heat attenuated DK-F25 mutant strain, no neutralizing antibodies were found. In contrast, when one of the virus isolates from the farm (homologous virus) was used in the 50%PNT, 90% of the fish were found to be positive. By examining a panel of different VHSV isolates in 50%PNT, it was demonstrated that the virus isolate used as test antigen could significantly affect the sensitivity and titre determination in 50%PNT for detection of rainbow trout antibodies against VHSV. When the sera were examined for the presence of VHSV antibodies by ELISA or WB, 61% were found to be positive. When conducting WB analysis, the viral glycoprotein was the protein most frequently recognized, followed by the viral nucleoprotein.     

Antibody response to VHS virus proteins in rainbow trout

The antibody response to viral haemorrhagic septicaemia virus proteins in rainbow trout surviving a disease outbreak under field conditions as well as animals immunised under laboratory conditions was analysed by immunoblotting, immunofluorescence and plaque neutralisation. No direct correlation between the serum reactivity in immunoblotting and the other serological tests was observed. Among sera from survivors from a disease outbreak in a farm, virus specific antibodies could be detected in most of the sera by immunofluorescence but only in a minority by immunoblotting. In fish injected with the individual viral proteins G, N, M1, or M2 under aquarium conditions, only the glycoprotein induced antibodies detectable by immunoblotting. Challenge of the fish with virulent virus indicated that only minor degrees of protective immunity had been induced. In sera from fish surviving the challenge, the neutralising activity was high. In immunoblotting however, a significant antibody reactivity was observed only in sera from fish primed with the glycoprotein. The results are discussed with respect to the immunogenicity of VHSV proteins in rainbow trout as well as the character of the epitopes recognised by antibodies induced in infected or immunised fish.     

Immunogenicity of a recombinant infectious hematopoietic necrosis virus glycoprotein produced in insect cells.

A recombinant infectious hematopoietic necrosis virus (IHNV) glycoprotein (G protein), produced in Spodoptera frugiperda (Sf9) cells following infection with a baculovirus vector containing the full-length (1.6 kb) glycoprotein gene, provided very limited protection in rainbow trout Oncorhynchus mykiss challenged with IHNV. Fish were injected intraperitoneally (i.p.) with Sf9 cells grown at 20 degrees C (RecGlow) or 27 degrees C (RecGhigh) expressing the glycoprotein gene. Various antigen (Ag) preparations were administered to adult rainbow trout or rainbow trout fry. Sera collected from adult fish were evaluated for IHNV neutralization activity by a complement-dependent neutralization assay. Anti-IHNV neutralizing activity was observed in sera, but the percent of fish responding was significantly lower (p < 0.05) in comparison to fish immunized with a low virulence strain of IHNV (LV-IHNV). A small number of fish immunized with RecGlow or RecGhigh possessed IHNV G protein specific antibodies (Abs) in their serum. Cumulative mortality (CM) of rainbow trout fry (mean weight, 1 g) vaccinated by i.p. injection of freeze/thawed Sf9 cells producing RecGlow was 18% in initial trials following IHNV challenge. This level of protection was significant (p < 0.05) but was not long lasting, and neutralizing Abs were not detected in pooled serum samples. When trout fry (mean weight, 0.6 g) were vaccinated with supernatant collected from sonicated Sf9 cells, Sf9 cells producing RecGlow, or Sf9 cells producing RecGhigh, CM averaged 46%. Protection was enhanced over negative controls, but not the positive controls (2% CM), suggesting that in the first trial soluble cellular proteins may have provided some level of non-specific protection, regardless of recombinant protein expression. Although some immunity was elicited in fish, and RecGlow provided short-term protection from IHNV, Ab-mediated protection could not be demonstrated. The results suggest that recombinant G proteins produced in insect cells lack the immunogenicity associated with vaccination of fish with an attenuated strain of IHNV.     

Experimental susceptibility of turbot Scophthalmus maximus to viral haemorrhagic septicaemia virus isolated from cultivated turbot

Juvenile pathogen-free turbot were infected with a viral haemorrhagic septicaemia virus (VHSV) isolate recovered from turbot cultivated on the island of Gigha, West Scotland. Mortality of 100% was recorded in fish infected via the intra-peritoneal (i.p.) route. Horizontal transmission of VHSV in sea water was demonstrated by cohabitation of naive fish with i.p. infected fish at a ratio of 1:1. The total cumulative average mortality in cohabiting fish was 60% by 60 d post-infection. Turbot infected via an immersion route exhibited a cumulative average mortality of 71% by the end of the experiment. VHSV identified by enzyme-linked immunosorbent assay (ELISA) was recovered from both organ (kidney and spleen) and brain samples of individual fish that died following infection by all experimental routes. These findings pose significant implications regarding the persistence of VHSV and its role in limiting natural populations of marine fish species. In addition, the establishment of infection models for the transmission of VHSV in sea water is of fundamental importance to the development of anti-VHSV vaccines in important commercial species such as turbot.     

Viral haemorrhagic septicaemia (VHS) outbreaks in Finnish rainbow trout farms

In Finland, viral haemorrhagic septicaemia virus (VHSV) was diagnosed for the first time in 2000 from 4 rainbow trout farms in brackish water. Since then the infection has spread and, by the end of 2004, VHSV had been isolated from 24 farms in 3 separate locations: 2 in the Baltic Sea and 1 in the Gulf of Finland. The pathogenicity of 3 of these isolates from 2 separate locations was analysed in infection experiments with rainbow trout fry. The cumulative mortalities induced by waterborne and intraperitoneal challenge were approximately 40 and 90 %, respectively. Pair-wise comparisons of the G and NV gene regions of Finnish VHSV isolates collected between 2000 and 2004 revealed that all isolates were closely related, with 99.3 to 100% nucleotide identity, which suggests the same origin of infection. Phylogenetic analysis revealed that they were closely related to the old freshwater isolates from rainbow trout in Denmark and to one old marine isolate from cod in the Baltic Sea, and that they were located close to the presumed ancestral source. As the Finnish isolates induce lower mortality than freshwater VHSV isolates in infection experiments, they could represent an intermediate stage of marine isolates evolving towards pathogenicity in rainbow trout.     

European freshwater VHSV genotype Ia isolates divide into two distinct subpopulations

Viral haemorrhagic septicaemia (VHS), caused by the novirhabdovirus VHSV, often leads to significant economic losses to European rainbow trout production. The virus isolates are divided into 4 distinct genotypes with additional subgroups including sublineage Ia, isolates of which are the main source of outbreaks in European rainbow trout farming. A significant portion of Danish rainbow trout farms have been considered endemically infected with VHSV since the first disease outbreak was observed in the 1950s. However, following a series of sanitary programs starting in 1965, VHSV has not been detected in Denmark since January 2009. Full-length G-genes of all Danish VHSV isolates that were submitted for diagnostic analyses in the period 2004-2009 were sequenced and analysed. All 58 Danish isolates from rainbow trout grouped with sublineage Ia isolates. Furthermore, VHSV isolates from infected Danish freshwater catchments appear to have evolved into a distinct clade within sublineage Ia, herein designated clade Ia-1, whereas trout isolates originating from other continental European countries cluster in another distinct clade, designated clade Ia-2. In addition, phylogenetic analyses indicate that VHSV Ia-1 strains have caused a few outbreaks in Germany and the UK. It is likely that viruses have been transmitted from infected site(s) out of the Danish environment, although a direct transmission pathway has not been identified. Furthermore, VHSV Ia-2 isolates seem to have been transmitted to Denmark at least once. Interestingly, one viral isolate possibly persisted in a Danish watershed for nearly 4 yr without detection whereas other subclades of VHSV isolates appear to have been eliminated, probably because of implemented eradication procedures.     

In vivo and in vitro analysis of the resistance against viral haemorrhagic septicaemia virus in Japanese flounder (Paralichthys olivaceus) precedingly infected with aquabirnavirus

The resistance of Japanese flounder (Paralichthys olivaceus Temminck et Schlegel) against a viral haemorrhagic septicaemia virus (VHSV) challenge induced by a preceding non-lethal aquabirnavirus (ABV) challenge was investigated through experimental dual-infections with different intervals between the two challenges. The non-specific protection conferred by the primary ABV infection against the secondary VHSV infection commenced at Day 3 and persisted up to Day 14 but vanished at Day 21 post-ABV challenge. The in vitro assay using HINAE (hirame natural embryo) cells demonstrated anti-VHSV activity in the serum of ABV-challenged flounder from Day 1 to Day 14 but not at Day 21 post-ABV challenge. A high expression of a Mx gene, a molecular marker of type I interferon(s) (IFN) occurred in the head kidneys of ABV-challenged flounder from Day 1 to Day 7. These results suggest that the non-specific protection against the secondary VHSV infection in flounder was due to IFN(s) induced by the primary ABV infection.     

Genetic relatedness among Japanese,American and European isolates of viral hemorrhagic septicemia virus (VHSV) based on partial G and P genes

Molecular virological analyses of 8 Japanese VHSV (viral hemorrhagic septicemia virus) isolates from wild and farmed Japanese flounder Paralichthys olivaceus were performed to investigate their genetic relatedness to American and European isolates of VHSV. Phylogenetic analyses based on the partial nucleotide sequences of G and P genes revealed that there are 2 genogroups of VHSV in Japan. The first one represented by the Obama25 isolate is closely related to the American isolates (Genogroup I) while the other, the KRRV9601 isolate, is closely related to the traditional European isolates (Genogroup III). The 2 types of Japanese VHSV showed differences in the relative mobility of the G protein and intensity of the antibody reaction on the P and M proteins. The Obama25 type of VHSV is widely distributed as a native virus in the coastal areas of western Japan and has been responsible for the occurrence of VHSV infection in farmed Japanese flounder while the KRRV9601 isolate is considered to have been introduced from a foreign country.     

Genotyping and pathogenicity of viral hemorrhagic septicemia virus from free-living turbot (Psetta maxima) in a Turkish coastal area of the Black Sea

Viral hemorrhagic septicemia (VHS) is one of the most serious fish viral diseases for cultured rainbow trout (Oncorhynchus mykiss), although VHS virus (VHSV) seems to be ubiquitous among marine fishes. In the present study, VHSV isolation was performed with free-living and cultured turbot (Psetta maxima) in the Trabzon coastal area of the Black Sea to evaluate participation of VHSV in mass mortalities of seed-produced turbot larvae. VHSV was detected in 14 of 66 free-living spawners (positive ratio, 21.2%), 1 of 65 free-living immature fish (1.5%) and 7 of 40 cultured brood stock (17.5%), respectively. Based on a partial glycoprotein gene nucleotide sequence, Turkish VHSV isolates were classified into the class I-e of genotype I and were the most closely related to the GE-1.2 isolate (>98% identity), which was found >20 years ago in Georgia. Thus, it was revealed that Turkish VHSV isolates were not introduced from European countries, it could be an indigenous type of VHSV distributing in the Black Sea environment. In pathogenicity tests, the Turkish isolates did not induce mortality in turbot larvae and rainbow trout fingerlings. Mass mortalities at a rate of approximately 90% occurred in turbot larvae produced by experimental seeding, although VHSV was not detected in any dead fish. Thus, it was concluded that mass mortality in the seed-produced turbot larvae was not caused by VHSV infection.     

Reversible inhibition of spreading of in vitro infection and imbalance of viral protein accumulation at low pH in viral hemorrhagic septicemia rhabdovirus, a salmonid rhabdovirus

The inhibition of viral hemorrhagic septicemia rhabdovirus (VHSV) in vitro infection by pHs of <7 (low pH) has been previously reported. Nevertheless, the details of the mechanism underlying this effect remain obscure. We present evidence showing that low-pH inhibition occurs during a viral postadsorption step. Thus, while VHSV bound, replicated within single cells, and presented its G protein on the membranes of infected cells at both low and physiological pHs, both cell-to-cell spreading of infection (as estimated by the appearance of foci of infected cells) and fusion (as estimated by a syncytium assay) were inhibited by this low pH. The decreased VHSV titers and the inhibition of both cell-to-cell spreading of infection and fusion could be reversed by adjusting the pH to 7.5 at any time during infection. This effect should be taken into account to avoid false negatives in the diagnosis of VHSV by cell culture. On the other hand, the cell-to-cell spreading of infection at pH 7.5 could be stopped at any time by reducing the pH to 6.5. Since at low pH there were changes in the protein G conformation and smaller and imbalanced amounts of N with respect to M1, M2, and G viral proteins, alterations of the assembly and/or budding of VHSV are most probably involved in the absence of newly released infective virions.     

Rhabdovirus infection induces public and private T cell responses in teleost fish.

Many viruses induce a strong T cell response that contributes to the elimination of infected cells presenting viral peptides by MHC molecules. The structure and expression of genes encoding molecules homologous to mammalian alphabeta TCRs have been recently characterized in rainbow trout and in several teleost species, but the alphabeta T cell response against pathogens has not been directly demonstrated. To study the modifications of the T cell repertoire during an acute viral infection in rainbow trout, we adapted the immunoscope methodology, which consists of spectratyping the complementarity-determining region 3 length of the TCRbeta chain. We showed that the naive T cell repertoire is polyclonal and highly diverse in the naive rainbow trout. Using viral hemorrhagic septicemia virus (VHSV), which provokes an acute infection in rainbow trout, we identified skewed complementarity-determining region 3 size profiles for several VbetaJbeta combinations, corresponding to T cell clonal expansions during primary and secondary response to VHSV. Both public and private T cell expansions were shown by immunoscope analysis of spleen cells from several infected individuals of a rainbow trout clone sharing the same genetic background. The public response to VHSV consisted of expansion of Vbeta4Jbeta1 T cell, which appeared early during the primary response and was strongly boosted during the secondary response.     

Shedding of viral hemorrhagic septicemia virus (Genotype IVb) by experimentally infected muskellunge (<Emphasis Type="Italic">Esox masquinongy</Emphasis>)

Previous experimental infection demonstrated that juvenile muskellunge (Esox masquinongy) can survive experimental infection of viral hemorrhagic septicemia virus, Genotype IVb (VHSV IVb) at a low concentration of exposure. Herein we report that survivors of experimental infection with VHSV IVb shed the virus into the surrounding environment for an extended period of time. When muskellunge were exposed to VHSV IVb by immersion at a concentration of 1,400 plaque forming units (PFU)/ml, VHSV IVb was detected in the water of surviving fish for up to 15 weeks postexposure (p.e.) with the highest levels of shedding occurring between weeks 1 and 5 p.e. We estimated that each juvenile muskellunge can shed upwards of 1.36×10(5) PFU/fish/h after initial exposure signifying the uptake and amplification of VHSV to several orders of magnitude above the original exposure concentration. Muskellunge surviving low concentration exposure were re-infected with VHSV IVb by immersion at week 22 p.e. at concentrations ranging from 0 to 10(6) PFU/ml. Viral shedding was detected in all re-exposed fish, including mock rechallenged controls up to 15 consecutive weeks. Rates of viral shedding were substantially higher following rechallenge in the first 5 weeks. The highest rate of viral shedding was approximately 4.6×10(6) PFU/fish/h and shedding did not necessarily correspond to the re-exposure VHSV concentration. The results of this study shed new light into the dynamics of VHSV IVb shedding in a highly susceptible host and provide useful insights to fishery managers to design effective control strategies to this deadly virus.     

Endocytosis and intracellular degradation of heterologous protein by eosinophilic granulocytes isolated from rainbow trout (Oncorhynchus mykiss) posterior intestine

Summry— Adherence capacity to tissue substrate, endocytosis capacity for heterologous proteins, and proteolytic activity were determined in intestinal granulocytes (EGCs) isolated from healthy adult rainbow trout. The percentage of cells that could adhere to a smooth plastic surface increased with increasing incubation time. Endocytosis was effective for heterologous (human immunoglobulin G, IgGh; ovine somatotropin, oST) but not homologous proteins (recombinant trout somatotropin, rtST). The activity of cathepsin D increased significantly after the endocytosis of a heterologous protein. Finaly, the analysis of immunoblots of homogenates of granulocytes incubated in the presence of the two different proteins was used to show the endocytosis and degradation of heterologous proteins. These results show that isolated EGCs can endocytose and degrade heterologous proteins.     

Experimental infection of rainbow trout Oncorhynchus mykiss with viral haemorrhagic septicaemia virus isolates from European marine and farmed fishes

The susceptibility of rainbow trout Oncorhynchus mykiss to infection with various isolates of viral haemorrhagic septicaemia virus (VHSV) was examined. A total of 8 experiments with rainbow trout ranging from 0.6 to 6.2 g was conducted for 139 isolates originating from wild marine fishes in European waters (115 isolates), farmed turbot from Scotland and Ireland (2 isolates), and farmed rainbow trout (22 isolates). The isolates were tested by immersion and/or intraperitoneal injection either as pooled or single isolates. The isolates from wild marine fishes did not cause mortality by immersion while some of the isolates caused mortality when injected. All VHSV isolates from farmed rainbow trout caused significant mortality by immersion. Currently, pathogenicity trials are the only way to differentiate VHSV isolates from wild marine fishes and farmed rainbow trout. The 2 farmed turbot isolates did not cause mortality by immersion, supporting the view that they originated from the marine environment.     

Leishmania tropica and Leishmania donovani: solid phase radioimmunoassay using leishmanial excreted factor

A radioimmunoassay for the quantitative determination of anti-leishmanial excreted factor (EF) antibody in rabbit sera was developed. The assay, using Leishmania tropica and Leishmania donovani promastigotes EF, purified by either extraction with phenol followed by fractionation on a Sephadex G-100 column or by the dissociation of EF antibody complexes, was shown to be sensitive and reproducible. Using monospecific anti-EF antibodies, levels of as low as 0.06-0.12 micrograms/ml of anti-EF IgG could be detected. The specificity of the assay was assessed by inhibition with homologous and heterologous EF. Only minor cross-reactivity with heterologous EF was observed, and as little as 2.5 micrograms/ml of EF could be detected. Sera from kala-azar patients showed only 1.8-3.1 times more anti-EF activity, as compared with uninfected controls. No specificity was observed with sera from kala-azar patients with regard to the type of EF used. Almost the same activity was obtained with both EF from L. tropica and L. donovani. No anti-EF antibodies were detected in sera from patients with cutaneous leishmaniasis.     

CCR5-reactive antibodies in seronegative partners of HIV-seropositive individuals down-modulate surface CCR5 in vivo and neutralize the infectivity of R5 strains of HIV-1 In vitro

Exposure to HIV does not necessarily result in infection. Because primary HIV infection is associated with CCR5-tropic HIV variants (R5), CCR5-specific Abs in the sera of HIV-seronegative, HIV-exposed individuals (ESN) might be associated with protection against infection. We analyzed sera from ESN, their HIV-infected sexual partners (HIV+), and healthy controls (USN) searching for CCR5-specific Abs, studying whether incubation of PBMC with sera could prevent macrophage inflammatory protein 1 beta (Mip1 beta) (natural ligand of CCR5) binding to CCR5. Results showed that Mip1 beta binding to CCR5 was not modified by sera of either 40 HIV+ or 45 USN but was greatly reduced by sera of 6/48 ESN. Binding inhibition was due to Abs reactive with CCR5. The CCR5-specific Abs neutralized the infectivity of primary HIV isolates obtained from the corresponding HIV+ partners and of R5-primary HIV strains, but not that of CXCR4-tropic or amphitropic HIV strains. Immunoadsorption on CCR5-transfected, but not on CXCR4-transfected, cells removed CCR5-specific and virus-neutralizing Abs. Epitope mapping on purified CCR5-specific Abs showed that these Abs recognize a conformational epitope in the first cysteine loop of CCR5 (aa 89-102). Affinity-purified anti-CCR5-peptide neutralized the infectivity of R5 strains of HIV-1. Anti-CCR5 Abs inhibited Mip1beta-induced chemotaxis of PBMC from healthy donors. PBMC from two ESN (with anti-CCR5 Abs) were CCR5-negative and could not be stimulated by Mip1beta in chemotaxis assays. These results contribute to clarifying the phenomenon of immunologic resistance to HIV and may have implications for the development of a protective vaccine.     

Induction of Neutralizing Antibodies to T-Cell Line-Adapted and Primary Human Immunodeficiency Virus Type 1 Isolates with a Prime-Boost Vaccine Regimen in Chimpanzees

Five chimpanzees were immunized by administration of one or more intranasal priming doses of one to three recombinant adenoviruses containing a gp160 insert from human immunodeficiency virus type 1 (HIV-1) MN (HIV-1_MN) followed by one or more boosts of recombinant HIV-1_SF2 gp120 delivered intramuscularly with MF59 adjuvant. This regimen resulted in humoral immune responses in three of five animals. Humoral responses included immunochemically active anti-HIV-1 antibodies (Abs) directed to recombinant gp120 and neutralizing Abs reactive with T-cell-line-adapted HIV-1_MN and HIV-1_SF2. In addition, neutralizing activity was detected to the two homologous primary isolates and to two of three heterologous primary isolates which, like the immunizing strains, can use CXCR4 as a coreceptor for infection. The three animals with detectable neutralizing Abs and a fourth exhibiting the best cytotoxic T-lymphocyte response were protected from a low-dose intravenous challenge with a cell-free HIV-1_SF2 primary isolate administered 4 weeks after the last boost. Animals were rested for 46 weeks and then rechallenged, without a boost, with an eightfold-higher challenge dose of HIV-1_SF2. The three animals with persistent neutralizing Abs were again protected. These data show that a strong, long-lived protective Ab response can be induced with a prime-boost regimen in chimpanzees. The data suggest that in chimpanzees, the presence of neutralizing Abs correlates with protection for animals challenged intravenously with a high dose of a homologous strain of HIV-1, and they demonstrate for the first time the induction of neutralizing Abs to homologous and heterologous primary isolates.     

Detection of neutralizing antibody to Egtved virus in rainbow trout (Salmo gairdneri) by plaque neutralization test with complement addition

The humoral immune response of trout to Egtved virus (VHS) was examined by means of a 50 % plaque neutralization test with complement addition. The test, wich has been adapted for use in microtitre plates, is documented with regard to specificity and sensitivity. Eight weeks after VHS infection by cohabitation at 13 °C neutralizing antibody could be detected in 100% of individually tagged experimental trout. Maximal litres of 10240 were found six weeks after infection. In 36% of the trout antibody was still detectable one year after infection. Neutralizing antibody could be detected also in trout infected with VHS under trout farm conditions. The percentage of positive sera ranged from 4 to 56.