Occurrence of viral haemorrhagic septicaemia virus (VHSV) in wild marine fish species in the coastal regions of Norway

This study was aimed at determining the occurrence of viral haemorrhagic septicaemia virus (VHSV) in selected stocks of wild fish species in the coastal regions of Norway. Six cruises were undertaken covering areas which included the coastal regions of northern Norway, the Norwegian Sea, the Barents Sea, and the Skagerrak area down to the Danish border. Collected, pooled samples of internal organs (kidney, spleen and heart) from 8395 fish were examined for the presence of VHSV by inoculation on BF-2 cells. Identification of virus was performed by a standard ELISA procedure with monoclonal antibody IP5B11, which is specific for the VHSV nucleocapsid protein (N-protein). VHSV was isolated from blue whiting and Norway pout in Skagerrak. No positive samples were detected in the northern coastal regions of Norway, the Norwegian Sea, or the Barents Sea. The findings indicate a very low occurrence of VHSV in the coastal regions of Norway. Geographically, the only positive samples were obtained from fish collected in areas where VHSV has previously been found in different species of fish.     

Use of a cDNA microarray to study immunity against viral hemorrhagic septicemia (VHS) in Japanese flounder (Paralichthys olivaceus) following DNA vaccination

Japanese flounder, Paralichthys olivaceus juveniles were vaccinated against viral hemorrhagic septicemia (VHS) by intramuscular injection of 10 microg of a plasmid DNA vector which encodes the viral hemorrhagic septicemia virus (VHSV) glycoprotein (G) gene under the control of the cytomegalovirus promoter. Experimental challenge of two viral doses (1 x 10(2) TCID50 and 1 x 10(3) TCID50) one month post-vaccination revealed that the G gene was able to induce protective immunity against VHS and this lasted until 21 days after the challenge. The VHSV G-protein gene DNA vaccine had a high protective efficiency, giving relative percentage survival (RPS) values of at least 93%. The defense mechanisms activated by the DNA vaccine were further elucidated by microarray analysis. Non-specific immune response genes such as NK, Kupffer cell receptor, MIP1-alpha and Mx1 protein gene were observed to be up-regulated by the VHSV G-protein DNA vaccine at 1 and 3 days post-immunization. Also, specific immune-related genes including the CD20 receptor, CD8 alpha chain, CD40 and B lymphocyte cell adhesion molecule were also up-regulated during that time. We observed significant up-regulation of some immune-related genes that are necessary for antiviral defense. Significant up- and/or down-regulation of unknown genes was also observed upon DNA vaccination. Our results confirm previous reports that the VHSV G gene elicits strong humoral and cellular immune responses which may play a pivotal role in protecting the fish during virus infections.     

Screening for Viral Hemorrhagic Septicemia Virus in Marine Fish along the Norwegian Coastal Line

Viral hemorrhagic septicemia virus (VHSV) infects a wide range of marine fish species. To study the occurrence of VHSV in wild marine fish populations in Norwegian coastal waters and fjord systems a total of 1927 fish from 39 different species were sampled through 5 research cruises conducted in 2009 to 2011. In total, VHSV was detected by rRT-PCR in twelve samples originating from Atlantic herring (Clupea harengus), haddock (Melanogrammus aeglefinus), whiting (Merlangius merlangus) and silvery pout (Gadiculus argenteus). All fish tested positive in gills while four herring and one silvery pout also tested positive in internal organs. Successful virus isolation in cell culture was only obtained from one pooled Atlantic herring sample which shows that today''s PCR methodology have a much higher sensitivity than cell culture for detection of VHSV. Sequencing revealed that the positive samples belonged to VHSV genotype Ib and phylogenetic analysis shows that the isolate from Atlantic herring and silvery pout are closely related. All positive fish were sampled in the same area in the northern county of Finnmark. This is the first detection of VHSV in Atlantic herring this far north, and to our knowledge the first detection of VHSV in silvery pout. However, low prevalence of VHSV genotype Ib in Atlantic herring and other wild marine fish are well known in other parts of Europe. Earlier there have been a few reports of disease outbreaks in farmed rainbow trout with VHSV of genotype Ib, and our results show that there is a possibility of transfer of VHSV from wild to farmed fish along the Norwegian coast line. The impact of VHSV on wild fish is not well documented.     

Differences in virulence of marine and freshwater isolates of viral hemorrhagic septicemia virus in vivo correlate with in vitro ability to infect gill epithelial cells and macrophages of rainbow trout (Oncorhynchus mykiss)

Two strains of viral hemorrhagic septicemia virus (VHSV) with known different virulence characteristics in vivo were studied (by a time course approach) for their abilities to infect and translocate across a primary culture of gill epithelial cells (GEC) of rainbow trout (RBT; Oncorhynchus mykiss). The strains included one low-virulence marine VHSV (ma-VHSV) strain, ma-1p8, and a highly pathogenic freshwater VHSV (fw-VHSV) strain, fw-DK-3592B. Infectivities toward trout head kidney macrophages were also studied (by a time course method), and differences in in vivo virulence were reconfirmed, the aim being to determine any correlation between in vivo virulence and in vitro infectivity. The in vitro studies showed that the fw-VHSV isolate infected and caused a cytotoxic effect in monolayers of GEC (demonstrating virulence) at an early time point (2 h postinoculation) and that the same virus strain had translocated over a confluent, polarized GEC layer by 2 h postinoculation. The marine isolate did not infect monolayers of GEC, and delayed translocation across polarized GEC was seen by 48 h postinoculation. Primary cultures of head kidney macrophages were also infected with fw-VHSV, with a maximum of 9.5% virus-positive cells by 3 days postinfection, while for the ma-VHSV strain, only 0.5% of the macrophages were positive after 3 days of culture. In vivo studies showed that the fw-VHSV strain was highly virulent for RBT fry and caused high mortality, with classical features of viral hemorrhagic septicemia. The ma-VHSV showed a very low level of virulence (only one pool of samples from the dead fish was VHSV positive). This study has shown that the differences in virulence between marine and freshwater strains of VHSV following the in vivo infection of RBT correlate with in vitro abilities to infect primary cultures of GEC and head kidney macrophages of the same species.     

Three dimensional morphology of rabies virus studied by cryo-electron tomography

The rabies virus (RABV) continues to be a worldwide health problem. RABV contains a single-stranded RNA genome that associates with the nucleoprotein N. The resulting ribonucleoprotein complex is surrounded by matrix protein M, lipid bilayer and glycoprotein G. RABV was reported to organize in bullet-like virions, but the role of each viral component in adopting this morphology is unclear. We present here a cryo-electron tomography study of RABV showing additional morphologies consisting in bullet-like virions containing a tubular, lipidic appendage having G-protein at its apex. In addition, there was evidence for an important fraction of pleomorphic particles. These pleomorphic forms differed in the amount of membrane-associated M-, M/N-protein providing interesting insight into its role in viral morphogenesis. In the absence of membrane-associated M-, M/N-protein viral morphology was almost spherical. Other images, showing straight membrane portions, correlate with the M-protein recruitment at the membrane independently of the presence of the G-protein. The viral membrane was found to contain a negative net charge indicating that M-, M/N-protein-membrane charge attraction drives this interaction.     

Temperature effects on immune response and hematological parameters of channel catfish Ictalurus punctatus vaccinated with live theronts of Ichthyophthirius multifiliis

This study evaluated the influence of temperature on the immune responses and hematological parameters in channel catfish Ictalurus punctatus immunized via intraperitoneal injection with live theronts of Ichthyophthirius multifiliis. Fish were distributed in 18 aquaria and received 9 treatments: 4 groups of fish were vaccinated with live theronts and maintained at constant temperature 15 °C, 20 °C, 25 °C and 30 °C; 3 groups of fish vaccinated and subjected to cycling temperature regime from 15-25 °C, 20-25 °C and 20-30 °C, changed 5 °C each day; 2 groups of fish were not vaccinated and served as controls at 25 °C, one with Ich challenge and the other without challenge. Non vaccinated fish and those vaccinated at 15 °C or 15-25 °C did not show anti-Ich antibodies in the serum 14 and 21 days post-immunization. The antibody levels were significantly higher from fish vaccinated at 25 °C, 30 °C, 20-25 °C and 20-30 °C compared to fish at 15 °C, 20 °C and 15-25 °C both 14 and 21 days post-immunization. At constant water temperature, fish vaccinated at 15 °C showed significantly higher mortality rate (67.8%, P < 0.05) than those vaccinated at 20 °C, 25 °C, and 30 °C (0-10.7% mortalities). At cycling water temperature, fish vaccinated at 15-25 °C showed significantly higher mortality rate (67.8%) than those vaccinated at 20-25 °C and 20-30 °C (P < 0.05). Twenty days after immunization fish vaccinated at 30 °C and 20-30 °C showed significant increase in the red blood cells, white blood cells, thrombocytes and monocytes. Six days after challenge with I. multifiliis theronts the fish showed decreased white blood cells, thrombocytes and monocytes. This study suggests that vaccinated catfish were severely impacted by low temperature, either at 15 °C constant temperature or at 15-25 °C cycling temperature. The fish showed no anti-Ich antibodies and suffered high mortality similar to non vaccinated control fish.     

Cyclical mechanical stretch-induced apoptosis in myocytes from young rats but necrosis in myocytes from old rats

Mechanical load as stimulus for apoptosis and necrosis could be responsible for the loss of cardiomyocytes. Ventricular myocytes from young (3 mo) and old (14-24 mo) rats underwent cyclical mechanical stretch (CMS; 5% elongation, 1 Hz) for 24 h. Spontaneous apoptosis was in myocytes from young rats 0.33 +/- 0.12% and from old rats 1.05 +/- 0.35% [Tdt-mediated dUTP nick-end labeling (TUNEL) assay]; associated with a decrease of Bcl-2. CMS increased the apoptosis to 0.58 +/- 0.18% in myocytes from young rats. Western blot analysis showed that CMS reduced Bcl-2 and increased p53 (young rats). Bax was not changed by CMS. These were confirmed by cytochrome c release (31 +/- 13%) and by the enrichment of cytosolic nucleosomes (11 +/- 8%). CMS did not influence the apoptosis in myocytes from old rats (TUNEL assay, Bcl-2, Bax, or p53). CMS did not cause necrosis in myocytes from young rats. CMS increased the number of necrotic cells by showing the cell membrane rupture in myocytes from old rats (50 +/- 13% 5-hexadecanoylaminofluorescein-positive and 38 +/- 6% propidium iodide-positive cells) as well as by measuring the lactate dehydrogenase release. The results suggest that CMS-induced apoptosis in myocytes of young rats but necrosis in myocytes from old rats, which could be attributed to more stress sensitivity of cells from old rats.     

Production of the 57 kDa major surface antigen by a non-agglutinating strain of the fish pathogen Renibacterium salmoninarum

Both the prevalence and tissue titer of viral hemorrhagic septicemia virus (VHSV) increased in Pacific herring Clupea pallasi following their introduction into net pens (pounds) used in the closed pound spawn-on-kelp (SOK) fishery in Prince William Sound, Alaska. VHSV was also found in water samples from inside and outside the SOK pounds after herring had been confined for several days; however, water samples taken near wild free-ranging, spawning herring either failed to test positive or tested weakly positive for virus. Little or no virus was found in tissue samples from free-ranging, spawning herring captured from the vicinity of the pounds, nor did the prevalence of VHSV increase following spawning as it did in impounded herring. The data indicated that increased prevalences of VHSV were correlated with confinement of herring for the closed pound SOK fishery and that infection was spread within the pounds through waterborne exposure to virus particles originating from impounded fish. In addition, pounds containing predominantly young fish had higher prevalences of VHSV, suggesting that older fish may be partially immune, perhaps as a result of previous infection with the virus. Operation of SOK pounds during spawning seasons in which young herring predominate may amplify the disease and possibly exacerbate the population fluctuations observed in wild herring stocks.     

Can VHS Virus Bypass the Protective Immunity Induced by DNA Vaccination in Rainbow Trout?

DNA vaccines encoding viral glycoproteins have been very successful for induction of protective immunity against diseases caused by rhabdoviruses in cultured fish species. However, the vaccine concept is based on a single viral gene and since RNA viruses are known to possess high variability and adaptation capacity, this work aimed at evaluating whether viral haemorrhagic septicaemia virus (VHSV), an RNA virus and member of Rhabdoviridae family, was able to evade the protective immune response induced by the DNA vaccination of rainbow trout. The experiments comprised repeated passages of a highly pathogenic VHSV isolate in a fish cell line in the presence of neutralizing fish serum (in vitro approach), and in rainbow trout immunized with the VHS DNA vaccine (in vivo approach). For the in vitro approach, the virus collected from the last passage (passaged virus) was as sensitive as the parental virus to serum neutralization, suggesting that the passaging did not promote the selection of virus populations able to bypass the neutralization by serum antibodies. Also, in the in vivo approach, where virus was passaged several times in vaccinated fish, no increased virulence nor increased persistence in vaccinated fish was observed in comparison with the parental virus. However, some of the vaccinated fish did get infected and could transmit the infection to naïve cohabitant fish. The results demonstrated that the DNA vaccine induced a robust protection, but also that the immunity was non-sterile. It is consequently important not to consider vaccinated fish as virus free in veterinary terms.     

Immunoelectron microscopic studies of the intracellular transport of the membrane glycoprotein (G) of vesicular stomatitis virus in infected Chinese hamster ovary cells

An immunoelectron microscopic study was undertaken to survey the intracellular pathway taken by the integral membrane protein (G-protein) of vesicular stomatitis virus from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane of virus-infected Chinese hamster ovary cells. Intracellular transport of the G-protein was synchronized by using a temperature-sensitive mutant of the virus (0-45). At the nonpermissive temperature (39.8 degrees C), the G-protein is synthesized in the cell infected with 0-45, but does not leave the rough endoplasmic reticulum. Upon shifting the temperature to 32 degrees C, the G-protein moves by stages to the plasma membrane. Ultrathin frozen sections of 0-45-infected cells were prepared and indirectly immunolabeled for the G-protein at different times after the temperature shift. By 3 min, the G-protein was seen at high density in saccules at one face of the Golgi apparatus. No large accumulation of G-protein-containing vesicles were observed near this entry face, but a few 50-70-mm electron-dense vesicular structures labeled for G-protein were observed that might be transfer vesicles between the rough endoplasmic reticulum and the Golgi complex. At blebbed sites on the nuclear envelope at these early times there was a suggestion that the G-protein was concentrated, these sites perhaps serving as some of the transitional elements for subsequent transfer of the G-protein from the rough endoplasmic reticulum to the Golgi complex. By 3 min after its initial asymmetric entry into the Golgi complex, the G-protein was uniformly distributed throughout all the saccules of the complex. At later times, after the G-protein left the Golgi complex and was on its way to the plasma membrane, a new class of G-protein-containing vesicles of approximately 200-nm diameter was observed that are probably involved in this stage of the transport process. These data are discussed, and the further prospects of this experimental approach are assessed.     

Rainbow trout surviving infections of viral haemorrhagic septicemia virus (VHSV) show lasting antibodies to recombinant G protein fragments

Rainbow trout antibodies (Abs) binding to recombinant fragments (frgs) derived from the protein G of the viral haemorrhagic septicemia virus (VHSV)-07.71 strain, could be detected by ELISA (frg-ELISA) in sera from trout surviving laboratory-controlled infections. Abs were detected not only by using sera from trout infected with the homologous VHSV isolate but also with the VHSV-DK-201433 heterologous isolate, which had 13 amino acid changes. Sera from healthy trout and/or from trout surviving infectious haematopoietic necrosis virus (IHNV) infection, were used to calculate cut-off absorbances to differentiate negative from positive sera. Specific anti-VHSV Abs could then be detected by using any of the following frgs: frg11 (56-110), frg15 (65-250), frg16 (252-450) or G21-465. While high correlations were found among the ELISA values obtained with the different frgs, no correlations between any frg-ELISA and complement-dependent 50% plaque neutralization test (PNT) titres could be demonstrated. Between 4 and 10 weeks after VHSV infection, more trout sera were detected as positives by using heterologous frg-ELISA rather than homologous PNT. Furthermore, the percentage of positive sera detected by frg11-ELISA increased with time after infection to reach 100%, while those detected by complement-dependent PNT decreased to 29.4%, thus confirming that the lack of neutralizing Abs does not mean the lack of any anti-VHSV Abs in survivor trout sera. Preliminary results with sera from field samples suggest that further refinements of the frg-ELISA could allow detection of anti-VHSV trout Abs in natural outbreaks caused by different heterologous VHSV isolates. The homologous frg-ELISA method could be useful to follow G immunization attempts during vaccine development and/or to best understand the fish Ab response during VHSV infections. The viral frgs approach might also be used with other fish species and/or viruses.     

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.     

Protection against Atlantic halibut nodavirus in turbot is induced by recombinant capsid protein vaccination but not following DNA vaccination

Fish nodaviruses (betanodaviruses) are small, non-enveloped icosahedral single-stranded positive-sense RNA viruses that can cause viral encephalopathy and retinopathy (VER) in a number of cultured marine teleost species, including Atlantic halibut (Hippoglossus hippoglossus). A recombinant protein vaccine and a DNA vaccine were produced, based on the same capsid-encoding region of the Atlantic halibut nodavirus (AHNV) genome, and tested for protection in juvenile turbot (Scophthalmus maximus). Vaccine efficacy was demonstrated in the fish vaccinated with recombinant capsid protein but not in the DNA-vaccinated fish, despite the fact that in vivo expression of the DNA vaccine-encoded antigen was confirmed by RNA in situ hybridisation and immunohistochemistry. Combined DNA and recombinant vaccine administration did not improve the effect of the latter. Surprisingly, fish vaccinated with 50 microg recombinant protein demonstrated a threefold lower survival rate than the two groups that received 10 microg recombinant protein. Neither the recombinant protein vaccine nor the DNA vaccine induced anti-viral antibodies 9 weeks after immunisation, while antibodies reactive with the recombinant protein were detectable mainly in fish vaccinated with 50 microg recombinant protein. The study also demonstrates evidence of viral replication inside the myocytes of intramuscularly challenged fish.     

Heterologous exchanges of the glycoprotein and the matrix protein in a Novirhabdovirus

Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are two salmonid rhabdoviruses replicating at low temperatures (14 to 20 degrees C). Both viruses belong to the Novirhabdovirus genus, but they are only distantly related and do not cross antigenically. By using a recently developed reverse-genetic system based on IHNV (S. Biacchesi et al., J. Virol. 74:11247-11253, 2000), we investigated the ability to exchange IHNV glycoprotein G with that of VHSV. Thus, the IHNV genome was modified so that the VHSV G gene replaced the complete IHNV G gene. A recombinant virus expressing VHSV G instead of IHNV G, rIHNV-Gvhsv, was generated and was shown to replicate as well as the wild-type rIHNV in cell culture. This study was extended by exchanging IHNV G with that of a fish vesiculovirus able to replicate at high temperatures (up to 28 degrees C), the spring viremia of carp virus (SVCV). rIHNV-Gsvcv was successfully recovered; however, its growth was restricted to 14 to 20 degrees C. These results show the nonspecific sequence requirement for the insertion of heterologous glycoproteins into IHNV virions and also demonstrate that an IHNV protein other than the G protein is responsible for the low-temperature restriction on growth. To determine to what extent the matrix (M) protein interacts with G, a series of chimeric pIHNV constructs in which all or part of the M gene was replaced with the VHSV counterpart was engineered and used to recover the respective recombinant viruses. Despite the very low percentage (38%) of amino acid identity between the IHNV and VHSV matrix proteins, viable chimeric IHNVs, harboring either the matrix protein or both the glycoprotein and the matrix protein from VHSV, were recovered and propagated. Altogether, these data show the extreme flexibility of IHNV to accommodate heterologous structural proteins.