Kinetics of Mx expression in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar L.) parr in response to VHS-DNA vaccination

The duration of the Mx mRNA response to an intramuscular injection of the viral haemorrhagic septicaemia virus (VHSV) glycoprotein (G) gene DNA vaccine as well as to the control plasmid was determined in rainbow trout at 14 degrees C over a period of 11 weeks. The Mx response was detectable on day 7, peaked on day 14 and returned to pretreatment levels on day 21 and thereafter. No increase in Mx expression was detectable to the control plasmid. In further experiments, the kinetics of the Mx response were compared in rainbow trout and Atlantic salmon parr kept at 10 degrees C and injected with the DNA vaccine or the synthetic double-stranded RNA, poly I:C. In both species there was a rapid response to poly I:C detectable from day 1, reaching maximum from days 3 to 9 and decreasing to background level by day 12. The peak level and return to background was reached slightly later in salmon. In both species the response to the VHS/DNA vaccine was slower to begin, not being detectable on days 1 and 3, but elevated levels were found on day 6. However, in the salmon parr, the peak level was on day 6 and the signal disappeared by day 12, while in the rainbow trout, the response peaked at day 12 and lasted until day 21. The kinetics of the Mx response to the VHS/DNA vaccine in rainbow trout correlate with the early non-specific protection against VHS in this species following vaccination. It is speculated that the more transient Mx response in Atlantic salmon parr to the DNA vaccine may be related to the innate resistance of salmon to VHS.     

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.     

Viral hemorrhagic septicemia (VHS) of nonsalmonids

Viral hemorrhagic septicemia (VHS) is a viscerotropic disease of fish that can cause enormous losses in European rainbow trout populations. Although previously thought to be species specific, recent reports have indicated that other nonsalmonid fish species, such as pike, whitefish, grayling, turbot, and herring, can become mortally infected with VHS. This paper reviews several cases of natural VHS-outbreaks in nonsalmonids. Pike, whitefish, grayling, turbot and Pacific herring infected with VHS displayed symptoms typical of hemorrhagic septicemia. The isolation and serological identification of the viruses from all of the infected fish species revealed a close relation to the Egtved-virus strain F1. The virus isolates from diseased pike and rainbow trout were capable of infecting their respective host fish. Experimental infections of pike, whitefish, and grayling fry with Egtved virus strain F1 resulted in high mortalities, with symptoms typical of VHS. Although experiments with older fish suggested an age-dependent decrease in susceptibility, the virus could be isolated from most of the infected fish after several months of experimental infection (carrier status). Histological and electron microscopical findings were comparable to those seen in VHS-infected rainbow trout. The susceptibility of nonsalmonid species to VHS infection is disconcerting with respect to the potential losses of the economically important turbot, Pacific herring, and whitefish, as well as the ecologically valuable grayling which is threatened by extinction. If the VHS susceptible nonsalmonid species can also support the propagation of the Egtved-virus, it could acquire carrier-status and, as a result, represent a “natural” reservoir for the virus.     

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.     

Indirect Fluorescent Antibody Techniques for Demonstration of Trout Viruses and Corresponding Antibody

Two variations of the indirect fluorescent antibody technique (FAT) have been utilized in the work concerning two important virus diseases of trout, viral haemorrhagic septicaemia (VHS) and infectious pancreatic necrosis (IPN). A “two layer” indirect FAT allowed demonstration of the respective viruses in cell cultures and a “three layer” indirect FAT allowed demonstration of trout antibody to the viruses. Antibody, by means of the latter technique, could be demonstrated only in artificially immunized trout.     

Rainbow trout offspring with different resistance to viral haemorrhagic septicaemia

To study immunological and immunogenetical parameters related to resistance against viral haemorrhagic septicaemia (VHS), attempts to make gynogenetic strains of rainbow trout selected for high and low resistance to VHS were initiated in 1988. The first gynogenetic generation of inbreeding resulted in the more resistant offspring E8 and the low resistance offspring K3; the K3 offspring having the same high mortality as the susceptible reference strain of outbred trout in infection trials. A second gynogenetic generation derived from the E8 strain resulted in some low resistance offspring, and two gynogenetic families in which all, or nearly all, fish survived challenge with VHS virus. In this study, an attempt to associate the distribution of different MHC class II genotypes with low and high resistance gynogenetic offspring was performed. Two different MHC haplotypes could be distinguished, and in both low and high resistance families all three genotypes were found, which could be explained by the fact that the mother fish carried the heterozygous genotype. Although no significant differences in MHC II genotypes were found between the high and low resistance offspring, a significantly different distribution of haplotypes in the low resistance offspring was observed, that could not be explained by a one- or two-locus model.     

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.     

DNA vaccination against viral haemorrhagic septicaemia (VHS) in rainbow trout: size,dose, route of injection and duration of protection-early protection correlates with Mx expression

Rainbow trout of different sizes (10 and 100g) were injected intramuscularly (i.m.) or intraperitoneally (i.p.) with different doses (range 10 ng-10 microg) of a viral haemorrhagic septicaemia (VHS)-DNA vaccine (pcDNA3vhsG). As controls, fish were injected with the pcDNA3 plasmid alone, or with inactivated VHS virus. Fish were challenged at different times post-vaccination (p.v.) to assess protection. At certain times p.v., serum samples were analysed for neutralising antibody and liver tissue was analysed for Mx mRNA expression. A DNA dose of 0.5 microg injected by the i.m. route induced protection in fish of all sizes in challenges performed either 1 or 4 weeks p.v. This dose also conferred effective protection up to 9 months p.v. in fish >100 g. With lower doses of DNA (0.1 and 0.01 microg) and challenge at 4 weeks p.v., 10 g fish were partially protected but protection was not observed in 100 g fish. Vaccination by the i.p. route induced no or lower levels of protection compared with the i.m. route. Fish vaccinated with 0.5 microg DNA i.m. had no detectable serum neutralising antibody (NAb) at 4 weeks p.v. (with the exception of a single 10 g fish) but antibody was detected at 8 weeks and 6 months p.v. but not at 9 months p.v. However, cohorts of these fish showed effective protection at all timepoints. Lack of detectable levels of NAb (at 9 weeks p.v.) despite partial protection in challenge at 4 weeks p.v. was also observed with 0.01 microg doses of DNA i.m. NAb was detected in sera of fish at 8 weeks after vaccination with 0.1 microg i.m. but not in fish vaccinated with doses of 0.01-0.5 microg i.p. Early protection (1 week p.v.) correlated with elevated Mx gene expression.     

Fish rhabdoviruses: comparative study of protein structure.

Proteins from four fish rhabdoviruses have been studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The viruses were: trout viral hemorrhagic septicemia (VHS), infectious hematopoietic necrosis virus (IHN), spring viremia virus of carp (SVC), and the pike fry rhabdovirus (PFR). For the two salmonid viruses (VHS-IHN), gel electrophoresis indicated the proteins, with molecular weights estimated to be 190,000, 80,000, 38,000, 25,000, and 19,000, respectively. The electrophoretic profile of the two other viruses (SVC-PFR) revealed four major proteins with molecular weights of 190,000 80,000 42,000 and 21,000, respectively. In this case a minor component with 50,000 daltons was found. For each virus only one protein was found to be glycosylated, i.e., the one with a molecular weight of 80,000. A major protein (molecular weight between 38,000 and 42,000) was found to be associated with the nucleocapsid. All these results revealed marked similarities in protein structure between the four fish rhabdoviruses and the previously well-characterized members of rhabdovirus group. However, one can distinguish two groups of viruses: the first one is composed of salmonid viruses (VHS and IHN) with a protein structure comparable to that of rabies virus and potato yellow dwarf virus; the second one is composed of carp and pike viruses, having a protein structure very similar to that of vesicular stomatitis virus.     

伪狂犬病病毒宿主关闭蛋白的基因克隆及结构分析

为研究伪狂犬病病毒 (pseudorabiesvires ,PRV)UL4 1基因编码的病毒宿主关闭蛋白 (VHS)的结构与功能 ,通过PCR扩增得到含UL4 1基因完整编码区的 1174bp片段 ,将该片段克隆到表达载体pGEX KG中GST下游 ,在大肠杆菌BL2 1(DE3)中实现了GST VHS融合蛋白的高效表达 .序列分析发现VHS蛋白具有 4个保守区 ,并且第 3个保守区与核酸内切酶结构域FEN 1(1A76 )高度同源 .PROSPECT软件预测的伪狂犬病病毒VHS蛋白三维结构中含有 10个α螺旋和 2 2个β折叠 ,与单纯疱疹病毒Ⅰ型的VHS蛋白三维结构十分相似 .     

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.     

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.     

Mode of Virus Rescue Determines the Acquisition of VHS Mutations in VP22-Negative Herpes Simplex Virus 1

It has been proposed that herpes simplex virus 1 with VP22 deleted requires secondary mutation of VHS for viability. Here we show that a replication-competent Δ22 virus constructed by homologous recombination maintains a wild-type (Wt) VHS gene and has no other gross mutations. By contrast, Δ22 viruses recovered from a bacterial artificial chromosome contain multiple amino acid changes within a conserved region of VHS. Hence, the mode of virus rescue influences the acquisition of secondary mutations.     

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.     

Diagnostic capacity for viral haemorrhagic septicaemia virus (VHSV) infection in rainbow trout (Oncorhynchus mykiss) is greatly increased by combining viral isolation with specific antibody detection

Detection of disease specific antibodies in farmed rainbow trout (Oncorhynchus mykiss) has been proposed as an alternative or supplement to the currently approved procedures for diagnosis and surveillance in this species. In samples from natural outbreaks of the disease viral haemorrhagic septicaemia (VHS) at two freshwater farms in southern Denmark serologic testing was used to broaden the diagnostic window from outbreak to diagnosis in the laboratory as compared to traditional procedures of isolation and identification of the virus. The serologic assay clearly increased the chance of detecting present or previous infections where the pathogen could not be isolated by standard methods (indicating older infections where the virus had been cleared). Our data allowed us to monitor the levels of neutralising antibodies in relation to the presence of the virus in fish experiencing two different types of outbreaks at two different farms. By sequence analysis of the viral glycoprotein from selected isolates we found no evidence for escape mutants having developed in the fish showing high titres of neutralising antibodies.     

Structural proteins of two salmonid rhabdoviruses.

Purified infectious hematopoietic necrosis (IHN) virus and the virus of haemorrhagic septicaemia (VHS) (Egtved virus) each contain five structural proteins which were designated L, G, N, M-1, and M-2. The IHN viral polypeptides have molecular weights estimated to be 157,000, 72,000, 40,000, 25,000 and 20,000, respectively, whereas those of VHS viral polypeptides are estimated to be 157,000 74,000, 41,000, 21,500, and 19,000, respectively. The carbohydrate composition of the glycoprotein (G) was confirmed by demonstrating selective incorporation of [3H]glucosamine into the designated G protein of both viruses. Phosphoproteins were identified by incorporation of [32P]orthophosphate into the N and M-1 proteins of IHN virus and into the N protein of VHS virus. The glycoprotein of each virus was selectively solubilized by treatment with Triton X-100 in low salt buffer, whereas the M-1, and M-2 proteins along with the G protein were solubilized by Ttition X-100 in 0.43 M NaCl. The protein composition of the salmonid rhabdoviruses resembles that of the rabies virus group more closely than the vesicular stomatitis virus group.     

Unstable lysogeny and pseudolysogeny in Vibrio harveyi siphovirus-like phage 1

Exposure of Vibrio harveyi (strain VH1114) to V. harveyi siphovirus-like phage 1 (VHS1) resulted in the production of a low percentage of lysogenized clones of variable stability. These were retrieved most easily as small colonies within dot plaques. Analysis revealed that VHS1 prophage was most likely carried by VH1114 as an episome rather than integrated into the host chromosome. In the late exponential growth phase, lysogenized VH1114 continuously produced VHS1 but also gave rise to a large number of cured progeny. The absence of phage DNA in the cured progeny was confirmed by the absence of VHS1 DNA in Southern blot and PCR assays. Curiously, these very stable, cured subclones did not show the parental phenotype of clear plaques with VHS1 but instead showed turbid plaques, both in overlaid lawns and in dot plaque assays. This phenotypic difference from the original parental isolate suggested that transient lysogeny by VHS1 had resulted in a stable genetic change in the cured clones. Such clones may be called pseudolysogens (i.e., false lysogens), since they have undergone transient lysogeny and have retained some resistance to full lytic phage development, despite the loss of viable or detectable prophage.