Infectious hematopoietic necrosis virus: monophyletic origin of European isolates from North American genogroup M.

Infectious hematopoietic necrosis virus (IHNV) was first detected in Europe in 1987 in France and Italy, and later, in 1992, in Germany. The source of the virus and the route of introduction are unknown. The present study investigates the molecular epidemiology of IHNV outbreaks in Germany since its first introduction. The complete nucleotide sequences of the glycoprotein (G) and non-virion (NV) genes from 9 IHNV isolates from Germany have been determined, and this has allowed the identification of characteristic differences between these isolates. Phylogenetic analysis of partial G gene sequences (mid-G, 303 nucleotides) from North American IHNV isolates (Kurath et al. 2003) has revealed 3 major genogroups, designated U, M and L. Using this gene region with 2 different North American IHNV data sets, it was possible to group the European IHNV strains within the M genogroup, but not in any previously defined subgroup. Analysis of the full length G gene sequences indicated that an independent evolution of IHN viruses had occurred in Europe. IHN viruses in Europe seem to be of a monophyletic origin, again most closely related to North American isolates in the M genogroup. Analysis of the NV gene sequences also showed the European isolates to be monophyletic, but resolution of the 3 genogroups was poor with this gene region. As a result of comparative sequence analyses, several different genotypes have been identified circulating in Europe.

Rapid and sensitive reverse transcriptase-polymerase chain reaction based detection and differential diagnosis of fish pathogenic rhabdoviruses in organ samples and cultured cells.

A reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed and applied to the detection and differentiation of viral haemorrhagic septicaemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) in organ samples and cultured cells, regardless of the serotype. This method was developed by selecting primer sets corresponding to highly conserved regions of the glycoprotein G-gene sequences of the 2 viruses. The very fast RNA extraction, reverse transcription and PCR permitted us to read the agarose gels within 7 to 9 h after samples, cultured cells and whole fish arrived, which is of great importance when there is reason to believe that VHSV or IHNV may be present. This is also the first report of a large-scale field trial comparing the RT-PCR assay in trout from 30 German fish farms (a total of 330 rainbow trout) with the usual virus isolation and identification method in order to evaluate the efficiency of the RT-PCR assay for general use in fish health management programs. RT-PCR followed by semi-nested PCR using RNA directly extracted from fish tissue turned out to be the most sensitive method. It recognized 9 fish farms as VHS-positive and 7 as IHN-positive. This is 3 VHS- and 4 IHN-farms more than detected by the traditional virus isolation method. By directly examining the tissue by means of a PCR test it was possible to detect viral RNA in acutely and subacutely to chronically diseased fish as well as in asymptomatic VHS/IHN-carrier fish. Therefore, this effective and powerful assay for detecting VHSV and IHNV by means of PCR has great advantages compared with the presently used procedures.

[Characterization of viral hemorrhagic septicemia (VHS) virus isolates in trout]. / Charakterisierung von Isolaten des Virus der Viralen Hämorrhagischen Septikämie (VHS) der Forellen.

Virus diseases of fish can seriously impair the economy of aquacultur. Control and prevention of fish diseases in the European Union (EU) are focussed on the viral haemorrhagic septicaemia (VHS) and the infectious haematopoeitic necrosis (IHN). The diagnosis of VHS and IHN is performed in the Federal Republic of Germany (FRG) on the basis of the legislation of the EU. Since 1994 we received an increasing number of VHS virus (VHSV) isolates which did not react with a commercially available anti-VHSV monoclonal antibody (MAb) in the indirect immuno fluorescence test. With our own MAb ID8, however, as well as with additional diagnostic methods these virus isolates could be identified. These isolates of rainbow trouts were designated as VHSV type "Wi". Electron microscopically all stages of rhabdovirus maturation could be detected. Morphologically the isolates were undistinguishable from other rhabdoviruses. By immuno electron microscopy using the MAb ID8 rhabdovirus nucleocapsid structures were demonstrated. The virulence of the new VHSV type Wi was not different from that of a VHSV isolate with conventional reaction patterns as well as of a VHSV laboratory strain.

Identification of the non-virion (NV) protein of fish rhabdoviruses viral haemorrhagic septicaemia virus and infectious haematopoietic necrosis virus.

Sequence analysis of a 795 nucleotide region of the fish rhabdovirus viral haemorrhagic septicaemia virus (VHSV) genome revealed one complete and one partial ORF of 369 and 153 nucleotides, respectively. The latter ORF probably encodes the amino-terminal part of the L (polymerase) protein. The former ORF potentially encodes a 122 amino acid protein. The location of this ORF as well as the size and deduced structure of the translation product indicate that it represents a homologue of the non-virion (NV) protein of the related infectious haematopoietic necrosis virus (IHNV). Antisera raised against prokaryotically expressed NV protein of VHSV and IHNV were used to detect NV expression in VHSV- and IHNV-infected cells by Western Blot and immunofluorescence analyses. We present here the sequence of the VHSV NV gene and demonstrate the presence of IHNV and VHSV NV proteins in virus-infected cells.

Recombinant infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus glycoprotein epitopes expressed in Aeromonas salmonicida induce protective immunity in rainbow trout (Oncorhynchus mykiss).

Fragments of the glycoprotein genes of viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) were cloned into a bacterial broad-host-range expression vector under the control of the plac promoter. Western blot (immunoblot) analysis with monoclonal antibodies specific to the glycoproteins demonstrated the inducible expression of the fusion proteins in Escherichia coli. Aeromonas salmonicida is the causative agent of furunculosis in salmonid fish. It was confirmed that an avirulent strain of A. salmonicida, A440, which contains a deletion in the structural gene for the paracrystalline surface protein array, will provide protective immunity against furunculosis when used as a live attenuated vaccine. The plasmid-encoded viral epitopes were then mobilized into A440 for use as a shuttle system for the expression of fragments of the glycoprotein genes of IHNV and VHSV. Vaccination of rainbow trout with A440 containing the viral epitopes resulted in the development of protective immunity against both VHSV and IHNV. This indicates that the use of cloned fragments of the glycoproteins and the use of A. salmonicida as a shuttle system constitute a feasible approach to fish vaccine development.

Complete genomic sequence of the fish rhabdovirus infectious haematopoietic necrosis virus.

The complete nucleotide sequence of the genome of the fish rhabdovirus infectious haematopoietic necrosis virus (IHNV) has been determined after cDNA cloning of the viral genomic RNA. Sequence analysis showed the presence of six open reading frames encoding the nucleoprotein N, the matrix proteins M1 and M2, the glycoprotein G, a so-called non-structural protein NV, and the RNA polymerase L. The genome organization is 3'N-M1-M2-G-NV-L 5'. The extreme 5' and 3' ends of the genome were sequenced after RNA ligation or RACE. Prokaryotic expression products of the open reading frames predicted to encode the matrix proteins M1 and M2, the glycoprotein G and the NV protein reacted with rabbit anti-IHNV serum thereby confirming their identity. This is the first complete nucleotide sequence of a fish rhabdovirus. Knowledge of the complete sequence is an essential prerequisite for future manipulation of the genome and also serves to provide gene- and protein specific reagents for use in further examination of the replication of the fish rhabdoviruses.

Differential diagnosis of fish pathogenic rhabdoviruses by reverse transcriptase-dependent polymerase chain reaction.

Reverse transcriptase-dependent polymerase chain reaction (RT-PCR) was applied to the detection and differentiation of viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) using primer pairs designed for the amplification of glycoprotein G-specific gene fragments of the two viruses. The products of 443 bp (VHS) and 548 bp (IHN), respectively, were amplified from the total RNA extracts of RTG-2 cells infected with a total of 9 different strains of either VHS virus or IHN virus. Restriction analysis using FokI, and DNA sequencing of the PCR products demonstrated specificity of the amplification. The RT-PCR amplification of VHSV or IHNV G-genes was found to be a simple, highly specific and sensitive method allowing differential diagnosis of VHS and IHN within 8 h.

In vivo labelling of viral proteins with 75selenomethionine.

A method is described for in vivo labelling of viral proteins with a gamma ray emitting radionuclide. The amino acid methionine labelled with 75Se instead of 35S will be accepted by cells in culture for protein synthesis. A high specific labelling of viral proteins is achieved which allows rapid detection of virus or viral proteins in gradient centrifugation and PAGE as well as further biochemical or serological analysis of the proteins. As an example hog cholera virus (family Togaviridae) was labelled with [75Se]methionine and analyzed in sucrose and CsCl gradients and in PAGE. The results were similar to those obtained by labelling of proteins with [35S]methionine.

Separation of alphavirus nucleocapsids from envelope fragments.

Methods for the preparation of alphavirus nucleocapsids and envelope fragments for biochemical, electron microscopical and serological investigations are described. Difficulties which have been encountered previously in attempts to isolate viral nucleocapsids for morphological studies, namely disruption of the fragile particles, have been overcome by treatment with formaldehyde.

Quantitative and rapid assays of togaviruses by immunofluorescence.

For the quantitative assay of selected togaviruses, suspensions of BHK cells were inoculated with virus and grown in spinner cultures. At intervals, dependent on the growth characteristics of the viruses, about 10(3) cells were centrifuged on to microscope slides and then stained with fluorescent antibody. For rapid demonstration (2--3 hr) of specific viral antigens, virus was bound in successive dilutions onto microscope slides and stained. Binding of specific anti-virus antibodies in both methods, was determined either by a second labelled antibody against complement or by labelled protein A. By these methods the determination of viral antigens (Sindbis, Semliki Forest, West Nile and hog cholera viruses) was independent from the source of immune sera.

Solid phase indirect radioimmunoassays for the rapid diagnosis of Sindbis virus antigen.

Indirect radioimmunoassays have been developed for the rapid detection of Sindbis virus. Dilutions of Sindbis virus from tissue culture fluids have been immobilized and allowed to react with rabbit anti-Sindbis virus antibodies. The bound antibodies were assayed either by 125I-labelled anti-rabbit IgG-antibodies or alternatively by addition of human complement and 125I-labelled anti-human C1q antibodies or 125I-labelled protein A.

Structural similarities of hog cholera virus with togaviruses.

Hog cholera virus grown in PK-15 cells was purified by centrifugation through a sucrose cushion followed by sucrose gradient centrifugation. Analysis of virus labeled externally with [3H]sodium borohydride on polyacrylamide gel electrophoresis revealed two glycoproteins, gp55 and gp46. A third structural polypeptide, p36, seems not to be glycosylated. The gp46 was also found in the virus-free supernatant of infected cells. It could be demonstrated by radioimmune precipitation of virus labeled with[35S]methionine that all three polypeptides are specific for hog cholera virions. Electron microscopically hog cholera virus appeared as a spherical particle with a diameter of 42 +/- 8 nm. The virus particles frequently displayed a fringe of projections with a length of about 6--8 nm. The similarities of hog cholera virus with Alphaviruses and Flaviviruses are discussed.

The structural components of hog cholera virus.

Hog cholera virus grown in PK-15 cells and SK-cells was labeled with [35S]methionine and [3H]uridine. At least 3 polypeptides were resolved by polyacrylamide gel electrophoresis after disruption of the virus with sodium dodecyl-sulfate. The molecular weights of the structural proteins were determined to be 55000 (p55), 46000 (gp46), and 36000 (p36). The molecular weight of the viral RNA was determined to be about 4 X 10(6) in polyacrylamide-agarose-gel electrophoresis. In sucrose gradients the RNA has a S20,w value of 40-45S.

Rabies group-specific ribonucleoprotein antigen and a test system for grouping and typing of rhabdoviruses.

Cell-associated ribonucleoprotein (RNP) was isolated from BHK-21 cells infected with several strains of rabies and rabies-related viruses. The RNP-antigen from rabies and related viruses induced the formation of complement-fixing, precipitating, and immunofluorescent antibodies, and proved to be the group-specific antigen common to all rabies viruses. Antigens of the envelope which induce virus-neutralizing antibodies are apparently determinative for the serotype of a virus as evidenced by two-way neutralization tests. A combination of these methods seems to be a useful approach to the serological grouping and typing of rhabdoviruses.