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.     

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.     

Spring viremia of carp (SVC)

Spring viremia of carp (SVC) is an important disease affecting cyprinids, mainly common carp Cyprinus carpio. The disease is widespread in European carp culture, where it causes significant morbidity and mortality. Designated a notifiable disease by the Office International des Epizooties, SVC is caused by a rhabdovirus, spring viremia of carp virus (SVCV). Affected fish show destruction of tissues in the kidney, spleen and liver, leading to hemorrhage, loss of water-salt balance and impairment of immune response. High mortality occurs at water temperatures of 10 to 17 degrees C, typically in spring. At higher temperatures, infected carp develop humoral antibodies that can neutralize the spread of virus and such carp are protected against re-infection by solid immunity. The virus is shed mostly with the feces and urine of clinically infected fish and by carriers. Waterborne transmission is believed to be the primary route of infection, but bloodsucking parasites like leeches and the carp louse may serve as mechanical vectors of SVCV. The genome of SVCV is composed of a single molecule of linear, negative-sense, single-stranded RNA containing 5 genes in the order 3'-NPMGL-5' coding for the viral nucleoprotein, phosphoprotein, matrix protein, glycoprotein, and polymerase, respectively. Polyacrylamide gel electrophoresis of the viral proteins, and sequence homologies between the genes and gene junctions of SVCV and vesicular stomatitis viruses, have led to the placement of the virus as a tentative member of the genus Vesiculovirus in the family Rhabdoviridae. These methods also revealed that SVCV is not related to fish rhabdoviruses of the genus Novirhabdovirus. In vitro replication of SVCV takes place in the cytoplasm of cultured cells of fish, bird and mammalian origin at temperatures of 4 to 31 degrees C, with an optimum of about 20 degrees C. Spring viremia of carp can be diagnosed by clinical signs, isolation of virus in cell culture and molecular methods. Antibodies directed against SVCV react with the homologous virus in serum neutralization, immunofluorescence, immunoperoxidase, or enzyme-linked immunosorbent assays, but they cross-react to various degrees with the pike fry rhabdovirus (PFR), suggesting the 2 viruses are closely related. However, SVCV and PFR can be distinguished by certain serological tests and molecular methods such as the ribonuclease protection assay.     

Replicative RNA synthesis and nucleocapsid assembly in vesicular stomatitis virus-infected permeable cells.

A permeable-cell system has been developed to study the replication of vesicular stomatitis virus. When vesicular stomatitis virus-infected BHK cells were permeabilized by lysolecithin treatment, they incorporated nucleoside triphosphates into RNA and amino acids into proteins at nearly normal rates. The viral mRNA's synthesized appeared normal in polarity, size distribution, and polyadenylation, and all five viral proteins were synthesized. Replication of the viral genome proceeded, and full-length RNA strands were synthesized in amounts and polarities resembling those found in intact cells. These full-length RNAs associated with viral N proteins to form RNase-resistant nucleocapsids of normal buoyant density. Permeable cells appear to represent ideal hosts for studying vesicular stomatitis virus replication since they closely mimic in vivo conditions while retaining much of the experimental flexibility of current in vitro systems.     

Oligonucleotide fingerprints of RNA species obtained from rhabdoviruses belonging to the vesicular stomatitis virus subgroup.

The relationships among the genomes of various rhabdoviruses belonging to the vesicular stomatitis virus subgroup were analyzed by an oligonucleotide fingerprinting technique. Of 10 vesicular stomatitis viruses, Indiana serotype (VSV Indiana), obtained from various sources, either no, few, or many differences were observed in the oligonucleotide fingerprints of the 42S RNA species extracted from standard B virions. Analyses of the oligonucleotides obtained from RNA extracted from three separate preparations of VSV Indiana defective T particles showed that their RNAs contain fewer oligonucleotides than the corresponding B particle RNA species. The fingerprints of RNA obtained from five VSV New Jersey serotype viruses were easily distinguished from those of the VSV Indiana isolates. Three of the VSV New Jersey RNA fingerprints were similar to each other but quite different from those of the other two viruses. The RNA fingerprints of two Chandipura virus isolates (one obtained from India and one from Nigeria) were also unique, whereas the fingerprint of Cocal virus RNA was unlike that of the serologically related VSV Indiana.     

Inactivation by gamma irradiation of animal viruses in simulated laboratory effluent

Several animal viruses were treated with gamma radiation from a 60Co source under conditions which might be found in effluent from an animal disease laboratory. Swine vesicular disease virus, vesicular stomatitis virus, and blue-tongue virus were irradiated in tissues from experimentally infected animals. Pseudorabies virus, fowl plague virus, swine vesicular disease virus, and vesicular stomatitis virus were irradiated in liquid animal feces. All were tested in animals and in vitro. The D10 values, that is, the doses required to reduce infectivity by 1 log10, were not apparently different from those expected from predictions based on other data and theoretical considerations. The existence of the viruses in pieces of tissue or in liquid feces made no difference in the efficacy of the gamma radiation for inactivating them. Under the "worst case" conditions (most protective for virus) simulated in this study, no infectious agents would survive 4.0 Mrads.     

Inactivation by gamma irradiation of animal viruses in simulated laboratory effluent.

Several animal viruses were treated with gamma radiation from a 60Co source under conditions which might be found in effluent from an animal disease laboratory. Swine vesicular disease virus, vesicular stomatitis virus, and blue-tongue virus were irradiated in tissues from experimentally infected animals. Pseudorabies virus, fowl plague virus, swine vesicular disease virus, and vesicular stomatitis virus were irradiated in liquid animal feces. All were tested in animals and in vitro. The D10 values, that is, the doses required to reduce infectivity by 1 log10, were not apparently different from those expected from predictions based on other data and theoretical considerations. The existence of the viruses in pieces of tissue or in liquid feces made no difference in the efficacy of the gamma radiation for inactivating them. Under the "worst case" conditions (most protective for virus) simulated in this study, no infectious agents would survive 4.0 Mrads.     

Sequence Determination of the (+) Leader RNA Regions of the Vesicular Stomatitis Virus Chandipura, Cocal, and Piry Serotype Genomes

Using 3'-end-labeled genome probes, cells infected with vesicular stomatitis virus Chandipura, Cocal, and Piry serotypes were shown to contain (+) leader RNAs of approximately 50 nucleotides in length. The nucleotide sequence of the leader RNA regions of these genomes was determined and compared with the previously reported sequences of both the (+) and (-) leader RNA regions of other vesicular stomatitis virus serotypes. Regions of strong conservation of nucleotide sequence among the various vesicular stomatitis virus serotypes suggest those nucleotides thought to be involved in control functions during vesicular stomatitis virus replication.