Detecting molecular adaptation at individual codons in the glycoprotein gene of the geographically diversified infectious hematopoietic necrosis virus, a fish rhabdovirus

Salmonid fishes, the principal hosts of the infectious hematopoietic necrosis virus (IHNV), are a candidate species for aquaculture in many countries. IHNV causes an acute disease resulting in severe economic loss in salmonid fish farming. Previous phylogenetic analyses revealed the existence of multiple genogroups of this virus throughout the geographical range of its host. Here, we report the importance of natural selection in shaping the evolution of certain codons at the surface glycoprotein (G-protein) gene of this virus. Maximum likelihood (ML)-based codon substitution analyses revealed that approximately 2.8% of the codons for the entire G-protein are shown to have higher nonsynonymous substitution per nonsynonymous site (dn) than the synonymous substitutions per synonymous site (ds) (dn/ds=omega>4.335). Thus, the data suggest that positive selection (omega>1) is the major driving force in the evolution of certain codons. However, majority of these positively selected sites cannot be mapped to the regions of antigenic determinants of IHNV. Based on the reports of previous studies, epitopes with positively selected sites are immunodominant and viruses can escape from immune responses by producing antigenic variation at positively selected sites, therefore, vaccines directed against these neutralizing epitopes of IHNV that consist of no positively selected sites will be more effective. Some of the positively selected sites showed radical change in amino acids with respect to their charge and polarity; however, it is unclear how these changes affect the fitness of the virus.     

Growth of the penaeid shrimp virus infectious hypodermal and hematopoietic necrosis virus in a fish cell line

An established fish cell line, epithelioma papillosum cyprini (EPC), originating from the carp (Cyprinus carpio) was found to be susceptible to the penaeid shrimp virus, infectious hypodermal and hematopoietic necrosis virus (IHHNV). This economically important pathogen of penaeid shrimp raised in mariculture has been previously limited to growth only in young post-larval shrimps. This is, to our knowledge, the first reported successful growth of the penaeid virus in a fish cell line; it promises to provide a sensitive and convenient cell culture system for the primary isolation and detection of IHHNV which will aid immensely the production of IHHNV-free penaeid shrimp stocks for mariculture. Of added significance is the replication of a crustacean virus in a fish cell line which has never before been reported. The use of this sensitive cell line will also greatly facilitate the characterization of the IHHN virus and other crustacean viruses.     

Viral coinfection in salmonids: infectious pancreatic necrosis virus interferes with infectious hematopoietic necrosis virus

Summary  Coinfection of farm-reared salmonids involving two viruses has been described, but there is no report on the interactions between viruses. Here we examine whether infectious pancreatic necrosis virus (IPNV) strain Sp interferes with the growth of infectious hematopoietic necrosis virus (IHNV) strain S46, a coinfected isolate from rainbow trout. When BF-2 cell culture was inoculated with S46 the infective titer of the IHNV fraction decreased by 3 log₁₀ units compared to the growth curve of IHNV in the single infection. RT-PCR assay confirmed this reduction, which after successive passages of the co-infected sample led to a decrease in IHNV mRNA and the absence of the specific PCR product for IHNV. Flow cytometry showed that only 13% of the cells inoculated with S46 strain were infected with IHNV at 48–72 h post infection, in contrast to the 50–80% of cells that were positive for IPNV. Exposure of cells to IHNV for 24 h before infection with IPNV did not affect the infective titers of either virus or the PCR results obtained in simultaneous coinfections. Moreover IHNV was not inhibited when the IPNV inoculum was reduced. So, a multiplicity of infection dependence was demonstrated for IPNV-IHNV interference; the RT-PCR assay described here was found to be a suitable technique for identifying and studying dual viral infections Received July 27, 1998 Accepted November 20, 1998     

A chlorophyll c2 analogue from the marine brown alga <Emphasis Type="Italic">Eisenia bicyclis</Emphasis> inactivates the infectious hematopoietic necrosis virus,a fish rhabdovirus

Summary. We screened in vitro antiviral activity against a salmonid pathogenic virus, infectious hematopoietic necrosis virus (IHNV), from the extracts of a total of 342 species of marine algae collected from the Japanese coastline. The anti-IHNV activity was found primarily in MeOH extracts, and the extract from one marine brown alga in particular, Eisenia bicyclis, showed high anti-IHNV activity. The anti-IHNV compound was isolated and purified as MC15 from the E. bicyclis extract, and the chemical structure was determined by several spectrometric analyses. The antiviral compound was proved to be a chlorophyll c2 derivative lacking the metal ion Mg²⁺. MC15 showed similar antiviral activity against other salmonid enveloped viruses such as Paralichthys olivaceus virus and Oncorhynchus masou virus, and stability against any pH and temperatures up to 100 °C. No cytotoxicity was observed at up to 5 μg/ml. The antiviral mechanism of MC15 appears to be direct inactivation of the viral particles. A time course study showed that the inactivation of IHNV was completed within 40 min when 200 PFU of IHNV was reacted with MC15 at 800 ng/ml.     

Genotyping of Korean isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene

Summary Glycoprotein (G) gene nucleotide sequences of four Korean isolates of infectious hematopoietic necrosis virus (IHNV) were analyzed to evaluate their genetic relatedness to worldwide isolates. All Korean isolates were closely related to Japanese isolates of genogroup JRt rather than to those of North American and European genogroups. It is believed that Korean IHNV has been most likely introduced from Japan to Korea by the movement of contaminated fish eggs. Among the Korean isolates, phylogenetically distinct virus types were obtained from sites north and south of a large mountain range, suggesting the possibility of more than one introduction of virus from Japan.     

Pathophysiology of infectious hematopoietic necrosis virus disease in rainbow trout: hematological and blood chemical changes in moribund fish.

Infectious hematopoietic necrosis (IHN) is a rhabdoviral disease of rainbow trout (Salmo gairdneri). Trout were injected with IHNV, and various hematological and biochemical measurements of clinically ill fish were compared to uninfected controls. Infected fish had reduced corpuscular counts, hemoglobin, and packed cell volume, but normal mean corpuscular volume, mean corpuscular hemoglobulin, and mean corpuscular hemoglobin concentration. The percentage of immature erythrocytes was increased, but the percentage of leukocytes was unchanged. Differential leukocyte counts showed a significant decrease in neutrophils, increase in lymphocytes, but no change in monocytes. Unidentifiable necrobiotic cells were prevelant in blood smears and hematopoietic tissue imprints. Plasma bicarbonate, chloride, calcium, phosphorus, bilirubin, and osmolality were signigicantly reduced, but plasma glucose and anterior kidney ascorbate were unchanged. Plasma pH increased and the alpha fractions of the serum proteins were altered. No change was found in plasma enzymes, except that a LDH isozyme was significantly increased. The alkali reserve was diminished and alterations in acid-base and fluid balance occurred. Death probably resulted from a severe electrolyte and fluid imbalance caused by renal failure.     

The haemagglutinating properties of infectious pancreatic necrosis virus

Summary The haemagglutinating properties of infectious pancreatic necrosis virus have been investigated. Erythrocytes from a wide range of animals were used in these tests but only cells derived from mice (Balb/c and Manchester strain) gave reproducible haemagglutination.The tests were carried out at various temperatures but haemagglutination only occurred at 4° and 22° C. The haemagglutination was pH dependent with an optimum being between pH 5.75 and 6.0.Variable results were obtained at the same pH values with erythrocytes derived from rats (Sprague Dawley) but the reaction in this case appeared to be related to individual batches of erythrocytes.With 1 Figure     

The structure of infectious pancreatic necrosis virus RNA

The RNA from infectious pancreatic necrosis virus has been purified and had a sedimentation velocity of 14S on sucrose gradients, a buoyant density of 1-60 g/ml in CS2SO4 and pyrimidine to purine ratios near unity. The RNA had the appearance of a linear double stranded molecule with an average length of 0-92 mum and a standard deviation of 0-07 mum when observed under the electron microscope using the Kleinschmidt protein film technique. This would correspond to a mol. wt. of 2-4 +/- 0-2 X 10(6). The RNase A resistance of IPN virus RNA exhibited a marked salt dependence; it was 92% resistant in 0-1 M-NaCl, but only 9% resistant, or less, in 0-*1 M-NaCl. The RNA was resistant to denaturation by boilding at NaCl concentrations of 0-04 M or higher, but did denature at lower concentrations. Polyacrylamide gel electrophoresis of the RNA indicated that two RNA species were present and the standard deviation of lengths in the electron microscope indicated that they could not differ by more than 4 X 10(5) in mol. wt.     

Isolation, purification and characterization of infectious hypodermal and hematopoietic necrosis virus (IHHNV) from penaeidshrimp

A protocol was developed for the isolation and purification of the infectious hypodermal and hematopoietic necrosis virus (IHHNV) of penaeid shrimps. Cesium chloride-banded virus obtained from three sources of infected shrimps was found to have a buoyant density of 1.33 g/cm3. Also, electron microscopical studies employing negative stain revealed isometric particles having a size of 19 +/- 1 nm. Colorimetric analysis of its nucleic acid type indicated that these particles contain only RNA.     

Structure of the glycoprotein gene of sonchus yellow net virus, a plant rhabdovirus

The nucleotide sequence of the glycoprotein (G) gene of sonchus yellow net virus (SYNV), a plant rhabdovirus, was determined from viral genomic and mRNA cDNA clones. The G cistron is 2045 nucleotides (nt) long and the G protein mRNA open reading frame (ORF), as determined from the cDNA sequence, contains 1896 nt and encodes a protein of 632 amino acids. Immunoblots with antibodies elecited against the purified glycoprotein from virus particles reacted with a fusion protein produced in Escherichia coli, indicating that the cloned ORF encodes the G protein. The 5' end of the G protein mRNA corresponds to nt 5111, relative to the 3' end of the viral (minus sense) genome, as determined by primer extension from mRNA isolated from infected plants, and extends to nt position 7155 on the genomic RNA. A 34-nt untranslated 5' leader sequence and a 115-nt untranslated 3' end flank the ORF on the mRNA. The gene junctions on either side of the G gene on the genomic RNA are identical to those previously described for other SYNV genes and are similar to sequences separating genes of animal rhabdoviruses. The predicted molecular weight of the G protein is 70,215 Da, a value less than the 77,000 Da estimated for the glycosylated G protein from virus particles. The deduced amino acid sequence of the SYNV G protein shares little direct relatedness with the G proteins of other rhabdoviruses, but appears to contain a similar signal sequence, a transmembrane anchor domain, and glycosylation signals. In addition, the SYNV G protein contains a putative nuclear targeting site near the carboxy terminus, which may be involved in transit to the nuclear membrane prior to morphogenesis.     

A new virus isolate from infectious hypodermal and hematopoietic necrosis virus (IHHNV)-infected penaeid shrimps

A new virus was isolated from infectious hypodermal and hematopoietic necrosis virus (IHHNV)-infected penaeid shrimps. The virus was isolated from two species of penaeid shrimps obtained from three different sources employing a previously developed cell-culture assay. Electron-microscopical studies of both purified virus and infected cells showed bullet-shaped particles identifying it as a rhabdovirus.