Production and characterization of a monoclonal antibody against a late gene encoded by grouper iridovirus 64L.

Viral envelope proteins play important roles in viral infection and assembly. The grouper iridovirus ORF 64L (GIV-64L) was predicted to encode an envelope protein and was conserved in all sequenced Ranaviruses. In this study, the complete nucleotide sequence of the GIV-64L gene (1215 bp) was cloned into the isopropyl ß-D-1-thiogalactopyranoside (IPTG) induction prokaryotic expression vector pET23a. The approximately 50.2 kDa recombinant GIV-64L-His protein was induced, purified and used as an immunogen to immunize BALB/c mice. Three monoclonal antibodies (mAbs), all IgG1 class antibodies against GIV-64L protein, were produced by enzyme-linked immunosorbent assay. Reverse transcription polymerase chain reaction analyses revealed GIV-64L to be a late gene when expressed in grouper kidney cells during GIV infection with cycloheximide (an inhibitor of protein synthesis) or cytosine arabinoside (an inhibitor of DNA synthesis) present. Finally, one of the established mAbs, GIV-64L-mAb-17, was used in Western blotting and an immunofluorescence assay, which showed that GIV-64L protein was expressed at 24 h post-infection and localized only in the cytoplasm in GIV-infected cells, packed into a whole virus particle. The presently characterized GIV-64L mAbs should have widespread applications in GIV immunodiagnostics and other research, and these results should offer important insights into the pathogenesis of GIV.

Infectious hematopoietic necrosis virus matrix protein inhibits host-directed gene expression and induces morphological changes of apoptosis in cell cultures.

Infectious hematopoietic necrosis virus (IHNV) infection in tissue culture cells has previously been shown to result in the shutdown of host protein synthesis, cell rounding, and cell death. We report here an investigation of the cytopathogenicity of the viral phosphoprotein (P or M1), matrix (M or M2), and nonvirion (NV) proteins in cultured fish cells. The expression of M alone potently inhibited reporter gene expression from a viral and an interferon (IFN)-inducible promoter, whereas P and NV did not produce a similar effect. Northern blot analysis further revealed a reduction in the steady-state level of reporter mRNA when the M gene was cotransfected into cells; conversely, M mRNA was not drastically reduced in the same cells. By immunofluorescence confocal microscopy, fragmented nuclei were found in some cells expressing M protein but not in cells expressing P, NV, or beta-galactosidase protein. Electron microscopy revealed the morphological changes associated with apoptosis in the M-transfected cells. Furthermore, IHNV infection was shown to produce DNA "laddering" in cultured cells. Taken together, these data suggested at least two functions for M protein in an IHNV infection: down regulation of host transcription and the induction of programmed cell death. In the course of these experiments, we also discovered that NV expression was associated with cell rounding, the first biological effect on cells to be attributed to the NV gene.

Truncated particles produced in fish surviving infectious hematopoietic necrosis virus infection: mediators of persistence?

Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus that produces an acute, lethal infection in rainbow trout (Oncorhynchus mykiss). Fish that survive infection cease to produce detectable infectious virus at approximately 46 days after infection, yet there is evidence that survivor fish continue to harbor virus particles (B. S. Drolet, P. P. Chiou, J. Heidel, and J. C. Leong, J. Virol. 69:2140-2147, 1995). In an effort to determine the biological function of these particles, the kidneys and livers from IHNV survivors were harvested and divided into samples for nested reverse transcriptase PCR analysis and explant culture. Sequences for the IHNV nucleoprotein and polymerase genes were detected in 50 and 89%, respectively, of the organs from survivor fish. When explant tissue cultures were infected with purified standard IHNV, the liver tissues from survivor fish produced up to 10-fold less virus than naive control fish liver tissues. In addition, immunosorbent electron microscopy analysis of the supernatant media from the cultured explants of survivor fish revealed truncated particles, whereas the control tissue supernatants contained only standard viral particles. These results suggest that the truncated IHNV particles observed in persistently infected fish are defective interfering particles that may mediate virus persistence.

Cloning of the rainbow trout (Oncorhynchus mykiss) Mx2 and Mx3 cDNAs and characterization of trout Mx protein expression in salmon cells.

Two rainbow trout (Oncorhynchus mykiss) Mx cDNAs were cloned by using RACE (rapid amplification of cDNA ends) PCR and were designated RBTMx2 and RBTMx3. The deduced RBTMx2 and RBTMx3 proteins were 636 and 623 amino acids in length with molecular masses of 72 and 70.8 kDa, respectively. These proteins, along with the previously described RBTMx1 protein (G. D. Trobridge and J. A. Leong, J. Interferon Cytokine Res. 15:691-702, 1995), have between 88.7 and 96.6% identity at the amino acid level. All three proteins contain the tripartite GTP binding domain and leucine zipper motif common to Mx proteins. A monospecific polyclonal antiserum to an Escherichia coli-expressed fragment of RBTMx3 was generated, and that reagent was found to react with all three rainbow trout Mx proteins. Subsequently, endogenous Mx production in RTG-2 cells induced with poly(IC) double-stranded RNA was detected by immunoblot analysis. The cellular localization of the rainbow trout proteins was determined by transient expression of the RBTMx cDNAs in CHSE-214 (chinook salmon embryo) cells. A single-cell transient-transfection assay was used to examine the ability of each Mx cDNA clone to inhibit replication of the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV). No significant inhibition in the accumulation of the IHNV nucleoprotein was observed in cells expressing either trout Mx1, Mx2, or Mx3 in transiently transfected cells.

The structural proteins of infectious pancreatic virus are not glycosylated.

The major capsid protein, VP2, of infectious pancreatic necrosis virus, a nonenveloped icosahedral virus, contains six N-glycosylation consensus sequences (Asn-X-[Thr/Ser]). Since VP2 contains the major virus-neutralizing epitopes, the possible role for glycosylation in capsid formation and antigenicity was examined. The carbohydrate content of the virion proteins was determined by chemical detection, pulse-chase experiments,[3H]mannose labeling, and alteration of protein migration on sodium dodecyl sulfate-polyacrylamide gels after tunicamycin treatment. No glycosylation of any virion protein was observed when the carbohydrate nature of the glycoprotein of infectious hematopoietic necrosis virus was detected.

Detection of truncated virus particles in a persistent RNA virus infection in vivo.

Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus which causes devastating epizootics of trout and salmon fry in hatcheries around the world. In laboratory and field studies, epizootic survivors are negative for infectious virus by plaque assay at about 50 days postexposure. Survivors are considered virus free with no sequelae and, thus, are subsequently released into the wild. When adults return to spawn, infectious virus can again be isolated. Two hypotheses have been proposed to account for the source of virus in these adults. One hypothesis contends that virus in the epizootic survivors is cleared and that the adults are reinfected with IHNV from a secondary source during their migration upstream. The second hypothesis contends that IHNV persists in a subclinical or latent form and the virus is reactivated during the stress of spawning. Numerous studies have been carried out to test these hypotheses and, after 20 years, questions still remain regarding the maintenance of IHNV in salmonid fish populations. In the study reported here, IHNV-specific lesions in the hematopoietic tissues of rainbow trout survivors, reared in specific-pathogen-free water, were detected 1 year after the epizootic. The fish did not produce infectious virus. The presence of viral protein detected by immunohistochemistry, in viral RNA by PCR amplification, and in IHNV-truncated particles by immunogold electron microscopy confirmed the presence of IHNV in the survivors and provided the first evidence for subclinical persistence of virus in the tissues of IHNV survivors.