Haploid-specific transcription of protamine-myc and protamine-T-antigen fusion genes in transgenic mice.

The protamines are small, basic, arginine-rich proteins synthesized postmeiotically in the testes. Analysis of the regulation of synthesis of the protamine mRNA and protein is restricted by the difficulty in culturing and manipulating the cells in which transcription and translation occur. To avoid these problems, we have produced transgenic mice carrying fusion genes in which sequences 5' to the mouse protamine-2 gene have been linked to exons 2 and 3 of the mouse c-myc gene and, separately, to the simian virus 40 (SV40) early region. We show here that the prot.myc gene is correctly regulated; transcription is detected only in the round spermatids. In one family of transgenic mice carrying the 5' protamine-SV40 T-antigen fusion gene, SV40 early-region mRNA accumulated to the highest level in the testes but was also detected in the thymuses, brains, hearts, and preputial glands of the animals. Although we have demonstrated specific transcription of these fusion genes in the round spermatids, we were not able to detect the SV40 T-antigen protein.

Molecular and biotechnological approaches to fish vaccines.

Global aquaculture is projected to grow from 10 million to 20 million metric tons by the year 2000. To meet this projection, the aquaculture industry must bring some of its infectious disease problems under control without relying on antibiotics and chemotherapeutics. Thus, vaccines for fish and shellfish are being sought by the industry. The very first commercial fish vaccine was a killed vibriosis bacterin which was very effective and relatively easy to produce. Vaccines for other bacterial, viral, and parasitic fish pathogens are proving more difficult to develop and researchers have begun to use molecular and biotechnological approaches to develop such vaccines. This review describes the vaccines that are now available as well as the vaccines that are being developed and includes a discussion of live, attenuated vaccines, immunoadjuvants, and subunit vaccines for fish.

Characterization of a rainbow trout Mx gene.

A full-length cDNA clone of a rainbow trout (Oncorhynchus mykiss) Mx gene was obtained using RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) amplification of RNA extracted from poly (I).(C)-induced rainbow trout gonad cells (RTG-2). Mx was previously identified in rainbow trout by Staeheli et al. by hybridization with a partial perch genomic Mx probe to induced rainbow trout mRNA. The 2.5 kb rainbow trout cDNA clone contains an open reading frame of 1863 nt (nucleotides) encoding a 621 amino acid protein. The deduced rainbow trout Mx protein is 70.6 kD and contains the characteristic tripartite GTP binding motif common to all Mx protein. Southern blot analysis with the rainbow trout Mx probe demonstrated the presence of Mx homologous genes in four other salmonid fish species, including chinook, coho, and kokanee salmon and brook trout. Poly (I).(C) treatment of both RTG-2 and chinook salmon cells (CHSE-214) induced two transcripts whose appearance was observed first at 24 h and as long as 72 h after treatment. Infection of rainbow trout with the salmonid rhabdovirus, IHNV (infectious hematopoietic necrosis virus), also induced the synthesis of Mx mRNA. A comparison of the rainbow trout Mx protein with other reported Mx proteins indicates that the piscine Mx is highly homologous to the mammalian Mx proteins.

Molecular characterization of the glycoproteins from two warm water rhabdoviruses: snakehead rhabdovirus (SHRV) and rhabdovirus of penaeid shrimp (RPS)/spring viremia of carp virus (SVCV).

We have determined the complete coding sequences for the glycoprotein (G) genes from two rhabdoviruses that infect warm water aquatic animals, the snakehead rhabdovirus (SHRV) and rhabdovirus of penaeid shrimp (RPS). Surprisingly, the G nucleotide sequence from RPS, a virus which has been isolated from diseased shrimp in Hawaii on numerous occasions, was over 99% identical to the G nucleotide sequence from spring viremia of carp virus (SVCV), a fish virus from Europe and Asia. This is the first report of SVCV isolation outside of Europe and Asia, and it is also the first report of SVCV infecting a non-vertebrate species. The G gene from SHRV was most closely related to the G genes from the three Novirhabdoviruses, viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), and hirame rhabdovirus (HIRRV), with 47, 37, and 36% amino acid identity, respectively. In addition, a phylogenetic analysis using the amino acid sequence from rhabdovirus G genes indicated that SHRV should be classified within the Novirhabdovirus genus. Finally, the SHRV-G gene was successfully expressed in mammalian cells under the control of the cytomegalovirus (CMV) promoter, establishing that it can potentially be used in the production of pseudotyped retroviruses designed to infect fish.

Molecular cloning of double-stranded RNA inducible Mx genes from Atlantic salmon (Salmo salar L.).

Mx proteins are induced by type I interferons in mice and humans and inhibit the replication of several negative-stranded RNA viruses. In this work Mx genes in Atlantic salmon were studied using the double stranded RNA, polyinosinic: polycytidylic acid (poly I:C), to induce interferon production. Northern blot analysis showed Mx mRNA in liver, head kidney and gills 2 days after poly I:C injection of fish, but not in untreated fish or fish injected with saline or LPS. Mx transcripts of 2.4 and 1.9 kb were detected in the liver. By screening of a cDNA library, three different full length Mx cDNA clones, ASMx1, ASMx2 and ASMx3, were identified and sequenced. The deduced ASMx proteins all contain 623 amino acids and show the tripartite GTP-binding motif typical of vertebrate Mx proteins. ASMx1 and ASMx2 may represent alleles of the same gene whereas ASMx3 represents a different gene. The deduced ASMx proteins showed 96 to 98% sequence identity with rainbow trout Mx1 and Mx3 and about 88% identity with rainbow trout Mx2 protein.

Development of DNA vaccines for salmonid fish.

Vaccination of fish against many different pathogenic organisms has made it possible to rear Atlantic salmon in net pen cages and produce fish commercially around the world. In fact, vaccine use is critical for the continued growth of the aquaculture industry and researchers are continually looking to develop new and improved vaccines for a wide variety of fish pathogens. Fish vaccines have been formulated from killed or attenuated pathogens, recombinant viral proteins or peptides, and most recently, plasmid DNA encoding viral proteins (1-7). The use of DNA vaccines for the control of viral diseases of fish is particularly appealing since this type of vaccine eliminates the need to purify the viral pathogen or immunoprotective antigen. Other advantages are the elimination of any possibility of reversion to virulence since the DNA vaccine encodes only a portion of the viral genome, the relative stability of the DNA preparation; the ease of DNA preparation; and, importantly, the elicitation of a robust immune response in fish.

Recombinant vaccines against infectious hematopoietic necrosis virus: production by the Caulobacter crescentus S-layer protein secretion system and evaluation in laboratory trials.

We report the development of an IHNV vaccine produced by a new protein production system based on the bacterium Caulobacter crescentus. The subunit vaccines that were tested contain a 184 amino acid segment of the IHNV glycoprotein in different fusion arrangements with the C. crescentus S-layer protein. Relative percent survival of 26 to 34% was demonstrated in rainbow trout fry for a vaccine that contained the 184 amino acid segment of the IHNV glycoprotein fused to the C-terminal one-quarter of the S-layer protein. Inclusion of the universal mammalian T-cell epitopes developed from the measles fusion protein or the tetanus toxin protein did not increase the effectiveness of the IHNV-G/S-layer recombinant protein.

Expression of the glycoprotein gene from a fish rhabdovirus by using baculovirus vectors.

A cDNA fragment containing the gene encoding the glycoprotein of infectious hematopoietic necrosis virus was inserted into Autographa californica baculovirus vectors under the control of the polyhedrin promoter. A 66-kilodalton protein, identical in size to the glycosylated glycoprotein of infectious hematopoietic necrosis virus, was expressed at high levels in Spodoptera frugiperda cells infected with the recombinant viruses. The expressed protein reacted with antiserum to the glycoprotein on Western blots (immunoblots).

The nucleocapsid gene of infectious hematopoietic necrosis virus, a fish rhabdovirus.

The complete nucleotide sequence of the infectious hematopoietic necrosis virus (IHNV) nucleocapsid gene has been determined using cDNA clones of genomic and messenger RNAs. Genomic clones were generated by using random DNA oligomers to prime cDNA synthesis and were mapped to their respective locations on the genome by the use of cDNA probes derived from viral mRNAs. Interesting features of the IHNV nucleocapsid gene sequence elucidated by the sequencing of these clones include short homologies with N genes of other rhabdoviruses at the 5' and 3' nontranslated termini of the mRNA, as well as an exceptionally long 5' noncoding region of the mRNA, suggesting a leader RNA may be coupled to the N mRNA. A comparison of the IHNV N protein coding sequence with other rhabdoviral N genes shows some homologies at the amino acid level which indicates the possible evolutionary relationship of these N proteins. The determination of the nucleotide sequences of IHNV genes and intergenic regions will be useful for studying the mechanisms of rhabdoviral transcription and replication.

Staphylococcal coagglutination, a rapid method of identifying infectious hematopoietic necrosis virus.

A staphylococcal coagglutination test was developed for the rapid detection of infectious hematopoietic necrosis virus (IHNV) in cell cultures and infected fish. The test could be completed in 15 min but required a minimum IHNV titer of 10(6) PFU/ml to obtain a positive reaction. All IHNV isolates, representing the five electropherotypes taken from a wide variety of species and different geographic ranges, caused coagglutination of Staphylococcus aureus cells sensitized with rabbit polyclonal serum against the Round Butte IHNV isolate. The coagglutination reaction was blocked by preincubation of IHNV with homologous antiserum, and IHNV did not cause coagglutination of S. aureus cells sensitized with normal rabbit serum. In specificity tests, cells sensitized with rabbit anti-IHNV serum or normal serum did not coagglutinate in the presence of infectious pancreatic necrosis virus, viral hemorrhagic septicemia virus, cell culture medium components, or media from cultures of cell lines of salmonid and nonsalmonid origin. Most importantly, the coagglutination test was able to detect and identify IHNV directly from experimentally infected rainbow trout fry, the organs of naturally infected adult kokanee salmon and winter steelhead trout, and ovarian fluids of the winter steelhead trout. The coagglutination test is very suitable for field use, since it is inexpensive, simple to interpret, sensitive, and rapid and requires no specialized equipment.

Comparison of representative strains of infectious hematopoietic necrosis virus by serological neutralization and cross-protection assays.

Infectious hematopoietic necrosis virus (IHNV) is a pathogen of young salmon and trout. Viral epizootics among these fish in private and public rearing facilities have been a problem in the northwestern United States from California to Alaska, and an IHNV vaccine has been sought by the aquaculture experts. Since an IHNV vaccine must be designed to immunize against all viral serotypes, an analysis of IHNV serotypes was made. A large number of viruses from widely separated geographic locations and different fish species had already been placed in one of five electropherotypes by the migration of the virion proteins in sodium dodecyl sulfate-polyacrylamide gels. Also, there was evidence that some of these virus isolates had differences in virulence for chinook salmon, rainbow trout, or kokanee salmon. Previous serological studies with polyclonal rabbit antisera and three IHNV isolates indicated that there was only one serotype (B. B. McCain, J. L. Fryer, and K. S. Pilcher, Proc. Soc. Exp. Biol. Med. 137:1042-1046, 1971). A substantial number of new IHNV isolations have been made since that study, and thus a more extensive comparison was made of 10 different IHNV isolates representing the five electropherotypes. This report shows that the glycoprotein from a single isolate of IHNV can induce a protective immune response in vivo to the five IHNV electropherotypes. Plaque reduction neutralization assays indicated that there was only one serotype. Thus, despite the differences observed in the migration of the structural proteins for IHNV isolated from separate geographic locations and different fish species, only one neutralizing virus type was identified.

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.

Gene expression in rainbow trout (Oncorhynchus mykiss) following intramuscular injection of DNA.

Expression of the firefly luciferase gene under the control of viral or fish promoters was observed in fish tissue after direct DNA injection of plasmid DNA. Plasmid DNA containing the firefly luciferase gene was injected into the skeletal muscle of rainbow trout (Oncorhynchus mykiss), and levels of luciferase activity were found to be dependent on the controlling promoter and the amount of injected DNA. Plasmids using the cytomegalovirus immediate early promoter (CMV-IEP) consistently produced the highest levels of luciferase activity. Maximal activity was observed five to seven days postinjection with 50 micrograms of DNA. This activity persisted in the tissues for as long as 115 days postinjection. When the DNA was examined up to two months postinjection, the predominant form was unreplicated, unintegrated DNA in linear and relaxed circular conformation. Expression of injected DNA was found predominantly within muscle cells along the injection path and in scattered muscle cells anterior to the injection site.

Normal human placentas contain RNA-directed DNA polymerase activity like that in viruses.

Extracts from over 100 normal human placentas have been examined for RNA-directed DNA polymerase (DNA nucleotidyltransferase, EC 2.7.7.7) activity. More than 80% of these placentas contained this enzyme activity, which banded at a density of 1.15-1.17 g/ml in sucrose. After heat treatment, this enzyme activity was shifted in density to 1.22-1.24 g/ml. The enzymatic activity was greater with (rC)n.(dG)12-18 than with (dC)n.(dG)12-18 and was not stimulated by (dG)12-18 alone. The product of the endogenous reaction, which was sensitive to RNase, had the characteristics of a small DNA associated with a large RNA by hydrogen bonding. Electron microscopic inspection of the material with a density of 1.15-1.17 g/ml revealed numerous retrovirus-like particles with central electron-dense cores and double-membraned envelopes. The enzyme may be associated with the retrovirus-lik particles noted in the trophoblast layer of some human placentas.

Production of recombinant snakehead rhabdovirus: the NV protein is not required for viral replication.

Snakehead rhabdovirus (SHRV) affects warm water fish in Southeast Asia and belongs to the genus Novirhabdovirus by virtue of its nonvirion gene (NV). Because SHRV grows best at temperatures between 28 and 31 degrees C, we were able to use the T7 expression system to produce viable recombinant SHRV from a cloned cDNA copy of the viral genome. Expression of a positive-strand RNA copy of the 11, 550-nucleotide SHRV genome along with the viral nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins resulted in the generation of infectious SHRV in cells preinfected with a vaccinia virus vector for T7 polymerase expression. Recombinant virus production was verified by detection of a unique restriction site engineered into the SHRV genome between the NV and L genes. Since we were now able to begin examining the function of the NV gene, we constructed a recombinant virus containing a nonsense mutation located 22 codons into the coding sequence of the NV protein. The NV knockout virus was produced at a concentration as high as that of wild-type virus in cultured fish cells, and the resulting virions appeared to be identical to the wild-type virions in electron micrographs. These initial studies suggest that NV has no critical function in SHRV replication in cultured fish cells.

Effect of passage in culture on a clone of BALB/c 3T3 cells transformed by simian virus 40.

Most simian virus 40 (SV40)-transformed BALB/c 3T3 clones employed for biochemical studies have been used without regard to passage level. To determine whether virus-induced properties are stable as a function of passage, we have extensively characterized one transformed clone, FNE, which was isolated after SV40 infection BALB/c 3T3 cells in factor-free medium. From the initial testing at passage 5 and for at least 50 subsequent subcultures, the cells stably maintained many transformed growth properties, including high saturation density, morphology, colony formation on contact-inhibited monolayers, tumorigenicity, and synthesis of viral-specific RNA. However, other properties varied as a function of passage. There was a slight decrease in viral genome equivalents per cell from 1.1 copy/cell at passage 5 to 0.7 copies at passage 40. Initially, the cells were negative for all type C virus; however, cells carried at low density for 13 to 20 passages (65 to 100 generations) began to release an endogenous type C virus that then persisted in the culture. Spontaneous release of type C virus did not occur in control BALB/c 3T3 cells carried under identical culture conditions for 90 passages. When the cultures were releasing type C viruses they stained uniformly and brightly positive for SV40 tumor (T) antigen by immunofluorescence, whereas T antigen staining was variable at early passage. These experiments suggest that subtle but perhaps important differences in viral gene expression can occur as a function of passage; they also demonstrate the importance of evaluating the interactions between SV40 and endogenous type C viruses.

Induction of Mx protein by interferon and double-stranded RNA in salmonid cells.

Mx protein is one of several antiviral proteins that are induced by the type I interferons (IFN), IFNalpha and beta, in mammals. In this work induction of a 76 kDa Mx protein by double-stranded RNA (dsRNA) or type I IFN-like activity in Atlantic salmon macrophages, Atlantic salmon fibroblast cells (AS cells) and in Chinook salmon embryo cells (CHSE-214) is reported. Type I IFN-like activity was produced by the stimulation of Atlantic salmon macrophages with the synthetic dsRNA polyinosinic polycytidylic acid (poly I:C). A correlation appeared to exist between Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) induced by IFN in CHSE-214 cells. Several observations in the present work suggest that, as in mammals, the induction of Mx protein by dsRNA in fish cells primarily occurs via induction of type I IFN. First, type I IFN-like activity but not poly I:C, induced Mx protein expression in CHSE-214 cells. These cells apparently lack the ability to produce IFN in response to poly I:C. Second, the putative IFN induced maximal Mx protein expression 48 h earlier than poly I:C in AS cells. Third, the peak expression of Mx protein in macrophages induced by poly I:C occurred after 48 h whereas peak in IFN-like activity was observed by 24 h after addition of poly I:C. The present work supports the notion of using Mx protein as a molecular marker for the production of putative type I IFN in fish.

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.

Sequence analysis of infectious pancreatic necrosis virus genome segment B and its encoded VP1 protein: a putative RNA-dependent RNA polymerase lacking the Gly-Asp-Asp motif.

The genome segment B sequence of infectious pancreatic necrosis virus was determined for both the Jasper and Sp serotypes. The sequences are 2784 and 2630 bp long, respectively, and contain a single large open reading frame encoding the VP1 protein, the putative RNA-dependent RNA polymerase (RdRp) of IPNV. The proteins exhibit an 88% homology with each other, but only 41% with infectious bursal disease virus (IBDV) VP1, another member of the Birnaviridae. Despite the low overall homology between the IPNV and IBDV VP1 proteins, homologous regions were detected within the central portion of the proteins. The carboxy-proximal regions of the VP1, which contain very low amino acid homology, displayed evidence of conservation in structural features such as a hydrophilic, highly basic domain. Consensus sequences associated with GTP-binding proteins and RdRps were also detected in VP1. However, unlike the RdRps associated with single-stranded plus RNA viruses, the birnavirus RdRp lacks the Gly-Asp-Asp motif characteristic of this enzyme family.