V. Gregory Chinchar

A Decade of Advances in Iridovirus Research

Publisher Summary This chapter focuses on the advances in iridovirus research over the past decade. It summarizes salient features of the iridovirus research studies, and discusses their importance to future work. As knowledge of iridovirus biology increases, so does the understanding of the taxonomic structure of the family. A new genus (Megalocytivirus) has recently been created to accommodate a number of viruses isolated from diseased fish in Southeast Asia, and an established one (Chloriridovirus) is about to be validated following the sequencing of the genome of the mosquito pathogen IIV-3. Genomic sequencing projects have clarified evolutionary relationships between iridoviruses and other families of large, nucleocytoplasmic DNA viruses. Iridoviruses appear situated at the center of a clade of DNA viruses that include the poxviruses, phycodnaviruses,ASFV, ascoviruses, and possibly a new giant virus of amoebae (mimivirus). Not all iridovirus infections result in mortal disease. The recognition that many iridovirus infections are chronic, especially among insects, should stimulate studies on the importance of such illnesses on host fitness. Sublethal effects have been demonstrated in infected insects, and theoretical studies suggest such effects have a major influence on the dynamics of host populations. However, the consequences of chronic disease on the demography (fecundity, fertility, death rate, etc.) of infected amphibian and fish populations are notable by their absence. The ability to detect iridovirus infections has advanced markedly with the adoption of highly sensitive loop-mediated isothermal amplification and multiplex polymerase chain reaction techniques for rapid diagnosis of infected individuals.

PATHOLOGY, ISOLATION, AND PRELIMINARY MOLECULAR CHARACTERIZATION OF A NOVEL IRIDOVIRUS FROM TIGER SALAMANDERS IN SASKATCHEWAN

An iridovirus was confirmed to be the cause of an epizootic in larval and adult tiger salamanders (Ambystoma tigrinum diaboli) from four separate ponds in southern Saskatchewan (Canada) during the summer of 1997. This organism also is suspected, based on electron microscopic findings, to be the cause of mortality of larval tiger salamanders in a pond over 200 km to the north during the same year. Salamanders developed a generalized viremia which resulted in various lesions including: necrotizing, vesicular and ulcerative dermatitis; gastrointestinal ulceration; and necrosis of hepatic, splenic, renal, lymphoid, and hematopoietic tissues. In cells associated with these lesions, large lightly basophilic cytoplasmic inclusions and vacuolated nuclei with marginated chromatin were consistently found. Virus was isolated from tissue homogenates of infected salamanders following inoculation of epithelioma papilloma cyprini (EPC) cells. The virus, provisionally designated Regina ranavirus (RRV), was initially identified as an iridovirus by electron microscopy. Subsequent molecular characterization, including partial sequence analysis of the major capsid protein (MCP) gene, confirmed this assignment and established that RRV was a ranavirus distinct from frog virus 3 (FV3) and other members of the genus Ranavirus. Intraperitoneal inoculation of 5 × 106.23 TCID50 of the field isolate caused mortality in inoculated salamanders at 13 days post infection. Field, clinical, and molecular studies jointly suggest that the etiological agent of recent salamander mortalities is a highly infectious novel ranavirus.

Development of leukocyte cell lines from the channel catfish (Ictalurus punctatus)

Techniques are described for the generation of channel catfish long term leukocyte cell lines. These techniques include the isolation of peripheral blood leukocytes, purification of B cells by anti-immunoglobulin panning, mitogen stimulation, and in vitro maintenance and cloning of leukocyte cultures. Once stimulated in vitro, channel catfish leukocytes proliferate continuously without the need for exogenous growth factors or feeder cells. The various leukocyte cultures generated are heterogeneous and contain mixtures of monocyte/macrophage-like, T-like, or B cells. Clonal cell lines can be obtained from these mixed cultures by limiting dilution cloning in the presence of conditioned medium. A critical component of the culture medium is the use of channel catfish serum which is required for supporting and maintaining in vitro leukocyte proliferation.

Identification and characterization of clonal NK-like cells from channel catfish (Ictalurus punctatus)

TcR alpha, beta, and gamma chain negative cytotoxic NK-like cells were cloned from alloantigen-stimulated PBL obtained from nai;ve channel catfish. Stimulation with allogeneic cells and growth promoting factors are required for their continued in vitro proliferation and cytotoxic activity. These granular cells kill not only the stimulating allogeneic cells, but also unrelated allogeneic targets by a perforin/granzyme-mediated apoptosis pathway. In addition, they are negative for markers that define neutrophils, monocytes/macrophages, and non-specific cytotoxic cells. Although these NK-like clones kill a number of different allogeneic targets, they display interclonal variation in cytotoxicity toward a panel of allogeneic targets, i.e. some clones have no apparent target specificity, while others display a target preference. In addition, flow cytometric analyses revealed that expression of a putative FcmuR, an LFA-1-like molecule, and a putative thymocyte/T cell antigen varies among the different clones, with no clear correlation between surface antigen expression and cytotoxic activity. Although not all clones express a putative FcmuR, it was noted that they all expressed an ITAM containing FcepsilonR gamma chain homolog. This finding suggests that the catfish FcepsilonR gamma chain may potentially be used as an accessory molecule for not only FcmuRs, but also for other unknown activation receptors. These results support the hypothesis that catfish NK-like cells are heterogeneous in terms of target specificities and cell surface phenotype.

Heterogeneity of Channel Catfish CTL with Respect to Target Recognition and Cytotoxic Mechanisms Employed

Two types of catfish alloantigen-dependent cytotoxic T cells were cloned from PBL from a fish immunized in vivo and stimulated in vitro with the allogeneic B cell line 3B11. Because these are the first clonal cytotoxic T cell lines derived from an ectothermic vertebrate, studies were undertaken to characterize their recognition and cytotoxic mechanisms. The first type of CTL (group I) shows strict alloantigen specificity, i.e., they specifically kill and proliferate only in response to 3B11 cells. The second type (group II) shows broad allogeneic specificity, i.e., they kill and proliferate in response to several different allogeneic cells in addition to 3B11. “Cold” target-inhibition studies suggest that group II CTL recognize their targets via a single receptor, because the killing of one allotarget can be inhibited by a different allotarget. Both types of catfish CTL form conjugates with and kill targets by apoptosis. Killing by Ag-specific cytotoxic T cells (group I) was completely inhibited by treatment with EGTA or concanamycin A, and this killing is sensitive to PMSF inhibition, suggesting that killing was mediated exclusively by the secretory perforin/granzyme mechanism. In contrast, killing by the broadly specific T cytotoxic cells (group II) was only partially inhibited by either EGTA or concanamycin A, suggesting that these cells use a cytotoxic mechanism in addition to that involving perforin/granzyme. Consistent with the presumed use of a secretory pathway, both groups of CTL possess putative lytic granules. These results suggest that catfish CTL show heterogeneity with respect to target recognition and cytotoxic mechanisms.

DIAGNOSTIC AND MOLECULAR EVALUATION OF THREE IRIDOVIRUS-ASSOCIATED SALAMANDER MORTALITY EVENTS

In 1998 viruses were isolated from tiger salamander larvae (Ambystoma tigrinum diaboli and A. tigrinum melanostictum) involved in North Dakota and Utah (USA) mortality events and spotted salamander (A. maculatum) larvae in a third event in Maine (USA). Although sympatric caudates and anurans were present at all three sites only ambystomid larvae appeared to be affected. Mortality at the North Dakota site was in the thousands while at the Utah and Maine sites mortality was in the hundreds. Sick larvae were lethargic and slow moving. They swam in circles with obvious buoyancy problems and were unable to remain upright. On the ventral surface, near the gills and hind limbs, red spots or swollen areas were noted. Necropsy findings included: hemorrhages and ulceration of the skin, subcutaneous and intramuscular edema, swollen and pale livers with multifocal hemorrhage, and distended fluid-filled intestines with areas of hemorrhage. Light microscopy revealed intracytoplasmic inclusions, suggestive of a viral infection, in a variety of organs. Electron microscopy of ultra thin sections of the same tissues revealed iridovirus-like particles within the inclusions. These viruses were isolated from a variety of organs, indicating a systemic infection. Representative viral isolates from the three mortality events were characterized using molecular assays. Characterization confirmed that the viral isolates were iridoviruses and that the two tiger salamander isolates were similar and could be distinguished from the spotted salamander isolate. The spotted salamander isolate was similar to frog virus 3, the type species of the genus Ranavirus, while the tiger salamander isolates were not. These data indicate that different species of salamanders can become infected and die in association with different iridoviruses. Challenge assays are required to determine the fish and amphibian host range of these isolates and to assess the susceptibility of tiger and spotted salamanders to heterologous virus isolates.

Channel catfish NK-like cells are armed with IgM via a putative FcμR

Two-color flow cytometry demonstrated that 4–8% of channel catfish PBL are positive for both F and G IgL chain isotypes, suggesting that they passively acquire serum IgM via a putative FcμR. These cells show spontaneous killing toward allogeneic targets, and in vitro stimulation of PBL with allogeneic cells results in an increase of double IgL chain positive cells with a concomitant increase in nonspecific cytotoxicity. Long-term cultures of alloantigen-stimulated PBL contain both sIgM+ and sIgM− cytotoxic cells that transcribe message for the catfish homolog of the FcϵR γ chain, but not for Igμ, and TCR-α,-β, or -γ chains. Immunoprecipitation of lysates from sIgM+ NK-like cells with anti-IgM co-immunoprecipitated a putative FcμR of ∼64 kDa. Finally, removal of IgM from sIgM+ NK-like cells and replacement with anti-hapten antibody enabled antibody-armed effectors to kill haptenated targets that were refractory to killing by effectors armed with normal IgM. This is the first report suggesting that teleost NK-like cells express a putative FcμR which participates in antibody-dependent cell-mediated cytotoxicity.

Experimental evidence that amphibian ranaviruses are multi-host pathogens

Abstract Infectious diseases, including those caused by ranaviruses (family Iridoviridae), are among the suspected causes of global amphibian declines. Like many pathogens, ranaviruses appear to infect multiple species. We examined several North American amphibian ranavirus isolates to improve our understanding of the effects these viruses have on host communities. Our study had two objectives. The first was to characterize isolates from epizootics affecting wild amphibian populations and compare them to previously described isolates. The second was to test whether amphibian ranaviruses infect ecologically relevant heterologous species, and if so, document the outcome of exposure. The combined results of restriction endonuclease (RE) digestion analyses, sequence analyses, and experimental challenge trials suggest that two amphibian ranaviruses, Ambystoma tigrinum virus (ATV)-like viruses and Frog Virus 3 (FV3)-like viruses, are distinct viral species with different ecologies. Characterizations revealed that several isolates with identical major capsid protein (MCP) gene sequences have distinctive RE profiles. This suggests that high degrees of similarity in MCP sequences may belie important differences among isolates, and we argue it is imperative to characterize ranavirus isolates beyond sequencing the MCP gene. Results from experimental exposure trials indicate that multiple host species may be involved in the ecology of ATV- and FV3-like viruses, and that each virus is capable of infecting several amphibian species that share breeding habitats. Additionally, field collections revealed FV3-like ranaviruses circulating in Wood Frogs (Rana sylvatica) and ATV-like ranaviruses circulating in Tiger Salamanders (Ambystoma tigrinum diaboli) in the same week at a single breeding pond, highlighting the ecological potential for transmission among different host species. Our research also corroborates a growing body of knowledge that suggests individual host populations may differ in their responses to ranavirus infection, a finding with complex conservation implications. Ultimately, experiments elucidating the dynamics of intra- and inter-specific transmission will be particularly important for understanding the roles that ranaviruses play in their host communities and the threat they pose to amphibian populations.

The molecular biology of frog virus 3 and other iridoviruses infecting cold-blooded vertebrates.

Frog virus 3 (FV3) is the best characterized member of the family Iridoviridae. FV3 study has provided insights into the replication of other family members, and has served as a model of viral transcription, genome replication, and virus-mediated host-shutoff. Although the broad outlines of FV3 replication have been elucidated, the precise roles of most viral proteins remain unknown. Current studies using knock down (KD) mediated by antisense morpholino oligonucleotides (asMO) and small, interfering RNAs (siRNA), knock out (KO) following replacement of the targeted gene with a selectable marker by homologous recombination, ectopic viral gene expression, and recombinant viral proteins have enabled researchers to systematically ascertain replicative- and virulence-related gene functions. In addition, the application of molecular tools to ecological studies is providing novel ways for field biologists to identify potential pathogens, quantify infections, and trace the evolution of ecologically important viral species. In this review, we summarize current studies using not only FV3, but also other iridoviruses infecting ectotherms. As described below, general principles ascertained using FV3 served as a model for the family, and studies utilizing other ranaviruses and megalocytiviruses have confirmed and extended our understanding of iridovirus replication. Collectively, these and future efforts will elucidate molecular events in viral replication, intrinsic and extrinsic factors that contribute to disease outbreaks, and the role of the host immune system in protection from disease.

Genomic organization and differential expression of channel catfish MHC class I genes.

Two clones, designated Icpu-UA/3 and Icpu-UA/26, were isolated from a genomic library prepared from a single homozygous gynogenetic channel catfish. Sequence analysis showed that each clone encoded a gene product containing features conserved among MHC class I molecules. The genomic organization of both clones indicated that each domain, with the exception of the cytoplasmic, was encoded by a separate exon. Moreover, like mammals, catfish cytoplasmic regions were encoded by three exons rather than two as previously described for other teleost MHC class I genes. Analysis of nucleotide sequences upstream of catfish class I genes revealed the presence of several regulatory motifs similar to those seen in mammalian class I genes. These included a TATA box, Enhancer B, Site alpha, ISRE, and GAS elements. To determine the functional significance of these elements, EMSAs and tissue expression assays were performed. EMSAs demonstrated that an Enhancer B element within Icpu-UA/26, and an imperfect Enhancer B element and/or a GC-rich region within Icpu-UA/3 were responsible for formation of specific DNA/protein complexes. Expression studies detected Icpu-UA/26 transcripts in all tissues tested, whereas Icpu-UA/3 encoded messages were seen in a limited number of tissues. These results define the intron/exon organization of catfish MHC class I genes, suggest that Icpu-UA/3 encodes a nonclassical gene, and provide the first functional evidence that upstream sequences, similar to those seen in mammalian class I genes, play important roles in regulating teleost MHC gene expression.