Heidi Morales

Innate Immune Responses and Permissiveness to Ranavirus Infection of Peritoneal Leukocytes in the Frog Xenopus laevis

ABSTRACT Ranaviruses such as frog virus 3 ([FV3] family Iridoviridae) are increasingly prevalent pathogens that infect reptiles, amphibians, and fish worldwide. Whereas studies in the frog Xenopus laevis have revealed the critical involvement of CD8 T-cell and antibody responses in host resistance to FV3, little is known about the role played by innate immunity to infection with this virus. We have investigated the occurrence, composition, activation status, and permissiveness to infection of peritoneal leukocytes (PLs) in Xenopus adults during FV3 infection by microscopy, flow cytometry, and reverse transcription-PCR. The total number of PLs and the relative fraction of activated mononucleated macrophage-like cells significantly increase as early as 1 day postinfection (dpi), followed by NK cells at 3 dpi, before the peak of the T-cell response at 6 dpi. FV3 infection also induces a rapid upregulation of proinflammatory genes including arginase 1, interleukin-1β, and tumor necrosis factor alpha. Although PLs are susceptible to FV3 infection, as evidenced by apoptotic cells, active FV3 transcription, and the detection of viral particles by electron microscopy, the infection is weaker (fewer infectious particles), more transitory, and involves a smaller fraction (less than 1%) of PLs than the kidney, the main site of infection. However, viral DNA remains detectable in PLs for at least 3 weeks postinfection, past the point of viral clearance observed in the kidneys. This suggests that although PLs are actively involved in anti-FV3 immune responses, some of these cells can be permissive and harbor quiescent, asymptomatic FV3.

XENOPUS LAEVIS: A POSSIBLE VECTOR OF RANAVIRUS INFECTION?

Frog virus 3 (FV3) or FV3-like viruses (Iridoviridae) infect a wide range of amphibian species, and they are becoming increasingly and causally associated with amphibian disease outbreaks worldwide. We have established the frog Xenopus laevis as an experimental model to study host defense and pathogenesis of FV3 infection. Although X. laevis adults usually clear FV3 infection within a few weeks, viral DNA has been detected in the kidneys several months after they had been experimentally infected; virus also has been detected in seemingly healthy nonexperimentally infected adults. Based on this information, we hypothesized that covert FV3 infection may occur in Xenopus. We first conducted a survey that detected FV3 by polymerase chain reaction (PCR) in the kidneys (the main site of FV3 infection) in a significant fraction of X. laevis raised in five different locations in the United States. Asymptomatic FV3 carriers also were detected by initiation of an acute systemic FV3 infection in frogs that had been immunosupressed by sublethal γ-irradiation. Finally, we focused on macrophages as a potential site for viral persistence, and we showed that FV3 can infect peritoneal macrophages in vitro as determined by reverse transcriptase-PCR detection of viral mRNAs. Unlike kidney cell lines that are readily killed by FV3, infected macrophages, like uninfected macrophages, survived up to 12 days. Viral transcription also was detected in macrophages from animals up to 12 days after infection. These results suggest that FV3 can become quiescent in resistant species such as Xenopus, thereby making these species potential viral reservoirs.

Characterization of Primary and Memory CD8 T-Cell Responses against Ranavirus (FV3) in Xenopus laevis

ABSTRACT In mammals, resistance to primary and secondary viral infections critically involves major histocompatibility complex class I-restricted cytotoxic CD8+ T lymphocytes (CTLs). Although many gene homologues involved in CTL function have been identified in all vertebrate classes, antiviral CTL responses have been poorly characterized for ectothermic vertebrates. Because of the threat of emerging wildlife viral diseases to global biodiversity, fundamental research on comparative viral immunity has become crucial. Ranaviruses (family Iridoviridae) are double-stranded DNA viruses possibly implicated in the worldwide decline of amphibian populations. We used the frog Xenopus laevis as a model to evaluate adaptive immune responses to the ranavirus frog virus 3 (FV3). FV3 infects the kidneys of adults but is cleared within 4 weeks, with faster clearance upon secondary infections. In vivo depletion of CD8+ T cells markedly decreases the survival of adults after viral infection. To further investigate the involvement of anti-FV3 CD8+ T-cell effectors in host resistance in vivo, we determined the proliferation kinetics of CD8+ T cells in the spleen by bromodeoxyuridine incorporation and their infiltration of kidneys by immunohistology. Upon primary infection, CD8+ T cells significantly proliferate in the spleen and accumulate in infected kidneys from day 6 onward, in parallel with virus clearance. Earlier proliferation and infiltration associated with faster viral clearance were observed during a secondary infection. These results provide in vivo evidence of protective antigen-dependent CD8+ T-cell proliferation, recognition, and memory in fighting a natural pathogen in Xenopus.

Localization and Differential Expression of Activation-Induced Cytidine Deaminase in the Amphibian Xenopus upon Antigen Stimulation and during Early Development

As in mammals, B cell maturation in the amphibian Xenopus involves somatic hypermutation (SHM) and class switch recombination to diversify the B cell receptor repertoire in response to Ag stimulation. Unlike mammals, however, the resulting increase in Ab affinity is poor in Xenopus, which is possibly related to the absence of germinal centers and a suboptimal selection mechanism of SHM. In mammals, both SHM and class switch recombination are mediated by the activation-induced cytidine deaminase enzyme and under Ag-dependent regulation. Given its evolutionary conservation in jawed vertebrates, we used activation-induced cytidine deaminase as a marker to monitor and localize B cell maturation in Xenopus upon immune responses and during early development. In adult, Xenopus laevis AID (XlAID) was detected mainly in the spleen, where cells expressing XlAID were preferentially distributed in follicular B cell zones, although some XlAID+ cells were also found in the red pulp. XlAID was markedly up-regulated in the spleen with different kinetics upon bacterial stimulation and viral infection. However, during secondary anti-viral response XlAID was also noticeably expressed by PBLs, suggesting that XlAID remains active in a subset of circulating B cells. During ontogeny, XlAID expression was detected as early as 5 days postfertilization in liver before the first fully differentiated B cells appear. Concomitant with appearance of mature B cells XlAID was up-regulated upon bacterial stimulation or viral infection at later larval stages. This study highlights the conserved involvement of XlAID during Ag-dependent B cell responses in Xenopus but also suggests another role in B cell differentiation earlier in ontogeny.

Phylogenetic Conservation of Glycoprotein 96 Ability to Interact with CD91 and Facilitate Antigen Cross-Presentation

Although the ability of gp96 to activate APCs and generate CD8 CTLs against peptides they chaperone through interaction with the endocytic receptors CD91 is supported by solid evidence, its biological relevance in immune surveillance is debated. We have used an evolutionary approach to determine whether gp96 interacts with receptors expressed on APCs and promotes MHC class I cross-presentation of minor histocompatibility Ags (H-Ags) to CTLs in the frog Xenopus. We show that in Xenopus gp96 binds the CD91 homolog at the surface of peritoneal leukocytes, and that this binding is inhibited by molar excess of unlabeled gp96 or the CD91 ligand α2-macroglobulin, by anti-CD91 Ab and by the specific CD91 antagonist receptor-associated protein. Surface binding followed by internalization of gp96 was confirmed by fluorescent microscopy. Furthermore, adoptive transfer of peritoneal leukocytes pulsed with as little as 800 ng of gp96 chaperoning minor H-Ags, but not minor H-Ag-free gp96, induces potent CD8 T cell infiltration and Ag-specific accelerated rejection of minor H-locus disparate skin grafts. Inhibition of gp96-CD91 interaction by pretreatment with anti-CD91 Ab and receptor-associated protein impairs both CD8 T cell infiltration and acute skin graft rejection. These data provide evidence of the conserved ability of gp96 to facilitate cross-presentation of chaperoned Ags by interacting with CD91. The persistence of this biological process for >350 million years that separate mammals and amphibians from a common ancestor strongly supports the proposition that gp96 and CD91 are critically involved in immune surveillance.

Mass mortality associated with a frog virus 3-like Ranavirus infection in farmed tadpoles Rana catesbeiana from Brazil

Ranaviruses (Iridoviridae) are increasingly associated with mortality events in amphibians, fish, and reptiles. They have been recently associated with mass mortality events in Brazilian farmed tadpoles of the American bullfrog Rana catesbeiana Shaw, 1802. The objectives of the present study were to further characterize the virus isolated from sick R. catesbeiana tadpoles and confirm the etiology in these outbreaks. Sick tadpoles were collected in 3 farms located in Goiás State, Brazil, from 2003 to 2005 and processed for virus isolation and characterization, microbiology, histopathology, and parasitology. The phylogenetic relationships of Rana catesbeiana ranavirus (RCV-BR) with other genus members was investigated by PCR with primers specific for the major capsid protein gene (MCP) and the RNA polymerase DNA-dependent gene (Pol II). Sequence analysis and multiple alignments for MCP products showed >99% amino acid identity with other ranaviruses, while Pol II products showed 100% identity. Further diagnostics of the pathology including histology and transmission electron microscopy confirmed the viral etiology of these mass deaths. As far as we know, this is the first report of a ranaviral infection affecting aquatic organisms in Brazil. Additionally, our results suggest that American bullfrogs may have served as a vector of transmission of this virus, which highlights the potential threat of amphibian translocation in the world distribution of pathogens.

Bacterial stimulation upregulates the surface expression of the stress protein gp96 on B cells in the frog Xenopus

Abstract The presence of the soluble intracellular heat shock protein gp96 (an endoplasmic reticulum resident protein) at the surface of certain cell types is an intriguing phenomenon whose physiological significance has been unclear. We have shown that the active surface expression of gp96 by some immune cells is found throughout the vertebrate phylum including the Agnatha, the only vertebrate taxon whose members (lamprey, hagfish) lack an adaptive immune system. To determine whether gp96 surface expression can be modulated by pathogens, we investigated the effects of in vitro stimulation by purified lipopolysaccharide (LPS) and the heat-killed gram-negative bacteria, Escherischia coli and Aeromonas hydrophilia. Purified Xenopus B cells are readily activated and markedly proliferate in vitro in response to the heat-killed bacteria but not to purified LPS. Furthermore, messenger ribonucleic acid, and intracellular and surface protein expressions of both gp96 and immunoglobulin were upregulated only after activation of B cells by heat-killed bacteria. These data are consistent with an ancestral immunological role of gp96 as an antigen-presenting or danger-signaling molecule, or both, interacting directly with antigen-presenting cells, T cells, or natural killer cells, (or all), to trigger or amplify immune responses.

In vivo and in vitro techniques for comparative study of antiviral T-cell responses in the amphibian Xenopus.

Activation of lymphocytes in mammals is often quantified by measuring the amount of proliferation during the expansion phase of an immune response. Bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assays are some of the techniques widely used in mammalian studies of pathogeninduced proliferation and provide a convenient way of quantifying the cellular response. We have extended the use of these proliferation assays to the amphibian Xenopus laevis. We have developed this species as a valuable comparative model to study immunity against a wellknown amphibian pathogen, Frog Virus 3 (FV3). Fluorescence activated cell sorting was used to assess the level of BrdU incorporation of lymphocytes in vivo and CFSE dilution in an in vitro activation assay. Both techniques have shown that splenic lymphocytes proliferate specifically upon FV3 challenge. This indicates that common methods for detection of proliferation upon immunologic challenge are easily applied to other vertebrate species, as it highlights the evolutionary conservation of the proliferative nature of immune responses throughout vertebrate phyla.