James K. Jancovich

Evidence for emergence of an amphibian iridoviral disease because of human-enhanced spread

Our understanding of origins and spread of emerging infectious diseases has increased dramatically because of recent applications of phylogenetic theory. Iridoviruses are emerging pathogens that cause global amphibian epizootics, including tiger salamander (Ambystoma tigrinum) die‐offs throughout western North America. To explain phylogeographical relationships and potential causes for emergence of western North American salamander iridovirus strains, we sequenced major capsid protein and DNA methyltransferase genes, as well as two noncoding regions from 18 geographically widespread isolates. Phylogenetic analyses of sequence data from the capsid protein gene showed shallow genetic divergence (< 1%) among salamander iridovirus strains and monophyly relative to available fish, reptile, and other amphibian iridovirus strains from the genus Ranavirus, suggesting a single introduction and radiation. Analysis of capsid protein sequences also provided support for a closer relationship of tiger salamander virus strains to those isolated from sport fish (e.g. rainbow trout) than other amphibian isolates. Despite monophyly based on capsid protein sequences, there was low genetic divergence among all strains (< 1.1%) based on a supergene analysis of the capsid protein and the two noncoding regions. These analyses also showed polyphyly of strains from Arizona and Colorado, suggesting recent spread. Nested clade analyses indicated both range expansion and long‐distance colonization in clades containing virus strains isolated from bait salamanders and the Indiana University axolotl (Ambystoma mexicanum) colony. Human enhancement of viral movement is a mechanism consistent with these results. These findings suggest North American salamander ranaviruses cause emerging disease, as evidenced by apparent recent spread over a broad geographical area.

Evidence for Multiple Recent Host Species Shifts among the Ranaviruses (Family Iridoviridae)

ABSTRACT Members of the genus Ranavirus (family Iridoviridae) have been recognized as major viral pathogens of cold-blooded vertebrates. Ranaviruses have been associated with amphibians, fish, and reptiles. At this time, the relationships between ranavirus species are still unclear. Previous studies suggested that ranaviruses from salamanders are more closely related to ranaviruses from fish than they are to ranaviruses from other amphibians, such as frogs. Therefore, to gain a better understanding of the relationships among ranavirus isolates, the genome of epizootic hematopoietic necrosis virus (EHNV), an Australian fish pathogen, was sequenced. Our findings suggest that the ancestral ranavirus was a fish virus and that several recent host shifts have taken place, with subsequent speciation of viruses in their new hosts. The data suggesting several recent host shifts among ranavirus species increase concern that these pathogens of cold-blooded vertebrates may have the capacity to cross numerous poikilothermic species barriers and the potential to cause devastating disease in their new hosts.

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.

Innate Immune Evasion Mediated by the Ambystoma tigrinum Virus Eukaryotic Translation Initiation Factor 2α Homologue

ABSTRACT Ranaviruses (family Iridoviridae, genus Ranavirus) are large, double-stranded DNA (dsDNA) viruses whose replication is restricted to ectothermic vertebrates. Many highly pathogenic members of the genus Ranavirus encode a homologue of the eukaryotic translation initiation factor 2α (eIF2α). Data in a heterologous vaccinia virus system suggest that the Ambystoma tigrinum virus (ATV) eIF2α homologue (vIF2αH; open reading frame [ORF] 57R) is involved in evading the host innate immune response by degrading the interferon-inducible, dsRNA-activated protein kinase, PKR. To test this hypothesis directly, the ATV vIF2αH gene (ORF 57R) was deleted by homologous recombination, and a selectable marker was inserted in its place. The ATVΔ57R virus has a small plaque phenotype and is 8-fold more sensitive to interferon than wild-type ATV (wtATV). Infection of fish cells with the ATVΔ57R virus leads to eIF2α phosphorylation, in contrast to infection with wtATV, which actively inhibits eIF2α phosphorylation. The inability of ATVΔ57R to prevent phosphorylation of eIF2α correlates with degradation of fish PKZ, an interferon-inducible enzyme that is closely related to mammalian PKR. In addition, salamanders infected with ATVΔ57R displayed an increased time to death compared to that of wtATV-infected salamanders. Therefore, in a biologically relevant system, the ATV vIF2αH gene acts as an innate immune evasion factor, thereby enhancing virus pathogenesis.

Size limitations in the filter chamber and digestive tract of nymphal and adult Bemisia tabaci whiteflies (Hemiptera: Aleyrodidae)

Abstract The molecular size limitations of the digestive system, including the filter chamber of immature and adult Bemisia tabaci (Gennadius) B biotype (=B. argentifolii), were studied by tracking the movement of fluorescent-labeled molecules and microspheres ingested by whiteflies. Soluble fluorescent molecules and labeled dextrans, ranging from 389 to 2,000,000 Da, were observed throughout the digestive tract of immatures 10–30 min after feeding was initiated. After removal of labeled molecules from the diet, fluorochromes were cleared from digestive system of immatures within 2 h. Fluorescent-labeled 0.1- and 0.2-μm microspheres were ingested by larvae and saturated the digestive system within 2 h after initiation of feeding. Large, 0.5-μm spheres were not observed in the digestive tract of immatures, probably because singly or as aggregates, they were too large to enter the stylet food canal. The smallest spheres examined, 0.02 μm, were not detectable in the digestive tract of immatures. Observations for whitefly adults were identical to those for larvae, with two exceptions. In adults, soluble fluorochromes were detectable1 h after feeding commenced, and 0.02-μm spheres were observed primarily in the esophagus, filter chamber, anterior midgut, and hindgut, but not in the posterior portions of the midgut. We hypothesize that most of the 0.02-μm spheres ingested by adult whiteflies were shunted directly to the hindgut by way of the filter chamber, effectively bypassing the midgut. This is, therefore, a feasible route for virions of the plant pathogenic genus Begomovirus, which are of similar size to the small microspheres and are transmitted in a circulative manner by B. tabaci.

Characterization of a PKR inhibitor from the pathogenic ranavirus, Ambystoma tigrinum virus, using a heterologous vaccinia virus system

Ambystoma tigrinum virus (ATV) (family Iridoviridae, genus Ranavirus) was isolated from diseased tiger salamanders (Ambystoma tigrinum stebbinsi) from the San Rafael Valley in southern Arizona, USA in 1996. Genomic sequencing of ATV, as well as other members of the genus, identified an open reading frame that has homology to the eukaryotic translation initiation factor, eIF2α (ATV eIF2α homologue, vIF2αH). Therefore, we asked if the ATV vIF2αH could also inhibit PKR. To test this hypothesis, the ATV vIF2αH was cloned into vaccinia virus (VACV) in place of the well-characterized VACV PKR inhibitor, E3L. Recombinant VACV expressing ATV vIF2αH partially rescued deletion of the VACV E3L gene. Rescue coincided with rapid degradation of PKR in infected cells. These data suggest that the salamander virus, ATV, contains a novel gene that may counteract host defenses, and this gene product may be involved in the presentation of disease caused by this environmentally important pathogen.

Immunization of Pigs by DNA Prime and Recombinant Vaccinia Virus Boost To Identify and Rank African Swine Fever Virus Immunogenic and Protective Proteins

ABSTRACT African swine fever virus (ASFV) causes an acute hemorrhagic fever in domestic pigs, with high socioeconomic impact. No vaccine is available, limiting options for control. Although live attenuated ASFV can induce up to 100% protection against lethal challenge, little is known of the antigens which induce this protective response. To identify additional ASFV immunogenic and potentially protective antigens, we cloned 47 viral genes in individual plasmids for gene vaccination and in recombinant vaccinia viruses. These antigens were selected to include proteins with different functions and timing of expression. Pools of up to 22 antigens were delivered by DNA prime and recombinant vaccinia virus boost to groups of pigs. Responses of immune lymphocytes from pigs to individual recombinant proteins and to ASFV were measured by interferon gamma enzyme-linked immunosorbent spot (ELISpot) assays to identify a subset of the antigens that consistently induced the highest responses. All 47 antigens were then delivered to pigs by DNA prime and recombinant vaccinia virus boost, and pigs were challenged with a lethal dose of ASFV isolate Georgia 2007/1. Although pigs developed clinical and pathological signs consistent with acute ASFV, viral genome levels were significantly reduced in blood and several lymph tissues in those pigs immunized with vectors expressing ASFV antigens compared with the levels in control pigs. IMPORTANCE The lack of a vaccine limits the options to control African swine fever. Advances have been made in the development of genetically modified live attenuated ASFV that can induce protection against challenge. However, there may be safety issues relating to the use of these in the field. There is little information about ASFV antigens that can induce a protective immune response against challenge. We carried out a large screen of 30% of ASFV antigens by delivering individual genes in different pools to pigs by DNA immunization prime and recombinant vaccinia virus boost. The responses in immunized pigs to these individual antigens were compared to identify the most immunogenic. Lethal challenge of pigs immunized with a pool of antigens resulted in reduced levels of virus in blood and lymph tissues compared to those in pigs immunized with control vectors. Novel immunogenic ASFV proteins have been identified for further testing as vaccine candidates.