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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., (Copyright © 2018 Jancovich et al.)

The iridovirus RNase III gene is one of 26 conserved core genes among the family Iridoviridae. Initial studies suggest this viral protein functions to suppress RNA interference pathways that may attack viral RNA during infection. Therefore, to determine if the Ambystoma tigrinum virus (ATV) RNase III-like gene (ORF 25R) can modulate the host innate immune response fish and human cells ectopically expressing 25R were treated with polyI:C and monitored for interferon synthesis and phosphorylation of eIF2α and PKR. We found a decrease in cellular IFN production and modulation of the PKR pathway. In addition, ATV deleted of the RNase III gene (ATVΔ25R) shows reduced pathogenicity in tiger salamanders. Collectively our data suggest that the ATV 25R protein is a pathogenesis factor that may function to help evade the host's immune response by masking activators of the IFN pathway., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (Copyright © 2017. Published by Elsevier Inc.)

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.

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.