title: Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA.
abstract: Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.
authors.affiliation: U.S. Geological Survey-Western Fisheries Research Center, Seattle, WA (Purcell, Pearman-Gillman, Thompson, Winton, Emmenegger)School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA (Thompson)U.S. Geological Survey-Marrowstone Marine Field Station, Nordland, WA (Gregg, Hart, Hershberger) mpurcell@usgs.gov.
authors.forename: Maureen K
authors.initials: MK
authors.lastname: Purcell
authors.affiliation: U.S. Geological Survey-Western Fisheries Research Center, Seattle, WA (Purcell, Pearman-Gillman, Thompson, Winton, Emmenegger)School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA (Thompson)U.S. Geological Survey-Marrowstone Marine Field Station, Nordland, WA (Gregg, Hart, Hershberger).
authors.forename: Schuyler
authors.initials: S
authors.lastname: Pearman-Gillman
authors.affiliation: U.S. Geological Survey-Western Fisheries Research Center, Seattle, WA (Purcell, Pearman-Gillman, Thompson, Winton, Emmenegger)School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA (Thompson)U.S. Geological Survey-Marrowstone Marine Field Station, Nordland, WA (Gregg, Hart, Hershberger).
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Thompson
authors.affiliation: U.S. Geological Survey-Western Fisheries Research Center, Seattle, WA (Purcell, Pearman-Gillman, Thompson, Winton, Emmenegger)School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA (Thompson)U.S. Geological Survey-Marrowstone Marine Field Station, Nordland, WA (Gregg, Hart, Hershberger).
authors.forename: Jacob L
authors.initials: JL
authors.lastname: Gregg
authors.affiliation: U.S. Geological Survey-Western Fisheries Research Center, Seattle, WA (Purcell, Pearman-Gillman, Thompson, Winton, Emmenegger)School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA (Thompson)U.S. Geological Survey-Marrowstone Marine Field Station, Nordland, WA (Gregg, Hart, Hershberger).
authors.forename: Lucas M
authors.initials: LM
authors.lastname: Hart
authors.affiliation: U.S. Geological Survey-Western Fisheries Research Center, Seattle, WA (Purcell, Pearman-Gillman, Thompson, Winton, Emmenegger)School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA (Thompson)U.S. Geological Survey-Marrowstone Marine Field Station, Nordland, WA (Gregg, Hart, Hershberger).
authors.forename: James R
authors.initials: JR
authors.lastname: Winton
authors.affiliation: U.S. Geological Survey-Western Fisheries Research Center, Seattle, WA (Purcell, Pearman-Gillman, Thompson, Winton, Emmenegger)School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA (Thompson)U.S. Geological Survey-Marrowstone Marine Field Station, Nordland, WA (Gregg, Hart, Hershberger).
authors.forename: Eveline J
authors.initials: EJ
authors.lastname: Emmenegger
authors.affiliation: U.S. Geological Survey-Western Fisheries Research Center, Seattle, WA (Purcell, Pearman-Gillman, Thompson, Winton, Emmenegger)School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA (Thompson)U.S. Geological Survey-Marrowstone Marine Field Station, Nordland, WA (Gregg, Hart, Hershberger).
authors.forename: Paul K
authors.initials: PK
authors.lastname: Hershberger
mesh_terms: D000818:Animals; D036022:Capsid Proteins / Q000235:genetics*; D004266:DNA Virus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D005393:Fish Diseases / Q000175:diagnosis* / Q000821:virology; D005399:Fishes; D007497:Iridoviridae / Q000145:classification / Q000235:genetics / Q000502:physiology*; D010802:Phylogeny; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D017422:Sequence Analysis, DNA / Q000662:veterinary
chemical_list: D036022:Capsid Proteins
keywords: Diagnostic validation; inclusion bodies; viral erythrocytic necrosis