title: Molecular identification of erythrocytic necrosis virus (ENV) from the blood of Pacific herring (Clupea pallasii).
abstract: Viral erythrocytic necrosis (VEN) is a condition affecting the red blood cells of more than 20 species of marine and anadromous fishes in the North Atlantic and North Pacific Oceans. Among populations of Pacific herring (Clupea pallasii) on the west coast of North America the disease causes anemia and elevated mortality in periodic epizootics. Presently, VEN is diagnosed by observation of typical cytoplasmic inclusion bodies in stained blood smears from infected fish. The causative agent, erythrocytic necrosis virus (ENV), is unculturable and a presumed iridovirus by electron microscopy. In vivo amplification of the virus in pathogen-free laboratory stocks of Pacific herring with subsequent virus concentration, purification, DNA extraction, and high-throughput sequencing were used to obtain genomic ENV sequences. Fragments with the highest sequence identity to the family Iridoviridae were used to design four sets of ENV-specific polymerase chain reaction (PCR) primers. Testing of blood and tissue samples from experimentally and wild infected Pacific herring as well as DNA extracted from other amphibian and piscine iridoviruses verified the assays were specific to ENV with a limit of detection of 0.0003 ng. Preliminary phylogenetic analyses of a 1448 bp fragment of the putative DNA polymerase gene supported inclusion of ENV in a proposed sixth genus of the family Iridoviridae that contains other erythrocytic viruses from ectothermic hosts. This study provides the first molecular evidence of ENV's inclusion within the Iridoviridae family and offers conventional PCR assays as a means of rapidly surveying the ENV-status of wild and propagated Pacific herring stocks.
authors.affiliation: U.S. Geological Survey, Western Fisheries Research Center (WFRC), 6505 NE 65th St., Seattle, WA 98115, USA. Electronic address: eemmenegger@usgs.gov.
authors.forename: Eveline J
authors.initials: EJ
authors.lastname: Emmenegger
authors.affiliation: U.S. Geological Survey, Western Fisheries Research Center (WFRC), 6505 NE 65th St., Seattle, WA 98115, USA.
authors.forename: Jolene A
authors.initials: JA
authors.lastname: Glenn
authors.affiliation: U.S. Geological Survey, Western Fisheries Research Center (WFRC), 6505 NE 65th St., Seattle, WA 98115, USA.
authors.forename: James R
authors.initials: JR
authors.lastname: Winton
authors.affiliation: U.S. Geological Survey, Western Fisheries Research Center (WFRC), 6505 NE 65th St., Seattle, WA 98115, USA.
authors.forename: William N
authors.initials: WN
authors.lastname: Batts
authors.affiliation: U.S. Geological Survey, WFRC, Marrowstone Marine Field Station, 616 Marrowstone Point Road, Nordland, WA 98358, USA.
authors.forename: Jacob L
authors.initials: JL
authors.lastname: Gregg
authors.affiliation: U.S. Geological Survey, WFRC, Marrowstone Marine Field Station, 616 Marrowstone Point Road, Nordland, WA 98358, USA.
authors.forename: Paul K
authors.initials: PK
authors.lastname: Hershberger
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004266:DNA Virus Infections / Q000662:veterinary*; D005393:Fish Diseases / Q000821:virology*; D005399:Fishes; D059014:High-Throughput Nucleotide Sequencing / Q000662:veterinary; D007497:Iridoviridae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D013047:Specific Pathogen-Free Organisms
chemical_list: D017931:DNA Primers
keywords: Erythrocytic necrosis virus; Fish disease; Herring; Iridovirus; VEN