Kinetics of Viral Load and Erythrocytic Inclusion Body Formation in Pacific Herring Artificially Infected with Erythrocytic Necrosis Virus

Abstract

Viral erythrocytic necrosis (VEN) is a condition that affects marine and anadromous fish species, including herrings and salmonids, in the Atlantic and Pacific oceans. Infection is frequently associated with severe anemia and causes episodic mortality among wild and hatchery fish when accompanied by additional stressors; VEN can be presumptively diagnosed by (1) light microscopic identification of a single characteristic-a round, magenta-colored, 0.8-μm-diameter inclusion body (IB) within the cytoplasm of erythrocytes and their precursors on Giemsa-stained blood films; or (2) observation (via transmission electron microscopy [TEM]) of the causative iridovirus, erythrocytic necrosis virus (ENV), within erythrocytes or their precursors. To better understand the kinetics of VEN, specific-pathogen-free Pacific herring Clupea pallasii were infected with ENV by intraperitoneal injection. At 1, 4, 7, 10, 14, 21, and 28 d postexposure, samples of blood, spleen, and kidney were collected and assessed (1) via light microscopy for the number of intracytoplasmic IBs in blood smears and (2) via TEM for the number of virions within erythrocytes. The mean prevalence of intracytoplasmic IBs in the blood cells increased from 0% at 0-4 d postexposure to 94% at 28 d postexposure. Viral load within circulating red blood cells peaked at 7 d postexposure, fell slightly, and then reached a plateau. However, blood cells observed within the kidney and spleen tissues demonstrated high levels of ENV between 14 and 28 d postexposure. The results indicate that the viral load within erythrocytes does not correlate well with IB prevalence and that the virus can persist in infected fish for more than 28 d.

Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA

“…The transient delay in inclusion body formation in our progression study was consistent with previous laboratory challenge studies and confinement studies in Pacific herring, which found detectable erythrocytic inclusion bodies appearing in a low percentage of cells 4-6 days after initiation of the epizootic. 9,12 Although DSe and DSp of blood smear analysis was similar to the qPCR assays, it should be noted that technical difficulties were encountered with the analysis of some blood films, where cell lysis occurred after inadvertent contact with seawater. We were not able to confidently analyze 20% of blood smear slides from the ENV-infected fish.…”

Molecular identification of erythrocytic necrosis virus (ENV) from the blood of Pacific herring (Clupea pallasii)

“…Fish were held in ambient seawater for the entire inoculation series with temperatures ranging from 9.7 to 13.2 8C. Injections occurred every 7-10 days, as this incubation period results in the highest viral titer in herring erythrocytes (Glenn et al, 2012). All herring used in the first five passages were 3 years of age.…”

“…Blood films were Giemsa stained as previously described by Glenn et al (2012) and inclusion body concentrations for each donor fish were determined. Average percent prevalence of inclusions in RBCs of the survivors for each challenge was calculated (Fig.…”

“…Disease outbreaks periodically occur in populations of Pacific herring, particularly juveniles, throughout coastal regions of the NE Pacific Ocean in both cultured and wild fish (Meyers et al, 1986;Hershberger et al, 2006Hershberger et al, , 2009. Laboratory challenges of chum salmon, pink salmon, and Pacific herring with blood or tissue homogenates from VENaffected fish have shown these species to be highly susceptible to infection and have reproduced essential features of the disease (Evelyn and Traxler, 1978;MacMillan and Mulcahy, 1979;Haney et al, 1992;Glenn et al, 2012). The disease is currently diagnosed by microscopic examination of Giemsa-stained blood smears for the presence of typical inclusion bodies within the cytoplasm of infected erythrocytes (AFS-FHS, 2012).…”

Iridovirus-Like Viruses in Erythrocytes of Lacertids from Portugal

“…morphologically similar to the icosahedral nucleo-cytoplasmic large DNA vi-ruses~NCLDVs; Iyer et al, 2001!. Cytoplasmic inclusion-producing NCLDV-like viruses also occur in fish and amphibian eryth-rocytes~Johnston, 1975;Sousa & Weigl, 1976;Walker & Sherburne, 1977;Gruya-Gray et al, 1989;Alves de Matos & Paperna, 1993b, Glenn & Emmenegger, 2012!, as well as in other reptiles, such as the peninsula ribbon snake Thamnophis sauritus sackenii~Wellehan et al, 2008!. Cytoplasmic inclusion-producing NCLDV-like viruses also occur in fish and amphibian eryth-rocytes~Johnston, 1975;Sousa & Weigl, 1976;Walker & Sherburne, 1977;Gruya-Gray et al, 1989;Alves de Matos & Paperna, 1993b, Glenn & Emmenegger, 2012!, as well as in other reptiles, such as the peninsula ribbon snake Thamnophis sauritus sackenii~Wellehan et al, 2008!.…”