Truncated particles produced in fish surviving infectious hematopoietic necrosis virus infection: mediators of persistence?

Abstract

Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus that produces an acute, lethal infection in rainbow trout (Oncorhynchus mykiss). Fish that survive infection cease to produce detectable infectious virus at approximately 46 days after infection, yet there is evidence that survivor fish continue to harbor virus particles (B. S. Drolet, P. P. Chiou, J. Heidel, and J. C. Leong, J. Virol. 69:2140-2147, 1995). In an effort to determine the biological function of these particles, the kidneys and livers from IHNV survivors were harvested and divided into samples for nested reverse transcriptase PCR analysis and explant culture. Sequences for the IHNV nucleoprotein and polymerase genes were detected in 50 and 89%, respectively, of the organs from survivor fish. When explant tissue cultures were infected with purified standard IHNV, the liver tissues from survivor fish produced up to 10-fold less virus than naive control fish liver tissues. In addition, immunosorbent electron microscopy analysis of the supernatant media from the cultured explants of survivor fish revealed truncated particles, whereas the control tissue supernatants contained only standard viral particles. These results suggest that the truncated IHNV particles observed in persistently infected fish are defective interfering particles that may mediate virus persistence.

FIG. 1

Mortality of rainbow trout challenged with the Rangen isolate of IHNV. (A) Triplicate tanks of 100 fish each were infected with 10⁵ PFU of IHNV per ml. The average mortality for each virus isolate is shown on the ordinate and is plotted against the number of days postexposure. The range in the mortalities did not vary significantly between tanks and thus is not shown. (B) Flowchart of experimental procedures for the processing of tissues from survivor and control fish for ISEM, plaque assay, and nested RT-PCR.

FIG. 2

(A) The schematic diagram of the IHNV genome indicates the location of the oligonucleotide primers and probes used for the RT-PCR experiments and Southern blotting. The IHNV genome is shown with its six viral genes in the order 3′-N-P-M-G-NV-L-5′. Arrowheads show the polarity of the primers, and circles represent the oligonucleotides used as end-labeled probes for hybridization analysis. (B) Southern blot analysis of nested RT-PCR products of the IHNV nucleoprotein and polymerase genes. Panels a and c (control) and b and d (survivor) show the nested RT-PCR products for the N gene and L gene. Samples are identified by fish number and tissue type (kidney [K] or liver [L]). (−), RNA sample from uninfected CHSE-214 tissue culture cells. The products were analyzed by Southern blotting with internal oligonucleotides specific for either the N or L gene. Numbers on the right are molecular sizes, in base pairs.

FIG. 3

Virus titers produced from liver and kidney tissues of IHNV survivor and control fish. Supernatants from explant cultures of liver and kidney tissues from survivor or control fish were used in plaque assays to assess the ability to replicate IHNV. The mean difference in virus production between the control and survivor livers was statistically significant (P < 0.0001). The data, with bars indicating standard errors, is shown for 27 different samples in the survivor and control groups.

FIG. 4

Immunosorbent electron micrographs of IHNV standard and truncated particles. Particles were selected by ISEM from a virus preparation grown in cell culture (A) and in control, uninfected fish liver explant culture (B). Truncated particles were selected from a virus preparation grown in cell culture (C) and in survivor fish liver explant culture (D). The EM grids were coated with a monoclonal antibody (136J) specific for the IHNV glycoprotein, incubated with the culture supernatant samples, washed with distilled water, and then examined by transmission EM. The white bar in each panel represents 50 nm.