Tissue Distribution of the Piscine Novirhabdovirus Genotype IVb in Muskellunge (Esox masquinongy)

Abstract

A novel sublineage of the piscine novirhabdovirus (synonym: viral hemorrhagic septicemia virus), genotype IVb, emerged in the Laurentian Great Lakes, causing serious losses in resident fish species as early as 2003. Experimentally infected juvenile muskellunge (Esox masquinongy) were challenged with VHSV-IVb at high (1 × 10⁵ PFU mL^-1), medium (4 × 10³ PFU mL^-1), and low (100 PFU mL^-1) doses. Samples from spleen, kidneys, heart, liver, gills, pectoral fin, large intestine, and skin/muscle were collected simultaneously from four fish at each predetermined time point and processed for VHSV-IVb reisolaton on Epitheliosum papulosum cyprini cell lines and quantification by plaque assay. The earliest reisolation of VHSV-IVb occurred in one fish from pectoral fin samples at 24 h post-infection. By 6 days post-infection (dpi), all tissue types were positive for VHSV-IVb. Statistical analysis suggested that virus levels were highest in liver, heart, and skin/muscle samples. In contrast, the kidneys and spleen exhibited reduced probability of virus recovery. Virus distribution was further confirmed by an in situ hybridization assay using a VHSV-IVb specific riboprobe. Heart muscle fibers, hepatocytes, endothelia, smooth muscle cells, and fibroblast-like cells of the pectoral fin demonstrated riboprobe labeling, thus highlighting the broad cellular tropism of VHSV-IVb. Histopathologic lesions were observed in areas where the virus was visualized.

Keywords: in situ hybridization; muskellunge; pathogenesis; tropism; viral hemorrhagic septicemia virus.

Figure 1

Load of viral hemorrhagic septicemia virus IVb (VHSV-IVb) determined by plaque assay, shown in log₁₀ plaque forming units (PFU) gram⁻¹ tissue in parallel tissue samples from four fish at predetermined time points. Fish were exposed VHSV-IVb concentrations of high (1 × 10⁵ PFU mL⁻¹), medium (4 × 10³ PFU mL⁻¹), and low (100 PFU mL⁻¹).

Figure 2

Combined total percentage of virus detections by viral plaque (black) among the high, medium, and low doses of infection. Non-detections are presented in gray. Concurrent, virus isolation by cell culture and confirmation of viral hemorrhagic septicemia virus IVb by reverse transcriptase-polymerase chain reaction was completed.

Figure 3

Primary and secondary lamellae of gills obtained from muskellunge (Esox masquinongy) exposed at the high dose (1 × 10⁶ plaque forming units ml⁻¹), 6 days post-infection. (a) Hematoxylin and eosin-stained sections reveal pyknotic nuclei of endothelia lining the primary lamellar space (black arrows). (b) In situ hybridization reveals intense riboprobe staining (blue) at the same sight. (Scale bar = 65 µm.)

Figure 4

Pectoral fin sections from muskellunge (Esox masquinongy) collected from fish exposed at the medium dose (4 × 10³ plaque forming units mL⁻¹), 6 days post-infection. (a) multifocal necrosis of fibroblasts adjacent to fin rays, characterized by pyknotic and fragmented nuclei (asterisks). (b) positive riboprobe staining (blue). (Scale bar = 70 µm.)

Figure 5

Liver sections from muskellunge (Esox masquinongy) exposed at the medium dose (4 × 10³ plaque forming units mL⁻¹), 28 days post-infection. (a) Multifocal hepatocellular necrosis (black arrows). (b) Positive riboprobe staining of hepatocytes (blue) (scale bar = 75 µm).

Figure 6

Heart sections from a muskellunge (Esox masquinongy) exposed at the medium dose (4 × 10³ plaque forming units mL⁻¹), 28 days post-infection. (a) Severe mononuclear infiltration between necrotic cardiac myocytes. (b) Positive riboprobe staining of myocardium. Note the lack of staining in the endo and pericardial layers. (c) Focally diffuse cardiac myositis. (d) Positive riboprobe staining in cardiac myocytes surrounding the area of inflammation and necrosis (scale bar a,b = 110 µm; c,d = 75 µm).

Figure 6

Heart sections from a muskellunge (Esox masquinongy) exposed at the medium dose (4 × 10³ plaque forming units mL⁻¹), 28 days post-infection. (a) Severe mononuclear infiltration between necrotic cardiac myocytes. (b) Positive riboprobe staining of myocardium. Note the lack of staining in the endo and pericardial layers. (c) Focally diffuse cardiac myositis. (d) Positive riboprobe staining in cardiac myocytes surrounding the area of inflammation and necrosis (scale bar a,b = 110 µm; c,d = 75 µm).

Figure 7

Kidney section of muskellunge (Esox masquinongy) exposed at the medium dose (4 × 10³ plaque forming units mL⁻¹), 6 days post-infection. (a) Degenerate renal tubular epithelium are swollen and vacuolated (arrowhead) or shrunken and necrotic with pyknotic nuclei (arrows). There are scattered foci of pyknotic nuclei and cellular debris within renal tubular lumena and interstitium (asterisks). (b) Positive riboprobe staining in areas of necrotic cellular debris and admixed with degenerate renal tubular epithelium (scale bar = 60 µm).

Figure 7

Kidney section of muskellunge (Esox masquinongy) exposed at the medium dose (4 × 10³ plaque forming units mL⁻¹), 6 days post-infection. (a) Degenerate renal tubular epithelium are swollen and vacuolated (arrowhead) or shrunken and necrotic with pyknotic nuclei (arrows). There are scattered foci of pyknotic nuclei and cellular debris within renal tubular lumena and interstitium (asterisks). (b) Positive riboprobe staining in areas of necrotic cellular debris and admixed with degenerate renal tubular epithelium (scale bar = 60 µm).

Figure 8

Section of meninges with subjacent gray matter of muskellunge (Esox masquinongy) exposed at the medium dose (4 × 10³ plaque forming units mL⁻¹), 6 days post-infection. (a) Spongiosis (arrows), gliosis (arrowheads), and lymphocytic infiltrates within the gray matter and minimally into the meninges (asterisks). (b) Positive riboprobe staining in areas of necrosis (scale bar = 60 µm).

Figure 8

Section of meninges with subjacent gray matter of muskellunge (Esox masquinongy) exposed at the medium dose (4 × 10³ plaque forming units mL⁻¹), 6 days post-infection. (a) Spongiosis (arrows), gliosis (arrowheads), and lymphocytic infiltrates within the gray matter and minimally into the meninges (asterisks). (b) Positive riboprobe staining in areas of necrosis (scale bar = 60 µm).

Figure 9

Longitudinal section of the posterior intestine in muskellunge exposed at the medium dose (4 × 10³ plaque forming units mL⁻¹), 6 days post-infection. (a) Single cell pyknosis smooth muscle (asterisk). (b) Positive riboprobe staining (blue) in smooth muscle cells intermixed in regions of unaffected regions. (Scale bar = 75 µm.)

Figure 9

Longitudinal section of the posterior intestine in muskellunge exposed at the medium dose (4 × 10³ plaque forming units mL⁻¹), 6 days post-infection. (a) Single cell pyknosis smooth muscle (asterisk). (b) Positive riboprobe staining (blue) in smooth muscle cells intermixed in regions of unaffected regions. (Scale bar = 75 µm.)

Figure 10

Skin and muscle sections of muskellunge (Esox masquinongy) exposed at the medium dose (4 × 10³ plaque forming units mL⁻¹), 6 days post-infection. (a) Mild endothelila degeneration characterized by pyknotic nuclei (black arrows). (b) Positive riboprobe staining observed in endothelial cells and at the base of the scales (black arrow) (scale bar = 90 µm).

Figure 10

Skin and muscle sections of muskellunge (Esox masquinongy) exposed at the medium dose (4 × 10³ plaque forming units mL⁻¹), 6 days post-infection. (a) Mild endothelila degeneration characterized by pyknotic nuclei (black arrows). (b) Positive riboprobe staining observed in endothelial cells and at the base of the scales (black arrow) (scale bar = 90 µm).