Murine coronavirus-induced hepatitis: JHM genetic background eliminates A59 spike-determined hepatotropism

Abstract

Recombinant murine coronaviruses, differing only in the spike gene and containing the strain A59 (moderately hepatotropic) and JHM (neurotropic) spike genes in the background of the JHM genome, were compared for the ability to replicate in the liver and induce hepatitis in weanling C57BL/6 mice. Interestingly, expression of the A59 spike glycoprotein within the background of the neurotropic JHM strain does not reproduce the A59 hepatotropic phenotype. Thus, the JHM genetic background plays a dominant role over the spike in the determination of hepatotropism.

FIG. 1.

Time course of released (A) and cell-associated (B) virus production in L2 cell cultures. L2 cells were infected in duplicate with RA59 (•), SJHM-RA59 (○), RJHM (♦), or SA59-RJHM (▴, ▵) at a multiplicity of infection of 1 PFU/cell. The data shown represent the mean titers of duplicate samples. In the case of SA59-RJHM, two independent recombinant viruses were analyzed. At the indicated times, virus titers were determined in cells and culture supernatants by plaque assay in L2 cells.

FIG. 2.

Viral loads in livers of C57BL/6 mice after intrahepatic inoculation with 500 (A) or 10⁵ to 10⁶ (B) PFU of recombinant virus RA59, SJHM-RA59, RJHM, or SA59-RJHM, at 5 day p.i. Viral titers were determined by plaque assay and are presented as the log₁₀ PFU per gram of liver. Each point represents viral titer of individual mice, and bars represent the log₁₀ of the mean in each group. The limit of detection was 200 PFU/g of liver. The numbers of mice examined per viral dose were as follows: RA59, SJHM-RA59, and RJHM, 5; SA59-RJHM, 10. RJHM and SA59-RJHM did not induce a productive infection in the liver, irrespective of the viral dose (P < 0.01 [Wilcoxon rank sum test]). RA59 replicated efficiently irrespective of the viral dose, whereas SJHM-RA59 replicated to a minimal extent after inoculation with 500 PFU (RA59 versus SJHM-RA59, P < 0.01 [Wilcoxon rank sum test]). SJHM-RA59 was able to replicate to an extent similar to that of RA59 only after inoculation of large viral doses.

FIG. 3.

Extent of hepatitis (scored as minimal [1], mild [2], moderate [3], or severe [4], as described in the text) and titers (log₁₀ PFU per gram) of recombinant viruses in livers of C57BL/6 mice after intrahepatic inoculation with 500 (A) or 10⁵ to 10⁶ (B, C, and D) PFU of RA59, SJHM-RA59, SA59-RJHM, or RJHM virus. The results are shown as percentages of mice with minimal, mild, moderate, and severe hepatitis. The numbers of mice examined per viral dose were as follows: RA59, SJHM-RA59, and RJHM, 5; SA59-RJHM, 10. (A) After inoculation with 500 PFU, RA59 induced moderate hepatitis whereas SJHM-RA59, like RJHM and SA59RJHM, caused minimal hepatocellular damage (RA59 versus SJHM-RA59, P < 0.01; RA59 versus RJHM or SA59-RJHM, P < 0.001 [Wilcoxon rank sum test]). (B) After inoculation with 10⁵ to 10⁶ PFU, RJHM and SA59-RJHM induced mild hepatitis whereas SJHM-RA59 caused the same extent of hepatitis as RA59 (JHM background versus A59 background, P < 0.001 [Wilcoxon rank sum test]). (C and D) Correlation of the viral load with the degree of hepatitis induced by each virus and the number of necrotic foci.

FIG. 4.

Histopathologic changes in the livers of mice infected intrahepatically with 10⁵ PFU of RA59 (A) or SJHM-RA59 (C), 10⁶ PFU of RJHM (B) or SA59-RJHM (D), or 500 PFU of SJHM-RA59 (E), compared with a mock-infected control (F), at day 5 p.i. (hematoxylin-and-eosin staining). (A) RA59 showed noticeable necrosis and inflammation, whereas RJHM (B) and SA59-RJHM (D) induced occasional foci of necrosis. SJHM-RA59 caused noticeable hepatocellular damage and inflammation after inoculation with 10⁵ PFU (C). In contrast, after inoculation with 500 PFU (E), SJHM-RA59 caused minimal changes in the liver. Insets (A to E) show inflammatory infiltrates, necrotic hepatocytes, and some apoptotic bodies. No signs of pathology were found in a mock-infected control (F). Magnifications: A to F, ×100; insets, ×400.

FIG. 5.

Immunohistochemical analysis of liver sections of C57BL/6 mice infected with recombinant virus RA59 at 10⁵ PFU (A), RJHM at 10⁶ PFU (B), SJHM-RA59 at 10⁵ PFU (C), SA59-RJHM at 10⁶ PFU (D), or SJHM-RA59 at 500 PFU (E), compared with a mock-infected control (F), at day 5 p.i. MHV was detected by immunolabeling with a MAb against the nucleocapsid protein of MHV as described in the text. Viral antigen always colocalized with necrotic areas. In contrast to RA59 (A), SJHM-RA59 (C and E) exhibited limited viral spreading. RJHM (B) and SA59-RJHM (D) showed isolated viral staining. No signs of viral antigen were found in the mock-infected control (F). Magnification, ×40.