Abstract
We have developed a defective-interfering (DI) RNA of mouse hepatitis virus (MHV) as a vector for expressing a variety of cellular and viral genes including the chloramphenicol acetyltransferase (CAT), hemagglutinin’esterase (HE), and gamma interferon. Here, we used the HE-expressing DI RNA for examining the role of HE protein in viral pathogenesis. The pseudorecombinant virus containing an expressed HE protein was generated by infecting cells with MHV-A59, which does not express HE, and transfecting the in vitro-transcribed DI RNA containing the HE gene. These pseudorecombinant viruses (DE-HE A59) were then inoculated intracerebrally into mice. Viruses recovered from cells infected with A59 and transfected with DI RNA expressing the CAT gene (DE-CAT A59) were used as a control. At various time points after inoculation, mice were observed for clinical symptoms. Tissues (brains and livers) were obtained for determining the replication of DI RNA by RT-PCR, virus replication by plaque assay, antigen expression by immunohistochemistry, and pathological changes. Results showed that all mice infected with DE-CAT A59 succumbed to infection by 9 days postinfection (d p.i). These data are identical to the pathogenesis of the parental A59 virus, demonstrating that inclusion of the DI RNA did not by itself alter pathogenesis. In contrast, only 40% of mice infected with DE-HE A59 succumbed to infection. The subgenomic mRNAs transcribed from the DI vector were detected at 1 and 2 d p.i. but not at subsequent time points, indicating that the genes in the DI vector were expressed only at an early stage of viral infection. No significant difference in virus replication in the brains was detected between these two groups of mice, suggesting that virus replication in brains was not affected by the expression of the HE. Histopathological examination showed only a small increase in the extent of inflammatory cell infiltration and reduced viral antigen in the mice infected with DE-HE A59. There was no difference in virus replication in the livers at 2 and 4 d p.i., but a 3 log10 reduction was detected in the livers of mice infected with DE-HE A59 at 6 d p.i. Histological examination showed a significant reduction in viral antigen, inflammation and necrosis in mice infected with DE-HE A59. These results indicate that the expression of HE from the DI vector altered the viral pathogenesis. This study thus demonstrates the usefulness of this system in studying the role of viral or cellular genes expressed locally at the sites of viral infection in viral pathogenesis.
Chapter PDF
Similar content being viewed by others
Keywords
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
References
Deregt, D., and Babiuk, L. A., 1987, Monoclonal antibodies to bovine Coronavirus: Characteristics and topographical mapping of neutralizing epitopes on the E2 and E3 glycoproteins, Virology 161: 410–420.
Deregt, D., Gifford, G.A., Khalid Ijaz, M., Watts, T.C., Gilchrist, J.E., Haines, D.M., and Babiuk, L.A., 1989, Monoclonal antibodies to bovine Coronavirus glycoproteins E2 and E3: demonstation of in vivo neutralizing activity, J. Gen. Virol. 70:993–998.
Fleming, J.O., Stohlman, S.A., Harmon, R.C., Lai, M.M.C., Frelinger, J.A., and Weiner, L.P., 1983, Antigenic relationship of murine coronaviruses: analysis using monoclonal antibodies to JHM (MHV-4) virus, Virology 131:296–307.
Gagnetan, S., Gout, O., Dubois-Dalcq, M., Rottier, P., Rossen, J., and Holmes, K.V., 1995, Interaction of mouse hepatitis virus (MHV) spike glycoprotein with receptor glycoprotein MHVR is required for infection with an MHV strain that expresses the hemagglutinin-esterase glycoprotein, J. Virol. 69: 889–895.
Herrler, G., Rott, R., Klenk, H.D., Müller, H.P., Shukda, A.K., and Schauer, R., 1985, The receptor-destroying enzyme of influenza C is virus neuraminidate-O-acetyl-esterase, EMBO J. 4:1503–1506.
Herrler, G., Rott, R., and Klenk, H.D., 1985, Neuraminic acid is involved in the binding of influenza C virus to erythrocytes, Virology 141: 144–147.
Hirano, N., Fujiwara, K., Hino, S., and Matsumoto, M., 1974, Replication and plaque formation of mouse hepatitis virus (MHV-2) in mouse cell line DBT culture, Arch. Gesamte Virusforsch. 44, 298–302.
Lai, M.M.C., 1992, RNA recombination in animal and plant viruses, Microbiol. Rev. 56: 61–79.
Lavi, E., Gilden, D.H., Highkin, M.K., Weiss, S.R., 1986, The organ tropism of mouse hepatitis virus A59 in mice is dependent on dose and route of inoculation, Lab. Ani. Sci. 36:130–135.
Lee, H.-J., Shieh, C.-K., Gorbalenya, A. E., Koonin, E. V., La Monica, N., Tuler, J., Bagdzyahdzhyan, A., and Lai, M. M. C, 1991, The complete sequence (22 kilobases) of murine Coronavirus gene 1 encoding the putative proteases and RNA polymerase, Virology 180: 567–582.
Liao, C.-L., and Lai, M. M. C, 1994, Requirement of the 5’-end genomic sequence as an upstream cis-acting element for Coronavirus subgenomic mRNA transcription, J. Virol. 68: 4727–4737.
Liao, C.-L., Zhang, X. M., and Lai, M. M. C, 1995, Coronavirus defective-interfering RNA as an expression vector: the generation of a pseudorecombinant mouse hepatitis virus expressing hemagglutinin-esterase, Virology 208: 319–327.
Lin, Y.-J., and Lai, M. M. C, 1993, Deletion mapping of a mouse hepatitis virus defective interfering RNA reveals the requirement of an internal and discontiguous sequence for replication, J. Virol. 67: 6110–6118.
Makino, S., Shieh, C.-K., Soe, L. H., Baker, S. C, and Lai, M. M. C, 1988, Primary structure and translation of a defective-interfering RNA of murine Coronavirus, Virology 166: 550–560.
Makino, S., Joo, M., and Makino, J. K., 1991, A system for study of Coronavirus mRNA synthesis: A regulated, expressed subgenomic defective-interfering RNA results from intergenic site insertion, J. Virol. 65: 6031–6041.
Yokomori, K., La Monica, N., Makino, S., Shieh, C.-K., and Lai, M.M.C., 1989, Biosynthesis, structure, and biological activities of envelope protein gp65 of murine Coronavirus, Virology 173: 683–691.
Yokomori, K., Baker, S.C., Stohlman, S.A., and Lai, M.M.C., 1992, Hemagglutinin-esterase (HE)-specific monoclonal antibodies alter the neuropathogenicity of mouse hepatitis virus, J. Virol. 66:2865–2874.
Yokomori, K., Asanaka, M., Stohlman, S.A., Makino, S., Shubin, R.A., Gilmore, W., Weiner, L.R, Wang, F.I., and Lai, M.M.C., 1995, Neuropathogenicity of mouse hepatitis virus JHM isolates differing in hemagglutinin-esterase protein expression, J. Neurovirol. 1:330–339.
Zhang, X. M., Liao, C.-L., and Lai, M. M. C, 1994, Coronavirus leader RNA regulates and initiates subgenomic mRNA transcription both in trans and in cis, J. Virol. 68: 4738–4746.
Zhang, X.M., Hinton, D.R., Cue, D., Stohlman, S., and Lai, M.M.C., 1997: Expression of gamma interferon by a Coronavirus defective-interfering RNA vector and its effect on viral replication, spread and pathogenicity, Virology. In press.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1998 Springer Science+Business Media New York
About this chapter
Cite this chapter
Zhang, X., Hinton, D., Park, S., Liao, CL., Lai, M.M.C., Stohlman, S. (1998). Using a Defective-Interfering RNA System to Express the HE Protein of Mouse Hepatitis Virus for Studying Viral Pathogenesis. In: Enjuanes, L., Siddell, S.G., Spaan, W. (eds) Coronaviruses and Arteriviruses. Advances in Experimental Medicine and Biology, vol 440. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-5331-1_67
Download citation
DOI: https://doi.org/10.1007/978-1-4615-5331-1_67
Publisher Name: Springer, Boston, MA
Print ISBN: 978-1-4613-7432-9
Online ISBN: 978-1-4615-5331-1
eBook Packages: Springer Book Archive