AiM  Vol.6 No.6 , May 2016
Japanese Encephalitis Virus Replication and Inhibitory Effect of shRNA in Mice
Abstract: Japanese Encephalitis Virus (JEV) is responsible for over 30,000 annual cases of encephalitis worldwide, causing 30% mortality. JEV is thus a continuing threat to public health, so development of new antiviral drugs against JEV is desirable. Here, we examined JEV replication in mouse and used a short hairpin RNA JRi as the antiviral agent. The features of virus replication in neuron and survival rates of mice infected with JEV were different between virus strains. The mice infected with the virulent JEV strain (JaGAr01) were injected with pJRi (100 μg/mouse) which produced shRNAJRi. The survival rates of mice treated at 3 days before, the same day and 3 days after JEV infection were 22%, 78% and 44%, respectively. In addition, we demonstrated that the injection of pJRi induced interferon (IFN) production in cells and mice. These results suggest that the replication of JEV can be efficiently inhibited by RNAi and innate immunity including IFN. These data mean that pJRi has the inhibitory activity against JEV infection in vivo, and could be used as an antiviral drug to treat JEV infection.
Cite this paper: Murakami, M. , Tasaki, T. , Nukuzuma, S. , Minato, H. , Nojima, T. , Ishigaki, Y. and Takegami, T. (2016) Japanese Encephalitis Virus Replication and Inhibitory Effect of shRNA in Mice. Advances in Microbiology, 6, 462-470. doi: 10.4236/aim.2016.66045.

[1]   Sumiyoshi, H., Mor, I.C., Fuke, I., Morita, K., Kuhara, S., Kondou, J., Kikuchi, Y., Nagamatsu, H. and Igarashi, A. (1987) Complete Nucleotide Sequence of the Japanese Encephalitis Virus Genome RNA. Virology, 161, 497-510.

[2]   Hashimoto, H., Nomoto, A., Watanabe, K., Mori, T., Takezawa, T., Aizawa, C., Takegami, T. and Hiramatsu, K. (1988) Molecular Cloning and Complete Nucleotide Sequence of the Genome of Japanese Encephalitis Virus Beijing-1 Strain. Virus Genes, 1, 305-317.

[3]   Takegami, T. (2003) Japanese Encephalitis. Virus, 53, 25-30.

[4]   Takegami, T., Tasaki, T., Murakami, M., Ishigaki, Y., Taniguchi, M., Nojima, T. and Nukuzuma, S. (2015) Japanese Encephalitis Virus Infection and Replication: Biological Roles of Nonstructural Protein NS4a and the 3’-Untranslated Region in Persistent Infection. In: Japanese Encephalitis, SMG-eBooks.

[5]   Managada, M.N.M. and Takegami, T. (1999) Molecular Characterization of the Japanese Encephalitis Virus Representative Immunotype Strain JaGAr01. Virus Research, 59, 101-112.

[6]   Fauci, A.S. and Morens, D.M. (2016) Zika Virus in the Americas-Yet Another Arbovirus Threat. The New England Journal of Medicine, 374, 601-606.

[7]   WHO Report (2013) Japanese Encephalitis: Status of Surveillance and Immunization in Asia and the Western Pacific. Weekly Epidemiological Record, 88, 357-364.

[8]   WHO Report (2015) Japanese Encephalitis Vaccines: WHO Position Paper. Weekly Epidemiological Record, 90, 69-88.

[9]   Takegami, T., Ishak, H., Miyamoto, C., Shirai, Y. and Kamimura, K. (2000) Isolation and Molecular Comparison of Japanese Encephalitis Virus in Ishikawa, Japan. Japan Journal of Infectious Disease, 53, 178-179.

[10]   Martinez, J., Patkaniowska, A., Urlaub, H., Luhrmann, R. and Tuschl, T. (2002) Single-Stranded Antisense siRNAs Guide Target RNA Cleavage in RNAi. Cell, 110, 563-574.

[11]   Kapadia, S. B., Brideau-Andersen, A. and Chisari, F. V. (2003) Interference of Hepatitis C Virus RNA Replication by Short Interfering RNAs. Proceedings of the National Academy of Sciences of the United States of America, 100, 2014-2018.

[12]   Murakami, M., Ota, T., Nukuzuma, S. and Takegami, T. (2005) Inhibitory Effect of RNAi on Japanese Encephalitis Virus Replication in Vitro and in Vivo. Microbiology and Immunology, 49, 1047-1056.

[13]   Takegami, T., Shimamura, E., Hira, I.K. and Koyama, J. (1998) Inhibitory Effect of Furanonaphtoquinone Derivatives on the Replication of Japanese Encephalitis Virus. Antiviral Research, 37, 37-45.

[14]   Lewis, D.L., Hagstrom, J.E., Loomis, A.G., Wolff, J.A. and Herweijer, H. (2002) Efficient Delivery of siRNA for Inhibition of Gene Expression in Postnatal Mice. Nature Genetics, 32, 107-108.

[15]   Takegami, T., Miyamoto, H., Nakamura, H. and Yasui, K. (1982) Biological Activities of the Structural Protein of Japanese Encephalitis Virus. Acta Virology, 26, 312-320.

[16]   Chiou, S.S. and Chen, W.J. (2001) Mutations in the NS3 Gene and 3’-NCR of Japanese Encephalitis Virus Isolated from an Unconventional Ecosystem and Implications for Natural Attenuation of the Virus. Virology, 289, 129-136.

[17]   Yamaguchi, Y., Nukui, Y., Kotaki, A., Sawabe, K., Saijo, M., Watanabe, H., Kurane, I., Takasaki, T. and Tajima, S. (2013) Characterization of a Serine-to-Asparagine Substitution at Position 123 in the Japanese Encephalitis Virus E Protein. Journal of General Virology, 94, 90-96.

[18]   Kato, F., Kotaki, A., Yamaguchi, Y., Shiba, H., Hosono, K., Seiya Harada, S., Saijo, M., Kurane, I., Takasaki, T. and Tajima, S. (2012) Identification and Characterization of the Short Variable Region of the Japanese Encephalitis Virus 3’ NTR. Virus Genes, 44, 191-197.

[19]   Nga, P.T., Parquet, M.C., Cuong, V.D., Ma, S.P., Hasebe, F., Inoue, S., Makino, Y., Takagi, M. and Morita, K. (2004) Shift in Japanese Encephalitis Virus (JEV) Genotype Circulating in Northern Vietnam: Implications for Frequent Introductions of JEV from South East Asia to East Asia. Journal of General Virology, 85, 1625-1631.

[20]   Bai, F., Wang, T., Pal, U., Bao, F., Gould, L.H. and Fikrig, E. (2005) Use of RNA Interference to Prevent Lethal Murine West Nile Virus Infection. Journal of Infectious Diseases, 191, 1148-1154.

[21]   Kumar, P., Lee, S.K., Shankar, P. and Manjunath, N. (2006) A Single siRNA Suppresses Fatal Encephalitis Induced by Two Different Flaviviruses. PLoS Medicine, 3, e96.

[22]   Wu, Z., Xue, Y., Wang, B., Du, J. and Jin, Q. (2011) Broad-Spectrum Antiviral Activity of RNA Interference against Four Genotypes of Japanese Encephalitis Virus Based on Single MicroRNA Polycistrons. PLoS ONE, 6, e26304.

[23]   Anantpadma, M. and Vrati, S. (2012) SiRNA-Mediated Suppression of Japanese Encephalitis Virus Replication in Cultured Cells and Mice. Journal of Antimicrobial Chemotherapy, 67, 444-451.

[24]   Shen, T., Liu, K., Miao, D., Cao, R. and Chen, P. (2014) Effective Inhibition of Japanese Encephalitis Virus Replication by shRNAs Targeting Various Viral Genes in Vitro and in Vivo. Virology, 454-455, 48-59.

[25]   Stein, D.A., Perry, S.T., Buck, M.D., Oehmen, C.S., Fischer, M.A., Poore, E., Smith, J.L., Lancaster, A.M., Hirsch, A.J., Slifka, M.K., Nelson, J.A., Shresta, S. and Früh, K. (2011) Inhibition of Dengue Virus Infections in Cell Cultures and in AG129 Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence. Journal of Virology, 85, 10154-10166.

[26]   Achazi, K., Patel, P., Paliwal, R., Radonic, A., Niedrig, M. and Donoso-Mantke, O. (2012) RNA Interference Inhibits Replication of Tick-Borne Encephalitis Virus in Vitro. Antiviral Research, 93, 94-100.

[27]   Liang, J.J., Liao, C.L., Liao, J.H., Lee, Y.L. and Lin, Y.L. (2009) A Japanese Encephalitis Virus Vaccine Candidate Strain Is Attenuated by Decreasing Its Interferon Antagonistic Ability. Vaccine, 27, 2746-2754.

[28]   Ye, J., Zhua, B., Fu, Z.F., Chen, H. and Cao, S. (2013) Immune Evasion Strategies of Flaviviruses. Vaccine, 31, 461-471.

[29]   Jin, R., Zhu, W., Cao, S., Chen, R., Jin, H., Liu, Y., Wang, S., Wang, W. and Xiao, G. (2013) Japanese Encephalitis Virus Activates Autophagy as a Viral Immune Evasion Strategy. PLoS ONE, 8, e52909.

[30]   Fadnis, P.R., Ravi, V., Desai, A., Turtle, L. and Solomon, T. (2013) Innate Immune Mechanisms in Japanese Encephalitis Virus Infection: Effect on Transcription of Pattern Recognition Receptors in Mouse Neuronal Cells and Brain Tissue. Viral Immunology, 26, 366-377.

[31]   Aoki, K., Shimada, S., Simantini, D.S., Tun, M.M.N., Buerano, C.C., Morita, K. and Hayasaka, D. (2014) Type-I Interferon Response Affects an Inoculation Dose-Independent Mortality in Mice Following Japanese Encephalitis Virus Infection. Virology Journal, 11, 105-112.

[32]   Figueiredo, M.L.G. and Figueiredo, L.T.M. (2014) Review on Infections of the Central Nervous System by St. Louis Encephalitis, Rocio and West Nile Flaviviruses in Brazil, 2004-2014. Advances in Microbiology, 4, 955-961.

[33]   Do, L.P., Bui, T.M., Hasebe, F., Morita, K. and Phan, N.T. (2015) Molecular Epidemiology of Japanese Encephalitis in Northern Vietnam, 1964-2011: Genotype Replacement. Virology Journal, 12, 51-58.