Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1992 Nov 1;89(21):10532–10536. doi: 10.1073/pnas.89.21.10532

Construction and characterization of chimeric tick-borne encephalitis/dengue type 4 viruses.

A G Pletnev 1, M Bray 1, J Huggins 1, C J Lai 1
PMCID: PMC50373  PMID: 1438242

Abstract

Dengue type 4 virus (DEN4) cDNA was used as a vector to express genes of the distantly related tick-borne encephalitis virus (TBEV). Full-length chimeric TBEV/DEN4 cDNAs were constructed by substituting TBEV genes coding for proteins such as capsid (C); pre-membrane, which is the precursor of membrane (M); envelope (E); or nonstructural protein NS1 for the corresponding DEN4 sequences. RNA transcripts prepared from cDNAs were used to transfect permissive simian cells. Two viable chimeric viruses that contained TBEV CME or ME genes were recovered. Compared with DEN4, chimeric TBE(ME)/DEN4 virus [designated vTBE(ME)/DEN4] produced larger plaques and grew to higher titer in simian cells. In contrast, vTBE(ME)/DEN4 produced smaller plaques on mosquito cells and grew to lower titer than DEN4. Analysis of viral RNA and proteins produced in vTBE(ME)/DEN4- and DEN4-infected mosquito or simian cells revealed that the chimera was restricted in its ability to enter and replicate in mosquito cells. In contrast, vTBE(ME)/DEN4 entered simian cells efficiently and its RNA was replicated more rapidly in these cells than was parental DEN4 RNA. Following intracerebral inoculation, vTBE(ME)/DEN4 caused fatal encephalitis in both suckling and adult mice, while nearly all mice inoculated by the same route with DEN4 did not develop disease. Unlike wild-type TBEV, vTBE(ME)/DEN4 did not cause encephalitis when adult mice were inoculated by a peripheral route. Adult mice previously inoculated with the chimera by a peripheral route were completely resistant to subsequent intraperitoneal challenge with 10(3) times the median lethal dose of TBEV, whereas mice previously inoculated with DEN4 were not protected. These findings indicate that (i) the TBEV M and E genes of the chimeric virus are major protective antigens and induce resistance to lethal TBEV challenge and (ii) other regions of the TBEV genome are essential for the ability of this virus to spread from a peripheral site to the brain. Success in constructing a viable TBEV/DEN4 chimera that retains the protective antigens of TBEV but lacks its peripheral invasiveness provides a strategy for the development of live attenuated TBEV vaccines.

Full text

PDF
10532

Images in this article

10534Image
on p.10534
10535Image
on p.10535
10535Image
on p.10535

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bray M., Lai C. J. Construction of intertypic chimeric dengue viruses by substitution of structural protein genes. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10342–10346. doi: 10.1073/pnas.88.22.10342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bray M., Zhao B. T., Markoff L., Eckels K. H., Chanock R. M., Lai C. J. Mice immunized with recombinant vaccinia virus expressing dengue 4 virus structural proteins with or without nonstructural protein NS1 are protected against fatal dengue virus encephalitis. J Virol. 1989 Jun;63(6):2853–2856. doi: 10.1128/jvi.63.6.2853-2856.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cahour A., Falgout B., Lai C. J. Cleavage of the dengue virus polyprotein at the NS3/NS4A and NS4B/NS5 junctions is mediated by viral protease NS2B-NS3, whereas NS4A/NS4B may be processed by a cellular protease. J Virol. 1992 Mar;66(3):1535–1542. doi: 10.1128/jvi.66.3.1535-1542.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Calisher C. H., Karabatsos N., Dalrymple J. M., Shope R. E., Porterfield J. S., Westaway E. G., Brandt W. E. Antigenic relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera. J Gen Virol. 1989 Jan;70(Pt 1):37–43. doi: 10.1099/0022-1317-70-1-37. [DOI] [PubMed] [Google Scholar]
  5. Chambers T. J., Hahn C. S., Galler R., Rice C. M. Flavivirus genome organization, expression, and replication. Annu Rev Microbiol. 1990;44:649–688. doi: 10.1146/annurev.mi.44.100190.003245. [DOI] [PubMed] [Google Scholar]
  6. Chu P. W., Westaway E. G. Characterization of Kunjin virus RNA-dependent RNA polymerase: reinitiation of synthesis in vitro. Virology. 1987 Apr;157(2):330–337. doi: 10.1016/0042-6822(87)90275-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Deubel V., Kinney R. M., Esposito J. J., Cropp C. B., Vorndam A. V., Monath T. P., Trent D. W. Dengue 2 virus envelope protein expressed by a recombinant vaccinia virus fails to protect monkeys against dengue. J Gen Virol. 1988 Aug;69(Pt 8):1921–1929. doi: 10.1099/0022-1317-69-8-1921. [DOI] [PubMed] [Google Scholar]
  8. Falgout B., Bray M., Schlesinger J. J., Lai C. J. Immunization of mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein NS1 protects against lethal dengue virus encephalitis. J Virol. 1990 Sep;64(9):4356–4363. doi: 10.1128/jvi.64.9.4356-4363.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Falgout B., Chanock R., Lai C. J. Proper processing of dengue virus nonstructural glycoprotein NS1 requires the N-terminal hydrophobic signal sequence and the downstream nonstructural protein NS2a. J Virol. 1989 May;63(5):1852–1860. doi: 10.1128/jvi.63.5.1852-1860.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Falgout B., Pethel M., Zhang Y. M., Lai C. J. Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. J Virol. 1991 May;65(5):2467–2475. doi: 10.1128/jvi.65.5.2467-2475.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grun J. B., Brinton M. A. Dissociation of NS5 from cell fractions containing West Nile virus-specific polymerase activity. J Virol. 1987 Nov;61(11):3641–3644. doi: 10.1128/jvi.61.11.3641-3644.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hori H., Lai C. J. Cleavage of dengue virus NS1-NS2A requires an octapeptide sequence at the C terminus of NS1. J Virol. 1990 Sep;64(9):4573–4577. doi: 10.1128/jvi.64.9.4573-4577.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Lai C. J., Zhao B. T., Hori H., Bray M. Infectious RNA transcribed from stably cloned full-length cDNA of dengue type 4 virus. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5139–5143. doi: 10.1073/pnas.88.12.5139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mackow E., Makino Y., Zhao B. T., Zhang Y. M., Markoff L., Buckler-White A., Guiler M., Chanock R., Lai C. J. The nucleotide sequence of dengue type 4 virus: analysis of genes coding for nonstructural proteins. Virology. 1987 Aug;159(2):217–228. doi: 10.1016/0042-6822(87)90458-2. [DOI] [PubMed] [Google Scholar]
  16. Mandl C. W., Kunz C., Heinz F. X. Presence of poly(A) in a flavivirus: significant differences between the 3' noncoding regions of the genomic RNAs of tick-borne encephalitis virus strains. J Virol. 1991 Aug;65(8):4070–4077. doi: 10.1128/jvi.65.8.4070-4077.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mason P. W., Pincus S., Fournier M. J., Mason T. L., Shope R. E., Paoletti E. Japanese encephalitis virus-vaccinia recombinants produce particulate forms of the structural membrane proteins and induce high levels of protection against lethal JEV infection. Virology. 1991 Jan;180(1):294–305. doi: 10.1016/0042-6822(91)90034-9. [DOI] [PubMed] [Google Scholar]
  18. Men R. H., Bray M., Lai C. J. Carboxy-terminally truncated dengue virus envelope glycoproteins expressed on the cell surface and secreted extracellularly exhibit increased immunogenicity in mice. J Virol. 1991 Mar;65(3):1400–1407. doi: 10.1128/jvi.65.3.1400-1407.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Morozova O. V., Belyavskaya N. A., Zaychikov E. F., Kvetkova E. A., Mustaev A. A., Pletnev A. G. Identification of RNA replicase subunits responsible for initiation of RNA synthesis of tick-borne encephalitis virus by affinity labelling. Biomed Sci. 1991;2(2):183–186. [PubMed] [Google Scholar]
  20. Morozova O. V., Mustaev A. A., Belyavskaya N. A., Zaychikov E. F., Kvetkova E. A., Wolf YuI, Pletnev A. G. Mapping of the region of the tick-borne encephalitis virus replicase adjacent to initiating substrate binding center. FEBS Lett. 1990 Dec 17;277(1-2):75–77. doi: 10.1016/0014-5793(90)80812-w. [DOI] [PubMed] [Google Scholar]
  21. Pletnev A. G., Yamshchikov V. F., Blinov V. M. Nucleotide sequence of the genome and complete amino acid sequence of the polyprotein of tick-borne encephalitis virus. Virology. 1990 Jan;174(1):250–263. doi: 10.1016/0042-6822(90)90073-z. [DOI] [PubMed] [Google Scholar]
  22. Preugschat F., Yao C. W., Strauss J. H. In vitro processing of dengue virus type 2 nonstructural proteins NS2A, NS2B, and NS3. J Virol. 1990 Sep;64(9):4364–4374. doi: 10.1128/jvi.64.9.4364-4374.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rice C. M., Grakoui A., Galler R., Chambers T. J. Transcription of infectious yellow fever RNA from full-length cDNA templates produced by in vitro ligation. New Biol. 1989 Dec;1(3):285–296. [PubMed] [Google Scholar]
  24. Ruiz-Linares A., Cahour A., Després P., Girard M., Bouloy M. Processing of yellow fever virus polyprotein: role of cellular proteases in maturation of the structural proteins. J Virol. 1989 Oct;63(10):4199–4209. doi: 10.1128/jvi.63.10.4199-4209.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Svitkin Y. V., Lyapustin V. N., Lashkevich V. A., Agol V. I. Differences between translation products of tick-borne encephalitis virus RNA in cell-free systems from Krebs-2 cells and rabbit reticulocytes: involvement of membranes in the processing of nascent precursors of flavivirus structural proteins. Virology. 1984 Jun;135(2):536–541. doi: 10.1016/0042-6822(84)90207-1. [DOI] [PubMed] [Google Scholar]
  26. Yasuda A., Kimura-Kuroda J., Ogimoto M., Miyamoto M., Sata T., Sato T., Takamura C., Kurata T., Kojima A., Yasui K. Induction of protective immunity in animals vaccinated with recombinant vaccinia viruses that express PreM and E glycoproteins of Japanese encephalitis virus. J Virol. 1990 Jun;64(6):2788–2795. doi: 10.1128/jvi.64.6.2788-2795.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Zhang Y. M., Hayes E. P., McCarty T. C., Dubois D. R., Summers P. L., Eckels K. H., Chanock R. M., Lai C. J. Immunization of mice with dengue structural proteins and nonstructural protein NS1 expressed by baculovirus recombinant induces resistance to dengue virus encephalitis. J Virol. 1988 Aug;62(8):3027–3031. doi: 10.1128/jvi.62.8.3027-3031.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Zhao B., Mackow E., Buckler-White A., Markoff L., Chanock R. M., Lai C. J., Makino Y. Cloning full-length dengue type 4 viral DNA sequences: analysis of genes coding for structural proteins. Virology. 1986 Nov;155(1):77–88. doi: 10.1016/0042-6822(86)90169-8. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES