Main Article Content


V.V. Keyer

National Center for Biotechnology, 13/5, Korgalzhyn road, Astana, 010000, Kazakhstan

A.B. Shevtsov

National Center for Biotechnology, 13/5, Korgalzhyn road, Astana, 010000, Kazakhstan

A.S. Kassymkhanova

National Center for Biotechnology, 13/5, Korgalzhyn road, Astana, 010000, Kazakhstan

A.Zh. Baltabekova

National Center for Biotechnology, 13/5, Korgalzhyn road, Astana, 010000, Kazakhstan

A.V. Shustov

National Center for Biotechnology, 13/5, Korgalzhyn road, Astana, 010000, Kazakhstan


Yellow fever virus (YFV) is a model representative in the genus Flavivirus, which along with the YFV includes other human pathogens such as the tick-borne encephalitis virus, Zika virus, etc. The flaviviruses are prevalent worldwide and pose a risk to the population of Kazakhstan. Although the YFV itself is not present in Kazakhstan, a vaccine strain of the YFV may serve as a convenient model to study aspects of molecular virology of the flaviviruses. The vaccine strain 17D of the YFV was used to construct cDNA copy of a full-length viral genome. The cDNA was cloned in an E.coli plasmid. The 5’-end of the genome is placed under control of SP6 RNA polymerase promoter. This allows producing the virus’ genomic RNA using in vitro transcription. The 3’-end of the genome is engineered to be fused with antigenomic ribozyme (RBZ) of the hepatitis D virus. The RBZ cleaves itself off upon transcription to yield the correct 3’-end of the YFV genome. Also, a gene encoding green fluorescent protein (GFP) was inserted into the YFV genome in a position preceding structural proteins. An activity of foot-and-mouth disease autoprotease 2A was utilized to cleave off the GFP from viral polyprotein to ensure correct processing of the YFV proteins. Precautions were undertaken to preserve the 5’-cyclization signal which important for replication.

The obtained molecular clone proved infectious. The virus (1612.YFV/GFP) was rescued from the plasmid by using in vitro transcription and RNA transfection. Thus an experimental system to produce recombinant flaviviruses was generated which may be advanced to produce live vaccines based on the YFV vector.


Yellow fever virus (YFV), Molecular clone, Flavivirus, Heterologous insert, Green fluorescent protein, HDV antigenomic ribozyme

Article Details


Yellow fever. Available at: URL.

Eliminate Yellow fever Epidemics (EYE): a global strategy, 2017-2026. Available at: URL.

Yellow fever outbreak Angola, Democratic Republic of the Congo and Uganda 2016-2017. Available at: URL.

Hay J., Yeh K.B., Dasgupta D., Shapieva Z., Omasheva G., Deryabin P., Nurmakhanov T., Ayazbayev T., Andryushchenko A., Zhunushov A., Hewson R., Farris C.M., Richards A.L. Biosurveillance in Central Asia: Successes and Challenges of Tick-Borne Disease Research in Kazakhstan and Kyrgyzstan. Front. Public Health., 2016, no. 4, pp. 35-40.

Grard G., Moureau G., Charrel R.N., Lemasson J.J., Gonzalez J.P., Gallian P., Gritsun T.S., Holmes E.C., Gould E.A., de Lamballerie X. Genetic characterization of tick-borne flaviviruses: new insights into evolution, pathogenetic determinants and taxonomy. Virology, 2007, no. 25, pp. 80-92.

Lim S.K., Lim J.K., Yoon I.K. An Update on Zika Virus in Asia. Infect. Chemother, 2017, no. 49, pp. 91-100.

Matthias Kalitzky, Peter Borowski. Molecular Biology of the Flavivirus. Horizon Scientific Press, 2006, 380 p.

Rastogi M., Sharma N., Singh S.K. Flavivirus NS1: a multifaceted enigmatic viral protein. Virol. J., 2016, no. 13, pp. 131. Crossref.

Tajima S., Takasaki T., Kurane I. Characterization of Asn130-to-Ala mutant of dengue type 1 virus NS1 protein. Virus Genes, 2008, no. 36, pp. 323-329.

Fan J., Liu Y., Yuan Z. Critical role of Dengue Virus NS1 protein in viral replication. Virol. Sin., 2014, no. 29, pp. 162-169.

Edeling M.A., Diamond M.S., Fremont D.H. Structural basis of Flavivirus NS1 assembly and antibody recognition. Proc. Natl. Acad. Sci. USA, 2014, no. 111, pp. 4285-4290.

Youn S., Li T., McCune B.T., Edeling M.A., Fremont D.H., Cristea I.M., Diamond M.S. Evidence for a genetic and physical interaction between nonstructural proteins NS1 and NS4B that modulates replication of West Nile virus. J. Virol., 2012, no. 8, pp. 7360-7371.

Youn S., Ambrose R.L., Mackenzie J.M., Diamond M.S. Non-structural protein-1 is required for West Nile virus replication complex formation and viral RNA synthesis. Virology Journal, no. 10, pp. 339. Crossref.

Chatel-Chaix L., Fischl W., Scaturro P., Cortese M., Kallis S., Bartenschlager M., Fischer B., Bartenschlager R. A combined genetic-proteomic approach identifies residues within Dengue virus NS4B critical for interaction with NS3 and viral replication. J. Virol., 2015, no. 89, pp. 7170-7186.

Scaturro P., Cortese M., Chatel-Chaix L., Fischl W., Bartenschlager R. Dengue Virus Non-structural Protein 1 Modulates Infectious Particle Production via Interaction with the Structural Proteins. PLoS Pathog., 2015, no. 11. 26562291.

Wu R.H., Tsai M.H., Tsai K.N., Tian J.N., Wu J.S., Wu S.Y., Chern J.H., Chen C.H., Yueh A. Mutagenesis of Dengue Virus Protein NS2A Revealed a Novel Domain Responsible for Virus-Induced Cytopathic Effect and Interactions between NS2A and NS2B Transmembrane Segments. J. Virol., 2017, no. 91. 28381578.

Leung J.Y., Pijlman G.P., Kondratieva N., Hyde J., Mackenzie J.M., Khromykh A.A. Role of nonstructural protein NS2A in flavivirus assembly. J. Virol., 2008, no. 82, pp. 4731-4741.

Xie X., Gayen S., Kang C., Yuan Z., Shi P-Y. Membrane Topology and Function of Dengue Virus NS2A Protein. J Virol., 2013, no. 87, pp. 4609-4622.

Kümmerer B., Rice C.M. Mutations in the yellow fever virus nonstructural protein NS2A selectively block production of infectious virus. J. Virol., 2002, no. 76, pp. 4773-4784.

Li K., Phoo W.W., Luo D. Functional interplay among the flavivirus NS3 protease, helicase, and cofactors. Virol. Sin., 2014, no. 29, pp. 74-85.

Luo D., Vasudevan S.G., Lescar J. The flavivirus NS2B-NS3 protease-helicase as a target for antiviral drug development. Antiviral. Res., 2015, no. 118, pp. 148-158.

Junaid M., Angsuthanasombat C., Wikberg J.E., Ali N., Katzenmeier G. Modulation of enzymatic activity of dengue virus nonstructural protein NS3 nucleoside triphosphatase/helicase by poly(U). Biochemistry (Mosc), 2013, no. 78, pp. 925-932.

Zou J., Xie X., Wang Q.Y., Dong H., Lee M.Y., Kang C., Yuan Z., Shi P.Y. Characterization of dengue virus NS4A and NS4B protein interaction. J. Virol., 2015, no. 89, pp. 3455-3470.

Apte-Sengupta S., Sirohi D., Kuhn R.J. Coupling of replication and assembly in flaviviruses. Curr. Opin. Virol., 2014, no. 9, pp. 134-142.

Zmurko J., Neyts J., Dallmeier K. Flaviviral NS4b, chameleon and jack-in-the-box roles in viral replication and pathogenesis, and a molecular target for antiviral intervention. Rev. Med. Virol., 2015, no. 25, pp. 205-223.

Klema V.J., Ye M., Hindupur A., Teramoto T., Gottipati K., Padmanabhan R., Choi K.H. Dengue Virus Nonstructural Protein 5 (NS5) Assembles into a Dimer with a Unique Methyltransferase and Polymerase Interface. PLoS Pathog., 2016, no. 12. 26895240.

Surana P., Satchidanandam V., Nair DT. RNA-dependent RNA polymerase of Japanese encephalitis virus binds the initiator nucleotide GTP to form a mechanistically important pre-initiation state. Nucleic Acids Res., 2014, no. 42, pp. 2758-2773.

Zhao Y., Soh T.S., Zheng J., Chan K.W., Phoo W.W., Lee C.C., Tay M.Y., Swaminathan K., Cornvik T.C., Lim S.P., Shi P.Y., Lescar J., Vasudevan S.G., Luo D. A crystal structure of the Dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication. PLoS Pathog., 2015, no. 11. 25775415.

Teramoto T., Balasubramanian A., Choi K.H., Padmanabhan R. Serotype-specific interactions among functional domains of dengue virus 2 nonstructural proteins (NS) 5 and NS3 are crucial for viral RNA replication. J. Biol. Chem., 2017, no. 292, pp. 9465-9479.

Le Breton M., Meyniel-Schicklin L., Deloire A., Coutard B., Canard B., de Lamballerie X., Andre P., Rabourdin-Combe C., Lotteau V., Davoust N. Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen. BMC Microbiol, 2011, no. 11, pp. 234. Crossref.

Azevedo A.S., Gonçalves A.J., Archer M., Freire M.S., Galler R., Alves A.M. The synergistic effect of combined immunization with a DNA vaccine and chimeric yellow fever/dengue virus leads to strong protection against dengue. PLoS One., 2013, no. 8. 26678487.