Citation: Zhao-rui Zhou, Man-li Wang, Fei Deng, Tian-xian Li, Zhi-hong Hu, Hua-lin Wang. Production of CCHF Virus-Like Particle by a Baculovirus-Insect Cell Expression System .VIROLOGICA SINICA, 2011, 26(5) : 338-346.  http://dx.doi.org/10.1007/s12250-011-3209-6

Production of CCHF Virus-Like Particle by a Baculovirus-Insect Cell Expression System

  • Corresponding author: Man-li Wang, Wangml@wh.iov.cn
  • Received Date: 29 June 2011
    Accepted Date: 31 August 2011
    Available online: 01 October 2011

    Fund Project: The Knowledge Innovation Program of the Chinese Academy of Sciences, Grant KSCX2-YW-N-065National Basic Research Priorities Program of China, Grant 2007National Key Basic Research Program (973 Program), Grant 2010CB530103the Knowledge Innovation Program of the Chinese Academy of Sciences, Grant KSCX2-EWG-8

  • Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is a tick-born virus of the Nairovirus genus within the Bunyaviridae family, which is widespread and causes high fatality. The nucleocapsid of CCHFV is comprised of N proteins that are encoded by the S segment. In this research, the N protein of CCHFV was expressed in insect cells using a recombinant baculovirus. Under an electron microscope, Virus-Like Particles (VLPs) with various size and morphology were observed in cytoplasmic vesicles in the infected cells. Sucrose-gradient purification of the cell lysate indicated that the VLPs were mainly located in the upper fraction after ultracentrifugation, which was confirmed by Western blot analysis and immuno-electron microscopy (IEM).

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    1. Barr J N, Wertz G W. 2004. Bunyamwera bunyavirus RNA synthesis requires cooperation of 3'-and 5'-terminal sequences. J Virol, 78: 1129-1138.
        doi: 10.1128/JVI.78.3.1129-1138.2004

    2. Chai J J, Xiao C E, Liu Y H, et al. 2004. Report of Investigation on Hemorrhagic Fever in Bachu County, Xinjiang in 1966 Ⅲ. Investigation an Epidemiology of Hemorrhagic Fever in Bachu. Endemic Disease Bulletin (in chinese), 19S: 15-22.

    3. Chinikar S, Ghiasi S M, Hewson R, et al. 2010. Crimean-Congo hemorrhagic fever in Iran and neighboring countries. J Clin Virol, 47: 110-114.
        doi: 10.1016/j.jcv.2009.10.014

    4. Deng F, Wang R R, Fang M G, et al. 2007. Proteomics analysis of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus identified two new occlusion-derived virus-associated proteins, HA44 and HA100. J Virol, 81: 9377-9385.
        doi: 10.1128/JVI.00632-07

    5. Deyde V M, Khristova M L, Rollin P E, et al. 2006. Crimean-Congo hemorrhagic fever virus genomics and global diversity. J Virol, 80: 8834-8842.
        doi: 10.1128/JVI.00752-06

    6. Elevli M, Ozkul A A, Civilibal M, et al. 2010. A newly identified Crimean-Congo hemorrhagic fever virus strain in Turkey. Int J Infect Dis, 14 Suppl (3): e213-216.

    7. Elliott R M. 1990. Molecular biology of the Bunyaviridae. J Gen Virol, 71: 501-522.
        doi: 10.1099/0022-1317-71-3-501

    8. Ergonul O. 2006. Crimean-Congo haemorrhagic fever. Lancet Infect Dis, 6: 203-214.
        doi: 10.1016/S1473-3099(06)70435-2

    9. Ergonul O. 2008. Treatment of Crimean-Congo hemorrhagic fever. Antiviral Res, 78: 125-131.
        doi: 10.1016/j.antiviral.2007.11.002

    10. Flick R. 2007. Chapter 4: Molecular Biology of the Crimean-Congo Hemorrhagic Fever Virus. In: Crimean-Congo Hemorrhagic Fever (O Ergonul, C A Whitehouse ed. ). Springer Netherlands, p 35-44.

    11. Frank S A. 2002. Immunology and Evolution of Infectious Disease. Princeton (NJ): Pricenton University Press, p94-108.

    12. Fu T M, Guan L, Friedman A, et al. 1999. Dose dependence of CTL precursor frequency induced by a DNA vaccine and correlation with protective immunity against influenza virus challenge. J Immunol, 162: 4163-4170.

    13. Ghodake R S, Babar R S, Patil S B, et al. 2011. CCHFV-an emerging infectious disease. Pharma science monitor: An international journal of pharmaceutical sciences, 1067-1087.

    14. Hewson R, Chamberlain J, Mioulet V, et al. 2004. Crimean-Congo haemorrhagic fever virus: sequence analysis of the small RNA segments from a collection of viruses world wide. Virus Res, 102: 185-189.
        doi: 10.1016/j.virusres.2003.12.035

    15. King L A, Possee R D. 1992. The baculovirus expression system: A laboratory guide. New York, NY (USA): Chapman & Hall. p158-159.

    16. Kunkel M, Lorinczi M, Rijnbrand R, et al. 2001. Self-assembly of nucleocapsid-like particles from recombinant hepatitis C virus core protein. J Virol, 75: 2119-2129.
        doi: 10.1128/JVI.75.5.2119-2129.2001

    17. Lazo L, Gil L, Lopez C, et al. 2010. Nucleocapsid-like particles of dengue-2 virus enhance the immune response against a recombinant protein of dengue-4 virus. Arch Virol, 155: 1587-1595.
        doi: 10.1007/s00705-010-0734-9

    18. Liu L, Celma C C, Roy P. 2008. Rift Valley fever virus structural proteins: expression, characterization and assembly of recombinant proteins. Virol J, 5: 82.
        doi: 10.1186/1743-422X-5-82

    19. Liu S J, Leng C H, Lien S P, et al. 2006. Immunological characterizations of the nucleocapsid protein based SARS vaccine candidates. Vaccine, 24: 3100-3108.
        doi: 10.1016/j.vaccine.2006.01.058

    20. Lopez C, Gil L, Lazo L, et al. 2009. In vitro assembly of nucleocapsid-like particles from purified recombinant capsid protein of dengue-2 virus. Arch Virol, 154: 695-698.
        doi: 10.1007/s00705-009-0350-8

    21. Ma B J, Wang H. 2000. The hacker of mount Tianshan— Crimean-Congo hemorrhagic fever virus. Foreign medical Sciences (Section of Virology) (in chinese), 7: 8-10.

    22. Midilli K, Gargılı A, Ergonul O, et al. 2007. Imported Crimean-Congo hemorrhagic fever cases in Istanbul. BMC Infectious Diseases, 7: 54.
        doi: 10.1186/1471-2334-7-54

    23. Ozdarendeli A, Canakoglu N, Berber E, et al. 2010. The complete genome analysis of Crimean-Congo hemorrhagic fever virus isolated in Turkey. Virus Res, 147: 288-293.
        doi: 10.1016/j.virusres.2009.11.009

    24. Reguera J, Weber F, Cusack S. 2010. Bunyaviridae RNA polymerases (L-protein) have an N-terminal, influenza-like endonuclease domain, essential for viral cap-dependent transcription. PLoS Pathogens, 6: 1-14.

    25. Saijo M, Qing T, Niikura M, et al. 2002. Recombinant nucleoprotein-based enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Crimean-Congo hemorrhagic fever virus. J Clin Microbiol, 40: 1587-1591.
        doi: 10.1128/JCM.40.5.1587-1591.2002

    26. Tang Q. 2006. Crimean-Congo hemorrhagic fever virus recently research progress. Chinese J Exp Clin Virol (in chinese), 20: 86-89.

    27. Wang M L, Tan Y, Yin F F, et al. 2008. The F-like protein Ac23 enhances the infectivity of the budded virus of gp64-null Autographa californica multinucleocapsid nucleopolyhedrovirus pseudotyped with baculovirus envelope fusion protein F. J Virol, 82: 9800-9804.
        doi: 10.1128/JVI.00759-08

    28. Wang M L, Tuladhar E, Shen S, et al. 2010. Specificity of baculovirus P6.9 basic DNA-binding proteins and critical role of the C terminus in virion formation. J Virol, 84: 8821-8828.
        doi: 10.1128/JVI.00072-10

    29. Wang R l, Jin N Y, Zhao B, et al. 2005. Expression of the Gene Encoding the N Protein of SARS Virus in E.coli and Its Usd in the Immunization of Hourse. Virologica Sinica (in chinese), 20: 582-585.

    30. Wasmoen T L, Kadakia N P, Unfer R C, et al. 1995. Protection of cats from infectious peritonitis by vaccination with a recombinant raccoon poxvirus expressing the nucleocapsid gene of feline infectious peritonitis virus. Adv Exp Med Biol, 380: 221-228.
        doi: 10.1007/978-1-4615-1899-0

    31. Wei P F, Luo Y J, Li T X, et al. 2010. Serial expression of the truncated fragments of the nucleocapsid protein of CCHFV and identification of the epitope region. Virologica Sinica, 25: 45-51.
        doi: 10.1007/s12250-010-3067-7

    32. Whitehouse C A. 2004. Crimean-Congo hemorrhagic fever. Antiviral Res, 64: 145-160.
        doi: 10.1016/S0166-3542(04)00163-9

    33. Wilson J A, Hart M K. 2001. Protection from Ebola virus mediated by cytotoxic T lymphocytes specific for the viral nucleoprotein. J Virol, 75: 2660-2664.
        doi: 10.1128/JVI.75.6.2660-2664.2001

    34. Xia H, Li P, Yang J, et al 2011. Epidemiological survey of Crimean-Congo hemorrhagic fever virus in Yunnan, China, 2008. Int J Infect Dis, 15: e459-463.
        doi: 10.1016/j.ijid.2011.03.013

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    Production of CCHF Virus-Like Particle by a Baculovirus-Insect Cell Expression System

      Corresponding author: Man-li Wang, Wangml@wh.iov.cn
    • State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
    Fund Project:  The Knowledge Innovation Program of the Chinese Academy of Sciences, Grant KSCX2-YW-N-065National Basic Research Priorities Program of China, Grant 2007National Key Basic Research Program (973 Program), Grant 2010CB530103the Knowledge Innovation Program of the Chinese Academy of Sciences, Grant KSCX2-EWG-8

    Abstract: Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is a tick-born virus of the Nairovirus genus within the Bunyaviridae family, which is widespread and causes high fatality. The nucleocapsid of CCHFV is comprised of N proteins that are encoded by the S segment. In this research, the N protein of CCHFV was expressed in insect cells using a recombinant baculovirus. Under an electron microscope, Virus-Like Particles (VLPs) with various size and morphology were observed in cytoplasmic vesicles in the infected cells. Sucrose-gradient purification of the cell lysate indicated that the VLPs were mainly located in the upper fraction after ultracentrifugation, which was confirmed by Western blot analysis and immuno-electron microscopy (IEM).

    • Crimean-Congo Haemorrhagic Fever virus (CCHFV) is a tick-born virus of the Nairovirus genus within the Bunyaviridae family. It is a virus with three segments of single-stranded, negative RNAs [7, 8, 32]. The three segments are named S (small, ~1.7 kb), M (middle, ~5.7 kb) and L (large, ~12 kb), principally encoding nucleocapsid protein (N protein, NP), glycoprotein and RNA-dependent RNA polymerase (RdRp), respectively [5, 7]. CCHFV has been found in more than 30 countries in Africa, Europe and Asia [23]. In China, it was first found in Xinjiang province in 1965 and outbreaks have been reported from time to time in Xinjiang [2, 21]. Apart from Xingjiang, CCHFV antibody positives were reported in animals or humans from several other provinces such as Yunnan, Qinghai and Sichuan [26, 34] and sporadic CCHF outbreaks have been recently reported in Turkey and Iran [3, 6, 22]. A CCHF outbreak poses a threat to public health because of the high mortality rate as well as the difficulties in treatment and prevention. 30-50% mortality is common and rates as high as 80% have also been reported [3, 13, 14, 32], mortality is related to the transmission routes and viral dose [3, 14, 32]. To date, the only known and used drug to treat CCHF is ribavirin [9] and there is no effective vaccine available against CCHFV [9].

      Viral envelope proteins are the first-choice for vaccine design. However, they are more prone to mutation to evade the host immune system [11]. The nucleocapsid (N) proteins are normally comparatively abundant and conserved in viruses, therefore N proteins have been widely tested as protective immunogens. The N protein of the feline infectious peritonitis virus (FIPV) was found to induce protective immunity in cats [30]; the N protein of Influenza virus, Ebola virus and avian coronavirus can elicit a strong cellular immune response [12, 19, 33]. These results indicate the nucleocapsid protein is a promising candidate for vaccine deve-lopment [17, 29]. The N protein of CCHFV is highly immunogenic [25, 31]. In this paper, the N protein of CCHFV was expressed by the baculovirus expression system, which is a candidate for protein overex-pression and is extensively used for vaccine development. By Western blot and IEM analysis, it was shown that CCHFV N protein can automatically assemble into Virus-Like Particles (VLPs) when over expressed in insect cells.

    • Spodoptera frugiperda cell line Sf9 cells were cultured at 27 ℃ in Grace's Insect Medium (Gibco-BRL) supplemented with 10% fetal bovine serum (FBS). AcMNPV bacmid bMON14272 (Invitrogen) used in the experiment was stored by our laboratory. The control virus Ac-egfp was previously constructed in our laboratory [27].

    • The S segment (GenBank accession no. FJ562093) of strain YL04057 CCHFV was kindly provided by Xinjiang CDC [31]. The complete NP ORF including the 5'-UTR and 3'-UTR was amplified using forward primer SF: 5'-TCTCAAAGAAACACGTGCCGC-3' and reverse primer SR: 5'-TCTCAAAGATATCGTT GCCGC-3' using Pyrobest DNA polymerase (TAKARA Bio Inc., Japan). The PCR product was extended with an A-tail by Taq DNA polymerase (Biostar, China), then cloned into a pGEM-T easy vector (Promega) and sequenced.

      For the construction of a recombinant bacmid, an hsp70-egfp-SV40 cassette was digested from the pKS-egfp-cmr vector (kindly provided by Prof. Just M. Vlak, Wageningen University) by XhoI and HindⅢ, blunted by Klenow Fragment (Takara) and inserted into the Bst1107I site of the transfer vector pFast-BacTMDual (Invitrogen), to give pFastBacDual-egfp. Then, the CCHFV S gene was digested from pGEM-T Easy vector by EcoRI and subcloned into pFast-BacDual-egfp, generating donor plasmid pFastBacDual-egfp-cchfvs. The donor plasmid pFastBacDual-egfp-cchfvs was introduced into AcBacmid via Tn7-mediated transposition (Bac-to-Bac, Invitrogen), resulting in recombinant bacmid Ac-egfp-cchfvs (Fig. 1). The recombinant bacmid was identified by PCR with M13 primers.

      Figure 1.  The schematic showing construction of recombinant virus Ac-egfp-cchfvs.

    • One μg of Ac-egfp-cchfvs DNA was transfected into 1×106 Sf9 cells using 12 μL lipofectin reagent (Invitrogen), according to the Bac-to-Bac Expression Systems manual (Invitrogen). At 120 h post transfection (p.t.), 1 mL supernatants were centrifuged at 3, 000×g for 5 min to remove cell debris, and then infected in a 75 cm2 cell culture flask (Corning) of Sf9 cells. Recombinant BVs were harvested at 168 h post infection (p.i.) and stored at 4 ℃ for usage. The titer of the recombinant virus was determined by an end-point dilution assay [15].

    • Sf9 cells were infected with Ac-egfp-cchfvs or control virus Ac-egfp at a MOI of 1 and 5, respectively. The cells were collected at 72 h p.i. and fixed for 16 h at 4 ℃ with 2.5% (W/V) glutaraldehyde in 0.1 mol/L PBS(pH7.2). The EM specimens were prepared according to Wang et al. (2010) [28] and recorded by transmission electron microscope (FEI Tecnai G2, operated at 200 kV).

    • Eight flasks (1×107 per each) of Sf9 cells were infected with Ac-egfp-cchfvs or Ac-egfp at a MOI of 1. The cells were collected at 72 h p.t. and broken by ultrasonic wave. The ultrasonic supernatants were layered on top of a 25% sucrose cushion, and spun for 2 h at 25, 000 × g. The pellets were dissolved in 0.1× TE buffer (pH 7.2) and further purified by a sedimen-tation trough with 25% to 60% sucrose gradients (28, 000 ×g, 3 h). Each band was collected separately.

    • The expression of the N protein in the infected Sf9 cells was examined by Western blot. Sf9 cells were infected with Ac-egfp or Ac-egfp-cchfvs at a MOI of 5 and cells were harvested at 72 h.p.i. The cell samples were disrupted in 4×SDS-PAGE sample buffer, and electrophoresed in 10% SDS-polyacrylamide gels. The proteins were transferred onto Hybond-N membranes (Amersham) by semi-dry electrophoresis. Western blot was also used to detect N proteins in different fractions after ultracentrifugation purification. Western blotting was performed according to Wei et al.[31] with a slight modification. The primary antibody, anti-CCHFV N polyclonal rabbit serum[31]was used at 1:1000 dilution, and the secondary antibody, goat anti-rabbit immunoglobulin conjugated with alkaline phosphatase was used at 1:2000 dilution. Finally, the signals were detected by NBT/BCIP (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate, SABC, China). Two control antibodies, anti-GP64 antibody (rabbit antiserum against AcMNPV major envelope protein, 1:2000 dilution) [27] and anti-VP39 antibody (rabbit antiserum against AcMNPV nucleocapsid protein, 1:5000 dilution) [4]were used as controls to detect AcMNPV virions in ultracentrifu-gation fractions.

    • For negative staining, each band from the sucrose-gradient ultracentrifugation was collected and samples were adsorbed onto carbon-coated copper 200 mesh grids for 2 h at room temperature (RT). Grids were transferred into a drop of negative stain (2% phosphomolybdic acid, pH6.5) for 1 min.

      IEM was performed according to Deng et al. [4]. Briefly, Nickel grids were floated for 2 h in a 20 μL drop of sample to adsorb the particles, and then the Grids were incubated with anti-CCHFV N polyclonal antibody (1:20 diluted in 5% [W/V] bovine serum albumin-Tris-Buffered-Saline [BSA-TBS]) for 2 h at room temperature (RT) in a wet chamber. Preimmune sera were used as negative controls. Grids were washed six times with 0.1 × TE buffer and then incubated for 2 h with 12-nm Colloidal Gold-AffiniPure goat anti rabbit immunoglobulin G (Jackson Immuno Research) diluted 1:50 at RT. Grids were then washed with 0.1 × TE buffer and stained as described above. Specimens were observed under a transmission electron microscope (FEI Tecnai G2) operated at 200 kV.

    • The recombinant bacmid Ac-egfp-cchfvs were generated as described in Materials and Methods and its genomic structure is shown in Fig. 1A. Positive clones were confirmed by PCR amplifications and sequencing (data not shown). Sf9 cells were transfected with the recombinant bacmid DNA. At 72 h p.t., green fluorescence was found in some cells (Fig. 2A), suggesting the successful transfection of recombinant bacmid DNA into the Sf9 cells. At 100 h p.t., fluorescence spread from cell to cell (Fig. 2B), indicating the propagation of infectious progeny recombinant Ac-egfp-cchfvs.

      Figure 2.  Transfection of the recombinant bacmid. Sf9 cells were transfected with Ac-egfp-cchfvs bacmid DNA. The EGFP expression was observed at 72 h p. t. (A) and 100 h p. t. (B).

    • To detect the expression of N proteins in Sf9 cells, Western blot analysis was conducted. As Fig. 3 shows, anti-CCHFV N antibody can detect a band corres-ponding to the predicted molecular mass (~54 kDa) of N protein in AcBac-egfp-cchfvs infected cells (Fig. 3, lane 3), but not in the control virus Ac-egfp infected Sf9 cells (Fig. 3, lane 2) or Sf9 cells control (Fig. 3, lane 1). Thus, Western analysis confirmed the correct expression of CCHFV N protein by the baculovirus-insect expression system.

      Figure 3.  Western blot analysis on the expression of CCHFV N protein in Sf9 cells. Lane 1, Sf9 cells control; lane 2, Sf9 cells infected with Ac-egfp; lane 3, Sf9 cells infected with Ac-egfp-cchfvs. M, pre-stained protein marker.

    • The nucleocapsid protein of some viruses, i.e. Dengue virus-2, Hepatitis C virus (HCV), can automatically assemble into Virus-like particles [16, 20]. To detect whether CCHFV N proteins can self-assemble into VLPs, Sf9 cells infected with Ac-egfp-cchfvs at different MOIs (1 or 5) were observed by transmission electron microscopy. A cluster of sphere-like particles, which appear like VLPs were found in vesicles (Fig. 4A and B, boxed region) in the cytoplasm of Sf9 cells both infected at MOI of 1 and 5. The numbers and density of sphere-like particles forms in Sf9 cells infected with a MOI of 5 seems to be higher than those infected at a MOI of 1 (Fig. 4 A' and B'). These particles varied both in morphology and size, ranging from 40 nm to 160 nm in diameter. In contrast, there were very few sphere-like particles present in the cytoplasmic vesicles in control virus Ac-egfp infected Sf9 cells, at an MOI of either 1 or 5 (data not shown).

      Figure 4.  Electron microscopy of Sf9 cells infected with Ac-egfp-cchfvs at a MOI of 1 (A) and 5 (B). A' and B' show an enlarged view of the boxed region of A and B, respectively.

      To further confirm whether the sphere-like particles were composed of CCHFV N proteins, Ac-egfp-cchfvs or Ac-egfp infected cell lysate was analyzed by sucrose-gradient ultracentrifugation, respectively. Three major bands, upper (~30% sucrose density), middle (between 35%-45%) and lower (between 50%-55%) were visible in Ac-egfp-cchfvs infected cell sample after centrifugation (Fig. 5A). However, only one band (~45%) was detected in the Ac-egfp infected cell sample, and EM analysis confirmed this band was composed mostly of rod-shape AcMNPV, not sphere-like particles (data not shown), and thus it was not subjected to further analyses. This result is in agreement with the above EM result that very few sphere-like particles were detected in Sf9 cells infected by Ac-egfp.

      Figure 5.  Western blot analyses of gradient-ultracentrifugation fractions. A: Schematic showing sucrose-gradient ultracentrifugation fractions of Sf9 cells infected with Ac-egfp-cchfvs. B and C: Western blot results of each ultracentrifugation fraction probed with anti-CCHFV N antibody (B) or anti-AcMNPV GP64 and VP39 antibodies (C). Lane 1, Sf9 cells infected with Ac-egfp; lane 2, Sf9 cells infected with Ac-egfp-cchfvs; lane 3, the upper band of cell lysate; lane 4, the middle band of cell lysate; Lane 5, the lower band of the cell lysate; M: pre-stained protein maker.

      Each centrifugation band in Ac-egfp-cchfvs infected cell samples was collected for Western and EM analysis. Western blot demonstrated the presence of CCHFV N protein in all three purified bands with most the abundance in the upper band (Fig. 5B, lane 3-5). It was also found in the positive control virus Ac-egfp-cchfvs infected cells (Fig. 5B, lane 2), but not in the negative control virus Ac-egfp infected cells (Fig. 5B, lane 1). Antibodies against AcMNPV budded virus (BVs) envelope fusion protein GP64 and viral nucleocapsid protein VP39 were also used for Western blot to detect baculoviruses in those samples (Fig. 5C). Both GP64 and VP39 were detected in the upper and middle bands (Fig. 5C, lane 3 and 4), suggesting the presence of AcMNPV BVs in upper and middle centrifugation fractions; VP39 but not GP64 was detected in lower band as well (Fig. 5C, lane 5), indicating the existence of AcMNPV occlusion derived virions (ODVs) in this band.

      Negative staining results demonstrated the upper band was mainly composed of sphere-like particles, with small amounts of AcMNPV BVs (Fig. 6A), and the lower band was mainly constituted of AcMNPV ODVs (data not shown). The EM results were in consistent with those of Western blot analyses. The upper band was further analyzed by IEM. As Fig. 6D showed, gold probes specifically surrounded the sphere-like particles when using anti-CCHFV N antiserum as the primary antibody. The pre-immune rabbit serum and no primary antibody were used as negative controls (Fig. 6B and C). These results confirmed the sphere-like particles formed in the Ac-egfp-cchfvs infected cells were made up of N protein. Thus, expressing CCHFV N protein by baculovirus can form VLPs in insect cells.

      Figure 6.  EM and IEM analyses of CCHFV VLPs (upper band from ultracentrifugation). A: Negative staining electron micrograph of CCHFV VLPs. B, C and D: IEM micrographs of CCHFV VLPs probed with no primary antibody (B), pre-immunized rabbit serum (C) and anti-CCHFV N antibody (D) as the primary antibody.

    • In this paper, we present evidence that VLPs are formed when Sf9 cells are infected with a recom-binant baculovirus expressing CCHFV N protein. After sucrose-gradient ultracentrifugation the VLPs were further confirmed by Western blot and IEM.

      As a major component of CCHFV virion, the nucleocapsid protein plays important roles during virus assembly and morphogenesis [32]. The N proteins bind with viral RNAs and interact with RdRp (RNA dependent RNA polymerase, also called L protein) to form RNP complexes[1, 10], then the RNPs are packaged with bilayer lipid membrane embedded with glycoproteins [24]. Our results indicated that N proteins may self-assemble into nucleocapsids in natural CCHFV infection. However, the specific mechanism of CCHFV assembly remains to be investigated.

      Many viral nucleocapsid proteins or envelope glycoproteins can spontaneously self-assemble into VLP. VLP has been developed as an ideal vaccine candidate, because it mimics the overall structures of a virus particle without containing infectious genetic materials, thus is safe and highly immunogenic. Among a variety of protein expression systems for VLP production, the baculovirus-insect system has many advantages, being safe, cheap and easy to scale-up. So far, more than 30 VLPs among 15 virus families have been produced by this system and demonstrated to be highly immunogenic and potential vaccine candidates, including members of bun-yaviridae family, Rift Valley fever virus (RVFV) and Hantaan virus[18]. Recently, the highly conserved region 235 to 305 aa of CCHFV (YL04057 strain) N protein was identified as a highly antigenic region[31]. Therefore, the VLP generated in this research may provide a promising vaccine candidate for CCHF prevention. Further experiments will be conducted to test the immunogenicity of CCHFV VLPs in animal models.

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