key: cord-1007790-bnd9lk8f authors: Zhou, Zhao-rui; Wang, Man-li; Deng, Fei; Li, Tian-xian; Hu, Zhi-hong; Wang, Hua-lin title: Production of CCHF virus-like particle by a baculovirus-insect cell expression system date: 2011-10-07 journal: Virol Sin DOI: 10.1007/s12250-011-3209-6 sha: 2c098d1f90a2be84f15db57229d3d8af5707c5f4 doc_id: 1007790 cord_uid: bnd9lk8f 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). 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 development [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 overexpression 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 One μg of Ac-egfp-cchfvs DNA was transfected into 1×10 6 Sf9 cells using 12 μL lipofectin reagent end-point dilution assay [15] . Eight flasks (1×10 7 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 sedimentation 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/5bromo-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 sucrosegradient ultracentrifugation was collected and samples were adsorbed onto carbon-coated copper 200 mesh grids for 2 h at room temperature (RT). Grids were IEM was performed according to Deng et al. [4] . 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 corresponding to the predicted molecular mass (~54 kDa) of N protein in AcBac-egfp-cchfvs infected cells (Fig. 3, Sf9 cells (Fig. 3, lane 2) or Sf9 cells control (Fig. 3, lane 1) . Thus, Western analysis confirmed the correct 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 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). 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 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 In this paper, we present evidence that VLPs are formed when Sf9 cells are infected with a recombinant 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 scaleup. 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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