Crimean-Congo haemorrhagic fever (CCHF) is a viral zoonotic disease with a high mortality rate in humans. The CCHF virus (CCHFV) is transmitted to humans through the bite of Ixodid ticks or by contact with blood or tissues of infected livestock. In addition to zoonotic transmission, CCHFV can be spread from person to person and is one of the rare haemorrhagic fever viruses able to cause nosocomial outbreaks in hospitals. Crimean-Congo haemorrhagic fever is a public health problem in many regions of the world such as Eastern Europe, Asia, the Middle East and Africa. In addition to clinical symptoms, the diagnosis of CCHF is based on the use of serological tests for the detection of immunoglobulin M and immunoglobulin G antibodies and on the use of molecular tools such as RT-PCR. From 1970 to 1978, serological and epidemiological studies were performed in humans and in livestock of Iran. After two decades and observations of CCHF in some provinces of Iran, a CCHF surveillance and detection system was established in 1999, leading to a dramatically decreased mortality rate from 20% (year 2000) to 2% (year 2007).
Crimean-Congo hemorrhagic fever virus (CCHFV) is a major cause of tick-borne viral hemorrhagic disease in the world. Despite of its importance as a deadly pathogen, there is currently no licensed vaccine against CCHF disease. The attachment glycoprotein of CCHFV (Gn) is a potentially important target for protective antiviral immune responses. To characterize the expression of recombinant CCHFV Gn in an insect-cell-based system, we developed a gene expression system expressing the full-length coding sequence under a polyhedron promoter in Sf9 cells using recombinant baculovirus. Recombinant Gn was purified by affinity chromatography, and the immunoreactivity of the protein was evaluated using sera from patients with confirmed CCHF infection. Codon-optimized Gn was successfully expressed, and the product had the expected molecular weight for CCHFV Gn glycoprotein of 37 kDa. In time course studies, the optimum expression of Gn occurred between 36 and 48 hours postinfection. The immunoreactivity of the recombinant protein in Western blot assay against human sera was positive and was similar to the results obtained with the anti-V5 tag antibody. Additionally, mice were subjected to subcutaneous injection with recombinant Gn, and the cellular and humoral immune response was monitored. The results showed that recombinant Gn protein was highly immunogenic and could elicit high titers of antigen-specific antibodies. Induction of the inflammatory cytokine interferon-gamma and the regulatory cytokine IL-10 was also detected. In conclusion, a recombinant baculovirus harboring CCHFV Gn was constructed and expressed in Sf9 host cells for the first time, and it was demonstrated that this approach is a suitable expression system for producing immunogenic CCHFV Gn protein without any biosafety concerns.
BackgroundCrimean-Congo hemorrhagic fever virus (CCHFV) is a member of the nairovirus, a genus in the Bunyaviridae family, which causes a life threatening disease in human. Currently, there is no vaccine against CCHFV and detailed structural analysis of CCHFV proteins remains undefined. The CCHFV M RNA segment encodes two viral surface glycoproteins known as Gn and Gc. Viral glycoproteins can be considered as key targets for vaccine development.ObjectivesThe current study aimed to investigate structural bioinformatics of CCHFV Gn protein and design a construct to make a recombinant bacmid to express by baculovirus system.Materials and MethodsTo express the Gn protein in insect cells that can be used as antigen in animal model vaccine studies. Bioinformatic analysis of CCHFV Gn protein was performed and designed a construct and cloned into pFastBacHTb vector and a recombinant Gn-bacmid was generated by Bac to Bac system.ResultsPrimary, secondary, and 3D structure of CCHFV Gn were obtained and PCR reaction with M13 forward and reverse primers confirmed the generation of recombinant bacmid DNA harboring Gn coding region under polyhedron promoter.ConclusionsCharacterization of the detailed structure of CCHFV Gn by bioinformatics software provides the basis for development of new experiments and construction of a recombinant bacmid harboring CCHFV Gn, which is valuable for designing a recombinant vaccine against deadly pathogens like CCHFV.
Rabies is a zoonotic fatal viral disease caused by the rabies virus of the genus Lyssavirus, and the family Rhabdoviridae. Challenge virus standard (CVS-11) strain of rabies virus is a key element in rabies reference laboratories, as many gold-standard tests depend on a suitable titer of this strain for interpretation of results. The present study investigated the optimal CVS-11 production in BSR cells (a clone of BHK-21). Methods: We analyzed the kinetic growth of BSR cells in a T-flask and inoculated BSR cells with different MOI of CVS-11 strain of rabies virus, and harvested the produced virus at different time points. Results: Our data showed that BSR cells had a doubling time of around 24-30 h, and at least 95% of cells kept their viability three days after culture. The virus reached the highest titer when the cells were infected at an MOI of 0.1 in DMEM medium, equal to 1.5 × 107 fluorescent focus units (FFU)/ml. Time-course analysis of CVS-11 titer showed that the highest titer was achieved around 72 h post-infection. All tests were performed in triplicate. Conclusion: Since producing the virus in mammalian cell culture is an expensive and complicated method, optimizing the virus production process may be an excellent strategy to lower the cost, save the laboratory resources and maximize productivity.
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