Development of molecular and serological assays for diagnosis and surveillance of Crimean-Congo haemorrhagic fever virus

[摘要] Crimean-Congo haemorrhagic fever virus (CCHFV) an arthropod-borne virus associated withhaemorrhagic disease in humans. The global distribution of CCHFV correlates with that of ticksfrom the Hyalomma genus. CCHFV infection is diagnosed by detection of viral nucleic acidusing reverse-transcription polymerase-chain-reaction (RT-PCR) or other molecular assays, byvirus isolation from infected cell culture or suckling mouse brain or by detection of anti-CCHFVantibodies using enzyme-linked immunosorbent assay (ELISA) or immunofluorescence assay(IFA). High biocontainment facilities are required for virus isolation and preparation of wholevirus native antigen for use in serological assays. Currently, treatment is limited to supportivetherapy. CCHFV is currently emerging and re-emerging in many regions, which emphasize therequirement for safe, reliable and inexpensive assays to increase diagnostic capacity and monitoremergence of the virus.A nucleic acid sequence-based amplification (NASBA) molecular assay for detection of CCHFVribonucleic acid (RNA) was developed. The assay can be performed without the requirement forsophisticated laboratory equipment. A commercially available enzyme mixture and buffer werecompared with a more cost effective and easier to obtain in-house enzyme mixture andamplification buffer. Specificity of the NASBA assays were determined by testing viral RNAextracted from Vero cell culture infected with genetically diverse southern African CCHFVstrains. A total of 41/48 samples tested were positive. Sensitivity of the NASBA assays wasdetermined using dilutions of viral RNA and transcribed RNA to detect minimal copy numberthat could be amplified. The NASBA assay was able to detect at least 3.7 RNA copies.Diagnostic application of the NASBA assays was investigated by amplifying RNA extractedfrom clinical samples and the results compared with two commercial real-time RT-PCR assays.A total of 20/22 samples tested positive using the NASBA whereas the commercially availableassays were able to amplify 22/22 samples. Subsequently, the inhibitory effect of sera on theamplification of CCHFV RNA using the NASBA assay was investigated using sera spiked withtranscribed RNA.Two expression systems were investigated for the expression of recombinant CCHFVnucleocapsid protein (NP) for use in serological assays. The baculovirus expression system wasinitially investigated. The open reading frame of the S segment of a CCHFV strain was codonoptimized for expression in insect cells. A pFastBac HT B transfer vector containing the optimized CCHFV NP gene was prepared and used to transform DH10Bac�?Escherichia colicells to transpose the optimized CCHFV NP gene to a bacmid. The recombinant bacmid wasutilized to transfect Spodoptera frugiperda 9 cells. The cell lysates were analysed, however, noexpression of the CCHFV NP could be confirmed. A mammalian expression system wassubsequently investigated. A pcDNATM 3.1D/V5-His-TOPO.CCHFV.NP construct was used totransfect baby hamster kidney-21 cells. Expression of CCHFV NP was detected in transientlytransfected cells using IFA and serum collected from a convalescent CCHFV patient.To profile the immune response against CCHF viral proteins, 15 sera collected fromconvalescent patients at various times after onset of illness were tested for antibody againstCCHFV NP and glycoproteins (GP) using commercially available slides. The antigen slideswere prepared from transfected cells expressing recombinant CCHFV NP and GP. Antibodyagainst CCHFV GP and NP were detected in all samples. End point titers of anti-CCHFV NPand GP were determined for two serum samples. Commercially available slides are expensiveand therefore have limited application for testing large numbers. Application of in-house antigenslides prepared from transfected cells expressing CCHFV NP were tested using IFA and 14 seracollected from convalescent CCHFV patients. All sera tested positive, suggesting thatpreparation of a stable cell line expressing CCHFV NP is warranted for application in detectionof antibody against CCHFV.

[发布机构] University of the Free State