Development of molecular and serological assays for diagnosis and surveillance of Crimean-Congo haemorrhagic fever virus
Loading...
Date
2015-05
Authors
Pieters, Danelle
Journal Title
Journal ISSN
Volume Title
Publisher
University of the Free State
Abstract
Crimean-Congo haemorrhagic fever virus (CCHFV) an arthropod-borne virus associated with
haemorrhagic disease in humans. The global distribution of CCHFV correlates with that of ticks
from the Hyalomma genus. CCHFV infection is diagnosed by detection of viral nucleic acid
using reverse-transcription polymerase-chain-reaction (RT-PCR) or other molecular assays, by
virus isolation from infected cell culture or suckling mouse brain or by detection of anti-CCHFV
antibodies using enzyme-linked immunosorbent assay (ELISA) or immunofluorescence assay
(IFA). High biocontainment facilities are required for virus isolation and preparation of whole
virus native antigen for use in serological assays. Currently, treatment is limited to supportive
therapy. CCHFV is currently emerging and re-emerging in many regions, which emphasize the
requirement for safe, reliable and inexpensive assays to increase diagnostic capacity and monitor
emergence of the virus.
A nucleic acid sequence-based amplification (NASBA) molecular assay for detection of CCHFV
ribonucleic acid (RNA) was developed. The assay can be performed without the requirement for
sophisticated laboratory equipment. A commercially available enzyme mixture and buffer were
compared with a more cost effective and easier to obtain in-house enzyme mixture and
amplification buffer. Specificity of the NASBA assays were determined by testing viral RNA
extracted from Vero cell culture infected with genetically diverse southern African CCHFV
strains. A total of 41/48 samples tested were positive. Sensitivity of the NASBA assays was
determined using dilutions of viral RNA and transcribed RNA to detect minimal copy number
that 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 extracted
from 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 available
assays were able to amplify 22/22 samples. Subsequently, the inhibitory effect of sera on the
amplification of CCHFV RNA using the NASBA assay was investigated using sera spiked with
transcribed RNA.
Two expression systems were investigated for the expression of recombinant CCHFV
nucleocapsid protein (NP) for use in serological assays. The baculovirus expression system was
initially investigated. The open reading frame of the S segment of a CCHFV strain was codon
optimized 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 coli
cells to transpose the optimized CCHFV NP gene to a bacmid. The recombinant bacmid was
utilized to transfect Spodoptera frugiperda 9 cells. The cell lysates were analysed, however, no
expression of the CCHFV NP could be confirmed. A mammalian expression system was
subsequently investigated. A pcDNATM 3.1D/V5-His-TOPO.CCHFV.NP construct was used to
transfect baby hamster kidney-21 cells. Expression of CCHFV NP was detected in transiently
transfected cells using IFA and serum collected from a convalescent CCHFV patient.
To profile the immune response against CCHF viral proteins, 15 sera collected from
convalescent patients at various times after onset of illness were tested for antibody against
CCHFV NP and glycoproteins (GP) using commercially available slides. The antigen slides
were prepared from transfected cells expressing recombinant CCHFV NP and GP. Antibody
against CCHFV GP and NP were detected in all samples. End point titers of anti-CCHFV NP
and GP were determined for two serum samples. Commercially available slides are expensive
and therefore have limited application for testing large numbers. Application of in-house antigen
slides prepared from transfected cells expressing CCHFV NP were tested using IFA and 14 sera
collected from convalescent CCHFV patients. All sera tested positive, suggesting that
preparation of a stable cell line expressing CCHFV NP is warranted for application in detection
of antibody against CCHFV.
Description
Keywords
Hemorrhagic fever, Serology, Hemorrhagic fever -- Molecular diagnosis, Arbovirus infections, Dissertation (M.Med.Sc. (Medical Microbiology and Virology))--University of the Free State, 2015, Tick-borne diseases