Abstract:
Marburg virus (MARV) is a zoonotic virus of significant potential public health concern in
Africa. Together with Ebola virus (EBOV), MARV belongs to the family Filoviridae and
causes a life-threatening haemorrhagic disease in humans and non-human primates. The
occurrence of large outbreaks of MARV disease (MVD) within the past two decades, as well
as the devastating EBOV outbreaks in West and Central Africa, indicates that filoviruses are a
much more significant public health threat than previously anticipated and can emerge at any
time without warning. These unprecedented outbreaks have emphasised the need for
surveillance in reservoir host populations and for safe and reliable surveillance tools and
diagnostic tests that may easily be performed in both laboratory and field settings. Egyptian
rousette bats (ERB; Rousettus aegyptiacus) are reservoir hosts for MARV, and there is a need
for understanding the dynamics of immune responses of these animals to MARV infection. This
knowledge can assist in predicting periods of increased transmission within bat colonies and in
turn, potential spillover events into human and other animal populations.
In this thesis, the development of indirect enzyme-linked immunosorbent assays (I-ELISA) for
the detection of specific anti-MARV immunoglobulin G (IgG) in bat sera is described. The IELISAs,
based on two recombinant MARV protein antigens (nucleoprotein and glycoprotein),
can be used without the need for high biocontainment facilities. Both I-ELISAs were found to
be robust and repeatable, with good sensitivity and specificity. Applying the I-ELISAs in
detecting IgG antibodies to MARV in sera collected from both wild-caught and experimentally
infected bats indicated that the assays are suitable methods for MARV serosurveillance, with the MARV GP-based I-ELISA demonstrating higher diagnostic performance compared to the
MARV NP-based assay.
Based on this knowledge, the MARV GP I-ELISA was applied in monitoring and characterising
the antibody responses of ERBs to MARV. Maternal antibodies to MARV were detected in
juvenile bats up to approximately five months after birth. In bats experimentally infected with
MARV, antibodies against the virus remained detectable in the majority of bats at 110 days
post-infection. Furthermore, antibodies to MARV remained detectable in 84% of naturally
exposed bats at least 11 months after capture, suggesting that bats develop long-term humoral
immunity in response to active infection with MARV. To test whether pre-existing immunity
in bats is protective against reinfection, 15 ERBs with differing levels of MARV-specific IgG
antibodies were inoculated with the Watsa isolate of the virus. Levels of anti-MARV IgG
antibodies increased swiftly from day 5 post inoculation. Viraemia was detected in 73% of
reinfected bats, and the challenge virus was isolated from the serum of one reinfected bat. Viral
ribonucleic acid was detected in the spleen (73% of bats), liver (47%) and lung (7%) at different
days post inoculation. These results suggest that primary infection of ERBs with MARV does
not induce sterilising humoral immunity; however, re-inoculation of previously infected bats
produced only localised infection, with an absence of the virus in tissues potentially involved
in viral transmission. Reinfection of previously infected bats is therefore not likely to be a key
factor driving MARV maintenance in nature.
The establishment of in-house capacity for the production of recombinant I-ELISA antigens as
described in this thesis will assist in the biosurveillance programme in South Africa aimed at
monitoring the presence and distribution of MARV infection in local bat populations. The
assays based on these antigens will also assist in monitoring the immune status of reservoir host
populations, predicting potential spillover events, implementing risk reduction strategies and
improving virus-host modelling studies. These tools will further contribute to the
characterisation of the antibody responses of ERBs to MARV, which may ultimately assist in
elucidating the mechanisms by which bats are able to combat clinical MARV disease.