DEVELOPMENT AND EVALUATION OF ENZYME-LINKED IMMUNOSORBENT ASSAYS FOR THE DETECTION OF ANTI-MARBURG VIRUS

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Marburg virus antigen detection tests

Antigen capture ELISAs are useful during the early stages of infection before convalescence occurs (8 to 10 days after the onset of symptoms) (Rougeron et al., 2015). Although antigen capture ELISAs are used widely by WHO reference laboratories in the diagnosis of MVD, these assays are all in-house and have not been thoroughly described in the literature. Saijo and colleagues (2005; 2006b) produced two clones of monoclonal antibodies to recombinant MARV Musoke NP in immunised mice and reported their efficacy in antigen capture ELISA format. The assay was reported to have a similar sensitivity to the RT-PCR described by Sanchez and colleagues (1999). Sherwood and colleagues (2007) described an antigen capture assay for MARV Musoke, Ravn and Angola that made use of NP-specific llama single domain antibodies. While the assay was not optimised, it was reported to be rapid, sensitive and specific, with no cross-reactivity occurring with other filovirus species.

Molecular techniques for the detection of Marburg virus nucleic acid

Molecular methods are currently the tools of choice for the diagnosis of MVD in WHO VHF reference laboratories, and are also used in MARV surveillance studies in bat populations (Amman et al., 2012; Paweska et al., 2018). Nucleic acid of MARV can be detected in blood from the third day after the onset of symptoms in humans, and may remain detectable in the blood up to the 16th day after the onset of symptoms (Martines et al., 2015). Experimental inoculation studies have shown that molecular methods are able to detect MARV RNA in the blood of bats from 1 to 12 days post-infection (p.i.) (Paweska et al., 2012; Amman et al., 2015; Paweska et al., 2015; Schuh et al., 2017a), in oral swabs, rectal swabs and urine specimens from 5 to 19 days p.i. (Amman et al., 2015; Schuh et al., 2017a) and in the liver, spleen and other tissues from 3 to 28 days p.i. (Paweska et al., 2012; Amman et al., 2015; Paweska et al., 2015). While these assays are important in confirming MARV infection in reservoir host bat populations during surveillance studies, the short period of viraemia and low levels of viral shedding make the detection of actively infected individuals in the wild difficult. Molecular assays should therefore be used in combination with serological assays for MARV surveillance.
As of 2018, the most recently published molecular assays for the detection of filovirus RNA include a consensus RT-PCR assay using a cocktail of primers targeting the L gene of filoviruses (Zhai et al., 2007), a qRT-PCR assay using five primers and three probes targeting the L gene of filoviruses (Panning et al., 2007), and a conventional RT-PCR assay using four primers targeting the NP gene of MARV and EBOV (Ogawa et al., 2011). The assays targeting the L-gene have been shown to be able to detect different strains of EBOV, SUDV, MARV, TAFV and RESTV with high analytical sensitivities (Panning et al., 2007; Zhai et al., 2007), while the assay targeting the NP gene was able to detect EBOV, MARV, SUDV, TAFV, RESTV and BDBV (Ogawa et al., 2011). A commercial kit (RealStar Filovirus Screen, Altona Diagnostics) based on the qRT-PCR method described by Panning and colleagues (2007) has also become available and has been shown to have a high diagnostic sensitivity with good differentiation between different filovirus species (Rieger et al., 2016). The major obstacles in designing an RT-PCR assay for filoviruses include the high genetic diversity between the different filovirus genera, and the inability to determine the clinical sensitivity of the assays due to the unavailability of well-characterised serum panels of patients infected with different filovirus species (Panning et al., 2007; Zhai et al., 2007).

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Virus isolation

Virus isolation in Vero E6 African green monkey kidney cells is the traditional gold standard technique to confirm the presence of MARV in a specimen. Virus isolation allows direct visualisation of MARV by electron microscopy within 1 week post inoculation. Although definitive, virus isolation methods require BSL-4 containment and are therefore restricted to laboratories outside of countries where MARV is endemic (Broadhurst et al., 2016).

CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW 
1.1 Introduction and thesis layout
1.2 History and taxonomy of Marburg virus
1.3 Morphology, genomic structure and genetic diversity of Marburg virus
1.4 Epidemiology of Marburg virus
1.5 Marburg virus host range and geographic distribution
1.6 Transmission of Marburg virus
1.7 Replication and dissemination of Marburg virus
1.8 Marburg virus immune evasion strategies and pathogenesis
1.9 Immunity and host immune responses to Marburg virus infection
1.10 Identification of Marburg virus
1.11 Significance and aims of this study
CHAPTER 2: DEVELOPMENT AND EVALUATION OF ENZYME-LINKED IMMUNOSORBENT ASSAYS FOR THE DETECTION OF ANTI-MARBURG VIRUS IMMUNOGLOBULIN G ANTIBODIES IN EGYPTIAN ROUSETTE BATS 
2.1 Introduction
2.2 Materials and methods
2.3 Results
2.4 Discussion
CHAPTER 3: ANTIBODY RESPONSES OF EGYPTIAN ROUSETTE BATS TO MARBURG VIRUS AND THEIR ROLE IN PROTECTION AGAINST INFECTION 
3.1 Introduction
3.2 Materials and methods
3.3 Results
3.4 Discussion
CHAPTER 4: FUTURE PERSPECTIVES AND CONCLUDING REMARKS 
REFERENCES

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