Development of a multiplex microsphere immunoassay for the detection of antibodies against highly pathogenic viruses in human and animal serum samples

Abstract

Surveillance of highly pathogenic viruses circulating in both human and animal populations is crucial to unveil endemic infections and potential zoonotic reservoirs. Monitoring the burden of disease by serological assay could be used as an early warning system for imminent outbreaks as an increased seroprevalance often precedes larger outbreaks. However, the multitude of highly pathogenic viruses necessitates the need to identify specific antibodies against several targets from both humans as well as from potential reservoir animals such as bats. In order to address this, we have developed a broadly reactive multiplex microsphere immunoassay (MMIA) for the detection of antibodies against several highly pathogenic viruses from both humans and animals. To this aim, nucleoproteins (NP) of Ebola virus (EBOV), Marburg virus (MARV) and nucleocapsid proteins (NP) of Crimean-Congo haemorrhagic fever virus, Rift Valley fever virus and Dobrava-Belgrade hantavirus were employed in a 5-plex assay for IgG detection. After optimisation, specific binding to each respective NP was shown by testing sera from humans and non-human primates with known infection status. The usefulness of our assay for serosurveillance was shown by determining the immune response against the NP antigens in a panel of 129 human serum samples collected in Guinea between 2011 and 2012 in comparison to a panel of 88 sera from the German blood bank. We found good agreement between our MMIA and commercial or in-house reference methods by ELISA or IIFT with statistically significant higher PLOS NEGLECTED TROPICAL DISEASES

Selective replication and vertical transmission of Ebola virus in experimentally infected Angolan free-tailed bats

“…However, this increase was not as dramatic in the TAFV cohort. For reference, we included previously validated positive AFB serum samples 26 as positive controls (POS) for orthoebolaviruses and MARV. Amongst all inoculation groups, EBOV elicited the highest Ab titres, with average MFIs at 10 dpi ( n = 2) more than twofold greater than the POS.…”

“…Briefly, 7 genotypes within the Lyssavirus genus were screened using a nested RT-PCR 53 ; filovirus active infection was screened using a RT-qPCR assay that has been validated to detect EBOV, SUDV, RESTV, TAFV, BDBV and MARV 54 . Previous exposure to filoviruses was screened using a previously validated Luminex assay 26 . Blood was collected from the cephalic wing vein using a sterile needle and a micropipette (p-100) and placed in 300 μl gel tubes (BD Microtainer®) for serum separation (5 min centrifugation at 8000 x g).…”

Rift Valley Fever Virus: An Overview of the Current Status of Diagnostics

“…Finally, the development of multiplex detection systems serves numerous purposes, such as surveillance, monitoring the presence of RVFV in animals or humans, diagnosis, confirming RVFV infection, and research, studying the biology of RVFV and developing new vaccines and treatments. Despite some limitations, such as complexity, elevated cost and training requirements, compared to traditional diagnostic methods, multiplex detection systems offer several advantages: sensitivity, detecting smaller amounts of viral antigens; specificity, distinguishing between different pathogens; and rapidity, delivering results relatively quickly [ 64 , 65 ].…”

“…Multiplex detection systems for the detection of antibodies against multiple highly pathogenic agents simultaneously are being developed, making them valuable tools for disease surveillance and diagnosis [ 64 , 65 ]. In the case of RVFV, a specific in situ hybridization (ISH) has been reported for the detection of viral RNA of several RVFV strains in different fixed tissues [ 66 ].…”

Multiplex Assay for Simultaneous Detection of Antibodies against Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein and Glycoproteins in Ruminants

“…Such an assay could be used to detect antibodies against all the CCHFV proteins in the same assay and to develop a DIVA assay depending on the CCHFV vaccine candidates. In addition, other targets from different pathogens could be included in the assay for the simultaneous surveillance of multiple pathogens ( 31 ). Multiplex assays reduce the time, labor, and sample volume requirements compared to individual ELISAs and could be very useful for surveillance studies, to control the spread of viruses and prevent future outbreaks, and to better understand the immune response induced by certain viruses.…”