Comparison of enzyme-linked immunosorbent assay systems using rift valley fever virus nucleocapsid protein and inactivated virus as antigens.

BACKGROUND: Rift Valley Fever (RVF) is a mosquito-borne viral zoonosis. To detect RVF virus (RVFV) infection, indirect immunoglobulin G (IgG) and immunoglobulin M (IgM) enzyme linked immunosorbent assays (ELISAs) which utilize recombinant RVFV nucleocapsid (RVFV-N) protein as assay antigen, have reportedly been used, however, there is still a need to develop more sensitive and specific methods of detection. METHODS: RVFV-N protein was expressed in Escherichia coli (E. coli) and purified by histidine-tag based affinity chromatography. This recombinant RVFV-N (rRVFV-N) protein was then used as antigen to develop an IgG sandwich ELISA and IgM capture ELISAs for human sera. Ninety six serum samples collected from healthy volunteers during the RVF surveillance programme in Kenya in 2013, and 93 serum samples collected from RVF-suspected patients during the 2006-2007 RVF outbreak in Kenya were used respectively, to evaluate the newly established rRVFV-N protein-based IgG sandwich ELISA and IgM capture ELISA systems in comparison with the inactivated virus-based ELISA systems. RESULTS: rRVFV-N protein-based-IgG sandwich ELISA and IgM capture ELISA for human sera were established. Both the new ELISA systems were in 100% concordance with the inactivated virus-based ELISA systems, with a sensitivity and specificity of 100%. CONCLUSIONS: Recombinant RVFV-N is a safe and affordable antigen for RVF diagnosis. Our rRVFV-N-based ELISA systems are safe and reliable tools for diagnosis of RVFV infection in humans and especially useful in large-scale epidemiological investigation and for application in developing countries.

Monoclonal Antibodies for Rift Valley Fever Virus Nucleocapsid: Application in IgG/IgM ELISA for Sero-Diagnosis.

INTRODUCTION: Rift Valley fever virus (RVFV) belonging to the Phenuiviridae family is responsible for a zoonotic disease called Rift Valley fever (RVF). Currently, RVFV has spread from Africa to Asia, and due to its ability to cause high mortality rates, it has significantly impacted human health and economic development in many societies. Highly specific and sensitive systems for sero-diagnosis of RVFV infection are needed for clinical use. METHOD: BALB/c mice were immunized with recombinant RVFV nucleocapsid (rRVFV-N) protein and the spleen cells fused with SP2/0 myeloma cells to create hybridoma cell lines. The secreted monoclonal antibodies (MAbs) were purified and characterized. Enzyme-linked immunosorbent assay (ELISA) systems for the detection of IgG and IgM using the new MAbs were established and evaluated. Serum samples from 96 volunteers and 93 patients of suspected RVF from Kenya were tested compared with the ELISA systems based on inactivated viruses and the rabbit polyclonal antibody. RESULT: Three monoclonal antibodies against rRVFV-N protein were established. The performance of the MAb-based sandwich IgG ELISA and the IgM capture ELISA perfectly matched the ELISA systems using the inactivated virus or the polyclonal antibody. CONCLUSIONS: Recombinant RVFV-N protein-specific MAbs were developed and they offer useful tools for RVFV studies. The MAb-based ELISA systems for detecting IgG and IgM offer safe and useful options for diagnosing RVFV infections in humans.

Global Outbreaks and Origins of a Chikungunya Virus Variant Carrying Mutations Which May Increase Fitness for Aedes aegypti: Revelations from the 2016 Mandera, Kenya Outbreak.

In 2016, a chikungunya virus (CHIKV) outbreak was reported in Mandera, Kenya. This was the first major CHIKV outbreak in the country since the global reemergence of this virus in Kenya in 2004. We collected samples and sequenced viral genomes from this outbreak. All Kenyan genomes contained two mutations, E1:K211E and E2:V264A, recently reported to have an association with increased infectivity, dissemination, and transmission in the Aedes aegypti vector. Phylogeographic inference of temporal and spatial virus relationships showed that this variant emerged within the East, Central, and South African lineage between 2005 and 2008, most probably in India. It was also in India where the first large outbreak caused by this virus appeared, in New Delhi, 2010. More importantly, our results also showed that this variant is no longer contained to India. We found it present in several major outbreaks, including the 2016 outbreaks in Pakistan and Kenya, and the 2017 outbreak in Bangladesh. Thus, this variant may have a capability of driving large CHIKV outbreaks in different regions of the world. Our results point to the importance of continued genomic-based surveillance and prompt urgent vector competence studies to assess the level of vector susceptibility and virus transmission, and the impact this might have on this variant's epidemic potential and global spread.

Human and entomologic investigations of chikungunya outbreak in Mandera, Northeastern Kenya, 2016.

Chikungunya is a reemerging vector borne pathogen associated with severe morbidity in affected populations. Lamu, along the Kenyan coast was affected by a major chikungunya outbreak in 2004. Twelve years later, we report on entomologic investigations and laboratory confirmed chikungunya cases in northeastern Kenya. Patient blood samples were received at the Kenya Medical Research Institute (KEMRI) viral hemorrhagic fever laboratory and the immunoglobulin M enzyme linked immunosorbent assay (IgM ELISA) was used to test for the presence of IgM antibodies against chikungunya and dengue. Reverse transcription polymerase chain reaction (RT-PCR) utilizing flavivirus, alphavirus and chikungunya specific primers were used to detect acute infections and representative PCR positive samples sequenced to confirm the circulating strain. Immature mosquitoes were collected from water-holding containers indoors and outdoors in the affected areas in northeastern Kenya. A total of 189 human samples were tested; 126 from Kenya and 63 from Somalia. 52.9% (100/189) tested positive for Chikungunya virus (CHIKV) by either IgM ELISA or RT-PCR. Sequence analysis of selected samples revealed that the virus was closely related to that from China (2010). 29% (55/189) of the samples, almost all from northeastern Kenya or with a history of travel to northern Kenya, tested positive for dengue IgM antibodies. Entomologic risk assessment revealed high house, container and Breteau indices of, 14.5, 41.9 and 17.1% respectively. Underground water storage tanks were the most abundant, 30.1%, of which 77.4% were infested with Aedes aegypti mosquitoes. These findings confirm the presence of active chikungunya infections in the northeastern parts of Kenya. The detection of dengue IgM antibodies concurrently with chikungunya virus circulation emphasizes on the need for improved surveillance systems and diagnostic algorithms with the capacity to capture multiple causes of arbovirus infections as these two viruses share common vectors and eco-systems. In addition sustained entomological surveillance and vector control programs targeting most productive containers are needed to monitor changes in vector densities, for early detection of the viruses and initiate vector control efforts to prevent possible outbreaks.

A household serosurvey to estimate the magnitude of a dengue outbreak in Mombasa, Kenya, 2013.

Dengue appears to be endemic in Africa with a number of reported outbreaks. In February 2013, several individuals with dengue-like illnesses and negative malaria blood smears were identified in Mombasa, Kenya. Dengue was laboratory confirmed and an investigation was conducted to estimate the magnitude of local transmission including a serologic survey to determine incident dengue virus (DENV) infections. Consenting household members provided serum and were questioned regarding exposures and medical history. RT-PCR was used to identify current DENV infections and IgM anti-DENV ELISA to identify recent infections. Of 1,500 participants from 701 households, 210 (13%) had evidence of current or recent DENV infection. Among those infected, 93 (44%) reported fever in the past month. Most (68, 73%) febrile infected participants were seen by a clinician and all but one of 32 participants who reportedly received a diagnosis were clinically diagnosed as having malaria. Having open windows at night (OR = 2.3; CI: 1.1-4.8), not using daily mosquito repellent (OR = 1.6; CI: 1.0-2.8), and recent travel outside of Kenya (OR = 2.5; CI: 1.1-5.4) were associated with increased risk of DENV infection. This survey provided a robust measure of incident DENV infections in a setting where cases were often unrecognized and misdiagnosed.