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.

The intersection of land use and human behavior as risk factors for zoonotic pathogen exposure in Laikipia County, Kenya.

A majority of emerging infectious diseases (EIDs) are zoonotic, mainly caused through spillover events linked to human-animal interactions. We conducted a survey-based human behavioral study in Laikipia County, Kenya, which is characterized by a dynamic human-wildlife-livestock interface. Questionnaires that assessed human-animal interactions, sanitation, and illnesses experienced within the past year were distributed to 327 participants among five communities in Laikipia. This study aimed to 1) describe variation in reported high-risk behaviors by community type and 2) assess the relationship between specific behaviors and self-reported illnesses. Behavioral trends were assessed in R via Fisher's exact tests. A generalized linear mixed model with Lasso penalization (GLMMLasso) was used to assess correlations between behaviors and participants' self-reported illness within the past year, with reported behaviors as independent variables and reported priority symptoms as the outcome. Reported behaviors varied significantly among the study communities. Participants from one community (Pastoralist-1) were significantly more likely to report eating a sick animal in the past year (p< 0.001), collecting an animal found dead to sell in the past year (p<0.0001), and not having a designated location for human waste (p<0.0001) when compared to participants from other communities. The GLMMLasso revealed that reports of an ill person in the household in the past year was significantly associated with self-reported illness. Sixty-eight percent of participants reported that bushmeat is available within the communities. Our study demonstrates community-level variation in behaviors that may influence zoonotic pathogen exposure. We further recommend development of targeted studies that explore behavioral variations among land use systems in animal production contexts.

Seroprevalence of yellow fever virus in selected health facilities in Western Kenya from 2010 to 2012.

Yellow fever (YF), which is caused by a mosquito-borne virus, is an important viral hemorrhagic fever endemic in equatorial Africa and South America. Yellow fever virus (YFV) is the prototype of the family Flaviviridae and genus Flavivirus. The aim of this study was to determine the seroprevalence of YFV in selected health facilities in Western Kenya during the period 2010-2012. A total of 469 serum samples from febrile patients were tested for YFV antibodies using in-house IgM-capture ELISA, in-house indirect IgG ELISA, and 50% focus reduction neutralization test (FRNT50). The present study did not identify any IgM ELISA-positive cases, indicating absence of recent YFV infection in the area. Twenty-eight samples (6%) tested positive for YFV IgG, because of either YFV vaccination or past exposure to various flaviviruses including YFV. Five cases were confirmed by FRNT50; of these, 4 were either vaccination or natural infection during the YF outbreak in 1992-1993 or another period and 1 case was confirmed as a West Nile virus infection. Domestication and routine performance of arboviral differential diagnosis will help to address the phenomenon of pyrexia of unknown origin, contribute to arboviral research in developing countries, and enhance regular surveillance.

A real-time reverse transcription loop-mediated isothermal amplification assay for the rapid detection of yellow fever virus.

Yellow fever, a mosquito-borne disease, is an important viral hemorrhagic fever in Africa and South America where it is endemic. Detection of yellow fever virus (YFV) in Africa remains a challenge due to a lack of highly specific tests. The aim of this study was to develop and optimize a rapid detection reverse transcription loop-mediated isothermal amplification (RT-LAMP) for YFV. The RT-LAMP was done isothermally at 62 °C using a real-time turbidimeter that allowed detection within 1h. Specificity of the RT-LAMP was determined using RNA from flaviviruses and other related viruses where only YFV RNA was detected: West Nile virus, dengue viruses, Japanese encephalitis virus, Rift Valley fever virus, and chikungunya virus. In addition, equal sensitivity was also observed when the RT-LAMP and the real-time RT-PCR were compared using YFV-spiked human serum samples with a detection limit of 0.29 PFU/ml. Two Kenyan YFV wild strains showed an equal detection limit as the vaccine strain 17D in this study. The RT-LAMP reduced the time of reaction from 3h to 1h and increased sensitivity tenfold compared to RT-PCR. Therefore, this test offers a simple, rapid and reliable diagnostic tool for yellow fever when there are outbreaks of acute hemorrhagic fever in Kenya and other African countries.