First detection of Rift Valley fever virus antibodies in non-human primates in Cameroon.

We tested for Rift Valley fever virus (RVFV) from at least 15 species of non-human primates. RVFV IgG/IgM antibodies were detected in 3.7% (2 out of 53) of chimpanzees (Pan troglodytes) and in 1.4% (1 out of 72) of unidentified non-human primate species. This study was the first investigation of RVFV in monkeys in Cameroon.

Unique Outbreak of Rift Valley Fever in Sudan, 2019.

We report a unique outbreak of Rift Valley fever in the Eldamar area, Sudan, May-July 2019, that resulted in 1,129 case-patients and 19 (1.7%) deaths. Patients exhibited clinical signs including fever (100%), headache (79%), and bleeding (4%). Most (98%) patients also reported death and abortions among their livestock.

The challenging management of Rift Valley Fever in humans: literature review of the clinical disease and algorithm proposal.

Rift Valley Fever (RVF) is an emerging zoonotic arbovirus with a complex cycle of transmission that makes difficult the prediction of its expansion. Recent outbreaks outside Africa have led to rediscover the human disease but it remains poorly known. The wide spectrum of acute and delayed manifestations with potential unfavorable outcome much complicate the management of suspected cases and prediction of morbidity and mortality during an outbreak. We reviewed literature data on bio-clinical characteristics and treatments of RVF human illness. We identified gaps in the field and provided a practical algorithm to assist clinicians in the cases assessment, determination of setting of care and prolonged follow-up.

Lesions and Cellular Tropism of Natural Rift Valley Fever Virus Infection in Young Lambs.

A clear distinction can be made regarding the susceptibility to and the severity of lesions in young lambs when compared to adult sheep. In particular, there are important differences in the lesions and tropism of Rift Valley fever virus (RVFV) in the liver, kidneys, and lymphoid tissues of young lambs. A total of 84 lambs (<6 weeks old), necropsied during the 2010 to 2011 Rift Valley fever (RVF) outbreak in South Africa, were examined by histopathology and immunohistochemistry (IHC). Of the 84 lambs, 71 were positive for RVFV. The most striking diagnostic feature in infected lambs was diffuse necrotizing hepatitis with multifocal liquefactive hepatic necrosis (primary foci) against a background of diffuse hepatocellular death. Lymphocytolysis was present in all lymphoid organs except for the thymus. Lesions in the kidney rarely progressed beyond hydropic change and occasional pyknosis or karyolysis in renal tubular epithelial cells. Viral antigen was diffusely present in the cytoplasm of hepatocytes, but this labeling was noticeably sparse in primary foci. Immunolabeling for RVFV in young lambs was also detected in macrophages, vascular smooth muscle cells, adrenocortical epithelial cells, renal tubular epithelial cells, renal perimacular cells, and cardiomyocytes. RVFV immunolabeling was also often present in capillaries and small blood vessels either as non-cell-associated viral antigen, as antigen in endothelial cells, or intravascular cellular debris. Specimens from the liver, spleen, kidney, and lungs were adequate to confirm a diagnosis of RVF. Characteristic lesions were present in these organs with the liver and spleen being the most consistently positive for RVFV by IHC.

Evaluation of an Indirect Enzyme-Linked Immunosorbent Assay Based on Recombinant Baculovirus-Expressed Rift Valley Fever Virus Nucleoprotein as the Diagnostic Antigen.

The increasing risk of Rift Valley fever virus (RVFV) infection as a global veterinary and public health threat demands the development of safe and accurate diagnostic tests. The aim of this study was to assess the suitability of a baculovirus expression system to produce recombinant RVFV nucleoprotein (N) for use as serodiagnostic antigen in an indirect enzyme-linked immunosorbent assay (ELISA). The ability of the recombinant N antigen to detect RVFV antibody responses was evaluated in ELISA format using antisera from sheep and cattle experimentally infected with two genetically distinct wild-type RVFV strains and sera from indigenous sheep and goat populations exposed to natural RVFV field infection in The Gambia. The recombinant N exhibited specific reactivity with the N-specific monoclonal antibody and various hyperimmune serum samples from ruminants. The indirect ELISA detected N-specific antibody responses in animals with 100% sensitivity compared to the plaque reduction neutralization test (6 to 21 days postinfection) and with 97% and 100% specificity in sheep and cattle, respectively. There was a high level of correlation between the indirect N ELISA and the virus neutralization test for sheep sera (R2 = 0.75; 95% confidence interval [CI] = 0.73 to 0.92) and cattle sera (R2 = 0.80; 95% CI = 0.67 to 0.97); in addition, the N-specific ELISA detected RVFV seroprevalence levels of 26.1% and 54.3% in indigenous sheep and goats, respectively, in The Gambia. The high specificity and correlation with the virus neutralization test support the idea of the feasibility of using the recombinant baculovirus-expressed RVFV N-based indirect ELISA to assess RVFV seroprevalence in livestock in areas of endemicity and nonendemicity.

Tracking Rift Valley fever: From Mali to Europe and other countries, 2016.

On 16 September 2016, the World Health Organization confirmed a Rift Valley fever (RVF) outbreak in Niger. Epidemiological surveillance was reinforced among the French Armed Forces deployed in Niger and bordering countries: Chad, Mali and Burkina Faso. On 26 October, a probable case of RVF was reported in a service member sampled in Mali 3 weeks earlier. At the time the result was reported, the patient was on vacation on Martinique. An epidemiological investigation was conducted to confirm this case and identify other cases. Finally, the case was not confirmed, but three suspected cases of RVF were confirmed using serological and molecular testing. RVF viral RNA was detectable in whole blood for 57 and 67 days after onset of symptoms for two cases, although it was absent from plasma and serum. At the time of diagnosis, these cases had already returned from Mali to Europe. The infectivity of other arboviruses in whole blood has already been highlighted. That RVF virus has been detected in whole blood that long after the onset of symptoms (67 days) raises the question of its potential prolonged infectivity. Because of exposure to tropical infectious diseases during deployment, military populations could import emerging pathogens to Europe.

Expression of Rift Valley fever virus N-protein in Nicotiana benthamiana for use as a diagnostic antigen.

BACKGROUND: Rift Valley fever virus (RVFV), the causative agent of Rift Valley fever, is an enveloped single-stranded negative-sense RNA virus in the genus Phlebovirus, family Bunyaviridae. The virus is spread by infected mosquitoes and affects ruminants and humans, causing abortion storms in pregnant ruminants, high neonatal mortality in animals, and morbidity and occasional fatalities in humans. The disease is endemic in parts of Africa and the Arabian Peninsula, but is described as emerging due to the wide range of mosquitoes that could spread the disease into non-endemic regions. There are different tests for determining whether animals are infected with or have been exposed to RVFV. The most common serological test is antibody ELISA, which detects host immunoglobulins M or G produced specifically in response to infection with RVFV. The presence of antibodies to RVFV nucleocapsid protein (N-protein) is among the best indicators of RVFV exposure in animals. This work describes an investigation of the feasibility of producing a recombinant N-protein in Nicotiana benthamiana and using it in an ELISA. RESULTS: The human-codon optimised RVFV N-protein was successfully expressed in N. benthamiana via Agrobacterium-mediated infiltration of leaves. The recombinant protein was detected as monomers and dimers with maximum protein yields calculated to be 500-558 mg/kg of fresh plant leaves. The identity of the protein was confirmed by liquid chromatography-mass spectrometry (LC-MS) resulting in 87.35% coverage, with 264 unique peptides. Transmission electron microscopy revealed that the protein forms ring structures of ~ 10 nm in diameter. Preliminary data revealed that the protein could successfully differentiate between sera of RVFV-infected sheep and from sera of those not infected with the virus. CONCLUSIONS: To the best of our knowledge this is the first study demonstrating the successful production of RVFV N-protein as a diagnostic reagent by Agrobacterium-mediated transient heterologous expression in N. benthamiana. Preliminary testing of the antigen showed its ability to distinguish RVFV-positive animal sera from RVFV negative animal sera when used in an enzyme linked immunosorbent assay (ELISA). The cost-effective, scalable and simple production method has great potential for use in developing countries where rapid diagnosis of RVFV is necessary.

Evaluation of Fluorescence Microsphere Immunoassay for Detection of Antibodies to Rift Valley Fever Virus Nucleocapsid Protein and Glycoproteins.

Rift Valley fever virus (RVFV) is a mosquito-borne, zoonotic virus that infects ruminants, including cattle, sheep, goats, camels, and buffalo. Multiplexing diagnostic assays that can simultaneously detect antibodies against multiple RVFV antigens offer a high-throughput test for disease surveillance and vaccine evaluations. We describe the improvement and evaluation of a previously developed fluorescence microsphere immunoassay (FMIA) for the detection of IgG and IgM antibodies against the RVFV glycoprotein (Gn) and the immunogenic nucleocapsid protein (Np). Well-characterized vaccinated and experimentally infected ruminant sera were used for the evaluation of the assay. Recombinant viral proteins were produced and then coupled to polystyrene magnetic beads for analysis using the Luminex MAGPIX system with xMAP technology. The FMIA was performed in parallel with virus neutralization tests. Our results revealed the highest median fluorescence intensity (MFI) values for the detection of IgG antibodies against RVFV Np, indicating that this antigen would be a good candidate for a screening assay. The Np and Gn targets could differentiate infected animals from animals vaccinated with a candidate subunit vaccine formulation based on the RVFV Gn and Gc proteins. The results presented in this report demonstrate that FMIA provides a rapid and robust serological diagnostic tool for the detection of antibodies against RVFV. The targets developed in this assay provide the basis for the development of a companion diagnostic test for an RVFV Gn/Gc subunit vaccine that is capable of differentiating infected from vaccinated animals (DIVA), as well as a multiplex serodiagnostic assay that can simultaneously screen for several ruminant diseases.

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.

Sero-prevalence and spatial distribution of Rift Valley fever infection among agro-pastoral and pastoral communities during Interepidemic period in the Serengeti ecosystem, northern Tanzania.

BACKGROUND: In the past two decades, Rift Valley Fever (RVF) outbreaks have been reported twice in Tanzania, with the most recent outbreak occurring in 2006/07. Given the ecology and climatic factors that support mosquito vectors in the Serengeti ecosystem, we hypothesized a continued transmission of RVF virus (RVFV) during interepidemic periods. This study was carried out to determine sero-prevalence, spatial distribution and factors associated with RVF in at-risk agro-pastoral and pastoral communities in the Serengeti Ecosystem in northern Tanzania. METHODS: A cross sectional study was carried out to establish the general exposure to RVFV by detecting anti-RVFV IgG and anti-RVFV IgM using ELISA techniques. The health facilities where human subjects were blood sampled concurrent with interviews included Bunda District Designated Hospital, Wasso DDH, Endulen hospital, Arash, Malambo, Olbabal, and Piyaya dispenaries (Ngorongoro district) and Nyerere DDH (Serengeti district) respectively. In addition, human subjects from Lamadi ward (Busega district) were recruited while receiving medical service at Bunda DDH. We conducted logistic regression to assess independent risk factor and mapped the hotspot areas for exposure to RVFV. RESULTS: A total of 751 subjects (males = 41.5%; females = 58.5%) with a median age of 35.5 years were enrolled at out-patient clinics. Of them, 34 (4.5, 95%CI 3.3-6.3%) tested positive for anti-RVFV IgG. Of the 34 that tested positive for anti-RVFV IgG, six (17.6%) tested positive for anti-RVFV IgM. Odds of exposure were higher among pastoral communities (aOR 2.9, 95% C.I: 1.21-6.89, p < 0.01), and agro-pastoral communities residing in Ngorongoro District (aOR 1.8, 95% C.I 1.14-3.39, p = 0.03). Hotspot areas for exposure to RVFV were Malambo, Olbalbal and Piyaya wards in Ngorongoro district, and Lamadi ward in Busega district. CONCLUSIONS: The study found both previous and recent exposure of RVFV in humans residing in the Serengeti ecosystem as antibodies against both IgG and IgM were detected. Detection of anti-RVF IgM suggests an ongoing transmission of RVFV in humans during inter-epidemic periods. Residents of Ngorongoro district were most exposed to RVFV compared to Bunda and Serengeti districts. Therefore, the risk of exposure to RVFV was higher among pastoral communities compared to farming communities.

Serological evidence of rift valley fever virus among acute febrile patients in Southern Mozambique during and after the 2013 heavy rainfall and flooding: implication for the management of febrile illness.

BACKGROUND: Rift Valley fever virus (RVFV) remains heavily neglected in humans in Mozambique, even though recent outbreaks were reported in neighboring countries in humans and several cases of RVFV in cattle were reported in several districts in Mozambique. FINDINGS: We conducted a cross sectional study during and after severe flooding that occurred in 2013 in Mozambique. Paired acute and convalescent serum samples were tested from febrile patients attending a primary health care unit in a suburban area of Maputo city for the presence of IgG and IgM antibodies against Rift Valley fever virus (RVFV) using enzyme-linked immunosorbent assay (ELISA). Seroconversion of IgG anti-RVFV was observed in 5 % (10/200) of convalescent patients and specific IgM anti-RVFV was detected in one acute patient (0.5 %; 1/200). All sera from acute patient tested negative by real time PCR. CONCLUSION: In conclusion, our results suggest that RVF represent an important but neglected cause of febrile illness following periods of flooding in southern Mozambique.

Notes from the Field: Rift Valley Fever Response - Kabale District, Uganda, March 2016.

On March 9, 2016, a male butcher from Kabale District, Uganda, aged 45 years, reported to the Kabale Regional Referral Hospital with fever, fatigue, and headache associated with black tarry stools and bleeding from the nose. One day later, a student aged 16 years from a different sub-county in Kabale District developed similar symptoms and was admitted to the same hospital. The student also had a history of contact with livestock. Blood specimens collected from both patients were sent for testing for Marburg virus disease, Ebola virus disease, Rift Valley fever (RVF), and Crimean Congo Hemorrhagic fever at the Uganda Virus Research Institute, as part of the viral hemorrhagic fevers surveillance program. The Uganda Virus Research Institute serves as the national viral hemorrhagic fever reference laboratory and hosts the national surveillance program for viral hemorrhagic fevers, in collaboration with the CDC Viral Special Pathogens Branch and the Uganda Ministry of Health.

Rift Valley fever in kidney transplant recipient returning from Mali with viral RNA detected in semen up to four months from symptom onset, France, autumn 2015.

A 29-year-old kidney transplant recipient returning from Mali was diagnosed with Rift Valley fever (RVF) in France in autumn 2015. The patient was immunosuppressed due to his renal transplant. IgM and IgG specific to RVF virus (RVFV) were detected in cerebrospinal fluid and blood up to two months after symptom onset, whereas in urine, RVFV genomic RNA was detected by RT-PCR up to three months, and in semen up to four months post symptom onset.

Generation and application of monoclonal antibodies against Rift Valley fever virus nucleocapsid protein NP and glycoproteins Gn and Gc.

Rift Valley fever virus (RVFV) is a vector-borne virus that causes high neonatal mortality in livestock and deadly haemorrhagic fever in humans. In this paper, we describe the generation of monoclonal antibodies (mabs) against all three structural proteins of RVFV (glycoproteins Gn and Gc and nucleocapsid protein NP). After immunization of BALB/c mice with individual recombinant proteins, a total of 45 clones secreting ELISA-reactive monoclonal antibodies against NP, Gn and Gc epitopes were obtained. Twelve clones were directed to NP, 28 to Gn, and 5 to Gc. Western blot analysis revealed that most of the mabs were reactive to linearized epitopes on recombinant as well as native virus proteins. Six mabs against NP, 21 against Gn and all mabs against Gc also detected conformational epitopes, as shown by indirect immunofluorescence on RVFV-infected cells. All of the mabs were evaluated for their use in a competition enzyme-linked immunosorbent assay (ELISA) for the detection of a RVFV infection. Several mabs were identified that competed with polyclonal rabbit serum, and one of them - mab Gn123, raised against Gn protein - was selected for a proof-of-principle study with field sera from a recent Rift Valley fever outbreak. The novel Gn-based competition ELISA demonstrated high performance, offering a promising alternative and addition to serological assays based on nucleocapsid protein.

Isolation of Rift Valley Fever Virus from Field Specimens Using Cell Culture Approaches.

Several techniques have been developed to diagnose Rift Valley fever infection. Viral isolation is one of the most difficult techniques to apply but offers great opportunities for further research. It is useful, for example, for the development of an accurate diagnostic test suitable for screening for Rift Valley fever virus infection, specific treatments by testing known antiviral molecules that act on the replication cycle to assess their therapeutic or even prophylactic potential, therapeutic applications, and vaccine candidates. Understanding how the virus replicates and interacts with the host cell and organism and identifying biomarkers of infection or new targets for the development of treatments are made possible through field virus isolates. Biosafety level 3 conditions are a pre-requisite for viral isolation by a trained staff member. Here, we describe the procedure to isolate Rift Valley fever virus from field samples by cell culture.

Analysis of Negative-Strand RNA Viruses by RT-qPCR: Rift Valley Fever Virus and Toscana Virus.

RT-qPCR allows the detection of viruses and the monitoring of viral replication. This technique was extensively employed during the SARS-CoV-2 pandemic, where it demonstrated its efficiency and robustness. Here we describe the analysis of Rift Valley fever and Toscana virus infections over time, achieved through the RT-qPCR quantification of the viral genome. We further elaborate on the method to discriminate between genomic and antigenomic viral RNAs by using primers specific for each strand during the reverse transcription step.

Detection and Diagnosis of Rift Valley Fever Virus.

Rift Valley fever virus (RVFV) is a globally important mosquito-borne virus that can also be directly transmitted via aerosolization of body fluids from infected animals. RVFV outbreaks cause mass mortality of young livestock and abortions in animals. In most severe human cases, the disease can progress to hemorrhagic fever and encephalitis, leading to death. RVF has a significant economic impact due to the loss of livestock that is a great challenge for people who depend on animals for income and food. Several vaccines are available for animal use, but none are yet licensed for use in human populations. This situation emphasizes the need to have robust and efficient diagnostic methods that can be used for early case confirmation, assessment of seroprevalence, and virus surveillance as well as vaccine efficacy evaluation. Despite the existence of different diagnostic methods for RVFV, we still have untimely reporting or underreporting of cases, probably due to lack of appropriate surveillance systems or diagnostic tools in some endemic countries. Here, we describe different methods available for detection and diagnosis of RVFV.

Use of Single-Domain Antibodies Against the NSm Protein for the Detection of Cells Infected by Rift Valley Fever Virus.

Single-domain antibodies, referred to as VHH (variable heavy chains of heavy chain-only antibodies) or in their commercial name as nanobodies, are potent tools for the detection of target proteins in biological samples. They have the advantage of being highly stable, specific, and sensitive, with affinities reaching the nanomolar range. We utilized this tool to develop a rapid detection method that discriminates cells infected with Rift Valley fever virus (RVFV), based on the intracellular detection of the viral nonstructural NSm protein localized on the outer membrane of mitochondria. Here we describe how NSm-specific VHHs have been produced, cloned, and characterized, highlighting their value in RVFV research and diagnosis. This work may also raise interest in other potential applications such as antiviral therapy.

Evaluation of commercial ELISA kits' diagnostic specificity for FAST diseases in wild animals.

Wild animals, sharing pathogens with domestic animals, play a crucial role in the epidemiology of infectious diseases. Sampling from wild animals poses significant challenges, yet it is vital for inclusion in disease surveillance and monitoring programmes. Often, mass surveillance involves serological screenings using enzyme-linked immunosorbent assay (ELISA) tests, typically validated only for domestic animals. This study assessed the diagnostic specificity of commercially available ELISA tests on 342 wild ruminant serum samples and 100 from wild boars. We evaluated three tests for foot-and-mouth disease: two for Peste des petits ruminants, two for Rift Valley fever and one for Capripox virus. Diagnostic specificity was calculated using the formula True Negative/(False Positive + True Negative). Cohen's kappa coefficient measured agreement between tests. Results showed high specificity and agreement across all tests. Specificity for foot-and-mouth disease (FMD) ranged from 93.89% for Prionics to 100% for IDEXX, with IDvet showing 99.6%. The highest agreement was between FMD IDvet and IDEXX at 97.1%. Rift Valley fever (RVF) tests, Ingezim and IDvet, achieved specificities of 100% and 98.83%, respectively. The optimal specificity was attained by retesting single reactors and inactivating the complement.Contribution: Commercially available ELISA kits are specific for foot-and-mouth disease and similar transboundary animal diseases and can be used for highly specific wild animal testing.

Monitoring RVFV Infection Using Bioluminescent Reporter Viruses In Vivo.

Rift Valley fever virus is able to infect multiple organs and cell types, and the course of infection varies between viral strains and between individuals in particular according to age, genetic background, and physiological status. Studies on viral and host factors involve detecting and quantifying viral load at multiple time points and in multiple tissues. While this is classically performed by genome quantification or viral titration, in vivo imaging techniques using recombinant viruses expressing a bioluminescent or fluorescent protein allow noninvasive longitudinal studies on the same group of mice over the entire course of disease and the detection of unsuspected sites of infection. Here, we describe the protocol to monitor and characterize mouse infection with Rift Valley fever virus by in vivo imaging using recombinant viruses expressing light-emitting reporter genes.