Rift Valley Fever Virus Antigen Research Articles

Rift Valley Fever Virus Infects the Posterior Segment of the Eye and Induces Inflammation in a Rat Model of Ocular Disease.

ABSTRACTPeople infected with the mosquito-borne Rift Valley fever virus (RVFV) can suffer from eye-related problems resulting in ongoing vision issues or even permanent blindness. Despite ocular disease being the most frequently reported severe outcome, it is vastly understudied compared to other disease outcomes caused by RVFV. Ocular manifestations of RVFV include blurred vision, uveitis, and retinitis. When an infected individual develops macular or paramacular lesions, there is a 50% chance of permanent vision loss in one or both eyes. The cause of blinding ocular pathology remains unknown in part due to the lack of a tractable animal model. Using 3 relevant exposure routes, both subcutaneous (SC) and aerosol inoculation of Sprague Dawley rats led to RVFV infection of the eye. Surprisingly, direct inoculation of the conjunctiva did not result in successful ocular infection. The posterior segment of the eye, including the optic nerve, choroid, ciliary body, and retina, were all positive for RVFV antigen in SC-infected rats, and live virus was isolated from the eyes. Proinflammatory cytokines and increased leukocyte counts were also found in the eyes of infected rats. Additionally, human ocular cell lines were permissive for Lrp1-dependent RVFV infection. This study experimentally defines viral tropism of RVFV in the posterior segment of the rat eye and characterizes virally-mediated ocular inflammation, providing a foundation for evaluation of vaccines and therapeutics to protect against adverse ocular outcomes.IMPORTANCE Rift Valley fever virus (RVFV) infection leads to eye damage in humans in up to 10% of reported cases. Permanent blindness occurs in 50% of individuals with significant retinal scarring. Despite the prevalence and severity of this outcome, very little is known about the mechanisms of pathogenesis. We addressed this gap by developing a rodent model of ocular disease. Subcutaneous infection of Sprague Dawley rats resulted in infection of the uvea, retina, and optic nerve along with the induction of inflammation within the posterior eye. Infection of human ocular cells induced inflammatory responses and required host entry factors for RVFV infection similar to rodents. This work provides evidence of how RVFV infects the eye, and this information can be applied to help mitigate the devastating outcomes of RVF ocular disease through vaccines or treatments.

Adaptation to a Multiplex Bead Assay and Seroprevalence to Rift Valley Fever N Protein: Nampula Province, Mozambique, 2013-2014.

Rift Valley fever virus (RVFV) is endemic in sub-Saharan Africa (SSA), with outbreaks reported in the Arabian Peninsula and throughout SSA. The natural reservoir for RVFV are ruminants, with livestock populations exceeding 50% exposure rates in some areas of SSA. Transmission to humans can occur through exposure to infected livestock products or multiple species of mosquito vectors. In 2013 and 2014, cross-sectional surveys occurred in two districts of Nacala-a-Velha and Mecubúri in northern Mozambique, and participants provided blood samples for later serological assays. IgG against the N protein of RVFV was detected through multiplex bead assay (MBA). Of the 2,278 persons enrolled between the two surveys and study sites, 181 (7.9%, 95% confidence interval (CI): 6.9%-9.1%) were found to be IgG seropositive with increasing seroprevalence with older age and significantly higher seroprevalence in Nacala-a-Velha (10.5%, 8.8%-12.5%) versus Mecubúri (5.7%, 4.5%-7.1%). Seroprevalence estimates were not significantly different between the 2013 and 2014 surveys. Significant spatial clustering of IgG positive persons were consistent among surveys and within the two districts, pointing toward the consistency of serology data for making population-level assumptions regarding RVFV seroprevalence. A subset of persons (n = 539) provided samples for both the 2013 and 2014 surveys, and a low percentage (0.81%) of these were found to seroconvert between these two surveys. Including the RVFV N protein in an MBA antigen panel could assist elucidate RVFV exposure in SSA. IMPORTANCE Due to sporadic transmission, human contact with Rift Valley Fever Virus (RVFV) is difficult to ascertain at a population level. Detection of antibodies against RVFV antigens assist in estimating exposure as antibodies remain in the host long after the virus has been cleared. In this study, we show that antibodies against RVFV N protein can be detected from dried blood spot (DBS) samples being assayed by multiplex bead assay. DBS from two districts in northern Mozambique were tested for IgG against the N protein, and 7.9% of all enrolled persons were seropositive. Older persons, males, and persons residing closer to the coast had higher RVFV N protein seroprevalence. Spatial clustering of IgG positive persons was noted in both districts. These results show low exposure rates to RVFV in these two northern districts in Mozambique, and the ability to perform serology for the RVFV N protein from dried blood samples.

Myeloid-like γδ T cell subset in the immune response to an experimental Rift Valley fever vaccine in sheep

γδ T cells are a numerically significant subset of immune cells in ruminants, where they may comprise up to 70 % of all peripheral blood mononuclear cells (PBMCs) in young animals and 25 % in adults. These cells can be activated through traditional TCR-dependent mechanisms, or alternatively in a TCR-independent manner by pattern recognition receptors and have been shown to uptake antigen, as well as process and present it to αβ T cells. We have identified a novel CD11b+ subset of γδ T cells in normal sheep peripheral blood. An increase in the frequency of these cells in sheep peripheral blood in response to immunization with an experimental recombinant subunit Rift Valley fever (RVF) vaccine was observed. However, injection of the vaccine adjuvant ISA-25VG alone without the recombinant RVF virus antigens demonstrated the same effect, pointing to an antigen-independent innate immune function of CD11b+ γδ T cells in response to the adjuvant. In vitro studies showed repeatable increases of CD11b-, CD14-, CD86-, CD40-, CD72-, and IFNγ- expressing γδ T cells in PBMCs after 24 h of incubation in the absence of a mitogen. Moreover, the majority of these myeloid-like γδ T cells were demonstrated to process exogenous antigen even in the absence of mitogen. ConA activation increased CD25- and MHCII- expression in γδ T cells, but not the myeloid associated receptors CD14 or CD11b or co-stimulatory molecules such as CD86 and CD40. Considering the role of CD11b and CD14 in the activation of innate immunity, we hypothesize that this subpopulation of sheep γδ T cells may function as innate antigen presenting and pro-inflammatory cells during immune responses. The results presented here also suggest that stress molecules and/or damage-associated molecular patterns may be involved in triggering antigen presenting and pro-inflammatory functions of γδ T cells, given their appearance in vitro in the absence of specific stimulation. Taken together, these data suggest that the early appearance of γδ T cells following adjuvant administration and their possible role in early activation of αβ T cell subsets may non-specifically contribute to augmented innate immunity and may promote strong initiation of the adaptive immune response to vaccines in general.

Reagents for detection of Rift Valley fever virus infection in sheep.

Rift Valley fever virus (RVFV) causes Rift Valley fever (RVF), resulting in morbidity and mortality in humans and ruminants. Evidence of transboundary outbreaks means that RVFV remains a threat to human health and livestock industries in countries that are free from the disease. To enhance surveillance capability, methods for detection of RVFV are required. The generation of reagents suitable for the detection of RVFV antigen in formalin-fixed, paraffin-embedded tissues from infected animals have been developed and are described herein. Recombinant nucleoprotein (rNP) was expressed in Escherichia coli and purified using immobilized metal ion affinity chromatography. Purified rNP was used as an immunogen to produce anti-NP polyclonal antisera in rabbits for use in detection of RVFV NP in experimentally infected animals by immunohistochemistry. Antisera raised in rabbits against rNP were able to recognize viral NP antigen in fixed infected Vero cell pellets and sheep liver. Therefore, the methods and reagents described herein are useful in assays for detection of RVFV infections in animals, for research and surveillance purposes.

First serological evidence of the Rift Valley fever Phlebovirus in Tunisian camels

Rift Valley fever (RVF) is a mosquito-borne zoonosis that severely impacts livelihoods, national and international economies, and human health. Few studies have investigated the prevalence of this infection in Tunisian livestock. The present report aimed to update the epidemiological status and identify the risk factors associated with this RVF virus infection in the one-humped dromedary camel from arid areas. A total of 470 sera of apparently healthy camels (Camelus dromedarius) were collected from six governorates from southern and central Tunisia. Samples were tested by a competitive Enzyme Linked Immunosorbent Assay (ELISA). An overall, 162 camels (34%, 95%CI: 0.1-0.4) were seropositive to RVF virus antigen. Logistic regression model revealed three potential risk factors associated with the infection. A meaningful high seropositivity was observed among aged camels (>10 years-old) (40%) (P=0.001; OR=3.367). Besides, camels raised in small flocks particularly intended for meat production showed a high level of seropositivity (37%) (P=0.013; OR=13.173). Animals having close contact with other ruminants showed high seroprevalence (37%) (P=0.022; OR=10.919). This report indicated that Tunisian one-humped dromedaries were exposed to this virus and may contribute to its dissemination among farmers and other livestock. Furthers studies are urgently required to isolate and characterize this virus, evaluate the potential risk of human infection particularly in farmers, veterinarians and slaughterhouse workers and finally to program a serious strategy for RVF control.

Characterization of Different Monoclonal and Polyclonal Antibodies for Immunohistological Detection of Rift Valley Fever Virus Antigens

Characterization of Different Monoclonal and Polyclonal Antibodies for Immunohistological Detection of Rift Valley Fever Virus Antigens

In Situ Hybridization for Detection of Rift Valley Fever Virus

Rift Valley fever virus (RVFV) is a mosquito‐borne zoonotic arbovirus with a tripartite genome consisting of a small, a medium (M), and a large (L) segment. This RNA virus causes frequent epidemics in Africa and the Arabian Peninsula. Outbreaks of Rift Valley fever typically result in abortion storms, the deaths of young ruminants and less often the deaths of adult ruminants. As a zoonotic virus, RVFV can also cause symptoms ranging from a mild flu‐like illness to encephalitis and death in humans. As such, RVFV‐infected tissues must be handled with caution. Formalin‐fixed, paraffin‐embedded (FFPE) tissues offer a safer format for researchers and diagnosticians testing for the presence of this virus. Currently, immunohistochemistry (IHC) serves as a confirmatory diagnostic test in South Africa alongside RT‐qPCR on fresh frozen tissue. However, PCR does not enable the ability to visualize the virus within a histological context and IHC has limitations in its ability to detect viral antigen in suboptimally preserved samples. These limitations create a need for more powerful confirmatory diagnostic tools. Here we demonstrate the use of RNAscope® in situ hybridization (ISH) technology for the detection of RVFV viral RNA (vRNA) in FFPE tissues.Samples tested included a broad set of previously published, confirmed positive field samples from sheep naturally infected during the 2010 South African outbreak. Prior, a DNA probe was designed to target the nucleotides between 20‐997 of the RVFV L segment (NC_014397.1) and the assay was validated on experimentally infected tissues. Here we tested this pan‐RVFV ISH assay on naturally infected liver, spleen, kidney, lung, thyroid, brain, skeletal and cardiac muscle, lymph node, salivary gland, adrenal gland, gastrointestinal tract, and male and female reproductive organs. We also compared ISH results to other tests, a previously established triplex FFPE RT‐qPCR targeting all three gene segments as well as IHC targeting the RVFV Gn glycoprotein. Finally, we were able to dual label samples with pan‐RVFV ISH and IHC for analysis of both RVFV RNA and protein distribution as well as RVFV RNA and cell type markers, providing finer grained detail regarding cellular tropism.Pan‐RVFV ISH appropriately labeled vRNA and this signal correlated with visible lesions in the various organs tested. Additionally, vRNA was detected in macrophages in the lymph nodes, a result previously seen by IHC. The positive ISH results mirrored those obtained by FFPE RT‐qPCR at the tissue block level. In comparison to IHC, ISH subjectively appears to produce a stronger and more reliable signal. This was observed in multiple organs where histopathology was present but RVFV antigen IHC was negative. We believe that this highly sensitive and specific ISH assay has potential as a RVFV confirmatory diagnostic test and research applications.Support or Funding InformationThis work was funded by the Department of Diagnostic Medicine, College of Veterinary Medicine, Kansas State University; the Department of Paraclinical Sciences, University of Pretoria, USDA Agricultural Research Services, and APHIS.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Some Virological Studies on Rift Valley Fever Virus in Camels

1. Abstract 1.1. Background: Rift Valley Fever Virus (RVFV) infected camels plays certain roles in transmitting pathogens to a new land. Egypt imports camels from the African countries. The camels arrive in Egypt by sea and border crossings with the Sudan and Libya. The present study performed on imported, non-vaccinated camels. This research aims to help in protection of camel from diseases. 1.2. <span style=font-size:10.0pt;font-family: times= new= roman,serif=>Materials and Methods: Blood, sera, portal blood and tissue specimens were collected and examined by virology techniques [Agar Gel Precipitation Tests (AGPT), Immune Fluorescence tests (IF), Enzyme Linked Immune Sorbent Assays (ELISA)] and histopathology. SNT (serum neutralization test) was applied. 1.3. Results: <span style=font-size: 10.0pt;color:black> RVFV antigen was detected in imported camels by AGPT and IF. IF test showed the inclusions and apoptotic bodies were giving special fluorescence that identical to histopathology changes. ELISA and SNT showing the presence of neutralizing antibodies against RVFV. 1.4. Conclusions: Camel plays certain roles in transmitting RVFV, but immune system weaken the virus under ordinary conditions. So that, other environmental factors needed to convert the weak virus into its virulence status. 2. Keywords: <span style=font-size:10.0pt; mso-bidi-font-weight:bold>Rift Valley Fever Virus; RVFV; RVF in Camels

Rift Valley Fever in Chronic Carrier and Liver Manifestations

Background: Rift Valley Fever virus (RVFV) causes certain type of hemorrhagic fever. In Egypt, during September 2017, RVFV was detected in samples from imported cattle (country of origin: the Sudan). Samples were collected from imported cattle from different localities in Cairo during October, November, December 2017.Material and methods: RVFV isolation in Swiss mice (6 days old) was by intra-peritoneal inoculation (I/P) with tissue homogenates, positive results were seen in second passages. Detection of RVFV antigen in samples was done by Agar Gel Precipitation test (AGPT) and the Immunofluorescence Technique (IF). However; reverse transcription real time polymerase chain reaction (RT-PCR) was applied and showed negative results for both samples and positive control Egyptian strain (Menya /Sheep/258) indicating non indicative information (false negative). IF tests were applied on paraffin sections and tissues impressions.Results: The pathological investigations showed chronic infection in the form of necrotic foci (necrogranulomas) and IF test was showing RVFV antigen in hepatocytes, lymphocytes, Kupffer cells (histiocytes of liver), polymorphnuclear cells and in macrophages. RVFV is necrotizing virus that use histiocytes to produce its destructive effects on infected tissues (hepatocytes eating virus), it has its specific and unique pathologic profile inside hepatic tissues. The infected cells produce the virus in large number which leaves cells in groups of millions of viral particles enclosed in certain type of capsule or membrane. The severity of RVFV infections of the liver indicates the virulence status of the virus.Conclusion: The pathologic changes indicate chronic disease. This study proves that RVFV causes chronic diseases and carriers are present in the endemic areas.

Evaluation of Fluorescence Microsphere Immunoassay for Antibody Detection to Rift Valley Fever Nucleocapsid Protein and Glycoproteins

Rift Valley Fever Virus (RVFV) is a zoonotic disease that infects ruminants including cattle, sheep, goats, camels and buffalo. There is a need to create a safe, rapid, and accurate assay that can simultaneously detect antibodies directed against multiple RVFV antigens. We describe the improvement and validation of a fluorescence microsphere immunoassay (FMIA) for the detection of IgG antibodies against the RVFV glycoproteins and the immunodominant nucleocapsid (N) protein. The purpose of this assay is to simultaneously screen and confirm sera from animals for positive or negative disease status. Well‐characterized vaccinated and experimentally infected sheep and calf sera were used for the evaluation of the assay. Recombinant viral proteins were produced then coupled to polystyrene magnetic beads for analysis using the Luminex MAGPIX system with xMAP technology. An unrelated viral antigen coupled control bead set was added to the reaction in order to account for non‐specific binding of antibodies to the antigen of the antigen‐bead complex. Median Fluorescence Intensity (MFI) results were converted to Sample/Positive ratios in order to standardize test results. Our results revealed highest MFI values for the detection of IgG antibodies against the RVFV N protein, indicating that this RVFV antigen could serve as a good candidate for a screening assay. The glycoprotein Gn target has been shown to differentiate vaccinated from non‐vaccinated animals in a candidate subunit vaccine formulation based on the RVFV Gn and Gc proteins. The FMIA demonstrated a high correlation (R2) of 0.91 when compared side by side with plaque reduction neutralization tests. The addition of further RVFV targets such as the non‐structural proteins NSs and NSm is currently under investigation. 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 will provide the basis for the development of a multiplex sero‐diagnostic assay system that can simultaneously screen for several ruminant diseases including transboundary animal diseases.Support or Funding InformationThis material is based upon work supported by the Kansas State NBAF Transition Funds, Kansas State University College of Veterinary Medicine, University of Pretoria Department of Veterinary Tropical Diseases, the USDA Agricultural Research project number 5430‐32000‐005‐00D and the U.S. Department of Homeland Security under Grant Award Number DHS‐2010‐ST‐061‐AG0001. The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the U.S. Department of Homeland Security.

Reverse Transcriptase Real Time PCR Detection of Rift Valley Fever Virus RNA in Formalin‐Fixed, Paraffin‐Embedded Tissues

Rift Valley Fever Virus (RVFV) is a mosquito‐borne zoonotic pathogen endemic in parts of Africa and the Arabian Peninsula. RVFV causes acute febrile disease with mass abortion and death in ruminants and occasional encephalitis and death in humans. In the US, RVFV is a BSL‐3Ag Select Agent and a foreign animal disease, requiring special containment laboratories for research work. Research goals include the study of RVFV pathogenesis and the development of novel diagnostic tools and disease prevention methods such as vaccines. Ongoing surveillance plays a critical role in prevention and control of outbreaks and virus spread. Formalin‐fixed, paraffin‐embedded tissues (FFPET) are non‐infectious diagnostic specimens, routinely used for histopathology and immunohistochemistry (IHC), however use of this diagnostic specimen for molecular detection of pathogen RNA is underinvestigated. The objective of this work is to develop and validate a protocol for use of FFPET for the sensitive and specific detection of RVFV RNA.Study samples were fresh‐frozen tissues (FFT) and FFPET (liver, spleen, lung, kidney) collected from sheep and calves experimentally infected with two RVFV strains (Kenya 2006 and Saudi Arabia 2001). Deparaffinization of paraffin whorls (5‐5μm thick), tissue lysis with proteinase K, and reversal of nucleic acid cross linking in heated alkaline buffer was performed in a single tube using commercial buffers (Qiagen). RNA was purified using automated magnetic bead extraction on the Taco mini (GeneReach) for FFPET or the Applied Biosystems reagents on the Kingfisher (FFT). Multiplex Reverse Transcriptase Real Time PCR (RT‐qPCR) for RVFV L, M and S gene segments was used to detect RVFV RNA using Quanta qScript XLT One‐Step RT‐qPCR ToughMix (FFPET) or AgPath RT‐qPCR mix (FFT). IHC for RVFV antigen was done with a mouse monoclonal anti‐RVFV nucleoprotein antibody and avidin‐biotin complex detection.To date, 18 FFPET samples have been tested. For samples that were both IHC and FFT RT‐qPCR positive (n=13), FFPET results were 100% positive. For FFT samples (n=5) that were not tested by RT‐qPCR but were IHC positive, the FFPET RVFV RT‐qPCR results were also positive. Testing of an additional 46 FFPE samples is presently underway.Using this newly developed single tube FFPET technique for nucleic acid recovery, followed by a rapid, low cost magnetic bead nucleic acid extraction allows for highly sensitive detection of RNA specific for RVFV. This novel method could strengthen routine diagnostics, surveillance and molecular epidemiological studies that are currently limited by the strict biosecurity requirements for RVFV samples. Moreover, this methodology could also be expanded for detection of RNA targets for additional high impact pathogens or for transcriptional studies that must relie on archival FFPET.Support or Funding InformationThis work was funded by startup funds provided to Dr. Davis by the Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University and uses archival animal study materials from research funded by grants from the Department of Homeland Security Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD), Grant No. 2010‐ST061‐AG0001, USDA Agricultural Services project 3020‐32000‐005‐00D and the Kansas Bioscience Authority.

Arabidopsis thaliana plants expressing Rift Valley fever virus antigens: Mice exhibit systemic immune responses as the result of oral administration of the transgenic plants

The zoonotic Rift Valley fever virus affects livestock and humans in Africa and on the Arabian Peninsula. The economic impact of this pathogen due to livestock losses, as well as its relevance to public health, underscores the importance of developing effective and easily distributed vaccines. Vaccines that can be delivered orally are of particular interest.Here, we report the expression in transformed plants (Arabidopsis thaliana) of Rift Valley fever virus antigens. The antigens used in this study were the N protein and a deletion mutant of the Gn glycoprotein. Transformed lines were analysed for specific mRNA and protein content by RT-PCR and Western blotting, respectively. Furthermore, the plant-expressed antigens were evaluated for their immunogenicity in mice fed the transgenic plants. After oral intake of fresh transgenic plant material, a proportion of the mice elicited specific IgG antibody responses, as compared to the control animals that were fed wild-type plants and of which none sero-converted.Thus, we show that transgenic plants can be readily used to express and produce Rift Valley Fever virus proteins, and that the plants are immunogenic when given orally to mice. These are promising findings and provide a basis for further studies on edible plant vaccines against the Rift Valley fever virus.

Rift Valley fever virus infection in golden Syrian hamsters.

Rift Valley fever virus (RVFV) is a formidable pathogen that causes severe disease and abortion in a variety of livestock species and a range of disease in humans that includes hemorrhagic fever, fulminant hepatitis, encephalitis and blindness. The natural transmission cycle involves mosquito vectors, but exposure can also occur through contact with infected fluids and tissues. The lack of approved antiviral therapies and vaccines for human use underlies the importance of small animal models for proof-of-concept efficacy studies. Several mouse and rat models of RVFV infection have been well characterized and provide useful systems for the study of certain aspects of pathogenesis, as well as antiviral drug and vaccine development. However, certain host-directed therapeutics may not act on mouse or rat pathways. Here, we describe the natural history of disease in golden Syrian hamsters challenged subcutaneously with the pathogenic ZH501 strain of RVFV. Peracute disease resulted in rapid lethality within 2 to 3 days of RVFV challenge. High titer viremia and substantial viral loads were observed in most tissues examined; however, histopathology and immunostaining for RVFV antigen were largely restricted to the liver. Acute hepatocellular necrosis associated with a strong presence of viral antigen in the hepatocytes indicates that fulminant hepatitis is the likely cause of mortality. Further studies to assess the susceptibility and disease progression following respiratory route exposure are warranted. The use of the hamsters to model RVFV infection is suitable for early stage antiviral drug and vaccine development studies.

Deletion of the NSm virulence gene of Rift Valley fever virus inhibits virus replication in and dissemination from the midgut of Aedes aegypti mosquitoes.

BackgroundPreviously, we investigated the role of the Rift Valley fever virus (RVFV) virulence genes NSs and NSm in mosquitoes and demonstrated that deletion of NSm significantly reduced the infection, dissemination, and transmission rates of RVFV in Aedes aegypti mosquitoes. The specific aim of this study was to further characterize midgut infection and escape barriers of RVFV in Ae. aegypti infected with reverse genetics-generated wild type RVFV (rRVF-wt) or RVFV lacking the NSm virulence gene (rRVF-ΔNSm) by examining sagittal sections of infected mosquitoes for viral antigen at various time points post-infection.Methodology and Principal Findings Ae. aegypti mosquitoes were fed an infectious blood meal containing either rRVF-wt or rRVF-ΔNSm. On days 0, 1, 2, 3, 4, 6, 8, 10, 12, and 14 post-infection, mosquitoes from each experimental group were fixed in 4% paraformaldehyde, paraffin-embedded, sectioned, and examined for RVFV antigen by immunofluorescence assay. Remaining mosquitoes at day 14 were assayed for infection, dissemination, and transmission. Disseminated infections were observed in mosquitoes as early as three days post infection for both virus strains. However, infection rates for rRVF-ΔNSm were statistically significantly less than for rRVF-wt. Posterior midgut infections in mosquitoes infected with rRVF-wt were extensive, whereas midgut infections of mosquitoes infected with rRVF-ΔNSm were confined to one or a few small foci.Conclusions/SignificanceDeletion of NSm resulted in the reduced ability of RVFV to enter, replicate, and disseminate from the midgut epithelial cells. NSm appears to have a functional role in the vector competence of mosquitoes for RVFV at the level of the midgut barrier.

The nucleocapsid protein of Rift Valley fever virus is a potent human CD8+ T cell antigen and elicits memory responses.

There is no licensed human vaccine currently available for Rift Valley Fever Virus (RVFV), a Category A high priority pathogen and a serious zoonotic threat. While neutralizing antibodies targeting the viral glycoproteins are protective, they appear late in the course of infection, and may not be induced in time to prevent a natural or bioterrorism-induced outbreak. Here we examined the immunogenicity of RVFV nucleocapsid (N) protein as a CD8+ T cell antigen with the potential for inducing rapid protection after vaccination. HLA-A*0201 (A2)-restricted epitopic determinants were identified with N-specific CD8+ T cells from eight healthy donors that were primed with dendritic cells transduced to express N, and subsequently expanded in vitro by weekly re-stimulations with monocytes pulsed with 59 15mer overlapping peptides (OLPs) across N. Two immunodominant epitopes, VT9 (VLSEWLPVT, N121–129) and IL9 (ILDAHSLYL, N165–173), were defined. VT9- and IL9-specific CD8+ T cells identified by tetramer staining were cytotoxic and polyfunctional, characteristics deemed important for viral control in vivo. These peptides induced specific CD8+ T cell responses in A2-transgenic mice, and more importantly, potent N-specific CD8+ T cell reactivities, including VT9- and IL9-specific ones, were mounted by mice after a booster vaccination with the live attenuated RVF MP-12. Our data suggest that the RVFV N protein is a potent human T cell immunogen capable of eliciting broad, immunodominant CD8+ T cell responses that are potentially protective. Understanding the immune responses to the nucleocapsid is central to the design of an effective RVFV vaccine irrespective of whether this viral protein is effective as a stand-alone immunogen or only in combination with other RVFV antigens.

Inactivation of infectious virus and serological detection of virus antigen in Rift Valley fever virus-exposed mosquitoes fixed with paraformaldehyde

Formaldehyde is routinely used to fix tissues in preparation for pathology studies, however concerns remain that treatment of tissues with cellular fixatives may not entirely inactivate infectious virus particles. This concern is of particular regulatory importance for research involving viruses that are classified as select agents such as Rift Valley fever virus (RVFV). Therefore, the specific aims of this study were to (1) assay RVFV-exposed Aedes aegypti mosquitoes fixed in 4% paraformaldehyde for the presence of infectious RVFV particles at various time points following infection and (2) demonstrate the utility of immunofluorescence assay (IFA) for the detection of RVFV antigen in various tissues of paraformaldehyde-fixed mosquitoes. Mosquitoes were administered an infectious blood meal containing one of two strains of RVFV, harvested at various time points following infection, intrathoracically inoculated with 4% paraformaldehyde, and fixed overnight at 4°C. The infection status of a subset of mosquitoes was verified by IFA on leg tissues prior to fixation, and infectivity of RVFV in fixed mosquito carcasses was determined by Vero cell plaque assay. Paraformaldehyde-fixed mosquitoes harvested 14 days post infection were also paraffin-embedded and sectioned for detection of RVFV antigen to particular tissues by IFA. None of the RVFV-exposed mosquitoes tested by Vero cell plaque assay contained infectious RVFV after fixation. Furthermore, incubation of mosquito sections with trypsin prior to antibody staining is recommended for optimal visualization of RVFV antigen in infected mosquito tissues by IFA.

Surface-enhanced Raman scattering (SERS) detection of multiple viral antigens using magnetic capture of SERS-active nanoparticles

A highly sensitive immunoassay based on surface-enhanced Raman scattering (SERS) spectroscopy has been developed for multiplex detection of surface envelope and capsid antigens of the viral zoonotic pathogens West Nile virus (WNV) and Rift Valley fever virus (RVFV). Detection was mediated by antibody recognition using Raman reporter-coated Au nanoparticles (GNPs) and paramagnetic nanoparticles (PMPs) conjugated with polyclonal antibodies specific for each antigen target, followed by 785nm laser excitation of magnetically concentrated GNP/antigen/PMP complexes. The discrimination of WNV and RVFV antigen detection in mixed Raman spectra was achieved by SERS enhancement of Raman spectra specific for the Raman reporter dyes Infrared 792 (IR-792) and Nile Blue (NB), respectively. Assay reactions containing dilutions of both target antigens yielded a reduction in the intensification of IR-792 and NB signature spectrum peaks and provided a conservative limit of detection of ∼5fg/ml for assays conducted in phosphate buffered saline buffer (PBS) and ∼25pg/ml for assays containing PBS spiked with fetal bovine serum. Based on the inherent simplicity of the assay, magnetic capture-based SERS assays afford promise as a biosensor platform that provides high-level multiplex detection sensitivity and can be adapted for portable diagnostic applications in limited resource settings.

Antigen-capture ELISA for the detection of Rift Valley fever virus nucleoprotein using new monoclonal antibodies

Monoclonal antibodies (MAbs) raised against the nucleoprotein (NP) of Rift Valley fever virus (RVFV) were developed, and an antigen-capture enzyme-linked immunosorbent assay (Ag-capture ELISA) system was developed for the detection of RVFV NP. The assay detected RVFV antigen from culture supernatants containing as little as 7.8-31.3 pfu per 100 μl. Reactivity with various truncated NPs indicated that MAb C10-54 bound only to the full-length NP, probably due to recognition of a conformational epitope, whereas MAbs G2-36 and D5-59 bound to a linear epitope ranging from amino acid residues 195-201 in the C-terminal region. Based on the alignments of the amino acid sequence of RVFV NP, the epitope regions of MAbs G2-36 and D5-59 were completely conserved among all RVFV strains. These results suggest that the MAbs are applicable to the Ag-capture ELISA for the diagnosis of RVFV infections.

Development and evaluation of one-step rRT-PCR and immunohistochemical methods for detection of Rift Valley fever virus in biosafety level 2 diagnostic laboratories

Rift Valley fever virus (RVFV) is a zoonotic insect transmitted virus endemic to Africa and the Arabian Peninsula. Infection causes abortions and high mortality in newborn ruminants. The overall human infection rate is <1%; however, fatality rates in those with severe clinical disease have been reported as high as 29%. The potential of RVFV as a bioterrorism agent and/or being accidentally introduced into North America is widely recognized. Currently, regional veterinary biosafety level 2 (BSL-2) diagnostic laboratories lack safe, modern, validated diagnostic tests to detect RVFV. An existing one-step real-time RT-PCR (rRT-PCR) assay was modified for quick virus inactivation for use in BSL-2 laboratories, evaluated on serum and tissue samples from experimentally infected lambs and calves, and compared to virus isolation. Viremia was detected in all inoculated sheep with titers reaching 10(6.5) plaque forming units/ml, or up to 10(10) viral RNA copies/ml. Viremia in calves was lower and not detected in all inoculated animals; however, all animals became transiently febrile and were infected as determined by rRT-PCR of tissues. Virus was isolated from rRT-PCR-positive liver and/or spleen in 33% of lamb and 41% of calf samples between 2 and 7 days post inoculation. For RVFV antigen detection, reagents are typically produced at BSL-3Ag or BSL-4 conditions and require inactivation and safety testing for use outside of containment. In this study, antiserum against recombinant RVFV-nucleocapsid (N) was produced to develop an immunohistochemical (IHC) assay which was subsequently evaluated on formalin fixed lamb and calf tissues at BSL-2 laboratory conditions. Antigen was detected by IHC in 79% of rRT-PCR-positive sheep and 70% of rRT-PCR-positive calf tissues tested. Once validated and approved by national regulatory agencies, these assays can be safely produced and distributed to regional diagnostic laboratories, providing capacity for early detection of RVFV in suspected ruminant samples.

Field Evaluation of A Wicking Assay for the Rapid Detection of Rift Valley Fever Viral Antigens In Mosquitoes1

Rift Valley fever virus (RVFV) causes outbreaks of severe disease in domestic ungulates as well as humans in Africa. There is a concern that outbreaks of Rift Valley fever may continue and that this virus may spread into regions where it had not previously been detected. Surveillance and rapid detection are critical to the initiation of an effective disease control program. Here we report on the field evaluation in Kenya of the VectorTest RVFV antigen assay, modeled on the VecTest assay for West Nile virus. The dipsticks provided results in <20 min, were easy to use, and did not require a laboratory with containment facilities. Although none of the field-collected mosquitoes were infected with RVFV, the dipstick provided a clear positive result with pools of field-collected mosquitoes spiked with a single positive, irradiated (to inactivate any infectious virus) mosquito. Similarly, the dipstick was able to detect virus from pools of mosquitoes captured during the RVFV outbreak in 2007. The RVFV dipstick assay was highly specific with only a single weak false positive out of 266 pools tested (specificity > 99.6%). The RVFV assay can provide a rapid, safe, easy-to-use preliminary test to alert public health personnel to the presence of RVFV in mosquitoes in a given area. Results from this assay will allow for more rapid medical threat assessments and the focusing of vector control measures on high-risk areas.