Letteratura scientifica selezionata sul tema "Rift Valley fever virus (RVFV)"

ABSTRACT Rift Valley fever (RVF) is an epizootic viral disease of sheep that can be transmitted from sheep to humans, particularly by contact with aborted fetuses. A capripoxvirus (CPV) recombinant virus (rKS1/RVFV) was developed, which expressed the Rift Valley fever virus (RVFV) Gn and Gc glycoproteins. These expressed glycoproteins had the correct size and reacted with monoclonal antibodies (MAb) to native glycoproteins. Mice vaccinated with rKS1/RVFV were protected against RVFV challenge. Sheep vaccinated with rKS1/RVFV twice developed neutralizing antibodies and were significantly protected against RVFV and sheep poxvirus challenge. These findings further document the value of CPV recombinants as ruminant vaccine vectors and support the inclusion of RVFV genes encoding glycoproteins in multivalent recombinant vaccines to be used where RVF occurs.

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.

Currently, there are no worldwide licensed vaccines for Rift Valley fever (RVF) that are both safe and effective. Development and evaluation of vaccines, diagnostics and treatments depend on the availability of appropriate animal models. Animal models are also necessary to understand the basic pathobiology of infection. Here, we report the use of an inbred MBT/Pas mouse model that consistently reproduces RVF disease and serves our purpose for testing the efficacy of vaccine candidates; an attenuated Rift Valley fever virus (RVFV) and a recombinant RVFV–capripoxvirus. We show that this model is relevant for vaccine testing.

Rift Valley fever (RVF) is a mosquito-borne viral disease, principally of ruminants, that is endemic to Africa. The causative Phlebovirus, Rift Valley fever virus (RVFV), has a broad host range and, as such, also infects humans to cause primarily a self-limiting febrile illness. A small number of human cases will also develop severe complications, including haemorrhagic fever, encephalitis and visual impairment. In parts of Africa, it is a major disease of domestic ruminants, causing epidemics of abortion and mortality. It infects and can be transmitted by a broad range of mosquitos, with those of the genus Aedes and Culex thought to be the major vectors. Therefore, the virus has the potential to become established beyond Africa, including in Australia, where competent vector hosts are endemic. Vaccines for humans have not yet been developed to the commercial stage. This review examines the threat of this virus, with particular reference to Australia, and assesses gaps in our knowledge that may benefit from research focus.

Rift Valley fever (RVF) is a vector-borne viral disease widespread in Africa. The primary cycle involves mosquitoes and wild and domestic ruminant hosts. Humans are usually contaminated after contact with infected ruminants. As many environmental, agricultural, epidemiological, and anthropogenic factors are implicated in RVF spread, the multidisciplinary One Health approach was needed to identify the drivers of RVF epidemics in Madagascar. We examined the environmental patterns associated with these epidemics, comparing human and ruminant serological data with environmental and cattle-trade data. In contrast to East Africa, environmental drivers did not trigger the epidemics: They only modulated local Rift Valley fever virus (RVFV) transmission in ruminants. Instead, RVFV was introduced through ruminant trade and subsequent movement of cattle between trade hubs caused its long-distance spread within the country. Contact with cattle brought in from infected districts was associated with higher infection risk in slaughterhouse workers. The finding that anthropogenic rather than environmental factors are the main drivers of RVF infection in humans can be used to design better prevention and early detection in the case of RVF resurgence in the region.

Abstract Rift Valley fever (RVF) is a zoonotic, vector-borne infectious disease of ruminants and camels transmitted mainly by the Aedes and Culex mosquito species. Contact with the blood or organs of infected animals may infect humans. Its etiological factor is the Rift Valley fever virus (RVFV) of the Phlebovirus genus and Bunyaviridae family. Sheep and goats are most susceptible to infection and newborns and young individuals endure the most severe disease course. High abortion rates and infant mortality are typical for RVF; its clinical signs are high fever, lymphadenitis, nasal and ocular secretions and vomiting. Conventional diagnosis is done by the detection of specific IgM or IgG antibodies and RVFV nucleic acids and by virus isolation. Inactivated and live-attenuated vaccines obtained from virulent RVFV isolates are available for livestock. RVF is endemic in sub-Saharan Africa and the Arabian Peninsula, but in the last two decades, it was also reported in other African regions. Seropositive animals were detected in Turkey, Tunisia and Libya. The wide distribution of competent vectors in non-endemic areas coupled with global climate change threaten to spread RVF transboundarily. The EFSA considers the movement of infected animals and vectors to be other plausible pathways of RVF introduction into Europe. A very low risk both of introduction of the virus through an infected animal or vector and of establishment of the virus, and a moderate risk of its transmission through these means was estimated for Poland. The risk of these specific modes of disease introduction into Europe is rated as very low, but surveillance and response capabilities and cooperation with the proximal endemic regions are recommended.

Rift Valley Fever (RVF) is a notifiable multiple species diseases in the OIE list, and causes human and agricultural losses in endemic regions. To develop the rapid method for detecting of RVF, 2 specific primers for reverse transcriptase polymerase chain reaction (RT-PCR) and 7 overlapping oligo primers were designed according to the nucleotide sequence information of RVFV published in GenBank, and a DNA fragment about 318 bp of the segment S was synthesized in vitro by overlap extension PCR to construct the recombinant plasmid pMD19-T-RVFVS. Then, the 2 specific primers were evaluated via a serial of tests, including reaction temperature optimization test, sensitivity and specificity tests. The results showed that the 2 designed primers are suitable for RVFV RT-PCR detection which is a rapid method with good specificity and sensitivity, the detection limit was approximately 85 copies of the cloned viral genomic fragments (pMD19-T-RVFVS) as well as resulted in no cross-reaction for peste des petits ruminants virus (PPRV), Epidemic encephalitis B virus, E.coli , Salmonella and Pasteurella multocida etc common pathogens isolated from ruminants detection.

Outbreaks of Rift Valley fever (RVF) occurred in Namibia in 2010 and 2011. Complete genome characterization was obtained from virus isolates collected during disease outbreaks in southern Namibia in 2010 and from wildlife in Etosha National Park in 2011, close to the area where RVF outbreaks occurred in domestic livestock. The virus strains were sequenced using Sanger sequencing (Namibia_2010) or next generation sequencing (Namibia_2011). A sequence-independent, single-primer amplification (SISPA) protocol was used in combination with the Illumina Next 500 sequencer. Phylogenetic analysis of the sequences of the small (S), medium (M), and large (L) genome segments of RVF virus (RVFV) provided evidence that two distinct RVFV strains circulated in the country. The strain collected in Namibia in 2010 is genetically similar to RVFV strains circulating in South Africa in 2009 and 2010, confirming that the outbreaks reported in the southern part of Namibia in 2010 were caused by possible dissemination of the infection from South Africa. Isolates collected in 2011 were close to RVFV isolates from 2010 collected in humans in Sudan and which belong to the large lineage containing RVFV strains that caused an outbreak in 2006–2008 in eastern Africa. This investigation showed that the RVFV strains circulating in Namibia in 2010 and 2011 were from two different introductions and that RVFV has the ability to move across regions. This supports the need for risk-based surveillance and monitoring.

Rift Valley fever virus (RVFV) belongs to the genus Phlebovirus, family Bunyaviridae, and carries single-stranded tripartite RNA segments. The virus is transmitted by mosquitoes and has caused large outbreaks among ruminants and humans in sub-Saharan African and Middle East countries. The disease is characterized by a sudden onset of fever, headache, muscle pain, joint pain, photophobia, and weakness. In most cases, patients recover from the disease after a period of weeks, but some also develop retinal or macular changes, which result in vision impairment that lasts for an undefined period of time, and severe disease, characterized by hemorrhagic fever or encephalitis. The virus also causes febrile illness resulting in a high rate of spontaneous abortions in ruminants. The handling of wild-type RVFV requires high-containment facilities, including biosafety level 4 or enhanced biosafety level 3 laboratories. Nonetheless, studies clarifying the mechanisms of the RVFV-induced diseases and preventing them are areas of active research throughout the world. By primarily referring to recent studies using several animal model systems, protein expression systems, and specific mutant viruses, this review describes the current knowledge about the mechanisms of pathogenesis of RVF and biological functions of various viral proteins that affect RVFV pathogenicity.

SUMMARYRift Valley fever virus (RVFV) is a mosquito-transmitted bunyavirus (genusPhlebovirus) associated with severe disease in livestock and fatal encephalitis or haemorrhagic fever in a proportion of infected humans. Although live attenuated and inactivated vaccines have been used in livestock, and on a limited scale in humans, there is a need for improved anti-RVFV vaccines. Towards this goal, Sindbis virus replicon vectors expressing the RVFV Gn and Gc glycoproteins, as well as the non-structural nsM protein, were constructed and evaluated for their ability to induce protective immune responses against RVFV. These replicon vectors were shown to produce the RVFV glycoproteins to high levelsin vitroand to induce systemic anti-RVFV antibody responses in immunized mice, as determined by RVFV-specific ELISA, fluorescent antibody tests, and demonstration of a neutralizing antibody response. Replicon vaccination also provided 100% protection against lethal RVFV challenge by either the intraperitoneal or intranasal route. Furthermore, preliminary results indicate that the replicon vectors elicit RVFV-specific neutralizing antibody responses in vaccinated sheep. These results suggest that alphavirus-based replicon vectors can induce protective immunity against RVFV, and that this approach merits further investigation into its potential utility as a RVFV vaccine.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease caused by a virus of the family Bunyaviridae, genus Phlebovirus. It is responsible for extensive outbreaks of disease in livestock in Africa with significant mortality and economic impact. Virus neutralization is considered the gold standard for confirming Rift Valley fever virus (RVFV) infection but the procedure is time consuming and expensive. Real-time reverse transcription-polymerase chain reaction (rRT-PCR), histopathology, and immunohistochemistry (IHC) are the diagnostic methods most often used in South Africa to confirm or exclude a diagnosis of RVF in necropsied animals. Validated estimates of diagnostic accuracy of these tests, in naturally infected livestock, however, have not been published. The objective of this study was to estimate the diagnostic sensitivity and specificity of rRT-PCR, histopathology, and IHC using Bayesian latent class methods in the absence of a gold standard. A secondary objective was to estimate stratum-specific values based on species, age, degree of specimen autolysis, and the presence/absence of tissue pigments. The Sensitivity (Se) and Specificity (Sp) of qRT-PCR were 97.4% (95% credibility interval (CI): 95.2% - 98.8%) and 71.7% (95% CI: 65% - 77.9%) respectively. The extraordinary analytical sensitivity of PCR makes this test very susceptible to false positive reactions, and thus reduced specificity. This is more likely during large-scale epidemics due to crosscontamination of specimens at necropsy facilities or testing laboratories. The Se and Sp of histopathology were 94.6% (95% CI: 91% - 97.2%) and 92.3% (95% CI: 87.6% - 95.8%) respectively. Single cases of RVF could be confused with acute poisoning with plants, bacterial septicaemias, and viral diseases such as infectious bovine rhinotracheitis and Wesselsbron disease. Most of these conditions, however, can be excluded using histological examination of the liver, special stains, bacterial culture, and toxicological or serological investigations. The Se and Sp of IHC were 97.6% (95% CI: 93.9% - 99.8%) and 99.4% (95% CI: 96.9% - 100%) respectively. Immunohistochemistry is highly specific because characteristic positive immunolabelling of the cytoplasm of hepatocytes can be correlated with the presence of hepatocellular injury typical for RVFV infection. False negative results are sometimes obtained with IHC because of reader error or loss of the antigenic epitopes due to advanced autolysis. Scant positive immunolabelling might be missed or viral proteins might be absent from sections of liver with advanced hepatocellular damage. The stratified analysis suggested differences in test accuracy in foetuses and severely autolysed specimens. The Sp of histopathology in foetuses (83.0%) was 9.3% lower than the value obtained for the sample population (92.3%). Lesions in some foetuses are more subtle and the typical eosinophilic intranuclear inclusions are often difficult to detect. In severely autolysed specimens, the Se of IHC decreased by 16.1% and the Sp of rRT-PCR by 17.4%. There is no plausible biological explanation for this decrease in the Sp of rRTPCR since the RNA of RVFV is resistant to degradation in autolysed tissues. Conversely, the antibody used to detect RVFV using IHC detects epitopes raised against nucleoproteins of the virus and it is possible that viral proteins become too widely dispersed and/or degraded in autolysed tissues to detect by light microscopy. It is possible that the marked decrease in Se of histopathology and IHC in severely autolysed specimens caused an apparent decrease in Sp of rRT-PCR, due to the latent class method. In conclusion, the high estimated Sp (99.4%) of IHC and the low Sp of rRT-PCR (71.3%) suggests that the definitive diagnosis or exclusion of RVF should not rely on a single PCR test and that IHC would be an effective confirmatory test for rRT-PCR positive field cases necropsied during an epidemic. Immunohistochemistry results from severely autolysed specimens, however, should be interpreted with caution and aborted foetuses in areas endemic for RVF should be screened using a variety of tests. The diagnostic Se and Sp of histopathology was much higher than expected confirming the value of routine post mortem examinations and histopathology of liver specimens. The most feasible RVF testing option in areas that do not have suitably equipped PCR laboratories, and where disease is often not detected in livestock until after human cases have been diagnosed, would be routine histopathology screening with IHC confirmation. Key Words: Rift Valley fever; Rift Valley fever virus; Bayesian; latent-class model; real-time reverse transcription-polymerase chain reaction; immunohistochemistry; histopathology; diagnosis; sensitivity; specificity.
Dissertation (MSc)--University of Pretoria, 2014.
gm2014
Paraclinical Sciences
unrestricted

Rift Valley fever phlebovirus (RVFV) is an arbovirus of medical and veterinary importance causing severe disease and mortality in humans and ruminants in endemic regions in Africa and the Arabian Peninsula. Understanding the capability, and limiting factors, of temperate British mosquitoes to support replication and transmission of RVFV is critical in order to understand the potential for RVFV establishment were it introduced to the UK. Using in vitro cell culture the effect of temperature on viral replication kinetics independently of the mosquito was investigated; demonstrating temperatures below 20 ̊C negatively affect RVFV replication. The full replication cycle was supported at 20 ̊C in vitro, and this was confirmed within in vivo mosquito experiments with wild-caught Aedes detritus demonstrating a transmission potential for RVFV at 20 ̊C and 25 ̊C. Experiments with two colonised lines of Cx. pipiens further demonstrated the transmission potential for RVFV by mosquitoes present in the UK. A novel RNA in situ hybridisation technique substantiated this result showing widespread dissemination of virus from the primary site of infection and evidence of secondary sites of replication within a single mosquito. Characterisation of the consensus sequence of RVFV propagated within these British mosquitoes in comparison to an in vivo mouse model showed potential for virus adaptation when switching between disparate hosts. Reproducible changes at the consensus level within each host had not previously been shown in early passages of RVFV in studies utilising in vitro models of replication. This suggests that RVFV replication generates genomic variation that may lead to adaptations that could promote potential survival in temperate regions. Taken together these findings indicate that transmission of RVFV within the UK by indigenous mosquitoes is possible. However, factors affecting mosquito survival including temperatures greater than 20 ̊C and ingestion of the higher virus dose (10^7 PFU/mL) will limit the likelihood of such events occurring.

Rift Valley fever virus (RVFV) is a mosquito-borne virus which has the ability to infect a large variety of animals including humans in Africa and Arabian Peninsula. The abortion rate among these animals are close to 100%, and young animals develop severe disease which often are lethal. In humans, Rift Valley fever (RVF) presents in most cases as a mild illness with influenza-like symptoms. However, in about 8% of the cases it progresses into a more severe disease with a high case fatality rate. Since there is such a high abortion rate among infected animals, a link between human miscarriage and RVFV has been suggested, but never proven. We could in paper I for the first time show an association between acute RVFV infection and miscarriage in humans. We observed an increase in pregnant women arriving at the Port Sudan Hospital with fever of unknown origin, and several of the patients experienced miscarriage. When we analysed their blood samples for several viral diseases we found that many had an acute RVFV infection and of these, 54% experienced a miscarriage. The odds of having a miscarriage was 7 times higher for RVFV patients compared to the RVFV negative women of which only 12% miscarried. These results indicated that RVFV infection could be a contributing factor to miscarriage. RVFV is an enveloped virus containing the viral glycoproteins n and c (Gn and Gc respectively), where Gn most likely is responsible for the initial cellular contact. The protein DC-SIGN on dendritic cells and the glycosaminoglycan heparan sulfate has been suggested as cellular receptors for RVFV, however other mechanisms are probably also involved in binding and entry. Charge is a driving force for molecular interaction and has been shown to be important for cellular attachment of several viruses, and in paper II we could show that when the charge around the cells was altered, the infection was affected. We also showed that Gn most likely has a positive charge at a physiological pH. When we added negatively charged molecules to the viral particles before infection, we observed a decreased infection efficiency, which we also observed after removal of carbohydrate structures from the cell surface. Our results suggested that the cellular interaction partner for initial attachment is a negatively charged carbohydrate. Further investigations into the mechanisms of RVFV cellular interactions has to be undertaken in order to understand, and ultimately prevent, infection and disease. There is currently no vaccine approved for human use and no specific treatments for RVF, so there is a great need for developing safe effective drugs targeting this virus. We designed a whole-cell based high-throughput screen (HTS) assay which we used to screen libraries of small molecular compounds for anti-RVFV properties. After dose-response and toxicity analysis of the initial hits, we identified six safe and effective inhibitors of RVFV infection that with further testing could become drug candidates for treatment of RVF. This study demonstrated the application of HTS using a whole-cell virus replication reporter gene assay as an effective method to identify novel compounds with potential antiviral activity against RVFV.

Doctor of Philosophy
Department of Diagnostic Medicine/Pathobiology
Wenjun Ma
Rift Valley fever virus (RVFV) is an enveloped, negative-sense, ssRNA virus with a tripartite genome that causes morbidity and mortality in both livestock and humans. Although RVFV is mainly circulating in mainland Africa, this arthropod-borne virus is a potential threat to the other parts of the world. No fully licensed vaccines for human or animal use in the U.S., and effective antiviral drugs have not been identified. As virulent RVFV strains are only handled in biosafety level (BSL) 3 or higher level facilities in the U.S., few laboratories have access to RVFV which limits antiviral development. However, it is crucial to develop effective antivirals to protect public and animal health. Animal models that reproduce Rift Valley fever are vital to identifying and developing antiviral compounds. The currently available attenuated RVFV strain, MP12, provides a BSL-2 challenge model virus for preliminary investigations of RVFV prior to using the virulent RVFV strains. All strains of RVFV have a highly conserved genome, indicating that antivirals or vaccines effective against any RVFV strain will most likely be effective for all RVFV strains. Therefore, we hypothesize that the MP12 is a suitable model virus that can be used for identification and evaluation of effective RVF antivirals. The first objective of this project was to establish a mouse model susceptible to MP12 infection. Based on the literature, we selected and screened six different strains of mice to test their susceptibilities to MP12. We found the STAT-1 knockout mice are the most susceptible to MP12 infection based on clinical symptoms, mortality, viremia, virus replication, histopathological, and immunochemical analyses. Importantly, these mice displayed acute-onset hepatitis and delayed-onset encephalitis similar to severe cases of human RVFV infection. Our second objective was to identify potential antiviral drugs in vitro. We developed and employed a cell-based assay using the recombinant MP12 virus expressing Renilla luciferase to screen a library of 727 small compounds purchased from National Institutes of Health. Of the compounds, 23 were identified and further tested for their inhibitory activities on the recombinant MP12 virus expressing green fluorescent protein. Further plaque reduction assays confirmed that two compounds inhibited replication of parental RVFV MP12 strain with limited cytotoxic effects. The 50% inhibitory concentrations using an MP12 multiplicity of infection (MOI) of 2 were 211.4 µM and 139.5 µM, respectively. Our third objective was to evaluate these two candidates, 6-azauridine and mitoxantrone, in vivo using our mouse model. After one-hour post MP12 infection via an intranasal route, treatment was given intranasally twice daily. Mice treated with placebo and 6-azauridine displayed severe weight loss and reached the threshold for euthanasia with obvious neurological signs, while mice treated with ribavirin (a known antiviral drug) or mitoxantrone showed delayed onset of disease. This result indicates that the mitoxantrone can improve the outcome of RVFV infection in our mouse model. The underlying mechanism of mitoxantrone to inhibit RVFV replication remains to be investigated. Our studies build the foundation for identification and development of antivirals against RVFV in a BSL-2 environment.

Rift Valley Fever (RVF) is an emerging infectious disease found in both livestock and humans. RVF is associated with high abortion and mortality rates in livestock and can be fatal in humans. As such, RVF is economically and socially significant to affected smallholder and subsistence farmers, those infected, and national livestock industries. However, Rift Valley Fever virus (RVFV) vaccines are not commercially available outside of endemic areas or for humans, and current vaccines are limited in their safety and efficacy. A plant-based, viral nanoparticle vaccine offers a more affordable alternative to conventional vaccines that is safe, rapidly producible, and easily scalable, better meeting the needs of impacted communities. This project focuses on assessing the potential of using a Nicotiana benthamiana plant expression system to generate recombinant tobacco mosaic virus (TMV) nanoparticles displaying RVFV glycoprotein epitopes. Eight TMV-RVFV glycoprotein constructs were designed. Five TMV-RVFV constructs were successfully cloned, and four recombinant TMV constructs were successfully expressed in planta. The antigenicity of these constructs was examined for their possible use in RVFV vaccine development.

Les malalties transmeses per vectors representen un alt percentatge de les malalties infeccioses en el món. Concretament, les malalties causades per arbovirus (arthropod-born viruses), que circulen en la naturalesa entre artròpodes (els seus vectors), i els hostes vertebrats (els seus reservorios), poden causar malalties greus en els hostes vertebrats, però no causen una patologia significativa en els vectors. Durant dècades les malalties causades per arbovirus van ser oblidades, ja que en la seva gran majoria estaven localitzades en zones en vies de desenvolupament. En l'actualitat, factors ambientals, ecològics i socioeconòmics, com el canvi climàtic i la globalització, han contribuït a l'emergència i reemergencia de les malalties arbovirales. El constant moviment de persones i mercaderies ha donat lloc a la colonització i establiment d'espècies d'exòtiques al nostre país, com el mosquit tigre (Aedes albopictus), el qual és transmissor de molts arbovirus (e.g. el virus del dengue, el virus Zika (ZIKV) o el virus chikungunya). El desenvolupament d'aquesta tesi es va centrar en realitzar estudis de competència vectorial per al ZIKV i en un estudi del transcriptoma de Culex pipiens després de ser exposat al phlebovirus de la febre de la Vall del Rift (RVFV) per a comprendre les interaccions el virus i els mosquits locals. Els capítols I i II es van focalitzar a estimar la competència vectorial per a ZIKV de diferents espècies de mosquits de camp presents al nostre país: Aedes albopictus, Aedes caspius i Culex pipiens. A més, es van desenvolupar experiments de transmissió vertical per a determinar si la generació de mosquits provinents de femelles infectades amb el ZIKV és capaç de disseminar-ho. Durant el desenvolupament d'aquests estudis, s'ha demostrat que els mosquits locals de l'espècie Ae. albopictus són vectors competents per al ZIKV. No obstant això, les espècies Cx. pipiens i Ae. caspius són refractàries per a aquest arbovirus. Respecte a l'experiment de transmissió vertical, es va demostrar que la progènie de les femelles inoculades amb el virus de manera intratoràcica va ser susceptible a la infecció del virus, però no van ser capaços de disseminar-lo. D'altra banda, el capítol III es va centrar en l'estudi de les interaccions a nivell molecular entre l'espècie de mosquit Cx. pipiens i RVFV, amb l'objectiu caracteritzar les alteracions a nivell molecular de l'expressió dels gens corresponents al sistema immune del mosquit durant la infecció per RVFV mitjançant una anàlisi del transcriptoma de novo. Com a resultat, es van obtenir 48 gens diferencialment expressats en els mosquits davant la presència del virus que servir de diana per a controlar la infecció, ja sigui per a desequilibrar la tolerància dels mosquits al virus com per a inhibir la infecció en els mosquits. Els resultats obtinguts de l'estudi de les alteracions del transcriptoma de mosquits de l'espècie Cx. pipiens exposats a RVFV estableixen les bases per a la realització de futurs estudis funcionals dels gens involucrats a controlar/permetre la infecció per RVFV. En el seu conjunt, el desenvolupament d'aquesta tesi incrementa el coneixement per a millorar el disseny d'estratègies eficients per a la vigilància de vectors transmissors del ZIKV i del RVFV.
Las enfermedades transmitidas por vectores representan un alto porcentaje de las enfermedades infecciosas en el mundo. Concretamente, las enfermedades causadas por arbovirus (arthropod-borne viruses), que circulan en la naturaleza entre artrópodos (sus vectores), y los hospedadores vertebrados (sus reservorios), pueden causar enfermedades graves en los hospedadores vertebrados, pero no causan una patología significativa en los vectores. Durante décadas las enfermedades causadas por arbovirus fueron olvidadas, ya que en su gran mayoría estaban localizadas en zonas en vías de desarrollo. En la actualidad, factores ambientales, ecológicos y socioeconómicos, como el cambio climático y la globalización, han contribuido a la emergencia y reemergencia de las enfermedades arbovirales. El constante movimiento de personas y mercancías ha dado lugar a la colonización y establecimiento de especies de exóticas en nuestro país, como el mosquito tigre (Aedes albopictus), el cual es transmisor de muchos arbovirus (e.g. el virus del dengue, el virus Zika (ZIKV) o el virus chikungunya). El desarrollo de esta tesis se centró en realizar estudios de competencia vectorial para el ZIKV y en un estudio del transcriptoma de Culex pipiens después de ser expuesto al phlebovirus de la fiebre del Valle del Rift (RVFV) para comprender las interacciones el virus y los mosquitos locales. Los capítulos I y II se focalizaron en estimar la competencia vectorial para ZIKV de diferentes especies de mosquitos de campo presentes en nuestro país: Aedes albopictus, Aedes caspius y Culex pipiens. Además, se desarrollaron experimentos de transmisión vertical para determinar si la generación de mosquitos provenientes de hembras infectadas con el ZIKV es capaz de diseminarlo. Durante el desarrollo de estos estudios, se ha demostrado que los mosquitos locales de la especie Ae. albopictus son vectores competentes para el ZIKV. Sin embargo, las especies Cx. pipiens y Ae. caspius son refractarias para este arbovirus. Con respecto al experimento de transmisión vertical, se demostró que la progenie de las hembras inoculadas con el virus de forma intratorácica fue susceptible a la infección del virus, pero no fueron capaces de diseminarlo. Por otro lado, el capítulo III se centró en el estudio de las interacciones a nivel molecular entre la especie de mosquito Cx. pipiens y RVFV, con el objetivo caracterizar las alteraciones a nivel molecular de la expresión de los genes correspondientes al sistema inmune del mosquito durante la infección por RVFV mediante un análisis del transcriptoma de novo. Como resultado, se obtuvieron 48 genes diferencialmente expresados en los mosquitos ante la presencia del virus que servir de diana para controlar la infección, ya sea para desequilibrar la tolerancia de los mosquitos al virus como para inhibir la infección en los mosquitos. Los resultados obtenidos del estudio de las alteraciones del transcriptoma de mosquitos de la especie Cx. pipiens expuestos a RVFV sientan las bases para la realización de futuros estudios funcionales de los genes involucrados en controlar/permitir la infección por RVFV. En conjunto, el desarrollo de esta tesis incrementa el conocimiento para mejorar el diseño de estrategias eficientes para la vigilancia de vectores transmisores del ZIKV y del RVFV.
Vector-borne diseases represent a 17 % of infectious diseases in the world. Among them, those diseases caused by arboviruses (arthropod-borne viruses), which circulate in the nature between arthropods (their vectors) and vertebrate hosts (their reservoirs), are currently provoking serious diseases in humans and animals. For decades, the arboviral diseases were neglected, since most of them were located in developing areas. Nowadays, environmental, ecological and socioeconomic factors (e.g., globalization and climate change) have contributed to the emergence and re-emergence of arboviral diseases. The constant movement of people and merchandise has allowed the colonization and establishment of exotic mosquito species in our country such as the tiger mosquito (Aedes albopictus), which is a potential vector of many arboviruses (e.g., dengue virus, Zika virus or chikungunya virus). This thesis focused on conducting vector competence and transmission studies in local mosquito species for Zika virus (ZIKV) and on the study of the Culex pipiens transcriptome alteration after being exposed to the Rift Valley fever phlebovirus (RVFV) in order to better understand how virus-vector interaction influences on ZIKV and RVFV transmission. Chapters I and II focused on estimating the vector competence for ZIKV of different field-collected mosquito species present in our country: Aedes albopictus, Aedes caspius and Culex pipiens. In addition, vertical transmission studies were performed to determine if the progeny of females infected with ZIKV were able to disseminate the virus. The results of these studies showed that local populations of Ae. albopictus were competent vectors for ZIKV and Cx. pipiens and Ae. caspius species were refractory for this arbovirus. Moreover, it was demonstrated that ZIKV was able to be transmitted to the progeny but the later could not disseminate the virus. Chapter III focused on the study of interactions between the Cx. pipiens mosquito species and RVFV at molecular level, with the aim to characterize the alterations in the expression of the mosquito genes related to the immune system during RVFV infection by analyzing de novo transcriptome. As a result, 48 immune differentially expressed genes in mosquitoes exposed to RVFV were altered, which could serve as potential targets to control the infection, either by unbalancing the mosquito tolerance to RVFV or by inhibiting the infection in mosquitoes. The results obtained on the Cx. pipiens transcriptome alterations due to exposure to RVFV pave the way for future functional studies about genes involved in the control/tolerance of RVFV infection. Overall, this thesis increased the knowledge to better design efficient strategies for ZIKV and RVFV surveillance and control.

The Adames strain of Punta Toro virus (PTV-A) causes acute hepatic disease in hamsters and mice similar to that seen in natural Rift Valley fever virus (RVFV) infection, while the Balliet strain (PTV-B) is apathogenic. The ability of PTV-A to suppress the interferon (IFN) response has been demonstrated in hamsters and is thought to be a contributing factor to PTV-A's pathogenicity in hamsters. PTV-B is not assumed to exhibit this IFN-antagonistic activity, as it stimulates production of significantly higher IFN-β levels. To elucidate the role of IFN in resistance of mice to PTV-B infection, we utilized mice deficient in a critical IFN signaling protein, STAT-1. We found that these mice were drastically more susceptible to PTV-B, which caused 100% lethality compared to 0% in their wild-type counterparts. STAT-1 deficient mice were also more susceptible to PTV-A, as these mice succumbed to infection significantly earlier than wild-type mice (p=0.0058). We sought to determine whether PTV-A's IFN-antagonistic mechanism is functional in mice by examining expression of IFN-β in primary macrophages infected with either strain. We found that IFN-β protein concentration is higher in samples taken from PTV-B-infected cells. We employed quantitative PCR arrays specific to IFN signaling and response pathways to evaluate changes in gene expression throughout the course of infection with either virus strain. We found several genes with differentially regulated expression between PTV-A- and PTV-B-infected macrophages, including Ifnβ1 and multiple Ifnα subtypes. Also, several genes coding for inflammatory and chemotactic molecules, Cxcl11, Cxcl10, Cxcl9, Vcam1, and Il6, demonstrated increased expression in PTV-B samples compared to PTV-A. Of particular interest, Isg20, a 3'-5' exonuclease with specificity for single-stranded RNA, was stimulated ~2-fold higher by PTV-B, and Iigp1, from the family of GTPases associated with host defense against intracellular pathogens, was stimulated ~2.7-fold higher by PTV-B. The individual functions of each of these genes in mouse resistance to PTV-B could be a focus of future studies to better understand essential host defense mechanisms to phleboviral infection.

Rift Valley fever (RVF) is an acute disease of domestic ruminants in mainland Africa and Madagascar, caused by a mosquito borne virus and characterized by necrotic hepatitis and a haemorrhagic state. Large outbreaks of the disease in sheep, cattle and goats occur at irregular intervals of several years when exceptionally heavy rains favour the breeding of the mosquito vectors, and are distinguished by heavy mortality among newborn animals and abortion in pregnant animals. Humans become infected from contact with tissues of infected animals or from mosquito bite, and usually develop mild to moderately severe febrile illness, but severe complications, which occur in a small proportion of patients, include ocular sequelae, encephalitis and fatal haemorrhagic disease. Despite the occurrence of low case fatality rates, substantial numbers of humans may succumb to the disease during large outbreaks. Modified live and inactivated vaccines are available for use in livestock, and an inactivated vaccine was used on a limited scale in humans with occupational exposure to infection. The literature on the disease has been the subject of several extensive reviews from which the information presented here is drawn, except where indicated otherwise (Henning 1956; Weiss 1957; Easterday 1965; Peters and Meegan 1981; Shimshony and Barzilai 1983; Meegan and Bailey 1989; Swanepoel and Coetzer 2004; Flick and Bouloy 2005). In September 2000, the disease appeared in south-west Saudi Arabia and adjacent Yemen, and the outbreak lasted until early 2001 (Al Hazmi et al. 2003; Madani et al. 2003; Abdo-Salem et al. 2006). The virus was probably introduced with infected livestock from the Horn of Africa, and it remains to be determined whether it has become endemic on the Arabian Peninsula.

The arboviruses are all single-stranded RNA viruses, although they belong to four different viral families. Several important human pathogens belong to the mosquito-borne arboviruses including yellow fever, Japanese encephalitis and Rift Valley Fever. They cause a wide range of illnesses from unrecognised infection to severe systemic disease with hemorrhagic complications and encephalitis with a high mortality similar range of illnesses is seen in infected animals.Arboviruses have several unique characteristics, these include; an ability to infect and be transmitted by mosquitos, ticks, midges, sand flies, bugs, fleas, blackflies and horseflies. They infect vertebrate hosts which may amplify virus for invertebrate vectors that feed on infected vertebrates. An ability to replicate in anthropods, with little pathology and in vertebrates often with significant pathology. Many arboviruses are Zoonotic.Control methods depend on the epidemiology of particular viruses, but epidemic vector control through control of insect breeding sites and the use of insecticide spraying have been successfully used in the past. Effective vaccines are available for yellow fever and Japanese encephalitis.

Rift Valley fever is a severe disease affecting both humans and animals. The Rift Valley fever virus can be transmitted by body fluids, and the most common way for humans to get infected is from animals. The virus is also vector-borne and can be transmitted by many species of mosquitoes. As with other vector-borne diseases, the epidemiology may vary in response to environmental changes. Here the effects of climate and land use changes on Rift Valley fever, as well as on other vector-borne diseases, are discussed. The effect of irrigation in East Africa on inter-epidemic transmission of RVF is discussed in greater detail, followed by recommendations for future research and actions.

Rift Valley fever is a severe disease affecting both humans and animals. The Rift Valley fever virus can be transmitted by body fluids, and the most common way for humans to get infected is from animals. The virus is also vector-borne and can be transmitted by many species of mosquitoes. As with other vector-borne diseases, the epidemiology may vary in response to environmental changes. Here the effects of climate and land use changes on Rift Valley fever, as well as on other vector-borne diseases, are discussed. The effect of irrigation in East Africa on inter-epidemic transmission of RVF is discussed in greater detail, followed by recommendations for future research and actions.