Publications by authors named "Francisco Llorente"

39 Publications

West Nile and Usutu virus infections in wild birds admitted to rehabilitation centres in Extremadura, western Spain, 2017-2019.

Vet Microbiol 2021 Apr 24;255:109020. Epub 2021 Feb 24.

Animal Health Department, Veterinary Faculty, University of Extremadura (UEx), Cáceres, Spain. Electronic address:

West Nile virus (WNV) is an emerging flavivirus transmitted generally by mosquitoes of Culex genus. It is maintained in an enzootic life cycle where birds act as reservoir hosts. Humans and horses are also susceptible to infection, and occasionally, they suffer from neurological complications. However, they do not transmit the virus to other vectors, behaving as dead-end hosts. Sporadic WNV outbreaks observed in horses and wild birds from Extremadura (western Spain) during 2016 and 2017 seasons prompted to carry out this survey in wild birds, focused on specimens coming from two wildlife rehabilitation centres. Between October 2017 and December 2019, samples from 391 wild birds, belonging to 56 different species were collected and analysed in search of evidence of WNV infection. The analysis of serum samples for WNV-specific antibodies by ELISA, whose specificity was subsequently confirmed by virus-neutralisation test (VNT) showed positive results in 18.23 % birds belonging to 18 different species. Pelecaniformes (33.33 %), Accipitriformes (25.77 %) and Strigiformes (22.92 %) orders had the higher seroprevalences. Remarkably, WNV-specific antibodies were found in a black stork for the first time in Europe. Analysis by real time RT-PCR in symptomatic birds confirmed the presence of WNV lineage 1 RNA in griffon vulture and little owls. Specificity analysis of ELISA positive and doubtful sera was performed by differential VNT titration against WNV and two other cross-reacting avian flaviviruses found in Spain: Usutu virus (USUV) and Bagaza virus (BAGV). Only four samples showed USUV-specific antibodies (1.04 %) corresponding to three species: Eurasian eagle-owl, griffon vulture and great bustard (first detection in Europe) whereas no samples were found reactive to BAGV. Differential VNT yielded undetermined flavivirus result in 16 samples (4.17 %). This is the first study carried out on wild birds from Extremadura (western Spain). It highlights the widespread circulation of WNV in the region and its co-circulation with USUV.
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http://dx.doi.org/10.1016/j.vetmic.2021.109020DOI Listing
April 2021

Evaluation of West Nile Virus Diagnostic Capacities in Veterinary Laboratories of the Mediterranean and Black Sea Regions.

Pathogens 2020 Dec 11;9(12). Epub 2020 Dec 11.

Centro de Investigación en Sanidad Animal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-CISA), 28130 Valdeolmos, Spain.

The increasing incidence of West Nile virus (WNV) in the Euro-Mediterranean area warrants the implementation of effective surveillance programs in animals. A crucial step in the fight against the disease is the evaluation of the capacity of the veterinary labs to accurately detect the infection in animal populations. In this context, the animal virology network of the MediLabSecure project organized an external quality assessment (EQA) to evaluate the WNV molecular and serological diagnostic capacities of beneficiary veterinary labs. Laboratories from 17 Mediterranean and Black Sea countries participated. The results of the triplex real time RT-PCR for simultaneous detection and differentiation of WNV lineage 1 (L1), lineage 2 (L2) and Usutu virus (USUV) were highly satisfactory, especially for L1 and L2, with detection rates of 97.9% and 100%, respectively. For USUV, 75% of the labs reported correct results. More limitations were observed for the generic detection of flaviviruses using conventional reverse-transcription polymerase chain reaction (RT-PCR), since only 46.1% reported correct results in the whole panel. As regards the serological panel, the results were excellent for the generic detection of WNV antibodies. More variability was observed for the specific detection of IgM antibodies with a higher percentage of incorrect results mainly in samples with low titers. This EQA provides a good overview of the WNV (and USUV) diagnostic performance of the involved veterinary labs and demonstrates that the implemented training program was successful in upgrading their diagnostic capacities.
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http://dx.doi.org/10.3390/pathogens9121038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763240PMC
December 2020

External quality assessment of Rift Valley fever diagnosis in 17 veterinary laboratories of the Mediterranean and Black Sea regions.

PLoS One 2020 28;15(9):e0239478. Epub 2020 Sep 28.

Centro de Investigación en Sanidad Animal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-CISA), Valdeolmos, Spain.

Rift Valley fever (RVF) is an arboviral zoonosis that primarily affects ruminants but can also cause illness in humans. The increasing impact of RVF in Africa and Middle East and the risk of expansion to other areas such as Europe, where competent mosquitos are already established, require the implementation of efficient surveillance programs in animal populations. For that, it is pivotal to regularly assess the performance of existing diagnostic tests and to evaluate the capacity of veterinary labs of endemic and non-endemic countries to detect the infection in an accurate and timely manner. In this context, the animal virology network of the MediLabSecure project organized between October 2016 and March 2017 an external quality assessment (EQA) to evaluate the RVF diagnostic capacities of beneficiary veterinary labs. This EQA was conceived as the last step of a training curriculum that included 2 diagnostic workshops that were organized by INIA-CISA (Spain) in 2015 and 2016. Seventeen veterinary diagnostic labs from 17 countries in the Mediterranean and Black Sea regions participated in this EQA. The exercise consisted of two panels of samples for molecular and serological detection of the virus. The laboratories were also provided with positive controls and all the kits and reagents necessary to perform the recommended diagnostic techniques. All the labs were able to apply the different protocols and to provide the results on time. The performance was good in the molecular panel with 70.6% of participants reporting 100% correct results, and excellent in the serological panel with 100% correct results reported by 94.1% of the labs. This EQA provided a good overview of the RVFV diagnostic capacities of the involved labs and demonstrated that most of them were able to correctly identify the virus genome and antibodies in different animal samples.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0239478PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7521712PMC
November 2020

Serological evidence of co-circulation of West Nile and Usutu viruses in equids from western Spain.

Transbound Emerg Dis 2020 Aug 27. Epub 2020 Aug 27.

Animal Health Department, Veterinary Faculty, University of Extremadura (UEx), Cáceres, Spain.

West Nile virus (WNV) is a mosquito-borne emerging virus in Europe with capacity to cause neurological complications such as encephalitis or meningoencephalitis in humans, birds or equids. In Spain, WNV is actively circulating in mosquitoes, birds and horses in different regions, but never has been deeply studied in Extremadura. Therefore, the aim of this study was to evaluate the seroprevalence of WNV in equids of those areas and to analyse the risk factors associated with exposure to the virus. A total of 199 out of 725 equids presented antibodies against WNV by competition ELISA (27.45%), while 22 were doubtful (3.03%). Anti-WNV IgM antibodies were detected in 16 equids (2.21%), and 3 animals were doubtful (0.41%). All ELISA-reactive positive/doubtful sera (N = 226) were further tested by micro-virus neutralization test (VNT), and a total of 143 horses were confirmed as positive for WNV, obtaining a seroprevalence of 19.72% in equids of western Spain. In addition, specific antibodies against USUV were confirmed in 11 equids. In 24 equids, a specific flavivirus species (detected by ELISA test) could not be determined. The generalized linear mixed-effects models showed that the significant risk factors associated with individual WNV infection in equids were the age (adults) and hair coat colour (light), whereas in USUV infections, it was the breed (pure). Data demonstrated that WNV and USUV are circulating in regions of western Spain. Given the high WNV seroprevalence found in equids from the studied areas, it is important to improve the surveillance programmes of public health to detect undiagnosed human cases and to establish a vaccination programme in equid herds in these regions.
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http://dx.doi.org/10.1111/tbed.13810DOI Listing
August 2020

The role of different Culex mosquito species in the transmission of West Nile virus and avian malaria parasites in Mediterranean areas.

Transbound Emerg Dis 2021 Mar 31;68(2):920-930. Epub 2020 Aug 31.

Estación Biológica de Doñana (EBD-CSIC), Seville, Spain.

Vector-borne diseases, especially those transmitted by mosquitoes, have severe impacts on public health and economy. West Nile virus (WNV) and avian malaria parasites of the genus Plasmodium are mosquito-borne pathogens that may produce severe disease and illness in humans and birds, respectively, and circulate in an endemic form in southern Europe. Here, we used field-collected data to identify the impact of Culex pipiens, Cx. perexiguus and Cx. modestus, on the circulation of both WNV and Plasmodium in Andalusia (SW Spain) using mathematical modelling of the basic reproduction number (R ). Models were calibrated with field-collected data on WNV seroprevalence and Plasmodium infection in wild house sparrows, presence of WNV and Plasmodium in mosquito pools, and mosquito blood-feeding patterns. This approach allowed us to determine the contribution of each vector species to pathogen amplification. Overall, 0.7% and 29.6% of house sparrows were positive to WNV antibodies and Plasmodium infection, respectively. In addition, the prevalence of Plasmodium was higher in Cx. pipiens (2.0%), followed by Cx. perexiguus (1.8%) and Cx. modestus (0.7%). Three pools of Cx. perexiguus were positive to WVN. Models identified Cx. perexiguus as the most important species contributing to the amplification of WNV in southern Spain. For Plasmodium models, R values were higher when Cx. pipiens was present in the population, either alone or in combination with the other mosquito species. These results suggest that the transmission of these vector-borne pathogens depends on different Culex species, and consequently, their transmission niches will present different spatial and temporal patterns. For WNV, targeted surveillance and control of Cx. perexiguus populations appear as the most effective measure to reduce WNV amplification. Also, preventing Culex populations near human settlements, or reducing the abundance of these species, are potential strategies to reduce WNV spillover into human populations in southern Spain.
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http://dx.doi.org/10.1111/tbed.13760DOI Listing
March 2021

A Duplex Quantitative Real-Time Reverse Transcription-PCR for Simultaneous Detection and Differentiation of Flaviviruses of the Japanese Encephalitis and Ntaya Serocomplexes in Birds.

Front Vet Sci 2020 21;7:203. Epub 2020 Apr 21.

Centro de Investigación en Sanidad Animal (CISA), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Valdeolmos-Alalpardo, Spain.

High impact, mosquito-borne flaviviruses such as West Nile virus (WNV), Usutu virus (USUV), Japanese encephalitis virus (JEV), Tembusu virus (TMUV), and Bagaza/Israel turkey meningoencephalomyelitis virus (BAGV/ITV) are emerging in different areas of the world. These viruses belong to the Japanese encephalitis (JE) serocomplex (JEV, WNV, and USUV) and the Ntaya serocomplex (TMUV and BAGV/ITV). Notably, they share transmission route (mosquito bite) and reservoir host type (wild birds), and some of them co-circulate in the same areas, infecting overlapping mosquito and avian population. This may simplify epidemiological surveillance, since it allows the detection of different infections targeting the same population, but also represents a challenge, as the diagnostic tools applied need to detect the whole range of flaviviruses surveyed, and correctly differentiate between these closely related pathogens. To this aim, a duplex real-time RT-PCR (dRRT-PCR) method has been developed for the simultaneous and differential detection of JE and Ntaya flavivirus serocomplexes. The method has been standardized and evaluated by analyzing a panel of 49 flaviviral and non-flaviviral isolates, and clinical samples of different bird species obtained from experimental infections or from the field, proving its value for virus detection in apparently healthy or suspicious animals. This new dRRT-PCR technique is a reliable, specific and highly sensitive tool for rapid detection and differentiation of JE and Ntaya flavivirus groups in either domestic or wild animals. This novel method can be implemented in animal virology diagnostic laboratories as screening tool in routine surveillance and in the event of bird encephalitis emergence.
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http://dx.doi.org/10.3389/fvets.2020.00203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7186316PMC
April 2020

Exposure of Wild Ungulates to the Usutu and Tick-Borne Encephalitis Viruses in France in 2009-2014: Evidence of Undetected Flavivirus Circulation a Decade Ago.

Viruses 2019 12 19;12(1). Epub 2019 Dec 19.

UMR (Unité mixte de recherche) Virologie, INRAE, Ecole Nationale Vétérinaire d'Alfort, ANSES, Université Paris-Est, 94700 Maisons-Alfort, France.

Flaviviruses have become increasingly important pathogens in Europe over the past few decades. A better understanding of the spatiotemporal distribution of flaviviruses in France is needed to better define risk areas and to gain knowledge of the dynamics of virus transmission cycles. Serum samples from 1014 wild boar and 758 roe deer from 16 departments (administrative units) in France collected from 2009 to 2014 were screened for flavivirus antibodies using a competitive ELISA (cELISA) technique. Serum samples found to be positive or doubtful by cELISA were then tested for antibodies directed against West Nile virus (WNV), Usutu virus (USUV), Bagaza virus (BAGV), and tick-borne encephalitis/Louping ill viruses (TBEV/LIV) by microsphere immunoassays (except BAGV) and micro-neutralization tests. USUV antibodies were detected only in southeastern and southwestern areas. TBEV/LIV antibodies were detected in serum samples from eastern, southwestern and northern departments. The results indicate continuous circulation of USUV in southern France from 2009 to 2014, which was unnoticed by the French monitoring system for bird mortality. The findings also confirm wider distribution of TBEV in the eastern part of the country than of human clinical cases. However, further studies are needed to determine the tick-borne flavivirus responsible for the seroconversion in southwestern and northern France.
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http://dx.doi.org/10.3390/v12010010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019733PMC
December 2019

Influence of flavivirus co-circulation in serological diagnostics and surveillance: A model of study using West Nile, Usutu and Bagaza viruses.

Transbound Emerg Dis 2019 Sep 28;66(5):2100-2106. Epub 2019 Jun 28.

Centro de Investigación en Sanidad Animal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-CISA), Valdeolmos, Spain.

This study aims at assessing the serological cross-reactions existing between three mosquito-borne flaviviruses with avian reservoirs co-circulating in Europe: West Nile (WNV), Usutu (USUV) and Bagaza (BAGV). The study is useful for a better interpretation of serological results in diagnostics and surveillance. Serum samples obtained from a natural host, the red-legged partridge (Alectoris rufa), experimentally infected with WNV, USUV or BAGV were analysed using two commercially available WNV competition ELISAs suitable for serological surveillance, and by the confirmatory virus neutralization test (VNT). The ELISAs examined showed different levels of specificity for WNV, as judged by cross-reaction observed with the other flaviviruses. By VNT, virus-specific antibodies were confirmed in 80%, 50% or 0% of sera from WNV-, BAGV-, or USUV-inoculated birds, respectively. The results indicate how the co-circulation of cross-reacting flaviviruses may affect the outcomes of WNV serological surveillance when applying currently available serological tools. On the one hand, the choice of the ELISA test for antibody screening should consider the differences found in specificity, since one test is more specific for WNV while the other one is more suitable for detection of a broader range of flavivirus antibodies. On the other hand, besides corroborating that cross-neutralization occurs between flaviviruses from different serocomplexes (WNV/USUV and BAGV), this study points out that cross-neutralization between WNV and USUV is not symmetric, and reveals the difficulty to identify USUV infections serologically. This finding indicates that actual USUV infections might be underestimated in the current diagnostic schemes.
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http://dx.doi.org/10.1111/tbed.13262DOI Listing
September 2019

Susceptibility and role as competent host of the red-legged partridge after infection with lineage 1 and 2 West Nile virus isolates of Mediterranean and Central European origin.

Vet Microbiol 2018 Aug 18;222:39-45. Epub 2018 Jun 18.

Centro de Investigación en Sanidad Animal-Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-CISA), Carretera Algete-El Casar s/n, 28130, Valdeolmos, Madrid, Spain; CIBER Epidemiologia y Salud Pública (CIBERESP), Madrid, Spain.

West Nile virus (WNV; genus Flavivirus; family Flaviviridae) is the aetiological agent of an emerging, mosquito-borne disease with great impact on human and animal health. Over the past 15 years, WNV has been responsible for large epidemics mainly in North America but also in Europe, where lineage 1 and more recently lineage 2 strains have caused an upsurge in the number of outbreaks with increased human infection and higher virulence for certain wild bird species. This study aimed to compare the course of infection of the lineage 1 WNV strains Israel/98 and Italy/08 and the lineage 2 strain Austria/08 in the red-legged partridge (Alectoris rufa), a gallinaceous bird indigenous to the Iberian Peninsula and widely distributed in Southern and Western Europe. After experimental inoculation, clinical and analytic parameters (viraemia, viral load, antibodies) were examined over a period of 15 days. All inoculated birds became viremic and showed clinical disease, with a morbidity rate of 100% and mortality rates between 22.2 and 55.5% depending on the virus strain. The red-legged partridge demonstrated to be a competent host for transmission of the three investigated WNV isolates with the highest competence index observed for the Italian strain. Likewise, this strain was the most pathogenic causing the highest viral loads in blood, organs, feathers and oral and cloacal secretions. These experimental results indicate that the red-legged partridge is highly susceptible to the infection with lineage 1 and 2 WNV strains and that this species may act as an amplifying host for both WNV lineages.
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http://dx.doi.org/10.1016/j.vetmic.2018.06.012DOI Listing
August 2018

A monoclonal antibody to DIII E protein allowing the differentiation of West Nile virus from other flaviviruses by a lateral flow assay.

J Virol Methods 2018 10 26;260:41-44. Epub 2018 Jun 26.

Inmunología y Genética Aplicada S.A. (INGENASA), Madrid, Spain.

West Nile Virus (WNV) belongs to the Flaviviridae family, genus Flavivirus, which includes other emerging arthropod-borne viruses (arboviruses) pathogenic for animals and/or humans. West Nile Virus is a genetically diverse RNA virus with at least 7 different recognized lineages. Following its recent introduction and subsequent expansion to the Americas, WNV is currently one of the most widely spread arboviruses in the world having recently re-emerged in the Mediterranean basin, Central and Eastern Europe. Laboratory tests are essential to confirm WNV infection and monoclonal antibodies represent useful tools for the development of diagnostic assays. A monoclonal antibody, 1D11, recognizing an epitope in the domain III of the envelope glycoprotein of WNV, was selected for this study. Its suitability to detect a range of WNV variants representative of its whole genetic range, and to differentiate it from other flaviviruses and arboviruses, was assessed by means of an immunochromatographic assay in an LFA format. A panel of cell culture supernatants infected with 9 different WNV isolates representing a wide range of genetic lineages, and 16 non-WNV arboviruses, including flaviviruses closely related to WNV, were tested. The mAb correctly detected all WNV strains, and did not react with any of the non-WNV arboviruses.
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http://dx.doi.org/10.1016/j.jviromet.2018.06.016DOI Listing
October 2018

Experimental infection of grey partridges with Bagaza virus: pathogenicity evaluation and potential role as a competent host.

Vet Res 2018 05 9;49(1):44. Epub 2018 May 9.

Centro de Investigación en Sanidad Animal (INIA-CISA), Ctra. Algete a El Casar, 28130, Valdeolmos, Spain.

Bagaza virus (BAGV; synonymous to Israel turkey meningoencephalomyelitis virus, ITV) is a relevant arthropod-borne epornitic flavivirus. In its first emergence in Europe (southern Spain, 2010) BAGV caused an outbreak, severely affecting red-legged partridges and common pheasants. The effects (pathogenicity, role as reservoir host) of BAGV in other European phasianids are unknown. To fill this gap, grey partridges were experimentally infected with BAGV. The clinical course of the disease was severe, with neurological signs, significant weight loss and 40% mortality. Low viral loads in the blood and the absence of contact transmission suggest a limited-if any-role on BAGV transmission for this European phasianid.
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http://dx.doi.org/10.1186/s13567-018-0536-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5941787PMC
May 2018

Mosquito community influences West Nile virus seroprevalence in wild birds: implications for the risk of spillover into human populations.

Sci Rep 2018 02 8;8(1):2599. Epub 2018 Feb 8.

Estación Biológica de Doñana (EBD-CSIC), Seville, Spain.

Mosquito community composition plays a central role in the transmission of zoonotic vector-borne pathogens. We evaluated how the mosquito community affects the seroprevalence of West Nile virus (WNV) in house sparrows along an urbanisation gradient in an area with the endemic circulation of this virus. We sampled 2544 birds and 340829 mosquitoes in 45 localities, analysed in 15 groups, each containing one urban, one rural and one natural area. WNV seroprevalence was evaluated using an epitope-blocking ELISA kit and a micro virus-neutralization test (VNT). The presence of WNV antibodies was confirmed in 1.96% and 0.67% of birds by ELISA and VNT, respectively. The VNT-seropositive birds were captured in rural and natural areas, but not in urban areas. Human population density was zero in all the localities where VNT-positive birds were captured, which potentially explains the low incidence of human WNV cases in the area. The prevalence of neutralizing antibodies against WNV was positively correlated with the abundance of the ornithophilic Culex perexiguus but negatively associated with the abundance of the mammophilic Ochlerotatus caspius and Anopheles atroparvus. These results suggest that the enzootic circulation of WNV in Spain occurs in areas with larger populations of Cx. perexiguus and low human population densities.
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http://dx.doi.org/10.1038/s41598-018-20825-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5805708PMC
February 2018

Absence of protection from West Nile virus disease and adverse effects in red legged partridges after non-structural NS1 protein administration.

Comp Immunol Microbiol Infect Dis 2018 Feb 9;56:30-33. Epub 2018 Jan 9.

Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria - Centro de Investigación en Sanidad Animal (INIA-CISA), Ctra Algete-El Casar s/n, 208130, Valdeolmos, Madrid, Spain; Centro de Investigación Biomédica en Red de Epidemiologia y Salud Pública (CIBERESP), Spain. Electronic address:

The red-legged partridge (Alectoris rufa) is a competent host for West Nile virus (WNV) replication and highly susceptible to WNV disease. With the aim to assess in this species whether the inoculation of non-structural protein NS1 from WNV elicits a protective immune response against WNV infection, groups of partridges were inoculated with recombinant NS1 (NS1 group) or an unrelated recombinant protein (mock group), and challenged with infectious WNV. A third group received no inoculation prior to challenge (challenge group). The NS1 group failed to elicit detectable antibodies to NS1 while in the mock group a specific antibody response was observed. Moreover, no protection against WNV disease was observed in the NS1 group, but rather, it showed significantly higher viral RNA load and delayed neutralizing antibody response, and suffered a more severe clinical disease, which resulted in higher mortality. This adverse effect has not been observed before and warrants further investigations.
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http://dx.doi.org/10.1016/j.cimid.2017.12.006DOI Listing
February 2018

Pathogenicity evaluation of twelve West Nile virus strains belonging to four lineages from five continents in a mouse model: discrimination between three pathogenicity categories.

J Gen Virol 2017 Apr;98(4):662-670

CIBER Epidemiología y Salud Pública (CIBERESP), Madrid, Spain.

Rodent models have been used extensively to study West Nile virus (WNV) infection because they develop severe neurological symptoms similar to those observed in human WNV neuroinvasive disease. Most of this research has focused on old lineage (L) 1 strains, while information about pathogenicity is lacking for the most recent L1 and L2 strains, as well as for newly defined lineages. In this study, 4-week-old Swiss mice were inoculated with a collection of 12 WNV isolates, comprising 10 old and recent L1 and L2 strains, the putative L6 strain from Malaysia and the proposed L7 strain Koutango (KOU). The intraperitoneal inoculation of 10-fold dilutions of each strain allowed the characterization of the isolates in terms of LD50, median survival times, ID50, replication in neural and extraneural tissues and antibody production. Based on these results, we classified the isolates in three groups: high virulence (all L1a strains, recent L2 strains and KOU), moderate virulence (B956 strain) and low virulence (Kunjin and Malaysian isolates). We determined that the inoculation of a single dose of 1000 p.f.u. would be sufficient to classify WNV strains by pathotype. We confirmed the enhanced virulence of the KOU strain with a high capacity to cause rapid systemic infection. We also corroborated that differences in pathogenicity among strains do not correlate with phylogenetic lineage or geographic origin, and confirmed that recent European and African WNV strains belonging to L1 and L2 are highly virulent and do not differ in their pathotype profile compared to the prototype NY99 strain.
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http://dx.doi.org/10.1099/jgv.0.000743DOI Listing
April 2017

Bagaza virus is pathogenic and transmitted by direct contact in experimentally infected partridges, but is not infectious in house sparrows and adult mice.

Vet Res 2015 Sep 4;46:93. Epub 2015 Sep 4.

Centro de Investigación en Sanidad Animal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-CISA), Ctra Algete-El Casar s/n, Valdeolmos, Spain.

Bagaza virus (BAGV) is a mosquito-borne flavivirus belonging to the Ntaya serocomplex. In 2010, a disease outbreak was reported in Cádiz (Southern Spain) affecting game birds (red-legged partridges and common pheasants). In this work, red-legged partridges were inoculated experimentally with infectious BAGV isolated from this outbreak in order to make a complete clinical and analytical assessment of the disease caused by the pathogen in this species. Viral load (by real-time RT-PCR) in blood, oral and cloacal swabs, and feathers, and neutralizing antibody titres (by VNT) were measured. In order to determine direct contact transmission, non-inoculated partridges were caged together with the inoculated ones. To assess infectiousness in other species, house sparrows and mice were also inoculated with the virus. All the inoculated partridges were clinically affected, and 30% of them died. All the infected individuals lost weight, with larger losses being recorded in females. Conversely, no mortality or disease symptoms were observed in the sparrows or mice. Remarkably, all the contact partridges acquired the infection by direct (non-vectored) transmission. This study confirms that the red-legged partridge is a susceptible host for BAGV infection, and that this pathogen is transmitted by direct contact. Long-lasting viral loads detected in calami of immature feathers demonstrate that feather sampling could be a useful strategy in active surveillance programs for early detection of BAGV.
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http://dx.doi.org/10.1186/s13567-015-0233-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4559182PMC
September 2015

Experimental infection of house sparrows (Passer domesticus) with West Nile virus strains of lineages 1 and 2.

Vet Microbiol 2014 Aug 10;172(3-4):542-7. Epub 2014 Jun 10.

Centro de Investigación en Sanidad Animal (CISA)-INIA, Ctra. Algete-El Casar s/n, 28130 Valdeolmos, Spain. Electronic address:

West Nile virus (WNV) is a zoonotic pathogen which is maintained in an enzootic cycle between mosquitoes and birds; humans, equines, other mammals and some bird species are dead-end hosts. Lineage 1 WNV strains have predominated in Europe since the 1960s. However, in 2004 lineage 2 strains emerged in Hungary and Russia, respectively, spreading since then to a number of neighbouring countries (e.g., Austria, Greece, Italy, Serbia and Romania). Wild bird mortality is a hallmark of North American WNV outbreaks, a feature uncommon in Europe. This study aimed to compare the course of infection of lineage 1 (NY99) and lineage 2 (Austria/2008) WNV strains in the house sparrow, a bird species common in Europe and North America. House sparrows were inoculated with either NY99 or Austria/2008 WNV strains, or sham-inoculated, and clinical and analytic parameters (viraemia, viral load, antibodies) were examined until 14 days after inoculation. Although all inoculated sparrows became infected, no mortality or clinical signs were observed due to the infection. However, the magnitude and duration of viraemia were higher for NY99 - than for Austria/2008 - infected birds. The house sparrow proved to be a competent host for both strains, although the competence index calculated for NY99 was higher than for Austria/2008. Viral load in tissues and swabs was also higher in NY99-inoculated sparrows. In conclusion, the house sparrow is a convenient avian model for studying host competence of WNV strains. The observed differences between NY99 and Austria/2008 strains might have important epidemiological consequences for disease incidence and dispersal capacity.
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http://dx.doi.org/10.1016/j.vetmic.2014.06.005DOI Listing
August 2014

Experimental infection of house sparrows (Passer domesticus) with West Nile virus isolates of Euro-Mediterranean and North American origins.

Vet Res 2014 Mar 19;45:33. Epub 2014 Mar 19.

Centro de Investigación en Sanidad Animal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (CISA-INIA), Ctra Algete-El Casar s/n, Valdeolmos, Spain.

West Nile virus (WNV) is a zoonotic arboviral pathogen transmitted by mosquitoes in a cycle involving wild birds as reservoir hosts. The virus has recently emerged in North America and re-emerged in Europe. North American WNV outbreaks are often accompanied by high mortality in wild birds, a feature that is uncommon in Europe. The reason for this difference is unknown, but the intrinsic virulence of the viruses circulating in each continent and/or the susceptibility to the disease of Palearctic as opposed to Nearctic wild bird species could play a role. To assess this question, experimental inoculations with four lineage 1 WNV strains, three from southern Europe (Italy/2008, Italy/2009 and Spain/2007) and one from North America (NY99) were performed on house sparrows (Passer domesticus), a wild passerine common in both continents. Non-significant differences which ranged from 0% to 25% were observed in mortality for the different WNV strains. Viremias lasted from 1 to 5-6 days post-inoculation (dpi) in all cases; individuals inoculated with NY99 had significantly higher titres than those inoculated with any of the Euro-Mediterranean strains. Remarkably, host competence was found to be higher for NY99 than for the other strains. Consequently, albeit being pathogenic for house sparrows, some Euro-Mediterranean strains had reduced capacity for replication in -and transmission from- this host, as compared to the NY99 strain. If applicable also to other wild bird host species, this relatively reduced transmission capacity of the Euro-Mediterranean strains could explain the lower incidence of this disease in wild birds in the Euro-Mediterranean area.
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http://dx.doi.org/10.1186/1297-9716-45-33DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3974220PMC
March 2014

Experimental infections of wild birds with West Nile virus.

Viruses 2014 Feb 13;6(2):752-81. Epub 2014 Feb 13.

Centro de Investigación en Sanidad Animal (CISA), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Valdeolmos (Madrid), 28130, Spain.

Avian models of West Nile virus (WNV) disease have become pivotal in the study of infection pathogenesis and transmission, despite the intrinsic constraints that represents this type of experimental research that needs to be conducted in biosecurity level 3 (BSL3) facilities. This review summarizes the main achievements of WNV experimental research carried out in wild birds, highlighting advantages and limitations of this model. Viral and host factors that determine the infection outcome are analyzed in detail, as well as recent discoveries about avian immunity, viral transmission, and persistence achieved through experimental research. Studies of laboratory infections in the natural host will help to understand variations in susceptibility and reservoir competence among bird species, as well as in the epidemiological patterns found in different affected areas.
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http://dx.doi.org/10.3390/v6020752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3939481PMC
February 2014

A novel quantitative multiplex real-time RT-PCR for the simultaneous detection and differentiation of West Nile virus lineages 1 and 2, and of Usutu virus.

J Virol Methods 2013 May 7;189(2):321-7. Epub 2013 Mar 7.

Centro de Investigación en Sanidad Animal, CISA-INIA, Valdeolmos, Spain.

An increase in activity of two mosquito-borne flaviviruses, West Nile virus (WNV) and Usutu virus (USUV), has been reported in Europe in recent years. The current epidemiological situation calls for RT-PCR methods that are able to detect not only the widespread lineage 1 (L1) WNV, but also lineage 2 (L2) WNV. In addition, the presence in Europe of the closely related USUV requires methods that can identify these three flaviviruses and permit an efficient and accurate differential diagnosis. Here we describe a new one-step real-time multiplex RT-PCR that detects and differentiates efficiently WNV-L1, WNV-L2 and USUV in a single reaction. The assay is based on different sets of primers and fluorogenic probes specific to each virus that are labelled with selective, non-overlapping fluorogen-quencher pairs. This enables the fluorescence emitted by each probe, characterized by distinct wavelengths, to be differentiated. This multiplex assay was very sensitive to all of the target viruses; in addition, there were no cross-reactions between the viruses and the assay did not react to any other phylogenetically or symptomatically related viruses. Quantitation was enabled through the use of in vitro-transcribed RNAs developed specifically for each virus as copy number standards. This new assay was validated using different types of experimental and field samples.
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http://dx.doi.org/10.1016/j.jviromet.2013.02.019DOI Listing
May 2013

Disruption of abscisic acid signaling constitutively activates Arabidopsis resistance to the necrotrophic fungus Plectosphaerella cucumerina.

Plant Physiol 2012 Dec 4;160(4):2109-24. Epub 2012 Oct 4.

Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Campus de Montegancedo, E-28223 Madrid, Spain.

Plant resistance to necrotrophic fungi is regulated by a complex set of signaling pathways that includes those mediated by the hormones salicylic acid (SA), ethylene (ET), jasmonic acid (JA), and abscisic acid (ABA). The role of ABA in plant resistance remains controversial, as positive and negative regulatory functions have been described depending on the plant-pathogen interaction analyzed. Here, we show that ABA signaling negatively regulates Arabidopsis (Arabidopsis thaliana) resistance to the necrotrophic fungus Plectosphaerella cucumerina. Arabidopsis plants impaired in ABA biosynthesis, such as the aba1-6 mutant, or in ABA signaling, like the quadruple pyr/pyl mutant (pyr1pyl1pyl2pyl4), were more resistant to P. cucumerina than wild-type plants. In contrast, the hab1-1abi1-2abi2-2 mutant impaired in three phosphatases that negatively regulate ABA signaling displayed an enhanced susceptibility phenotype to this fungus. Comparative transcriptomic analyses of aba1-6 and wild-type plants revealed that the ABA pathway negatively regulates defense genes, many of which are controlled by the SA, JA, or ET pathway. In line with these data, we found that aba1-6 resistance to P. cucumerina was partially compromised when the SA, JA, or ET pathway was disrupted in this mutant. Additionally, in the aba1-6 plants, some genes encoding cell wall-related proteins were misregulated. Fourier transform infrared spectroscopy and biochemical analyses of cell walls from aba1-6 and wild-type plants revealed significant differences in their Fourier transform infrared spectratypes and uronic acid and cellulose contents. All these data suggest that ABA signaling has a complex function in Arabidopsis basal resistance, negatively regulating SA/JA/ET-mediated resistance to necrotrophic fungi.
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http://dx.doi.org/10.1104/pp.112.200154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3510135PMC
December 2012

Antibodies to West Nile virus and related flaviviruses in wild boar, red foxes and other mesomammals from Spain.

Vet Microbiol 2012 Oct 19;159(3-4):291-7. Epub 2012 Apr 19.

Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM-JCCM), Ciudad Real, Spain.

Red foxes (Vulpes vulpes), wild boar (Sus scrofa) and Iberian pigs (Sus scrofa domestica) that are raised extensively outdoors, as well as other wild mesomammals from south central Spain and wild boar from Doñana National Park (DNP), were tested for antibodies against related flaviviruses by ELISA and for antibodies against WNV by VNT. Mean flavivirus seroprevalence according to ELISA was 20.4 ± 7.8% (21 out of 103) in red foxes, 12.6 ± 2.8% (69 out of 545) in wild boars, and 3.3±2.7% (6 out of 177) in Iberian pigs. A stone marten (Martes foina) also tested positive. Flavivirus seroprevalence in wild boar was significantly higher in DNP, and increased with age. Haemolysis of the serum samples limited interpretation of VNT to 28 samples, confirming WNV seroprevalence in one red fox, four Iberian pigs and nine wild boars. ELISA positive, microVNT negative samples suggest presence of non-neutralizing antibodies against WNV or antibodies to other antigenically related flaviviruses. Despite the importance of wetlands for flavivirus maintenance and amplification, WNV/flavivirus seroprevalence in wild boar and red foxes was not associated to wetland habitats. This is the first report of exposure of red foxes to WNV. With view to use of the tested species as sentinels for flavivirus activity, limited exposure of Iberian pigs that would be available for regular sampling, low numbers of foxes collected and concentration of wild boar harvest in the winter season are major drawbacks.
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http://dx.doi.org/10.1016/j.vetmic.2012.04.019DOI Listing
October 2012

Bagaza virus in partridges and pheasants, Spain, 2010.

Emerg Infect Dis 2011 Aug;17(8):1498-501

Laboratorio Central de Veterinaria, Algete, Spain.

In September 2010, an unusually high number of wild birds (partridges and pheasants) died in Cádiz in southwestern Spain. Reverse transcription PCR and virus isolation detected flavivirus infections. Complete nucleotide sequence analysis identified Bagaza virus, a flavivirus with a known distribution that includes sub-Saharan Africa and India, as the causative agent.
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http://dx.doi.org/10.3201/eid1708.110077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3381565PMC
August 2011

Phylogenetic relationships of Western Mediterranean West Nile virus strains (1996-2010) using full-length genome sequences: single or multiple introductions?

J Gen Virol 2011 Nov 20;92(Pt 11):2512-2522. Epub 2011 Jul 20.

Centro de Investigación en Sanidad Animal (CISA)-INIA, Ctra. Algete-El Casar s/n, 28130 Valdeolmos, Spain.

In recent years, West Nile virus (WNV) has re-emerged in the Western Mediterranean region. As a result, the number of complete WNV genome sequences available from this region has increased, allowing more detailed phylogenetic analyses, which may help to understand the evolutionary history of WNV circulating in the Western Mediterranean. To this aim, the present work describes six new complete WNV sequences from recent outbreaks and surveillance in Italy in 2008-2009 and in Spain in 2008 and 2010. Comparison with other sequences from different WNV clusters within lineage 1 (clade 1a) confirmed that all Western Mediterranean WNV isolates obtained since 1996 (except one from Tunisia, collected in 1997) cluster in a single monophyletic group (here called 'WMed' subtype). The analysis differentiated two subgroups within this subtype, which appear to have evolved from earlier WMed strains, suggesting a single introduction in the area, and further dissemination and evolution. Close similarities between WNV variants circulating in consecutive years, one in Spain, between 2007 and 2008, and another in Italy between 2008 and 2009, suggest that the virus possibly overwinters in Western Mediterranean sites. The NS3(249)-proline genotype, recently proposed as a virulence determinant for WNV, has arisen independently at least twice in the area. Overall, these results indicate that the frequent recurrence of outbreaks caused by phylogenetically homogeneous WNV in the Western Mediterranean since 1996 is consistent with a single introduction followed by viral persistence in endemic foci in the area, rather than resulting from independent introductions from exogenous endemic foci.
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http://dx.doi.org/10.1099/vir.0.033829-0DOI Listing
November 2011

Development and evaluation of a new epitope-blocking ELISA for universal detection of antibodies to West Nile virus.

J Virol Methods 2011 Jun 17;174(1-2):35-41. Epub 2011 Mar 17.

Centro de Investigación en Sanidad Animal (CISA)-INIA, Ctra Algete-El Casar, s/n, 28130 Valdeolmos, Madrid, Spain.

West Nile virus (WNV) is an emerging zoonotic pathogen with a wide range of hosts, including birds, horses and humans. The development and evaluation of the performance of a new enzyme-linked immunosorbent assay (ELISA) are described for rapid detection of WNV-specific antibodies in samples originating from an extensive range of vertebrates susceptible to WNV infection. The assay uses a monoclonal antibody (MAb) which binds whole virus particles and neutralizes infection in vitro by recognizing a neutralizing epitope within the envelope (E) glycoprotein of the virus. This MAb, labelled with horseradish peroxidase, was used to compete with WNV-specific serum antibodies for virus-binding in vitro. The epitope-blocking ELISA was optimized in a manner that enabled its validation with a number of experimental and field sera, from a wide range of wild bird species, and susceptible mammals. The new ELISA exhibited high specificity (79.5-96.5%) and sensitivity (100%), using the virus-neutralization test as reference standard. It also required a much lower volume of sample (10 μl per analysis) compared to other ELISAs available commercially. This new method may be helpful for diagnosis and disease surveillance, particularly when testing samples from small birds, which are available in limited amounts.
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http://dx.doi.org/10.1016/j.jviromet.2011.03.015DOI Listing
June 2011

Pathogenicity of two recent Western Mediterranean West Nile virus isolates in a wild bird species indigenous to Southern Europe: the red-legged partridge.

Vet Res 2011 Jan 18;42:11. Epub 2011 Jan 18.

Centro de Investigación en Sanidad Animal del Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (CISA-INIA), Ctra Algete-El Casar, s/n, 28130 Valdeolmos (Madrid), Spain.

West Nile virus (WNV) is an emerging zoonotic pathogen whose geographic spread and incidence in humans, horses and birds has increased significantly in recent years. WNV has long been considered a mild pathogen causing self-limiting outbreaks. This notion has changed as WNV is causing large epidemics with a high impact on human and animal health. This has been particularly noteworthy since its introduction into North America in 1999. There, native bird species have been shown to be highly susceptible to WNV infection and disease with high mortalities. For this reason, the effect of WNV infection in North American bird species has been thoroughly studied by means of experimental inoculations in controlled trials. To a lesser extent, European wild birds have been shown to be affected clinically by WNV infection. Yet experimental studies on European wild bird species are lacking. The red-legged partridge (Alectoris rufa) is a gallinaceous bird indigenous to the Iberian Peninsula, widely distributed in South Western Europe. It plays a key role in the Mediterranean ecosystem and constitutes an economically important game species. As such it is raised intensively in outdoor facilities. In this work, red-legged partridges were experimentally infected with two recent WNV isolates from the Western Mediterranean area: Morocco/2003 and Spain/2007. All inoculated birds became viremic and showed clinical disease, with mortality rates of 70% and 30%, respectively. These results show that Western Mediterranean WNV variants can be pathogenic for some European bird species, such as the red-legged partridge.
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http://dx.doi.org/10.1186/1297-9716-42-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3037891PMC
January 2011

Prevalence of neutralizing antibodies to West Nile virus in Eleonora's Falcons in the Canary Islands.

J Wildl Dis 2010 Oct;46(4):1321-4

Department of Ecology and Evolution, University of Lausanne CH-1015, Lausanne, Switzerland.

Birds are the major amplifying host for West Nile virus (WNV), a flavivirus that may affect humans and transmitted by bloodsucking vectors. Eleonora's Falcons (Falco eleonorae) migrate to the Canary Islands annually from WNV-endemic regions. To investigate the possible role of Eleonora's Falcons in the circulation of WNV, we measured WNV-specific antibodies in 81 falcons captured in 2006. None of the nestlings but 14.8% of the adults had WNV-neutralizing antibodies. RT-PCR did not detect flaviviruses in nonculicine ectoparasites (n=231) of the falcons. These findings suggest that WNV infection did not occur locally, but rather on the wintering grounds or during migration.
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http://dx.doi.org/10.7589/0090-3558-46.4.1321DOI Listing
October 2010

West Nile virus antibodies in wild birds, Morocco, 2008.

Emerg Infect Dis 2009 Oct;15(10):1651-3

Estacion Biologica de Donana, Consejo Superior de Investigaciones Científicas, Seville, Spain.

To determine circulation of West Nile virus (WNV) during nonepidemic times, we serosurveyed wild birds of Morocco in 2008. We found antibodies against WNV in 12 (3.5%) birds, against Usutu virus in 1 (0.3%), and against both in 2 (0.6%). High WNV prevalence among juvenile birds suggests local virus circulation among resident birds.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2866403PMC
http://dx.doi.org/10.3201/eid1510.090340DOI Listing
October 2009

Characterization of West Nile virus isolates from Spain: new insights into the distinct West Nile virus eco-epidemiology in the Western Mediterranean.

Virology 2009 Dec 15;395(2):289-97. Epub 2009 Oct 15.

Centro de Investigación en Sanidad Animal del Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Ctra Algete-El Casar, s/n, 28130, Valdeolmos (Madrid), Spain.

West Nile virus (WNV) is a mosquito-borne flavivirus which causes important morbidity and mortality in birds, horses and humans. In the Western Mediterranean region, WNV causes sporadic, self-limited outbreaks, with few or no human cases. Here we report the characterization of two recent Western Mediterranean WNV isolates, obtained in Spain in 2007 from two golden eagles. Complete genome sequence comparisons revealed high identity between these isolates and close relationship with other Western Mediterranean WNV strains isolated since 1996. Phylogenetic analysis within this group indicated that two distinct phylogenetic groups have emerged from earlier strains. Pathogenicity analysis in mice showed that the Spanish isolate is less pathogenic than other strains either from the Western Mediterranean (Morocco 2003) or from North America (NY'99). Changes in amino acid position NS3-249 (claimed as a virulence marker) did not influence the pathogenicity observed.
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http://dx.doi.org/10.1016/j.virol.2009.09.013DOI Listing
December 2009