Publications by authors named "Sylvie Lecollinet"

62 Publications

Serological evidence of West Nile virus infection in human populations and domestic birds in the Northwest of Morocco.

Comp Immunol Microbiol Infect Dis 2021 Apr 5;76:101646. Epub 2021 Apr 5.

Institut Pasteur du Maroc, Service de Parasitologie et des Maladies Vectorielles, Place Louis Pasteur, Casablanca 20360, Morocco. Electronic address:

West Nile virus (WNV) was recently detected in Culex pipiens mosquitoes in Morocco. The aim of this study was to evaluate the seroprevalence of WNV in humans and in domestic birds in two regions of Morocco by the detection of IgG antibodies. Blood samples were obtained from 91 human patients and 92 domestic birds from September to December 2019. All study samples were tested using competitive enzyme-linked immunosorbent assay (cELISA) and WNV neutralization tests (VNT) were performed on positive sera. Of all samples, 4 (4.39 %) humans and 4 (4.34 %) birds were found to be seropositive for flaviviruses by the cELISA test. The VNT revealed that three of the four human samples detected positive by cELISA contained neutralizing antibodies against WNV. Two bird samples were confirmed positive by VNT. These results show a significant seroprevalence of anti-WNV antibodies and therefore suggest the active circulation and exposure of human and bird populations in the northwest of Morocco.
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http://dx.doi.org/10.1016/j.cimid.2021.101646DOI Listing
April 2021

[Which tools for monitoring emerging arboviruses within their mammalian hosts and arthropod vectors ?]

Virologie (Montrouge) 2021 Feb;25(1):12-28

Anses, Inrae, École nationale vétérinaire d'Alfort, UMR Bipar, Laboratoire de Santé animale, Maisons-Alfort, F-94700, France.

Arboviruses are viruses transmitted to humans and/or animals by hematophagous arthropods. They have a significant economic and public health impact. Given the number of arboviruses already identified and their great genetic variability, it is essential to have highly flexible tools for their monitoring. Arbovirus circulation within animal populations can be demonstrated by direct and/or indirect screening of a specific virus within vertebrate hosts and/or arthropod vectors. Viruses have great adaptive capacities that enable them to emerge into new geographic areas and/or cross species barriers. Over the decades, arbovirus monitoring has considerably evolved due to innovations in detection technologies. The objectives of this review are to list and assess (i) the current tools for direct or indirect screening for arboviruses, (ii) the new generation tools that best meet expectations in terms of optimal arbovirus monitoring and (iii) the potentials for improved arbovirus monitoring.
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http://dx.doi.org/10.1684/vir.2021.0880DOI Listing
February 2021

Differential neurovirulence of Usutu virus lineages in mice and neuronal cells.

J Neuroinflammation 2021 Jan 6;18(1):11. Epub 2021 Jan 6.

Pathogenesis and Control of Chronic Infections, Université de Montpellier, INSERM, EFS, Montpellier, France.

Background: Usutu virus (USUV) is an emerging neurotropic arthropod-borne virus recently involved in massive die offs of wild birds predominantly reported in Europe. Although primarily asymptomatic or presenting mild clinical signs, humans infected by USUV can develop neuroinvasive pathologies (including encephalitis and meningoencephalitis). Similar to other flaviviruses, such as West Nile virus, USUV is capable of reaching the central nervous system. However, the neuropathogenesis of USUV is still poorly understood, and the virulence of the specific USUV lineages is currently unknown. One of the major complexities of the study of USUV pathogenesis is the presence of a great diversity of lineages circulating at the same time and in the same location.

Methods: The aim of this work was to determine the neurovirulence of isolates from the six main lineages circulating in Europe using mouse model and several neuronal cell lines (neurons, microglia, pericytes, brain endothelial cells, astrocytes, and in vitro Blood-Brain Barrier model).

Results: Our results indicate that all strains are neurotropic but have different virulence profiles. The Europe 2 strain, previously described as being involved in several clinical cases, induced the shortest survival time and highest mortality in vivo and appeared to be more virulent and persistent in microglial, astrocytes, and brain endothelial cells, while also inducing an atypical cytopathic effect. Moreover, an amino acid substitution (D3425E) was specifically identified in the RNA-dependent RNA polymerase domain of the NS5 protein of this lineage.

Conclusions: Altogether, these data show a broad neurotropism for USUV in the central nervous system with lineage-dependent virulence. Our results will help to better understand the biological and epidemiological diversity of USUV infection.
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http://dx.doi.org/10.1186/s12974-020-02060-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789689PMC
January 2021

Molecular Determinants of West Nile Virus Virulence and Pathogenesis in Vertebrate and Invertebrate Hosts.

Int J Mol Sci 2020 Nov 30;21(23). Epub 2020 Nov 30.

UMR 1161 Virology, ANSES, INRAE, ENVA, ANSES Animal Health Laboratory, EURL for Equine Diseases, 94704 Maisons-Alfort, France.

West Nile virus (WNV), like the dengue virus (DENV) and yellow fever virus (YFV), are major arboviruses belonging to the genus. WNV is emerging or endemic in many countries around the world, affecting humans and other vertebrates. Since 1999, it has been considered to be a major public and veterinary health problem, causing diverse pathologies, ranging from a mild febrile state to severe neurological damage and death. WNV is transmitted in a bird-mosquito-bird cycle, and can occasionally infect humans and horses, both highly susceptible to the virus but considered dead-end hosts. Many studies have investigated the molecular determinants of WNV virulence, mainly with the ultimate objective of guiding vaccine development. Several vaccines are used in horses in different parts of the world, but there are no licensed WNV vaccines for humans, suggesting the need for greater understanding of the molecular determinants of virulence and antigenicity in different hosts. Owing to technical and economic considerations, WNV virulence factors have essentially been studied in rodent models, and the results cannot always be transported to mosquito vectors or to avian hosts. In this review, the known molecular determinants of WNV virulence, according to invertebrate (mosquitoes) or vertebrate hosts (mammalian and avian), are presented and discussed. This overview will highlight the differences and similarities found between WNV hosts and models, to provide a foundation for the prediction and anticipation of WNV re-emergence and its risk of global spread.
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http://dx.doi.org/10.3390/ijms21239117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7731113PMC
November 2020

Evidence of Exposure to USUV and WNV in Zoo Animals in France.

Pathogens 2020 Nov 30;9(12). Epub 2020 Nov 30.

Pathogenesis and Control of Chronic Infections, University of Montpellier, INSERM, EFS, 34000 Montpellier, France.

West Nile virus (WNV) and Usutu virus (USUV) are zoonotic arboviruses. These flaviviruses are mainly maintained in the environment through an enzootic cycle involving mosquitoes and birds. Horses and humans are incidental, dead-end hosts, but can develop severe neurological disorders. Nevertheless, there is little data regarding the involvement of other mammals in the epidemiology of these arboviruses. In this study, we performed a serosurvey to assess exposure to these viruses in captive birds and mammals in a zoo situated in the south of France, an area described for the circulation of these two viruses. A total of 411 samples comprising of 70 species were collected over 16 years from 2003 to 2019. The samples were first tested by a competitive enzyme-linked immunosorbent assay. The positive sera were then tested using virus-specific microneutralization tests against USUV and WNV. USUV seroprevalence in birds was 10 times higher than that of WNV (14.59% versus 1.46%, respectively). Among birds, greater rhea () and common peafowl () exhibited the highest USUV seroprevalence. Infections occurred mainly between 2016-2018 corresponding to a period of high circulation of these viruses in Europe. In mammalian species, antibodies against WNV were detected in one dama gazelle () whereas serological evidence of USUV infection was observed in several , especially in African wild dogs (). Our study helps to better understand the exposure of captive species to WNV and USUV and to identify potential host species to include in surveillance programs in zoos.
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http://dx.doi.org/10.3390/pathogens9121005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760666PMC
November 2020

Contrasted Epidemiological Patterns of West Nile Virus Lineages 1 and 2 Infections in France from 2015 to 2019.

Pathogens 2020 Oct 30;9(11). Epub 2020 Oct 30.

UMR 1161 Virology, ANSES, INRAE, ENVA, ANSES Animal Health Laboratory, EURL for Equine Diseases, 94704 Maisons-Alfort, France.

Since 2015, annual West Nile virus (WNV) outbreaks of varying intensities have been reported in France. Recent intensification of enzootic WNV circulation was observed in the South of France with most horse cases detected in 2015 ( = 49), 2018 ( = 13), and 2019 ( = 13). A WNV lineage 1 strain was isolated from a horse suffering from West Nile neuro-invasive disease (WNND) during the 2015 episode in the Camargue area. A breaking point in WNV epidemiology was achieved in 2018, when WNV lineage 2 emerged in Southeastern areas. This virus most probably originated from WNV spread from Northern Italy and caused WNND in humans and the death of diurnal raptors. WNV lineage 2 emergence was associated with the most important human WNV epidemics identified so far in France (n = 26, including seven WNND cases and two infections in blood and organ donors). Two other major findings were the detection of WNV in areas with no or limited history of WNV circulation (Alpes-Maritimes in 2018, Corsica in 2018-2019, and Var in 2019) and distinct spatial distribution of human and horse WNV cases. These new data reinforce the necessity to enhance French WNV surveillance to better anticipate future WNV epidemics and epizootics and to improve the safety of blood and organ donations.
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http://dx.doi.org/10.3390/pathogens9110908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692118PMC
October 2020

Serological evidence of West Nile virus infection among birds and horses in some geographical locations of Iran.

Vet Med Sci 2021 01 28;7(1):204-209. Epub 2020 Aug 28.

Department of virology, Arboviruses and Insect Vectors, Institut Pasteur, Paris, France.

Recent expansion of arboviruses such as West Nile (WNV), Usutu (USUV), and tick-borne encephalitis (TBEV) over their natural range of distribution needs strengthening their surveillance. As common viral vertebrate hosts, birds and horses deserve special attention with routine serological surveillance. Here, we estimated the seroprevalence of WNV, USUV and TBEV in 160 migrating/resident birds and 60 horses sampled in Mazandaran, Golestan, North Khorasan, Kordestan provinces and Golestan province of Iran respectively. ELISA results showed that of 220 collected samples, 32 samples (14.54%), including 22 birds and 10 horses, were positive. Microsphere immunoassay results showed that 16.7% (10/60) of horse blood samples collected in Golestan province were seropositive against WNV (7; 11.7%), Flavivirus (2; 3.3%) and seropositive for USUV or WNV (1; 1.7%). Furthermore, micro virus neutralization tests revealed that four of seven ELISA-positive bird blood samples were seropositive against WNV: two Egyptian vultures, and one long-legged buzzard collected in Golestan province as well as a golden eagle collected in North Khorasan province. No evidence of seropositivity with TBEV was observed in collected samples. We showed that WNV, responsible for neuroinvasive infection in vertebrates, is circulating among birds and horses in Iran, recommending a sustained surveillance of viral infections in animals, and anticipating future infections in humans.
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http://dx.doi.org/10.1002/vms3.342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7840194PMC
January 2021

Study of Usutu virus neuropathogenicity in mice and human cellular models.

PLoS Negl Trop Dis 2020 04 23;14(4):e0008223. Epub 2020 Apr 23.

Pathogenesis and Control of Chronic Infections, University of Montpellier, INSERM, EFS, Montpellier, France.

Usutu virus (USUV), an African mosquito-borne flavivirus closely related to West Nile virus, was first isolated in South Africa in 1959. USUV emerged in Europe two decades ago, causing notably massive mortality in Eurasian blackbirds. USUV is attracting increasing attention due to its potential for emergence and its rapid spread in Europe in recent years. Although mainly asymptomatic or responsible for mild clinical signs, USUV was recently described as being associated with neurological disorders in humans such as encephalitis and meningoencephalitis, highlighting the potential health threat posed by the virus. Despite this, USUV pathogenesis remains largely unexplored. The aim of this study was to evaluate USUV neuropathogenicity using in vivo and in vitro approaches. Our results indicate that USUV efficiently replicates in the murine central nervous system. Replication in the spinal cord and brain is associated with recruitment of inflammatory cells and the release of inflammatory molecules as well as induction of antiviral-responses without major modulation of blood-brain barrier integrity. Endothelial cells integrity is also maintained in a human model of the blood-brain barrier despite USUV replication and release of pro-inflammatory cytokines. Furthermore, USUV-inoculated mice developed major ocular defects associated with inflammation. Moreover, USUV efficiently replicates in human retinal pigment epithelium. Our results will help to better characterize the physiopathology related to USUV infection in order to anticipate the potential threat of USUV emergence.
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http://dx.doi.org/10.1371/journal.pntd.0008223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7179837PMC
April 2020

Serological Evidence of West Nile and Usutu Viruses Circulation in Domestic and Wild Birds in Wetlands of Mali and Madagascar in 2008.

Int J Environ Res Public Health 2020 03 18;17(6). Epub 2020 Mar 18.

UMR 1161 Virology, ANSES, INRA, ENVA, ANSES Animal Health Laboratory, EURL for Equine Diseases, 94704 Maisons-Alfort, France.

The geographical distribution and impact on animal and human health of both West Nile and Usutu viruses, two flaviviruses of the Japanese encephalitis complex, have been increasing during the past two decades. Both viruses circulate in Europe and Africa within a natural cycle between wild birds and mosquitoes, mainly from the genus. We retrospectively analyzed sera from domestic and wild birds sampled in 2008 in two wetlands, namely the Inner Niger Delta, Mali, and the Lake Alaotra area, Madagascar. Sera were first tested using a commercial ID Screen West Nile Competition Multi-species ELISA kit. Then, positive sera and sera with insufficient volume for testing with ELISA were tested with a Microneutralization Test. In Mali, the observed seroprevalence in domestic birds was 28.5% [24.5; 32.8] , 3.1 % [1.8; 5.2] , 6.2% [3.4; 10.2] and 9.8 % [7.3; 12.8] , for West Nile virus (WNV), Usutu virus (USUV), undetermined flavivirus, and WNV/USUV respectively. Regarding domestic birds of Madagascar, the observed seroprevalence was 4.4 % [2.1; 7.9] for WNV, 0.9% [0.1; 3.1] for USUV, 1.3% [0.5; 2.8] for undetermined flavivirus, and null for WNV/USUV. Among the 150 wild birds sampled in Madagascar, two fulvous whistling-ducks () were positive for WNV and two for an undetermined flavivirus. One white-faced whistling-duck () and one Hottentot teal () were tested positive for USUV. African and European wetlands are linked by wild bird migrations. This first detection of USUV-as well as the confirmed circulation of WNV in domestic birds of two wetlands of Mali and Madagascar-emphasizes the need to improve the surveillance, knowledge of epidemiological patterns, and phylogenetic characteristics of flavivirus in Africa, particularly in areas prone to sustained, intense flavivirus transmission such as wetlands.
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http://dx.doi.org/10.3390/ijerph17061998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7142923PMC
March 2020

Pathological modeling of TBEV infection reveals differential innate immune responses in human neurons and astrocytes that correlate with their susceptibility to infection.

J Neuroinflammation 2020 Mar 3;17(1):76. Epub 2020 Mar 3.

UMR1161 Virologie, Anses, INRAE, Ecole Nationale Vétérinaire d'Alfort, Université Paris-Est, Maisons-Alfort, France.

Background: Tick-borne encephalitis virus (TBEV) is a member of the Flaviviridae family, Flavivirus genus, which includes several important human pathogens. It is responsible for neurological symptoms that may cause permanent disability or death, and, from a medical point of view, is the major arbovirus in Central/Northern Europe and North-Eastern Asia. TBEV tropism is critical for neuropathogenesis, yet little is known about the molecular mechanisms that govern the susceptibility of human brain cells to the virus. In this study, we sought to establish and characterize a new in vitro model of TBEV infection in the human brain and to decipher cell type-specific innate immunity and its relation to TBEV tropism and neuropathogenesis.

Method: Human neuronal/glial cells were differentiated from neural progenitor cells and infected with the TBEV-Hypr strain. Kinetics of infection, cellular tropism, and cellular responses, including innate immune responses, were characterized by measuring viral genome and viral titer, performing immunofluorescence, enumerating the different cellular types, and determining their rate of infection and by performing PCR array and qRT-PCR. The specific response of neurons and astrocytes was analyzed using the same approaches after enrichment of the neuronal/glial cultures for each cellular subtype.

Results: We showed that infection of human neuronal/glial cells mimicked three major hallmarks of TBEV infection in the human brain, namely, preferential neuronal tropism, neuronal death, and astrogliosis. We further showed that these cells conserved their capacity to mount an antiviral response against TBEV. TBEV-infected neuronal/glial cells, therefore, represented a highly relevant pathological model. By enriching the cultures for either neurons or astrocytes, we further demonstrated qualitative and quantitative differential innate immune responses in the two cell types that correlated with their particular susceptibility to TBEV.

Conclusion: Our results thus reveal that cell type-specific innate immunity is likely to contribute to shaping TBEV tropism for human brain cells. They describe a new in vitro model for in-depth study of TBEV-induced neuropathogenesis and improve our understanding of the mechanisms by which neurotropic viruses target and damage human brain cells.
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http://dx.doi.org/10.1186/s12974-020-01756-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053149PMC
March 2020

Evidence of circulation of West Nile virus in Culex pipiens mosquitoes and horses in Morocco.

Acta Trop 2020 May 20;205:105414. Epub 2020 Feb 20.

Institut Pasteur du Maroc, Service de Parasitologie et des Maladies Vectorielles, Place Louis Pasteur, Casablanca 20360, Morocco. Electronic address:

West Nile virus (WNV) is one of the most widely distributed mosquito-borne viruses in the world. In North Africa, it causes human cases of meningoencephalitis with fatalities in Algeria and in Tunisia, whereas only horses were affected in Morocco. The aims of this study were to detect WNV in mosquitoes and to determine seroprevalence of WNV in Moroccan horses by the detection of IgG antibodies. A total of 1455 mosquitoes belonging to four different species were grouped by collection site, date, and sex with 10 specimens per pool and tested for 38 arboviruses using a high-throughput chip based on the BioMark Dynamic array system. Out of 146 mosquito pools tested, one pool was positive for WNV. This positive pool was confirmed by real time RT-PCR. The serosurvey showed that 33.7% (31/92) of horses were positive for competitive enzyme-linked immunosorbent assay (cELISA) test. The flavivirus-sphere microsphere immnoassay (MIA) test, targeting three flaviviruses (WNV, Usutu virus (USUV) and Tick borne encephalitic virus (TBEV)) showed that 23 sera out of 31 were positive for WNV, two for USUV, two for USUV or WNV, and four for an undetermined flavivirus. Virus neutralization tests with USUV and WNV showed that 28 of 31 sera were positive for WNV and all sera were negative for USUV. This study reports, for the first time, the detection of WNV from Culex pipiens mosquitoes in Morocco and its circulation among horses. This highlights that the detection of arboviruses in mosquitoes could serve as an early warning signal of a viral activity to prevent future outbreaks in animals and humans.
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http://dx.doi.org/10.1016/j.actatropica.2020.105414DOI Listing
May 2020

First serological evidence of West Nile virus infection in wild birds in Northern Algeria.

Comp Immunol Microbiol Infect Dis 2020 Apr 7;69:101415. Epub 2020 Jan 7.

UMR1161 Virologie, INRAE, Ecole Nationale Vétérinaire d'Alfort, ANSES, Université Paris-Est, Maisons-Alfort, Paris, France.

While the epidemiology of Flaviviruses has been extensively studied in most of the Mediterranean basin, little is known about the current situation in Algeria. In order to detect the circulation of West Nile (WNV) and Usutu viruses (USUV) in Kabylia, 165 sera were collected from two wild birds species, namely the long distance migrant Turdus philomelos (song thrush) (n = 92) and the resident Passer domesticus (house sparrow) (n = 73). A total of 154 sera were first analyzed by commercial competition ELISA. WNV and USUV micro-neutralization tests were performed on all c-ELISA positive sera and all samples with poor volume. Overall, 7.8 % (CI95 %: 3.5-11.9) were positive by c-ELISA. Positive results were detected in 12.5 % (CI95 %:5.6-19.4) of song thrushes and 1.5 % (CI95 %: 0.0-4.5) for sparrow. Micro-neutralization tests revealed an overall seroprevalence of 6.7 % for WNV (CI95 %: 2.9-10.3), Neutralizing antibodies were found in 8.7 % (CI95 %: 3.0-14.4) for song thrushes and in 4.1 % (CI95 %: 0.0-8.7) of sparrows. The current study demonstrates significant seroprevalence of WNV antibodies in wild birds in Algeria.
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http://dx.doi.org/10.1016/j.cimid.2020.101415DOI Listing
April 2020

Viral Equine Encephalitis, a Growing Threat to the Horse Population in Europe?

Viruses 2019 12 24;12(1). Epub 2019 Dec 24.

RESPE (Réseau d'épidémio-surveillance en pathologie équine), 14280 Saint-Contest, France.

Neurological disorders represent an important sanitary and economic threat for the equine industry worldwide. Among nervous diseases, viral encephalitis is of growing concern, due to the emergence of arboviruses and to the high contagiosity of herpesvirus-infected horses. The nature, severity and duration of the clinical signs could be different depending on the etiological agent and its virulence. However, definite diagnosis generally requires the implementation of combinations of direct and/or indirect screening assays in specialized laboratories. The equine practitioner, involved in a mission of prevention and surveillance, plays an important role in the clinical diagnosis of viral encephalitis. The general management of the horse is essentially supportive, focused on controlling pain and inflammation within the central nervous system, preventing injuries and providing supportive care. Despite its high medical relevance and economic impact in the equine industry, vaccines are not always available and there is no specific antiviral therapy. In this review, the major virological, clinical and epidemiological features of the main neuropathogenic viruses inducing encephalitis in equids in Europe, including rabies virus (), Equid herpesviruses (), Borna disease virus () and West Nile virus (), as well as exotic viruses, will be presented.
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http://dx.doi.org/10.3390/v12010023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019608PMC
December 2019

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

Remodeling of the Actin Network Associated with the Non-Structural Protein 1 (NS1) of West Nile Virus and Formation of NS1-Containing Tunneling Nanotubes.

Viruses 2019 09 27;11(10). Epub 2019 Sep 27.

Université de Lyon, University Claude Bernard Lyon 1, INRA, EPHE, IVPC, UMR754, Viral Infections & Comparative Pathology, Cedex 07, 69366 Lyon, France.

The cellular response to the recombinant NS1 protein of West Nile virus (NS1) was studied using three different cell types: Vero E6 simian epithelial cells, SH-SY5Y human neuroblastoma cells, and U-87MG human astrocytoma cells. Cells were exposed to two different forms of NS1: (i) the exogenous secreted form, sNS1, added to the extracellular milieu; and (ii) the endogenous NS1, the intracellular form expressed in plasmid-transfected cells. The cell attachment and uptake of sNS1 varied with the cell type and were only detectable in Vero E6 and SH-SY5Y cells. Addition of sNS1 to the cell culture medium resulted in significant remodeling of the actin filament network in Vero E6 cells. This effect was not observed in SH-SY5Y and U-87MG cells, implying that the cellular uptake of sNS1 and actin network remodeling were dependent on cell type. In the three cell types, NS1-expressing cells formed filamentous projections reminiscent of tunneling nanotubes (TNTs). These TNT-like projections were found to contain actin and NS1 proteins. Interestingly, similar actin-rich, TNT-like filaments containing NS1 and the viral envelope glycoprotein E were also observed in WNV-infected Vero E6 cells.
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http://dx.doi.org/10.3390/v11100901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832617PMC
September 2019

Novel Function of Bluetongue Virus NS3 Protein in Regulation of the MAPK/ERK Signaling Pathway.

J Virol 2019 08 30;93(16). Epub 2019 Jul 30.

UMR Virologie, INRA, École Nationale Vétérinaire d'Alfort, ANSES, Université Paris-Est, Maisons-Alfort, France

Bluetongue virus (BTV) is an arbovirus transmitted by blood-feeding midges to a wide range of wild and domestic ruminants. In this report, we showed that BTV, through its nonstructural protein NS3 (BTV-NS3), is able to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, as assessed by phosphorylation levels of ERK1/2 and the translation initiation factor eukaryotic translation initiation factor 4E (eIF4E). By combining immunoprecipitation of BTV-NS3 and mass spectrometry analysis from both BTV-infected and NS3-transfected cells, we identified the serine/threonine-protein kinase B-Raf (BRAF), a crucial player in the MAPK/ERK pathway, as a new cellular interactor of BTV-NS3. BRAF silencing led to a significant decrease in the MAPK/ERK activation by BTV, supporting a model wherein BTV-NS3 interacts with BRAF to activate this signaling cascade. This positive regulation acts independently of the role of BTV-NS3 in counteracting the induction of the alpha/beta interferon response. Furthermore, the intrinsic ability of BTV-NS3 to bind BRAF and activate the MAPK/ERK pathway is conserved throughout multiple serotypes/strains but appears to be specific to BTV compared to other members of genus. Inhibition of MAPK/ERK pathway with U0126 reduced viral titers, suggesting that BTV manipulates this pathway for its own replication. Altogether, our data provide molecular mechanisms that unravel a new essential function of NS3 during BTV infection. Bluetongue virus (BTV) is responsible of the arthropod-borne disease bluetongue (BT) transmitted to ruminants by blood-feeding midges. In this report, we found that BTV, through its nonstructural protein NS3 (BTV-NS3), interacts with BRAF, a key component of the MAPK/ERK pathway. In response to growth factors, this pathway promotes cell survival and increases protein translation. We showed that BTV-NS3 enhances the MAPK/ERK pathway, and this activation is BRAF dependent. Treatment of MAPK/ERK pathway with the pharmacologic inhibitor U0126 impairs viral replication, suggesting that BTV manipulates this pathway for its own benefit. Our results illustrate, at the molecular level, how a single virulence factor has evolved to target a cellular function to increase its viral replication.
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http://dx.doi.org/10.1128/JVI.00336-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6675888PMC
August 2019

Flaviviruses in migratory passerines during spring stopover in a desert oasis.

Zoonoses Public Health 2019 08 14;66(5):495-503. Epub 2019 May 14.

Unité de Recherche 'Ecologie de la Faune Terrestre', UR17ES44, Faculté des Sciences, Université de Gabès, Gabès, Tunisia.

Bird migration has long been hypothesized as the main mechanism for long-distance dispersal of flaviviruses, but the role of migratory birds in flaviviruses spillover is not well documented. In this study, we investigated the eco-epidemiology of West Nile virus (WNV) and Usutu virus (USUV) in trans-Saharan passerines during their spring stopover in southern Tunisian oases. To do, we combined oral swab analysis and serological tools to assess whether migratory birds could be reaching these stopover sites while infectious or have been previously exposed to viruses. All sampled birds tested negative for oral swab analysis. However, anti-WNV and anti-USUV antibodies were detected in 32% and 1% of tested birds, respectively. Among WNV-seropositive species, the Golden oriole (Oriolus oriolus) showed the highest anti-WNV occurrence probability. In this species, anti-WNV occurrence was twice larger in males than females. Inter-specific and intraspecific morphological, physiological and behavioural differences could explain these results. Although our findings did not show evidence for passerines migrating while infectious, they did not exclude an existing enzootic WNV transmission cycle in Tunisian oases. Further investigations including larger samples of migratory birds are needed for a better understanding of this issue.
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http://dx.doi.org/10.1111/zph.12584DOI Listing
August 2019

Evaluation of the Antiviral Activity of Sephin1 Treatment and Its Consequences on eIF2α Phosphorylation in Response to Viral Infections.

Front Immunol 2019 12;10:134. Epub 2019 Feb 12.

Université de Toulouse, ENVT, INRA, UMR 1225, Toulouse, France.

The guanabenz derivative Sephin1 has recently been proposed to increase the levels of translation initiation factor 2 (eIF2α) phosphorylation by inhibiting dephosphorylation by the protein phosphatase 1-GADD34 (PPP1R15A) complex. As phosphorylation of eIF2α by protein kinase R (PKR) is a prominent cellular antiviral pathway, we evaluated the consequences of Sephin1 treatment on virus replication. Our results provide evidence that Sephin1 downregulates replication of human respiratory syncytial virus, measles virus, human adenovirus 5 virus, human enterovirus D68, human cytomegalovirus, and rabbit myxoma virus. However, Sephin1 proved to be inactive against influenza virus, as well as against Japanese encephalitis virus. Sephin1 increased the levels of phosphorylated eIF2α in cells exposed to a PKR agonist. By contrast, in virus-infected cells, the levels of phosphorylated eIF2α did not always correlate with the inhibition of virus replication by Sephin1. This work identifies Sephin1 as an antiviral molecule in cell culture against RNA, as well as DNA viruses belonging to phylogenetically distant families.
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http://dx.doi.org/10.3389/fimmu.2019.00134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379315PMC
January 2020

Utility of examining fallen stock data to monitor health-related events in equids: Application to an outbreak of West Nile Virus in France in 2015.

Transbound Emerg Dis 2019 May 6;66(3):1417-1419. Epub 2019 Mar 6.

Dozulé Laboratory for Equine Diseases, ANSES, Goustranville, France.

Few studies about the use of quantitative equine mortality data for monitoring purposes are available. Our study evaluated the utility of monitoring emerging equine diseases using mortality data collected by rendering plants. We used approaches involving modelling of historical mortality fluctuations and detection algorithm methods to analyse changes in equine mortality in connection with the West Nile Virus (WNV) outbreak that occurred between July and September 2015 along the Mediterranean coast of France. Two weeks after the first equine WNV case was detected by clinical surveillance, detection algorithms identified excess mortality. The temporal distribution of this excess mortality suggested that it was related to the WNV outbreak, which may helped to assess the impact of the WNV epizootic on equine mortality. The results suggest that real-time follow-up of mortality could be a useful tool for equine health surveillance.
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http://dx.doi.org/10.1111/tbed.13150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6850354PMC
May 2019

Serological evidence of infection with dengue and Zika viruses in horses on French Pacific Islands.

PLoS Negl Trop Dis 2019 02 7;13(2):e0007162. Epub 2019 Feb 7.

UMR 1161 Virology, ANSES, INRA, ENVA, ANSES Animal Health Laboratory, EURL for equine diseases, Maisons-Alfort, France.

New Caledonia and French Polynesia are areas in which arboviruses circulate extensively. A large serological survey among horses from New Caledonia and French Polynesia was carried out to investigate the seroprevalence of flaviviruses in the horse population. Here, 293 equine sera samples were screened for flaviviruses using a competitive enzyme-linked immunosorbent assay (cELISA). The positive sera were then confirmed using a flavivirus-specific microsphere immunoassay (MIA) and seroneutralization tests. This serosurvey showed that 16.6% (27/163) and 30.8% (40/130) of horses were positive for cELISA tests in New Caledonia and French Polynesia, respectively, but the MIA technique, targeting only flaviviruses causing neuro-invasive infections in humans and horses (i.e. West Nile virus [WNV], Japanese encephalitis virus [JEV] and tick-borne encephalitis virus [TBEV]), showed negative results for more than 85% (57/67) of the cELISA-positive animals. Seroneutralization tests with the main flaviviruses circulating in the South Pacific revealed that 6.1% (10/163; confidence interval [95% CI] 3.0%-11.0%) of sera in New Caledonia and 7.7% (10/130; 95% CI 3.8%-13.7%) in French Polynesia were positive for dengue virus serotype 1 (DENV1) and 4.3% (7/163; 95% CI 1.7%-8.6%) in New Caledonia and 15.4% (20/130, 95% CI 9.7%-22.8%) in French Polynesia were found positive for Zika virus (ZIKV). Seroprevalence of the JEV and WNV flaviviruses on the 293 samples from both island groups were comparatively much lower (less than 2%). This seroprevalence study in the horse population shows that horses can be infected with dengue and Zika viruses and that these infections lead to seroconversions in horses. The consequences of these infections in horses and their role in ZIKV and DENV epidemiological cycles are two issues that deserve further investigation.
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http://dx.doi.org/10.1371/journal.pntd.0007162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382171PMC
February 2019

Emerging Mosquito-Borne Threats and the Response from European and Eastern Mediterranean Countries.

Int J Environ Res Public Health 2018 12 7;15(12). Epub 2018 Dec 7.

UMR BIPAR, Animal Health Laboratory, ANSES, INRA, Ecole Nationale Vétérinaire d'Alfort, Université Paris-Est, 94700 Maisons-Alfort, France.

Mosquito-borne viruses are the cause of some of the greatest burdens to human health worldwide, particularly in tropical regions where both human populations and mosquito numbers are abundant. Due to a combination of anthropogenic change, including the effects on global climate and wildlife migration there is strong evidence that temperate regions are undergoing repeated introduction of mosquito-borne viruses and the re-emergence of viruses that previously were not detected by surveillance. In Europe, the repeated introductions of West Nile and Usutu viruses have been associated with bird migration from Africa, whereas the autochthonous transmission of chikungunya and dengue viruses has been driven by a combination of invasive mosquitoes and rapid transcontinental travel by infected humans. In addition to an increasing number of humans at risk, livestock and wildlife, are also at risk of infection and disease. This in turn can affect international trade and species diversity, respectively. Addressing these challenges requires a range of responses both at national and international level. Increasing the understanding of mosquito-borne transmission of viruses and the development of rapid detection methods and appropriate therapeutics (vaccines / antivirals) all form part of this response. The aim of this review is to consider the range of mosquito-borne viruses that threaten public health in Europe and the eastern Mediterranean, and the national response of a number of countries facing different levels of threat.
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http://dx.doi.org/10.3390/ijerph15122775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6313739PMC
December 2018

Monitoring Anti-NS1 Antibodies in West Nile Virus-Infected and Vaccinated Horses.

Biomed Res Int 2018 25;2018:8309816. Epub 2018 Sep 25.

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

West Nile virus (WNV) is a zoonotic arboviral pathogen affecting humans, birds, and horses. Vaccines are available for veterinary use, which efficiently prevent the infection in horses. Most common diagnostic tools rely on the identification of the agent (RT-PCR, virus isolation), or on the detection of antibodies (IgM and IgG) recognizing structural proteins of the virus or neutralizing virus infection in cell cultures (virus-neutralization tests). The recent emergence of WNV in different parts of the world has resulted in an increase in the vaccination of horses in many countries. Methods for differentiation between infected and vaccinated animals ("DIVA" assays) would be useful for surveillance and control purposes but are still not available. A usual approach in this regard is the use of antibodies to nonstructural proteins as markers of nonvaccinated, infected animals, and the nonstructural NS1 protein of WNV has been proposed as a candidate for such a marker. The aim of this study was to test the hypothesis that NS1 can be a useful antigen in DIVA assays for differentiating WNV vaccinated and infected horses in field conditions. For that, we examined serum samples from either vaccinated and infected horses both from experimental infections/vaccinations (under controlled conditions) and from the field, exposed to natural infection or vaccinated in response to a risk of infection. The overall conclusion of the study is that NS1 antigen can effectively differentiate WNV infected from vaccinated horses in experimental (controlled) conditions, but this differentiation might be difficult depending on the conditions prevailing in the field.
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http://dx.doi.org/10.1155/2018/8309816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176291PMC
January 2019

[Epidemiological surveillance of Usutu virus in avifauna].

Virologie (Montrouge) 2018 10;22(5):261-263

UMR 1161 Virologie, Anses, INRA, ENVA, LNR West Nile, Laboratoire de santé animale de Maisons-Alfort, 94704 Maisons-Alfort, France.

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http://dx.doi.org/10.1684/vir.2018.0751DOI Listing
October 2018

[Usutu virus: the phantom menace].

Med Sci (Paris) 2018 Aug-Sep;34(8-9):709-716. Epub 2018 Sep 19.

Pathogenesis and control of chronic infections, Université de Montpellier, Inserm, EFS, 60, rue de Navacelle, 34000 Montpellier, France.

Usutu virus, an arbovirus discovered in Africa in 1959, has spread over a large part of Europe over the last twenty years causing significant bird mortality as reported in France since 2015. The zoonotic risk, associated with this succession of avian epizootics in Europe, deserves to be taken into account even if human cases remain rare to date. Human infections are most often asymptomatic or present a benign clinical expression. However, neurological complications such as encephalitis or meningoencephalitis have been described. In addition, the recent description of an atypical case of facial paralysis in France suggests that the clinical spectrum of infections caused by Usutu virus is not fully characterized. Finally, the recent history of other arboviral outbreaks invites the scientific community to be extremely vigilant.
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http://dx.doi.org/10.1051/medsci/20183408018DOI Listing
January 2019

Assessment of reproducibility of a VP7 Blocking ELISA diagnostic test for African horse sickness.

Transbound Emerg Dis 2019 Jan 2;66(1):83-90. Epub 2018 Aug 2.

The Pirbright Institute, Pirbright, UK.

The laboratory diagnosis of African horse sickness (AHS) is important for: (a) demonstrating freedom from infection in a population, animals or products for trade (b) assessing the efficiency of eradication policies; (c) laboratory confirmation of clinical diagnosis; (d) estimating the prevalence of AHS infection; and (e) assessing postvaccination immune status of individual animals or populations. Although serological techniques play a secondary role in the confirmation of clinical cases, their use is very important for all the other purposes due to their high throughput, ease of use and good cost-benefit ratio. The main objective of this study was to support the validation of AHS VP7 Blocking ELISA up to the Stage 3 of the World Animal Health Organization (OIE) assay validation pathway. To achieve this, a collaborative ring trial, which included all OIE Reference Laboratories and other AHS-specialist diagnostic centres, was conducted in order to assess the diagnostic performance characteristics of the VP7 Blocking ELISA. In this trial, a panel of sera of different epidemiological origin and infection status was used. Through this comprehensive evaluation we can conclude that the VP7 Blocking ELISA satisfies the OIE requirements of reproducibility. The VP7 Blocking ELISA, in its commercial version is ready to enter Stage 4 of the validation pathway (Programme Implementation). Specifically, this will require testing the diagnostic performance of the assay using contemporary serum samples collected during control campaigns in endemic countries.
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http://dx.doi.org/10.1111/tbed.12968DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6378617PMC
January 2019

Seroprevalence of horses to Coxiella burnetii in an Q fever endemic area.

Vet Microbiol 2018 Feb 13;215:49-56. Epub 2017 Nov 13.

University of Lyon, VetAgroSup, Marcy L'Etoile, France; EPIA, UMR 0346, Epidemiologie des maladies animales et zoonotiques, INRA, VetAgroSup, 63122 Saint-Genès Champanelle, France. Electronic address:

Coxiella burnetii can infect many animal species, but its circulation dynamics in and through horses is still unclear. This study evaluated horse exposure in an area known to be endemic for ruminants and humans. We assessed antibody prevalence in horse serum by ELISA, and screened by qPCR horse blood, ticks found on horses and dust from stables. Horse seroprevalence was 4% (n = 335, 37 stables) in 2015 and 12% (n = 294, 39 stables) in 2016. Of 199 horses sampled both years, 13 seroconverted, eight remained seropositive, and one seroreverted. Seropositive horses were located close to reported human cases, yet none displayed Q fever-compatible syndromes. Coxiella DNA was detected in almost 40% of collected ticks (n = 59/148 in 2015; n = 103/305 in 2016), occasionally in dust (n = 3/46 in 2015; n = 1/14 in 2016) but never in horse blood. Further studies should be implemented to evaluate if horses may be relevant indicators of zoonotic risk in urban and suburban endemic areas.
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http://dx.doi.org/10.1016/j.vetmic.2017.11.012DOI Listing
February 2018

Improved reliability of serological tools for the diagnosis of West Nile fever in horses within Europe.

PLoS Negl Trop Dis 2017 Sep 15;11(9):e0005936. Epub 2017 Sep 15.

University Paris Est, UMR 1161 Virology, ANSES, INRA, Ecole Nationale Vétérinaire d'Alfort, ANSES Animal Health Laboratory, EURL on Equine Diseases, Maisons-Alfort, France.

West Nile Fever is a zoonotic disease caused by a mosquito-borne flavivirus, WNV. By its clinical sensitivity to the disease, the horse is a useful sentinel of infection. Because of the virus' low-level, short-term viraemia in horses, the primary tools used to diagnose WNV are serological tests. Inter-laboratory proficiency tests (ILPTs) were held in 2010 and 2013 to evaluate WNV serological diagnostic tools suited for the European network of National Reference Laboratories (NRLs) for equine diseases. These ILPTs were designed to evaluate the laboratories' and methods' performances in detecting WNV infection in horses through serology. The detection of WNV immunoglobulin G (IgG) antibodies by ELISA is widely used in Europe, with 17 NRLs in 2010 and 20 NRLs in 2013 using IgG WNV assays. Thanks to the development of new commercial IgM capture kits, WNV IgM capture ELISAs were rapidly implemented in NRLs between 2010 (4 NRLs) and 2013 (13 NRLs). The use of kits allowed the quick standardisation of WNV IgG and IgM detection assays in NRLs with more than 95% (20/21) and 100% (13/13) of satisfactory results respectively in 2013. Conversely, virus neutralisation tests (VNTs) were implemented in 33% (7/21) of NRLs in 2013 and their low sensitivity was evidenced in 29% (2/7) of NRLs during this ILPT. A comparison of serological diagnostic methods highlighted the higher sensitivity of IgG ELISAs compared to WNV VNTs. They also revealed that the low specificity of IgG ELISA kits meant that it could detect animals infected with other flaviviruses. In contrast VNT and IgM ELISA assays were highly specific and did not detect antibodies against related flaviviruses. These results argue in favour of the need for and development of new, specific serological diagnostic assays that could be easily transferred to partner laboratories.
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http://dx.doi.org/10.1371/journal.pntd.0005936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617233PMC
September 2017

Deleterious effect of Usutu virus on human neural cells.

PLoS Negl Trop Dis 2017 Sep 5;11(9):e0005913. Epub 2017 Sep 5.

Pathogenesis and Control of Chronic Infections, Université de Montpellier, INSERM, EFS, Montpellier, France.

In the last decade, the number of emerging Flaviviruses described worldwide has increased considerably. Among them Zika virus (ZIKV) and Usutu virus (USUV) are African mosquito-borne viruses that recently emerged. Recently, ZIKV has been intensely studied due to major outbreaks associated with neonatal death and birth defects, as well as neurological symptoms. USUV pathogenesis remains largely unexplored, despite significant human and veterinary associated disorders. Circulation of USUV in Africa was documented more than 50 years ago, and it emerged in Europe two decades ago, causing massive bird mortality. More recently, USUV has been described to be associated with neurological disorders in humans such as encephalitis and meningoencephalitis, highlighting USUV as a potential health threat. The aim of this study was to evaluate the ability of USUV to infect neuronal cells. Our results indicate that USUV efficiently infects neurons, astrocytes, microglia and IPSc-derived human neuronal stem cells. When compared to ZIKV, USUV led to a higher infection rate, viral production, as well as stronger cell death and anti-viral response. Our results highlight the need to better characterize the physiopathology related to USUV infection in order to anticipate the potential threat of USUV emergence.
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http://dx.doi.org/10.1371/journal.pntd.0005913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5600396PMC
September 2017

Accuracy estimation of an indirect ELISA for the detection of West Nile Virus antibodies in wild birds using a latent class model.

J Virol Methods 2017 10 27;248:202-206. Epub 2017 Jul 27.

Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna. Via Antonio Bianchi, 7. 25126 Brescia, Italy.

West Nile virus (WNV) and Usutu virus (USUV), genus Flavivirus, are members of the Japanese encephalitis virus antigenic complex, and are maintained primarily in an enzootic cycle between mosquitoes and birds. WNV is zoonotic, and poses a threat to public health, especially in relation to blood transfusion. Serosurveillance of wild birds is suitable for early detection of WNV circulation, although concerns remain to be addressed as regards i) the type of test used, whether ELISA, virus neutralization test (VNT), plaque reduction neutralization test (PRNT), ii) the reagents (antigens, revealing antibodies), iii) the different bird species involved, and iv) potential cross-reactions with other Flaviviruses, such as USUV. The authors developed an indirect IgG ELISA with pan-avian specificity using EDIII protein as antigen and a monoclonal antibody (mAb 1A3) with broad reactivity for avian IgG. A total of 140 serum samples were collected from juvenile European magpies (Pica pica) in areas where both WNV and USUV were co-circulating. The samples were then tested using this in-house ELISA and VNT in parallel. Estimation of test accuracy was performed using different Bayesian two latent class models. At a cut-off set at an optical density percentage (OD%) of 15, the ELISA showed a posterior median of diagnostic sensitivity (DSe) of 88% (95%PCI: 73-99%) and a diagnostic specificity (DSp) of 86% (95%PCI: 68-99%). At this cut-off, ELISA and VNT (cut-off 1/10) performances were comparable: DSe=91% (95%PCI: 79-99%), and DSp=77% (95%PCI: 59-98%). With the cut-off increased to 30 OD%, the ELISA DSe dropped to 78% (95%PCI: 52-99%), and the DSp rose to 94% (95%PCI: 83-100%). In field conditions, the cut-off that yields the best accuracy for the ELISA appears to correspond to 15 OD%. In areas where other Flaviviruses are circulating, however, it might be appropriate to raise the cut-off to 30 OD% in order to achieve higher specificity and reduce the detection of seropositive birds infected by other Flaviviruses, such as USUV.
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http://dx.doi.org/10.1016/j.jviromet.2017.07.010DOI Listing
October 2017