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.

Abstract: 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.

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

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.

Spot the difference-development of a syndrome based protein microarray for specific serological detection of multiple flavivirus infections in travelers.

BACKGROUND: The family Flaviviridae, genus Flavivirus, holds many of the world's most prevalent arboviral diseases that are also considered the most important travel related arboviral infections. In most cases, flavivirus diagnosis in travelers is primarily based on serology as viremia is often low and typically has already been reduced to undetectable levels when symptoms set in and patients seek medical attention. Serological differentiation between flaviviruses and the false-positive results caused by vaccination and cross-reactivity among the different species, are problematic for surveillance and diagnostics of flaviviruses. Their partially overlapping geographic distribution and symptoms, combined with increase in travel, and preexisting antibodies due to flavivirus vaccinations, expand the need for rapid and reliable multiplex diagnostic tests to supplement currently used methods. GOAL: We describe the development of a multiplex serological protein microarray using recombinant NS1 proteins for detection of medically important viruses within the genus Flavivirus. Sera from clinical flavivirus patients were used for primary development of the protein microarray. RESULTS: Results show a high IgG and IgM sensitivity and specificity for individual NS1 antigens, and limited cross reactivity, even within serocomplexes. In addition, the serology based on this array allows for discrimination between infection and vaccination response for JEV vaccine, and no cross-reactivity with TBEV and YFV vaccine induced antibodies when testing for antibodies to other flaviviruses. CONCLUSION: Based on these data, multiplex NS1-based protein microarray is a promising tool for surveillance and diagnosis of flaviviruses.

Animal viral diseases and global change: bluetongue and West Nile fever as paradigms.

Environmental changes have an undoubted influence on the appearance, distribution, and evolution of infectious diseases, and notably on those transmitted by vectors. Global change refers to environmental changes arising from human activities affecting the fundamental mechanisms operating in the biosphere. This paper discusses the changes observed in recent times with regard to some important arboviral (arthropod-borne viral) diseases of animals, and the role global change could have played in these variations. Two of the most important arboviral diseases of animals, bluetongue (BT) and West Nile fever/encephalitis (WNF), have been selected as models. In both cases, in the last 15 years an important leap forward has been observed, which has lead to considering them emerging diseases in different parts of the world. BT, affecting domestic ruminants, has recently afflicted livestock in Europe in an unprecedented epizootic, causing enormous economic losses. WNF affects wildlife (birds), domestic animals (equines), and humans, thus, beyond the economic consequences of its occurrence, as a zoonotic disease, it poses an important public health threat. West Nile virus (WNV) has expanded in the last 12 years worldwide, and particularly in the Americas, where it first occurred in 1999, extending throughout the Americas relentlessly since then, causing a severe epidemic of disastrous consequences for public health, wildlife, and livestock. In Europe, WNV is known long time ago, but it is since the last years of the twentieth century that its incidence has risen substantially. Circumstances such as global warming, changes in land use and water management, increase in travel, trade of animals, and others, can have an important influence in the observed changes in both diseases. The following question is raised: What is the contribution of global changes to the current increase of these diseases in the world?

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

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.

More recent swine vesicular disease virus isolates retain binding to coxsackie-adenovirus receptor, but have lost the ability to bind human decay-accelerating factor (CD55).

Swine vesicular disease virus (SVDV) evolved from coxsackie B virus serotype 5 (CVB5) in the recent past, crossing the species barrier from humans to pigs. Here, SVDV isolates from early and recent outbreaks have been compared for their capacity to utilize the progenitor virus receptors coxsackie-adenovirus receptor (CAR) and decay-accelerating factor (DAF; CD55). Virus titre of CVB5 and SVDV isolates It'66 and UK'72 on human HeLa cells was reduced by pre-incubation with either anti-DAF or anti-CAR antibodies; however, recent SVDV isolates R1072, R1120 and SPA'93 did not infect HeLa cells lytically. CVB5 and SVDV infection of the pig cell line IB-RS-2 was inhibited completely by anti-CAR antibodies for all isolates, and no reduction was observed following pre-incubation of cells with anti-pig DAF antibodies. Expression of human DAF in the pig cell line IB-RS-2 enhanced the virus titre of early SVDV isolates by 25-fold, but had no effect on recent SVDV isolate titre. Binding of radiolabelled CVB5 to IB-RS-2 cells was increased seven- to eightfold by expression of human DAF and binding of early SVDV isolates was increased 1.2-1.3-fold, whereas no increase in binding by recent SVDV isolates was mediated by human DAF expression. Addition of soluble hDAF-Fc inhibited CVB5, but not SVDV, infection of pig cells. Pre-incubation of all viruses with soluble hCAR-Fc blocked infection of IB-RS-2 pig cells completely; titration of the amount of soluble hCAR-Fc required to block infection revealed that early isolate UK'72 was the least susceptible to inhibition, and the most recent isolate, SPA'93, was the most susceptible.

Structure of swine vesicular disease virus: mapping of changes occurring during adaptation of human coxsackie B5 virus to infect swine.

The structure of swine vesicular disease virus (SVDV) was solved and refined at a 3.0-A resolution by X-ray crystallography to gain information about the role of sequence changes that occurred as this virus evolved from the parental human pathogen coxsackievirus B5 (CVB5). These amino acid substitutions can be clustered in five distinct regions: (i) the antigenic sites, (ii) the hydrophobic pocket of the VP1 beta-sandwich, (iii) the putative CAR binding site, (iv) the putative heparan sulfate binding site, and (v) the fivefold axis. The VP1 pocket is occupied by a branched pocket factor, apparently different from that present in the closely related virus CVB3 and in other picornaviruses. This finding may be relevant for the design of new antiviral compounds against this site. Density consistent with the presence of ions was observed on the fivefold and threefold axes. The structure also provided an accurate description of the putative receptor binding sites.

Crystallization and preliminary X-ray analysis of swine vesicular disease virus (SVDV).

Three different crystal forms of the swine vesicular disease virus (SVDV), isolate SPA/2/'93, were obtained by the hanging-drop vapour-diffusion technique using ammonium sulfate and sodium/potassium phosphate as precipitants. Monoclinic crystals, space group C2, with unit-cell parameters a = 473.7, b = 385.3, c = 472.8 A, beta = 100.4 degrees, contain one virus pArticle in the crystal asymmetric unit and diffract to 3.0 A resolution. A second type of crystals had a cubic morphology and diffracted beyond 2.6 A resolution. These crystals belong to a primitive orthorhombic space group, with unit-cell parameters a = 319.6, b = 353.8, c = 377.7 A, and contain half a virus pArticle in the asymmetric unit. A third type of crystals, with a prismatic shape and belonging to space group I222, was also obtained under similar crystallization conditions. These latter crystals, with unit-cell parameters a = 318.3, b = 349.9, c = 371.7 A, diffract to at least 3.0 A resolution and contain 15 protomers per asymmetric unit; this requires that three perpendicular crystal twofold axes coincide with three of the viral pArticles' dyad axes.

Foot-and-mouth disease virus: biology and prospects for disease control.

Foot-and-mouth disease virus (FMDV) is the causative agent of a disease that constitutes one of the main animal health concerns, as evidenced by the devastating outbreaks that occurred in different areas of the world over the last few years. In this review, we summarise important features of FMDV, aspects of its interactions with cells and hosts as well as current and new strategies for FMD control by vaccination.