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coronavirus interactions with the cellular autophagy machinery.the covid-19 pandemic, caused by the sars-cov-2 virus, is the most recent example of an emergent coronavirus that poses a significant threat to human health. virus-host interactions play a major role in the viral life cycle and disease pathogenesis, and cellular pathways such as macroautophagy/autophagy prove to be either detrimental or beneficial to viral replication and maturation. here, we describe the literature over the past twenty years describing autophagy-coronavirus interactions. there ...202032964796
abortion in donkeys associated with salmonella abortus equi infection.salmonella enterica subspecies enterica serovar abortus equi (s. abortus equi) is well known as the aetiological agent of equine abortion. however, abortion caused by s. abortus equi has not been previously described in donkeys.201930868638
identification of a divergent genotype of equine arteritis virus from south american donkeys.a novel equine arteritis virus (eav) was isolated and sequenced from feral donkeys in chile. phylogenetic analysis indicates that the new virus and south african asinine strains diverged at least 100 years from equine eav strains. the results indicate that asinine strains belonged to a different eav genotype.201728921885
equine arteritis virus elicits a mucosal antibody response in the reproductive tract of persistently infected stallions.equine arteritis virus (eav) has the ability to establish persistent infection in the reproductive tract of the stallion (carrier) and is continuously shed in its semen. we have recently demonstrated that eav persists within stromal cells and a subset of lymphocytes in the stallion accessory sex glands in the presence of a significant local inflammatory response. in the present study, we demonstrated that eav elicits a mucosal antibody response in the reproductive tract during persistent infecti ...201728814389
epidemiology and molecular detection of equine herpesviruses in western algeria in 2011.an episode of acute equine respiratory infection was reported in western algeria (tiaret province) between february and march 2011, affecting a large population of horses. nasal swabs (n=100) were taken from horses aged between 1 and 27 years, presenting with cough and mucopurulent nasal discharge. the prevalence of equine respiratory virus infections was examined using quantitative polymerase chain reaction (qpcr). one, or more, of four equine respiratory viruses were detected in the nasal swab ...201728757025
development and characterization of an infectious cdna clone of equine arteritis virus.development and characterization of several infectious cdna clones of equine arteritis virus (eav) have been described in the literature. here we describe the assembly of the full-length infectious cdna clone of the virulent bucyrus strain (vbs; atcc vr-796) of eav in a plasmid vector. this system allows generation of infectious in vitro-transcribed (ivt) rna from the linearized plasmid that can be transfected or electroporated into mammalian cells to produce infectious recombinant progeny virus ...201728508211
equine infectious anemia prevalence in feral donkeys from northeast brazil.equine infectious anemia virus (eiav) is an important cause of morbidity and mortality throughout the world. although the virus infects all members of the equidae the vast majority of studies have been conducted in horses (equus caballus) with comparatively little information available for other equid species. brazil has one of the most abundant donkey (e. asinus) populations of any nation although the economic importance of these animals is declining as transportation becomes increasingly mecha ...201728460747
equine arteritis virus has specific tropism for stromal cells and cd8(+) t and cd21(+) b lymphocytes but not for glandular epithelium at the primary site of persistent infection in the stallion reproductive tract.equine arteritis virus (eav) has a global impact on the equine industry as the causative agent of equine viral arteritis (eva), a respiratory, systemic, and reproductive disease of equids. a distinctive feature of eav infection is that it establishes long-term persistent infection in 10 to 70% of infected stallions (carriers). in these stallions, eav is detectable only in the reproductive tract, and viral persistence occurs despite the presence of high serum neutralizing antibody titers. carrier ...201728424285
equine viral arteritis in breeding and sport horses in central spain.equine viral arteritis (eva) may have a high economic impact on breeding stud farms due to the occurrence of eva-associated abortion outbreaks and the ability of the virus to persist in carrier stallions. while the consequences of eva in premises with sport horses are usually less severe, the first confirmed outbreak of eva in spain occurred in a riding club in barcelona, but no data on the seroprevalence of eva in sport horses have been reported in spain. given the importance of both spanish pu ...201728161600
domain organization and evolution of the highly divergent 5' coding region of genomes of arteriviruses, including the novel possum nidovirus.in five experimentally characterized arterivirus species, the 5'-end genome coding region encodes the most divergent nonstructural proteins (nsp's), nsp1 and nsp2, which include papain-like proteases (plps) and other poorly characterized domains. these are involved in regulation of transcription, polyprotein processing, and virus-host interaction. here we present results of a bioinformatics analysis of this region of 14 arterivirus species, including that of the most distantly related virus, wob ...201728053107
allelic variation in cxcl16 determines cd3+ t lymphocyte susceptibility to equine arteritis virus infection and establishment of long-term carrier state in the stallion.equine arteritis virus (eav) is the causative agent of equine viral arteritis (eva), a respiratory, systemic, and reproductive disease of horses and other equid species. following natural infection, 10-70% of the infected stallions can become persistently infected and continue to shed eav in their semen for periods ranging from several months to life. recently, we reported that some stallions possess a subpopulation(s) of cd3+ t lymphocytes that are susceptible to in vitro eav infection and that ...201627930647
structural biology of the arterivirus nsp11 endoribonucleases.endoribonuclease (nendou) is unique and conserved as a major genetic marker in nidoviruses that infect vertebrate hosts. arterivirus nonstructural protein 11 (nsp11) was shown to have nendou activity and play essential roles in the viral life cycle. here, we report three crystal structures of porcine reproductive and respiratory syndrome virus (prrsv) and equine arteritis virus (eav) nsp11 mutants. the structures of arterivirus nsp11 contain two conserved compact domains: the n-terminal domain ( ...201727795409
detection of equine arteritis virus by two chromogenic rna in situ hybridization assays (conventional and rnascope(®)) and assessment of their performance in tissues from aborted equine fetuses.equine arteritis virus (eav) is the causative agent of equine viral arteritis, a respiratory and reproductive disease of equids. eav infection can induce abortion in pregnant mares, fulminant bronchointerstitial pneumonia in foals, and persistent infection in stallions. here, we developed two rna in situ hybridization (ish) assays (conventional and rnascope(®) ish) for the detection of viral rna in formalin-fixed paraffin-embedded (ffpe) tissues and evaluated and compared their performance with ...201627541817
nonlinear hierarchical modeling of experimental infection data.in this paper, we propose a nonlinear hierarchical model (nlhm) for analyzing longitudinal experimental infection (ei) data. the nlhm offers several improvements over commonly used alternatives such as repeated measures analysis of variance (rm-anova) and the linear mixed model (lmm). it enables comparison of relevant biological properties of the course of infection including peak intensity, duration and time to peak, rather than simply comparing mean responses at each observation time. we illus ...201627435656
dual infections of equine herpesvirus 1 and equine arteritis virus in equine respiratory mucosa explants.equine herpesvirus 1 (ehv-1) and equine arteritis virus (eav) induce respiratory problems and abortion in horses and are considered as two serious threats to equine industry. both ehv-1 and eav misuse patrolling leukocytes in the upper respiratory tract to breach the basement membrane (bm) and to migrate to blood vessels. so far, the behavior and impact of a double infection in the respiratory mucosa of a horse are unknown. in the present study, the outcome of double infections with ehv-1 and th ...201627117322
biogenesis and architecture of arterivirus replication organelles.all eukaryotic positive-stranded rna (+rna) viruses appropriate host cell membranes and transform them into replication organelles, specialized micro-environments that are thought to support viral rna synthesis. arteriviruses (order nidovirales) belong to the subset of +rna viruses that induce double-membrane vesicles (dmvs), similar to the structures induced by e.g. coronaviruses, picornaviruses and hepatitis c virus. in the last years, electron tomography has revealed substantial differences b ...201627071852
development and evaluation of a reverse transcription-insulated isothermal polymerase chain reaction (rt-iipcr) assay for detection of equine arteritis virus in equine semen and tissue samples using the pockit™ system.equine arteritis virus (eav) is the causative agent of equine viral arteritis (eva), a respiratory and reproductive disease of horses. most importantly, eav induces abortion in pregnant mares and can establish persistent infection in up to 10-70% of the infected stallions, which will continue to shed the virus in their semen. the objective of this study was to develop and evaluate a reverse transcription insulated isothermal polymerase chain reaction (rt-iipcr) for the detection of eav in semen ...201627036504
equine arteritis virus uses equine cxcl16 as an entry receptor.previous studies in our laboratory have identified equine cxcl16 (eqcxcl16) to be a candidate molecule and possible cell entry receptor for equine arteritis virus (eav). in horses, the cxcl16 gene is located on equine chromosome 11 (eca11) and encodes a glycosylated, type i transmembrane protein with 247 amino acids. stable transfection of hek-293t cells with plasmid dna carrying eqcxcl16 (hek-eqcxcl16 cells) increased the proportion of the cell population permissive to eav infection from <3% to ...201626764004
conserved arginine residues in the carboxyl terminus of the equine arteritis virus e protein may play a role in heparin binding but may not affect viral infectivity in equine endothelial cells.equine arteritis virus (eav), the causative agent of equine viral arteritis, has relatively broad cell tropism in vitro. in horses, eav primarily replicates in macrophages and endothelial cells of small blood vessels. until now, neither the cellular receptor(s) nor the mechanism(s) of virus attachment and entry have been determined for this virus. in this study, we investigated the effect of heparin on eav infection in equine endothelial cells (eecs). heparin, but not other glycosaminoglycans, c ...201626739582
further evaluation and validation of a commercially available competitive elisa (celisa) for the detection of antibodies specific to equine arteritis virus (eav).the purpose of this study was to further evaluate and validate two commercially available equine arteritis virus (eav) competitive elisas (original and enhanced celisas) using archived equine sera from experimentally inoculated animals and field sera submitted for laboratory diagnosis. first, the original and subsequently enhanced celisas were compared with the virus neutralisation test (vnt) using a panel of archived serum samples from experimentally inoculated animals. then, the enhanced celis ...201626733051
intrinsic, extrinsic and endoplasmic reticulum stress-induced apoptosis in rk13 cells infected with equine arteritis virus.the modulation of the expression of caspases by viruses influences the cell survival of different cell types. equine arteritis virus (eav) induces apoptosis of bhk21 and vero cell lines, but it is not known whether eav induces apoptosis in rk13 cells, a common cell line routinely used in eav diagnosis and research. in this study, we determined that caspase-3 expression was triggered after infection of rk13 cells with eav in a time- and dose-dependent manner. we also detected caspase-8 and caspas ...201626732484
development and characterization of a synthetic infectious cdna clone of the virulent bucyrus strain of equine arteritis virus expressing mcherry (red fluorescent protein).strains of equine arteritis virus (eav) differ in their virulence phenotypes, causing anywhere from subclinical infections to severe disease in horses. here, we describe the in silico design and de novo synthesis of a full-length infectious cdna clone of the horse-adapted virulent bucyrus strain (vbs) of eav encoding mcherry along with in vitro characterization of the progeny virions (eav svbsmcherry) in terms of host-cell tropism, replicative capacity and stability of the mcherry coding sequenc ...201626711457
reorganization and expansion of the nidoviral family arteriviridae.the family arteriviridae presently includes a single genus arterivirus. this genus includes four species as the taxonomic homes for equine arteritis virus (eav), lactate dehydrogenase-elevating virus (ldv), porcine respiratory and reproductive syndrome virus (prrsv), and simian hemorrhagic fever virus (shfv), respectively. a revision of this classification is urgently needed to accommodate the recent description of eleven highly divergent simian arteriviruses in diverse african nonhuman primates ...201626608064
arterivirus rna-dependent rna polymerase: vital enzymatic activity remains elusive.all rna viruses encode an rna-dependent rna polymerase (rdrp), which in arteriviruses is expressed as the c-terminal domain of nonstructural protein 9 (nsp9). previously, potent primer-dependent rdrp activity has been demonstrated for the homologous polymerase subunit (nsp12) of the distantly related coronaviruses. the only previous study focusing on the in vitro activity of nsp9 of an arterivirus (equine arteritis virus; eav) reported weak de novo polymerase activity on homopolymeric rna templa ...201626499043
enhanced sensitivity of an antibody competitive blocking enzyme-linked immunosorbent assay using equine arteritis virus purified by anion-exchange membrane chromatography.in an effort to improve a competitive blocking enzyme-linked immunosorbent assay (celisa) for antibody detection to equine arteritis virus (eav), antigen purified by anion-exchange membrane chromatography capsule (aec) was evaluated. virus purification by the aec method was rapid and easily scalable. a comparison was made between virus purified by the aec method with that obtained by differential centrifugation based on the following: 1) the relative purity and quality of eav glycoprotein 5 (gp5 ...201526462762
a longitudinal study of poor performance and subclinical respiratory viral activity in standardbred trotters.while clinical respiratory disease is considered a main cause of poor performance in horses, the role of subclinical respiratory virus infections is less clear and needs further investigation.201526392904
encephalomyocarditis virus 3c protease relieves traf family member-associated nf-κb activator (tank) inhibitory effect on traf6-mediated nf-κb signaling through cleavage of tank.traf family member-associated nf-κb activator (tank) is a negative regulator of canonical nf-κb signaling in the toll-like receptor- and b-cell receptor-mediated signaling pathways. however, functions of tank in viral infection-mediated nf-κb activation remain unclear. here, we reported that tank was cleaved by encephalomyocarditis virus 3c at the 197 and 291 glutamine residues, which depends on its cysteine protease activity. in addition, encephalomyocarditis virus 3c impaired the ability of ta ...201526363073
identification of a novel conserved b cell epitope in the n protein of equine arteritis virus (bucyrus strain).the nucleocapsid (n) protein is the most conserved structural protein in equine arteritis virus (eav). this study aimed to identify the minimal conserved b cell epitope on the eav n protein. the purified n protein was used to immunize mice for preparing monoclonal antibody (mab). the reactivity of mab was evaluated by western blot and immunofluorescence assay. moreover, 11 overlapping peptides (named mbp-n1 to mbp-n11) were designed to localize the linear antigenic epitope within the n protein. ...201526331346
discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the rna polymerase-containing protein of all nidoviruses.rna viruses encode an rna-dependent rna polymerase (rdrp) that catalyzes the synthesis of their rna(s). in the case of positive-stranded rna viruses belonging to the order nidovirales, the rdrp resides in a replicase subunit that is unusually large. bioinformatics analysis of this non-structural protein has now revealed a nidoviral signature domain (genetic marker) that is n-terminally adjacent to the rdrp and has no apparent homologs elsewhere. based on its conservation profile, this domain is ...201526304538
partial sequence of a novel virus isolated from pelodiscus sinensis hemorrhagic disease.outbreaks of hemorrhagic syndrome-like disease with high mortality rates have frequently occurred in pelodiscus sinensis farms. the purpose of this study was to investigate the pathogen through challenge infection assays and partial sequencing of the genome of the pathogen. a 453-bp amplicon was obtained by random pcr using the nucleic acid extracted from the tissue homogenate filtrate and showed 32% identity at the amino acid level with the replicase polyprotein of the porcine reproductive and ...201526279281
seroprevalence and factors associated with seropositivity to equine arteritis virus in spanish purebred horses in spain.equine viral arteritis (eva), a disease caused by infection with the equine arteritis virus (eav), is present in many european countries. in spain, the last confirmed outbreak was reported in 1992 and there is a paucity of seroprevalence studies. the disease has a major impact on the equine breeding industry, which is mainly represented by spanish purebred (sp) horses in spain.201626278700
variability of non-structural proteins of equine arteritis virus during persistent infection of the stallion.the genetic stability of orf1a encoding non-structural proteins nsp1, nsp2, nsp3 and nsp4 of equine arteritis virus (eav) has been analysed for nearly seven years in a persistently infected stallion of the malopolska breed. between november 2004 and june 2011, 11 semen samples were collected. viral rna extracted from semen of this carrier stallion was amplified, sequenced and compared with the sequences of the other known strains of eav. sequence analysis of orf1a showed 84 synonymous and 16 non ...201526172173
arterivirus nsp12 versus the coronavirus nsp16 2'-o-methyltransferase: comparison of the c-terminal cleavage products of two nidovirus pp1ab polyproteins.the 3'-terminal domain of the most conserved orf1b in three of the four families of the order nidovirales (except for the family arteriviridae) encodes a (putative) 2'-o-methyltransferase (2'-o-mtase), known as non structural protein (nsp) 16 in the family coronaviridae and implicated in methylation of the 5' cap structure of nidoviral mrnas. as with coronavirus transcripts, arterivirus mrnas are assumed to possess a 5' cap although no candidate mtases have been identified thus far. to address t ...201526041874
in vivo assessment of equine arteritis virus vaccine improvement by disabling the deubiquitinase activity of papain-like protease 2.arteriviruses are a family of positive-stranded rna viruses that includes the prototypic equine arteritis virus (eav) and porcine reproductive and respiratory syndrome virus (prrsv). although several vaccines against these viruses are commercially available there is room for improvement, especially in the case of prrsv. the ability of arteriviruses to counteract the immune response is thought to decrease the efficacy of the current modified live virus vaccines. we have recently shown that the de ...201525975520
prevalence of equine viral arteritis in algeria.in order to determine the prevalence of equine viral arteritis in algeria, 268 sera from non-vaccinated horses were collected from the western and eastern regions. serological analysis of the sera, which were collected from 2009 to 2011, was performed using the virus neutralisation test, as described by the world organisation for animal health. overall, 20 sera (7.46%) were seropositive, 152 (56.71%) were negative and 96 sera (35.82%) were cytotoxic. equine arteritis virus (eav) seroprevalence w ...201425812220
the equine arteritis virus isolate from the 2010 argentinian outbreak.a semen sample from a stallion infected during the 2010 equine arteritis virus (eav) outbreak was received for viral isolation prior to castration of the animal. the virus was identified using a polyclonal antibody immunofluorescence test. reverse-transcription polymerase chain reaction (rt-pcr) was used to amplify a region of the gp5 gene with primers gl105f and gl673r. the pcr products were purified and sequences of both strands were determined in a megabace™1000 with inner primers cr2 and eav ...201425812217
proteolytic processing of the porcine reproductive and respiratory syndrome virus replicase.the porcine reproductive and respiratory syndrome virus (prrsv) replicase polyproteins pp1a and pp1ab are proteolytically processed by four proteases encoded in orf1a. in this study, a large set of prrsv replicase cleavage products were identified and pp1a cleavage sites were verified by using a combination of bioinformatics, proteomics, immunoprecipitation, and site-directed mutagenesis. for genotype 1 prrsv (isolate sd01-08), proteomic analysis identified h180/s181, g385/a386, and g1446/a1447 ...201525557977
re-emergence of a genetic outlier strain of equine arteritis virus: impact on phylogeny.equine arteritis virus (eav) is the causative agent of equine viral arteritis (eva), a respiratory and reproductive disease of equids, which is notifiable in some countries including the great britain (gb) and to the oie. herein, we present the case of a persistently infected stallion and the phylogenetic tracing of the virus strain isolated. discussing eav occurrence and phylogenetic analysis we review features, which may aid to harmonise and enhance the classification of eav.201525527462
antiviral activity of a bacillus sp. p34 peptide against pathogenic viruses of domestic animals.p34 is an antimicrobial peptide produced by a bacillus sp. strain isolated from the intestinal contents of a fish in the brazilian amazon basin with reported antibacterial activity. the aim of this work was to evaluate the peptide p34 for its in vitro antiviral properties against canine adenovirus type 2 (cav-2), canine coronavirus (ccov), canine distemper virus (cdv), canine parvovirus type 2 (cpv-2), equine arteritis virus (eav), equine influenza virus (eiv), feline calicivirus (fcv) and felin ...201425477947
equine viral arteritis.equine arteritis virus (eav), the causative agent of equine viral arteritis (eva), is a respiratory and reproductive disease that occurs throughout the world. eav infection is highly species-specific and exclusively limited to members of the family equidae, which includes horses, donkeys, mules, and zebras. eva is an economically important disease and outbreaks could cause significant losses to the equine industry. the primary objective of this article is to summarize current understanding of ev ...201425441113
[development of pcr methods for detection of eav infection].the goal of this work was the development of suitable (real-time) rt-pcr techniques for fast and sensitive diagnosis of eav and for molecular-epidemiological characterisation of viral strains, as an alternative to virus isolation. to this purpose two conventional rt-pcr methods and one real-time rt-pcr were adapted to detect the broadest possible spectrum of viral strains. several dilutions with bucyrus strain showed a 100-fold higher sensitivity of real-time rt-pcr and heminested rt-pcr compare ...201425359114
combination of an unbiased amplification method and a resequencing microarray for detecting and genotyping equine arteritis virus.this study shows that an unbiased amplification method applied to equine arteritis virus rna significantly improves the sensitivity of the real-time reverse transcription-quantitative pcr (rt-qpcr) recommended by the world organization for animal health. twelve viral rnas amplified using this method were hybridized on a high-density resequencing microarray for effective viral characterization.201525339390
membrane proteins of arterivirus particles: structure, topology, processing and function.arteriviruses, such as equine arteritis virus (eav) and porcine reproductive and respiratory syndrome virus (prrsv), are important pathogens in veterinary medicine. despite their limited genome size, arterivirus particles contain a multitude of membrane proteins, the gp5/m and the gp2/3/4 complex, the small and hydrophobic e protein and the orf5a protein. their function during virus entry and budding is understood only incompletely. we summarize current knowledge of their primary structure, memb ...201425278143
modulation of innate immune signaling by nonstructural protein 1 (nsp1) in the family arteriviridae.arteriviruses infect immune cells and may cause persistence in infected hosts. inefficient induction of pro-inflammatory cytokines and type i ifns are observed during infection of this group of viruses, suggesting that they may have evolved to escape the host immune surveillance for efficient survival. recent studies have identified viral proteins regulating the innate immune signaling, and among these, nsp1 (nonstructural protein 1) is the most potent ifn antagonist. for porcine reproductive an ...201425262851
possible involvement of coaxially stacked double pseudoknots in the regulation of -1 programmed ribosomal frameshifting in rna viruses.-1 programmed ribosomal frameshifting (prf) in viruses is often stimulated by a pseudoknot downstream from the slippery sequence. at the prf junction of hiv-1, transmissible gastroenteritis virus (tgev), barmah forest virus (bfv), fort morgan virus (fmv), and equine arteritis virus (eav), we identified potential double pseudoknots in either a tandem mode or embedded mode. in viruses with tandem pseudoknots (5'pk & 3'pk), the slippery sequence is encompassed in the 5'pk. the ribosome needs to unw ...201525204560
semen quality of stallions challenged with the kentucky 84 strain of equine arteritis virus.equine arteritis virus (eav) is the causal agent of equine viral arteritis (eva), a respiratory and reproductive disease of equids. some strains of eav can cause fever, leukopenia, and dependent edema of the limbs, scrotum, and preputium in the acutely infected stallion. we hypothesized that fever and scrotal edema observed during the acute phase of the infection, but not the presence of eav, have an adverse effect on semen quality. a group of seven stallions were intranasally inoculated with th ...201425156969
how viruses hijack the erad tuning machinery.an essential step during the intracellular life cycle of many positive-strand rna viruses is the rearrangement of host cell membranes to generate membrane-bound replication platforms. for example, nidovirales and flaviviridae subvert the membrane of the endoplasmic reticulum (er) for their replication. however, the absence of conventional er and secretory pathway markers in virus-induced er-derived membranes has for a long time hampered a thorough understanding of their biogenesis. recent report ...201424990995
the recent european isolate (08p178) of equine arteritis virus causes inflammation but not arteritis in experimentally infected ponies.in the last two decades, outbreaks of equine viral arteritis (eva) have been reported in europe, but little is known about these european isolates of equine arteritis virus (eav). eav european strain (08p178, eu-1 clade) isolated from one of these recent outbreaks is able to cause clinical signs on experimental infection. the aim of the present study was to investigate the microscopical lesions induced by this isolate after experimental infection of ponies. animals were killed at 3, 7, 14 and 28 ...201424975896
equine arteritis virus does not induce interferon production in equine endothelial cells: identification of nonstructural protein 1 as a main interferon antagonist.the objective of this study was to investigate the effect of equine arteritis virus (eav) on type i interferon (ifn) production. equine endothelial cells (eecs) were infected with the virulent bucyrus strain (vbs) of eav and expression of ifn-β was measured at mrna and protein levels by quantitative real-time rt-pcr and ifn bioassay using vesicular stomatitis virus expressing the green fluorescence protein (vsv-gfp), respectively. quantitative rt-pcr results showed that ifn-β mrna levels in eecs ...201424967365
biogenesis of non-structural protein 1 (nsp1) and nsp1-mediated type i interferon modulation in arteriviruses.type i interferons (ifns-α/β) play a key role for the antiviral state of host, and the porcine arterivirus; porcine reproductive and respiratory syndrome virus (prrsv), has been shown to down-regulate the production of ifns during infection. non-structural protein (nsp) 1 of prrsv has been identified as a viral ifn antagonist, and the nsp1α subunit of nsp1 has been shown to degrade the creb-binding protein (cbp) and to inhibit the formation of enhanceosome thus resulting in the suppression of if ...201424928046
experiences with infectious cdna clones of equine arteritis virus: lessons learned and insights gained.the advent of recombinant dna technology, development of infectious cdna clones of rna viruses, and reverse genetic technologies have revolutionized how viruses are studied. genetic manipulation of full-length cdna clones has become an especially important and widely used tool to study the biology, pathogenesis, and virulence determinants of both positive and negative stranded rna viruses. the first full-length infectious cdna clone of equine arteritis virus (eav) was developed in 1996 and was a ...201424913633
development of a peptide elisa for the diagnosis of equine arteritis virus.a peptide-based indirect elisa was developed to detect antibodies against equine arteritis virus (eav). two peptides for epitope c of protein gp5 and fragment e of protein m were designed, synthesized, purified and used as antigens either alone or combined. ninety-two serum samples obtained from the 2010 equine viral arteritis outbreak, analyzed previously by virus neutralization, were evaluated by the elisa here developed. the best resolution was obtained using peptide gp5. the analysis of the ...201424803114
signal peptide cleavage from gp3 enabled by removal of adjacent glycosylation sites does not impair replication of equine arteritis virus in cell culture, but the hydrophobic c-terminus is essential.the disulphide-linked gp2/3/4 spike of equine arteritis virus (eav) is essential for virus entry. we showed recently that in transfected cells carbohydrates attached adjacent to the signal peptide of gp3 inhibit cleavage. here we confirm this unique phenomenon in recombinant viruses with disabled glycosylation sites. surprisingly, the infectivity of eav containing gp3 with cleaved signal peptide was not impaired and gp3 with cleaved signal peptide associates with gp2/4 in virus particles. in con ...201424556360
equine arteritis virus gp5 protein induces apoptosis in cultured insect cells.equine arteritis virus (eav) has been shown to induce apoptosis in vitro but the induction of this mechanism has not been previously associated with any viral gene product. in this work, we found a cytotoxicity effect of the eav gp5 protein on baculovirus-insect cells and a low yield of protein recovery. besides, different morphological features by electron transmission microscopy, dna fragmentation in agarose gel, tunel analysis and caspase 3 activity were found. all these findings indicate tha ...201424518298
validation of an improved competitive enzyme-linked immunosorbent assay to detect equine arteritis virus antibody.the objective of the present study was to validate a previously described competitive enzyme-linked immunosorbent assay (celisa) to detect antibody to equine arteritis virus (eav) based on gp5-specific nonneutralizing monoclonal antibody (mab) 17b7(9) using the world organization for animal health (oie)-recommended protocol, which includes the following 5 in-house analyses. 1) the assay was calibrated with the oie-designated reference serum panel for eav; 2) repeatability was evaluated within an ...201324202992
serological markers of bornavirus infection found in horses in iceland.in a stable of eight horses in northern iceland, six horses presented with clinical signs, such as ataxia and reduced appetite, leading to euthanasia of one severely affected horse. serological investigations revealed no evidence of active equine herpes virus type 1 infection, a common source of central nervous system disease in horses, nor equine arteritis virus and west nile virus. another neurotropic virus, borna disease virus, was therefore included in the differential diagnosis list.201324180621
inhibition of virus replication and induction of human tetherin gene expression by equine ifn-α1.type i interferons (ifns) play important roles in the defense of host cells against viral infection by inducing the expression of a diverse range of antiviral factors. ifns from different animals likely share similar features with human ifns, and some of them have cross-species activities. equine ifn-α was proved effective in both equine and human cells. however, the previous studies mostly focused on the inhibition of virus induced cytopathic effects. in this study, we used virus-specific assay ...201324144682
co-translational processing of glycoprotein 3 from equine arteritis virus: n-glycosylation adjacent to the signal peptide prevents cleavage.signal peptide cleavage and n-glycosylation of proteins are co-translational processes, but little is known about their interplay if they compete for adjacent sites. here we report two unique findings for processing of glycoprotein 3 of equine arteritis virus. glycoprotein 3 (gp3) contains an n-terminal signal peptide, which is not removed, although bioinformatics predicts cleavage with high probability. there is an overlapping sequon, nntt, adjacent to the signal peptide that we show to be glyc ...201324142700
identification of target cells of a european equine arteritis virus strain in experimentally infected ponies.currently, little is known on the cellular pathogenesis of equine arteritis virus (eav). the purpose of the present study was to identify the target cells in ponies experimentally inoculated with eav 08p178 (eu, clade-1). eav-target organs (respiratory tissues with associated lymphoid tissues and large intestines), collected at 3 and 7 days post inoculation (dpi) and with virus titers≥10(5.0) tcid50/g, were processed with double immunofluorescence staining for the simultaneous detection of eav n ...201323993255
arterivirus molecular biology and pathogenesis.arteriviruses are positive-stranded rna viruses that infect mammals. they can cause persistent or asymptomatic infections, but also acute disease associated with a respiratory syndrome, abortion or lethal haemorrhagic fever. during the past two decades, porcine reproductive and respiratory syndrome virus (prrsv) and, to a lesser extent, equine arteritis virus (eav) have attracted attention as veterinary pathogens with significant economic impact. particularly noteworthy were the 'porcine high fe ...201323939974
equine arteritis virus.equine arteritis virus (eav) is the causative agent of equine viral arteritis (eva), a respiratory and reproductive disease of equids. there has been significant recent progress in understanding the molecular biology of eav and the pathogenesis of its infection in horses. in particular, the use of contemporary genomic techniques, along with the development and reverse genetic manipulation of infectious cdna clones of several strains of eav, has generated significant novel information regarding t ...201323891306
identification of a conserved b-cell epitope in the equine arteritis virus (eav) n protein using the pepscan technique.the nucleocapsid (n) gene of equine arteritis virus (eav) is highly conserved between isolates, and the n protein is an important antigen that induces immunity when horses are infected with eav. this study describes the identification of a linear b-cell epitope on the n protein using the pepscan technique with a monoclonal antibody (mab) 2b1 directed against the n protein. the n protein was divided into 11 overlapping peptides, each containing 16 amino acids associated with six overlapping amino ...201323813249
evidence for absence of equine arteritis virus in the horse population of new zealand.to summarise investigation and laboratory data collected between 2001 and 2011 to provide evidence that equine arteritis virus is not present in the horse population of new zealand.201323611669
development and use of a polarized equine upper respiratory tract mucosal explant system to study the early phase of pathogenesis of a european strain of equine arteritis virus.the upper respiratory tract mucosa represents the first line of defense, which has to be overcome by pathogens before invading the host. considering the economic and ethical aspects involved in using experimental animals for pathogenesis studies, respiratory mucosal explants, in which the tissue's three-dimensional architecture is preserved, may be ideal alternatives. different respiratory mucosal explant cultures have been developed. however, none of them could be inoculated with pathogens sole ...201323537375
sequence analysis of orfs 5, 6 and 7 of equine arteritis virus during persistent infection of the stallion--a 7-year study.nucleotide and amino acid sequences of orfs 5, 6 and 7 of eav during persistent infection in the stallion of the malopolska breed were analysed in the study. a total of 11 blood and semen samples were collected between 2004 and 2011. the titre of specific eav antibodies in this carrier stallion was maintained at a high level throughout the study and was equal approximately 1:128. the sequence analysis of orf5 showed 16 variable sites including 12 with synonymous substitutions and 4 with non-syno ...201323490558
comparison of an improved competitive enzyme-linked immunosorbent assay with the world organization for animal health-prescribed serum neutralization assay for detection of antibody to equine arteritis virus.equine arteritis virus (eav) causes contagious equine viral arteritis, characterized by fever, anorexia, conjunctivitis, nasal discharge, dependent edema, abortion, infrequent death in foals, and establishment of the carrier state in stallions. the world organization for animal health (oie) defines a horse as seropositive if the serum neutralization (sn) antibody titer is ≥1:4 to eav. however, determining the sn titer is time-consuming and requires specific laboratory facilities, equipment, and ...201323404482
deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells.protein ubiquitination regulates important innate immune responses. the discovery of viruses encoding deubiquitinating enzymes (dubs) suggests they remove ubiquitin to evade ubiquitin-dependent antiviral responses; however, this has never been conclusively demonstrated in virus-infected cells. arteriviruses are economically important positive-stranded rna viruses that encode an ovarian tumor (otu) domain dub known as papain-like protease 2 (plp2). this enzyme is essential for arterivirus replica ...201323401522
single-layer centrifugation reduces equine arteritis virus titre in the semen of shedding stallions.several countries have adopted strategies for preventing and/or controlling equine viral arteritis based on vaccination and restricting the breeding activities of carrier stallions. however, in some cases, carrier stallions are only identified after they have transmitted virus to a mare. therefore, a mechanism for separating virus from spermatozoa in the semen of carrier stallions would facilitate control measures for preventing disease transmission. in this study, the use of several modificatio ...201323190015
an autophagy-independent role for lc3 in equine arteritis virus replication.equine arteritis virus (eav) is an enveloped, positive-strand rna virus. genome replication of eav has been associated with modified intracellular membranes that are shaped into double-membrane vesicles (dmvs). we showed by immuno-electron microscopy that the dmvs induced in eav-infected cells contain double-strand (ds)rna molecules, presumed rna replication intermediates, and are decorated with the autophagy marker protein microtubule-associated protein 1 light chain 3 (lc3). replication of eav ...201323182945
cyclophilin inhibitors block arterivirus replication by interfering with viral rna synthesis.virus replication strongly depends on cellular factors, in particular, on host proteins. here we report that the replication of the arteriviruses equine arteritis virus (eav) and porcine reproductive and respiratory syndrome virus (prrsv) is strongly affected by low-micromolar concentrations of cyclosporine a (csa), an inhibitor of members of the cyclophilin (cyp) family. in infected cells, the expression of a green fluorescent protein (gfp) reporter gene inserted into the prrsv genome was inhib ...201223152531
nf-κb activation by equine arteritis virus is myd88 dependent and promotes viral replication.nf-κb, a family of transcription factors involved in different cell functions and immune responses is targeted by viruses. the mechanism of nf-κb signalling and its role in replication of eav have not been investigated. we demonstrate that eav infection in bhk-21 cells activates nf-κb, and this activation was found to be mediated through the myd88 pathway. infection of ikkβ(-/-) murine embryo fibroblasts (mefs), which are deficient in nf-κb signalling, resulted in lower virus titre, less cytopat ...201323151818
comparative analysis of orf5 nucleotide sequences and amino acid sequences of the gp5 protein of equine arteritis virus (eav) detected in the semen of stallions from eastern poland.the purpose of this study was to conduct a comparative analysis of the orf5 gene fragment nucleotide sequences and the gp5 protein amino acid sequences formed on this matrix, for the equine arteritis virus (eav) strains isolated from the semen of infected stallions from eastern poland. the study covered 41 stallions whose blood serum tested positive for antigens specific to the eav. the presence of eav genetic material was shown in material from 5 horses, in one of which permanent presence of vi ...201323116636
equine arteritis virus induced cell death is associated with activation of the intrinsic apoptotic signalling pathway.equine arteritis virus (eav) causes a respiratory and reproductive disease in horses, equine viral arteritis. though cell death in infection with eav is considered to occur by apoptosis, the underlying molecular mechanism has not been extensively elucidated. we investigated the expression of mrna of pro-apoptotic and caspase genes during eav infection in bhk21 cells, a well-established cell type for eav replication. using a sybr green real-time pcr, mrna of p53, bax, caspase 3 and caspase 9 were ...201323079113
evidence that in vitro susceptibility of cd3+ t lymphocytes to equine arteritis virus infection reflects genetic predisposition of naturally infected stallions to become carriers of the virus.we investigated the correlation between in vitro susceptibility of cd3(+) t lymphocytes to equine arteritis virus (eav) infection and establishment of persistent infection among 14 stallions following natural infections. the data showed that carrier stallions with a cd3(+) t lymphocyte susceptibility phenotype to in vitro eav infection may be at higher risk of becoming carriers than those that lack this phenotype (p = 0.0002).201222933293
development and characterization of an infectious cdna clone of the modified live virus vaccine strain of equine arteritis virus.a stable full-length cdna clone of the modified live virus (mlv) vaccine strain of equine arteritis virus (eav) was developed. rna transcripts generated from this plasmid (peavrmlv) were infectious upon transfection into mammalian cells, and the resultant recombinant virus (rmlv) had 100% nucleotide identity to the parental mlv vaccine strain of eav. a single silent nucleotide substitution was introduced into the nucleocapsid gene (peavrmlvb), enabling the cloned vaccine virus (rmlvb) to be dist ...201222739697
chimeric viruses containing the n-terminal ectodomains of gp5 and m proteins of porcine reproductive and respiratory syndrome virus do not change the cellular tropism of equine arteritis virus.equine arteritis virus (eav) and porcine reproductive and respiratory syndrome virus (prrsv) are members of family arteriviridae; they are highly species specific and differ significantly in cellular tropism in cultured cells. in this study we examined the role of the two major envelope proteins (gp5 and m) of eav and prrsv in determining their cellular tropism. we generated three viable eav/prrsv chimeric viruses by swapping the n-terminal ectodomains of these two proteins from prrsv ia1107 str ...201222739441
isolation of equine rhinitis a virus from a horse semen sample.semen from an apparently healthy 4-year-old american quarter horse was submitted to the national veterinary services laboratories for equine arteritis virus isolation. visual inspection of the semen sample upon arrival noted it was unusually yellow in color. the semen sample was inoculated onto cell monolayers, and cytopathic effect was observed 5 days postinoculation. the resultant isolate tested negative for equine arteritis virus, and was subsequently identified as equine rhinitis a virus. eq ...201222621949
folding and oligomerization of the gp2b/gp3/gp4 spike proteins of equine arteritis virus in vitro.equine arteritis virus (eav) is a small, positive-stranded rna virus. the glycoproteins gp2b, gp3 and gp4 form a heterotrimer in the viral envelope, which is required for cell entry of eav. we describe expression of the ectodomains of the proteins in e. coli and their refolding from inclusion bodies. after extraction of inclusion bodies and dialysis, gst-, but not his-tagged proteins, refold into a soluble conformation. however, when dialyzed together with gst-gp3 or with gst-gp4, his-gp2b and h ...201222590679
ela-dra polymorphisms are not associated with equine arteritis virus infection in horses from argentina.polymorphisms at major histocompatibility complex (mhc) genes have been associated with resistance/susceptibility to infectious diseases in domestic animals. the aim of this investigation was to evaluate whether polymorphisms of the dra gene the equine lymphocyte antigen is associated with susceptibility to equine arteritis virus (eav) infection in horses in argentina. the equine dra gene was screened for polymorphisms using pyrosequencing® technology which allowed the detection of three ela-dra ...201222534582
clinical and virological outcome of an infection with the belgian equine arteritis virus strain 08p178.equine viral arteritis (eva) is an infectious disease with variable clinical outcome. outbreaks, causing important economic losses, are becoming more frequent. currently, there is a shortage of pathogenesis studies performed with european strains. in the present study, eight seronegative ponies were experimentally inoculated with the belgian strain of equine arteritis virus (eav) 08p178 (eu-1 clade) and monitored daily for clinical signs of eva. nasopharyngeal swabs, ocular swabs, bronchoalveola ...201222306037
arterivirus minor envelope proteins are a major determinant of viral tropism in cell culture.arteriviruses are enveloped positive-strand rna viruses for which the attachment proteins and cellular receptors have remained largely controversial. arterivirus particles contain at least eight envelope proteins, an unusually large number among rna viruses. these appear to segregate into three groups: major structural components (major glycoprotein gp5 and membrane protein [m]), minor glycoproteins (gp2a, gp3, and gp4), and small hydrophobic proteins (e and the recently discovered orf5a protein ...201222258262
Emergence of novel equine arteritis virus (EAV) variants during persistent infection in the stallion: Origin of the 2007 French EAV outbreak was linked to an EAV strain present in the semen of a persistently infected carrier stallion.During the summer of 2007, an outbreak of equine viral arteritis (EVA) occurred in Normandy (France). After investigation, a link was suggested between an EAV carrier stallion (A) and the index premise of the outbreak. The full-length nucleotide sequence analysis of a study reference strain (F27) isolated from the lung of a foal revealed a 12,710 nucleotides EAV genome with unique molecular hallmarks in the 5'UTR leader sequence and the ORF1a sequence encoding the non-structural protein 2. The e ...201122209234
Ultrastructural characterization of arterivirus replication structures: Reshaping the endoplasmic reticulum to accommodate viral RNA synthesis.Virus-induced membrane structures support the assembly and function of positive-stranded RNA virus replication complexes. The replicase proteins of arteriviruses are associated with double-membrane vesicles (DMVs), which were previously proposed to derive from endoplasmic reticulum (ER). Using electron tomography, we performed an in-depth ultrastructural analysis of cells infected with the prototypic arterivirus equine arteritis virus (EAV). We established that the outer membranes of EAV-induced ...201122190716
assessment of correlation between in vitro cd3(+) t cell susceptibility to eav infection and clinical outcome following experimental infection.in a recent study, we demonstrated that the virulent bucyrus strain (vbs) of eav could infect in vitro a small population of cd3(+) t lymphocytes from some but not all horses. furthermore, we have shown that a common haplotype is associated with this in vitro cd3(+) t cell susceptibility/resistance phenotype to eav infection. in this study, we investigated whether the differences in the susceptibility or resistance of cd3(+) t cells in vitro correlate with the outcome and severity of clinical si ...201122177968
prevalence of antibodies to equine viruses in the netherlands.summary the prevalence of antibodies to various viruses was investigated in a series of serum samples collected from horses in the netherlands between 1963 and 1966 and from 1972 onwards. neutralizing antibodies to equine rhinopneumonitis virus, equine arteritis virus and to equine rhinovirus types 1 and 2 were detected in respectively 76%, 14%, 66% and 59% of the equine serum samples tested. the observed incidence of serum samples positive to equine adenovirus in the complement fixation tes ...197922039753
Genome-wide association study among four horse breeds identifies a common haplotype associated with in vitro CD3+ T cell susceptibility/resistance to equine arteritis virus infection.Previously, we have shown that horses could be divided into susceptible and resistant groups based on an in vitro assay using dual-color flow cytometric analysis of CD3+ T cells infected with equine arteritis virus (EAV). Here, we demonstrate that the differences in in vitro susceptibility of equine CD3+ T lymphocytes to EAV infection have a genetic basis. To investigate the possible hereditary basis for this trait, we conducted a genome-wide association study (GWAS) to compare susceptible and r ...201121994447
validation of an internally controlled one-step real-time multiplex rt-pcr assay for the detection and quantitation of dengue virus rna in plasma.dengue is mosquito-borne virus infection that annually causes ôê+50 million clinically apparent cases worldwide. an internally controlled one-step real-time multiplex rt-pcr assay was developed for detection and quantitation of denv rna in plasma sample by using specific primers and fluorogenic taqman probes. all primers and probes targeted sequences near the 3' end of the ns5 gene. the method comprised two multiplex assays and was validated for sensitivity, specificity, linearity, reproducibili ...201121843553
evaluation of two magnetic bead-based viral nucleic acid purification kits and three real-time rt-pcr reagent systems in two taqman(r) assays for equine arteritis virus detection in semen.this study showed that under specifically defined conditions with respect to nucleic acid extraction method and testing reagents, a previously described real-time rt-pcr assay (t1 assay) provides sensitivity equal to or higher than virus isolation for the detection of equine arteritis virus in semen.201121832018
eca20 microsatellite polymorphisms in equine viral arteritis-infected horses from argentina.we investigated the association of equine arteritis virus (eav) infection and three short tandem repeat (str) polymorphisms located within or in close proximity to equine lymphocyte antigen (ela) region. we used a case-control design as a first approach before proceeding to select candidate genes. one hundred and sixty-five silla argentino horses were taken in 2002 from positive serological detections of eav in argentina, to determine whether str genotypes were correlated to genetic susceptibili ...201121812763
structure and genetic analysis of the arterivirus nonstructural protein 7{alpha}arterivirus replicase polyproteins are cleaved into at least 13 mature nonstructural proteins (nsps) and in particular the nsp5-8 region is subject to a complex processing cascade. the function of the largest subunit from this region, nsp7, which is further cleaved into nsp7α and nsp7ß, is unknown. using nmr spectroscopy, we determined the solution structure of nsp7α of equine arteritis virus, revealing an interesting unique fold for this protein but as such providing little clue to its possible ...201121561912
development of an antigen-capture elisa for the detection of equine influenza virus nucleoprotein.an antigen-capture enzyme-linked immunosorbent assay (ac-elisa) was developed for the detection of the equine influenza virus (eiv), employing monoclonal and polyclonal antibodies against the a/equine/xingjiang/2007 (h3n8) nucleoprotein (np). immunoglobulin g antibodies were purified and used as capture or detector antibodies. the specificity of the optimized ac-elisa was evaluated using eiv, equine herpesvirus 1 (ehv-1), equine herpesvirus 4 (ehv-4), equine arteritis virus (eav) and japanese en ...201121536075
serological survey of equine viral diseases in mongolia.three hundred sera were collected from horses in various parts of mongolia in 2007 and seroepidemiological surveys for several equine viruses performed on them. equid herpesvirus 1 and equine rhinitis a virus were prevalent, and equine arteritis virus and equid herpesvirus 3 were detected over a wide area though their rates of antibody-positivity were not high. equine infectious anemia was distributed locally. the rates of horses antibody-positive for japanese encephalitis virus and equine influ ...201121447048
evaluation of the safety of vaccinating mares against equine viral arteritis during mid or late gestation or during the immediate postpartum period.to determine whether it is safe to vaccinate pregnant or postpartum mares with a commercial modified-live virus vaccine against equine viral arteritis (eva). design-randomized controlled study. animals-73 mares and their foals.201121401431
tenacity of mammalian viruses in the gut of leeches fed with porcine blood.leech therapy is currently considered to be of high therapeutic value in medicine. however, feeding leeches with fresh animal blood during the maintenance and reproduction phase bears the risk of transmission of zoonotic viruses to the patient. we hypothesize that this would be abolished by subjecting leeches to quarantine measures prior to use. the required duration of quarantine would depend on the maximum survival time of pathogens in contaminated leeches. in order to be able to estimate this ...201121372183
infection of embryos following insemination of donor mares with equine arteritis virus infective semen.the objective was to evaluate the potential risks associated with embryo transfer from mares bred with equine arteritis virus (eav) infective semen. twenty-six mares were embryo donors, whereas 18 unvaccinated and eav antibody seronegative mares were embryo recipients. of the 26 donor mares, 15 were unvaccinated and seronegative for antibodies to eav and 11 were vaccinated for the first time with a commercially available modified live virus vaccine against eva before breeding and subsequent embr ...201121345485
discovery of a small arterivirus gene that overlaps the gp5 coding sequence and is important for virus production.the arterivirus family (order nidovirales) of single-stranded, positive-sense rna viruses includes porcine respiratory and reproductive syndrome virus and equine arteritis virus (eav). their replicative enzymes are translated from their genomic rna, while their seven structural proteins are encoded by a set of small, partially overlapping genes in the genomic 3'-proximal region. the latter are expressed via synthesis of a set of subgenomic mrnas that, in general, are functionally monocistronic ( ...201121307223
equine disease surveillance: quarterly summary. 201121257557
characterization of equine humoral antibody response to the nonstructural proteins of equine arteritis virus.equine arteritis virus (eav) replicase consists of two polyproteins (pp1a and pp1ab) that are encoded by open reading frames (orfs) 1a and 1b of the viral genome. these two replicase polyproteins are posttranslationally processed by three orf 1a-encoded proteinases to yield at least 13 nonstructural proteins (nsp1 to nsp12, including nsp7α and 7β). these nsps are expressed in eav-infected cells, but the equine immune response they induce has not been studied. therefore, the primary purpose of th ...201021147938
zn(2+) inhibits coronavirus and arterivirus rna polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture.increasing the intracellular zn(2+) concentration with zinc-ionophores like pyrithione (pt) can efficiently impair the replication of a variety of rna viruses, including poliovirus and influenza virus. for some viruses this effect has been attributed to interference with viral polyprotein processing. in this study we demonstrate that the combination of zn(2+) and pt at low concentrations (2 µm zn(2+) and 2 µm pt) inhibits the replication of sars-coronavirus (sars-cov) and equine arteritis virus ...201021079686
description of the first recorded major occurrence of equine viral arteritis in france.the vast majority of equine arteritis virus (eav) infections are inapparent or relatively mild, but may occasionally cause outbreaks of equine viral arteritis. the event observed in france during the summer of 2007 was the most important seen in the country, with mortality and disruption of economic activity.201021039801
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