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development and partial validation of a recombinant e2-based indirect elisa for detection of specific igm antibody responses against classical swine fever virus.detecting classical swine fever virus specific antibody responses is critical for prevention and control of csf. in this study, a δe2-based indirect elisa was developed to detect specific igm antibodies against csfv. the optimized conditions that were determined experimentally are: a δe2 antigen concentration of 0.5 μg/ml, a serum sample dilution of 1/100 incubated at 37°c for 1.5 h, and a hrp conjugated rabbit anti-pig igm dilution of 1/50,000 incubated at 37°c for 1 h. three hundred clinical s ...201323500647
hemoglobin subunit beta interacts with the capsid protein and antagonizes the growth of classical swine fever virus.the capsid (c) protein of the flaviviridae family members is involved in nucleocapsid formation and virion assembly. however, the influence of c protein-interacting partners on the outcome of pestivirus infections is poorly defined. in this study, hemoglobin subunit beta (hb) was identified as a c protein-binding protein by glutathione s-transferase pulldown and subsequent mass spectrometry analysis of pk-15 cells, which are permissive cells for classical swine fever virus (csfv). coimmunoprecip ...201323487454
conformational and thermal stability improvements for the large-scale production of yeast-derived rabbit hemorrhagic disease virus-like particles as multipurpose vaccine.recombinant virus-like particles (vlp) antigenically similar to rabbit hemorrhagic disease virus (rhdv) were recently expressed at high levels inside pichia pastoris cells. based on the potential of rhdv vlp as platform for diverse vaccination purposes we undertook the design, development and scale-up of a production process. conformational and stability issues were addressed to improve process control and optimization. analyses on the structure, morphology and antigenicity of these multimers we ...201323460801
approaches to define the viral genetic basis of classical swine fever virus virulence.classical swine fever (csf), a highly contagious disease of pigs caused by the classical swine fever virus (csfv), can lead to important economic losses in the pig industry. numerous csfv isolates with various degrees of virulence have been isolated worldwide, ranging from low virulent strains that do not result in any apparent clinical signs to highly virulent strains that cause a severe peracute hemorrhagic fever with nearly 100% mortality. knowledge of the molecular determinants of csfv virul ...201323415391
sequencing approach to analyze the role of quasispecies for classical swine fever.classical swine fever virus (csfv) is a positive-sense rna virus with a high degree of genetic variability among isolates. high diversity is also found in virulence, with strains covering the complete spectrum from avirulent to highly virulent. the underlying genetic determinants are far from being understood. since rna polymerases of rna viruses lack any proof-reading activity, different genome variations called haplotypes, occur during replication. a set of haplotypes is referred to as a viral ...201323415390
complete genome sequences of classical swine fever virus isolates belonging to a new subgenotype, 2.1c, from hunan province, china.two isolates of a new classical swine fever virus (csfv) subgenotype, 2.1c (hnly-2011 and hnsd-2012), were recently isolated from pigs in hunan province, china. the most significant difference in the amino acid sequences of the polyproteins from subgenotypes 2.1a and 2.1b is an spa → tpv amino acid substitution at positions 777 and 779 in the e2 protein.201323405338
cp7_e2alf oral vaccination confers partial protection against early classical swine fever virus challenge and interferes with pathogeny-related cytokine responses.the conventional c-strain vaccine induces early protection against classical swine fever (csf), but infected animals cannot be distinguished from vaccinated animals. the cp7_e2alf marker vaccine, a pestivirus chimera, could be a suitable substitute for c-strain vaccine to control csf outbreaks. in this study, single oral applications of cp7_e2alf and c-strain vaccines were compared for their efficacy to induce protection against a csf virus (csfv) challenge with the moderately virulent bas-rhin ...201323398967
bovine viral diarrhea virus (bvdv) infections in pigs.cattle are the natural hosts of bovine viral diarrhea virus (bvdv), which causes mucosal disease, respiratory and gastrointestinal tract infections, and reproductive problems in cattle. however, bvdv can also infect goats, sheep, deer, and pigs. the prevalence of bvdv infection in pig herds has substantially increased in the last several years, causing increased economic losses to the global pig breeding industry. this article is a summary of bvdv infections in pigs, including a historical overv ...201323587625
intra-host variation structure of classical swine fever virus ns5b in relation to antiviral therapy.classical swine fever (csf) is one of most important diseases of the suidea with severe social economic consequences in case of outbreaks. antivirals have been demonstrated, in recent publications, to be an interesting alternative method of fighting the disease. however, classical swine fever virus is an rna virus which presents a challenge as intra-host variation and the error prone rna dependent rna polymerase (rdrp) could lead to the emergence/selection of resistant variants hampering further ...201323511203
two newly developed e(rns)-based elisas allow the differentiation of classical swine fever virus-infected from marker-vaccinated animals and the discrimination of pestivirus antibodies.new generations of classical swine fever virus (csfv) marker vaccines have recently been developed in order to make emergency vaccination in case of a csf outbreak more feasible. however, the application of a marker vaccine is dependent on the availability of an accompanying discriminatory test allowing differentiation of infected from vaccinated animals (diva). cp7_e2alf, the most promising live marker vaccine candidate currently available, is a genetically modified bovine viral diarrhea virus ...201322902189
development of a suspension microarray for the genotyping of african swine fever virus targeting the snps in the c-terminal end of the p72 gene region of the genome.african swine fever virus (asfv) causes one of the most dreaded transboundary animal diseases (tads) in suidae. african swine fever (asf) often causes high rates of morbidity and mortality, which can reach 100% in domestic swine. to date, serological diagnosis has the drawback of not being able to differentiate variants of this virus. previous studies have identified the 22 genotypes based on sequence variation in the c-terminal region of the p72 gene, which has become the standard for categoriz ...201322776009
preliminary mapping of non-conserved epitopes on envelope glycoprotein e2 of bovine viral diarrhea virus type 1 and 2.bovine viral diarrhea virus (bvdv) belongs together with classical swine fever virus (csfv) and border disease virus (bdv) to the genus pestivirus in the flaviviridae family. bvdv has been subdivided into two different species, bvdv1 and bvdv2 based on phylogenetic analysis. subsequent characterization of both strains revealed major antigenic differences. because the envelope glycoprotein e2 is the most immunodominant protein for all pestiviruses, the present study focused on epitope mapping by ...201323838147
numerical taxonomy of the genus pestivirus: new software for genotyping based on the palindromic nucleotide substitutions method.the genus pestivirus from the family flaviviridae is represented by four established species; bovine viral diarrhea virus 1 (bvdv-1); bovine viral diarrhea virus 2 (bvdv-2); border disease virus (bdv); and classical swine fever virus (csfv); as well a tentative species from a giraffe. the palindromic nucleotide substitutions (pns) in the 5' untranslated region (utr) of pestivirus rna has been described as a new, simple and practical method for genotyping. new software is described, also named pn ...201323684846
identification and characterization of mimotopes of classical swine fever virus e2 glycoprotein using specific anti-e2 monoclonal antibodies.classical swine fever virus (csfv) shares high nucleic acid and amino acid sequence homology with the other members of the pestivirus genus, namely bovine viral diarrhea virus and border disease virus. all three viruses are able to infect swine and generate cross reactive antibodies, which is problematic during differential diagnosis for classical swine fever (csf). toward the development of a new generation of csf specific diagnostic tools, monoclonal antibodies specific for csfv were mapped us ...201323583685
hobi-like viruses: an emerging group of pestiviruses.the genus pestivirus is composed of 4 important pathogens of livestock: bovine viral diarrhea virus 1 and 2 (bvdv-1 and bvdv-2), classical swine fever virus (csfv), and border disease virus of sheep (bdv). bvdv are major pathogens of cattle, and infection results in significant economic loss worldwide. a new putative pestivirus species, tentatively called "hobi-like," "bvdv-3," or "atypical pestiviruses," was first identified in europe in fetal bovine serum (fbs) imported from brazil. hobi-like ...201323345268
molecular diagnosis of african swine fever by a new real-time pcr using universal probe library.a highly sensitive and specific real-time pcr method was developed for the reliable and rapid detection of african swine fever virus (asfv). the method uses a commercial universal probe library (upl) probe combined with a specifically designed primer set to amplify an asfv dna fragment within the vp72 coding genome region. the detection range of the optimized upl pcr technique was confirmed by analysis of a large panel (n = 46) of asfv isolates, belonging to 19 of the 22 viral p72 genotypes desc ...201322394449
laboratory methods to study african swine fever virus.we summarize findings of comparative studies in different cells cultures susceptible to asfv infection, through the analysis of virus components and infectious virus particles production, as alternative means to grow field and laboratory asfv strains. we also provide different methods to assay the infectivity of asfv samples and to purify the infective virus particles. finally we describe the general strategy to construct virus deletion mutants that can be engineered to obtain attenuated asfv st ...201323041357
african swine fever virus transcription.african swine fever virus (asfv), a large, enveloped, icosahedral dsdna virus, is currently the only known dna-containing arbovirus and the only recognized member of the family asfarviridae. its genome encodes more than 150 open reading frames that are densely distributed, separated by short intergenic regions. asfv gene expression follows a complex temporal programming. four classes of mrnas have been identified by its distinctive accumulation kinetics. gene transcription is coordinated with dn ...201323041356
comparative evaluation of novel african swine fever virus (asf) antibody detection techniques derived from specific asf viral genotypes with the oie internationally prescribed serological tests.the presence of antibodies against african swine fever (asf), a complex fatal notifiable oie disease of swine, is always indicative of previous infection, since there is no vaccine that is currently used in the field. the early appearance and subsequent long-term persistence of antibodies combined with cost-effectiveness make antibody detection techniques essential in control programmes. recent reports appear to indicate that the serological tests recommended by the oie for asf monitoring are mu ...201322944073
identification and utility of innate immune system evasion mechanisms of asfv.the interferon (ifn) system is an early innate anti-virus host defense mechanism that takes place shortly after entry of the pathogen and long before the onset of adaptive immunity. thus, african swine fever virus (asfv), as an acute and persistent virus in pigs, is predicted to have evolved multiple genes for the manipulation and evasion of interferon. although, asfv is known to interfere with signaling pathways controlling the transcription of cytokines, surprisingly no individual virus gene m ...201323165138
particle tracking analysis for the intracellular trafficking of nanoparticles modified with african swine fever virus protein p54-derived peptide.previous studies showed that the cytoplasmic transport of nanoparticles to the nucleus is driven by a vesicular sorting system. artificial approaches for targeting a microtubule-associating motor complex is also a challenge. we describe herein the development of a liposomal nanoparticle, the surface of which is modified with stearylated octa-arginine (str-r8), and a dynein light chain (lc8)-associated peptide derived from an african swine fever virus protein p54 (p54(149-161)) with polyethyleneg ...201323164937
a comparative analysis on the synonymous codon usage pattern in viral functional genes and their translational initiation region of asfv.the synonymous codon usage pattern of african swine fever virus (asfv), the similarity degree of the synonymous codon usage between this virus and some organisms and the synonymous codon usage bias for the translation initiation region of viral functional genes in the whole genome of asfv have been investigated by some simply statistical analyses. although both gc12% (the gc content at the first and second codon positions) and gc3% (the gc content at the third codon position) of viral functional ...201323161403
antibody-mediated neutralization of african swine fever virus: myths and facts.almost all viruses can be neutralized by antibodies. however, there is some controversy about antibody-mediated neutralization of african swine fever virus (asfv) with sera from convalescent pigs and about the protective relevance of antibodies in experimentally vaccinated pigs. at present, there is no vaccine available for this highly lethal and economically relevant virus and all classical attempts to generate a vaccine have been unsuccessful. this failure has been attributed, in part, to what ...201323159730
african swine fever virus controls the host transcription and cellular machinery of protein synthesis.throughout a viral infection, the infected cell reprograms the gene expression pattern in order to establish a satisfactory antiviral response. african swine fever virus (asfv), like other complex dna viruses, sets up a number of strategies to evade the host's defense systems, such as apoptosis, inflammation and immune responses. the capability of the virus to persist in its natural hosts and in domestic pigs, which recover from infection with less virulent isolates, suggests that the virus disp ...201323154157
african swine fever virus replication and genomics.african swine fever virus (asfv) is a large icosahedral dna virus which replicates predominantly in the cytoplasm of infected cells. the asfv double-stranded dna genome varies in length from about 170 to 193 kbp depending on the isolate and contains between 150 and 167 open reading frames. these are closely spaced and read from both dna strands. the virus genome termini are covalently closed by imperfectly base-paired hairpin loops that are present in two forms that are complimentary and inverte ...201323142553
african swine fever virus eradication in africa.african swine fever was reported in domestic pigs in 26 african countries during the period 2009-2011. the virus exists in an ancient sylvatic cycle between warthogs (phacochoerus africanus) and argasid ticks of the ornithodoros moubata complex in many of the countries reporting outbreaks and in two further countries in the region. eradication of the virus from the countries in eastern and southern africa where the classic sylvatic cycle occurs is clearly not an option. however, the virus has be ...201323142552
virological diagnosis of african swine fever--comparative study of available tests.the rapid and reliable detection of african swine fever virus (asfv) is essential both for timely implementation of control measures to prevent the spread of disease, and to differentiate african swine fever (asf) from other pig disease with similar clinical presentations. many virological tests are currently available for the detection of asfv (live virus), antigen and genome, including virus isolation, elisa, fluorescent antibody, polymerase chain reaction (pcr) and isothermal assays. in recen ...201323131492
african swine fever virus serodiagnosis: a general review with a focus on the analyses of african serum samples.african swine fever (asf) is an infectious disease that causes heavy mortality in domestic pigs. at present there is no vaccine against asf, and eradication in countries where the disease is endemic is based only on competent diagnosis programs and the sacrifice of infected animals. due to the presence of natural attenuated strains, certain infection conditions may result in reduced mortality. in these situations, the disease can be diagnosed by detection of specific antibodies. the use of class ...201323131491
epidemiology of african swine fever virus.african swine fever virus used to occur primarily in africa. there had been occasional incursions into europe or america which apart from the endemic situation on the island of sardinia always had been successfully controlled. but following an introduction of the virus in 2007, it now has expanded its geographical distribution into caucasus and eastern europe where it has not been controlled, to date. african swine fever affects domestic and wild pig species, and can involve tick vectors. the ab ...201323123296
african swine fever virus infection in ornithodoros ticks.african swine fever virus (asfv) is an arbovirus which is vectored by soft ticks of the ornithodoros spp. and in the sylvatic cycle infects wart hogs and bush pigs. asfv infection of domestic swine causes a high mortality disease. on the other hand, asfv infection of the tick can result in a high-titered and persistent infection depending upon the asfv isolate and the tick combination. recently, morphological, classical virology (titration) and recombinant asfv have been used to study the cellul ...201323085123
african swine fever virus morphogenesis.this review summarizes recent structural and molecular biology studies related to the morphogenesis of african swine fever virus (asfv). asfv possesses icosahedral morphology and is constituted by four concentric layers: the central nucleoid, the core shell, the inner envelope and the icosahedral capsid. the extracellular virus acquires an external envelope by budding through the plasma membrane. the genes coding for 19 of the 54 structural proteins of the asfv particle are known and the localiz ...201323059353
prevalence of african swine fever virus in apparently healthy domestic pigs in uganda.african swine fever (asf) is a contagious viral disease which can cause up to 100% mortality among domestic pigs leading to serious socio-economic impact on people's livelihoods. asf is endemic in uganda and there is paucity of information on the epidemiology of the disease. the major aim of this study was to determine the seroprevalence and prevalence of african swine fever virus (asfv) in apparently healthy slaughter pigs at wambizi slaughterhouse in kampala city, uganda. we also estimated the ...201324369729
how dna polymerase x preferentially accommodates incoming datp opposite 8-oxoguanine on the template.the modified base 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxog) is a common dna adduct produced by the oxidation of dna by reactive oxygen species. kinetic data reveal that dna polymerase x (pol x) from the african swine fever virus incorporates adenine (datp) opposite to oxog with higher efficiency than the non-damaged g:c basepair. to help interpret the kinetic data, we perform molecular dynamics simulations of pol x/dna complexes, in which the template base opposite to the incoming dntp (dctp, d ...201324314086
pathogenesis of highly virulent african swine fever virus in domestic pigs exposed via intraoropharyngeal, intranasopharyngeal, and intramuscular inoculation, and by direct contact with infected pigs.to investigate the pathogenesis of african swine fever virus (asfv), domestic pigs (n=18) were challenged with a range (10(2)-10(6) 50% hemadsorbing doses (had50)) of the highly virulent asfv-malawi strain by inoculation via the intraoropharyngeal (iop), intranasopharyngeal (inp), or intramuscular (im) routes. a subsequent contact challenge experiment was performed in which six iop-inoculated donor pigs were allowed to have direct contact (dc) with six naïve pigs for exposure times that varied f ...201324076499
african swine fever virus: on the move and dangerous. 201324038277
comprehensive phylogenetic reconstructions of african swine fever virus: proposal for a new classification and molecular dating of the virus.african swine fever (asf) is a highly lethal disease of domestic pigs caused by the only known dna arbovirus. it was first described in kenya in 1921 and since then many isolates have been collected worldwide. however, although several phylogenetic studies have been carried out to understand the relationships between the isolates, no molecular dating analyses have been achieved so far. in this paper, comprehensive phylogenetic reconstructions were made using newly generated, publicly available s ...201323936068
development and validation of a multiplex, real-time rt pcr assay for the simultaneous detection of classical and african swine fever viruses.a single-step, multiplex, real-time polymerase chain reaction (rt-pcr) was developed for the simultaneous and differential laboratory diagnosis of classical swine fever virus (csfv) and african swine fever virus (asfv) alongside an exogenous internal control rna (ic-rna). combining a single extraction methodology and primer and probe sets for detection of the three target nucleic acids csfv, asfv and ic-rna, had no effect on the analytical sensitivity of the assay and the new triplex rt-pcr was ...201323923045
a nanoparticle-assisted pcr assay to improve the sensitivity for rapid detection and differentiation of wild-type pseudorabies virus and gene-deleted vaccine strains.nanoparticle-assisted polymerase chain reaction (nanopcr) is a novel method for the rapid amplification of dna and has been adopted for the detection of virus because of its simplicity, rapidity, and specificity. a nanopcr assay was developed to detect and differentiate wild-type and gene-deleted pseudorabies virus (prv). three pairs of primers for nanopcr developed in this study were selected from conserved regions of prv, producing specific amplicons of 431 bp (gb), 316 bp (ge), and 202 bp (gg ...201323872268
involvement of the reparative dna polymerase pol x of african swine fever virus in the maintenance of viral genome stability in vivo.the function of the african swine fever virus (asfv) reparative dna polymerase, pol x, was investigated in the context of virus infection. pol x is a late structural protein that localizes at cytoplasmic viral factories during dna replication. using an asfv deletion mutant lacking the pol x gene, we have shown that pol x is not required for virus growth in vero cells or swine macrophages under one-step growth conditions. however, at a low multiplicity of infection, when multiple rounds of replic ...201323824796
phytochemical analysis and in-vitro anti-african swine fever virus activity of extracts and fractions of ancistrocladus uncinatus, hutch and dalziel (ancistrocladaceae).african swine fever (asf), a highly contagious fatal acute haemorrhagic viral disease of pigs currently has no treatment or vaccination protocol and it threatens the pig industry worldwide. recent outbreaks were managed by farmers with ethnoveterinary preparations with various claims of effectiveness.201323777548
prospects for development of african swine fever virus vaccines.african swine fever virus is a large dna virus which can cause an acute haemorrhagic fever in pigs resulting in high mortality. no vaccine is available, limiting options for control. the virus encodes up to 165 genes and virus particles are multi-layered and contain more than 50 proteins. pigs immunised with natural low virulence isolates or attenuated viruses produced by passage in tissue culture and by targeted gene deletions can be protected against challenge with virulent viruses. cd8+ cells ...201323689892
transmission rate of african swine fever virus under experimental conditions.african swine fever (asf) is a highly lethal, viral disease of swine. no vaccine is available, so controlling an asf outbreak is highly dependent on zoosanitary measures, such as stamping out infected herds and quarantining of affected areas. information on asf transmission parameters could allow for more efficient application of outbreak control measures. three transmission experiments were carried out to estimate the transmission parameters of two asf virus isolates: malta'78 (in two doses) an ...201323664069
antigen targeting to apc: from mice to veterinary species.antigen delivery to receptors expressed on antigen presenting cells (apc) has shown to improve immunogenicity of vaccines in mice. an enhancement of cytotoxic t lymphocyte (ctl), helper t cell or humoral responses was obtained depending on the type of apc and the surface molecule targeted. although this strategy is being also evaluated in livestock animals with promising results, some discrepancies have been found between species and pathogens. the genetic diversity of livestock animals, the dif ...201323648645
quantification of airborne african swine fever virus after experimental infection.knowledge on african swine fever (asf) transmission routes can be useful when designing control measures against the spread of asf virus (asfv). few studies have focused on the airborne transmission route, and until now no data has been available on quantities of asf virus (asfv) in the air. our aim was to validate an air sampling technique for asf virus (asfv) that could be used to detect and quantify virus excreted in the air after experimental infection of pigs. in an animal experiment with t ...201323608475
bacmam immunization partially protects pigs against sublethal challenge with african swine fever virus.lack of vaccines and efficient control measures complicate the control and eradication of african swine fever (asf). limitations of conventional inactivated and attenuated virus-based vaccines against african swine fever virus (asfv) highlight the need to use new technologies to develop efficient and safe vaccines against this virus. with this aim in mind, in this study we have constructed bacmam-shapq, a baculovirus based vector for gene transfer into mammalian cells, expressing a fusion protei ...201323428670
in vitro antiviral activity of fluoroquinolones against african swine fever virus.african swine fever (asf) is a viral swine disease against which neither an effective vaccine nor a treatment is available. the antiviral effect of thirty fluoroquinolones on the infectivity of african swine fever virus (asfv) was screened in vitro. there was a severe reduction of the cytopathic effect in asfv-infected vero cells when exposed to six independent fluoroquinolones, or to some of their combinations, from an early phase of infection. moreover, after 7-day treatments, asfv genome coul ...201323415476
asfv in tanzania: asymptomatic pigs harbor virus of molecular similarity to georgia 2007.in 2011 african swine fever virus (asfv) genome was detected in asymptomatic pigs in field samples in mbeya, tanzania. the aim of this paper is to partly characterize the virus that was harbored in these pigs and furthermore to confirm, by a second sampling, the latest occurrence of asfv in the study area. asfv genome was detected in serum from 10 out of 127 healthy european/crossbreed pigs. asfv dna was polymerase chain reaction (pcr) amplified and sequenced from sera with high viral loads usin ...201323398669
host dna damage response facilitates african swine fever virus infection.studies with different viral infection models on virus interactions with the host cell nucleus have opened new perspectives on our understanding of the molecular basis of these interactions in african swine fever virus (asfv) infection. the present study aims to characterize the host dna damage response (ddr) occurring upon in vitro infection with the asfv-ba71v isolate. we evaluated protein levels during asfv time-course infection, of several signalling cascade factors belonging to ddr pathways ...201323398667
african swine fever virus conquers new territories. 201323376880
detection of african swine fever virus-like sequences in ponds in the mississippi delta through metagenomic sequencing.metagenomic characterization of water virome was performed in four mississippi catfish ponds. although differing considerably from african swine fever virus (asfv), 48 of 446,100 sequences from 12 samples were similar enough to indicate that they represent new members in the family asfarviridae. at present, asfv is the only member of asfarviridae, and this study presents the first indication of a similar virus in north america. at this point, there is no indication that the identified virus(es) ...201323338931
standardization of pathological investigations in the framework of experimental asfv infections.african swine fever is still one of the major viral diseases of swine for which a commercial vaccine is lacking. for the design and development of such preventive products, researchers involved in african swine fever virus (asfv) vaccinology need standardized challenge protocols and well characterized clinical, pathological and immunological responses of inbreed and outbreed pigs to different viral strains and vaccine-like products. the different approaches used should be assessed by immunologis ...201323313935
african swine fever virus organelle rearrangements.like most viruses african swine fever virus (asfv) subsumes the host cell apparatus in order to facilitate its replication. asfv replication is a highly orchestrated process with a least four stages of transcription, immediate-early, early, intermediate and late. as the infective cycle progresses through these stages most if not all of the organelles that comprise a nucleated cell are modified, adapted or in some cases destroyed. the entry of the virus is receptor-mediated, but the precise mecha ...201323291273
a179l, a new viral bcl2 homolog targeting beclin 1 autophagy related protein.autophagy is a relevant cellular defense mechanism that directly eliminates intracellular pathogens and has a crucial role for innate and adaptive immune responses. some viruses have developed tools to counteract this cellular response. a179l, the viral bcl2 homolog of african swine fever virus, interacts with proapoptotic bcl2 family proteins to inhibit apoptosis. here we report that this gene manipulates autophagy by interacting with beclin 1 through its bh3 homology domain. at subcellular lev ...201323228131
comparison of african swine fever virus prevalence and risk in two contrasting pig-farming systems in south-west and central kenya.we describe a horizontal survey of african swine fever virus (asfv) prevalence and risk factors associated with virus infection in domestic pigs in two contrasting production systems in kenya. a free range/tethering, low input production system in ndhiwa district of south-western kenya is compared with a medium input stall fed production system in kiambu district of central kenya. analysis of variance (anova) of data derived from cluster analysis showed that number of animals, number of breeding ...201323219357
cellular immunity in asfv responses.african swine fever virus (asfv) infection usually results in an acute haemorrhagic disease with a mortality rate approaching 100% in domestic pigs. however, pigs can survive infection with less-virulent isolates of asfv and may become chronically infected. surviving animals are resistant to challenge with homologous or, in some cases, closely related isolates of the virus indicating that pigs can develop protective immunity against asfv. during asymptomatic, non-virulent asfv infections natural ...201323201582
innate immunity correlates with host fitness in wild boar (sus scrofa) exposed to classical swine fever.constitutive humoral immunity (chi) is thought to be a first-line of protection against pathogens invading vertebrate hosts. however, clear evidence that chi correlates with host fitness in natural conditions is still lacking. this study explores the relationship between chi, measured using a haemagglutination-haemolysis assay (hahl), and resistance to classical swine fever virus (csfv) among wild boar piglets. the individual dynamics of hahl during piglet growth was analysed, using 423 serum sa ...201324260286
deletion of virulence associated genes from attenuated african swine fever virus isolate our t88/3 decreases its ability to protect against challenge with virulent virus.african swine fever virus (asfv) causes an acute haemorrhagic disease of domestic pigs against which there is no effective vaccine. the attenuated asfv strain our t88/3 has been shown previously to protect vaccinated pigs against challenge with some virulent strains including our t88/1. two genes, dp71l and dp96r were deleted from the our t88/3 genome to create recombinant virus our t88/3δdp2. deletion of these genes from virulent viruses has previously been shown to reduce asfv virulence in dom ...201323725691
proteome-wide screening reveals immunodominance in the cd8 t cell response against classical swine fever virus with antigen-specificity dependent on mhc class i haplotype expression.vaccination with live attenuated classical swine fever virus (csfv) vaccines induces a rapid onset of protection which has been associated with virus-specific cd8 t cell ifn-γ responses. in this study, we assessed the specificity of this response, by screening a peptide library spanning the csfv c-strain vaccine polyprotein to identify and characterise cd8 t cell epitopes. synthetic peptides were pooled to represent each of the 12 csfv proteins and used to stimulate pbmc from four pigs rendered ...201324376799
autocatalytic cleavage within classical swine fever virus ns3 leads to a functional separation of protease and helicase.classical swine fever virus (csfv) is a positive-stranded rna virus belonging to the genus pestivirus within the flaviviridae family. pivotal for processing of a large portion of the viral polyprotein is a serine protease activity within nonstructural protein 3 (ns3) that also harbors helicase and ntpase activities essential for rna replication. in csfv-infected cells, ns3 appears as two forms, a fully processed ns3 of 80 kda and the precursor molecule ns2-3 of 120 kda. here we report the identi ...201323986594
assessment of the phenotype and functionality of porcine cd8 t cell responses following vaccination with live attenuated classical swine fever virus (csfv) and virulent csfv challenge.vaccination with live attenuated classical swine fever virus (csfv) induces solid protection after only 5 days, which has been associated with virus-specific t cell gamma interferon (ifn-γ) responses. in this study, we employed flow cytometry to characterize t cell responses following vaccination and subsequent challenge infections with virulent csfv. the cd3(+) cd4(-) cd8(hi) t cell population was the first and major source of csfv-specific ifn-γ. a proportion of these cells showed evidence for ...201323966552
molecular characterization and phylogenetic study of african swine fever virus isolates from recent outbreaks in uganda (2010-2013).african swine fever (asf) is a highly lethal and economically significant disease of domestic pigs in eastern africa particularly in uganda where outbreaks regularly occur. sequence analysis of variable genome regions have been extensively used for molecular epidemiological studies of african swine fever virus (asfv) isolates. by combining p72, p54 and pb602l (cvr), a high level resolution approach is achieved for viral discrimination. the major aim of this study therefore, was to investigate th ...201323914918
prevalence of swine viral and bacterial pathogens in rodents and stray cats captured around pig farms in korea.in 2008, 102 rodents and 24 stray cats from the areas around 9 pig farms in northeast south korea were used to determine the prevalence of the following selected swine pathogens: ten viral pathogens [porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev), rotavirus, classical swine fever virus (csfv), porcine circovirus type 2 (pcv2), encephalomyocarditis virus (emcv), porcine reproductive and respiratory syndrome virus (prrsv), porcine parvovirus (ppv), pseudorabies ...201323892461
efficient generation of recombinant rna viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cdna.infectious cdna clones are a prerequisite for directed genetic manipulation of rna viruses. here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (csfvs) from full-length cdnas present within bacterial artificial chromosomes (bacs) is described. this strategy allows manipulation of viral cdna by targeted recombination-mediated mutagenesis within bacteria.201324262008
increase in chemokines cxcl10 and ccl2 in blood from pigs infected with high compared to low virulence african swine fever virus isolates.modulation of the expression of chemokines and chemokine receptors in whole blood was compared following infection of pigs with high and low virulence isolates of african swine fever virus. levels of mrnas for ccl2, ccl3l1, ccl4, cxcl10, ccr1 and ccr5 were significantly increased in at least one time point following infection in two experiments and ccl5, ccr9 and cxcr4 mrna were significantly increased in one of the experiments. the results showed that greatest fold increases in mrnas for cxcl10 ...201324083897
autophagy enhances the replication of classical swine fever virus in vitro.autophagy plays an important role in cellular responses to pathogens. however, the impact of the autophagy machinery on classical swine fever virus (csfv) infection is not yet confirmed. in this study, we showed that csfv infection significantly increases the number of autophagy-like vesicles in the cytoplasm of host cells at the ultrastructural level. we also found the formation of 2 ubiquitin-like conjugation systems upon virus infection, including lc3-i/lc3-ii conversion and atg12-atg5 conjug ...201324262968
efficient sensing of infected cells in absence of virus particles by plasmacytoid dendritic cells is blocked by the viral ribonuclease e(rns.).plasmacytoid dendritic cells (pdc) have been shown to efficiently sense hcv- or hiv-infected cells, using a virion-free pathway. here, we demonstrate for classical swine fever virus, a member of the flaviviridae, that this process is much more efficient in terms of interferon-alpha induction when compared to direct stimulation by virus particles. by employment of virus replicon particles or infectious rna which can replicate but not form de novo virions, we exclude a transfer of virus from the d ...201323785283
analysis of a pair of end+ and end- viruses derived from the same bovine viral diarrhea virus stock reveals the amino acid determinants in npro responsible for inhibition of type i interferon production.the exaltation of newcastle disease virus (end) phenomenon is induced by the inhibition of type i interferon in pestivirus-infected cells in vitro, via proteasomal degradation of cellular interferon regulatory factor (irf)-3 with the property of the viral autoprotease protein n(pro). reportedly, the amino acid residues in the zinc-binding trash motif of n(pro) determine the difference in characteristics between end-phenomenon-positive (end(+)) and end-phenomenon-negative (end(-)) classical swine ...201425648277
integrin β3 is required in infection and proliferation of classical swine fever virus.classical swine fever (csf) is a highly infectious fatal pig disease, resulting in huge economic loss to the swine industry. integrins are membrane-bound signal mediators, expressed on a variety of cell surfaces and are known as receptors or co-receptors for many viruses. however, the role of integrin β3 in csfv infection is unknown. here, through quantitive pcr, immunofluorescence (ifc) and immunocytohistochemistry (icc), we revealed that st (swine testicles epithelial) cells have a prominent a ...201425340775
interaction of csfv e2 protein with swine host factors as detected by yeast two-hybrid system.e2 is one of the envelope glycoproteins of pestiviruses, including classical swine fever virus (csfv) and bovine viral diarrhea virus (bvdv). e2 is involved in several critical functions, including virus entry into target cells, induction of a protective immune response and virulence in swine. however, there is no information regarding any host binding partners for the e2 proteins. here, we utilized the yeast two-hybrid system and identified fifty-seven host proteins as positive binding partners ...201424416391
molecular characterization of african swine fever virus from domestic pigs in northern tanzania during an outbreak in 2013.african swine fever (asf) is an acute, highly contagious and deadly viral hemorrhagic fever of domestic pigs caused by african swine fever virus (asfv), a double-stranded dna virus of the family asfarviridae. in this study, molecular diagnosis and characterization of outbreak asfv in northern tanzania, was performed on spleen, lymph node, kidney, and heart samples collected in june and july 2013 from domestic pigs that died during a hemorrhagic disease outbreak. confirmatory diagnosis of asf was ...201424996815
modular framework to assess the risk of african swine fever virus entry into the european union.the recent occurrence and spread of african swine fever (asf) in eastern europe is perceived as a serious risk for the pig industry in the european union (eu). in order to estimate the potential risk of asf virus (asfv) entering the eu, several pathways of introduction were previously assessed separately. the present work aimed to integrate five of these assessments (legal imports of pigs, legal imports of products, illegal imports of products, fomites associated with transport and wild boar mov ...201424992824
dynamics of african swine fever virus shedding and excretion in domestic pigs infected by intramuscular inoculation and contact transmission.african swine fever virus (asfv) is a highly virulent swine pathogen that has spread across eastern europe since 2007 and for which there is no effective vaccine or treatment available. the dynamics of shedding and excretion is not well known for this currently circulating asfv strain. therefore, susceptible pigs were exposed to pigs intramuscularly infected with the georgia 2007/1 asfv strain to measure those dynamics through within- and between-pen transmission scenarios. blood, oral, nasal an ...201425256695
the medical and veterinary role of ornithodoros erraticus complex ticks (acari: ixodida) on the iberian peninsula.argasid ticks of the ornithodoros erraticus complex are associated with traditional pig-farming practices on the iberian peninsula and are also found elsewhere in north africa, west africa, and western asia. the ticks associated with pig farming on the iberian peninsula are the only biological vectors of african swine fever virus (asfv) known to occur in europe, and their ecology makes them an extremely effective reservoir of both asfv and the borrelia species which cause tick-borne relapsing fe ...201425424252
alternative sampling strategies for passive classical and african swine fever surveillance in wild boar--extension towards african swine fever virus antibody detection. 201425448452
evaluation of hemostaseological status of pigs experimentally infected with african swine fever virus.african swine fever is a highly contagious hemorrhagic disease of pigs caused by african swine fever virus (asfv). hemorrhages are the most frequently reported lesions in acute and subacute forms of asf. hemorrhagic lesions are accompanied by impaired hemostasis, which includes thrombocytopenia and changes in the coagulation system. in the present study, experimental infection was conducted to elucidate whether a highly virulent asfv genotype ii circulating in the trans-caucasus and eastern euro ...201425239678
expression library immunization can confer protection against lethal challenge with african swine fever virus.african swine fever is one of the most devastating pig diseases, against which there is no vaccine available. recent work from our laboratory has demonstrated the protective potential of dna vaccines encoding three african swine fever viral antigens (p54, p30, and the hemagglutinin extracellular domain) fused to ubiquitin. partial protection was afforded in the absence of detectable antibodies prior to virus challenge, and survival correlated with the presence of a large number of hemagglutinin- ...201425210179
genetic variation among african swine fever genotype ii viruses, eastern and central europe.african swine fever virus (asfv) was first reported in eastern europe/eurasia in 2007. continued spread of asfv has placed central european countries at risk, and in 2014, asfv was detected in lithuania and poland. sequencing showed the isolates are identical to a 2013 asfv from belarus but differ from asfv isolated in georgia in 2007.201425148518
drivers and risk factors for circulating african swine fever virus in uganda, 2012-2013.we explored observed risk factors and drivers of infection possibly associated with african swine fever (asf) epidemiology in uganda. representative sub-populations of pig farms and statistics were used in a case-control model. indiscriminate disposal of pig viscera and waste materials after slaughter, including on open refuse dumps, farm-gate buyers collecting pigs and pig products from within a farm, and retention of survivor pigs were plausible risk factors. wire mesh-protected windows in pig ...201425066802
suitability of faeces and tissue samples as a basis for non-invasive sampling for african swine fever in wild boar.a challenging aspect of asfv control in wild boar populations is the design and implementation of effective surveillance and monitoring programmes, both for early warning, and to determine the ongoing epidemiological situation in an infected population. testing blood samples requires invasive sampling strategies like hunting or capture of wild boar. besides being biased towards healthy animals, such strategies are also linked to further spread of the virus. non-invasive sampling strategies would ...201425017975
no evidence of african swine fever virus replication in hard ticks.african swine fever (asf) is caused by african swine fever virus (asfv), a tick-borne dna virus. soft ticks of the genus ornithodoros are the only biological vectors of asfv recognized so far. although other hard ticks have been tested for vector competence, two commonly found tick species in europe, ixodes ricinus and dermacentor reticulatus, have not been assessed for their vector competence for asfv. in this study, we aimed to determine whether virus replication can occur in any of these two ...201424980962
modern adjuvants do not enhance the efficacy of an inactivated african swine fever virus vaccine preparation.african swine fever (asf) is among the most devastating viral diseases of pigs. in recent years, the disease has spread alarmingly. despite intensive research activities, promising vaccine candidates are still lacking. for this reason, a study was undertaken to re-assess inactivated asfv preparations with state-of-the-art adjuvants. inactivated preparations of asf virus (asfv) "armenia08" were adjuvanted with either polygen™ or emulsigen(®)-d, respectively, and used to immunize six weaner pigs t ...201424877766
a novel bromodeoxyuridine-resistant wild boar lung cell line facilitates generation of african swine fever virus recombinants.manipulation of african swine fever virus (asfv) genomes, in particular those from field strains, is still a challenge. we have shown recently that generation of a green-fluorescent-protein-expressing, thymidine-kinase-negative (tk-) mutant of the low-pathogenic african swine fever virus field strain nhv was supported by a tk- vero cell line. since nhv, like other asfv field strains, does not replicate well in vero cells, a bromodeoxyuridine (brdu)- resistant cell line derived from wild boar lun ...201424793370
arboviruses pathogenic for domestic and wild animals.the objective of this chapter is to provide an updated and concise systematic review on taxonomy, history, arthropod vectors, vertebrate hosts, animal disease, and geographic distribution of all arboviruses known to date to cause disease in homeotherm (endotherm) vertebrates, except those affecting exclusively man. fifty arboviruses pathogenic for animals have been documented worldwide, belonging to seven families: togaviridae (mosquito-borne eastern, western, and venezuelan equine encephaliliti ...201424751197
a new nanopcr molecular assay for detection of porcine bocavirus.nanoparticle-assisted polymerase chain reaction (nanopcr) is a novel method for the rapid amplification of dna and has been used for the detection of virus. for detection of porcine bocavirus (pbov), a sensitive and specific nanopcr assay was developed with a pair of primers that were designed based on ns1 gene sequences available in genbank. under the optimized conditions of the pbov nanopcr assay, the nanopcr assay was 100-fold more sensitive than a conventional pcr assay. the lower detection ...201424642242
how a low-fidelity dna polymerase chooses non-watson-crick from watson-crick incorporation.a dogma for dna polymerase catalysis is that the enzyme binds dna first, followed by mgdntp. this mechanism contributes to the selection of correct dntp by watson-crick base pairing, but it cannot explain how low-fidelity dna polymerases overcome watson-crick base pairing to catalyze non-watson-crick dntp incorporation. dna polymerase x from the deadly african swine fever virus (pol x) is a half-sized repair polymerase that catalyzes efficient dg:dgtp incorporation in addition to correct repair. ...201424617852
correlation of cell surface marker expression with african swine fever virus infection.the expression of surface markers on african swine fever virus (asfv) infected cells was evaluated to assess their involvement in infection. previous findings indicated cd163 expression was correlated with asfv susceptibility. however, in this study the expression of porcine cd163 on cell lines did not increase the infection rate of these cells indicating other factors are likely to be important in determining susceptibility to infection. on adherent porcine bone marrow (pbm) cells the expressio ...201424398227
first molecular assessment of the african swine fever virus status of ornithodoros ticks from swaziland.african swine fever (asf) is an economically significant haemorrhagic disease of domestic pigs. it is caused by the african swine fever virus (asfv), a deoxyribonucleic acid (dna)arbovirus. argasid ticks of the genus ornithodoros, which are widely distributed throughout southern africa, play a primary role in virus maintenance and spread within the endemic sylvatic cycle. the asf status of swaziland is unknown, but this land-locked country is surrounded by asf-positive countries, has a burgeonin ...201426304186
[real-time pcr kits for the detection of the african swine fever virus].the results obtained using the diagnostic kit based on real-time polymerase chain reaction to detect the dna of the african swine fever in the pathological material, as well as in the culture fluid, are presented. a high sensitivity and specificity for detection of the dna in the organs and tissues of animals was shown to be useful for detection in the european union referentiality reagent kits for dna detection by real time pcr of asfv. more rapid and effective method of dna extraction using co ...201425895212
pestiviruses.pestiviruses cause economically important diseases among domestic ruminants and pigs, but they may also infect a wide spectrum of wild species of even-toed ungulates (artiodactyla). bovine viral diarrhea virus (bvdv) and border disease virus of sheep infect their hosts either transiently or persistently. cellular and humoral immunotolerance to the infecting strain is a unique feature of persistent infection (pi) by ruminant pestiviruses. persistence, caused by transplacental infection early in f ...201425384138
first isolation of border disease virus in japan is from a pig farm with no ruminants.the first isolation of border disease virus (bdv) in japan was from a pig farm of the farrow-to-finishing type that kept no small ruminants or cattle. the infection was detected in the course of sero-surveillance for classical swine fever virus (csfv) in japan. the infected pigs had no clinical symptoms of csfv or other disease; nevertheless, a high sero-positive rate of 58.5% was identified. a persistently infected pig with the bdv was found and suspected to be the cause of sero-prevalence in t ...201424735918
identification and epidemiology of a rare hobi-like pestivirus strain in bangladesh.the genus pestivirus of the family flaviviridae consists of four recognized species: bovine viral diarrhoea virus 1 (bvdv-1), bovine viral diarrhoea virus 2 (bvdv-2), classical swine fever virus and border disease virus. a new putative pestivirus species tentatively named as either 'hobi-like pestivirus' or bvdv-3 has recently been identified in brazil, italy and thailand. despite reports of serological evidence of bvdv in bangladesh, the types of the virus circulating in cattle have not been id ...201424650238
n pro of bungowannah virus exhibits the same antagonistic function in the ifn induction pathway than that of other classical pestiviruses.bungowannah virus is the most divergent atypical pestivirus that had been detected up to now, and does not fit into any of the four approved species: bovine viral diarrhea virus type 1 (bvdv-1) and type 2 (bvdv-2), classical swine fever virus (csfv) and border disease virus (bdv). however, the presence of n(pro) and e(rns) coding regions, which are unique to pestiviruses, provides clear evidence of a pestivirus. nevertheless, the amino acid identity of bungowannah virus n(pro) and bvdv-1 n(pro) ...201424398226
comparison of conventional rt-pcr, reverse-transcription loop-mediated isothermal amplification, and sybr green i-based real-time rt-pcr in the rapid detection of bovine viral diarrhea virus nucleotide in contaminated commercial bovine sera batches.bovine viral diarrhea virus (bvdv) can contaminate biological products produced in bovine or porcine cells or manufactured using bovine sera. a rapid, specific, sensitive, and practical method of detecting bvdv in bio-products is needed. the purpose of this study was to compare three assays with respect to their ability to accurately detect bvdv in biological samples, namely reverse-transcription loop-mediated isothermal amplification (rt-lamp), sybr green i-based real-time rt-pcr, and conventio ...201425019170
substituted 2,6-bis(benzimidazol-2-yl)pyridines: a novel chemical class of pestivirus inhibitors that targets a hot spot for inhibition of pestivirus replication in the rna-dependent rna polymerase.2,6-bis(benzimidazol-2-yl)pyridine (bbp/csfa-0) was identified in a cpe-based screening as a selective inhibitor of the in vitro bovine viral diarrhea virus (bvdv) replication. the ec50-values for the inhibition of bvdv-induced cytopathic (cpe) effect, viral rna synthesis and the production of infectious virus were 0.3±0.1μm, 0.05±0.01μm and 0.3±0.04μm, respectively. furthermore, bbp/csfa-0 inhibits the in vitro replication of the classical swine fever virus (csfv) with an ec50 of 0.33±0.25μm. b ...201424680957
pore-forming activity of pestivirus p7 in a minimal model system supports genus-specific viroporin function.viroporins are small integral membrane proteins functional in viral assembly and egress by promoting permeabilization. blocking of viroporin function therefore constitutes a target for antiviral development. classical swine fever virus (csfv) protein p7 has been recently regarded as a class ii viroporin. here, we sought to establish the determinants of the csfv p7 permeabilizing activity in a minimal model system. assessment of an overlapping peptide library mapped the porating domain to the c-t ...201424189547
efficacy of marker vaccine candidate cp7_e2alf against challenge with classical swine fever virus isolates of different genotypes.classical swine fever (csf) is among the most important viral disease of domestic and feral pigs and has a serious impact on animal health and pig industry. in most countries with industrialized pig production, prophylactic vaccination against csf is banned, and all efforts are directed towards eradication of the disease, e.g. by culling of infected herds and animal movement restrictions. nevertheless, emergency vaccination remains an option to minimize the socio-economic impact of outbreaks. fo ...201424411658
porcine circovirus type 2 decreases the infection and replication of attenuated classical swine fever virus in porcine alveolar macrophages.recently, it has been noted that porcine circovirus type 2 (pcv2) infection adversely affects the protective efficacy of lapinized philippines coronel (lpc) vaccine, an attenuated strain of classical swine fever virus (csfv), in pigs. in order to investigate the possible mechanisms of the pcv2-derived interference, an in vitro model was established to study the interaction of lpc virus (lpcv) and pcv2 in porcine alveolar macrophages (ams). the results showed that pcv2 reduced the lpcv infection ...201424370262
development of a loop-mediated isothermal amplification assay combined with a lateral flow dipstick for rapid and simple detection of classical swine fever virus in the field.classical swine fever (csf) is a highly contagious viral disease and may cause heavy economic loss to farmers. the rapid, simple and accurate diagnosis of the disease at the frontline, for example on the farms of concern is crucial for disease control. this study describes the development and evaluation of a new loop-mediated isothermal amplification (lamp) assay coupled with lateral flow dipstick (lfd) for the detection of classical swine fever virus (csfv). this rt-lamp-lfd assay combines the ...201424300833
beta-actin interacts with the e2 protein and is involved in the early replication of classical swine fever virus.the e2 glycoprotein of classical swine fever virus (csfv) is involved in viral infection and induction of neutralizing antibodies. little is known about how e2 interacts with host cells. to understand the cellular factors involved in viral replication cycle, e2 was used as bait in yeast two-hybrid screens, resulting in the identification of β-actin as a potential e2-interacting partner. e2-β-actin interaction was confirmed by co-immunoprecipitation, gst pulldown, laser confocal and bimolecular f ...201424184320
a promising multiple-epitope recombinant vaccine against classical swine fever virus.classical swine fever (csf) is a highly contagious and often fatal disease of swine. it is caused by classical swine fever virus (csfv), one of the members of the genus pestivirus of the flaviviridae family. the development of a safe and effective vaccine against the csf is critical to pandemic control, this article shows a tandem-repeat multiple-epitope recombinant vaccine can protect pigs from csfv challenge. that was composed as following: two copies each of glycoprotein e2 residues 693-707, ...201424269058
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