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first molecular identification and characterization of classical swine fever virus isolates from nepal.classical swine fever (csf) is a major constraint to pig production worldwide, and in many developing countries, the epidemiological status is unknown. here, for the first time, molecular identification and characterization of csfv isolates from two recent outbreaks in nepal are presented. analysis of full-length e2-encoding sequences revealed that these isolates belonged to csfv subgenotype 2.2 and had highest genetic similarity to isolates from india. hence, for csfv, nepal and india should be ...201322975985
a t-cell epitope on ns3 non-structural protein enhances the b and t cell responses elicited by dendrimeric constructions against csfv in domestic pigs.it has been recently reported by our group that dendrimeric constructs combining b- and t-cell epitopes from classical swine fever virus (csfv) provided partial protection against experimental infection. this research evaluated four newly designed constructions while taking into account our previous work, including the direct implication that a t-cell epitope from the ns3 protein contributes to the generation of the immune response against csfv. to this end, the dendrimeric constructions, includ ...201222959286
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
efficacy of marker vaccine candidate cp7_e2alf in piglets with maternally derived c-strain antibodies.marker vaccines offer the possibility to differentiate classical swine fever (csf) infected from csf vaccinated animals based on serology and their implementation will ensure free trade with pigs. therefore, new generations of promising marker vaccines have been developed, among them the chimeric vaccine cp7_e2alf. however, in populations previously vaccinated with live attenuated vaccines like the c-strain, passive immunity through maternal antibodies can interfere with efficacy of cp7_e2alf va ...201222939909
in vitro infection with classical swine fever virus inhibits the transcription of immune response genes.classical swine fever virus (csfv) can evade the immune response and establish chronic infection under natural and experimental conditions. some genes related to antigen processing and presentation and to cytokine regulation are known to be involved in this response, but the precise mechanism through which each gene responds to csfv infection remains unclear.201222925563
prevalence of classical swine fever virus in domestic pigs in south korea: 1999-2011.the major policy for eradication of classical swine fever (csf) in south korea has focused on the implementation of compulsory vaccination of the susceptible pig population. a vaccine strain of csf virus, the lom strain, is used to maintain high herd seroconversion, a practice complementary to the 'stamping-out policy' and restriction of animal movement during disease outbreaks. to survey for the prevalence of csf in domestic pigs in south korea over the past 13 years (1999-2011), we tested 4 19 ...201322925439
sequential deletion of genes from the african swine fever virus genome using the cre/loxp recombination system.a method has been established to sequentially delete combinations of genes from the asfv genome to test the effect on virus replication and host responses to infection. initially the asfv genes mgf505 2r and mgf505 3r and a truncated mgf360 9l gene were deleted from the genome of the tissue-culture adapted asfv strain ba71v and replaced with bacteriophage loxp sequences flanking the beta-glucuronidase (gus) marker gene to create recombinant virus vδmgf-gus. subsequently the gus gene was removed ...201222902236
classical swine fever virus isolates from cuba form a new subgenotype 1.4.identification and classification of classical swine fever virus (csfv) on the basis of nucleotide sequencing and phylogenetic analysis have become an important tool to study the epidemiology and to control csf disease. according to phylogenetic analyses of short sequences from the 5'nontranslated region (150 nt) and the e2 (190 nt), most csfv isolates from south and central america have been assorted to the subgenotypes 1.1 and 1.3, while csfv isolates from cuba have been allocated to subgenoty ...201322902191
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
new insights into the antigenic structure of the glycoprotein e(rns) of classical swine fever virus by epitope mapping.the e(rns) glycoprotein of classical swine fever virus (csfv) has been studied in detail concerning biochemical and functional properties, whereas less is known about its antigenic structure. in order to define epitopes recognized by csfv-specific antibodies, the binding sites of seven e(rns)-specific monoclonal antibodies were investigated. mapping experiments using chimeric e(rns) proteins, site-directed mutagenesis and an overlapping peptide library identified one antigenic region located bet ...201222868042
detection of antibodies against classical swine fever virus in fecal samples from wild boar.classical swine fever (csf) is a contagious viral disease that affects pigs. wild boars can play an important epidemiological role in csf outbreaks. in the past decades, studies conducted in many countries have reported that the csf virus (csfv) may persist in wild boar populations. the existence of csfv in the free-ranging wild boar populations was indirectly confirmed by determining the prevalence of antibodies against csfv in the serum of hunted wild boars. however, analyzing sero-prevalence ...201222841406
production and characterization of monoclonal antibodies against a local isolate of classical swine fever virus.monoclonal antibodies (mabs) against a classical swine fever virus (csfv; subgenogroup 1:1) isolate from assam, india were produced and characterized. four fusions of myeloma cells (sp2/0ag) were made with spleenocytes of 8-10 weeks old balb/c mice immunized with the viral antigen. several hybridoma clones secreting antibodies to the virus were obtained after four fusions, but five hybridoma clones secreting antibody specific to the virus could be stabilized. all the mabs belong to the igg2a iso ...201222822524
classical swine fever virus ns5a regulates viral rna replication through binding to ns5b and 3'utr.in this report, classical swine fever virus (csfv) ns5a inhibit viral rna replication when its concentration reached and surpassed the level of ns5b. three amino acid fragments of csfv ns5a, 137-172, 224-268 and 390-414 individually were shown to be essential to ns5b binding. the former two fragments were independently necessary for regulation of viral rna replication and correlated with ns5b and 3'utr binding activity. we also found that amino acids w143, v145, p227, t246, p257, k399, t401, e40 ...201222795973
african swine fever virus excretion patterns in persistently infected animals: a quantitative approach.the continuing circulation of african swine fever (asf) in russia and in the trans-caucasian countries has led to increased efforts in characterizing the epidemiology of asf. for a better insight in epidemiology, quantitative data on virus excretion is required. until now, excretion data has mainly focused on the initial stages of the disease. in our study we have studied asf virus (asfv) excretion dynamics in persistently infected animals. for this purpose, virus excretion through different rou ...201222795261
outbreaks of highly pathogenic porcine reproductive and respiratory syndrome in jiangxi province, china.in 2007, herds of pigs in jiangxi province, china experienced outbreaks of a severe form of suspected porcine reproductive and respiratory syndrome (prrs) characterized by high fever, high morbidity and mortality in animals of different ages. 152 swine sera and 42 tissues (consisting of liver, lung, lymph node and kidney) from five herds of pigs were collected. pigs were diagnosed as infected with a highly pathogenic form of the prrs virus (prrsv) based on elisa and reverse transcriptase polymer ...201222784793
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
the atf6 branch of unfolded protein response and apoptosis are activated to promote african swine fever virus infection.african swine fever virus (asfv) infection induces apoptosis in the infected cell; however, the consequences of this activation on virus replication have not been defined. in order to identify the role of apoptosis in asfv infection, we analyzed caspase induction during the infection and the impact of caspase inhibition on viral production. caspases 3, 9 and 12 were activated from 16 h post-infection, but not caspase 8. indeed, caspase 3 activation during the early stages of the infection appear ...201222764100
a real-time pcr to detect and analyze virulent emcv loads in sows and piglets.a real-time polymerase chain reaction with sybr green was developed for the detection and quantification of encephalomyocarditis virus (emcv) in porcine tissues; the method uses two primers specific for the 3d gene. the detection limit of this assay was 22 gene copies/reaction, equivalent to 0.001 tcid(50)/ml. the assay was linear over a 10(7) dilution range of template concentrations and was specific for emcv; it did not amplify other porcine pathogens (porcine circovirus 2, porcine reproductiv ...201222752806
identification of conformational epitopes and antigen-specific residues at the d/a domains and the extramembrane c-terminal region of e2 glycoprotein of classical swine fever virus.envelope glycoprotein e2 of classical swine fever virus (csfv) is the major antigen that induces neutralizing antibodies in infected pigs. previous studies revealed that both conformation-dependent and linear epitopes are most present within domains b/c/d/a in the n-terminal half of e2. however, studies of antigenicity beyond the b/c domains remain limited. this study revealed that conformational epitopes were present on the d/a domains as well as the proximal c-terminal of e2, since the mutatio ...201222727685
african swine fever virus uses macropinocytosis to enter host cells.african swine fever (asf) is caused by a large and highly pathogenic dna virus, african swine fever virus (asfv), which provokes severe economic losses and expansion threats. presently, no specific protection or vaccine against asf is available, despite the high hazard that the continued occurrence of the disease in sub-saharan africa, the recent outbreak in the caucasus in 2007, and the potential dissemination to neighboring countries, represents. although virus entry is a remarkable target for ...201222719252
classical swine fever virus ns5a protein localizes to endoplasmic reticulum and induces oxidative stress in vascular endothelial cells.classical swine fever virus (csfv) causes a severe disease of pigs that is characterized by hemorrhage, disseminated intravascular coagulation, and leucopenia. until now, the role of the nonstructural protein 5a (ns5a) produced by csfv in the pathogenesis of csf is not well known. in this study, we investigated the function of csfv ns5a by examining its role in the induction of oxidative stress and related intracellular events. stable swine umbilical vein endothelial cell lines expressing csfv n ...201222718084
improved strategy for phylogenetic analysis of classical swine fever virus based on full-length e2 encoding sequences.molecular epidemiology has proven to be an essential tool in the control of classical swine fever (csf) and its use has significantly increased during the past two decades. phylogenetic analysis is a prerequisite for virus tracing and thus allows implementing more effective control measures. so far, fragments of the 5´ntr (150 nucleotides, nt) and the e2 gene (190 nt) have frequently been used for phylogenetic analyses. the short sequence lengths represent a limiting factor for differentiation o ...201222676246
modulation of translation initiation efficiency in classical swine fever virus.modulation of translation initiation efficiency on classical swine fever virus (csfv) rna can be achieved by targeted mutations within the internal ribosome entry site (ires). in this study, cdnas corresponding to the wild-type (wt) or mutant forms of the ires of csfv strain paderborn were amplified and inserted into dicistronic reporter plasmids encoding fluc and rluc under the control of a t7 promoter. the mutations were within domains ii, iiid(1), and iiif of the ires. the plasmids were trans ...201222674994
selection of classical swine fever virus with enhanced pathogenicity reveals synergistic virulence determinants in e2 and ns4b.classical swine fever virus (csfv) is the causative agent of classical swine fever (csf), a highly contagious disease of pigs. there are numerous csfv strains that differ in virulence, resulting in clinical disease with different degrees of severity. low-virulent and moderately virulent isolates cause a mild and often chronic disease, while highly virulent isolates cause an acute and mostly lethal hemorrhagic fever. the live attenuated vaccine strain gpe(-) was produced by multiple passages of t ...201222674973
genetic typing of classical swine fever virus isolates from china.the e2 genes of 73 classical swine fever virus (csfv) originated from csf suspected cases in different regions of china were genetically characterized and compared with reference csf viruses. all chinese viruses that characterized were segregated into two major groups and subdivided into four subgroups. most of isolates (61.6%) belonged to group 2 and were further divided into three subgroups: subgroup 2.1, 2.2 and 2.3. subgroup 2.1 was the largest subgroup which contained 46.6% of isolates, whi ...201322672483
[quantitative detection of anti-classical swine fever virus sirna expression in cells by stem-loop rt-qpcr].rna interference (rnai) is a promising technology in development of specific antiviral therapy, but the quantitative detection of small interfering rna (sirna) expressed in vivo is the main challenge to assess its antiviral effect. in order to detect the sirna molecules (sin1 and sin2) particularly expressed in cells to inhibit the replication of classical swine fever virus (csfv), serial specific stem-loop primers were designed and synthesized. two of them (slp-n1-6 and slp-n2-8) were selected ...201222667106
real-time analysis of the interaction of a multiple-epitope peptide with antibodies against classical swine fever virus using surface plasmon resonance.the e2 envelope glycoprotein is the major immunodominant protein of classical swine fever virus (csfv), and can induce neutralizing antibodies and protective immune responses in infected swine. we developed a tandem-repeat multiple-epitope recombinant protein that contains two copies of each of the regions of e2 spanned by residues 693-704, 770-780, and 826-843, coupled by two copies of the region spanned by residues 1446-1460 of the csfv nonstructural protein ns2-3. the chemically synthesized g ...201222634031
withdrawn: development of an rt-pcr-rflp assay for the detection and differentiation of wild-type and vaccine strains of classical swine fever virus.this article has been withdrawn at the request of the author(s). the publisher apologizes for any inconvenience this may cause. the full elsevier policy on article withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.201222609180
early pathogenesis of classical swine fever virus (csfv) strains in danish pigs.host-virus interactions play an important role for the clinical outcome of classical swine fever virus (csfv) infections in pigs. strain virulence, host characteristics and environment are all factors that markedly influence disease severity. we tested csfv strains of varying virulence in an experimental set-up, reducing the influence of host and environmental factors. thus, weaner pigs were inoculated with one of 4 csfv strains in order to compare the pathogenesis for a 3-week-period after infe ...201222608103
african swine fever virus strain georgia 2007/1 in ornithodoros erraticus ticks. 022607706
diagnosis of porcine teschovirus encephalomyelitis in the republic of haiti.in february and march 2009, approximately 1,500 backyard pigs of variable age became sick, and approximately 700 of them died or were euthanized in the lower artibonite valley and the lower plateau of the republic of haiti. the main clinical sign was posterior ataxia followed by paresis and/or paralysis on the second or third day of illness. no gross lesions were observed at postmortem examinations. the morbidity and mortality were approximately 60% and 40%, respectively. diagnostic samples (who ...201222604774
positive selection pressure on the b/c domains of the e2-gene of classical swine fever virus in endemic areas under c-strain vaccination.in cuba, classical swine fever (csf) has become an endemic disease with several outbreaks each year, despite the implemented vaccination program. interestingly, a trend towards a milder presentation of the disease has been observed among the animals during the last years. this study aimed to assess positive selection pressure acting on partial e2 gene of csf viruses to gain insights into the mechanisms governing virulence and the driving forces of classical swine fever virus (csfv) evolution in ...201222580241
development of multiplex pcr for simultaneous detection of six swine dna and rna viruses.uniplex and multiplex reverse transcription-polymerase chain reaction (rt-pcr) and pcr protocols were developed and evaluated subsequently for its effectiveness in detecting simultaneously single and mixed infections in swine. specific primers for three dna viruses and three rna viruses, including classical swine fever virus (csfv), porcine reproductive and respiratory syndrome virus (prrsv), japanese encephalitis virus (jev), porcine circovirus type 2 (pcv2), porcine pseudorabies virus (prv) an ...201222575688
neoplastic transformation of t lymphocytes through transgenic expression of a virus host modification protein.virus host evasion genes are ready-made tools for gene manipulation and therapy. in this work we have assessed the impact in vivo of the evasion gene a238l of the african swine fever virus, a gene which inhibits transcription mediated by both nf-κb and nfat. the a238l gene has been selectively expressed in mouse t lymphocytes using tissue specific promoter, enhancer and locus control region sequences for cd2. the resulting two independently derived transgenic mice expressed the transgene and dev ...201222558084
tick-borne viruses in europe.the aim of this review is to present briefly background information on 27 tick-borne viruses ("tiboviruses") that have been detected in europe, viz flaviviruses tick-borne encephalitis (tbev), louping-ill (liv), tyuleniy (tyuv), and meaban (meav); orthobunyaviruses bahig (bahv) and matruh (mtrv); phleboviruses grand arbaud (gav), ponteves (ptvv), uukuniemi (uukv), zaliv terpeniya (ztv), and st. abb's head (sahv); nairoviruses soldado (solv), puffin island (piv), avalon (avav), clo mor (cmv), cri ...201222526290
prevalence of and risk factors associated with viral and bacterial pathogens in farmed european wild boar.the aim of this study was to estimate in farmed european wild boars the prevalence of and risk factors associated with a range of common porcine viral and bacterial infections, namely, porcine parvovirus (ppv), porcine circovirus type 2 (pcv2), swine influenza virus (siv), aujeszky's disease virus (adv), classical swine fever virus (csfv), swine vesicular disease virus (svdv), coronavirus causing transmissible gastroenteritis (tgev), porcine reproductive and respiratory syndrome virus (prrsv), m ...201222516920
characterization of c-strain "riems" tav-epitope escape variants obtained through selective antibody pressure in cell culture.classical swine fever virus (csfv) c-strain "riems" escape variants generated under selective antibody pressure with monoclonal antibodies and a peptide-specific antiserum in cell culture were investigated. candidates with up to three amino acid exchanges in the immunodominant and highly conserved linear tav-epitope of the e2-glycoprotein, and additional mutations in the envelope proteins erns and e1, were characterized both in vitro and in vivo.it was further demonstrated, that intramuscular im ...201222515281
baculovirus expression and antigenic characterization of classical swine fever virus e2 proteins.genes encoding a major structural glycoprotein, e2, of classical swine fever viruses (csfv) brescia (subgroup 1.2), paderborn (subgroup 2.1) and kanagawa (subgroup 3.4) were constructed by removing the transmembrane domain and adding a c-terminal 6 histidine (his) tag. all the e2 constructs were efficiently expressed in a baculovirus system as 53-kda glycosylated proteins that were identified in western blots by their reaction with anti-his and csfv-specific antibodies. these proteins were used ...201322510427
novel approach for the generation of recombinant african swine fever virus from a field isolate using gfp expression and 5-bromo-2'-deoxyuridine selection.generation of african swine fever virus (asfv) recombinants has so far relied mainly on the manipulation of virus strains which had been adapted to growth in cell culture, since field isolates do not usually replicate efficiently in established cell lines. using wild boar lung cells (wsl) which allow for propagation of asfv field isolates, a novel approach for the generation of recombinant asfv directly from field isolates was developed which includes the integration into the viral thymidine kin ...201222498259
classical swine fever virus p7 protein is a viroporin involved in virulence in swine.the nonstructural protein p7 of classical swine fever virus (csfv) is a small hydrophobic polypeptide with an apparent molecular mass of 6 to 7 kda. the protein contains two hydrophobic stretches of amino acids interrupted by a short charged segment that are predicted to form transmembrane helices and a cytosolic loop, respectively. using reverse genetics, partial in-frame deletions of p7 were deleterious for virus growth, demonstrating that csfv p7 function is critical for virus production in c ...201222496228
a serosurvey for brucella suis, classical swine fever virus, porcine circovirus type 2, and pseudorabies virus in feral swine (sus scrofa) of eastern north carolina.as feral swine (sus scrofa) populations expand their range and the opportunity for feral swine hunting increases, there is increased potential for disease transmission that may impact humans, domestic swine, and wildlife. from september 2007 to march 2010, in 13 north carolina, usa, counties and at howell woods environmental learning center, we conducted a serosurvey of feral swine for brucella suis, pseudorabies virus (prv), and classical swine fever virus (csfv); the samples obtained at howell ...201222493123
high virulence of african swine fever virus caucasus isolate in european wild boars of all ages. 201222469497
the core protein of classical swine fever virus is dispensable for virus propagation in vitro.core protein of flaviviridae is regarded as essential factor for nucleocapsid formation. yet, core protein is not encoded by all isolates (gbv- a and gbv- c). pestiviruses are a genus within the family flaviviridae that affect cloven-hoofed animals, causing economically important diseases like classical swine fever (csf) and bovine viral diarrhea (bvd). recent findings describe the ability of ns3 of classical swine fever virus (csfv) to compensate for disabling size increase of core protein (rie ...201222457622
molecular characterization of african swine fever virus isolates originating from outbreaks in the russian federation between 2007 and 2011.african swine fever is one of the most important viral diseases of pigs and which caused significant economic damage on the pig production worldwide. nowadays, it is still present on the african continent, in transcaucasus countries (tcc), on island of sardinia and in russia. outbreaks of the disease have been reported in russia for the last four years, affected especially the southern federal district of the country. since 2010, a new outbreak area has been observed in the northwestern federal ...201222445729
a novel pcr-based method for high throughput prokaryotic expression of antimicrobial peptide genes.to facilitate the screening of large quantities of new antimicrobial peptides (amps), we describe a cost-effective method for high throughput prokaryotic expression of amps. eddie, an autoproteolytic mutant of the n-terminal autoprotease, npro, from classical swine fever virus, was selected as a fusion protein partner. the expression system was used for high-level expression of six antimicrobial peptides with different sizes: bombinin-like peptide 7, temporin g, hexapeptide, combi-1, human hista ...201222439858
establishment and characterization of an infectious cdna clone of a classical swine fever virus lom strain.classical swine fever virus (csfv) causes a highly contagious disease among swine that has an important economic impact worldwide. csfv strain lom is an attenuated virus of low virulent strain of miyagi isolated from japan in 1956. eight dna fragments representing the genome of the csfv strain lom were obtained by rt-pcr. these were used to determine the complete nucleotide sequence and construct a full-length cdna clone which was called flc-lom. sequence analysis of the recombinant clone (flc-l ...201222437540
the 3'-terminal hexamer sequence of classical swine fever virus rna plays a role in negatively regulating the ires-mediated translation.the 3' untranslated region (utr) is usually involved in the switch of the translation and replication for a positive-sense rna virus. to understand the 3' utr involved in an internal ribosome entry site (ires)-mediated translation in classical swine fever virus (csfv), we first confirmed the predicted secondary structure (designated as sli, slii, sliii, and sliv) by enzymatic probing. using a reporter assay in which the luciferase expression is under the control of csfv 5' and 3' utrs, we found ...201222432046
comparative [corrected] codon usage between the three main viruses in pestivirus genus and their natural susceptible livestock.classical swine fever virus, bovine viral diarrhea virus (bvdv), and border disease virus can cause serious livestock diseases. the relative synonymous codon usage value, the "effective number of codons" (enc), the ratio of k(s) value to k(a) value and the principle component analysis were employed to analyze the genetic characteristics of open reading frame (orf) and the four genes (the n(pro), erns, e1, e2 genes) of the three viruses and the relationship of codon usage pattern between each vir ...201222395914
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
pathology of porcine peripheral white blood cells during infection with african swine fever virus.african swine fever virus (asfv) is the causative agent of african swine fever (asf) that is the significant disease of domestic pigs. several studies showed that asfv can influence on porcine blood cells in vitro. thus, we asked ourselves whether asfv infection results in changes in porcine blood cells in vivo. a series of experiments were performed in order to investigate the effects of asfv infection on porcine peripheral white blood cells. nine pigs were inoculated by intramuscular injection ...201222373449
characterisation of vaccine-induced, broadly cross-reactive ifn-γ secreting t cell responses that correlate with rapid protection against classical swine fever virus.live attenuated c-strain classical swine fever viruses (csfv) provide a rapid onset of protection, but the lack of a serological test that can differentiate vaccinated from infected animals limits their application in csf outbreaks. since immunity may precede antibody responses, we examined the kinetics and specificity of peripheral blood t cell responses from pigs vaccinated with a c-strain vaccine and challenged after five days with a genotypically divergent csfv isolate. vaccinated animals di ...201222366027
classical swine fever virus diagnostics and vaccine production in insect cells. 199622358490
n(pro) of classical swine fever virus prevents type i interferon-mediated priming of conventional dendritic cells for enhanced interferon-α response.a hallmark of acute classical swine fever is the high interferon (ifn)-α levels found in the serum early after infection, followed by an inflammatory cytokine storm. plasmacytoid dendritic cells (pdcs) represent the only known cell type that produces ifn-α upon classical swine fever virus (csfv) infection in vitro. in primary target cells of the virus the viral protein n(pro) inhibits the induction of type i ifn via the degradation of irf3. we hypothesized that the early systemic pdc-derived ifn ...201222313263
yeast expressed classical swine fever e2 subunit vaccine candidate provides complete protection against lethal challenge infection and prevents horizontal virus transmission.classical swine fever (csf) caused by the classical swine fever virus (csfv) is a highly contagious swine disease resulting in large economical losses worldwide. the viral envelope glycoprotein e(rns) and e2 are major targets for eliciting antibodies against csfv in infected animals. a pichia pastoris yeast expressed e2 protein (ye2) has been shown to induce a protective immune response against csfv challenge. the purpose of this study is to determine the optimal dose of ye2 and its efficacy on ...201222300723
development of multiple elisas for the detection of antibodies against classical swine fever virus in pig sera.the major immunogenic proteins (e(rns), e2 and ns3) of classical swine fever virus (csfv) (shimen strain) were expressed in e. coli and purified by affinity chromatography. the recombinant antigens were applied to develop multiple enzyme-linked immunosorbent assays (elisas) for the detection of specific antibodies in pig sera. optimum cut-off values were determined by receiver operating characteristic (roc) analysis after testing 201 sera of vaccinated pigs and 64 negative sera of unvaccinated p ...201222270806
classical swine fever virus ns5a protein interacts with 3'-untranslated region and regulates viral rna synthesis.to investigate the function of classical swine fever virus (csfv) ns5a protein, the experiments for viral rna synthesis and viral replication were performed in the co-presence of ns5a and ns5b. results showed that small concentrations of ns5a stimulated, large concentrations of ns5a inhibited, viral rna synthesis and viral replication. affinity chromatography experiments and uv-crosslinking assays revealed that csfv ns5a and ns5b bound its cognate 3'utr and that ns5a had higher affinity than ns5 ...201222261205
classical swine fever virus ns5b protein suppresses the inhibitive effect of ns5a on viral translation by binding to ns5a.in order to investigate molecular mechanisms of internal ribosome entry site (ires)-mediated translation in classical swine fever virus (csfv), an important pathogen of pigs, expression level was evaluated in the context of genomic rnas and reporter rna fragments. all data showed that ns5a protein has an inhibitory effect on ires-mediated translation and ns5b proteins suppress the inhibitory effect of ns5a on viral translation but csfv ns5b gdd mutants do not. furthermore, gst pull-down assay an ...201222258858
effects of astragalus polysaccharide on immune responses of porcine pbmc stimulated with prrsv or csfv.astragalus polysaccharide (aps) has been used as an immunomodulator that can enhance immune responses, whereas the immunomodulatory effects of aps on porcine peripheral blood mononuclear cells (pbmcs) exposed to porcine reproductive and respiratory syndrome virus (prrsv) and classical swine fever virus (csfv) have not been investigated.201222253710
challenge of pigs with classical swine fever viruses after c-strain vaccination reveals remarkably rapid protection and insights into early immunity.pre-emptive culling is becoming increasingly questioned as a means of controlling animal diseases, including classical swine fever (csf). this has prompted discussions on the use of emergency vaccination to control future csf outbreaks in domestic pigs. despite a long history of safe use in endemic areas, there is a paucity of data on aspects important to emergency strategies, such as how rapidly csfv vaccines would protect against transmission, and if this protection is equivalent for all viral ...201222235283
interferon status and white blood cells during infection with african swine fever virus in vivo.african swine fever virus (asfv) is the causative agent of african swine fever that is the significant disease of domestic pigs, with high rates of mortality. asfv is double-stranded dna virus whose genes encode some proteins that are implicated in the suppression of host immune response. in this study, we have modeled in vivo infection of asfv for determination of interferon (ifn) status in infected pigs. we measured the level of ifn-α, -β and -γ by enzyme-linked immunosorbent assay and showed ...201122226551
virus survival in slurry: analysis of the stability of foot-and-mouth disease, classical swine fever, bovine viral diarrhoea and swine influenza viruses.farm slurry can be highly contaminated with viral pathogens. the survival of these pathogens within slurry is important since this material is often distributed onto farm land either directly or after heat treatment. there is clearly some risk of spreading pathogens in the early stages of an outbreak of disease before it has been recognized. the survival of foot-and-mouth disease virus, classical swine fever virus, bovine viral diarrhoea virus and swine influenza virus, which belong to three dif ...201122226541
development of an immunochromatographic strip for rapid detection of antibodies against classical swine fever virus.the genes encoding the erns and e2 antigen epitopes of classical swine fever virus (csfv) were expressed as a chimeric protein in escherichia coli bl21 by pet expression system. the antigenicity of the expressed protein cnc2 was identified by indirect enzyme-linked immunoabsorbant assay (elisa) and immunoblot with anti-csfv antibodies. based on the cnc2 protein, an immunochromatographic strip was developed to evaluate the antibody titer of serum samples from swine vaccinated with csfv vaccine ra ...201122207080
genetic variation of border disease virus species strains.the 5´-untranslated region of pestivirus strains isolated from domestic and wild animals were analysed to determine their taxonomic status according to nucleotide changes in the secondary genomic structure using the palindromic nucleotide substitutions (pns) method. a total of 131 isolates out of 536 pestivirus strains evaluated, were clustered as border disease virus (bdv) species. the bdv strains were further divided into at least 8 genotypes or subspecies. thirty-two isolates from small rumin ...201122194225
SARS Coronavirus nsp1 Protein Induces Template-Dependent Endonucleolytic Cleavage of mRNAs: Viral mRNAs Are Resistant to nsp1-Induced RNA Cleavage.SARS coronavirus (SCoV) nonstructural protein (nsp) 1, a potent inhibitor of host gene expression, possesses a unique mode of action: it binds to 40S ribosomes to inactivate their translation functions and induces host mRNA degradation. Our previous study demonstrated that nsp1 induces RNA modification near the 5'-end of a reporter mRNA having a short 5' untranslated region and RNA cleavage in the encephalomyocarditis virus internal ribosome entry site (IRES) region of a dicistronic RNA template ...201122174690
development of a loop-mediated isothermal amplification method for rapid detection of porcine boca-like virus.the porcine boca-like virus (pbo-likev) was recently discovered in swedish pigs with post-weaning multisystemic wasting syndrome (pmws). in this study, a loop-mediated isothermal amplification (lamp) assay was developed for rapid, specific and sensitive detection of pbo-likev. a set of four primers specific for six regions of pbo-likev vp1/2 genes was designed with the online software. the reaction temperature and time were optimized to 65°c and 60min, respectively. lamp products were detected b ...201122172971
characterization of african swine fever virus caucasus isolate in european wild boars.since 2007, african swine fever has spread from the caucasus region. to learn more about the dynamics of the disease in wild boars (sus scrofa), we conducted experiments by using european wild boars. we found high virulence of caucasus isolates limited potential for establishment of endemicity.201122172247
methods for growing and titrating african swine fever virus: field and laboratory samples.growing african swine fever virus (asfv) isolates obtained mainly from the field, but also engineered in the laboratory, is a critical step for diagnosis, titration, or virus infection studies. this unit describes a set of methods and protocols to produce and titrate any asfv strain in cell cultures. the procedures include (1) basic techniques to prepare virus-sensitive target cells; (2) strategies for growth, concentration, and purification of virus stocks; and (3) the semi-quantitative (end di ...201122161547
porcine circovirus type 2 (pcv2) infection decreases the efficacy of an attenuated classical swine fever virus (csfv) vaccine.abstract: the lapinized philippines coronel (lpc) vaccine, an attenuated strain of classical swine fever virus (csfv), is an important tool for the prevention and control of csfv infection and is widely and routinely used in most csf endemic areas, including taiwan. the aim of this study was to investigate whether pcv2 infection affects the efficacy of the lpc vaccine. eighteen 6-week-old, cesarean-derived and colostrum-deprived (cdcd), crossbred pigs were randomly assigned to four groups. a tot ...201122129109
Clustering of classical swine fever virus isolates by codon pair bias.ABSTRACT:201122126254
disinfection of foot-and-mouth disease and african swine fever viruses with citric acid and sodium hypochlorite on birch wood carriers.transboundary animal disease viruses such as foot-and-mouth disease virus (fmdv) and african swine fever virus (asfv) are highly contagious and cause severe morbidity and mortality in livestock. proper disinfection during an outbreak can help prevent virus spread and will shorten the time for contaminated agriculture facilities to return to food production. wood surfaces are prevalent at these locations, but there is no standardized method for porous surface disinfection; commercial disinfectant ...201122115968
a new cloning system based on the opri lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulations.the conjugation of antigens with ligands of pattern recognition receptors (prr) is emerging as a promising strategy for the modulation of specific immunity. here, we describe a new escherichia coli system for the cloning and expression of heterologous antigens in fusion with the opri lipoprotein, a tlr ligand from the pseudomonas aeruginosa outer membrane (om). analysis of the opri expressed by this system reveals a triacylated lipid moiety mainly composed by palmitic acid residues. by offering ...201222115954
small rho gtpases and cholesterol biosynthetic pathway intermediates in african swine fever virus infection.the integrity of the cholesterol biosynthesis pathway is required for efficient african swine fever virus (asfv) infection. incorporation of prenyl groups into rho gtpases plays a key role in several stages of asfv infection, since both geranylgeranyl and farnesyl pyrophosphates are required at different infection steps. we found that rho gtpase inhibition impaired virus morphogenesis and resulted in an abnormal viral factory size with the accumulation of envelope precursors and immature virions ...201122114329
detection of foot-and-mouth disease virus rna by reverse transcription loop-mediated isothermal amplification.a reverse transcription loop-mediated isothermal amplification (rt-lamp) assay was developed for foot-and-mouth disease virus (fmdv) rna. the amplification was able to finish in 45 min under isothermal condition at 64°c by employing a set of four primers targeting fmdv 2b. the assay showed higher sensitivity than rt-pcr. no cross reactivity was observed from other rna viruses including classical swine fever virus, swine vesicular disease, porcine reproductive and respiratory syndrome virus, japa ...201122070774
A novel dendrimeric peptide induces high level neutralizing antibodies against classical swine fever virus in rabbits.The amino acid sequence (TAVSPTTLR, 829-837aa) on the glycoprotein E2 of classical swine fever virus (CSFV) is a conserved and linear neutralizing epitope. In the present study, two peptides were constructed based the core sequence of this neutralizing epitope, the dendrimeric peptide (Th-B(4)) containing four copies of B cell epitope fused to one copy of promiscuous T helper (Th) cell epitope and the peptide Th-B containing a single copy of B cell epitope fused to one copy of Th cell epitope. T ...201122051445
rapid typing of foot-and-mouth disease serotype asia 1 by reverse transcription loop-mediated isothermal amplification.a reverse transcriptase loop-mediated isothermal amplification (rt-lamp) assay was rapidly used to detect serotype asia 1 of foot-and-mouth disease virus (fmdv) within 45 min at 61°c. all fmdv serotype asia 1 reference strains were positive by rt-lamp, while other viruses such as fmdv serotypes o, c, a and classical swine fever virus, swine vesicular disease virus, porcine reproductive and respiratory syndrome virus and japanese encephalitis virus remained negative. furthermore, fmdv sreotype as ...201122040459
neurological disorders, virus persistence and hypomyelination in calves due to intra-uterine infections with bovine virus diarrhoea virus.summary the clinical and pathological findings after a natural intra-uterine infection with bvd-virus in a friesian dairy herd are described. the virological and serological aspects will be discussed in a separate paper (30). in a period of 4 years, 11 calves were hum with the following nervous symptoms: more or less serious incoördination, tremor, oscillating nystagmus, and a negative blinking reflex. the pupillary and sucking reflexe's were normal. no ocular defects, such as lenticular opacity ...198322040145
microbial ecology of swine farms and prrs vaccine vaccination strategies.the present study investigated the microbial ecology and vaccination strategies against porcine reproductive and respiratory syndrome (prrs) in field condition. four representative farms with a history of prrs were included in this study. over the almost 3-year period, the average detection rate was 68.9%, making prrsv the first most frequently detected virus, followed by porcine circovirus type 2 (pcv2) (64.2%), pseudorabies virus (prv) (11.03%) and classical swine fever virus (csfv) (4.41%). s ...201122014373
RNA self-cleavage activated by ultraviolet light-induced oxidation.A novel UV-C-light-induced ribozyme activity was discovered within the highly structured 5'-genomic regions of both Hepatitis C Virus (HCV) and the related Classic Swine Fever Virus (CSFV). Cleavage is mediated by exposure to UV-C light but not by exogenous oxygen radicals. It is also very selective, occurring at base positions HCV C(79) and CSFV A(45) in some molecules and at the immediately adjacent 5'-positions HCV U(78) and CSFV U(44) in others. Among other reaction products, the majority of ...201121989404
Characterization of the monoclonal antibody against classical swine fever virus glycoprotein E(rns) and its application to an indirect sandwich ELISA.Classical swine fever virus (CSFV) E(rns) is an envelope glycoprotein possessing RNase activity. The E(rns)-based enzyme-linked immunosorbent assay (ELISA) has been considered a discriminating diagnostic test for differentiating infected from vaccinated animals. The purpose of this study was to produce a specific monoclonal antibody (MAb) to E(rns) for further developing an indirect sandwich ELISA. The MAb CW813 was shown to specifically recognize both the monomer and dimer forms of Pichia pasto ...201121972132
effects of glycosylation on antigenicity and immunogenicity of classical swine fever virus envelope proteins.classical swine fever virus (csfv) harbors three envelope glycoproteins (e(rns), e1 and e2). previous studies have demonstrated that removal of specific glycosylation sites within these proteins yielded attenuated and immunogenic csfv mutants. here we analyzed the effects of lack of glycosylation of baculovirus-expressed e(rns), e1, and e2 proteins on immunogenicity. interestingly, e(rns), e1, and e2 proteins lacking proper post-translational modifications, most noticeable lack of glycosylation, ...201121968199
mechanism of collapse of endoplasmic reticulum cisternae during african swine fever virus infection.infection of cells with african swine fever virus (asfv) can lead to the formation of zipper-like stacks of structural proteins attached to collapsed endoplasmic reticulum (er) cisternae. we show that the collapse of er cisternae observed during asfv infection is dependent on the viral envelope protein, j13lp. expression of j13lp alone in cells is sufficient to induce collapsed er cisternae. collapse was dependent on a cysteine residue in the n-terminal domain of j13lp exposed to the er lumen. l ...201221951707
detection of porcine circovirus type 1 in commercial porcine vaccines by loop-mediated isothermal amplification.a loop-mediated isothermal amplification (lamp) method with a real-time monitoring system was developed for the detection of porcine circovirus type 1 (pcv1) in commercial swine vaccines. this method was highly specific for pcv1. no cross-reaction to porcine circovirus type 2, porcine parvovirus, pseudorabies virus, classical swine fever virus, and porcine reproductive and respiratory syndrome virus was observed. the analytical sensitivity of the lamp for pcv1 dna was 10 copies/μl in the case of ...201121948286
Development and inter-laboratory validation study of an improved new real-time PCR assay with internal control for detection and laboratory diagnosis of African swine fever virus.A real-time polymerase chain reaction (PCR) assay for the rapid detection of African swine fever virus (ASFV), multiplexed for simultaneous detection of swine beta-actin as an endogenous control, has been developed and validated by four National Reference Laboratories of the European Union for African swine fever (ASF) including the European Union Reference Laboratory. Primers and a TaqMan(®) probe specific for ASFV were selected from conserved regions of the p72 gene. The limit of detection of ...201121946285
antigenic mimicking with cysteine-based cyclized peptides reveals a previously unknown antigenic determinant on e2 glycoprotein of classical swine fever virus.envelope glycoprotein e2 of classical swine fever virus (csfv) is the major antigen that induces neutralizing antibodies in infected pigs. the conformational epitope(s) on b/c domains were mapped to the n-terminal 90 residues of e2 between amino acids 690 and 779 (chang et al., 2010a). to mimic the conformational epitopes, a set of synthetic cyclized peptides spanning the b/c domains of e2 were used to react with monoclonal antibodies (mabs) against e2 and with swine anti-csfv polyclonal sera. a ...201121945636
experimental infection of bama miniature pigs with a highly virulent classical swine fever virus.currently, larger domestic pigs are only animals widely used in vaccine evaluation and pathogenicity study of classical swine fever virus (csfv). this study was aimed to create an alternative animal experimental infection model of csfv.201121943299
immunogenicity of recombinant classic swine fever virus cd8(+) t lymphocyte epitope and porcine parvovirus vp2 antigen coexpressed by lactobacillus casei in swine via oral vaccination.classical swine fever virus (csfv) and porcine parvovirus (ppv) are highly contagious pathogens, resulting in enormous economic losses in pig industries worldwide. because vaccines play an important role in disease control, researchers are seeking improved vaccines that could induce antiviral immune responses against csfv and ppv at the mucosal and systemic levels simultaneously. in this study, a genetically engineered lactobacillus strain coexpressing the csfv-specific cytotoxic t lymphocyte (c ...201121940406
diagnostic value of meat juice in early detection of classical swine fever virus infection.to evaluate the diagnostic potential of meat juice for early detection of classical swine fever virus (csfv), meat juice and serum samples from pigs experimentally infected with different strains of csfv were compared for virus load. from all samples, viral rna was extracted by automated procedure before real-time reverse transcription polymerase chain reaction analysis was performed. viral rna was detected in meat juice, but at a lower level than in corresponding serum. sensitivity was calculat ...201121908364
classical swine fever virus detection: results of a real-time reverse transcription polymerase chain reaction ring trial conducted in the framework of the european network of excellence for epizootic disease diagnosis and control.the current study reports on a real-time reverse transcription polymerase chain reaction (real-time rt-pcr) ring trial for the detection of classical swine fever virus (csfv) genomic rna undertaken by 10 european laboratories. all laboratories were asked to use their routine in-house real-time rt-pcr protocols and a standardized protocol commonly used by the friedrich-loeffler-institute (fli) on a panel of well-characterized samples. in general, all participants produced results within the accep ...201121908363
development, optimization, and validation of a classical swine fever virus real-time reverse transcription polymerase chain reaction assay.classical swine fever (csf) is an economically devastating disease of pigs. instrumental to the control of csf is a well-characterized assay that can deliver a rapid, accurate diagnosis prior to the onset of clinical signs. a real-time fluorogenic-probe hydrolysis (taqman) reverse transcription polymerase chain reaction (rt-pcr) for csf was developed by the united states department of agriculture (usda) at the plum island animal disease center (csf piadc assay) and evaluated for analytical and d ...201121908362
Comparison of methods for improved RNA extraction from blood for early detection of Classical swine fever virus by real-time reverse transcription polymerase chain reaction.Classical swine fever (CSF) is a highly contagious disease of pigs. Early detection of the Classical swine fever virus (CSFV) in infected animals and routine surveillance is important for a rapid response and control of an outbreak of CSF. The current study investigated whole blood as a clinical specimen for the detection of CSFV by real-time reverse transcription polymerase chain reaction (real-time RT-PCR) in experimentally infected pigs. The virus was detectable in pre-clinical animals in who ...201121908315
a novel spatial and stochastic model to evaluate the within and between farm transmission of classical swine fever virus: ii validation of the model.a new, recently published, stochastic and spatial model for the evaluation of classical swine fever virus (csfv) spread into spain has been validated by using several methods. internal validity, sensitivity analysis, validation using historical data, comparison with other models and experiments on data validity were used to evaluate the overall reliability and consistency of the model. more than 100 modifications in input data and parameters were evaluated. outputs were obtained after 1000 itera ...201121899960
[antigenic diversity of african swine fever viruses].data on the seroimmunotypic and hemadsorbing characteristics of african swine fever virus (asf) are summarized. according to the results of immunological sampling in pigs and those of hemagglutination inhibition test, the known asfv strains and isolates were divided into 11 groups, 8 were characterized as seroimmunogroups having their specific reference strains. a 110-140-kd asfv serotype-specific nonstructural major glycoprotein was identified. it is suggested that it is the glycoprotein that c ...201121899069
A novel alphavirus replicon-vectored vaccine delivered by adenovirus induces sterile immunity against classical swine fever.Low efficacy of gene-based vaccines due to inefficient gene delivery and expression has been major bottleneck of their applications. Efforts have been made to improve the efficacy, such as gene gun and electroporation, but the strategies are difficult to put into practical use. In this study, we developed and evaluated an adenovirus-delivered, alphavirus replicon-vectored vaccine (chimeric vector-based vaccine) expressing the E2 gene of classical swine fever virus (CSFV) (rAdV-SFV-E2). Rabbits i ...201121888938
Application of GP5 protein to develop monoclonal antibody against porcine reproductive and respiratory syndrome virus.In this study, a panel of monoclonal antibodies (mAbs) against Porcine reproductive and respiratory syndrome virus(PRRSV), named as 8C9 and4B4, were produced by fusing SP2/0 myeloma cells and spleen cells of BALB/c mice immunized with the PRRSV (TCID(50)=5.5), screened by the indirect ELISA and subjected to several limiting dilutions. mAbs were then identified by biological characterization. Among the two fusion cell strains, 8C9 belonged to the IgG1 subclass and 4B4 belonged to the IgG2a subcla ...201121847758
Rapid and sensitive detection of PRRSV by a reverse transcription-loop-mediated isothermal amplification assay.A real-time monitoring reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the sensitive and specific detection of prototypic, prevalent North American porcine reproductive and respiratory syndrome virus (PRRSV) strains. As a higher sensitivity and specificity method than reverse transcription polymerase chain reaction (RT-PCR), the RT-LAMP method only used a turbidimeter, exhibited a detection limit corresponding to a 10(-4) dilution of template RNA ex ...201121847756
quantitative risk assessment for the introduction of african swine fever virus into the european union by legal import of live pigs.the recent incursion and spread of african swine fever virus (asfv) in the russian federation and caucasus region, close to european union (eu) borders, have increased the concerns regarding the probability of asfv introduction into the eu. there are many potential routes of asfv entry into eu, but here we specifically aimed to assess the probability of asfv introduction by legal trade of pigs, which historically has been one of the most important ways of exotic diseases introduction into the eu ...201121831148
virus load in pigs affected with different clinical forms of classical swine fever.summary classical swine fever (csf) is an endemic disease in india, but the real magnitude of the problem is not known as only outbreaks of acute csf are reported and many cases of chronic and clinically inapparent forms of the disease, which manifest a confusing clinical picture, remain undiagnosed. the real status of classical swine fever virus (csfv) infection can only be known by testing pigs with highly specific and sensitive diagnostic assays. to obtain the baseline prevalence of csfv infe ...201121827642
african swine fever virus p72 genotype ix in domestic pigs, congo, 2009.african swine fever virus p72 genotype ix, associated with outbreaks in eastern africa, is cocirculating in the republic of the congo with west african genotype i. data suggest that viruses from eastern africa are moving into western africa, increasing the threat of outbreaks caused by novel viruses in this region.201121801650
multiple linear b-cell epitopes of classical swine fever virus glycoprotein e2 expressed in e.coli as multiple epitope vaccine induces a protective immune response.abstract: classical swine fever is a highly contagious disease of swine caused by classical swine fever virus,an oie list a pathogen.epitope-based vaccines is one of the current focuses in the development of new vaccines against classical swine fever virus (csfv). two b-cell linear epitopes re2-ba from the e2 glycoprotein of csfv, re2-a (cfrrekpfphrmdcvtttvened, aa 844-865) and re2-b (ckedyryaisstneigllgagglt, aa 693-716), were constructed and heterologously expressed in escherichia coli as mult ...201121801433
chemical disinfection of high-consequence transboundary animal disease viruses on nonporous surfaces.disinfection is a critical part of the response to transboundary animal disease virus (tadv) outbreaks by inactivating viruses on fomites to help control infection. to model the inactivation of tadv on fomites, we tested selected chemicals to inactivate foot and mouth disease virus (fmdv), african swine fever virus (asfv), and classical swine fever virus (csfv) dried on steel and plastic surfaces. for each of these viruses, we observed a 2 to 3 log reduction of infectivity due to drying alone. w ...201121798759
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