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[bovine diarrhea virus: an update].bovine viral diarrhea virus (bvdv) is a pathogen of cattle, member of the family flaviviridae, genus pestivirus, which also includes classical swine fever virus (csfv, or hog cholera virus), and border disease virus of sheep (bdv). it causes important economical losses associated mainly with reproductive failure. pestiviruses are small enveloped viruses, with a diameter of about 40 nm. the nucleocapsid is probably icosahedral . the genome consists of a single stranded positive rna, encoding appr ...19979229725
proteolytic processing in african swine fever virus: evidence for a new structural polyprotein, pp62.we have identified an open reading frame (orf), cp530r, within the ecori c' fragment of the african swine fever virus (asfv) genome that encodes a polyprotein of 62 kda (pp62). antisera raised against different regions of orf cp530r recognized a polypeptide of 62 kda in asfv-infected cells during the late phase of virus replication, after the onset of viral dna synthesis. pulse-chase experiments showed that polyprotein pp62 is posttranslationally processed to give rise to two proteins of 35 kda ...19979223468
protein cell receptors mediate the saturable interaction of african swine fever virus attachment protein p12 with the surface of permissive cells.previous studies have demonstrated that the entry of african swine fever virus (asfv) into vero cells and swine macrophages is mediated by saturable binding sites located on the plasma membrane. the asfv protein p12 has been implicated in virus attachment to the host cell, but the cellular component responsible for the interaction with the virus is largely unknown. we have studied the binding of recombinant p12 and asfv to different cell lines. permissive cells were able to bind p12 in saturable ...19979213394
african swine fever virus-specific cytotoxic t lymphocytes recognize the 32 kda immediate early protein (vp32).african swine fever (asf) virus-specific cytotoxic t lymphocyte (ctl) activity has been studied in a model in which sla inbred minipigs were experimentally infected with an attenuated isolate of the virus. the ctl assays were performed using alveolar macrophages as target cells. the specific lysis is mediated by purified cd8+ lymphocytes but not by cd4+ cells and can be blocked by incubation with anti-sla class i monoclonal antibodies. the purified cd8+ population produced high levels of interfe ...19979213386
blocking antibodies inhibit complete african swine fever virus neutralization.a persistent non-neutralized african swine fever virus (asfv) fraction is found with most convalescent swine sera in in vitro neutralization assays. to study this phenomenon, antisera from convalescent pigs infected with different virus isolates and showing complete or incomplete virus neutralization were used. different experiments determined that incomplete neutralization of asfv is caused neither by virus aggregation, nor low affinity or stability of virus-antibody complexes. additionally, at ...19979213385
polymerase chain reaction amplification of segments of pestivirus genomes.reverse transcription followed by polymerase chain reaction (pcr) amplification of a region of the viral genome at the 3' end of the glycoprotein(s) gene was employed with the aim of determining its applicability as a diagnostic tool for pestiviruses. candidate primers were designed, from homologous segments detected by comparison between the sequences of strains nadl, osloss and alfort. a segment of 634 base pairs on the glycoprotein gene was targeted for amplification. segments of five pestivi ...19919210946
differentiation of pestiviruses by a hog cholera virus-specific genetic probe.a hog cholera virus (hcv)-specific genetic probe has been generated after cloning of the genomic viral rna. this probe distinguished between hcv and the closely related bovine viral diarrhoea virus (bvdv). furthermore, it detected a broad spectrum of hcv strains and isolates which differ in their phenotype such as virulence.19919210943
cdna probes for the detection of pestiviruses.probes were prepared from genomic rna of hog cholera virus (hcv) after synthesis of cdna and cloning. six probes were selected according to their place on the viral genome determined by sequencing and comparison with bvdv sequence. these probes were hybridized with two strains of hcv (alfort and nord), two strains of bovine viral diarrhea (bvdv) (nadl, new york) and four strains of border disease (bd) (lyon 1, lyon 2, aveyron, iemvt). this panel of six probes seem to be able to differentiate pes ...19919210941
bvd monoclonal antibodies: relationship between viral protein specificity and viral strain specificity.seventeen monoclonal antibodies raised against bovine viral diarrhoea virus were divided into three groups on the basis of radioimmunoprecipitation results. seven monoclonal antibodies precipitated a polypeptide of 80kd and defined four domains, all of which showed considerable conservation amongst the 180 pestivirus strains and isolates examined. nine monoclonal antibodies, including six with virus neutralizing activity, precipitated a 53kd polypeptide and all appeared to be directed towards a ...19919210925
molecular characterization of hog cholera virus.an efficient tissue culture system was established which allowed to obtain substantial quantities of hog cholera virus (hcv) from the cell free tissue culture supernatant. after preparation of viral rna and cdna synthesis, the complete hcv genome was cloned and sequenced. comparison with published bvdv sequences revealed a surprisingly high homology between hcv and bvdv at both the nucleotide and the amino acid level. in addition host cellular sequences were identified in bvdv genomes. the genom ...19919210921
development of microscopic lesions in splenic cords of pigs infected with african swine fever virus.acute forms of african swine fever are characterized by hemorrhagic lesions in the lymphoid organs. this paper reports the evolution of lesions in the splenic cords of pigs inoculated with african swine fever (asf) virus (strain malawi'83). ultrastructural examination of the splenic cords of the infected pigs revealed numerous macrophages attached to the muscle cells harboring virus replication center and cytopathic effects at 3 dpi (days post-infection). from 5 dpi, the splenic cords contained ...19979172845
influence of breed-related factors on the course of classical swine fever virus infection. 19979172299
[swine plague: symptoms, epizootiology and diagnosis].the clinical signs, laboratory diagnosis and modes of transmission of classical swine fever are described. special attention is paid to combinations of signs of disease which should arise suspicion to the veterinary practitioner in the present epizootic.19979157622
a bir motif containing gene of african swine fever virus, 4cl, is nonessential for growth in vitro and viral virulence.an african swine fever virus (asfv) gene with similarity to viral and cellular inhibitor of apoptosis genes (iap) has been described in the african isolate malawi lil-20/1 (orf 4cl) and a cell-culture-adapted european virus, ba71v (orf a224l). the similarity of the asfv gene to genes involved in inhibiting cellular apoptosis suggested the gene may regulate apoptosis in asfv-infected cells and thus may function in asfv virulence and/or host range. sequence analysis of additional african and europ ...19979143281
characterization of the african swine fever virus structural protein p14.5: a dna binding protein.the gene encoding the structural protein p14.5 of african swine fever virus (asfv) has been mapped and sequenced. this gene, designated e120r, is located in the sa/l h/ecorl e restriction fragment of the asfv genome and is predicted to encode a protein of 120 amino acids with a molecular weight of 13.4 kda. northern-blot analysis showed that e120r is transcribed at late times during the viral replication cycle. the e120r gene product has been expressed in escherichia coli, purified, and used as ...19979123862
inhibition of apoptosis by the african swine fever virus bcl-2 homologue: role of the bh1 domain.the function of the african swine fever virus (asfv) bcl-2 homologue, gene a179l, in the regulation of apoptosis was investigated using as a model system the human myeloid leukemia cell line k562 induced to die by apoptosis with inhibitors of macromolecular synthesis, a process that is prevented by overexpression of human bcl-2. it is shown that transfection of k562 cells with the asfv a179l gene protects these cells from apoptotic cell death induced by a combination of cycloheximide and actinom ...19979123849
neutralization susceptibility of african swine fever virus is dependent on the phospholipid composition of viral particles.in this study we have investigated the generation of african swine fever (asf) virus variants resistant to neutralizing antibodies after cell culture propagation. all highly passaged asf viruses analyzed were resistant to neutralization by antisera from convalescent pigs or antibodies generated against individual viral proteins which neutralized low-passage viruses. a molecular analysis of neutralizable and nonneutralizable virus isolates by sequencing of the genes encoding for neutralizing prot ...19979123824
subcellular changes in the tonsils of pigs infected with acute african swine fever virus.a study of the pathogenesis of acute african swine fever (asf) was carried out in pigs inoculated with a highly virulent strain of asf virus to determine the sequential development of the subcellular changes in a particular lymphoepithelial organ, the tonsil. the apoptosis of the lymphocytes and the inhibition of lymphocyte proliferation were the main changes that occurred in the tonsillar lymphoid structures. this may explain the early lymphopenia observed in acute asf. moreover, vascular chang ...19979112739
genetic analysis of classical swine fever virus isolates from a small geographic area.twenty-eight field csfv samples were isolated from outbreaks of csf which have occurred in three small geographic areas in slovakia in the period of december 1993-november 1994. all the organ homogenates were positive by virus isolation and rt-pcr assay using general pestivirus 324/326 primers selected from 5'-noncoding (5'-nc) genomic region. specific discrimination of csfv was confirmed by the cleavage of amplicons using bgl1 and ava 1. four viral isolates (a, b, c, e) were selected for a comp ...19979091287
immunohistochemical detection of hog cholera viral glycoprotein 55 in paraffin-embedded tissues.formalin-fixed, paraffin embedded tissues obtained from 40 pigs inoculated with a field isolate of hog cholera virus were examined for the presence of gp55, a major structural protein of the virus envelope, using a monoclonal antibody-based immunohistochemical test with the avidin-biotin-peroxidase complex method. immunoreactivity was detected in hog cholera virus-infected tissues but not in control pigs tissues, african swine fever virus-infected tissues, or bovine viral diarrhea virus-infected ...19979087919
african swine fever virus trans-prenyltransferase.the present study describes the characterization of an african swine fever virus gene homologous to prenyltransferases. the gene, designated b318l, is located within the ecori b fragment in the central region of the virus genome, and encodes a polypeptide with a predicted molecular weight of 35,904. the protein is characterized by the presence of a putative hydrophobic transmembrane domain at the amino end. the gene is expressed at the late stage of virus infection, and transcription is initiate ...19979083080
[intercellular interactions in swine bone marrow cell culture and ppk-66b cell line infected with african swine fever virus]. 19969081441
african swine fever virus infection of bone marrow: lesions and pathogenesis.the effects of african swine fever (asf) virus infection on bone marrow hematopoiesis and microenvironment were determined by studying the sequential development of ultrastructural lesions of bone marrow and blood cell changes. eight pigs (two pigs/infected group) were inoculated by intramuscular route with 10(5) 50% hemadsorbing doses (had50) of the malawi'83 asf virus isolate. two uninfected pigs were used as controls. ultrastructural changes developed by day 3 postinoculation (pi), persisted ...19979066076
differentiation of classical swine fever virus from ruminant pestiviruses by reverse transcription and polymerase chain reaction (rt-pcr).a reverse transcriptase-polymerase chain reaction (rt-pcr) assay was designed to allow the differentiation of pestiviruses by the expected size of the amplified fragments. one oligonucleotide primer, conserved amongst pestiviruses, and two others specific for either classical swine fever virus (csfv) or bovine viral diarrhea virus (bvdv), were designed from the 5' non-coding region of the genome. csfv infected cultures (10 strains) amplified a fragment of an expected size of 200 bp; bvdv culture ...19969054133
a set of african swine fever virus tandem repeats shares similarities with sar-like sequences.a group of cross-hybridizing dna segments contained within the ecori restriction fragments u', x and j of a vero cell-adapted strain (ba71v) of african swine fever virus (asfv) were mapped and sequenced. analysis of the nucleotide sequence revealed the presence of a set of long internal repeated sequences composed of five types of tandemly repeat units of about 200 bp. these tandem repeats contain a g-rich core of 10-14 nucleotides surrounded by regions with a high a + t content distributed in o ...19959049318
modulation of immune cell populations and activation markers in the pathogenesis of african swine fever virus infection.african swine fever (asf) virus induces immune cell alterations that may be detected by changes in peripheral blood cells phenotypic antigens and activation markers which were examined by flow cytometry, analyzing both cell proportion and/or expression intensity of superficial antigens. these studies were conducted in pigs with experimental acute of chronic asf infection to determine whether changes among important surface activation markers and phenotypic antigens, and their correlative lymph n ...19979037734
genetic characterization of ruminant pestiviruses: sequence analysis of viral genotypes isolated from sheep.historically, the genus pestivirus was believed to contain three species of viruses; bovine viral diarrhea virus (bvdv), border disease virus (bdv) and classical swine fever virus (csfv). however, based on limited sequence analysis of a small number of pestiviral isolates from domestic livestock, evidence has recently emerged indicating that at least four distinct genotypes exist. in an attempt to gain a better understanding of the degree of viral variation among ruminant pestiviruses, the entir ...19979037733
assembly of african swine fever virus: role of polyprotein pp220.polyprotein processing is a common strategy of gene expression in many positive-strand rna viruses and retroviruses but not in dna viruses. african swine fever virus (asfv) is an exception because it encodes a polyprotein, named pp220, to produce several major components of the virus particle, proteins p150, p37, p34, and p14. in this study, we analyzed the assembly pathway of asfv and the contribution of the polyprotein products to the virus structure. electron microscopic studies revealed that ...19979032369
detection and identification of ruminant and porcine pestiviruses by nested amplification of 5' untranslated cdna regions.based on published gene sequences of bovine viral diarrhoea virus (bvdv) type i and classical swine fever virus (csfv), genus- and species-specific primers were designed to detect and identify pestivirus cdna sequences in a nested polymerase chain reaction (pcr). the pcr primers were validated using cdna synthesized from 146 pestivirus isolates, comprising representatives of all four so far described genotypes (bvdv type i, bvdv type ii, csfv and border disease virus), as well as others of uncer ...19979029529
high level expression of the major antigenic african swine fever virus proteins p54 and p30 in baculovirus and their potential use as diagnostic reagents.at present, the eradication of african swine fever (asf) in affected countries is based only on an efficient diagnosis program because of the absence of an available vaccine. the highly antigenic asf virus proteins p54 and p30, encoded by genes e183l and cp204l respectively, were expressed in baculovirus for diagnostic purposes. a sequence comparison analysis of these genes from different field virus strains which are geographically diverse and isolated in different years, revealed that both gen ...19979029527
internal entry of ribosomes is directed by the 5' noncoding region of classical swine fever virus and is dependent on the presence of an rna pseudoknot upstream of the initiation codon.bicistronic rnas containing the 373-nucleotide-long 5' nontranslated region (ntr) of the classical swine fever virus (csfv) genome as intercistronic spacer were used to show the presence of an internal ribosome entry site (ires) in the 5' end of the csfv genome. by coexpression of the poliovirus 2a protease it was demonstrated that the csfv 5' ntr-driven translation is independent of the presence of functional eukaryotic initiation factor eif-4f. deletion analysis indicated that the 5' border of ...19978985370
gene homology between orf virus and smallpox variola virus.about 47% identity was observed between the deduced amino acid sequences of a protein encoded by a gene of the parapoxvirus orf virus (ov) strain nz2 and a 6 kda protein of unknown function reported to be produced by an open reading frame expressed early after infection by the orthopoxvirus western reserve vaccinia virus (vac); the open reading frame is absent from vac strain copenhagen. examination of sequences reported for variola virus (var) strains bangladesh, india, congo- 1970, somalia- 19 ...19968972571
expression in e. coli and purification of the active autoprotease p20 of classical swine fever virus.the genes for flaviviridae structural proteins are located at the 5' terminus of the genome, while the 3' terminus contains the genes for the non-structural proteins. the first protein product of the large orf of pestiviruses, the p20 protein, is however a non-structural protein which possess an autoproteolytic activity. here we report the cloning of the p20/p14 genes behind the strong trc promotor and expression of the p20 at high levels in e. coli. the autoprotease p20 was responsible for its ...19968972567
an african swine fever virus virulence-associated gene nl-s with similarity to the herpes simplex virus icp34.5 gene.we described previously an african swine fever virus (asfv) open reading frame, 23-nl, in the african isolate malawi lil 20/1 whose product shared significant similarity in a carboxyl-terminal domain with those of a mouse myeloid differentiation primary response gene, myd116, and the herpes simplex virus neurovirulence-associated gene, icp34.5 (m. d. sussman, z. lu, g. kutish, c. l. afonso, p. roberts, and d. l. rock, j. virol. 66:5586-5589, 1992). the similarity of 23-nl to these genes suggeste ...19968971015
intermediate class of mrnas in african swine fever virus.a transcriptional analysis of the african swine fever virus (asfv) i226r and i243l genes is presented. steady-state kinetics and transfection experiments showed the existence of a new temporal class of asfv mrnas transcribed from these genes, with the characteristics of the poxvirus intermediate transcripts. transcription of the i226r gene gave rise to intermediate and late mrnas that started from different sites, while the i243l gene produced early, intermediate, and late mrnas, also starting f ...19968970983
an ikappab homolog encoded by african swine fever virus provides a novel mechanism for downregulation of proinflammatory cytokine responses in host macrophages.cytokines stimulate inflammatory defenses against viral infections. in order to evade host defenses, viruses have developed strategies to counteract antiviral cytokines. african swine fever virus (asfv) is a large, double-stranded dna virus that infects macrophages. this study demonstrates that asfv effectively inhibited phorbol myristic acid-induced synthesis of antiviral, proinflammatory cytokines alpha interferon, tumor necrosis factor alpha, and interleukin-8 in infected macrophages as asses ...19968970976
involvement of the endoplasmic reticulum in the assembly and envelopment of african swine fever virus.african swine fever (asf) virus is a large enveloped dna virus assembled in the cytoplasm of cells. in this study, the membrane compartments involved in the envelopment of asf virus were investigated. a monoclonal antibody recognizing p73, the major structural protein of asf virus, was generated to analyze the binding of p73 to membranes during the assembly of the virus. approximately 50% of the intracellular pool of p73 associated with membranes as a peripheral membrane protein. binding was rap ...19968970959
evaluation of a gp 55 (e2) recombinant-based elisa for the detection of antibodies induced by classical swine fever virus.an enzyme-linked immunosorbent assay (elisa) for the detection of antibodies against classical swine fever virus (csfv) based on the competition of the serum antibodies with a csfv-specific monoclonal antibody directed against the viral glycoprotein gp 55 (e2), has been evaluated. a total of 553 sera obtained from pigs experimentally infected in groups with different pestiviruses were included in this study. the elisa was applied to a group of sera collected from pigs prior to pestivirus inocula ...19968968126
evaluation of nucleic acid amplification methods for the detection of hog cholera virus.a blind panel was tested in a diagnostic evaluation of a reverse transcription (rt) polymerase chain reaction (pcr) method for detecting hog cholera virus (hcv) from pig tissues. the capability of the rt-pcr test to discriminate between hcv and related pestiviruses, bovine viral diarrhea virus (bvdv), and those viruses causing similar diseases in swine, including african swine fever virus (asfv) and pseudorabies virus (prv), was also considered. nucleic acid extraction involved either kit-based ...19968953524
the pathogenic role of pulmonary intravascular macrophages in acute african swine fever.recent studies of pulmonary intravascular macrophages have led to the re-examination of the mechanisms giving rise to alveolar oedema. a highly virulent isolate of african swine fever virus was replicated in pulmonary intravascular macrophages, interstitial and alveolar macrophages, fibroblasts and neutrophils. the alveolar oedema-characteristic of acute forms of african swine fever-and the vascular changes observed, which consisted of the formation of fibrin microthrombi in septal capillaries a ...19968938846
expression and characterization of part of hog cholera virus non-structural proteins.in a preceding paper, the molecular cloning and partial nucleotide sequence of the alfort strain of hog cholera virus (hcv) was described. to study the genetic organization of the 3'-end of the hcv genome, which encodes some of the non-structural proteins, a cdna fragment (s2.20) of 849 nucleotides was subcloned into the bacterial expression vector pgex-3x and expressed in escherichia coli as a s2.20-glutathione-s-transferase fusion protein (s2.20-gst). this protein was used to produce hcv-speci ...19968928577
classical swine fever virus (csfv) envelope glycoprotein e2 containing one structural antigenic unit protects pigs from lethal csfv challenge.envelope glycoprotein e2, formerly called e1 or gp51-54, of classical swine fever virus (csfv) expressed in insect cells protects swine from classical swine fever. monoclonal antibodies directed against epitopes of domains b and c and subdomain a1 are neutralizing. the domains are located on two structural antigenic units in a proposed model of the antigenic structure of e2. one unit consists of nonconserved antigenic domains b and c and the other contains highly conserved antigenic domain a. we ...19968922467
african swine fever virus gene a179l, a viral homologue of bcl-2, protects cells from programmed cell death.the african swine fever virus (asfv) open reading frame a179l, which is similar to the human proto-oncogene bcl-2, has been cloned and expressed in vaccinia virus under control of the peil synthetic early/late promoter. the a179l gene product prevented cell death in hela and bsc-40 cells doubly infected with another recombinant vaccinia virus expressing the interferon-induced double-stranded rna-activated protein kinase (p68 kinase), which activates a rapid cell death characteristic of apoptosis ...19968918551
epidemiological characteristics of an outbreak of classical swine fever in an area of high pig density.the objective of this study was to analyse an outbreak of classical swine fever under a policy of non-vaccination, intensive surveillance and eradication in an area of high pig density. the virus was found in 52 herds, where some 90,000 pigs were slaughtered. the clinical signs were vague and the reports of suspect herds generally coincided with increased mortality. the interval between the first occurrence of clinical signs and the report of a suspect herd was shorter when the disease was first ...19968903020
strong sequence conservation of african swine fever virus p72 protein provides the molecular basis for its antigenic stability.the major capsid protein p72 of african swine fever virus (asfv) has long been considered an important immunodominant antigen for serologic diagnosis. here we describe the cloning and sequence analysis of two p72-coding genes from asfv strains uganda (uga) and dominican republic-2 (dr2). sequence comparison of these genes, together with those from two other asfv strains (ba71v and e70), demonstrated that the p72 proteins are highly conserved (97.8% to 100% amino acid sequence identity) in strain ...19968893801
african swine fever: morphopathology of a viral haemorrhagic disease.twelve miniature pigs were infected experimentally with the moderately-virulent spanish isolate of african swine fever virus e75. the disease was reproduced acutely and subacutely, and six days after infection the animals showed severe clinical and pathological signs characterised by diffuse haemorrhages in lymphoid and non-lymphoid organs. an immunohistopathological study, using two different methods, and a monoclonal antibody (mab) (18bg3) were used to analyse the distribution of the virus pro ...19968888559
granulocyte-macrophage colony stimulating factor promotes prolonged survival and the support of virulent infection by african swine fever virus of macrophages generated from porcine bone marrow and blood.long-surviving cultures of non-adherent cells of the monocyte-macrophage lineage were established from the bone marrow and blood of weanling pigs by culturing cells from these tissues in the presence of recombinant porcine granulocyte-macrophage colony stimulating factor (gm-csf). the cells increased in number, principally during the first 4 weeks of culture, bound monoclonal antibodies recognizing porcine macrophage antigens and avidly phagocytosed latex particles. the gm-csf generated mononucl ...19968887499
phylogenetic analysis of pestivirus based on the 5'-untranslated region.a phylogenetic tree of pestiviruses constructed by analyzing their 5'-untranslated region (utr) indicated that the genetic relatedness between border disease virus and hog cholera virus is much closer than that between genotypes of various bovine diarrhoea viruses. this suggests that these viruses are host variants within a single species, which can be distinguished by comparison of secondary structures at three variable loci in the 5'-utr.19968886099
a common structural core in the internal ribosome entry sites of picornavirus, hepatitis c virus, and pestivirus.cap-independent translations of viral rnas of enteroviruses and rhinoviruses, cardioviruses and aphthoviruses, hepatitis a and c viruses (hav and hcv), and pestivirus are initiated by the direct binding of 40s ribosomal subunits to a cis-acting genetic element termed the internal ribosome entry site (ires) or ribosome landing pad (rlp) in the 5' noncoding region (5'ncr). rna higher ordered structure models for these ires elements were derived by a combined approach using thermodynamic rna foldin ...19968879130
characterisation of transcription factors in african swine fever virus. 19968878959
expression of ubiquitin, actin, and actin-like genes in african swine fever virus infected cells.northern blot hybridisation was used to study the accumulation of specific cellular mrnas (ubiquitin and actin) in vero cells infected with african swine fever virus (asfv). asfv modulates the cytoplasmic levels of ubiquitin and actin mrnas throughout infection. before viral dna replication, degradation of ubiquitin mrnas is dependent on de novo protein synthesis, since treatment with cycloheximide (ch) allowed the accumulation of ubiquitin mrnas, while treatment with cytosine arabinoside (arac) ...19968873409
genetic variability of classical swine fever virus.the genetic variability of classical swine fever virus was studied by comparative nucleotide sequence analysis of 76 virus isolates, collected during a half century from three continents. parts of the e2 (gp55) and the polymerase gene coding regions of the viral genome were amplified by rt-pcr and dna fragments of 254 and 207 bp, respectively, were sequenced. the comparative sequence analysis of the e2 region revealed two main phylogenetic groups of csfv, indicating that the virus apparently evo ...19968864203
assembly of african swine fever virus: quantitative ultrastructural analysis in vitro and in vivo.african swine fever virus is an icosahedral double-standed dna virus which replicates in the cytoplasm of porcine monocytic cells. progeny virus particles, like poxviruses and iridoviruses, are assembled in cytoplasmic inclusions, termed virus factories. the formation of these structures in susceptible cells infected in vitro with pathogenic and attenuated asfv isolates has been studied by semiquantitative and qualitative electron microscopy. recognizable virus factory elements were detected by ...19968862402
classical swine fever virus-specific cytotoxic t lymphocytes and identification of a t cell epitope.classical swine fever virus (csfv)-specific cytotoxic t lymphocytes (ctl) were derived from peripheral blood mononuclear leukocytes of immunized nih-minipigs (mhc d/d haplotype) after in vitro restimulation with infectious csfv. their cytotoxic activity was determined against csfv-infected target cells obtained from simian virus 40 (sv40) large t antigen-transfected immortalized kidney cells of a syngeneic miniature swine. experiments with separated effector cell populations revealed that the cs ...19958847509
production of monoclonal antibodies against classical swine fever virus and their use for antigenic characterization of japanese isolates.twenty monoclonal antibodies (mabs) were produced against the virulent ald or vaccinal gpe- strains of classical swine fever (csf) virus. the mabs were divided into 7 groups on the basis of the difference in the reaction spectrum to various csf and bovine viral diarrhea-mucosal disease (bvd-md) viruses. the panel of the mabs could classify 20 csf viruses into 6 types, and could discriminate between not only csf and bvd-md viruses but also the vaccinal gpe- strain and the field isolates of csf vi ...19968844614
evolution of viral dna-dependent rna polymerases.the dna-dependent rna polymerase (ddrp or rnap) is an essential enzyme of transcription of replicating systems of prokaryotic and eukaryotic organisms as well as cytoplasmic dna viruses. ddrps are complex multisubunit enzymes consisting of 8-14 subunits, including two large subunits and several smaller polypeptides (small subunits). an extensive search between the amino acid sequences of the known largest subunit of dna-dependent rna polymerases (rpo1) of different organisms indicates that all t ...19958828152
detection of antibodies against classical swine fever virus in swine sera by indirect elisa using recombinant envelope glycoprotein e2.recombinant envelope protein e2 (gp55) of classical swine fever virus (csfv) strain alfort/187 was evaluated as an alternative to whole virus as elisa antigen for the detection of antibodies against csfv. a glycosylated and a non-glycosylated form of e2 was expressed in the baculovirus system. six histidine residues added at the carboxy terminus of each of the recombinant proteins allowed purification by nickel-chelate affinity chromatography. comparison of the antigenic properties of the two pr ...19968828121
implementation of internal laboratory quality control procedures for the monitoring of elisa performance at a regional veterinary laboratory.quality control (qc) procedures for antigen detection enzyme-linked immunosorbent assays (elisas) for hog cholera (hc) virus, foot and mouth disease (fmd) virus, and an antibody detection elisa for fmd virus were established at a regional veterinary laboratory in northern thailand. a recently developed computer software package, qcel, was used to facilitate management and analysis of qc data. the program was used to assess test performance by producing shewhart-cusum control charts which monitor ...19968828117
comparison of the protective efficacy of recombinant pseudorabies viruses against pseudorabies and classical swine fever in pigs; influence of different promoters on gene expression and on protection.the glycoprotein e (ge) locus in the genome of pseudorabies virus (prv) was used as an insertion site for the expression of glycoprotein e1 of classical swine fever virus (csfv). transcription of e1 in the recombinants m401, m402 or m403 was regulated by the gd promoter of prv, the immediate early gene promoter of human cytomegalovirus, or the ge promoter of prv, respectively. groups of four pigs were vaccinated once intramuscularly with 10(6) plaque forming units (p.f.u.) of the recombinant vir ...19968821642
immunohistopathological study of african swine fever (strain e-75)-infected bone marrow.a study was made of the action of african swine fever virus (asfv) on the bone marrow of 12 miniature pigs inoculated intramuscularly with the moderately virulent asfv isolate e75 and killed 2 to 12 days after infection. a sequential description is provided of the histological lesions of the bone marrow in the experimental animals, which developed haemorrhagic lesions from 6 days after inoculation onwards. immunohistochemical techniques were used to demonstrate the viral protein vp73 and immunog ...19968814534
apoptosis: a mechanism of cell killing and lymphoid organ impairment during acute african swine fever virus infection.induction of programmed cell death has been described during infection with many different viruses. we have investigated the influence of african swine fever virus (asfv) on apoptosis of different cell populations during in vitro and in vivo infection. we observed apoptosis in asfv-infected monocyte/macrophage and peripheral blood mononuclear cell cultures. apoptosis was demonstrated in these cells by dna fragmentation, dna staining and dna-associated histone fraction detection assays. flow cyto ...19968811021
fine mapping of a c-terminal linear epitope highly conserved among the major envelope glycoprotein e2 (gp51 to gp54) of different pestiviruses.envelope glycoprotein e2 (gp51 to gp54) is the major neutralizing antigen of pestiviruses, which include classical swine fever virus (csfv), bovine viral diarrhoea virus (bvdv), and border disease virus (bvd). previous studies carried out using a panel of monoclonal antibodies raised against csfv strain brescia have revealed the existence of four antigenic domains, a to d, of the e2 protein, all of which are located at the n-terminal half of the molecule. here we report the detailed mapping, usi ...19968806512
expression and characterization of an rna capping enzyme encoded by chlorella virus pbcv-1.we report that the a103r protein of chlorella virus pbcv-1 is an mrna capping enzyme that catalyzes the transfer of gmp from gtp to the 5' diphosphate end of rna. this is a two-step reaction in which the enzyme first condenses with gtp to form a covalent enzyme-gmp intermediate and then transfers the gmp to an rna acceptor to form a gpppn cap. purified recombinant al03r is a 38-kda monomer that lacks rna (guanine-7-) methyltransferase activity. with respect to its size, amino acid sequence, and ...19968794301
virus association with lymphocytes in acute african swine fever.this paper reports the presence of mature viral particles within the lymphocytes of samples taken from pigs inoculated with a highly virulent african swine fever (asf) virus isolate (malawi 83), and the adhesion of the lymphocytes to macrophages containing the virus replication sites. virus replication in lymph-node medullar tissue macrophages was observed from 3 days post inoculation (pi). at 3 days pi, transmission electron microscopy revealed hemadsorption in some infected macrophages. at 5 a ...19968767892
neutralizing antibodies to different proteins of african swine fever virus inhibit both virus attachment and internalization.african swine fever virus induces in convalescent pigs antibodies that neutralized the virus before and after binding to susceptible cells, inhibiting both virus attachment and internalization. a further analysis of the neutralization mechanisms mediated by the different viral proteins showed that antibodies to proteins p72 and p54 are involved in the inhibition of a first step of the replication cycle related to virus attachment, while antibodies to protein p30 are implicated in the inhibition ...19968764090
viral antigen and b and t lymphocytes in lymphoid tissues of gnotobiotic piglets infected with hog cholera virus.four 4-day-old gnotobiotic piglets infected intranasally with the kanagawa/74 strain of hog cholera virus (hcv) did not develop severe illness over a period of 3 weeks. large amounts of hcv were isolated from the lymphoid tissues and serum at necropsy. after the acute phase, hyperplasia of histiocytes and plasmacytopoiesis were observed in two pigs (killed 14 and 21 days after inoculation). the number of cd4+ and cd8+ t lymphocytes increased significantly and their location was consistent with t ...19968762583
cytocidal infection of hog cholera virus in porcine bone marrow stroma cell cultures.porcine bone marrow stroma cell (bmsc) cultures producing cells of granulocyte-lineage were established. hog cholera (hc) virus ald and alfort strains replicated in the porcine bmsc cultures showing distinct cytopathic effect (cpe). the differentiation of granulocyte-lineage cells in the cultures ceased after infection with hc virus. polyclonal antibody against the ald strain inhibited completely the development of cpe of the both ald and alfort strains. monoclonal antibodies (mabs) specific to ...19958748554
six antigenic groups within the genus pestivirus as identified by cross neutralization assays.antigenic differences between pestivirus isolates of ruminant and porcine origin were characterized by neutralization assays. first, six different clusters of pestiviruses were identified by clustering cross-neutralization results of 13 pestivirus strains tested against 23 sera. cluster i consisted of four strains of bovine viral diarrhoea virus (bvdv) of bovine origin and two bvdv isolates of porcine origin. cluster ii consisted of one sheep isolate and two porcine bdv isolates. cluster iii con ...19958748547
molecular cloning and nucleotide sequence determination of three envelope genes of classical swine fever virus taiwan isolate p97.a strain of classical swine fever virus (csfv) has been isolated in taiwan. the cdna coding for three envelope glycoproteins e1, e2 and e3 were molecularly cloned from purified viral particles using the reverse transcription-polymerase chain reaction (rt-pcr) method and sequence-specific primers. the resulting pcr products (1113 bp for e1. 699 bp for e2 and 567 bp for e3) were cloned into the smai site of puc19 and then subjected to dna sequence analysis. data showed that nucleotide sequence of ...19968738176
the structural protein p54 is essential for african swine fever virus viability.protein p54, one of the most antigenic structural african swine fever virus (asfv) proteins, has been localized by immuno-electron microscopy in the replication factories of infected cells, mainly associated with membranes and immature virus particles. attempts to inactivate the p54 gene from asfv by targeted insertion of beta-galactosidase selection marker was uniformly unsuccessful, suggesting that this gene is essential for virus viability. to demonstrate that, we inserted in the tk (thymidin ...19968725112
[pcr: a look behind the scenes at bovine viral diarrhea virus].as indicated by their nucleotide sequences bovine viral diarrhea viruses (bvd-virus) are genetically quite heterogenous. despite differences in the nucleotide sequence in the ns3 region, we found that the derived amino acid sequence of all viral strains analyzed was identical and differed from that of classical swine fever viruses, thus allowing differentiation between bovine and porcine pestiviruses. remarkably, the bvd viral strains were isolated over a time period of some 40 years in europe, ...19968720729
experimental quantification of transmission of genetically engineered pseudorabies virus.there is concern that live pseudorabies virus (prv) vaccine or prv vector vaccine strains may spread from vaccinated to unvaccinated pigs. moreover, it is feared that recombining prv vaccine strains with related vaccine or wild-type strains may lead to spread and survival of recombinant prv. to learn more about to what extent different prv vaccine strains could spread we used a previously described experimental model to study the transmission of intranasally inoculated prv mutant strains under e ...19958701591
classical swine fever virus diversity and evolution.by analysing the nucleotide sequence data generated from both the e2 (gp55) and the ns5b genes of classical swine fever virus (csfv), in addition to previously published data from the 5'ncr, we were able to divide 115 csfv isolates into two major groups, five subgroups and two disparate isolates. further discrimination was possible by analysis of sequence data from the e2 region. the three sequencing based methods were compared to monoclonal antibody (mab) typing and to limited restriction enzym ...19968683221
swine and ruminant pestiviruses require the same cellular factor to enter bovine cells.pestiviruses initiate infection of susceptible cells by receptor-mediated endocytosis. cellular plasma membrane or endosomal molecules involved in translocation of these viruses into the cytosol have not been unequivocally identified. we reported previously that a mutant cell line derived from madin-darby bovine kidney (mdbk) cells, termed crib-1, was resistant to infection with bovine viral diarrhoea virus. crib-1 cells were also resistant to infection with classical swine fever virus and borde ...19968683219
an african swine fever virus bc1-2 homolog, 5-hl, suppresses apoptotic cell death.here, we show that the african swine fever virus 5-hl gene is a highly conserved viral gene and contains all known protein domains associated with bcl-2 activity, including those involved with dimerization, mediating cell death, and protein-binding functions, and that its protein product, p21, suppresses apoptotic cell death in the mammalian lymphoid cell line fl5.12. thus, 5-hl is a true functional viral member of the bcl-2 gene family.19968676523
amino acid tandem repeats within a late viral gene define the central variable region of african swine fever virus.the central variable region (cvr) of the african swine fever virus (asfv) genome is contained within the 9-rl open reading frame (orf). orf 9-rl of the asfv isolate malawi lil-20/1 predicts a protein of 614 amino acids with amino- and carboxy-terminal hydrophobic regions and a centrally located hydrophilic region. the cvr of the genome, located centrally within this orf, is 372 bp and contains a 132-bp direct repeat. the translated cvr within orf 9-rl contains 31 tandem tetramers, predominantly ...19968659112
processing in the pestivirus e2-ns2 region: identification of proteins p7 and e2p7.the pestivirus genome encodes a single polyprotein which is subject to co- and posttranslational processing by cellular and viral proteases. the map positions of all virus-encoded proteins are known with the exception of a hypothetical peptide (p?) which interlinks the glycoprotein e2 and the nonstructural protein ns2-3 approximately between amino acid positions 1060 and 1130. expression studies with recombinant vaccinia viruses bearing a set of c-terminally truncated e2-p?-ns2-encoding sequence ...19968648755
nucleotide sequence of classical swine fever virus strain alfort/187 and transcription of infectious rna from stably cloned full-length cdna.the complete nucleotide sequence of the genome of classical swine fever virus (csfv) strain alfort/187 was determined from three cdna libraries constructed by cloning of dna fragments obtained from independent sets of reverse transcription and pcr. the cdna fragments were then assembled and inserted downstream of a t7 promoter in a p15a-derived plasmid vector to obtain the full-length cdna clone pa187-1. the first nucleotide of the csfv genome was positioned at the transcription start site of th ...19968648680
comparative sequence analysis of the 5' noncoding region of classical swine fever virus strains from europe, asia, and america.polymerase chain reaction was utilized to determine the sequence of a 280 base pair fragment from cdnas of the 5' noncoding region of 29 isolates of classical swine fever virus. phylogenetic analysis of the sequences revealed low level genomic variation that correlated with the geographic origins of the isolates.19968645113
a structural dna binding protein of african swine fever virus with similarity to bacterial histone-like proteins.here we describe an african swine fever virus (asfv) protein encoded by the open reading frame 5-ar that shares structural and functional similarities with the family of bacterial histone-like proteins which include histone-like dna binding proteins, integration host factor, and bacillus phage spo1 transcription factor, tf1. the asfv 5-ar gene was cloned by pcr and expressed in e. coli. monospecific antiserum prepared to the 5-ar bacterial expression product specifically immunoprecipitated a pro ...19968634022
role of monocytes in the up-regulation of the early activation marker cd69 on b and t murine lymphocytes induced by microbial mitogens.cd69 is an early marker of lymphoid cell activation. the authors report on an up-regulation of cd69 in splenic b and t cells of c57bl/6 mice after administration of lipopolysaccharide (lps) or microbial immunosuppressive/mitogenic (ism) proteins produced by c. albicans (p43) and african swine fever virus (p36). this up-regulation of cd69 was observed 6 and 24 h after mitogenic treatments. the same pattern of increased cd69 expression was observed in the lymph nodes of mice treated with p43 or lp ...19968633195
classical swine fever virus: recovery of infectious viruses from cdna constructs and generation of recombinant cytopathogenic defective interfering particles.the 5'- and 3'-terminal sequences of the genomic rna from classical swine fever virus (csfv) were determined, and the resulting information was used for construction of full-length csfv cdna clones. after transfection of in vitro-transcribed rna derived from a cdna construct, infectious csfv was recovered from porcine cells. to confirm the de novo generation of infectious csfv from cloned dna, a genetically tagged csfv was constructed. in comparison with parental csfv, the recombinant viruses we ...19968627678
evolutionary relationships among large double-stranded dna viruses that infect microalgae and other organisms as inferred from dna polymerase genes.in order to examine genetic relatedness among viruses that infect microalgae, dna polymerase gene (dna pol) fragments were amplified and sequenced from 13 virus clones that infect three genera of distantly related microalgae (chlorella strains nc64a and pbi, micromonas pusilla and chrysochromulina spp.). phylogenetic trees based on dna pol sequences and hybridization of total genomic dna showed similar branching patterns. genetic relatedness calculated from the hybridization and sequence data sh ...19968623526
in vivo replication of african swine fever virus (malawi '83) in neutrophils.the presence of virus replication centers in neutrophils from pigs inoculated with a highly virulent strain of african swine fever virus is described for the first time in vivo. virus antigens were observed from 3 days post-inoculation (dpi) onwards by means of an immunohistochemical technique. at this time (3 dpi), transmission electron microscopy studies revealed the presence of large amounts of neutrophils in the vascular lumens. at 5 and 7 dpi, neutrophils with phagosomes frequently containe ...19968620189
functional and immunological properties of the baculovirus-expressed hemagglutinin of african swine fever virus.a recombinant baculovirus harboring the hemagglutinin (ha) gene of african swine fever virus, with homology to the t-lymphocyte surface antigen cd2, was constructed. the efficient expression of the ha gene was determined by immunofluorescence and western blot studies on insect cells infected with the recombinant baculovirus. the baculovirus-expressed ha showed hemadsorption and erythrocyte-agglutinating activities characteristic of the cd2 homolog protein induced by the virus in infected macroph ...19968615037
characterization of the african swine fever virion protein j18l.the african swine fever virus (asfv) open reading frame (orf) that is named jl8l in the malawi (lil20/1) isolate and e199l in the ba71v isolate encodes a cysteine rich protein of 195 amino acids with a predicted molecular mass of 21.7 kda and a hydrophobic domain near the c terminus. there are several possible motifs for glycosylation, phosphorylation and myristoylation. rabbit antisera and monoclonal antibodies raised against a recombinant asfv j18l protein expressed as a fusion protein with gl ...19968609490
application of a monoclonal antibody recognizing protein p30 to detect african swine fever virus-infected cells in peripheral blood.monoclonal antibody (mab) 174f11.8 recognizes an epitope of the african swine fever (asf) virus-induced protein, p30, a protein expressed on the plasma membrane of infected cells. this mab has been used to analyze infected cell populations in peripheral blood of experimentally inoculated pigs with a virulent or attenuated asf virus. flow cytometric analysis of peripheral blood at different days postinfection using this mab, showed expression of p30 mainly in the monocyte/macrophage cell lineage. ...19958609199
differentiation of classical swine fever virus (csfv) strains using monoclonal antibodies against structural glycoproteins.two panels of monoclonal antibodies (mabs) against the classical swine fever virus (csfv) envelope glycoproteins e2 (12 mabs) and e0 (11 mabs) were established and tested by immunoperoxidase binding assay against 135 pestivirus strains and isolates. variability of the binding pattern was demonstrated for csfv and also for bovine viral diarrhea virus (bvdv) strains and isolates. the panels of mabs against e2 and e0 led to very different reactivity patterns. particular mabs against e2 reacted with ...19958604543
fusion activity of african swine fever virus towards target membranes: lipid dependence and effect of dehydrating agents.fusion of african swine fever virus (asfv) with model membranes was monitored by a fluorescence dequenching assay. asfv was able to fuse with liposomes of various compositions. fusion was more extensive with liposomes made of negatively charged phospholipids, and reduced in the presence of the neutral phospholipid phosphatidylcholine (pc). fusion activity of asfv was ph-dependent, the extent of fusion increasing with decreasing ph for all target membranes. these results are consistent with the i ...19958595388
reverse transcription combined with polymerase chain reaction as a detection method for pestiviral infections.an assay based on reverse transcription coupled with the polymerase chain reaction (rt-pcr) was used for the detection of hog cholera virus (hcv) and bovine virus diarrhoea virus (bvdv) in cell culture. in this study, a precipitate of the supernatants derived from cell cultures infected with hcv and bvdv was used in rt reactions, in place of extracted viral rna. both rt and pcr were performed using recombinant thermus thermophilus (rtth) dna polymerase. the specificity of the rt-pcr products was ...19958593411
evaluation of the enzyme-linked immunosorbent assay for the rapid screening and detection of classical swine fever virus antigens in the blood of pigs.a workshop was convened, at which seven enzyme-linked immunosorbent assays (elisas) were compared with virus isolation for the detection of viraemia in serial blood samples collected from six pigs at up to fourteen days after inoculation with classical swine fever virus. all elisas were of the double antibody sandwich type, using monoclonal and/or polyclonal antibodies to detect a variety of viral proteins in leukocytes, or in anti-coagulated blood or serum. compared to virus isolation, specific ...19958593401
serological study of pigs for antibody against african swine fever virus in two areas of southern malawi.a serological survey was conducted in july 1991 on domestic pigs in two areas of southern malawi which were severely affected by the african swine fever (asf) epizootic in 1989-1991. sixty-six of the 216 owners questioned reported having witnessed asf in their pigs. forty-seven owners had pigs with antibodies against asf virus, and the overall prevalence of pigs with anti-asf virus antibodies was found to be 12.4%, in 445 pigs sampled in 35 villages. spread of asf was thought to occur principall ...19958593400
a serological study on infection patterns, control and persistence of classical swine fever in infected farms in the philippines.in 3 farrow-to-finish farms with enzootic classical swine fever (csf) all sick pigs were culled and an intensive vaccination program was introduced. boars and cows were vaccinated every 6 months and piglets at the age of 6 and 8 weeks. the infection was monitored on clinical grounds and by means of serological examination of the farms. serological results reflected well the clinical situation in the farms. high titers and high percentage of animals with positive titers were observed in infected ...19958593302
classical swine fever in wild boar (sus scrofa)--experimental infections and viral persistence.a pregnant wild boar and two wild boar weaners were inoculated intranasally with a field isolate of classical swine fever virus (csfv) recently derived from a diseased domestic pig. the clinical, pathological and haematological findings noted in the young wild boars were comparable to those in domestic weaner pigs inoculated with the same virus isolate. both wild boars showed the acute haemorrhagic form of csf, one animal died 18 days post inoculation (p. i.) and the second one had to be euthani ...19958591736
molecular evolution and secondary structural conservation in the b-cell lymphoma leukemia 2 (bcl-2) family of proto-oncogene products.the nature of the bcl-2 family of proto-oncogenes was analyzed by sequence alignment, secondary structure prediction, and phylogenetic techniques. phylogenies were inferred from both the nucleic acid and amino acid sequences of the human, murine, rat, and chicken sequences for bcl-2 and bcl-x, human mcl1, murine a1, the nematode caenorhabditis elegans and caenorhabditis briggsiae ced-9 proteins, and the sequences bhrf1 from epstein-barr and lmw5-hl from african swine fever viruses. both sequence ...19958587122
[diagnosis of contagious diseases in animals using pcr].the pcr is used for diagnostic purposes as it allows to detect infections agents within a much shorter time than by cultural isolation. in addition, it can detect non-infectious viruses and bacteria in clinical samples. these advantages are important factors in the diagnosis of highly contagious animal diseases (mainly caused by viruses) since a rapid laboratory diagnosis will allow to take immediate disease control actions. pcr is routinely used at the institute of african and classical swine f ...19958584867
isolation of bovine viral diarrhea virus-like pestiviruses from roe deer (capreolus capreolus).cytopathogenic pestiviruses were isolated from two seronegative free-ranging roe deer (capreolus capreolus) from northern germany (schleswig-holstein): an adult female and a young buck collected on 6 december 1990 and 26 july 1991, respectively. the two isolates were identified by polymerase chain reaction as pestiviruses. however, they were negative when primers specific for bovine virus diarrhea virus or classical swine fever virus were used, indicating that the two isolates might belong to a ...19958583645
characterization of the hog cholera virus 5' terminus.hog cholera virus (hocv) 5' terminus of the ald and gpe(-) strains were analyzed by using rapid amplification of cdna end method (5'race). an additional nine nucleotides were found at the 5' termini of genomic rna in the ald and gpe(-) strains of hocv. these nine nucleotides were also conserved in bvdv and were suggested to form a hairpin structure at the 5' terminus by computer-assisted analysis. it seems possible that the secondary structure and/or the 5' terminus sequence has a significant ro ...19958560779
baculovirus expression and affinity purification of protein e2 of classical swine fever virus strain alfort/187.the genome region encoding the major envelope glycoprotein e2 (gp55) of the classical swine fever virus (csfv) strain alfort/187 was cloned and sequenced. the e2 gene, either with or without additional authentic 5'-terminal sequences coding for two variants of a putative signal sequence, was used to construct recombinant baculoviruses expressing the respective glycosylated and nonglycosylated e2 protein in insect cells. the signal sequences mediated glycosylation in insect cells, but no efficien ...19958560771
the african swine fever virus iap homolog is a late structural polypeptide.the analysis of the complete nucleotide sequence of the african swine fever virus genome has revealed the existence of a number of genes potentially capable of modifying the host's response to the virus infection. in this report, we describe the results of the characterization of the a224l gene that encodes a novel member of the family of apoptosis inhibitors known as iap proteins. a224l is expressed during the late phase of the infectious cycle, and its polypeptide product is assembled into vir ...19958553574
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