pulmonary lesions in lambs experimentally infected with ovine adenovirus 5 strain rts-42.twelve lambs were inoculated transtracheally and intranasally with mastadenovirus ovi 5 strain rts-42 and killed sequentially. pulmonary lesions were studied by light and electron microscopy. four lambs served as sham inoculated controls. pulmonary lesions consisted of multifocal areas of bronchiolitis and alveolitis associated with necrosis and sloughing of isolated type i and type ii alveolar epithelial cells and nonciliated bronchiolar epithelial cells. this was followed rapidly by hyperplasi ...19863022454
new human adenovirus associated with respiratory illness: candidate adenovirus type 39.a new human adenovirus was isolated from a stool specimen from a 2-year-old el salvadorian male hospitalized with severe respiratory illness. the virus, strain d335, is a typical adenovirus by routine classification tests and bears little resemblance to the other 38 adenovirus serotypes by standard hemagglutination inhibition and serum neutralization tests. the only significant cross-reaction observed was between adenovirus types 39 and 13, bilaterally, by the hemagglutination inhibition test. t ...19826179962
sequence analysis of bovine adenovirus type 2 early region 1 and pix gene.bovine adenoviruses (bavs) cause both respiratory and/or enteral diseases in cattle and are widespread throughout the world. we have sequenced the extreme left end (0 to 12.2 mu) of the bav2 genome. partial analysis of the nucleotide sequence revealed 12 potential open reading frames (orfs) which could encode for polypeptides of 50 or more amino acids. these orfs were compared to those of the early region 1 (e1) proteins and hexon-associated protein ix (pix) from other adenoviruses (ads). the 4 ...19947698885
candidate adenoviruses 40 and 41: fastidious adenoviruses from human infant stool.about 200 antigenically related adenoviruses were isolated from cases of infantile diarrhoea in the netherlands and north-west germany. the viruses were fastidious and failed to replicate serially in human diploid fibroblasts and in primary human embryonic kidney cells. a number of strains were established in hela, hep-2, graham (293), cynomolgus monkey kidney, and chang conjunctival cells. the viruses were mammalian adenoviruses by the usual criteria. no relationship to the 39 known human adeno ...19836306161
sequence of ovine adenovirus homologs for 100k hexon assembly, 33k, pviii, and fiber genes: early region e3 is not in the expected location.ovine adenovirus oav287 was previously isolated from sheep in western australia. as a first step in characterizing the genome of this virus we have determined the sequence of its genome between map units 65 and 81. this region was expected to contain the nonessential e3 region which, in other adenoviruses, lies between the genes encoding the pviii and fiber proteins, although its size and complexity varies. oav287 genes coding for the hexon assembly, 33k, pviii, and fiber proteins were identifie ...19957778275
a p53 mutation in exon 5 associated with adenovirus transformation.the adenovirus type 5 (ad5) 55-kda e1b oncoprotein has been shown to form complexes with the p53 tumor suppressor protein. these complexes are thought to interfere with normal p53 activity and may be responsible for the paucity of p53 mutations in cells transformed by these viruses. this report describes an example of a p53 mutation in exon 5 in an ad5-transformed cell line that exhibited less expression of e1b 55-kda protein and a longer tumor-latency phenotype than another ad5-transformed cell ...19957818760
induction of transformation progression in type 5 adenovirus-transformed rat embryo cells by a cloned protein kinase c beta 1 gene and reversal of progression by 5-azacytidine.protein kinase c (pkc) is a key component in signal transduction in eucaryotic cells and when specific pkc isoforms are over-expressed in immortal mammalian cells they can induce transformation-associated properties. in the present study we demonstrate that a cloned pkc beta 1 gene can induce an enhanced expression of the transformed phenotype in type 5 adenovirus (ad5)-transformed rat embryo (re) cells (clone e11), a process termed transformation progression. e11 cells expressing the pkc beta 1 ...19947510863
adenovirus endopeptidases. 19947845235
sequence and structural analysis of murine adenovirus type 1 hexon.the genomic region encoding the major capsid protein (hexon) of murine adenovirus type 1 (mav-1) has been isolated and sequenced. the sequence predicts a 908 residue mav-1 hexon protein and is flanked by a portion of the upstream pvi gene and the downstream endoproteinase gene. the order of these genes and their location in the middle of the genome are the same as those found in other adenoviruses sequenced to date. multiple sequence alignment with the other five known hexon protein sequences re ...19947509368
adenovirus 38: a new human adenovirus species of subgenus d.from the stool of a dutch girl, a new virus (lj) was isolated that by morphological, biological, and immunological criteria was a human adenovirus. because of its hemagglutinating activity for rat and human red blood cells and its dna restriction enzyme pattern, the virus was classified as a member of subgenus d. it was distinct in neutralization tests from all other human adenoviruses. the hemagglutinin was antigenically similar to that of adenoviruses 13 and 39, but the dna differed from that ...19846094388
identification of ovine adenovirus types five and six in an epizootic of respiratory tract disease in recently weaned lambs.eight hundred fourteen polypay-cross lambs, 32 to 35 days old, were housed in an isolation barn under semiconfinement conditions. the lambs were observed twice daily for clinical signs of respiratory tract disease. during the 8-week observation period, an epizootic of respiratory tract disease developed, in which the combined morbidity and mortality was approximately 13%. the overall mortality was 4.1%, of which 57.6% was attributable to pneumonia. pasteurella haemolytica was isolated from 11 (5 ...19873038805
sequence analysis of the putative e3 region of bovine adenovirus type 2.adenoviruses are nonenveloped icosahedral-shaped particles. the double-stranded viral dna genome contains four major early transcription units, designated e1 (a and b), e2 (a and b), e3 and e4, which are expressed in a regulated manner soon after infection. the gene products of the region e3, shown to be nonessential for viral replication in vitro, are believed to be involved in counteracting host immunosurveillance. human adenovirus type 5 dna sequences of transcription units l4 and l5 adjacent ...19947698883
restriction endonuclease analysis and physical mapping of the genome of porcine adenovirus type 5.the hnf61 and hnf70 isolates of porcine adenovirus type 5 (pav-5) were cultivated in pk-15 cells, and viral dna was extracted from the infected cells by a modified hirt procedure. the dnas were digested by each of 9 restriction endonucleases, and fragments representing the entire genomes were cloned. based on the sizes of the restriction enzyme fragments, the genome of each isolate was estimated to be 33.2 kb. physical maps for the 9 restriction endonucleases were constructed. the physical maps ...19957483821
isolation and partial characterization of a human adenovirus type 4 temperature-sensitive mutant (mastadenovirus h 4 tsl).a random selection procedure was used to isolate a temperature-sensitive (ts) mutant of human adenovirus type 4 (mastadenovirus h 4 tsl) from nitrous acid mutagenized virus stock. the mutant displayed restricted growth at the nonpermissive temperature of 39 degrees c. analysis of the mutant grown at 39 degrees c, by two-dimensional immunoelectrophoretic analysis, showed the mutant to be defective in the expression of the penton base and fibre structural components. the mutant was, however, capab ...19846713306
characteristics of mastadenovirus h 8, the causative agent of epidemic keratoconjunctivitis.adenovirus 8 (ad 8, strain 1127) has the general structural features of an adenovirus; it produces the genus-specific hexon antigen. the fiber length of 12 nm corresponds to that of other subgenus d adenoviruses. in hemagglutination-inhibition, ad 8 is related to ad9 and ad 15/h9. the slow multiplication, compared to most other subgenus d viruses, was shown not to be caused by an extended growth cycle, but appears to be due to an inefficient virus release into the medium. after restriction analy ...19836312928
susceptibility and signs associated with mouse adenovirus type 1 infection of adult outbred swiss swiss outbred mice from two sources had a nearly 6,000-fold difference in susceptibility to mouse adenovirus type 1-induced disease. this difference was not attributable to differential organ tropism. signs associated with mouse adenovirus type 1 infection that have not been previously reported are described at the clinical, gross pathological, and histological levels.19957494327
should the criteria for species distinction in adenoviruses be reconsidered? evidence from canine adenoviruses 1 and 2.four strains of canine adenovirus type 1 (cav-1) and two strains of canine adenovirus type 2 (cav-2) were examined by restriction enzyme analysis. in all cases, cav-1 could be readily differentiated from cav-2. additionally, monoclonal antibodies were prepared against the mirandola and manhattan strains of cav-1 and cav-2, respectively. 18 of 36 monoclonal antibodies from the cav-1 fusion and 77 of 160 monoclonal antibodies from the cav-2 fusion were type-specific by an indirect fluorescent-anti ...19853884549
seroepidemiologic survey for antibodies to selected viruses in the respiratory tract of lambs.a serologic study was conducted to determine the prevalence of antibodies to, and infection rate of, mastadenovirus ovi 5, m ovi 6, parainfluenza-3 (pi-3) virus, bovine herpesvirus-1 (bhv-1), respiratory syncytial virus (rsv), bovine viral diarrhea (bvd) virus, and ovine progressive pneumonia (opp) virus in lambs at a ram lamb growth-rate test station. for 2 consecutive years, serum samples were prepared from blood collected from 1- to 2-month-old ram lambs as they entered the test station (1st ...19853002217
adenoviridae.the elevation of the genus adenovirus to a family named adenoviridae is described. this family at the present stage contains two genera, mammalian adenoviruses--mastadenovirus--and avian adenoviruses--aviadenovirus. the two genera are separated primarily on the basis of difference in immunological properties of the virions.19761002415
spatial and temporal control of gene therapy using ionizing radiation.activation of transcription of the egr-1 gene by x-rays is regulated by the promoter region of this gene. we linked the radiation-inducible promoter region of the egr-1 gene to the gene encoding the radiosensitizing and tumoricidal cytokine, tumour necrosis factor-alpha (tnf-alpha) and used a replication-deficient adenovirus to deliver the egr-tnf construct to human tumours growing in nude mice. combined treatment with ad5.egr-tnf and 5,000 cgy (rad) resulted in increased intratumoral tnf-alpha ...19957585181
immunological relationship among human adenoviruses of subgenus d.antigenic relationships between adenoviruses of subgenus d were determined by neutralization tests in hela cell cultures by cpe inhibition. for cross-testing, several antisera of the same species were tested against the prototype viruses. 39 wild strains belonging to 12 different virus species were also studied. marked variation in the degree of cross-neutralization between individual sera of the same species was often observed. however, virus strains within a species mostly showed identical ser ...19826180704
disseminated adenovirus infection associated with cutaneous and visceral hemorrhages in a nursing unusual adenovirus infection in a conventionally raised nursing pig was characterized clinically by hemorrhagic diathesis of the skin and microscopically by disseminated viral inclusions in the endothelial, interstitial, and epithelial cells of the skin, kidney, spleen, liver, heart, and small and large intestines. this disease was confirmed ultrastructurally by demonstration of typical adenoviral particles in the nuclei of endothelial cells in the myocardium and of interstitial cells in the ...19957483222
restriction endonuclease analysis and molecular cloning of porcine adenovirus type 3.the 6618 strain of porcine adenovirus type 3 was cultivated in swine testis cells, and viral dna was extracted from the infected cells by a modified hirt procedure. the dna was digested by each of 13 restriction endonucleases, and the number of cleavage sites which were identified varied from 1 to 17. the size of the porcine adenovirus type 3 genome, estimated from the sizes of the restriction enzyme fragments, was approximately 35 kb. fragments representing the entire genome were cloned. physic ...19938150597
inoculation of lambs with ovine adenovirus 5 (mastadenovirus ovi 5) strain rts-42.twelve 1-week-old colostrum-deprived lambs inoculated with the rts-42 strain of mastadenovirus ovi 5 were killed and necropsied (2 lambs/day) on postinoculation days (pid) 2, 4, 6, 8, 12, and 21. four noninoculated lambs were killed and necropsied (2/day) on pid 6 and 12. virus was isolated from nasal secretions and feces on pid 1 to pid 6, from tracheal fluids and lung tissue of lambs necropsied on pid 2, 4, and 6, and from lung tissue from 1 lamb necropsied on pid 8. virus was not recovered fr ...19863008604
evasion of host immune responses by tumours and viruses.viruses and tumours use various mechanisms to avoid immune surveillance. oncogenic viruses have achieved a balance with the immune system through evolutionary time to ensure long-term persistence. mutations that promote escape mechanisms favouring tumour growth to the detriment of host survival through reproductive age offer no selective advantage and will not generally be maintained in the viral genome that persists in nature. conventional (non-oncogenic) and tumour viruses interact with variou ...19947796674
sequence analysis of putative pviii, e3 and fibre regions of porcine adenovirus type 3.sequence analysis of a region of the genome of porcine adenovirus type 3 from map unit 79.5 to map unit 92 was performed. homology studies revealed genes coding for the hexon-associated protein pviii on the left and for the fibre protein on the right of the sequenced region. by analogy with the genomic organization of other adenoviruses, the 1179 bp sequence between the pviii and fibre open reading frames, extending from map unit 81.3 to map unit 84.7, was identified as the equivalent of the e3 ...19957625129
comparison of the inverted terminal repetition sequences from five porcine adenovirus serotypes.the nucleotide sequences of the region of inverted terminal repetition from representative strains of all five porcine adenovirus (pav) serotypes were determined and analyzed. the first 17 nucleotides of this region were identical in pav-1 to 3 and pav-5, and 10 bp of identical sequence was found in all the pavs. the closest relationships were among pav-1 to 3, which shared more common sequences than the other serotypes. pav-4 had the longest inverted terminal repeat reported for any adenovirus. ...19957676637
[comparative study of the hexons in adenoviruses of the genus mastadenovirus]. 19862478865
preparation of serotype specific antiserum against porcine adenoviruses in mice.serotyping of porcine adenoviruses needs type-specific antiserum. the injections of purified virions to spf balb/c mice, followed by an intraperitoneal injection of ehrlich ascitic tumour cells, gave enough ascitic fluid containing practically applicable type-specific antibody.19968722316
intertype specific epitopes on the hexons of adenovirus types 1, 2, 5 and 6.the intertype specificities of the epitopes present on the surface of the hexon types belonging to human adenovirus (ad h) subgenus c were studied. the reactivity pattern (rp) and the titres of ascitic fluids containing monoclonal antibodies (mabs) were determined in elisa. from three panels of mabs 38 directed against ad h1, 17 directed against ad h 35 and 6 mabs directed against bovine adenovirus type 2 were studied. twelve of the mabs proved to be genus or type specific. the rest of the mabs ...19968806946
antigenic homogeneity among the adenovirus hexon types of subgenus c.antigenic relationships of hexons of human adenovirus (ad h) types 1, 2, 5 and 6 of subgenus c were studied with 61 monoclonal antibodies (mabs) raised against human ad h1, ad h35 and bovine adenovirus 2. the reactivity pattern (rp) and the titers of the mabs were determined in indirect elisa. in previous experiments with hexons of different subgenera 49 mabs displayed numerous different intertype specificities besides genus specific and type specific ones. with the four hexon types of subgenus ...19957646361
molecular cloning and physical mapping of porcine adenovirus types 1 and 2.the 25r strain of porcine adenovirus type 1 (pav-1) and the a47 strain of pav-2 were propagated in st cells, and dna was extracted from the infected cells by a modified hirt method. the dna of each virus was digested by each of nine restriction endonucleases, and restriction enzyme fragments representing the entire genome were cloned. the genomic size of each virus was approximately 33 kb. physical maps for the nine restriction endonucleases were constructed from the results of double digestion ...19957646345
isolation of porcine adenovirus as a candidate of 5th serotype.a strain of cytopathic virus, named strain tg/k79, was isolated from the brain of a newborn piglet, pure hampshire breed, which died shortly after birth. the physicochemical property of virus was considered to be that of the family adenoviridae. a significant difference between our isolated and 4 reference porcine adenoviruses was demonstrated by cross-seroneutralization test. differences between tg/k79 and other porcine adenoviruses were also seen in electrophoretic patterns of viral dna in aga ...19957608865
adenovirus e1a expression controlled by the red pigment promoter in transgenic mice: a model for developmental abnormalities of the eye.four transgenic founder mice were produced that carry the adenovirus e1a gene under control of the human red pigment promoter. two of the founder animals passed the transgene to progeny, and one line was bred to homozygosity. the line of mice homozygous for the transgene expressed e1a-specific rna at a low level in their eyes; no expression could be detected in other tissues that were tested. either the founder animals or their progeny exhibited developmental abnormalities of their eyes, includi ...19947848907
lmp-associated proteolytic activities and tap-dependent peptide transport for class 1 mhc molecules are suppressed in cell lines transformed by the highly oncogenic adenovirus 12.expression of class i major histocompatibility complex antigens on the surface of cells transformed by adenovirus 12 (ad12) is generally very low, and correlates with the in vivo oncogenicity of this virus. in primary embryonal fibroblasts (h-2b) that express transgenic swine class i antigen (pd1), ad12-mediated transformation results in inhibition in transport of newly synthesized class i molecules, as well as significant reduction in transporter associated with antigen presentation (tap) gene ...19968627162
the refined crystal structure of hexon, the major coat protein of adenovirus type 2, at 2.9 a resolution.the crystal structure of hexon, the major coat protein from adenovirus type 2, has been refined at 2.9 a resolution. hexon is a homo-trimer (molecular mass 3 x 109,077 da) and crystallizes in the cubic space group p2(1)3, with a cell edge of 150.5 a. there are four molecules in the unit cell so that the crystallographic asymmetric unit contains one subunit of the trimer. the electron density in most regions is well-defined and 880 amino acid residues, of the 967 in this unusually long polypeptid ...19947932702
modulation of e-cadherin localization in cells expressing wild-type e1a 12s or hypertransforming mutants.the adenovirus e1a 12s gene can immortalize primary epithelial cells such that they retain expression of epithelial cell characteristics. e1a 12s can also cooperate with an activated ras oncogene to cause tumorigenic transformation of primary cells. specific substitution and deletion mutants of e1a 12s cooperate more efficiently with ras to produce foci with a hypertransformed phenotype, wherein the cells are less differentiated and exhibit invasive properties. loss of epithelial differentiation ...19958547227
adenovirus protein-protein interactions: molecular parameters governing the binding of protein vi to hexon and the activation of the adenovirus 23k protease.a variety of recombinant proteins derived from protein pvi of human adenovirus type 2 (ad2) were analysed for their ability to bind ad2 hexon in vitro. as pvi is also required for activation of the adenovirus-coded protease, the same pvi derivatives were assessed for their ability to activate recombinant adenovirus-coded 23k protease. two regions, between amino acid residues 48-74 and 233-239 of pvi, were required for the interaction with hexon. these regions are highly conserved amongst mastade ...19957636476
sequence and sequence analysis of e1 and pix regions of the bav3 genome.bovine adenovirus type 3 (bav3) is a dna virus that causes respiratory and gastrointestinal disorders in cattle. we have sequenced the extreme left end of bav3 genome (0-11.7 map units). partial analysis of the nucleotide sequence revealed 19 potential open reading frames (orfs) that could encode for polypeptides of 50 or more amino acids. four of these orfs show homology to known adenovirus polypeptides. the four bav3 orfs are located in approximately the same area as the ad5 e1a, e1b, and pix ...19938150592
sequence of the mouse adenovirus serotype-1 dna encoding the precursor to capsid protein vi.the nucleotide sequence predicted to encode the precursor to virion structural protein vi (previ) of mouse adenovirus (ad) serotype-1 (mav-1) was determined. the 237-amino-acid sequence has 45% identity and 66% similarity to the human ad serotype-2 previ sequence. there is a marked conservation at the c terminus, the last eleven residues of which may be necessary for activating the ad endoproteinase, and at the n terminus, including the consensus endoproteinase cleavage site.19957835716
genetic relationship between mouse adenovirus-2 (strain k87) and human adenovirus-2.the dna of mouse adenovirus strain k87 (mad-2) was cloned and mapped with restriction endonucleases bglii, clai, ecori, hindiii and sphi. large differences were found between the mad-2 and mad-1 (strain fl) dna molecules in terms of number and location of restriction sites. the mad-2 genome also appeared as larger in size than the mad-1 genome (34.72 kb vs. 30.14 kb). our results confirm the existence of two distinct adenovirus species in the mouse. hybridization experiments, on the other hand, ...19948125249
molecular cloning, physical mapping and cross-hybridization of the murine adenovirus type 1 and type 2 genomes.murine adenovirus (mad) type 1 strain fl and type 2 strain k87 genomes were cloned into plasmid pat153 as hindiii restriction fragments. the mad-1 and mad-2 dna genomes, 30.10 kb and 34.71 kb in length respectively, were mapped using bglii, clai, ecori, hindiii and sphi restriction endonuclease cleavage sites. in view of the large differences found between the mad-1 and mad-2 genomes in terms of the number and location of restriction sites, cross-hybridization experiments were performed. homolog ...19948207397
the fibre of bovine adenovirus type 3 is very long but bent.the sequence of the fibre of bovine adenovirus type 3 (bad3) predicts an extremely long structure due to a large number of 15-residue repeats in the fibre shaft, the tail and head domains being similar in size to the human adenovirus fibres. the length of the fibre was confirmed using negative-strain electron microscopy of bad3 pentons (fibre plus penton base). the fibre was found to be bent in several discrete places and the bending sites appear to correspond with irregular repeats in the shaft ...19948046411
sequence analysis of bovine adenovirus type 3 early region 3 and fibre protein genes. 19938277294
conservation of dna sequence in the predicted major late promoter regions of selected mastadenoviruses.the major late promoter (mlp) of the subgroup c human adenoviruses is a preeminent model for the study of the mechanisms of basal and activated transcription, both in vivo and in vitro. however, while the structure and function of the human virus mlp has been the subject of extensive investigation, the conservation of the various promoter elements among the adenoviruses from different species has not been examined. conservation of specific elements would strongly suggest the importance and unive ...19968661390
the protease of adenovirus serotype 2 requires cysteine residues for both activation and catalysis.sequence analysis and site-directed mutagenesis were used to study the mechanisms of activation and catalysis of the adenovirus type 2 (ad2) protease. primary structure alignments of proteases from 12 serotypes and previously elucidated inhibition profiles were used to target residues for mutagenesis. all conserved serine and cysteine residues were mutated separately and following expression in escherichia coli their activity in a synthetic peptide assay was compared to that of wild-type recombi ...19947931163
pathology and immunogenicity in the cotton rat (sigmodon hispidus) model after infection with a bovine adenovirus type 3 recombinant virus expressing the firefly luciferase gene.the histopathology of adenovirus pneumonia in cotton rats (sigmodon hispidus) due to bovine adenovirus type 3-luciferase recombinant virus (bad3-luc), which has a 0.7 kb deletion from the early region 3 (e3) replaced with the firefly luciferase gene, was compared with that produced by the parental wild-type (wt) bovine adenovirus type 3 (bad3). after intranasal inoculation of cotton rats with 3 x 10(7) p.f.u. of bad3-luc, the infectious virus titres in the lungs at various times post-infection w ...19968558115
interaction of mouse adenovirus type 1 early region 1a protein with cellular proteins prb and p107.we demonstrated functional associations between mouse adenovirus type 1 (mav-1) early region 1a (e1a) protein and both the mouse retinoblastoma protein (prb) and the mouse prb-related protein, p107. interactions between mav-1 e1a and mouse prb or mouse p107 proteins were examined in infected cell lysates using a mouse embryonic fibroblast cell line infected with wild-type and mutant mav-1 viruses. using a polyclonal antibody to mav-1 e1a, exogenously added mouse prb or mouse p107 was coimmunopre ...19968862413
porcine adenoviruses types 1, 2 and 3 have short and simple early e-3 regions.the nucleotide sequence of the e-3 region genes, the hexon associated protein pviii genes, and part of the fiber genes coding for the n-terminal tail regions, of porcine adenovirus (pav) types 1 and 2 were determined. the sizes of the e-3 regions were found to be 1162 and 1222 bp, respectively. the five open reading frames (orf) encoded within the sequenced regions of pav types 1 and 2 shared a high degree of homology with the published sequences of the corresponding orfs of pav-3. the e-3 regio ...19968864200
detection of homologous dna sequences in animal adenoviruses by polymerase chain reaction.a primer pair originally designed for the universal detection of human adenovirus (hav) serotypes of all subgenera was modified then tested and found feasible for the detection of different bovine, ovine, and porcine adenovirus (bav, oav, and pav, respectively) serotypes. apparently, in the examined viruses, parts of the dna sequence coding for the basal part of the hexon protein are conserved enough for being applicable in polymerase chain reaction (pcr) as primers. positive amplification could ...19968908748
molecular characterization of a caprine adenovirus.restriction endonuclease site maps were constructed for the genome of a caprine adenovirus (gadv), strain nc90-7261, which was isolated in 1990 from a 3-year-old goat with encephalitis. genomic gadv dna was digested with seven restriction endonucleases (re). genomic dna libraries of gadv were constructed by cloning bamhi and hindiii restriction fragments into a plasmid vector. using cloned gadv genomic fragments as probes in southern blot hybridizations, an re site map was constructed. the posit ...19969078469
adenovirus-mediated gene therapy for experimental spinal cord tumors: tumoricidal efficacy and functional outcome.we evaluated the efficacy of adenoviral-mediated gene therapy of experimental spinal cord tumors and the functional outcome after this treatment. spinal cord tumors were generated in the thoracic region of the spinal cord in fischer 344 rats by stereotaxic intramedullary injection of 1 x 10(4) 9l gliosarcoma cells. seven days after tumor cell injection, a replication-defective adenoviral vector carrying the herpes simplex virus thymidine kinase gene (adv-tk) or a control adenoviral vector carryi ...19958590067
close phylogenetic relationship between egg drop syndrome virus, bovine adenovirus serotype 7, and ovine adenovirus strain 287.a cloned egg drop syndrome (eds) virus genomic dna fragment containing the protease gene has been identified and the complete nucleotide sequence of the protease and partial nucleotide sequence of the hexon genes has been determined. phylogenetic analysis of the protease gene has revealed eds virus to be genetically more closely related to bovine adenovirus type 7 (bav-7) and ovine adenovirus isolate 287 (oav287) than either of these two viruses are to other members of the genus mastadenovirus o ...19979123875
characterization of adenovirus hexons by their epitope composition.for the investigation of epitope composition of different adenovirus hexon types sixty-one mouse ascitic fluids containing monoclonal antibodies (mabs) developed in three different panels were used. the distinction and marking of the different epitopes recognized by the mabs were carried out by the determination of the composite cross-reactivity pattern, the titer and the correlation coefficient of all the 61 mabs with 21 different hexon types representing all the six human subgenera, as well as ...19968920823
sequence analysis of the fiber genomic region of a porcine adenovirus predicts a novel fiber protein.the complete nucleotide sequence of the putative fiber protein of a porcine adenovirus isolate, nadc-1, was determined. the coding sequence for the fiber protein was found to be 2112 nucleotides, predicting a 703 amino acid protein with a calculated molecular mass of 76,681 da. the coding sequence is located between 86 and 92.5 map units. a polyadenylation signal was found 44 bases downstream of the stop codon. northern hybridization analysis identified a band at 2.4 kb, which likely includes th ...19958837892
analysis of the hexon gene sequence of bovine adenovirus type 4 provides further support for a new adenovirus genus (atadenovirus).the putative hexon gene of bovine adenovirus type 4 (bav-4), encoding 910 amino acid residues, has been identified and sequenced. a characteristic codon usage biased towards the use of at-rich triplets was observed. comparative analysis with other hexon sequences detected a high level of amino acid identity in the regions corresponding to the pedestals of the hexon. substitutions, insertions and deletions were identified mainly in the variable regions forming the loops which are exposed on the o ...19989634088
mechanisms of unusually high antioxidant activity of rsv-sr-transformed cells and of its suppression by activated p21ras.we have previously demonstrated that hamster embryo fibroblasts (hefs) transformed by rous sarcoma virus, schmidt-ruppin strain (rsv-sr) are highly resistant to damage by h202 (h2o2r), (in contrast to hefs transformed spontaneously, or by bovine adenovirus and sv40), while n-ras transfection of rsv-sr transformants leads to suppression of pp6ov-scr and of h2o2r. in this study we have examined (1) mechanisms of antioxidant activity (aoa) of hefs transformed by these agents and (2) the possible ro ...19968647644
unique genome arrangement of an ovine adenovirus: identification of new proteins and proteinase cleavage sites.the completed sequence and genome organization of oav287, a serologically distinct ovine adenovirus, is described. the genome of 29,544 bp has inverted terminal repeats that are only 46 bp in length. many oav genes are identified by their homology with other adenovirus (ad) sequences but three groups of reading frames show little homology. one group at the left-hand end of the genome probably represents the e1a/e1b regions. two others, on the complementary strand at the right-hand end of the gen ...19968659111
characterization of bovine adenovirus type 3 early region 2b.we have determined the nucleotide sequence of a 6999 base pair region of bovine adenovirus-3 covering map units 9.0 to 29.17, which contained the adenovirus homologs of iva2 protein and the dna replication proteins, precursor of terminal protein and dna polymerase proteins. analysis of the sequence for cis-acting elements suggests that transcripts of dna polymerase and precursor of terminal protein are 3' co-terminal. in addition, this region also contains major late promoter sequence. the seque ...19989654686
nucleotide and amino acid sequence analysis of the porcine adenovirus 23k protein.the genomic location of the viral encoded protease (23k) of porcine adenovirus serotype 3 (pav3) was determined and the appropriate fragment cloned and sequenced. an open reading frame (orf) coding for a polypeptide of 203 amino acids and a calculated molecular weight of 23.3 kda was found. the orf was situated in a position similar to that of the human adenovirus 23k, that is, between a putative stop codon for the hexon gene and the polyadenylation signal, aataaa, for the late region 3. amino a ...19968912929
lesions in lambs experimentally infected with ovine adenovirus serotype 6 and pasteurella haemolytica.twenty-five colostrum-deprived lambs reared in isolation were inoculated with a us variant of ovine adenovirus serotype 6 (oav-6) strain rts-151, pasteurella haemolytica, or a combination of the 2 agents. although severe pulmonary lesions were caused by each agent, the lesions were more severe and lasted longer with the combined infection. lesions induced by oav-6 alone developed 6-9 days after inoculation and lasted for 15 days, the length of the experiment. the lesions were characterized by su ...19968844571
hydropathic characteristics of adenovirus hexons.the complete nucleotide sequence and the predicted amino acid sequence of the adenovirus type 7 hexon gene were determined. the hydropathy of the hexon proteins from human adenovirus types 2, 3, 4, 5, 7, 12, 16, 40, 41, and 48, bovine adenovirus type 3, murine adenovirus type 1, and avian adenovirus types 1 and 10 was analysed. the presence of purines and pyrimidines in the second position of the codons was correlated to hydrophilicity and hydrophobicity, respectively. comparison of the hydrophi ...19979267445
efficacy of adenovirus-vectored respiratory syncytial virus vaccines in a new ferret the absence of an adequate small animal model for testing the efficacy of adenovirus-vectored respiratory syncytial virus (rsv) vaccines, a ferret model was established for this purpose. recombinant adenovirus types 4, 5 and 7 expressing the rsv fusion glycoprotein (f), the attachment glycoprotein (g) or both f and g were constructed previously. these recombinants contain a deletion of a large portion of the e3 region of the respective adenovirus vector. in addition, an ad7(e3+)f recombinant ...19948085377
subcloning and restriction enzyme mapping of bovine adenovirus type 2.bovine adenoviruses (bavs), causing both respiratory and/or enteral diseases in cattle, have been isolated in many countries all over the world. in this study we report on the molecular cloning of the internal ecori fragments spanning 20.6-90.5%, and the internal sali fragments spanning 3.1-65.2% of the bav type 2 (bav2; strain no. 19) genome into the plasmid vector puc19. moreover, the subcloning of the bav2 genome facilitated the construction of a detailed physical restriction endonuclease map ...19938294184
selective sites of adenovirus (foreign) dna integration into the hamster genome: changes in integration patterns.we investigated whether, upon the integration of multiple copies of adenovirus type 12 (ad12) dna into an established mammalian (hamster) genome, the pattern of foreign dna insertion would remain stable or change with consecutive passages of cells in culture. by the injection of purified ad12 into newborn hamsters, tumors were induced, cells from these tumors were cultivated, and five independent cell lines, ht5, h201/2, h201/3, h271, and h281, were established. these cell lines carried differen ...19948254728
construction and characterization of hexon-chimeric adenoviruses: specification of adenovirus serotype.this study has used the strategy of gene replacement to characterize the contribution of the adenovirus (ad) capsid protein hexon to serotype definition. by replacing the ad type 5 (ad5) hexon gene with sequences from ad2, we have changed the type specificity of the chimeric virus. the type-determining epitopes are primarily associated with loop 1 of hexon and, to a much lesser degree, with loop 2. in spite of the serotype distinctiveness of the chimeric hexon viruses, epitope similarity between ...19989811772
construction and transfection of ovine adenovirus genomic clones to rescue modified viruses.the genome of ovine adenovirus oav287 has an arrangement which is unique among known adenoviruses. to facilitate further experimentation on the structure and function of this genome, plasmids containing a complete clone of the genome were constructed. the cloned viral genome was released from plasmids by restriction enzyme digestion as an intact linear molecule with authentic 5' termini. transfection of the linear dna into cells which supported replication produced infectious virus. mutation of ...19968659113
cloning and sequencing of the bovine adenovirus type 2 early region 4.bovine adenovirus type 2 (bav2) is a medium size double-stranded dna virus which infects both bovine and ovine species, resulting in mild respiratory and gastrointestinal disorders. to better understand the virus and its growth characteristics in madin-darby bovine kidney (mdbk) cells, we have cloned and sequenced the extreme right-end segment of the bav2 genome (90.5-100 map units). analysis of the nucleotide sequence revealed 40 potential open reading frames (orfs) with coding capacity for pol ...19979055813
adenovirus protein-protein interactions: hexon and protein vi.a variety of mastadenoviruses were denatured, their polypeptides separated by electrophoresis on sds-polyacrylamide gels and transferred to nitrocellulose. the immobilized polypeptides were washed, incubated with buffers containing hexons from human adenoviruses (ad) types 2, 5 and 12 and the location of bound hexons was detected with anti-hexon antibodies. it was found that hexons from any of the three human adenovirus types bound to protein vi from all the mastadenoviruses examined. furthermor ...19947996131
nucleotide sequence of ovine adenovirus tripartite leader sequence and homologues of the iva2, dna polymerase and terminal proteins.ovine adenovirus oav287 was previously isolated from sheep in western australia. here we describe a portion of its genome between map units 10.3 and 31.7 which includes major orfs for homologues of the iva2 polypeptide and the dna replication proteins, terminal protein and dna polymerase, as well as the n-terminal portion of the 52/55-kda polypeptide. in addition, as a prelude to possible adaptation of this virus as a vector we have mapped the elements which make up the tripartite leader sequenc ...19968921842
transcriptional organization of the avian adenovirus celo.a detailed map of the transcriptional organization of the celo virus genome was produced. recent computer analysis of celo virus has indicated the presence of 38 putative open reading frames (orfs). this study, based on analysis of the transcriptional products of celo in vitro, confirmed the presence of rnas for 26 of these 38 orfs. all of the results were obtained by cdna isolation or specific reverse transcriptase pcr. observation of orf transcription kinetics postinfection revealed the existe ...19989765475
expression from cardiomyocyte-specific promoter after adenovirus-mediated gene transfer in vitro and in vivo.adenoviruses are very attractive vectors for gene transfer into the cardiac muscle; however, their promiscuous tissue tropism, leading to an ectopic expression of the transgene, is a considerable practical limitation. to restrict expression of a reporter gene in cultured cardiomyocytes and in the heart of the rat, we have constructed a recombinant adenovirus (ad-mlc2 beta gal) containing the beta-galactosidase gene under the control of the rat ventricle-specific cardiac myosin light chain 2 (mlc ...19979181118
the involvement of brain-derived neurotrophic factor in hippocampal long-term potentiation revealed by gene targeting experiments.brain-derived neurotrophic factor (bdnf) is a member of the ngf gene family, which has been shown to influence the survival and differentiation of specific classes of neurons in vitro and in vivo. the possibility that neurotrophins are also involved in processes of neuronal plasticity has only recently begun to receive attention. to determine whether bdnf has a function in processes like long-term potentiation (ltp), we produced a strain of mice with a deletion in the coding sequence of the bdnf ...19969116659
development of porcine adenovirus-3 as an expression vector.porcine adenovirus-3 (pav-3) was developed as an expression vector using homologous recombination in escherichia coli bj 5183. as a prerequisite, the complete genome of pav-3 was first introduced as a paci restriction fragment into a bacterial plasmid. the plasmid, when paci restricted and transfected into swine testicular cells, produces an infectious virus. the potential of this procedure was demonstrated by the construction of several pav-3 recombinants. part of the e3 region, which is noness ...199910091994
dissociative expression of adenoviral-mediated e. coli lacz gene between ischemic and reperfused rat brains.a replication-defective adenoviral vector containing the e. coli lacz gene was directly injected into the ischemic or reperfused cerebral cortex of rats. an administration of adenoviral vector showed a slight to moderate expression of the lacz gene in the cerebral cortex of the middle cerebral artery (mca) region until 2 days after the mca occlusion. in contrast, expression of the lacz gene was not observed, or only minimally so, in the reperfused brain until 2 days after a 90 min of transient m ...19979153640
completion of the dna sequence of mouse adenovirus type 1: sequence of e2b, l1, and l2 (18-51 map units).the dna sequence of 9991 nt, corresponding to 18-51 map units of mouse adenovirus type 1 (mav-1), was determined, completing the sequence of the larsen strain of mav-1. the length of the complete mav-1 genome is 30,946 nucleotides, consistent with previous experimental estimates. the 18-51 map unit region encodes early region 2b proteins necessary for adenoviral replication as well as late region l1 and l2 structural and packaging proteins. sequence comparison in this region with human adenoviru ...19979381795
distribution of mouse adenovirus type 1 in intraperitoneally and intranasally infected adult outbred situ nucleic acid hybridization and immunohistochemistry were used to determine the histological localization of mouse adenovirus type 1 (mav-1) during acute infection of adult mice infected either intraperitoneally or intranasally with 1,000 pfu of wild-type virus. organ samples were collected from days 1 to 17 postinfection for the intraperitoneally infected mice and from days 1 to 13 for the intranasally infected mice. endothelial cells of the brain and spinal cord showed extensive evidenc ...19989445021
nucleotide sequence, genome organization, and transcription map of bovine adenovirus type 3.the complete dna sequence of bovine adenovirus type 3 is reported here. the size of the genome is 34,446 bp in length with a g+c content of 54%. all the genes of the early and late regions are present in the expected locations of the genome. however, the late-region genes are organized into seven families, instead of five as they are in human adenovirus type 2. the deduced amino acid sequences of open reading frames (orfs) in the late regions and early region 2 (e2) and for iva2 show higher degr ...19989445040
nucleotide and amino acid sequence analysis of the 100k protein of a serotype 3 porcine adenovirus.the genomic region between map units 69 and 78 of a type 3 porcine adenovirus (pav3) was sequenced and analysed. an open reading frame (orf) of 2514 nucleotides encoding a polypeptide of 838 amino acids and approximately 94.1 kda was found. the size and location of the orf suggested it was the pav3 homologue of the 100k gene and this was confirmed by nucleotide sequence comparison with the 100k of human adenovirus type 2. amino acid sequence alignment of the predicted polypeptide with the sequen ...19979522122
survey for antibodies to bovine adenoviruses in six- to nine-month-old feedyard determine the prevalence of antibody to bovine adenovirus (badv) serotypes 1-8 and 10 in calves at a farm and after 5 weeks in a feedyard.19989858410
sequence analysis of the terminal protein precursor coding regions from bovine adenovirus serotypes 2 and part of our efforts to develop bovine adenoviruses into a vector system, we sequenced the region predicted to encode the terminal protein precursor of either bovine adenoviruses type 2 and bovine adenoviruses type 3. we examined the regions containing the terminal protein precursors and showed that the bovine adenovirus early region 2b had an identical organization to all adenoviruses so far examined. the bovine adenovirus terminal protein precursors and those of other adenoviruses were also ...19979612727
the complete nucleotide sequence of the egg drop syndrome virus: an intermediate between mastadenoviruses and aviadenoviruses.the complete nucleotide sequence of an avian adenovirus, the egg drop syndrome (eds) virus, was determined. the total genome length is 33,213 nucleotides, resulting in a molecular weight of 21.9 x 10(6). the gc content is only 42.5%. between map units 3.5 and 76.9, the distribution of open reading frames with homology to known genes is similar to that reported for other mammalian and avian adenoviruses. however, no homologies to adenovirus genes such as e1a, pix, pv, and e3 could be found. outsi ...19979375018
a proposal for a new (third) genus within the family adenoviridae.this article presents a proposal for the establishment of a new adenovirus genus to accommodate certain bovine, ovine, and avian adenoviruses with special characteristics which differentiate them from members of the existing genera mastadenovirus and aviadenovirus. this proposal has been developed from earlier versions with advice from the adenovirus study group of the international committee on taxonomy of viruses (ictv).19989638153
sequencing and phylogenetic analysis of the protease gene, and genetic mapping of bovine adenovirus type 10 define its relatedness to other bovine adenoviruses.the complete genome of a bovine adenovirus (bav) type 10 isolate was molecularly cloned and partially sequenced. the encoded proteins were predicted by computer analysis of the dna sequences of the ends or the entire length of the cloned viral fragments, and thus a rough genetic map was constructed. the protease gene of bav-10 was completely sequenced and used in phylogenetic analysis. based on the results of the phylogenetic analysis, and the location and presence of certain genes thought to be ...19989712509
evidence for transudation of specific antibody into the middle ears of parenterally immunized chinchillas after an upper respiratory tract infection with adenovirus. 19979067660
[adenovirus long-term expression of thrombopoietin in vivo: a new model for myeloproliferative syndrome and osteomyelofibrosis].using a new adenoviral vector (ad) construct, we expressed human thrombopoietin (tpo) cdna (adtpo) in mice with various inherited immune deficiency syndromes such as nude, scid and nod-scid mice. immune normal balb/c mice and a vector construct without tpocdna (adnull), respectively, were used for controls. all animals (3 per group) were treated with a single application of 10(9) pfu (plaque forming unit) of ad (adtpo or adnull) intraperitoneally on day 0. four to 5 weeks following adtpo adminis ...19989824887
in vivo adenovirus-mediated gene transfer to newborn rat retinal pigment epithelial cells.a successful surgical access to the subretinal space is critical for achieving adenovirus-mediated gene transfer to the retinal pigment epithelial (rpe) cells or photoreceptor cells. we report a novel surgical approach allowing an efficient delivery of recombinant replication-deficient adenoviral vectors into the subretinal space of newborn rats. our data suggest that this method may be useful for infecting reproducibly large area of the rpe cell layer of normal newborn rats and should be applic ...19979309253
sequence, transcriptional analysis, and deletion of the bovine adenovirus type 1 e3 region.the early 3 (e3) transcriptional unit of human adenoviruses (hav) encodes proteins that modulate host antiviral immune defenses. hav e3 sequences are highly variable; different hav groups encode phylogenetically unrelated proteins. the role of the e3 region of many human and animal adenoviruses is unknown because the sequences are unrelated to previously characterized viruses and the functions of proteins encoded by these regions have not been studied. we sequenced a portion of the bovine adenov ...19989581789
pathological and in situ hybridisation findings in calves experimentally infected with bovine adenovirus type 4.three colostrum deprived calves were inoculated intravenously with a strain of bovine adenovirus type 4 (bav-4) isolated from a calf with acute fibrinous enteritis. the calves were monitored clinically and submitted to post mortem examination at 5, 6 and 10 days post inoculation (p. i.) respectively. the 3 animals showed raise in temperature and diarrhoea. acute enteritis was observed in calves submitted to post mortem examination at 5 and 6 days p. i. in these animals viral dna was specifically ...19989618984
distribution, persistency, toxicity, and lack of replication of an e1a-deficient adenoviral vector after intracardiac delivery in the cotton rat.adenoviral vectors were inoculated via intracardiac injection into 5- to 1o-week-old cotton rats (sigmodon hispidus) to evaluate the effects of systemic delivery. cotton rats were chosen as a model because they are semipermissive to the replication of human adenoviruses. the vector used was adv.rsv-tk, a replication-deficient adenovirus with a herpes simplex virus thymidine kinase gene inserted in the e1 region. vector doses were 3 x 10(8), 3 x 10(9), and 3 x 10(10) viral particles per animal wi ...19989917091
mouse adenovirus type 1 early region 1a is dispensable for growth in cultured fibroblasts.mouse adenovirus type 1 (mav-1) mutants with deletions of conserved regions of early region 1a (e1a) or with point mutations that eliminate translation of e1a were used to determine the role of e1a in mav-1 replication. mav-1 e1a mutants expressing no e1a protein grew to titers comparable to wild-type mav-1 titers on mouse fibroblasts (3t6 fibroblasts and fibroblasts derived from rb+/+, rb+/-, and rb-/- transgenic embryos). to test the hypothesis that e1a could induce a quiescent cell to reenter ...19989658071
characterization of early region 1 and pix of bovine adenovirus-3.the mrnas from early region 1 (e1) and pix of bovine adenovirus type 3 (bav-3) have been studied by northern blot, s1 nuclease, and cdna analysis and transcriptional maps for the regions were constructed. the transcriptional map for the e1 region of bav-3 is different from those of mouse and human adenoviruses for which transcriptional maps for the regions have been constructed. the e1a region of bav-3 is located between 0.8 and 10.5 map units and several different transcripts are produced from ...19999918888
construction and characterization of e3-deleted bovine adenovirus type 3 expressing full-length and truncated form of bovine herpesvirus type 1 glycoprotein gd.using the homologous recombination machinery of e. coli, a 1.245-kb deletion was introduced in the e3 region of bovine adenovirus 3 (bav3) genomic dna cloned in a plasmid. transfection of the restriction enzyme-excised, linear e3-deleted bav3 genomic dna into primary fetal bovine retina cells produced infectious virus (bav3. e3d), suggesting that all the e3-specific open reading frames are nonessential for virus replication in vitro. using a similar approach, we constructed replication-competent ...19989770436
efficient, repeated adenovirus-mediated gene transfer in mice lacking both tumor necrosis factor alpha and lymphotoxin alpha.the efficiency of adenovirus-mediated gene transfer is now well established. however, the cellular and the humoral immune responses triggered by vector injection lead to the rapid elimination of the transduced cells and preclude any efficient readministration. the present investigation focuses on the role of tumor necrosis factor alpha (tnf-alpha), a proinflammatory cytokine, and the related cytokine lymphotoxin alpha (ltalpha), in mounting an immune reaction against recombinant adenovirus vecto ...19989811684
group d adenoviruses infect primary central nervous system cells more efficiently than those from group c adenovirus-mediated gene transfer to central nervous system cells is inefficient. we found that wild-type group d viruses, or recombinant adenovirus type 2 (ad2) (group c) modified to contain ad17 (group d) fiber, were more efficient in infecting primary cultures of neurons. together with studies on primary vascular endothelial cells and tissue culture cell lines, our results indicate that there is not a universally applicable adenovirus serotype for use as a gene transfer vector.19999971839
generation of infectious genome of bovine adenovirus type 3 by homologous recombination in bacteria.the widely used technique of generating adenovirus vectors by homologous recombination in mammalian cells is usually not very efficient. this communication describes a simple method of generating a plasmid containing the full-length genome of an adenovirus by homologous recombination in bacteria. following transfection of a suitable mammalian cell line with the full-length adenovirus genome, infectious virus progeny could easily be generated. using this technique the generation of adenovirus rec ...199910092136
normal development, wound healing, and adenovirus susceptibility in beta5-deficient mice.integrins have been shown to play important roles in embryonic development, wound healing, metastasis, and other biological processes. alphavbeta5 is a receptor for rgd-containing extracellular matrix proteins that has been suggested to be important in cutaneous wound healing and adenovirus infection. to examine the in vivo function of this receptor, we have generated mice lacking beta5 expression, using homologous recombination in embryonic stem cells. mice homozygous for a null mutation of the ...200010629031
lesions and transmission of experimental adenovirus hemorrhagic disease in black-tailed deer fawns.adenovirus infection was the cause of an epizootic of hemorrhagic disease that is believed to have killed thousands of mule deer (odocoileus hemionus) in california during the latter half of 1993. a systemic vasculitis with pulmonary edema and hemorrhagic enteropathy or a localized vasculitis associated with necrotizing stomatitis/pharyngitis/glossitis or osteomyelitis of the jaw were common necropsy findings in animals that died during this epizootic. to study transmission of adenovirus infecti ...199910098637
transcription mapping and characterization of 284r and 121r proteins produced from early region 3 of bovine adenovirus type 3.we established the transcription map of early region (e) 3 of bovine adenovirus 3 (bav-3) by northern blot, s1 nuclease protection assays, cdna sequencing, and rt-pcr analysis. five major classes of mrnas were identified, which shared the 3' ends. four classes of mrnas transcribed from the e3 promoter also shared the 5' end, while one major class of mrna transcribed from the major late promoter contained a tripartite leader sequence at the 5' end. these five transcripts have the potential to enc ...199910191200
identification of transcripts and promoter regions of ovine adenovirus oav287.the ovine adenovirus isolated oav287 represents a new group of adenoviruses that are distinct from the mast- and aviadenoviruses by several criteria, including genome arrangement. the oav major late promoter and some late transcripts were previously mapped. to better define the probable coding sequences and to identify the approximate location of early promoters a partial transcription map of the genome was elucidated using a pcr-based approach. this was possible because the complete nucleotide ...19989614874
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