Isolation and genome characterization of porcine reproductive and respiratory syndrome virus in P.R. China.
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Amino Acid Sequence↗
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Failure to consider the antigenic diversity of porcine reproductive and respiratory syndrome (PRRS) virus isolates may lead to misdiagnosis.
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Animals↗
[Isolation and comparative study of highly reproductive recombinant influenza A viruses with a high antibody sensitivity].
The possibility of generating avid and highly reproductive recombinants of influenza A virus (H3N2, H3N1) using strain A/PR/8/34 (H1N1) as a donor of high reproductive activity was demonstrated. In the process of recombination, the transmission of the gene responsible for synthesis of avid hemagglutinin H3 from one virus variant to another provides for high avidity of recombinants. However, a possible influence of other influenza A virus genes on the manifestation of avidity cannot be ruled out.
Antibodies, Viral↗
[Reproduction of the anaphylactic reaction of isolated human smooth muscle organs and the effect of several adrenotropic substances].
A comparative study was carried out of passive sensitization of the lungs, bronchi, small and large intestine by the blood sera of patients sensitive to ambrosia pollen. Passive sensitization and subsequent anaphylactic contraction were reproduced on the isolated bronchi, lungs and the ileum. Development of this reaction was less constant in the large intestine, jejunum and the appendix. The value of anaphylactic reaction of human smooth muscle displayed significant changes with the treatment of the muscles by agents selectively acting on the beta-adrenergic receptors of these organs.
Anaphylaxis↗
[Reproduction of tobacco mosaic virus in isolated rat liver mitochondria].
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Animals↗
A study of a herpesvirus isolated from dairy cattle with a history of reproductive disorders.
Three strains of herpesvirus were recovered from cows with vulvovaginitis. The three isolates (85/BH 16TV, 85/BH 17TV, 85/BH 18TV), when compared by cross serum neutralization (SN) tests, were found to be antigenically identical. They were serologically distinct from infectious bovine rhinotracheitis (IBR) virus and Bovid herpesvirus 2 (BHV2), while they cross reacted with bovine herpesvirus DN-599. Besides the serologic aspects, the three isolates appeared to share common biological, physical and morphological properties with the newly recognized bovine herpesviruses, of which DN-599 is a representative strain.
Animals↗
Dermatitis in broilers caused by Escherichia coli: isolation of Escherichia coli from field cases, reproduction of the disease with Escherichia coli O78:K80 and conclusions under consideration of predisposing factors.
A dermatitis in broiler chickens, especially on the caudal back, thighs and around the cloaca is observed more frequently in the last years. The skin is swollen at sites of inflammation and a fibrineous plaque extends between muscle and subcutis. No clinical signs are visible in the living flock but the disease causes economical losses because of degrading and rejection of carcasses. Studies of literature and own field observations suggest that Escherichia (E.) coli is involved in the development of the dermatitis. The following serotypes were isolated from field cases: O78:K80 (3X), O2:K56 (2X), O127:K63 (3X), O9:K57 (1X), O140 (2X); two isolates could not be identified. The reproduction of the dermatitis was successful by infection via feather follicles with E. coli O78:K80. The density of broilers kept on farms may contribute to the outbreak of dermatitis by violation of the skin followed by infection of the injuries. Massage of the infected sites by close contact of birds and insufficient hygiene may support the development of the disease.
Animals↗
Sequence analysis of the nucleocapsid protein gene of the porcine reproductive and respiratory syndrome virus Taiwan MD-001 strain.
The 3'-portion of the genome from a Taiwan isolate of porcine reproductive and respiratory syndrome (PRRS) virus, strain MD-001, was cloned and sequenced. The resultant 549 nucleotides contained an open reading frame with a coding capacity of 123 amino acids (predicted Mr 13,600). The predicted protein corresponds to the nucleocapsid protein, the gene product of ORF7. Comparative sequence analysis of several known PRRSV strains indicated that this protein showed the highest degree of amino acid similarity to the US VR2332 and the Canadian IAF-Exp91 strains (92.7%) and the least to the Dutch Lelystad strain (56.5%). The phylogenic trees constructed on the basis of the known PRRSV nucleotide sequences indicated that MD-001 strain belongs to the North American strain cluster and that it is distinct from the European virus.
Amino Acid Sequence↗
Characterization of amino Acid efflux from isolated soybean cells.
Cells from reproductive soybean (Glycine max [L.] Merr.) plants were isolated using a mechanical-enzymic technique that produced a high yield of uniform, physiologically active cells. Cells were incubated in a pH 6.0 buffered solution and subjected to various treatments in order to determine the nature of net amino acid efflux. Total net amino acid (ninhydrinreactive substances) efflux was not affected by the following conditions: (a) darkness, (b) aeration, (c) K(+) concentrations of 0.1, 1.0, 10, or 100 millimolar and (d) pH 4, 5, 6, 7, or 8. The Q(10) for net amino acid efflux between 10 degrees C and 30 degrees C was 1.6. Thus, it seems that net amino acid efflux requires neither current photosynthetic energy nor a pH/ion concentration gradient. Amino acid analyses of the intra-and extracellular fractions over time showed that each amino acid was exported linearly for at least 210 minutes, but that export rate was not necessarily related to internal amino acid pools. Amino acids that were exported fastest were alanine, lysine, leucine, and glycine. Addition of the inhibitor p-chloromercuriphenyl sulfonic acid, 3(3,4-dichlorophenyl)-1,1-dimethylurea, or carbonylcyanide p-trifluoromethoxyphenylhydrazone increased the rate of total amino acid efflux but had specific effects on the efflux of certain amino acids. For example, p-chloromercuriphenyl sulfonic acid greatly enhanced efflux of gamma-aminobutyric acid, which is not normally exported rapidly even though a high concentration normally exists within cells. The data suggest that net amino acid efflux is a selective diffusional process. Because net efflux is the result of simultaneous efflux and influx, we propose that efflux is a facilitated diffusion process whereas influx involves energy-dependent carrier proteins.
Journal Article↗
Production, characterization and reactivity of monoclonal antibodies to porcine reproductive and respiratory syndrome virus.
This report describes the preparation of six monoclonal antibodies (MAbs) raised against a British isolate of porcine reproductive and respiratory syndrome virus (PRRSV), their characterization in terms of protein specificity and their reactivity with different PRRS viruses from Europe and the USA. Radioimmunoprecipitation and Western blotting studies of MAb reactivity with proteins from cell lysates of infected cells and purified virus revealed that four of the six MAbs (WBE1 and WBE4-6) precipitated a 15 kDa viral protein. Further studies using in vitro translated products of the Lelystad virus genome showed that this protein was the product of ORF7, the putative nucleocapsid protein. The specificity of another MAb, WBE2, was found to be for a 45 kDa protein, determined to be the product of ORF3 and demonstrated to be present in purified virion preparations. The protein specificity of the sixth MAb, WBE3 could not be determined. Thirty-three PRRSV isolates from Europe and the USA were grown in alveolar macrophages and examined by immunoperoxidase staining, using the panel of six MAbs. All European isolates were recognized by the four MAbs specific for the putative nucleocapsid, but the viruses showed different patterns of reactivity with WBE2 and WBE3. Furthermore, these two MAbs stained only a small proportion of the cells infected with certain isolates, suggesting that a single isolate may be antigenically heterogeneous. No MAbs bound to US isolates, indicating a consistent antigenic difference between the putative nucleocapsid of US and European isolates. Detergent extraction of cell lysate antigen abrogated the binding of WBE1-3, suggesting that the epitopes are conformation dependent.
Animals↗
Differentiation of North American and European porcine reproductive and respiratory syndrome virus genotypes by in situ hybridization.
Non-radioactive probes that can detect specifically North American and European isolates of porcine reproductive and respiratory syndrome virus (PRRSV) in formalin-fixed paraffin-embedded tissues by in situ hybridization were developed. These probes allow the differentiation between North American and European genotypes of the PRRS virus as well as the detection of both genotypes. Two amplified cDNA products generated by polymerase chain reaction (PCR), one from the cDNA of the Canadian PRRSV LHVA-93-3 isolate and the second one from the European Lelystad isolate, and labelled with digoxigenin were utilized as probes. The LHVA-93-3 derived probe was found to detect Canadian and USA PRRSV isolates in infected cells, while the Lelystad derived probe hybridized only with European isolates. The specificity of both probes was also demonstrated on formalin-fixed tissues collected from PRRSV infected pigs. Furthermore, by combining the LHVA-93-3 (North American) probe and the Lelystad (European) probe, successful detection of both PRRSV genotypes in fixed tissues could be achieved.
Animals↗
Localization and fine mapping of antigenic sites on the nucleocapsid protein N of porcine reproductive and respiratory syndrome virus with monoclonal antibodies.
The purpose of this study was to analyze the antigenic structure of the nucleocapsid protein N of the Lelystad virus isolate of porcine reproductive and respiratory syndrome virus (PRRSV) and to identify antigenic differences between this prototype European isolate and other North American isolates. To do this, we generated a panel of monoclonal antibodies (mAbs) directed against the N protein of Lelystad virus and tested them in competition assays with other N-specific mAbs described previously (Drew et al., 1995; Nelson et al., 1993; van Nieuwstadt et al., 1996). Four different competition groups of mAbs were identified. Pepscan analysis with solid-phase dodecapeptides was used to identify specific antigenic regions in the N protein that were bound by the mAbs. In this pepscan analysis, we found that the mAb of the first competition group reacted with linear peptides whose core sequences consisted of amino acids 2-12 (site A), the mAbs of the second group reacted with peptides whose core sequences consisted of amino acids 25-30 (site B), and the mAb of the third group reacted with peptides whose core sequences consisted of amino acids 40-46 (site C). However, the fourth group of mAbs binding to an antigenic region, provisionally designated as domain D, reacted very weakly or did not react at all with solid-phase dodecapeptides. To further characterize the structure of the epitopes in domain D, we produced chimeric constructs composed of the N protein sequences of Lelystad virus and another arterivirus lactate dehydrogenase-elevating virus, which was used because its N protein has similarity in amino acid sequence and hydropathicity profile but does not react with our mAbs. When the mAbs specific to domain D were tested for binding to the chimeric N proteins expressed by Semliki Forest virus, we found that the regions between amino acids 51-67 and amino acids 80-90 are involved in the formation or are part of the epitopes in domain D. Therefore, we conclude that the N protein contains four distinct antigenic regions. The epitopes mapped to sites A-C are linear, whereas the epitopes mapped to domain D are more conformation dependent or discontinuous. Sites A and C contain epitopes that are conserved in European but not in North American isolates; site B contains epitopes that are conserved in European and North American isolates; and site D contains epitopes that are either conserved or not conserved in European and North American isolates. The antigenic regions identified here might be important for the development of diagnostic test for PRRSV in particular tests that discriminate between different antigenic types of PRRSV.
Amino Acid Sequence↗
Determination of 5'-leader sequences from radically disparate strains of porcine reproductive and respiratory syndrome virus reveals the presence of highly conserved sequence motifs.
We determined the untranslated 5'-leader sequence for three different isolates of porcine reproductive and respiratory syndrome virus (PRRSV): pathogenic European- and American-types, as well as an American-type vaccine strain. 5'-leader from European- and American-type PRRSV differed in length (220 and 190 nt, respectively), and exhibited only approximately 50% nucleotide homology. Nevertheless, highly conserved areas were identified in the leader of all 3 PRRSV isolates, which constitute candidate motifs for binding of protein(s) involved in viral replication. These comparative data provide a priori knowledge for mutational identification of virulence determinants in the 5' nontranslated part of the PRRSV genome.
5' Untranslated Regions↗
Herpesvirus type 2: isolation from seminal vesicle and testes.
Reproductive tissues from 10 recent male cadavers were examined. Herpesvirus type 2 was isolated from testes, seminal vesicle, or both in 4 cases. This is the first report of the isolation of herpesvirus type 2 from human seminal vesicular tissue. The data support previous evidence that herpesvirus type 2 can be isolated from the reproductive tissues of males without active or prior infection and suggest that these tissues may serve as a reservoir for transmission of this virus. The study also documents, for the first time, the ability to culture herpesvirus type 2 in organ explants from cadaveric reproductive tissues with the subsequent release of infectious virus.
Adolescent↗
Genetic, phenotypic, and behavioral variation in North American sylvatic isolates of Trichinella.
Two restriction endonucleases (Cla I and Hpa II) produced polymorphic repetitive DNA profiles which were used in a clustering analysis to quantify the level of genetic variation among 14 North American sylvatic isolates of T5 Trichinella. Differences in genetic profiles reflected phenotypic differences in parasite reproductive success as measured by an isolate's reproductive capacity index in natural hosts. Two genetically distinct isolates of the T5 genotype and T. spiralis were used to infect white-footed mice Peromyscus leucopus for a behavioral analysis. Three behavioral characteristics were tabulated from videotapes: time to emerge from cage, time spent inside cage, and number of rears (index of exploratory activity). Mice infected with 400 T. spiralis larvae showed a significant decrease in exploratory activity and significant increases in time to emerge and time spent inside cage, whereas mice infected with 200 larvae displayed no differences in behavior. Mice infected with a T5 isolate from a raccoon (R9) also displayed decreased exploratory activity. In contrast, mice infected with the other T5 isolate from a coyote (C26) showed relatively constant rearing levels but spent less time inside their cages during the late period of the infection. Results suggest that different genetic strains of Trichinella induce varied types of behavioral modifications upon their hosts.
Animals↗
Association between genetic sequence homology of Porcine reproductive and respiratory syndrome virus and geographic distance between pig sites.
The objective of this study was to investigate whether geographic distance was correlated with genetic homology among isolates of Porcine reproductive and respiratory syndrome virus (PRRSV) from a single pork-producing company. We analyzed geographic distance, temporal distance, and percentage similarity in the PRRSV nucleotide sequence among 62 farms, applying the Mantel test for correlation between distance matrices and PRRSV sequencing. Genomic similarity had a significant (P < 0.01) negative (rM = -0.217) correlation with geographic distance. These findings indicate that, under the conditions of this study, the greater the distance between farms, the less the genetic homology among PRRSV isolates.
Animals↗
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) disrupts early morphogenetic events that form the lower reproductive tract in female rat fetuses.
In female rats, in uteroexposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during critical periods of organogenesis causes a permanent thread of tissue, consisting of a core of mesenchyme surrounded by keratinized epithelia, across the vaginal opening. The objective of the current study was to determine the earliest time after exposure to TCDD during fetal development that morphological changes in the development of the lower reproductive tract could be detected. In addition, the spatio-temporal expression of several growth factors within the developing reproductive tract was investigated to provide insight into the mechanism of action involved in TCDD-induced vaginal thread formation. Pregnant rats received a single oral dose of 1.0 microg TCDD/kg on gestation day (GD) 15. Dams were sacrificed on GD 17, 18, 19, and 21 and individual reproductive tracts were isolated from female fetuses. As early as GD 18, TCDD produced distinct abnormalities in the female reproductive tract. The width of mesenchyme separating the Mullerian ducts was significantly greater in TCDD-exposed female GD 18 and 19 fetuses and the zone of unfused Mullerian ducts was substantially increased on GD 19 and 21. TCDD induced alterations within the developing reproductive tract in the subcellular and temporal expression of transforming growth factor-beta3 (TGF-beta3) and epidermal growth factor receptor (EGFR). DNA array analysis suggested effects on several genes expressed on GD 18 and 19.
Abnormalities, Drug-Induced↗
Genetic variation of Chinese PRRSV strains based on ORF5 sequence.
Thirteen isolates of porcine reproductive and respiratory syndrome virus (PRRSV) from different provinces of China were studied and compared with several PRRSV isolates from other countries. Phylogenetic analysis shows that all Chinese isolates of PRRSV in this study belong to the American genotype, except for one strain, B13, which clustered as a European genotype. Sequence analysis revealed that PRRSV Chinese isolates of the American genotype were highly similar in the ORF5 sequence and could be classified into two subclades. One contains PRRSV isolates that are more closely related to the American vaccine strain MLV Resp and its parent strain VR-2332, and the other contains ones only distantly related to them. Within the Chinese isolates slight genetic variation occurred, and some strains may originate directly from the vaccine virus.
Amino Acid Sequence↗