The effect of chemical anti-inhibitors on fibrinolytic enzymes and inhibitors

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 uncertain classification. PCR products of the predicted size were amplified from all viruses with the genus-specific primers. All 53 cattle isolates, including 5 typed antigenically as BVDV type II were amplified by the internal BVDV-specific primers, but not the CSFV-specific primers. The same result was found for other BVDV type I and II viruses isolated from sheep and pigs. Seventy-seven CSF viruses were amplified by their respective internal primers. Available information strongly indicate that 4 CSF viruses also amplified by the BVDV-specific primers had been contaminated with BVDV in cell cultures. Border disease viruses were mostly not detected by the BVDV-specific primers, but were detected weakly by the CSFV-specific primer pair. Using carrier RNA for extraction of viral RNA, the sensitivity of detection of the single and nested PCR was, respectively, 5 and 50 times higher than obtained with a cell culture assay. The RT-PCR also detected BVDV in all of 15 commercial batches of fetal calf serum examined, and verified three earlier diagnoses of CSFV by detecting specific gene sequences in 30 year old frozen archival organ samples.     

Development of PCR tests for the detection of bovine herpesvirus-1, bovine respiratory syncytial viruses and pestiviruses

The development of PCR assays for detection of BHV-1, BRSV, BVDV and another pestiviruses is summarized. A polymerase chain reaction assay based on primers selected from the viral gI glycoprotein gene detected 3 fg pure BHV-1 DNA, 0.1-1.0 TCID50 or a single infected cell. No amplification was observed with DNA from BHV-2, BHV-3, BHV-4, OHV-1 or OHV-2. However, a fragment of the correct size (468 bp) was amplified using DNA from herpesviruses isolated from reindeer, red deer and goat. The PCR assay was able to detect virus in nasal swabs 1-14 days after experimental infection of cattle and there was a good correlation when PCR was compared to virus isolation for the detection of BHV-1 in clinical field samples. Detection of BHV-1 in fetal bovine serum and semen samples was also successful. PCR detecting a broad range of BVDV, BDV and HCV was developed. Of six sets of primers selected from different parts of the pestivirus genome the best results were provided by a pair 324/326 from the highly conserved 5'-non-coding region which gave an amplification with all 129 isolates tested. This panel consisted of 79 isolates from cattle, 33 from pigs and 17 from sheep. Differentiation between viruses was achieved by cleavage of the PCR-amplified products (288 bp) with the restriction endonucleases AvaI and BglI. The BVDV products were cleaved by AvaI, HCV by BglI and AvaI. Both enzymes, AvaI and BglI, did not cut the BDV products. A nested polymerase chain reaction assay was developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein. The sensitivity of PCR assay was 0.1 TCID50. No cross reaction was observed with nine heterologous respiratory viruses. PCR products of bovine and human RSV strains were discriminated using endonuclease ScaI, which specifically cleaved products of BRSV. PCR assay detected BRSV in nasal swabs collected from cattle in the acute stage of respiratory disease. In vitro amplification detected 31 positive samples of 35 while immunofluorescence only 23 samples.     

Classical swine fever in 1993 in Switzerland: molecular-epidemiologic characterization of the virus isolate

RT-PCR followed by direct nucleotide sequencing of the amplified cDNA was carried out to analyse most of the 5' nontranslated region (5'NTR) of classical swine fever virus (CSFV) isolates from the five 1993 disease outbreaks in Switzerland. Sequence data were compared to other CSFV strains, and dendrograms were constructed in order to determine the phylogenetic relationship of the Swiss virus strains. Dendrograms formed by the analysis of different parts of the 5'NTR were compared. It was shown that all Swiss isolates were related to other CSFV strains involved in disease outbreaks in Europe in the 1990s. Two of the isolates were indistinguishable from a CSFV strain isolated from wild boar meat imported from China into Austria in 1993. The risk of introducing classical swine fever by improperly treated swill and, in particular by importing wild boar meat is discussed.     

Investigation of film curing stages by dielectric analysis and physical characterization

A previously published sequence of the 23S rRNA gene of Coxiella burnetii has been reported to contain an intervening sequence of 444 base pairs (bp). The sequence information on the intervening sequence and the 23S rRNA gene was exploited to develop a specific PCR-based assay for C. burnetii. A primer set was designed that amplified a 477-bp fragment encompassing part of the intervening sequence and part of the 23S rDNA. From all of nine C. burnetii strains tested, a fragment of the expected size was amplified. As predicted from the published sequence, restriction endonuclease digestion of the PCR product from the Coxiella strains with RsaI produced two distinct fragments approximately 210- and 270-bp in size. The PCR-based method showed a detection limit of 10(2) bacteria as determined by visualization of the amplicon on an agarose gel. When experimentally infected blood was analyzed, the detection limit was 10(3) bacteria. No visible amplicons were observed when 41 bacterial strains, representing 29 species other than C. burnetii, were tested. The presence of the DNA in all bacterial samples was confirmed by amplification of a 350-bp fragment of the 16S rDNA using two universal primers. The described method proved to be specific for C. burnetii and may become a rapid and sensitive diagnostic assay for C. burnetii. The results also demonstrate that the intervening sequence within the 23S rRNA gene is generally found among isolates of C. burnetii.     

The changing face of work in the United States: implications for individual, institutional, and societal survival

To study the identification and phylogeny of pathogenic isolates of Aspergillus, we designed primers from known cytochrome b amino acid sequences. Using these primers, 426 bp fragments of a mitochondrial (mt) cytochrome b gene were amplified by polymerase chain reaction (PCR), directly sequenced, and compared among Aspergillus fumigatus, A. flavus, A. niger, A. terreus and Emericella nidulans. Except for E. nidulans, all strains produced the 426 bp fragment by PCR. The E. nidulans strains demonstrated both an intron-presence fragment ( approximately 1500 bp) and intron-absence fragment (426 bp). Species-specific nucleotides were found in each of the five species. Based on sequence analysis, the strains were further divided into several groups within each species. When a 142-amino-acid sequence was estimated from the 426 bp nucleotide sequence using the yeast mt genetic code, the amino acid sequences showed no difference among strains of the individual species. DNA-based phylogenetic and amino acid-based trees were constructed. In conclusion, the DNA sequences of the cytochrome b gene may be of use in identification of pathogenic Aspergillus species and the amino acid-based tree suitable for discussing their phylogenetic relationships.     

Saccharomyces species assignment by long range ribotyping

Type strains of 10 genotypically distinct Saccharomyces species are differentiated by ribosomal DNA restriction fragment analysis (ribotyping). The full length of the chromosomal ribosomal repeat was amplified in two parts, the 18SrDNA including both ITS region (2600 bp) and the 25SrDNA (3300 bp). Restriction fragments generated by 9 enzymes from these two products yield characteristic patterns, by which unknown Saccharomyces isolates are assigned to the type strains. For convenient separation and detection only fragments longer than 200 bp were monitored. In contrast to molecular differentiation methods of highest resolution as RAPD-PCR or fingerprinting, the results from ribotyping are absolutely reproducible and thereby suitable for databases. The phylogeny computed from the discrete character matrix for presence/absence of fragments by the PHYLIP program package is in complete accordance to the phylogeny derived from ribosomal RNA sequence analysis. By this the field of application of the long range ribotyping can be regarded basically as equal to DNA sequence analysis of the same locus. Because distant relationships are recognized, misidentified genera were detected upon the species assignment. This cannot be done by methods of higher resolution like RAPD-PCR or fingerprinting.     

The changing mole. Additional warning signs of malignant melanoma

The causal involvement of bovine viral diarrhoea virus (BVDV) and border disease virus (BDV) infection in bovine and ovine abortion and perinatal mortality remain unclear. From 1992 until 1994, 213 bovine and 31 ovine foetuses as well as 36 calves and 25 lambs which had died perinatally were investigated. Tissue samples were tested for the presence of pestiviruses and serum or fluid from the body cavities were analysed for the presence of pestivirus antibodies. Detection of pestiviruses was performed by (i) cell culture isolation, (ii) antigen ELISA and (iii) immunohistochemical staining for viral antigen. For antibody-testing an indirect ELISA was used. In nine bovine foetuses and in two calves BVDV was isolated. Pestiviruses, most likely BDV were detected in one ovine foetus and three lambs. In 6% of the bovine and 11% of the ovine foetuses anti-pestivirus antibodies were detected. However, clinical features and history of bovine cases did not show a correlation with the diagnostic results, In contrast, the presence of central nervous system signs in neonatal lambs and the detection of BDV was correlated.     

Antigenicity of poly(dA-dT) . poly(dA-dT) photocrosslinked with 8-methoxypsoralen

Hepatitis C virus (HCV) shows substantial nucleotide sequence diversity distributed throughout the viral genome, with many variants showing only 68-79% overall sequence homology. This has led to problems in diagnosis of HCV using commercial immunoassays. Based on clustering of homologous sequences, various genotypes and subtypes of HCV have been described from different geographical regions. In the present study, 11 isolates from India were genotyped using sequence comparison for part of the non-structural (NS5) and structural (core) regions. Parts of the genome covering 451 bp (nt 9-459) of the core gene and a 249 bp fragment (nt 7959-8207) of the NS5 gene were reverse transcribed and amplified using nested polymerase chain reaction (RT-PCR). The amplified fragments were cloned and sequenced. The classification into genotypes was done on the basis of phylogenetic analysis. Four isolates showed sequence homology to type 1b. Two of the isolates were classified as type 3a. One isolate was classified as type 3b and the remaining four isolates were found to be variants of type 3 but did not belong to any designated subtype. On the basis of phylogenetic analysis two of the unclassified isolates were put into a new subtype of 3 named as 3g. In one of these variants, parts of a 5'-noncoding (5' NCR; 204 bp), envelope-E1 (435 bp), and NS3 (502 bp) regions were also amplified, cloned, and sequenced. This study demonstrates the type 3 variants including a new subtype (3g) to be the major cause of HCV infection in India.     

Partial sequencing of hog cholera virus Alfort strain genome and its comparison with other pestivirus strains

After molecular RNA cloning of the Alfort strain (Alfort/LCRV) of hog cholera virus (HCV), the nucleotide sequence of about 70% of the total genome was determined. This sequence was compared with homologous parts of previously published pestivirus genomes. The average homology with another clone of the Alfort strain (Alfort/FRC) was found to be lower (86.1%) than with Brescia strain of HCV (94.3%), while, compared with NADL, Osloss and SD-1 (3 different strains of bovine viral diarrhea virus, BVDV), the average homology was 67%. Although the amino-acid sequences show a higher degree of conservation, they had a similar degree of homology (92.7, 96.7, 69, 68.2 and 69%, respectively). Partial sequence comparison also revealed that Alfort/LCRV strain was more closely related to Alfort 187 (98.6% for the nucleotides and amino acids) and Weybridge (97.3% for the nucleotides and 96.1% for the amino acids) strains of HCV than it was to Alfort/FRC. These results may indicate that the Alfort/FRC strain has undergone more genomic variations during its historical passage. Genomic relationships among the pestiviruses are discussed.     

Spotted fever group rickettsiosis and vectors in Kanagawa prefecture

Primer pairs for PCR were designed from the gene encoding the 17,000-molecular-weight genus-common antigen of Rickettsia japonica, Rickettsia rickettsii, Rickettsia conorii, Rickettsia typhi and Rickettsia prowazekii. Primers R1, R2 were designed for amplifying the genomic DNA from spotted fever group (SFG) rickettsiae and epidemic typhus rickettsiae. Primers Rj5, Rj10 were designed for amplifying the genomic DNA from only R. japonica. Using the primers R1, R2, about a 540-bp fragment was observed by amplifying the genomic DNA from R. japonica, R. rickettsii, R. conorii, Thai tick typhus TT-118, Rickettsia sibirica, Rickettsia montana, Rickettsia askari, R. typhi, R. prowazekii and Katayama strain isolated from the patient infected with SFG rickettsiae. Using the primers Rj5, Rj10, the 357-bp fragment was observed by amplifying the genomic DNA from R. japonica and Katayama strain. Therefore, the Katayama strain was identified to belong to R. japonica. With primers R1, R2 and Rj5, Rj10, 537 bp and 357 bp bands were amplified from blood of the patients infected with SFG rickettsiae in Kanagawa prefecture. These findings indicate that the causative agent of SFG rickettsiosis in these two patients was R. japonica. The ticks, Ixodes ovatus and Haemaphysalis flava, were collected by out field research in Kanagawa prefecture. With primers R1, R2 and Rj5, Rj10, 537 bp and 357 bp were amplified from these ticks. This indicates that I. ovatus and H. flava were the vector of R. japonica in Kanagawa prefecture. Also, with the primers R1, R2, about a 540 bp fragment was amplified but with primers Rj5, Rj10, no fragments were amplified from I. ovatus and H. flava. Therefore, these ticks may have SFG rickettsiae other than R. japonica and epidemic typhus rickettsiae.     

Immunohistochemical diagnosis of pestivirus infection associated with bovine and ovine abortion and perinatal death

OBJECTIVE: To establish a reliable, rapid, economical method for detection of pestivirus infection in bovine and ovine fetuses and to examine participation of these viruses in abortions and neonatal mortality. ANIMALS: 213 bovine and 31 ovine fetuses, as well as 36 newborn calves and 25 lambs, which had died within 3 days after birth, were tested for bovine viral diarrhea virus (BVDV) and border disease virus by use of different methods. PROCEDURE: Detection of BVDV in fetuses was performed by immunohistochemical methods, using a panel of monoclonal antibodies against pestivirus antigens on cryostat and paraffin sections and by virus isolation in cell culture; in some instances, an antigencapture ELISA was performed. Results of the various methods were compared. RESULTS: Sensitivity of BVDV detection by immunohistochemical methods and virus isolation in cell culture was equal; however, it decreased in association with autolysis. In autolytic fetuses, use of formalin-fixed, paraffin-embedded brain sections was the most favorable method. Antigen detection by ELISA was less sensitive. CONCLUSIONS: Immunohistochemical analysis of cryostat sections of brain, skin, thyroid gland, abomasum, and placenta is a rapid, sensitive method for detecting pestiviruses in fetuses. In the presence of advanced autolysis, this method used on formalin-fixed, paraffin-embedded brain sections is recommended over the other described methods.     

A new approach to utilize PCR-single-strand-conformation polymorphism for 16S rRNA gene-based microbial community analysis

Single-strand-conformation polymorphism (SSCP) of DNA, a method widely used in mutation analysis, was adapted to the analysis and differentiation of cultivated pure-culture soil microorganisms and noncultivated rhizosphere microbial communities. A fragment (approximately 400 bp) of the bacterial 16S rRNA gene (V-4 and V-5 regions) was amplified by PCR with universal primers, with one primer phosphorylated at the 5' end. The phosphorylated strands of the PCR products were selectively digested with lambda exonuclease, and the remaining strands were separated by electrophoresis with an MDE polyacrylamide gel, a matrix specifically optimized for SSCP purposes. By this means, reannealing and heteroduplex formation of DNA strands during electrophoresis could be excluded, and the number of bands per organism was reduced. PCR products from 10 of 11 different bacterial type strains tested could be differentiated from each other. With template mixtures consisting of pure-culture DNAs from 5 and 10 bacterial strains, most of the single strains could be detected from such model communities after PCR and SSCP analyses. Purified bands amplified from pure cultures and model communities extracted from gels could be reamplified by PCR, but by this process, additional products were also generated, as detected by further SSCP analysis. Profiles generated with DNAs of rhizosphere bacterial communities, directly extracted from two different plant species grown in the same field site, could be clearly distinguished. This study demonstrates the potential of the selected PCR-single-stranded DNA approach for microbial community analysis.     

Ruminant pestiviruses

The ruminant pestiviruses, bovine virus diarrhoea virus (BVDV) and border disease virus (BDV) are highly successful and important pathogens which infect ruminant species worldwide. Although the serological relationships among ruminant pestiviruses require further clarification, there is growing evidence for two antigenic groups, one of which predominates in cattle and one in sheep. The success of pestiviruses stems from the ability of the non-cytopathic (NCP) biotype of the virus to cross the placenta and establish a persistent infection (PI) in the developing foetus. This biotype should be regarded as the 'normal' biotype with the cytopathic (CP) biotype being an abnormal virus that is usually isolated only from PI animals dying from mucosal disease. Recent molecular evidence points to CP viruses arising from their NCP counterparts by recombination events that include the insertion of host RNA and/or the duplication of viral RNA sequences. However, the biological mechanism through which CP viruses kill cells remains unknown. Virtually all CP and NCP viruses cause only mild, transient clinical symptoms in healthy adult animals and stimulate a protective immune response. Despite the urgent requirement for a safe, effective vaccine, there is still no commercial vaccine that has been shown to immunize dams so that foetal infection is prevented. In the absence of an effective vaccine, reliable diagnostic techniques are essential to implement effective control measures. There is now a range of monoclonal antibody-based enzyme-linked immunosorbent assays for identifying PI or convalescent animals. These tests are specific, rapid, sensitive and reliable but may themselves become redundant as they are superceded by ever-increasing molecular biology-based techniques.