Taxonomy of genus Hepacivirus. Application of palindromic nucleotide substitutions for the determination of genotypes of human hepatitis C virus species.

The palindromic nucleotide substitutions (PNS) in the 5'-untranslated region (UTR) of Pestivirus RNA have been described as a new, simple and practical method for genotyping. Given the genetic relatedness between Pestivirus and hepatitis C virus species, the application of the method was investigated preliminarily on 180 isolates, including reference strains. The keys for hepatitis C virus identification have been determined at the genus, species, genotype and subtype levels. Secondary structure nucleotide substitutions were characteristics to the genus included in a complex stem-loop structure composed of 112-115 nucleotides. Due to the worldwide importance of hepatitis C virus, and the difficulties encountered in the control of the disease, it is, therefore, important to understand the genetic aspects of the virus. The application of the PNS method might represent an additional useful tool for determining the genetic variations among hepatitis C virus strains. The identification of viral types or subtypes based on genetic changes should improve our understanding of hepatitis C virus and might provide markers for biological differences, such as virulence, and improve understanding of the evolution of the virus.

Characteristics of the 5' untranslated region of wisent (Bison bonasus) and reindeer (Rangifer tarandus) Pestivirus isolates.

The 5'-untranslated region (5'-UTR) of the pestivirus strains isolated from wisent and reindeer suffering from severe diarrhoea in the Duisburg zoo in Germany were amplified by reverse transcription-polymerase chain reaction and sequenced for comparison with those of other pestiviruses from cattle, sheep, goats and swine. Phylogenetic trees constructed from the primary nucleotide sequences of these strains demonstrated that the 5'-UTR of both the wisent and reindeer isolates were identical and suggested that the isolates were allocated to a new cluster of Border disease virus (BDV). The BDV strains were further divided into at least three genotypes or subspecies by phylogenetic analysis and a newly proposed method based on palindromic nucleotide substitutions was used at the variable regions in the 5'-UTR.

Genotypes of Pestivirus RNA detected in anti-influenza virus vaccines for human use.

Nine polyvalent human influenza virus vaccines were tested by reverse transcriptase-polymerase chain reaction (RT-PCR) for the presence of pestivirus RNA. Samples were selected from manufacturers in Europe and the USA. Three samples of the nine vaccines tested (33.3%) gave positive results for pestivirus RNA. The 5'-untranslated genomic region sequence of the contaminant pestivirus RNA was analysed based on primary nucleotide sequence homology and on secondary sequence structures characteristic to genotypes. Two sequences belonged to Pestivirus type-1 (bovine viral diarrhoea virus [BVDV]) species, genotypes BVDV-1b and BVDV-1e. These findings confirm previous reports, suggesting an improvement in preventive measures against contamination of biological products for human use.

Characterisation of genotypes among bovine viral diarrhoea virus 2 strains according to palindromic nucleotide substitutions in the 5 untranslated genomic region.

Pestivirus bovine viral diarrhea virus 2 (BVDV-2) strains from 61 isolates from cattle and sheep, and from some adventitious contaminants of biologicals, have been assessed using the palindromic nucleotide substitutions (PNS) method at three variable loci (V1, V2 and V3) located delin the 5' untranslated region (UTR) of genomic RNA. This genotyping procedure is new, simple and practical. Two characteristics of the base pairings common to BVDV-2 species, a C-G or U-A pairing at the V1 locus, and a G*U pairing at the V2 locus, were observed in isolates tested. The PNS method showed six genotypes: BVDV-2a, BVDV-2b, BVDV-2c, BVDV- 2d, BVDV-2e and BVDV-2f. Twenty-five strains showed the BVDV-2a genotype specific combination of three base pairings (A-U in position 1 and C-G or U*G in position 18 in V1 and U-A or U*G in position 4 in V2). Ten strains were identified by a single C-G pairing in position 4 from the bottom of the V2 stem region, characteristic to genotype BVDV-2b. Three strains were assigned to genotype BVDV-2c, due to their recognition by a G*U pairing at the bottom of the V1 stem region. A U-A pairing, characteristic of the BVDV-2d genotype when found in position 18 of the V1 stem region, was observed in fourteen strains. Genotype BVDV- 2e, present in only six South American cattle isolates, was characterized by G-C pairing in position 12, by U-A pairing in position 16 and G_G or G-_A bulges in position 18 in the V1 region. One strain from Argentina was classified as genotype BVDV-2f, showing: A-U pairing in position 9 and 12, U-A in position 16 and G_A bulge in position 18 in V1 region. Two strains were not characterized due to incomplete sequence of V1 locus.

Genotypic characteristics of bovine viral diarrhea virus 2 strains isolated in northern Italy.

Two strains of Bovine viral diarrhea virus 2 (BVDV-2) were isolated from calves in northern Italy. Variations in the 5'-untranslated region (UTR) of the genome were studied by primary structure alignment and neighbor-joining method based phylogenetic tree analyses and by palindromic nucleotide substitutions at the three variable loci in the 5'-UTR. Genetic analysis indicated their appurtenance to genovar BVDV-2a. Nucleotide sequence at the 5'-UTR of strain BS-95-II, one of the Italian isolates from healthy calves, showed 98% homology to that of the Japanese isolate OY89, a cytopathic strain derived from cattle with mucosal disease.

Genotypes of pestivirus RNA detected in live virus vaccines for human use.

Live virus vaccines for human use, 29 monovalent vaccines against measles, mumps, rubella or polio, eight polyvalent vaccines against measles-mumps-rubella and one bacterial polyvalent vaccine against Streptococcus pneumoniae, were tested by reverse transcriptase-nested PCR for the presence of petivirus or pestivirus RNA. Twenty-four samples were selected from European manufacturers, ten were from U.S.A. and four from Japan. Five (13.1%) out of 38 tested samples were positive for pestivirus RNA. Three vaccines (rubella and two measles) were from Europe and two (mumps and rubella) from Japan. The 5'-untranslated genomic region of the contaminant pestivirus RNA were amplified by reverse transcription-PCR and sequenced. Analyses based on primary nucleotide sequence homology and on secondary structures, characteristic to genotypes, revealed that the cDNA sequences belonged to bovine viral diarrhea virus (BVDV). A cDNA sequence, detected from one measles sample, belonged to BVDV-1b genotype. Pestiviral cDNA detected from the Japanese mumps and rubella vaccine samples, belonged to the BVDV genotypes 1a and 1c, respectively. Analysis on two cDNA sequences detected from measles and rubella vaccine samples from Europe showed their appurtenance to a new genotype, BVDV-1d. These findings indicate that contamination by animal pestivirus may occur in biological products for human use.

Comparison of partial ribosomal DNA sequences of Babesia gibsoni occurring in Miyazaki prefecture, Japan.

Nucleotide sequences of ribosomal DNA (rDNA) of Babesia (B.) gibsoni occurring in Miyazaki, western Japan, were examined using blood samples obtained from seven dogs suffering from natural canine babesiosis. DNA isolated from these blood samples was subjected to the polymerase chain reaction (PCR). The nucleotide sequences of the PCR products were determined and compared with other rDNA sequences of B. gibsoni isolated from Asia, Europe and U.S.A. Although homology values between our isolates and those isolated from Europe and U.S.A. were both 84.0%, respectively, our isolates were identical to the Asian types. In conclusion, B. gibsoni occurring in Miyazaki was revealed to have the genotype Asia 1 or Asia 2 from a comparison of the partial rDNA sequences.

Detection of babesia species from infected dog blood by polymerase chain reaction.

Polymerase chain reaction (PCR) was first applied to diagnosis of canine babesiosis in Japan. Blood samples from 13 dogs suffering from canine babesiosis were used for examination of specificity and sensitivity of the PCR diagnosis. Of the 13 dogs, three were experimentally infected, and ten were naturally infected with Babesia species in west part of Japan. We designed a nested PCR to amplify the babesial small subunit ribosomal RNA gene and found that only the nested PCR produced a visual band, which were not apparent by the first-round PCR to the positive samples. Specificity of the nested PCR was confirmed by amplification after the second-round PCR. Sensitivity of the nested PCR was examined by diluting the blood samples from infected and uninfected dogs. The nested PCR was found to show positive results on the most diluted blood at 0.0001% parasitemia. These results indicate that the nested PCR is highly sensitive and useful for diagnosis of canine babesiosis.

Monoclonal antibody against Mycoplasma fermentans-specific aminoglycoglycerolipid.

Previously, we reported that Mycoplasma fermentans has specific antigens (phosphocholine-containing glycoglycerolipids: GGPL-I and GGPL-III) and discussed the possibility of their pathogenic role. In this paper, we report the characterization of a monoclonal antibody (MF-III-1) specific to GGPL-III (phosphocholine-containing aminoglycoglycerolipid) using methods of electron microscopy, immunofluorescence cell surface staining, laser scanning microscopy, immunoelectron microscopy, and thin-layer chromatography immunostaining. The MF-III-1 antibody specifically recognized M. fermentans attached to the surface of HTLV-I-infected human helper T-cells, and it did not cross-react with other lipids nor with human T-cell antigens. Since MF-III-1 distinguishes GGPL-III from GGPL-I, the binding site may include a serinol (2-amino-1,3-propanediol) residue of GGPL-III. MF-III-1 is useful for the in vitro study of M. fermentans, and may also be useful as a tool for the study of the involvement of M. fermentans in human diseases.

Quantitative analysis of microfilarial periodicity of Dirofilaria immitis in cats.

Microfilarial periodicity of Dirofilaria immitis in the venous blood of infected cats was analyzed by a trigonometric model. Cats were infected by subcutaneous transplantation with 120-day-old juvenile D. immitis. Microfilariae in the blood were first observed 98 days after transplantation. Blood was collected at 4h intervals for a 24h period, and examinations were repeated five times in two cats. The calculated periodicity index was 75.1 and 50.3 in these two cats. The estimated hour of peak microfilarial density ranged from 1.00 to 2.84h. Thus, the periodicity of microfilariae of D. immitis in the blood of cats was characterized as nocturnally sub-periodic.

Comparison of the 16S-23S rRNA intergenic spacer regions among strains of the Mycoplasma mycoides cluster, and reassessment of the taxonomic position of Mycoplasma sp. bovine group 7.

Nucleotide sequence analysis of the 16S-23S rRNA intergenic spacer regions of six type or reference strains belonging to the Mycoplasma mycoides cluster and of Mycoplasma putrefaciens suggested the presence of two subclusters. One subcluster comprised M. mycoides subsp. mycoides small colony (SC) type, M. mycoides subsp. mycoides large colony (LC) type and M. mycoides subsp. capri, whereas the second subcluster comprised Mycoplasma capricolum subsp. capricolum, M. capricolum subsp. capripneumoniae and Mycoplasma sp. bovine group 7. The type strains from M. mycoides subsp. mycoides SC and M. mycoides subsp. capri had identical spacer sequences. The existence of two subclusters was supported by predicted secondary structures of the analysed region. The nucleotide variations in the loop domains of the secondary structures might be a useful genetic marker to distinguish between the two subclusters. The secondary structure differences delineated the differences between the two subclusters more clearly than the nucleotide sequence alignments, which only showed a small number of differences, and some of these were common to both clusters. The data also provided evidence in favour of a reclassification of Mycoplasma sp. bovine group 7 as another subspecies of M. capricolum.

Giraffe strain of pestivirus: its taxonomic status based on the 5'-untranslated region.

The 5'-untranslated region (5'-UTR) of the 'Giraffe' strain of pestivirus was sequenced for comparison with those of other pestiviruses from cattle, sheep, goats, and swine. A phylogenetic tree constructed with these strains suggested that the 'Giraffe' strain was allocated to a new taxon. This observation was also confirmed by a newly proposed method based on palindromic nucleotide substitutions (PNS) at the three variable regions in the 5'-UTR. Other reported pestivirus strains isolated from deer were assigned as bovine viral disease virus (BVDV)-1 according to the PNS as well as phylogenetic analysis, suggesting that BVDV-1 strains can cross-infect deer as well as cattle, sheep, goats, and swine, and that wild deer may serve as a reservoir of BVDV-1. We also identified the genovar of a deer isolate, SH9/11, as BVDV-1c by the PNS method.

Ovine pestiviruses: their taxonomic status revealed by palindromic nucleotide substitutions.

We examined previously identified border disease virus (BDV) strains by using a newly proposed genotyping procedure based on palindromic nucleotide substitutions (PNS) in the 5'-untranslated region (UTR), and found 22 (41.5%) out of 53 strains of BDV in the nucleotide sequence databases are not of BDV. All the 22 ovine pestivirus strains were allocated to the BVDV species according to the PNS, and were compared with reference strains of pestivirus 1 (BVDV-Ia,-Ib, and-Ic genovars), pestivirus 2 (BVDV-II genovar), pestivirus 3 (BDV) and pestivirus 4 (CSFV), respectively. Ten strains (Weybridge, A553, B1056, D771/1, D861, D1120/1, D1432/P, Q1161/1, Q1161/2, 114817) showed a palindromic structure in the 5'-UTR characteristic to the BVDV-Ia genovar, three strains (7535, 7546, 7548) were characteristic to the BVDV-Ib genovar, and nine strains (BD-78, 59386, SCP, Lees, C413, 167237, 168149, 173157, 175375) belonged to the BVDV-II genovar.

Differentiation of two biovars of Ureaplasma urealyticum based on the 16S-23S rRNA intergenic spacer region.

The 16S-23S rRNA intergenic spacer regions of 14 strains representing the 14 serovars of Ureaplasma urealyticum were amplified by PCR and sequenced for genetic differentiation between the two biovars Parvo and T960. Although the spacer region of the Parvo and T960 biovars comprised 302 nucleotides and lacked spacer tRNA genes, 15 nucleotides were different between the two biovars. The four nucleotide sequences of the 16S-23S rRNA intergenic spacer region of serovars 1, 3, 6, and 14 in the Parvo biovar were found to be identical. Similarly, the 10 nucleotide sequences of the 16S-23S rRNA intergenic spacer region of serovars 2, 4, 5, and 7 to 13 in the T960 biovar were found to be identical. The nucleotide sequence of the T960 biovar contains multiple restriction sites for restriction endonuclease SspI, which allows differentiation of the T960 biovar from the Parvo biovar.

Genetic variation in the 5' end and NS5B regions of classical swine fever virus genome among Japanese isolates.

Sixteen clinical strains of classical swine fever virus (CSFV) isolated in Japan were subjected to analyses of nucleotide sequence variations in the 5' end and NS5B regions of the genome. These isolates were divided into three genovars, CSFV-1, CSFV-2 and CSFV-3, based on palindromic nucleotide substitutions at the three variable loci in the 5' untranslated region (UTR). Phylogenetic trees constructed from nucleotide sequences in the 5'-UTR and NS5B gene indicated that the CSFV strains were divided into three clusters, I, II and III. CSFV strains included in clusters I, II and III were identical to those in the CSFV-1, CSFV-2 and CSFV-3 genovars, respectively.

Genetic relationships among mycoplasmas based on the 16S-23S rRNA spacer sequence.

The nucleotide sequences of the spacer regions between the 16S and 23S rRNA genes of 20 Mycoplasma species were determined following amplification by PCR. Although the spacer regions lacked spacer tRNA genes, they contained the box B and box A sequences in this order from the 5' terminus. The sequence alignment indicated that the 20 species were divided into four clusters, the M. pneumoniae, M. hominis, M. hyorhinis and M. fermentans clusters, and a single floating species, M. hyopneumoniae.

A novel method for pestivirus genotyping based on palindromic nucleotide substitutions in the 5'-untranslated region.

A simple and practical method was developed for pestivirus genotyping based on analysis of the secondary structures in the 5'-untranslated region (UTR). Three stable stem-loop structures, V1, V2 and V3, predicted by computer in the 5'-UTR, included strictly conserved consensus base-pairings which are shared by all the genotypes of pestivirus or are characteristic to each genotype of pestivirus. On the basis of the palindromic nucleotide substitution at the secondary structural level, six genotypes have been identified among pestivirus strains, irrespective of the cytopathic and non-cytopathic biotypes. They are genotypes Ia, Ib, Ic and II in bovine viral diarrhea virus, genotype III in border disease virus, and genotype IV in classical swine fever virus. The stable stem-loop structures, which were maintained by palindromic nucleotide substitutions in the stem region, may represent references for the classification and identification of pestivirus species and/or genotypes.

Phosphocholine-containing glycoglycerolipids (GGPL-I and GGPL-III) are species-specific major immunodeterminants of Mycoplasma fermentans.

Mycoplasma fermentans has unique glycoglycerophospholipids (GGPLs: GGPL-I and GGPL-III). Previously, the structure of these lipids was determined as phosphocholine-6'-alpha-glucopyranosyl-(1'-3)-1, 2-diacyl-glycerol (GGPL-I) and 1"-phosphocholine-2"-aminodihydroxypropane-3"-phospho-6'-alph++ + a- glucopyranosyl-(1'-3)-1, 2-diacyl-glycerol (GGPL-III). Thin-layer chromatography (TLC) immunostaining showed that the GGPLs were main lipid-antigens of the M. fermentans species. Anti-M. fermentans serum stained mainly the GGPLs, but the other anti-mycoplasma sera (anti-M. arginini, anti-M. hyorhinis, anti-M. pneumonia, anti-M. primatum, and anti-Acholeplasma laidlawii, anti-M. hominis, anti-M. orale, and M. salivarium) stained neither GGPL-I nor GGPL-III. The TLC analysis of glycolipids and phospholipids of various human related mycoplasmas showed clearly that GGPLs are specifically expressed in M. fermentans species. GGPL-I and GGPL-III ranged from 1.6 to 28% and from an undetectable level to 35% of total phospholipids, respectively. Although there was heterogeneity among the amounts of GGPL-I or GGPL-III of M. fermentans strains, all of the M. fermentans strains had GGPL-I and/or GGPL-III. These observations showed that GGPL structures are species-specific immunodeterminants of M. fermentans. The fact that the GGPLs are main phospholipid components of the M. fermentans species means the M. fermentans has a unique choline metabolic pathway. This observation may raise phylogenetic interest.

Genotypic analysis of the 5'-untranslated region of a pestivirus strain isolated from human leucocytes.

The 5'-untranslated genomic region of the pestivirus strain Europa, originated in human leucocytes and previously identified as bovine diarrhea virus (BVDV), was amplified by reverse transcription-PCR and sequenced. Analyses based on primary nucleotide sequence homology and on secondary palindromic sequence structures characteristic to genotypes revealed that this human isolate should be assigned to a novel genotype of pestivirus, type Ic. This newly emerged genotype was related to, but distinguishable from the three known BVDV genotypes, Ia, Ib and II. Three other bovine field isolates of BVDV originated from Germany were also found to belong to this new genotype Ic. Within pestivirus genotype Ic strains, the overall nucleotide sequence homology was 95-96%, and 88-92%, 88-90% and 77-79% with the other BVDV genotypes Ia, Ib and II, respectively. With the strains from border disease virus (genotype III) and hog cholera virus (genotype IV), homologies were less than 75%.

Detection of Mycoplasma in avian live virus vaccines by polymerase chain reaction.

The polymerase chain reaction (PCR) was evaluated to detect mycoplasma contamination of avian live virus vaccines. The specificity of the primers showed that 34 strains belonging to nine species of avian mycoplasma DNA could be detected. The sensitivity of PCR to detect mycoplasma DNA was 10(0.2) colony forming units (cfu) of Mycoplasma synoviae and 10(0.7) cfu of Mycoplasma gallisepticum. When M. synoviae and M. gallisepticum were spiked into several avian live virus vaccines, PCR gave a positive reaction except for the avian pox and the avian encephalomyelitis vaccines which were prepared from organ homogenates. Short-term incubation of avian encephalomyelitis vaccines improved the sensitivity of PCR to detect both M. synoviae and M. gallisepticum. Therefore, PCR, combined with the short-term incubation, were shown to be most effective in detecting mycoplasma contamination in all of avian live virus vaccines.