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PubMedDate not supplied

Detection of avian reovirus RNA and comparison of a portion of genome segment S3 by polymerase chain reaction and restriction enzyme fragment length polymorphism.

A reverse transcription-polymerase chain reaction (RT-PCR) was established to amplify a 672-base pairs fragment on the segment S3 of avian reovirus (ARV). The amplified fragments were detected in nine strains of ARV as well as three tendon tissue specimens, indicating that the primer regions were well conserved. The RT-PCR was able to detect as low as 0.2 pg using an ethidium bromide stained gel. The detection limit could be enhanced further to 0.04 pg by hybridisation after southern transfer. The amplified DNA fragments from nine ARV strains and two tissue specimens showed different restriction enzyme cleavage patterns. Analysis of the data revealed that these 11 strains were classified into four groups. The results suggest that PCR followed by restriction enzyme analysis may provide a simple and rapid method for the characterisation of ARV isolates.

Animals↗
PubMed2002

Detection of reovirus by reverse transcription-polymerase chain reaction using primers corresponding to conserved regions of the viral L1 genome segment.

A rapid, reverse transcription-polymerase chain reaction (RT-PCR) procedure for the detection of reovirus RNA in cell culture is described. Total nucleic acids are extracted from a small volume of cell culture supernatant and reverse transcribed using random hexamers. An aliquot of cDNA is then utilized in nested PCR. The PCR primers correspond to sequences conserved between prototype reovirus strains type 1 Lang, type 2 Jones, and type 3 Dearing, as well as those of several reovirus field-isolate strains. Reactions are analyzed by agarose gel electrophoresis, and samples showing a band of the appropriate size in the first and second amplification, or in the second amplification alone, are designated as positive. This protocol allows for the rapid and sensitive detection of reovirus in cell culture. The RT-PCR methods described below can easily be adapted to the amplification of reovirus from other media, including preserved tissues, clinical specimens, and water.

Buffers↗
PubMed2000

Identification and differentiation of the nine African horse sickness virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2.

This paper describes the first RT-PCR for discrimination of the nine African horse sickness virus (AHSV) serotypes. Nine pairs of primers were designed, each being specific for one AHSV serotype. The RT-PCR was sensitive and specific, providing serotyping within 24 h. Perfect agreement was recorded between the RT-PCR and virus neutralization for a coded panel of 56 AHSV reference strains and field isolates. Serotyping was achieved successfully with live and formalin-inactivated AHSVs, with isolates of virus after low and high passage through either tissue culture or suckling mouse brain, with viruses isolated from widely separated geographical areas and with viruses isolated up to 37 years apart. Overall, this RT-PCR provides a rapid and reliable method for the identification and differentiation of the nine AHSV serotypes, which is vital at the start of an outbreak to enable the early selection of a vaccine to control the spread of disease.

African Horse Sickness↗
PubMed1999

[Cloning and sequencing analysis of rice stripe virus genome segment 4 of Chinese isolate Y].

The cDNA fragment covering full-length sequence of RStV RNA4 of Yunnan isolate in China was obtained by RT-PCR. The PCR-derived fragment was then cloned into vector pCRII. The cloned cDNA was sequenced. Comparison of the nucleotide and deduced amino acid sequences with those of the Japanese isolate T was made. The results showed that at the nucleotides level, vORF, vcORF and the intergenic region had 94.9%, 94.1% and 86.1% identity respectively, the 5'-untranslational region was exactly the same as that of Japanese isolate T, while the 3'-terminal sequence had 96.1% identity, differing by two nucleotides; at the amino acid level, vORF and vcORF had 99.4% and 98.3% identity respectively. Therefore, as well as being exactly the same size for the two isolates, the amino acid sequences of the coding regions and the 5'- and 3'-terminal sequences were well conserved. Our results indicated that the Chinese isolate is closely related to the Japanese isolate T.

Amino Acid Sequence↗
PubMed2005

Genome segment RNA-1 of a flat apple isolate of Cherry rasp leaf virus: nucleotide sequence analysis and RT-PCR detection.

The sequence of the RNA-1 of a flat apple isolate of Cherry rasp leaf virus (CRLV-FA) was determined using overlapping cDNA fragments. CRLV-FA RNA-1 consists of 6992 nucleotides (nt), excluding a 3' poly (A) tail. A single open reading frame (ORF) consisting of 6705 nt was identified. This ORF encodes a putative polyprotein consisting of 2235 amino acid (aa) residues, approximately 249.6 kDa. When compared to CRLV-pot (potato isolate) RNA-1 ORF, 2 deletions of 5 aa and 10 aa (total 15 aa) were observed at the variable N-terminus of the protease cofactor of CRLV-FA. Non-coding regions were identified at the 5'-(142 nt) and 3'-end (145 nt). CRLV-FA and CRLV-pot are isolates of the same virus with identity levels for the RNA-1 associated nt and deduced aa of 94% and 95%, respectively. RT-PCR targeting CRLV-FA RNA-1 appear to be of similar sensitivity and just as reliable as RT-PCR targeting RNA-2.

Amino Acid Sequence↗
PubMed2001

Identification of barley genome segments introgressed into wheat using PCR markers.

Barley has several important traits that might be used in the genetic improvement of wheat. For this report, we have produced wheat-barley recombinants involving barley chromosomes 4 (4H) and 7 (5H). Wheat-barley disomic addition lines were crossed with 'Chinese Spring' wheat carrying the phlb mutation to promote homoeologous pairing. Selection was performed using polymerase chain reaction (PCR) markers to identify lines with the barley chromosome in the ph1b background. These lines were self pollinated, and recombinants were identified using sequence-tagged-site (STS) primer sets that allowed differentiation between barley and wheat chromosomes. Several recombinant lines were isolated that involved different STS-PCR markers. Recombination was confirmed by allowing the lines to self pollinate and rescreening the progeny via STS-PCR. Progeny testing confirmed 9 recombinants involving barley chromosome 4 (4H) and 11 recombinants involving barley chromosome 7 (5H). Some recombinants were observed cytologically to eliminate the possibility of broken chromosomes. Since transmission of the recombinant chromosomes was lower than expected and since seed set was reduced in recombinant lines, the utility of producing recombinants with this method is uncertain.

DNA, Plant↗
PubMed2006

Bluetongue virus detection by two real-time RT-qPCRs targeting two different genomic segments.

The detection of the bluetongue virus (BTV) by conventional methods is especially difficult and labour-intensive. Molecular diagnosis is also complex because of the high genetic diversity between and within the 24 serotypes of BTV. In the present study, two laboratories joined forces to develop and validate two new RT-qPCRs detecting and amplifying BTV segments 1 and 5. The 2 assays detect strains from all 24 serotypes. They both have a detection limit of 0.01 ECE50 and all 114 samples from BTV-free goats, sheep and cattle were negative. The two assays resulted in similar C(t) values when testing biological samples collected in sheep infected experimentally with a field strain of BTV from the Mediterranean basin. On average, the C(t) values obtained with the 2 methods applied to the 24 serotypes were not significantly different from each other, but some moderate to high differences were seen with a few strains. Therefore these two methods are complementary and could be used in parallel to confirm the diagnosis of a possible new introduction of BTV. An RT-qPCR amplifying a fragment of the beta-actin mRNA was also developed and validated as internal control for the bluetongue specific assays. The three assays described allow a reliable and rapid detection of BTV.

Animals↗
PubMed2002

Multitarget ribozyme against the S1 genome segment of reovirus possesses novel cleavage activities and is more efficacious than its constituent mono-ribozymes.

Two hammerhead motif containing ribozymes (Rzs) were constructed through recombinant techniques that were directed to cleave at the conserved sites of the reovirus S1 gene segment which encodes the cell attachment protein sigma1. The two mono-ribozymes 553 and 984 cleaved the target RNA in a sequence specific manner, and Rz-553 being the more efficient. When the mono-Rzs were combined in direct tandem to make it a multitarget-Rz, very efficient cleavage of the S1 RNA was achieved that retained the specificity of the two mono-ribozymes. This cleavage was, as expected, Mg(++)-dependent but protein-independent. Almost complete cleavage of the S1 RNA was observed with multitarget ribozyme alone. Although S1-Rz-984 cleaved the short S1 synthetic RNA, it failed to cleave the full length S1 RNA (1.4 kb). On the contrary, Rz-553 cleaved the short synthetic RNA as well as the full length S1 RNA with equal efficiency. Full length S1 RNA was, however, cleaved efficiently by the multitarget-ribozyme-S1-Rz-984-553 that cleaved at both the target sites. Thus, hybridization of one ribozyme (Rz-553) to a full length S1 RNA potentially opened up the 984-Rz target site that was otherwise inaccessible to the mono-Rz-984. Multitarget ribozyme expressing mammalian cells showed reduced amounts of S1 RNA that correlated well with the levels of reovirus sigma1 protein. Potential uses of such multitarget-Rzs are discussed.

Base Sequence↗
PubMed2001

The genome segment B encoding the RNA-dependent RNA polymerase protein VP1 of very virulent infectious bursal disease virus (IBDV) is phylogenetically distinct from that of all other IBDV strains.

A full-length cDNA clone of the segment B of the very virulent infectious bursal disease virus (IBDV) strain BD 3/99 was constructed and the full-length nucleotide sequence was established. The nucleotide sequence encoding VP1, an RNA-dependent RNA polymerase, of BD 3/99 was aligned with that of 17 other IBDV strains including six very virulent, three classical virulent, five classical attenuated, one antigenic variant and two serotype 2 strains. The VP1 genes of all very virulent strains were 97.5% to 99.8% identical. With the exception of an atypical Australian strain, 002-73, all of the classical virulent or attenuated and antigenic variant strains were also 97.5% to 100% identical. Serotype 2 strains showed only 4-6% divergence from serotype 1 classical virulent or attenuated strains; in contrast, however, the very virulent strains were 10.5% to 12.5% divergent from the classical virulent or attenuated strains as well as serotype 2 strains. Analysis of the deduced amino acid sequence of VP1 revealed 17 common, including 8 unique amino acid substitutions in the very virulent strains. In the phylogenetic tree the very virulent strains formed a distinct cluster and all other strains including classical virulent, attenuated and antigenic variant strains and even serotype 2 strains were grouped together. It is suggested that the VP1 of very virulent IBDV is phylogenetically distinct from that of all other IBDV strains and probably originated from a hitherto unidentified source.

Amino Acid Sequence↗
PubMed2003

A novel strategy for analysis of gene homologues and segmental genome duplications.

Transformation-associated recombination (TAR) cloning allows selective isolation of a desired chromosomal region or gene from complex genomes. The method exploits a high level of recombination between homologous DNA sequences during transformation in the yeast Saccharomyces cerevisiae. We investigated the effect of nonhomology on the efficiency of gene capture and found that up to 15% DNA divergence did not prevent efficient gene isolation. Such tolerance to DNA divergence greatly expands the potential applications of TAR cloning for comparative genomics. In this study, we were able to use the technique to isolate nonidentical chromosomal duplications and gene homologues.

Centromere↗
PubMed2000

Using alu J elements as molecular clocks to trace the evolutionary relationships between duplicated HLA class I genomic segments.

The class I region of the major histocompatibility complex contains two subgenomic blocks (250-350 kb each), known as the alpha and beta blocks. These blocks contain members of multicopy gene families including HLA class I, HERV-16 (previously called P5 sequences), and PERB11 (MIC). We have previously shown that each block consists of imperfect duplicated segments (duplicons) containing linked members of different gene families, retroelements and transposons that have coevolved as part of two separate evolutionary events. Another region provisionally designated here as the kappa block is located between the alpha and the beta blocks and contains HLA-E, -30, and -92, HERV-16 (P5.3), and PERB11.3 (MICC) within about 250 kb of sequence. Using Alu elements to trace the evolutionary relationships between different class I duplicons, we have found that (a) the kappa block contains paralogous (duplicated) Alu J sequences and other retroelement patterns more in common with the beta than the alpha block; (b) the retroelement pattern associated with the HLA-E duplicon is different from all other HLA class I duplicons, indicating a more complex evolution; (c) the HLA-92 duplicon, although substantially shorter, is closely related in sequence to the HLA-B and -C duplicons; (d) two of the six paralogous Alu J elements within the HLA-B and -C duplicons are associated with the HLA-X duplicon, confirming their evolutionary relationships within the beta block; and (e) the paralogous Alu J elements within the alpha block are distinctly different from those identified within the beta and kappa blocks. The sequence conservation and location of duplicated (paralogous) Alu J elements in the MHC class I region show that the beta and kappa blocks have evolved separately from the alpha block beginning at a time before or during the evolution of Alu J elements in primates.

Alu Elements↗