BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm

<p>Abstract</p> <p>Background</p> <p>Bovine leukemia virus (BLV) is closely related to human T-cell leukemia virus (HTLV) and is the etiological agent of enzootic bovine leukosis, a disease characterized by a highly extended course that often involves persistent lymphocytosis and culminates in B-cell lymphomas. BLV provirus remains integrated in cellular genomes, even in the absence of detectable BLV antibodies. Therefore, to understand the mechanism of BLV-induced leukemogenesis and carry out the selection of BLV-infected animals, a detailed evaluation of changes in proviral load throughout the course of disease in BLV-infected cattle is required. The aim of this study was to develop a new quantitative real-time polymerase chain reaction (PCR) method using Coordination of Common Motifs (CoCoMo) primers to measure the proviral load of known and novel BLV variants in clinical animals.</p> <p>Results</p> <p>Degenerate primers were designed from 52 individual BLV long terminal repeat (LTR) sequences identified from 356 BLV sequences in GenBank using the CoCoMo algorithm, which has been developed specifically for the detection of multiple virus species. Among 72 primer sets from 49 candidate primers, the most specific primer set was selected for detection of BLV LTR by melting curve analysis after real-time PCR amplification. An internal BLV TaqMan probe was used to enhance the specificity and sensitivity of the assay, and a parallel amplification of a single-copy host gene (the bovine leukocyte antigen <it>DRA </it>gene) was used to normalize genomic DNA. The assay is highly specific, sensitive, quantitative and reproducible, and was able to detect BLV in a number of samples that were negative using the previously developed nested PCR assay. The assay was also highly effective in detecting BLV in cattle from a range of international locations. Finally, this assay enabled us to demonstrate that proviral load correlates not only with BLV infection capacity as assessed by syncytium formation, but also with BLV disease progression.</p> <p>Conclusions</p> <p>Using our newly developed BLV-CoCoMo-qPCR assay, we were able to detect a wide range of mutated BLV viruses. CoCoMo algorithm may be a useful tool to design degenerate primers for quantification of proviral load for other retroviruses including HTLV and human immunodeficiency virus type 1.</p

Altered balance of inhibitory and active Fc gamma receptors in murine autoimmune glomerulonephritis

Mag is an MRL-derived glomerulonephritis susceptibility locus that includes the Fcgr2b and Fcgr3 genes encoding the inhibitory Fc gamma receptor IIB (FcγRIIB) and active FcγRIII, respectively. We measured changes in gene balance in three B6.MRLc1 congenic mouse strains containing the 82-86, 92–100 and 100 cM regions of the MRL chromosome 1. We found that only the strain that has 92-100 (which includes Fcgr loci) developed glomerulonephritis. These congenic mice had splenomegaly, elevated blood urea nitrogen, anti-dsDNA antibodies and higher urinary albumin excretion compared to the parental strain C57BL/6(B6). Prior to the development of glomerulonephritis, large CD3- (T cell) and B220- (B cell) positive areas were identified in the spleens of B6.MRLc1(92–100) mice. Both Fc receptors were found in mesangial and dendritic cells; important sites of immune-complex clearance and antigen presentation. The FcγRIII-positive areas were more prominent in the congenic strain. Fcgr2b mRNA was lower in the B6.MRLc1(92–100) kidney and spleen than in those organs of the B6 mice while Fcgr3 expression and the Fcgr3 to Fcgr2b mRNA ratio was higher in the congenic strain kidneys, spleen and thymus than in those of the B6 prior to and at an early stage of glomerulonephritis. We conclude that the imbalance of inhibitory and active Fc gamma receptors influences the pathogenesis of glomerulonephritis

Higher Sensitivity in Induction of Apoptosis in Fibroblast Cell Lines Derived from LEC Strain Rat to Ultraviolet B Radiation

LECラット由来繊維芽細胞のUVBに対する感受性をコロニー形成法を用いて調べた結果、LECラット細胞はWKAHラット細胞に比べてUVBに対して高い感受性を示した。D37値からLECラット細胞ではWKAH細胞の1.5倍以上高い感受性が認められた。ラジカルスキャベンジャーとして働く0.5MDMSOの存在下で細胞にUVBを照射するとLEC、WKAH細胞ともにUVB照射のみと比べて生存率に大きな違いは認められなかった。この結果からUVB照射によって生じるフリーラジカルはUVB誘発細胞死にはあまり関与しないと考えられる。LECラット細胞においてUVB照射後のアポトーシスをフローサイトメーターで検出したところ、照射線量に依存して増加した。一方、WKAHラット細胞ではUVB照射後アポトーシスの増加は認められなかった。これらの結果からLECラット細胞のUVBに対する高い感受性はUVB誘発アポトーシスに依拠していることが示唆された

Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription

A method for the isolation of genomic fragments of RNA virus based on cDNA representational difference analysis (cDNA RDA) was developed. cDNA RDA has been applied for the subtraction of poly(A)(+) RNAs but not for poly(A)(−) RNAs, such as RNA virus genomes, owing to the vast quantity of ribosomal RNAs. We constructed primers for inefficient reverse transcription of ribosomal sequences based on the distribution analysis of hexanucleotide patterns in ribosomal RNA. The analysis revealed that distributions of hexanucleotide patterns in ribosomal RNA and virus genome were different. We constructed 96 hexanucleotides (non-ribosomal hexanucleotides) and used them as mixed primers for reverse transcription of cDNA RDA. A synchronous analysis of hexanucleotide patterns in known viral sequences showed that all the known genomic-size viral sequences include non-ribosomal hexanucleotides. In a model experiment, when non-ribosomal hexanucleotides were used as primers, in vitro transcribed plasmid RNA was efficiently reverse transcribed when compared with ribosomal RNA of rat cells. Using non-ribosomal primers, the cDNA fragments of severe acute respiratory syndrome coronavirus and bovine parainfluenza virus 3 were efficiently amplified by subtracting the cDNA amplicons derived from uninfected cells from those that were derived from virus-infected cells. The results suggest that cDNA RDA with non-ribosomal primers can be used for species-independent detection of viruses, including new viruses

No Significant Differences Were Observed in the Amounts of DNA Strand Breaks Produced by Copper between Male and Female Liver Cells of Long-Evans Cinnamon（LEC) Strain Rats

肝炎を自然発症するLECラットの肝細胞に蓄積する銅による酸化損傷と考えられるDNA単鎖切断の量をコメット法で検出した。LECラットの肝臓では4週齢でコントロールとして用いたWKAHの30倍の銅が蓄積していた。4週齢から15週齢では雄、雌ともに週齢に依存して肝臓の銅の蓄積量は増加したが、WKAHラットでは週齢による増加は認められなかった。LECラットの肝臓でのDNA単鎖切断の量は10週齢から15週齢まで週齢に依存して急激に増加した。肝炎発症は雌が雄に比べて頻度が高いことが報告されているが、肝細胞における雌のDNA単鎖切断の量は雄に比べて大きな違いは認められなかった

Isolation of Novel Adenovirus from Fruit Bat (Pteropus dasymallus yayeyamae)

Isolation of Novel Adenovirus from Fruit Ba

Hepatic iron Accumulation Is Not Directly Associated with Induction of DNA Strand Breaks in the Liver Cells of Long-Evans Cinnamon（LEC) Rats

肝炎を自然発症するLECラットにおいてDNA単鎖切断に及ぼす銅の蓄積の影響を調べた。LECラットの肝臓において銅と鉄は4週齢から15週齢に蓄積した。低鉄食で飼育したラットでは鉄の蓄積は認められなかったが、銅の蓄積は普通食のラットと同様量認められた。肝細胞のDNA単鎖切断の量はcomet法を用いて検出した。テールが認められた細胞の頻度とテールの長さの平均値を求めたところ、低鉄食を与えたLECラットの肝細胞と普通食を与えたLECラットの間にほとんど違いは認められなかった。これらの結果からLECラットの肝細胞におけるDNA損傷の誘発には鉄の蓄積は関与していないことが示唆された

Postmortem and helminthological examination of seabirds killed by oil spilled at Ishikari, Hokkaido, Japan, in November 2004

Postmortems and helminthological examinations were performed on beached seabirds killed by oil spilled from a grounded freighter at Ishikari, Hokkaido, Japan in November 13, 2004. The carcasses were covered with crude oil, but they had adequate subcutaneous fat levels. Gross pathological findings consisting of gastric ulcers, pulmonary edema, enlarged spleens and blackish liquid contents in the digestive tracts suggested that they had a rapid progression to death caused by a loss of ascending force, hypothermia and dehydration. Although the visceral organs had degenerated, no direct evidence of mortality caused by ingesting oil was observed. However, extensive acute inflammatory reactions caused by large numbers of mature and immature nematodes (Contracaecum rudolphii) deeply penetrating the gastric walls was observed in two of the birds. Helminthological investigations were conducted on 21 birds from six species, namely: Phalacrocorax capillatus, Aythya marila, Cerorhinca monocerata, Synthliboramphus antiquus, Aethia cristatella, and Brachyramphus perdix. Thirteen helminth species were obtained and identified, including eight nematodes (Eucoleus contortus, Baruscapillaria mergi, B. rudolphii, Amidostomum acutum, C. rudolphii, Tetrameres fissispina, Cosmocephalus obvelatus and Stegophorus stercorarii), three trematodes (Aporchis sp., Hyptiasmus sp. and Renicola sp.), one cestode (Diorchis nyrocae) and one acanthocephalan species (Andracantha phalacrocoracis). Of these, B. rudolphii from S. antiquus, C. rudolphii and Renicola sp. from A. cristatella, and C. obvelatus and Renicola sp. from B. perdix were first host records. Additionally, B. rudolphii was the first geographical record of this species in Japan

Radioprotective Effect of Alk(en)yl Thiosulfates Derived from Allium Vegetables against DNA Damage Caused by X-Ray Irradiation in Cultured Cells: Antiradiation Potential of Onions and Garlic

To evaluate a radioprotective effect of sodium n-propyl thiosulfate (NPTS) and sodium 2-propenyl thiosulfate (2PTS) derived from onions and garlic, respectively, rat hepatoma H4IIE cells and mouse lymphoma L5178Y cells were preincubated with each of these compounds for 48 hours at 37°C before receiving 10 Gy of X-ray irradiation. Cell damage caused by the irradiation was quantified as comet tail moment, which represents the degree of DNA damage. X-ray-induced DNA damage was significantly decreased in both H4IIE and L5178Y cells by micromolar concentrations of NPTS and 2PTS compared with the control without the compounds. The protective effect was more potent with 2PTS than NPTS. Onions and garlic have antiradiation potential

Trials for Risk Assessment of Japanese Encephalitis Based on Serologic Surveys of Wild Animals

Flavivirus Encephaliti