First case of molecularly identified and genetically characterized human T-cell leukemia virus type 2 infection in a pregnant woman in non-endemic Japan.

Human T-cell leukemia virus type 2 (HTLV-2) is non-endemic in Japan unlike the related HTLV type 1. Previously, although HTLV-2-seropositivity was identified via western blotting in one male blood donor in Japan, there have been no reports of HTLV-2 provirus detection by nucleic acid testing. In this report, one Japanese pregnant woman was clinically diagnosed as being HTLV-2-infected with a line immunoassay for specific antibodies after primary testing through prenatal screening in Japan. In genomic DNA of her peripheral blood mononuclear cells, HTLV-2 proviral genome was detected by nucleic acid testing (three methods) with quantitative polymerase chain reaction. The full-genome sequence of this strain was successfully determined. The identified virus was interestingly characterized as a presumed progenitor of subtypes a and c by recombination region and phylogenetic tree analyses. In conclusion, the present infection is, to our knowledge, the first case of molecularly identified and genetically characterized HTLV-2 infection found via prenatal screening in non-endemic Japan.

Establishment of a novel diagnostic test algorithm for human T-cell leukemia virus type 1 infection with line immunoassay replacement of western blotting: a collaborative study for performance evaluation of diagnostic assays in Japan.

BACKGROUND: The reliable diagnosis of human T-cell leukemia virus type 1 (HTLV-1) infection is important, particularly as it can be vertically transmitted by breast feeding mothers to their infants. However, current diagnosis in Japan requires a confirmatory western blot (WB) test after screening/primary testing for HTLV-1 antibodies, but this test often gives indeterminate results. Thus, this collaborative study evaluated the reliability of diagnostic assays for HTLV-1 infection, including a WB-based one, along with line immunoassay (LIA) as an alternative to WB for confirmatory testing. RESULTS: Using peripheral blood samples from blood donors and pregnant women previously serologically screened and subjected to WB analysis, we analyzed the performances of 10 HTLV-1 antibody assay kits commercially available in Japan. No marked differences in the performances of eight of the screening kits were apparent. However, LIA determined most of the WB-indeterminate samples to be conclusively positive or negative (an 88.0% detection rate). When we also compared the sensitivity to HTLV-1 envelope gp21 with that of other antigens by LIA, the sensitivity to gp21 was the strongest. When we also compared the sensitivity to envelope gp46 by LIA with that of WB, LIA showed stronger sensitivity to gp46 than WB did. These findings indicate that LIA is an alternative confirmatory test to WB analysis without gp21. Therefore, we established a novel diagnostic test algorithm for HTLV-1 infection in Japan, including both the performance of a confirmatory test where LIA replaced WB on primary test-reactive samples and an additional decision based on a standardized nucleic acid detection step (polymerase chain reaction, PCR) on the confirmatory test-indeterminate samples. The final assessment of the clinical usefulness of this algorithm involved performing WB analysis, LIA, and/or PCR in parallel for confirmatory testing of known reactive samples serologically screened at clinical laboratories. Consequently, LIA followed by PCR (LIA/PCR), but neither WB/PCR nor PCR/LIA, was found to be the most reliable diagnostic algorithm. CONCLUSIONS: Because the above results show that our novel algorithm is clinically useful, we propose that it is recommended for solving the aforementioned WB-associated reliability issues and for providing a more rapid and precise diagnosis of HTLV-1 infection.

Development of reference material with assigned value for human T-cell leukemia virus type 1 quantitative PCR in Japan.

Quantitative PCR (qPCR) of human T-cell leukemia virus type 1 (HTLV-1) provirus is used for HTLV-1 testing and for assessment of risk of HTLV-1-related diseases. In this study, a reference material was developed for standardizing HTLV-1 qPCR. Freeze-dried TL-Om1 cells diluted with Jurkat cells were prepared and an assigned value for proviral load (PVL) of 2.71 copies/100 cells was determined by digital PCR. Nine Japanese laboratories using their own methods evaluated the PVLs of this reference material as 1.08-3.49 copies/100 cells. The maximum difference between laboratories was 3.2-fold. Correcting measured PVLs by using a formula incorporating the assigned value of this reference material should minimize such discrepancies.

Proviral Features of Human T Cell Leukemia Virus Type 1 in Carriers with Indeterminate Western Blot Analysis Results.

Western blotting (WB) for human T cell leukemia virus type 1 (HTLV-1) is performed to confirm anti-HTLV-1 antibodies detected at the initial screening of blood donors and in pregnant women. However, the frequent occurrence of indeterminate results is a problem with this test. We therefore assessed the cause of indeterminate WB results by analyzing HTLV-1 provirus genomic sequences. A quantitative PCR assay measuring HTLV-1 provirus in WB-indeterminate samples revealed that the median proviral load was approximately 100-fold lower than that of WB-positive samples (0.01 versus 0.71 copy/100 cells). Phylogenic analysis of the complete HTLV-1 genomes of WB-indeterminate samples did not identify any specific phylogenetic groups. When we analyzed the nucleotide changes in 19 HTLV-1 isolates from WB-indeterminate samples, we identified 135 single nucleotide substitutions, composed of four types, G to A (29%), C to T (19%), T to C (19%), and A to G (16%). In the most frequent G-to-A substitution, 64% occurred at GG dinucleotides, indicating that APOBEC3G is responsible for mutagenesis in WB-indeterminate samples. Moreover, interestingly, five WB-indeterminate isolates had nonsense mutations in Pol and/or Tax, Env, p12, and p30. These findings suggest that WB-indeterminate carriers have low production of viral antigens because of a combination of a low proviral load and mutations in the provirus, which may interfere with host recognition of HTLV-1 antigens.

Standardization of Quantitative PCR for Human T-Cell Leukemia Virus Type 1 in Japan: a Collaborative Study.

Quantitative PCR (qPCR) analysis of human T-cell leukemia virus type 1 (HTLV-1) was used to assess the amount of HTLV-1 provirus DNA integrated into the genomic DNA of host blood cells. Accumulating evidence indicates that a high proviral load is one of the risk factors for the development of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, interlaboratory variability in qPCR results makes it difficult to assess the differences in reported proviral loads between laboratories. To remedy this situation, we attempted to minimize discrepancies between laboratories through standardization of HTLV-1 qPCR in a collaborative study. TL-Om1 cells that harbor the HTLV-1 provirus were serially diluted with peripheral blood mononuclear cells to prepare a candidate standard. By statistically evaluating the proviral loads of the standard and those determined using in-house qPCR methods at each laboratory, we determined the relative ratios of the measured values in the laboratories to the theoretical values of the TL-Om1 standard. The relative ratios of the laboratories ranged from 0.84 to 4.45. Next, we corrected the proviral loads of the clinical samples from HTLV-1 carriers using the relative ratio. As expected, the overall differences between the laboratories were reduced by half, from 7.4-fold to 3.8-fold on average, after applying the correction. HTLV-1 qPCR can be standardized using TL-Om1 cells as a standard and by determining the relative ratio of the measured to the theoretical standard values in each laboratory.

Preparation of rat gingival mitochondria with an improved isolation method.

In order to establish a method of obtaining rat gingival mitochondria (Mt), Mt fractions were prepared in various combinations of homogenizing time with collagenase concentration. Rat gingival tissues were excised, minced, treated with collagenase, homogenized, and subjected to differential centrifugation rates. Both the respiratory control ratio (RCR) and adenosine diphosphate/oxygen (ADP/O) ratio of the Mt fraction prepared in a combination of 40, 50, or 60 sec homogenization with collagenase in a concentration range of 0.115%-0.130% (w/v) were measured. The values for the RCR and ADP/O ratio of the Mt fraction obtained in an optimal condition was 1.80 ± 0.05 and 1.65 ± 0.03, respectively. These results suggest that Mt of fairly high quality can be obtained through this refined combination of the homogenizing time and collagenase concentration.

Polystyrene microgel amphiphiles with maltohexaose. Synthesis, characterization, and potential applications.

4-Vinylbenzyl maltohexaoside peracetate (1) was copolymerized with divinylbenzene (DVB) using 1-phenyl-1-(2',2',6',6'-tetramethyl-1'-piperidinyloxy)ethane (2) in m-xylene. The copolymerizations were performed at 138 degrees C for 20 h using the mole fraction of 1 in the total feed of 1 and DVB (F1: [1]/[1]+[DVB]) varying from 0.11 to 0.38, affording polymeric products in yields ranging from 32 to 40%. The characterizations by linear PS-calibrated size exclusion chromatography (SEC), dynamic laser light scattering (DLS) measurements, and 1H NMR spectroscopy indicated that the product was assignable to the cross-linked poly(4-vinylbenzyl maltohexaoside peracetate) particle which is able to produce stable solutions, i.e., the PSt microgel with acetyl maltohexaose, 3. The specific rotations ([alpha]D23, c = 1.0 CHCl3) of 3 ranged from +43.3 degrees to +85.6 degrees. The average molar masses determined by the static laser light scattering (SLS) measurement of 3, M(w,SLS)'s, were from 64,700 to 118,000, which were calculated using the respective refractive index increments, dn/dc's, ranging from 0.03387 to 0.08340. The apparent numbers of the 1, 2, and DVB units in 3, N1, N2, and N(DVB), which were estimated from the respective [alpha]D23 values, M(w,SLS)'s, and real yields, ranged from 22 to 35, from 7 to 26, and from 146 to 506, respectively. The deacetylation of 3 was achieved by treatment with sodium methoxide in dry 1,4-dioxane to produce the PSt microgel with maltohexaose as the hydrophilic segment, 4, as a white solid. The solubility of 4 in various solvents was examined, indicating that a hydrophilic property was effectively introduced. Notably, 4 gave clear solutions in the mixed solvent of 1,4-dioxane and H2O. The ability to solubilize fullerite (mixture of fullerenes, C60/C70 = ca. 9/1) in aqueous solutions was examined according to the literature method. Approximately, 100 mg of 4 (1.7 micromol) solubilizes 1.3 mg of fullerite (1.7 micromol).