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Background To reduce the risk of human parvovirus B19 (B19V) transmission through contaminated blood for transfusion and plasma-derived products, the Japanese Red Cross (JRC) Blood Centers introduced B19V antigen screening by chemiluminescent enzyme immunoassay (CLEIA-B19V) in 2008. Study Design and Methods Donor samples that were positive by CLEIA-B19V screening were tested for B19V DNA. The sensitivity of CLEIA-B19V was tested using samples of all three genotypes and B19V DNA–positive donations. B19V DNA–positive donations and pooled plasma were quantitatively assayed for B19V DNA. B19V DNA–positive donations were phylogenetically analyzed by polymerase chain reaction direct sequencing. Results The sensitivity of CLEIA-B19V was inferred to be approximately 6.3 log IU/mL with the genotype samples and 6.4 log IU/mL with B19V DNA–positive donor samples. Of 417 CLEIA-B19V–positive samples from 1,035,560 donations in Hokkaido, Japan, 101 were positive for B19V DNA. The 198 strains of B19V DNA–positive donations in Hokkaido over the past 15 years clustered exclusively with Genotype 1. After introduction of CLEIA-B19V, the viral load for B19V DNA in all 772 pooled plasma for fractionation from donors in nationwide Japan did not exceed 4 log IU/mL. Conclusion CLEIA-B19V can detect all three genotypes of B19V (viral load >6.3 log IU/mL) and limit the viral load (

【背景】日赤では，輸血用血液や血漿分画製剤原料へのヒトパルボウイルスB19（B19V）の混入を防止するため，化学発光酵素免疫測定法（chemiluminescence enzyme immunoassay：CLEIA）によるB19V抗原スクリーニング検査を実施している．近年，B19Vは3種の遺伝子型に分類されたため，米国食品医薬品局（FDA）は，血漿分画製剤製造工程中の検査として，すべての遺伝子型が検出可能な核酸増幅検査を導入するよう勧告した．しかし，本邦で流行しているB19Vの遺伝子型や，CLEIA法がB19Vの遺伝子型すべてを検出可能かは不明である．【対象と方法】既報を改変したB19Vユニバーサルreal-time PCR法（U-PCR法）およびCLEIA法で，3種類のB19V遺伝子型を含むWHOパネルを測定し，それぞれの検出感度を評価した．また，過去13年間の北海道内献血者から検出されたB19V陽性検体96例を用いて，U-PCR法による検出および遺伝子型を調査した．【成績】U-PCR法およびCLEIA法のいずれの方法でも，3種類のB19Vが検出可能であった．U-PCR法の検出感度は1型，2型および3型に対して，それぞれ13.6，9.4および14.6IU/ml以上であった．またCLEIA法の検出感度はいずれの遺伝子型についても約6.3 log10IU/ml以上と推測された．B19V陽性96例全例がU-PCR法で陽性となり，遺伝子型はすべて1型であった．【結論】CLEIA法は3種類のB19Vすべてを検出可能であった．また過去13年間に道内で検出されたB19Vは1型だけであった．

ヒトパルボウイルスB19 (以下B19) に感染した人の献血によって, B19が輸血用血液や分画原料血漿に混入し, 更にその製剤を介して患者にB19が感染する可能性がある. その危険性を減少させるため, 日本赤十字社ではReceptor-mediated hemagglutination assay (RHA) 法による献血者B19スクリーニングを導入した. それにより分画プール血漿中に混入するB19量は大幅に減少した. しかしFDAなどは原料プール血漿に混入するB19DNA量を104geq/mL (IU/mL) 以下とするよう勧告しており, RHA法の検出限界である1010IU/mLでは, この要求を十分に満たすものではない. 今回我々は新しく感度のよいchemiluminescence enzyme immunoassay for screening B19 (CLEIA-B19抗原) を開発した. B19DNA陽性パネル (n=152) をCLEIA-B19抗原で測定した結果, RHA法より4Log高感度であることがわかった. また20プールNAT陰性の671検体をCLEIA-B19抗原で測定した結果, 1例のみ偽陽性となった. 以上のことよりCLEIA-B19抗原は感度特異性に優れ, 献血者B19スクリーニングに有用であると考えられた.

Infectious hepatitis B virus (HBV), namely Dane particles (DPs), consists of a core nucleocapsid including genome DNA covered with an envelope of hepatitis B surface antigen (HBsAg). We report the synthesis, structure, and HBV-trapping capability of multilayered protein nanotubes having an anti-HBsAg antibody (HBsAb) layer as an internal wall. The nanotubes were prepared using an alternating layer-by-layer assembly of human serum albumin (HSA) and oppositely charged poly-L-arginine (PLA) into a nanoporous polycarbonate (PC) membrane (pore size, 400 nm), followed by depositions of poly-L-glutamic acid (PLG) and HBsAb. Subsequent dissolution of the PC template yielded (PLA/HSA)(2)PLA/PLG/HBsAb nanotubes (AbNTs). The SEM measurements revealed the formation of uniform hollow cylinders with a 414 ± 16 nm outer diameter and 59 ± 4 nm wall thickness. In an aqueous medium, the swelled nanotubes captured noninfectious spherical small particles of HBsAg (SPs); the binding constant was 3.5 × 10(7) M(-1). Surprisingly, the amount of genome DNA in the HBV solution (HBsAg-positive plasma or DP-rich solution) decreased dramatically after incubation with the AbNTs (-3.9 log order), which implies that the infectious DPs were completely entrapped into the one-dimensional pore space of the AbNTs.

Hepatitis B virus DNA (HBV-DNA) in 391 anti-HBc positive blood samples were tested by nested polymerase chain reaction. DNA was prepared from pellets of plasma samples after ultra-centri fugation.Of 135 HBsAg weakly positive samples, 127 (93.3%) were HBV-DNA positive. Anti-HBc was the only HB maker for 156 samples, 57 of which (36.50) were HBV-DNA positive, whereas only 6 (3.8%) were positive if the samples were not concentrated. Of 100 anti-HBs positive donors, 6 were HBV-DNA positive. The positive rate of HBV-DNA tended to be high in the samples with high titre of anti-HBc, especially when the anti-HBc was the only HB marker or when HBsAg was positive only by EIA.Thus, it is reasonable to exclude blood units with high titre of anti-HBc even if HBsAg is undetectable in order to prevent post-transfusion hepatitis B.

The hemagglutination inhibition (HI) method was evaluated as a screening test of antibodies to hepatitis B core antigens (anti-HBc).Of 1000 donors samples tested, 272 were HI-positive which included all the anti-HBc positive samples by EIA. Five samples were HBsAg positive only by EIA, which were also HI-positive.The HI titer of HBsAg positve samples were mostly 26 or higher, although some of the HBeAg positive samples were HI-negative.The HI method could be adapted to PK7100, an automated pretransfusion screening system. Of the 2523 samples tested for anti-HBc by PK7100, 694 were positive, most of which were confirmed by the manual HI test. Eighteen samples were positive only by the PK method probably due to carry-over.Finally, we developed the SC-HI for PK7100. The SC-HI cells are double-labeled cells with anti-HBc and HBsAg so that the anti-HBc and anti-HBs are simultaneously detectable.The HI method is an useful method for detecting HBV carriers, when it is supplemented by RPHA for HBsAg and PHA for anti-HBs.

The cellular receptor for hepatitis B virus (HBV) infection has not yet been identified. The purpose of this study was to address the possibility of participation by desialylated HBV and the asialoglycoprotein receptor (ASGP-R) exclusively expressed on liver parenchymal cells, in infection. Assays for viral binding and entry were performed by culturing a hepatoblastoma cell line, HepG2, and HBV particles derived from the HBV carrier in the presence or absence of neuraminidase (NA). Viral binding and entry were clearly enhanced in the presence of NA, and the enhancement of the binding could be blocked by asialo-fetuin and ethylenediamine-tetraacetic acid (EDTA). In addition, covalently closed circular (CCC)-DNA, as a marker of infectivity, was detected in the presence of NA, but not in its absence. The optimal concentration of NA raised infectivity more than 1000 times. We concluded that this method makes it feasible to evaluate the infectivity of HBV in vitro and that ASGP-R may be a specific HBV receptor once viral particles are desialylated.

BACKGROUND : Studies were conducted using samples from early and late-stage HBV-infected persons to determine the pool size at which PCR had better sensitivity than a sensitive HBsAg chemoluminescence immunoassay (CLIA-HBsAg). STUDY DESIGN AND METHODS: HBV seroconversion panels were tested for HBsAg by CLIA and for HBV DNA by nested PCR (95% hit rate: 100 copies/mL); PCR was carried out at various dilutions. HBV serologically positive samples that were detected from the simultaneous screening of 540,161 routine whole-blood donations using CLIA-HBsAg and agglutination assays were also characterized for additional markers of HBV infection. RESULTS: In 9 of 10 HBV seroconversion panels, PCR had better sensitivity than CLIA-HBsAg at dilutions of 1-in-25 or lower. Of 65 CLIA-only confirmed-positive donor samples (agglutination assay-negative), 8 represented early infection, 2 of which were PCR positive at a 1-in-50 dilution but negative at a 1-in-100 dilution. Only 2 of 47 samples from probable late-stage HBV infection that were positive on CLIA only were PCR positive with 0.1-mL sample volume and the S-region primer; the remaining 45 samples required a 1.0-mL sample input and C-region primer for increased PCR positivity. The remaining 10 CLIA-only confirmed-positive donor samples were from HBV vaccine recipients. None of the 12 CLIA- and HBsAg-negative donor samples that were strongly anti-HBc reactive could be detected by PCR at any dilution; all 12 were PCR positive when undiluted, but 4 required a 1.0-mL input volume for PCR positivity. CONCLUSION: For the detection of samples representing early-stage HBV infection, PCR at dilutions of 1-in-25 or lower (equivalent to a pool of ≤25 members) had greater sensitivity than CLIA-HBsAg. In contrast, samples from late-stage HBV infection were detected by PCR only with undiluted samples (0.1-mL or 1.0-mL input volumes), regardless of CLIA-HBsAg reactivity. Therefore, although NAT using minipools of 25 donations or less may be effective for the detection of early-stage HBV infection, it may not be effective for the detection of persistent HBV infection.