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VIRAL PATHOGENESIS AND IMMUNITY

Detection of Hepatitis G Virus Replication Sites by Using Highly Strand-Specific Tth-Based Reverse Transcriptase PCR

Tomasz Laskus, Marek Radkowski, Lian-Fu Wang, Hugo Vargas, Jorge Rakela
Tomasz Laskus
Division of Transplantation Medicine, Thomas E. Starzl Transplantation Institute, University of Pittsburgh, and
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Marek Radkowski
Division of Transplantation Medicine, Thomas E. Starzl Transplantation Institute, University of Pittsburgh, and
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Lian-Fu Wang
Division of Transplantation Medicine, Thomas E. Starzl Transplantation Institute, University of Pittsburgh, and
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Hugo Vargas
Division of Gastroenterology and Hepatology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania
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Jorge Rakela
Division of Transplantation Medicine, Thomas E. Starzl Transplantation Institute, University of Pittsburgh, and
Division of Gastroenterology and Hepatology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania
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DOI: 10.1128/JVI.72.4.3072-3075.1998
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    Fig. 1.

    Sensitivity and specificity of RT-PCR using the Tth assay. Synthetic positive and negative strands were generated by in vitro runoff transcription with T7 RNA polymerase from a vector (pGEM-3Z) containing the 5′ untranslated sequence of HCV and serially diluted in water. The number of target template copies was calculated from optical density readings. A positive-sense primer was present during cDNA synthesis, after which the enzyme was inactivated by chelation with Mn2+ and then negative-sense primer was added. Samples were amplified as described in the text. Twenty microliters (20%) of the reaction mixture was fractionated on agarose, transferred to a nylon membrane by Southern blotting, and subsequently hybridized to a 32P-labeled probe. When 1 or 6 μg of total cellular RNA extracted from normal human liver tissue was added, the sensitivity of the reactions was lowered by no more than 1 log, while the specificity of the assay was not affected.

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    Fig. 2.

    Detection of negative-strand HCV RNA in various tissue samples from patient 2 by the Tth-based strand-specific RT-PCR assay and by the non-strand-specific MMLV RT-based assay. The amount of RNA loaded into each reaction mixture was 1 μg; in the case of serum (S), it corresponded to 20 μl. The examined tissues included liver (Lv), spleen (Sp), bone marrow (BM), lymph node (LN), pancreas (Pn), thyroid (Th), adrenal gland (AG), kidney (Kd), lung (Lg), muscle (Ms), Skin (Sk), and spinal cord (SC). Positive or sensitivity controls (lane P) consisted of end-point dilutions of the correct synthetic strand (10 genomic eq for MMLV RT assay and 100 genomic eq for the Tth assay), and negative controls (lane N) consisted of RNA extracted from livers from uninfected subjects. While the majority of samples were positive by the MMLV RT-based RT-PCR, only bone marrow and spleen samples were positive by the strand-specific Tth-based assay.

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  • Table 1.

    Detection of positive and negative strands of HGV RNA in serum samples and PBMCs in six HIV-infected patients

    PatientCD4 cells/mm3HGV RNA titera in:
    Serum (genomic eq/ml)PBMCs (genomic eq/2 × 106 to 3 × 106 cells)
    Positive strandNegative strandPositive strandNegative strand
    11325 × 107 Negb 103 Neg
    22705 × 105 Neg102 Neg
    31985 × 104 Neg102 Neg
    43095 × 104 Neg102 Neg
    55455 × 103 Neg101 Neg
    6335 × 103 Neg101 Neg
    • ↵a The presence and titers of the positive strand were determined by the MMLV RT-based assay, while the presence of the negative strand was determined by the Tth-based assay.

    • ↵b Neg, negative.

  • Table 2.

    Detection of positive (+) and negative (−) strands of HGV RNA in serum samples and various tissues from four patients with AIDS

    PatientHGV RNA titera in:
    Serum (genomic eq/ml)Tissue (genomic eq of RNA/μg)
    LiverSpleenBone marrowLymph nodePancreasThyroidAdrenal glandKidneyLungMuscleSkinSpinal cord
    +−+−+−+−+−+−+−+−+−+−+−+−+−
    15 × 104 NPNNDND103 102 103 N102 N101 N101 N101 N102 N101 NNDND101 N
    25 × 104 N102 N103 102 105 103 101 N101 NPNPN101 N101 NNN101 NNN
    35 × 105 N103 102 103 102 103 P101 NPNPN101 N101 N101 NNN101 NNN
    45 × 104 N102 NNDND102 N103 N102 N101 N102 NPN102 N101 NNDNDNN
    • ↵a The presence and titers of the positive strand were determined by the MMLV RT-based assay, while the presence and titers of the negative strand were determined by the Tth-based assay. N, negative; P, positive in 6 μg of total RNA; ND, not done.

  • Table 3.

    Detection of positive and negative strands of HCV RNA in serum and liver tissue samples from four patients with AIDS

    PatientHCV RNA titera in:
    Serum (genomic eq/ml)Liver tissue (genomic eq of RNA/μg)
    Postive strandNegative strandPositive strandNegative strand
    15 × 105 Negb 107 106
    25 × 103 Neg105 104
    35 × 104 Neg104 102
    45 × 103 Neg103 102
    • ↵a The presence and titers of the positive strand were determined by the MMLV RT-based assay, while the presence and titers of the negative strand were determined by the Tth-based assay.

    • ↵b Neg, negative.

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Detection of Hepatitis G Virus Replication Sites by Using Highly Strand-Specific Tth-Based Reverse Transcriptase PCR
Tomasz Laskus, Marek Radkowski, Lian-Fu Wang, Hugo Vargas, Jorge Rakela
Journal of Virology Apr 1998, 72 (4) 3072-3075; DOI: 10.1128/JVI.72.4.3072-3075.1998

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Detection of Hepatitis G Virus Replication Sites by Using Highly Strand-Specific Tth-Based Reverse Transcriptase PCR
Tomasz Laskus, Marek Radkowski, Lian-Fu Wang, Hugo Vargas, Jorge Rakela
Journal of Virology Apr 1998, 72 (4) 3072-3075; DOI: 10.1128/JVI.72.4.3072-3075.1998
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KEYWORDS

AIDS-Related Opportunistic Infections
Flaviviridae
Hepatitis, Viral, Human
Polymerase Chain Reaction
RNA, Viral
RNA-Directed DNA Polymerase
virus replication

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