Equal Amounts of Intracellular and Virion‐Enclosed Hepatitis C Virus RNA Are Associated with Peripheral‐Blood Mononuclear Cells In Vivo

Abstract

The substantial and patient-specific amounts of intracellular HCV RNA found by the present study support a concept of low-level replication in PBMCs. There was no evidence for persistent HCV infection in PBMCs after clearance of viremia in plasma.

Clearance of hepatitis C virus RNA from the peripheral blood mononuclear cells of blood donors who spontaneously or therapeutically control their plasma viremia

“…This conclusion is supported by a recent analysis that similarly failed to detect PBMC-associated HCV RNA in 9 spontaneous and 2 treatment-induced aviremic patients. 16 A greater than 90% rate of clearance of HCV RNA in the liver of sustained virological response also indicates that a long-lived hepatic reservoir is unlikely to exist, [37][38][39] although another report detected hepatic HCV RNA in 50% of spontaneously aviremic seropositive subjects. 22 The slow decrease in anti-HCV antibody titers in subjects with spontaneously cleared viremia [40][41][42][43][44] as well as the complete seroreversion detected in 7% of transfusion-transmitted infections 5 may also reflect an absence of ongoing antigenic stimulation, indirectly supporting clearance of infection in persons who test HCV RNA-negative in plasma.…”

“…[7][8][9][10][11][12][13][14] The contribution of PBMC-based HCV replication to total viremia remains unclear, however, as the level of negative-strand HCV RNA in PBMC is very low with respect to that in liver. [15][16][17][18] The continued presence of viral RNA in the PBMC of subjects who had either spontaneously cleared their plasma viremia or cleared viremia following antiviral therapy has recently been reported, raising concerns that PBMC may serve as a long-lived HCV reservoir capable of rekindling systemic infection. [19][20][21][22] In this study, plasma and corresponding PBMC were prepared from recalled confirmed HCV-seropositive blood donors who had tested either HCV RNA-positive or HCV RNA-negative in their original index donation.…”

Productive Human Immunodeficiency Virus Type 1 Infection in Peripheral Blood Predominantly Takes Place in CD4/CD8 Double-Negative T Lymphocytes

“…Selective isolation of extracellular, vRNAex associated with PBMC was performed as previously described (19,20,22,33). The procedure included the disintegration of cells by repeated freezing and thawing followed by nuclease and protease digestion of intracellular RNAs and relied on the physical resistance of small viral envelopes to this treatment (22,30,33).…”

“…The procedure included the disintegration of cells by repeated freezing and thawing followed by nuclease and protease digestion of intracellular RNAs and relied on the physical resistance of small viral envelopes to this treatment (22,30,33). In brief, pellets were thawed and resuspended in 130 l phosphate-buffered saline containing 10 mM Tris HCl, pH 7.5, 1 mM MgCl 2 , 1 mg/ml DNase I (Roche), and 1.8 mg/ml RNase A (QIAGEN).…”

Rational design of HIV-1 fluorescent hydrolysis probes considering phylogenetic variation and probe performance

“…To confirm this concept, SNRs of individual FH-probes were calculated by dividing fluorescence at the end of the amplification by fluorescence during the initial cycles of amplification. To avoid interference of qPCR by possible primer dimer amplification and the resulting reduction of fluorescence in reactions with low copy numbers (Kaiser et al, 2006), high amounts of template (3x10 6 copies HXB2 DNA) were used. As shown in Figure 2a and …”