Introduction
Sites of HCV replication in vivo
HCV can bind and enter extrahepatic cells
Detection of extrahepatic HCV replication by PCR
Initial results with PCR
Patients, Special groups | Results for negative strand by PCR | Ratio of +/- strands | References |
---|---|---|---|
HCC | 5/5 L, 0/5 S | 1 to 100 L | [26] |
Hemophilia | 14/27 P, 20/27 PBMC | [148] | |
Hemophilia | 14/19 S, 14/14 PBMC | [149] | |
HIV-HCV co-infected | 4/6 PBMC, 0/6 P | 8-50 PBMC | [150] |
IFN α-treated | 3/11 P untreated; 1/11 treated PBMC | [151] | |
IFN α-treated | 10/10 untreated; 7/9 nonresponders, 2/6 responders PBMC | [152] | |
Mixed cryoglobulinemia | 4/6 PBMC, 5/7 BM | [153] | |
NHL | 6/8 B | [154] | |
Normal livers | 4/4 L, 4/4 PBMC | [155] | |
Oral lichen planus, oral cancer | 3/19 Oral lichen planus; 5/7 Oral cancer | [74] | |
Chronic | 0/5 S, 1/5 PBMC | [156] | |
Chronic | 7/13 PBMC untreated, 5/8 PBMC treated | [157] | |
Chronic | 15/15 L, 0/13 P, 2/3 PBMC, 5/9 BM | [158] | |
Chronic | 0/3 S, 3/3 PBML | [159] | |
Chronic | L, PBML, B positive (no numbers); Not T, NK | [160] | |
Chronic | 8/9 L, 5/9 S | 10-1000 L; 1-10 S | [25] |
Chronic | 0/19 PBMC, M, T, NK; 3/19 B, 3/19 DC | [73] | |
Chronic | 1/1 L, 10/11 S | [161] | |
Chronic | 28/51 B, 0/14 T, 1/24 M, 19/47 PML | [162] | |
Chronic | 3/5 S, 5/5 PBMC, 3/3 B, 3/3 T, 2/3 M | [163] |
Problems with regular PCR
Improved PCR methods for the detection of HCV replication
Patients, Special groups | Corrections to regular PCR | Results for negative strand by PCR | Ratio of +/- strands | References |
---|---|---|---|---|
Acute, chronic | RNase A | Chronic: 6/12 PBMC; Incubation and acute 0 PBMC (no + or - strands) | [164] | |
Acute, chronic, IFN α-treated | Heat for 30 min, Core | 14/35 chronic PBMC, 1/19 acute PBMC | [165] | |
Different genotypes | Core | 3/11 PBMC, 3/10 CD15+ granulocytes; | [166] | |
1/10 CD14+ M, 2/9 CD19+ B, 0/7 CD3+ T | ||||
IFN α-treated | Heat 60 min | Untreated 0/18 S, 13/15 PBML; treated 0/7 S, 3/3 PBML | [167] | |
IFN α-treated | Heat 30 min | Untreated: 16/17 L, 5/14 S, 7/13 PBMC; | [168] | |
Treated, Nonresponders 9/10 L, 3/15 S, 3/7 PBMC | ||||
LT | Heat 1 hr, Core, 3 RT tested | 16/20 L, 0/32 S, 2/26 PBMC, 0/6 BM | 10 to 100 PBMC | [96] |
LT | Core and tag | 5/5 L | 3 to 340 L | [95] |
Mothers | Heat 60 min, RNase, low RT | 5/13 PBMC that transmitted to child; 0/13 that didn't transmit HCV | [30] | |
Occult | Heat 30 min | 2/5 PBMC | [169] | |
Peripheral neuropathy | Tag | 0/9 muscle, 0/3 nerve | [170] | |
Chronic | Chemically modify 3' of RNA | 9/10 L, 5/10 S, 1/10 PBMC | [171] | |
Chronic | Heat 2 hr, RNase H | 0/106 P; 33/83 PBMC (40%) | [172] | |
Chronic | Heat 98 2 hr | 0/7 P, 3/7 PBMC | 1 to 100 PBMC | [173] |
Chronic | Core, RNase, Heat > 20 min | 4/8 PBMC | [174] | |
Chronic | Core and tag | 8/10 L, 0/65 S or P, 1/30 PBMC, 0/12 LT PBMC | [175] | |
Chronic | Tag | L 2/2, 1/6 P, 1/6 DC, 0/6 CD4+, CD8+, CD14+ M, CD19+ B | 300-600 L | [176] |
Chronic | Tag | 29/29 S, 11/29 PBMC, 6/29 PMNG | [177] | |
Chronic | Tag | 3/3 L, 0/3 S | 100 L | [178] |
Chronic | Heat 45 min | 43/45 L, 0/45 S, 37/45 PBMC | [179] | |
Chronic | Tag, RNase A, RNase H | 1/1 L | 60 L | [180] |
Chronic | NS5 | Chronic: 8/8 UTr, 1/3 Tr PBMC; Resolved: 1/10 UTr, 1/1 Tr PBMC; | up to 1000 | [58] |
Chronic: 6/8 UTr, 3/4 Tr CD4+ T; 6/8 UTr, 1/7 Tr CD8+ T; | ||||
Resolved: 6/10 Tr CD4+ T; 1/3 UTr, 2/4 Tr CD8+ T; | ||||
Chronic: 3/4 UTr B; 3/4 UTr M; Resolved: 4/5 UTr B; 2/3 UTr M | ||||
Chronic | Heat 30 min | 13/33 S | [16] | |
Chronic | Heat 30 min | 9/9 L, 0/9 S, 0/9 PBMC | [181] |
Patients, special group | Results for negative strand by PCR | Ratio of +/- strands | References |
---|---|---|---|
Cadavers | 0/6 S, 1/4 lymph; 3/6 CNS | 10 CNS to 100 S | [84] |
Co-infected, cadavers | 7/8 L, 5/8 lymph, 5/8 pancreas, 2/8 adrenal & thyroid, 1/8 BM & spleen | 10-100 Various | [29] |
Co-infected | 15/20 PBMC | [45] | |
Co-infected | 0/10 S, 6/10 PBMC, 4/10 M, 2/10 CD4+ & CD8+ T cells, 1/10 CD19+ B, 2/6 M cultured | 10-100 Various | [70] |
Co-infected women | 13/47 S, 17/48 PBMC | [104] | |
Co-infected women | 61/144 PBMC; 78/315 total specimens | [182] | |
HCV recovered | 9 of 12 PBMC (mitogen stimulated in vitro) | [32] | |
Humans, Chimps | 2/2 S, 0/10 PBMC | 10 L; >= 1000 S | [183] |
Ischemic heart disease | 8/10 Carotid plaque | [184] | |
Lichen planus | 3/3 Skin | [185] | |
Lichen planus/MC+Cutaneous Vasculitis | 0/19 Skin | [186] | |
LT | 42/44, as soon as 7 days post-LT | [94] | |
LT | 0/9 PBMC before transplantation and 3/9 PBMC after | [97] | |
Lymphoproliferative | 4/16 B | [187] | |
Mixed cryoglobulinemia | 6/46 PBMC, 5/46 BM | [188] | |
Occult | 10/10 L, 5/6 PBMC | 1-40 L, 1-2 PBMC | [189] |
Occult | 56/62 L of HCV+ positive, 27/44 L of HCV+ negative | [33] | |
Occult | 11/18 PBMC | 10-100 PBMC | [36] |
SVR | 15/19 L; 12/13 PBMC | 6 PBMC, 2.6 L | [57] |
Chronic | 5/30 in B, 2/30 non-B cells | [190] | |
Chronic | 4 of 17 Oral | [191] | |
Chronic | 6/6 L, 0/6 S | 10-100 L | [89] |
Chronic | 3/3 L | 100-1000 L | [192] |
Chronic | 0/47 Leukocyte, 3/3 Liver | 41 L | [193] |
Chronic | 2/2 L, HCV-strand declines in concentration with increasing time before storage | [194] | |
Chronic | 57/61 L | 10-1000 L | [195] |
Chronic | 5'UTR, Heat 60 min, RNase: 7/9 PBMC, 0/9 S; rTth: 2/3 L, 0/4 S, 0/4 PBMC | [196] | |
Chronic | 4/4 liver, 4/8 PBMC | [197] | |
Chronic | 0/33 P, 0/33 PBMC | [67] | |
Chronic | 39/48 L | 10 L | [198] |
Studies | ||||
---|---|---|---|---|
Cell types or tissue
|
Regular PCR
|
Corrected PCR
|
rTth PCR
|
Totals
|
L = liver | 6/6 | 13/13 | 13/13 | 32/32 |
S = serum | 3/6 | 6/11 | 3/6 | 12/23 |
P = plasma | 2/4 | 1/3 | 0/1 | 3/8 |
PBMC | 10/11 | 14/16 | 12/15 | 36/42 |
PBML | 3/3 | 2/2 | 0/1 | 5/6 |
B = B cells/lymphocytes | 5/5 | 3/4 | 3/3 | 11/12 |
T = T cells/lymphocytes | 1/4 | 7/11 | 2/2 | 10/17 |
M = Monocytes/Macrophages | 1/2 | 3/4 | 2/2 | 6/8 |
DC = Dendritic cells | 1/1 | 1/1 | 2/2 | |
BM = Bone marrow | 2/2 | 0/1 | 2/2 | 4/5 |
Granulocytes, PMNG | 2/2 | 2/2 | ||
Oral tissues | 3/3 | 1/1 | 4/4 | |
Carotid plaque | 1/1 | 1/1 | ||
Skin | 1/2 | 1/2 | ||
Lymph nodes | 2/2 | 2/2 | ||
Pancreas | 1/1 | 1/1 | ||
Spleen | 1/1 | 1/1 | ||
Thyroid | 1/1 | 1/1 | ||
Adrenal | 1/1 | 1/1 | ||
CNS, brain, nerve | 0/1 | 1/1 | 1/2 | |
Muscle | 0/1 | 0/1 | ||
NK | 0/2 | 0/2 | ||
Total for all tissues
|
37/47
|
53/72
|
47/56
|
137/175
|
Percent | 78.7 | 73.6 | 83.9 | 78.3 |
Total extrahepatic tissues
|
31/41
|
40/59
|
34/43
|
105/143
|
Percent | 75.6 | 67.8 | 79.1 | 73.4 |
Studies with > = 1 positive result
|
21/21
|
22/23
|
28/30
|
71/75
|
Studies with > = 1 positive extrahepatic result
|
20/21
|
18/21
|
19/25
|
57/67
|
Estimated ratios of positive to negative HCV strands
Conclusions from PCR results
Detection of extrahepatic HCV replication by other methods
Detection of HCV RNA using in situ hybridization
Patients, special groups | HCV region of genome | Results for negative strand | References |
---|---|---|---|
Co-infected | 5'UTR-core-E1 | 42/63 liver; Some only hepatocytes, some (portal) monocytes/sinusoidal/biliary | [55] |
End-stage liver disease | 5'UTR-core-E1 | 10/20 perihepatic lymph nodes | [41] |
HCC | 5'UTR, core, NS4B | 4/9 HCC hepatocytes; 7/10 Non-HCC | [199] |
HCC | 5'UTR | Hepatocytes positive, some mononuclear, no biliary or mesenchymal | [200] |
LT | 5'UTR | 2/11 LT patients, 2/11 PBMC, 3/3 mitogen stimulated PBMC | [40] |
MC | 5'UTR, core | 12/12 hepatocytes | [201] |
NHL, MC | 5'UTR | Ductal and acinar salivary epithelial cells | [42] |
Occult | 5'UTR | 11/18 PBMC | [36] |
Oral lichen planus | 5'UTR | 14/19 oral epithelia | [44] |
Sjogren's syndrome or Chronic sialadenitis | 5'UTR | 8/8 salivary gland | [43] |
35% of genome used | Hepatocytes not infected-macrophages or lymphocytes; No - strand detected | [202] | |
SVR | 5'UTR | 15/19 Liver | [57] |
Chronic | core-E1 | 8 of 8 hepatocytes | [56] |
Chronic | core-E1 | Quantitative image analysis; hepatocytes, some Kuppfer; +/- ratio 3-20 | [147] |
Chronic | 7 regions | Some hepatocytes positive for negative strands | [203] |
Chronic | 5'UTR | 24/28 liver negative strand; 14/17 PBMC negative strand; | [37] |
Chronic | 5'UTR | 9/20 liver-perinuclear in hepatocytes; mononuclear cells had only positive strands | [204] |
Chronic | 5'UTR | 4 of 11, all HIV co-infected-hepatocytes and mononuclear cells | [205] |
Chronic | NS5 | 4/10 H nuclei; 6/10 H cytoplasm; 4/10 M; 0/10 Bile duct epithelial | [54] |
Chronic | 5'UTR | FISH, positive strands only: 15-70% of liver cells positive, 0.1 to 4% PBMC positive | [39] |
Chronic | 5'UTR, core | 4/5 liver, 5/20 PBMC, 0/20 BM | [38] |
Chronic | 5'UTR, core, NS4B | 4/10 hepatocytes; 2/4 some lymphocytes | [206] |
Chronic | 5'UTR | 17/19 hepatocytes | [34] |
Detection of HCV proteins using Immunohistochemistry and immunofluorescence
Patients, special groups | Method | Proteins | Evidence for NS proteins | References |
---|---|---|---|---|
Acute/chronic | IF | NS3, NS4 | NS4 but not NS3 in hepatocytes | [207] |
Acute/chronic, IFN α-treated | IF | NS5 | 17/20 PBMC positive for NS5, all in cytoplasm | [165] |
Co-infected, cadavers | IHC | NS5A | Astrocytes and microglial cells in CNS (macrophages) positive | [47] |
Co-infected | SUSHI | NS5A | CD14+CD16++ (DC) and CD14++CD16++ (macrophages); not CD14++CD16- (monocytes) | [45] |
Co-infected | IHC | NS3 | Liver: NS3 more associated with replication than core; | [55] |
End-stage liver disease | IHC | NS3 | NS3 stained lymph nodes-probably B cells, but likely T and other cells | [41] |
HCC | IHC | Core, E2, NS4, NS5 | Hepatocytes positive, some mononuclear | [200] |
HCC/Cardiomyolitis | IHC | Core, NS4B | Core, NS4B-mostly in macrophages | [109] |
NHL, MC | IHC | Core, NS4 | Core salivary parotid epithelial, none for NS4 | [42] |
MC | IHC | Core, NS4 | Both liver and skin positive in some samples were positive | [208] |
MC | RIBA (Ortho) | NS4 | 17/37 Sera positive for anti-NS4 antibodies | [209] |
MC | IHC | Core, E2, NS4, NS5 | 3/12 NS4, 5/12 NS5 kidneys; 2/12, 3/12 capillaries; 2/12, 4/12 blood vessels | [49] |
Chronic | IHC | NS3, NS5a | 3/4 Liver; 4/10 Intestinal with NS3, 3 also with NS5a | [48] |
Chronic | IHC | NS4 | Hepatocytes, not monocytes, not endothelial, not bile duct epithelium | [210] |
Chronic | IF | Core, NS4 | 4/11 monocytes and B; 3/11 T cells in BM; 1/11 T in PBMC | [46] |
Chronic | IHC | NS4 | 6/14 livers; hepatocytes stained, no mention of non-hepatocytes | [206] |
Chronic | IHC | NS4 | NS4 mainly in hepatocytes, some Kupffer, bile duct, mononuclear | [211] |
Chronic | IF | E2, NS3, NS4 NS5 | NS3: mostly CD8+ T, CD20+ B, some CD4+ T. No CD14+ cells found | [108] |
Chronic | Confocal | NS5 | CD4+, CD8+, B, and M all stained positive (0.02-0.1%) for NS5 | [58] |
Chronic | IHC | Core, NS4 | 9/12 for both Core and NS4 in hepatocytes and some monocytes | [201] |
Other methods
Percentages of cells infected in vivo
Different cell types could contain different HCV variants
Patients, special groups | Cell types investigated | Regions studied | Methods | References |
---|---|---|---|---|
a. Liver, PBMC, other
| ||||
End-stage liver disease | L, S, PBMC, PLN | HVR1 | Seq | [41] |
LT | L, S, PBMC, PLN | HVR1 | HMA, Seq | [101] |
LT | L, S, PBMC, PLN | HVR1 | Seq | [66] |
IFN α-treated and SVR occult | L, S, PBMC | 5'UTR | Seq | [61] |
IFN α-treated and SVR occult | L, S, PBMC, PLN | HVR1 | Seq | [100] |
Chronic | L, P, PBMC | HVR1 | Seq | [60] |
Chronic | L, S, PBMC | HVR1 | Clonal frequency analysis, Seq | [212] |
Chronic | L, S, PBMC | HVR1 | Seq | [196] |
Chronic | L, S, P, PBMC, monocytic DC | 5'UTR, HVR1 | Seq | [78] |
Chronic | L, P, PBMC | 5'UTR | INNO-LiPA | [62] |
b. Liver, other
| ||||
Cadavers | L, S, CNS, lymph | 5'UTR, HVR1 | Seq | [79] |
Co-infected | L, P, CNS | 5'UTR, E1 | Seq | [80] |
Chronic | L, S | 5'UTR, E2-NS2 | Seq | [86] |
Chronic | L, S | E2-NS2 | Seq | [87] |
Chronic | L, S | E2-NS2 | Seq | [88] |
Chronic | L, P, CNS | 5'UTR, E1 | Seq | [81] |
Chronic | L, S | 5'UTR | SSCP, Seq | [89] |
Chronic | L, PBMC, monocytic DC | 5'UTR | Seq | [77] |
c. PBMC, other extrahepatic
| ||||
Co-infected | P, PBMC, CSF | 5'UTR | Seq, INNO-LiPA | [75] |
Co-infected | S, PBMC, M, B, CD4+ T, CD8+ T | 5'UTR | SSCP, Seq | [70] |
Co-infected | S, PBMC | HVR1 | Seq | [105] |
Co-infected women | P, PBMC, Genital-cervicovaginal lavage fluid (CVL) | 5'UTR | SSCP, Seq | [90] |
LT | S, PBMC | 5'UTR | SSCP, Seq | [98] |
LT | P, PBMC | HVR1 | Seq | [99] |
LT | S, PBMC | 5'UTR | SSCP, Seq | [213] |
LT | S, PBMC | NS5B | Seq | [66] |
MC | P, PBMC, cryoprecipitate | 5'UTR, HVR1 | Seq | [65] |
Chronic | P, PBMC | 5'UTR, NS5B | Seq | [214] |
Chronic | P, PBMC | 5'UTR, HVR1 | Seq | [69] |
Chronic | P, PBMC | 5'UTR, NS5B | SSCP, Seq, INNO-LiPA | [63] |
Chronic | S, PBMC, CSF | 5'UTR | SSCP, Seq | [82] |
Chronic | P, PBMC | 5'UTR | SSCP, Seq | [67] |
Chronic | S, PBMC | HVR1,2+ | Seq | [64] |
d. Other extrahepatic
| ||||
Cadavers | S, CNS (brain autopsies) | 5'UTR | SSCP, Seq | [84] |
Co-infected | S, CSF | 5'UTR, HVR1 | SSCP | [83] |
Co-infected women | P, Genital-cervical cytobrush | 5'UTR, HVR1 | Seq | [91] |
Co-infected men | P, Semen | HVR1 | Seq | [92] |
LT | S, Brain | 5'UTR | SSCP, Seq | [85] |
Oral lichen planus, oral cancer | Oral lichen planus, oral cancer | 5'UTR, HVR1 | Seq | [74] |
Chronic | S, peripheral blood DC | HVR1 | Seq | [72] |
Chronic | P, BMC | 5'UTR | SSCP, Seq, INNO-LiPA | [76] |
Chronic | P, B, M, T | HVR1 | Seq | [71] |
Chronic | P, peripheral blood DC | HVR1 | Seq | [73] |
Chronic | P, M, B, CD4+ T, CD8+ T | 5'UTR | Seq | [58] |