Abstract
Peripheral blood mononuclear cells (PBMC) harbored TT virus (TTV) of genotypes (3 and 4) different from those (1 and 2) of free virions in plasma of the same individuals. PBMC may act as a reservoir, and TTV of particular genotypes might have tropism for hematopoietic cells.
TT virus (TTV) is a nonenveloped, single-stranded, and circular DNA virus with a genomic length of approximately 3.8 kb (8, 9, 14, 15) and is provisionally classified in the Circoviridae family (7). TTV is blood borne (10) and is excreted into feces via secretion from the liver into the bile (11, 26). Hence, the extensive spread of TTV in the general population of examined countries is caused not only by parenteral transmission via transfusion or illicit intravenous drugs but also by nonparenteral transmission through fecal-oral infection. TTV has an extremely wide range of sequence divergence by which at least 16 genotypes are classified (17). The nucleotide sequence of TTV is conserved to a higher extent in the untranslated region (UTR) than in coding regions, such as the N22 region in open reading frame 1 (15). As a result, TTV DNA is detected more frequently by PCR with UTR primers (UTR PCR) than with N22 primers (N22 PCR) (4, 5, 17, 22). UTR PCR detects TTV DNA of essentially all 16 genotypes, while N22 PCR detects primarily TTV DNA of genotypes 1 to 4 (11, 13, 14, 17). Mixed infection with TTV of distinct genotypes is common in healthy individuals and patients (1, 2, 17).
In previous studies, TTV DNA has been detected in peripheral blood mononuclear cells (PBMC) from infected individuals (13, 19). Genotypes can differ between PBMC and plasma from the same individuals (13). For further defining the presence of TTV in PBMC, the viral DNA was detected by UTR PCR and N22 PCR in paired plasma and PBMC samples from 108 healthy individuals in Japan. Furthermore, genotypes 1 to 4 were detected by PCR with type-specific primers in paired plasma and PBMC samples to find any differences in the distribution of genotypes between them.
TTV DNA in plasma and PBMC from healthy individuals, detected by UTR PCR and N22 PCR.
Individuals were selected who were negative for hepatitis B surface antigen (HBsAg) or antibody to hepatitis C virus and whose alanine aminotransferase levels were within the normal range (<45 U/liter) in Japan. There were 108 such individuals with the age (mean ± standard deviation [SD]) of 31.9 ± 12.7 years (range, 16 to 69 years), comprised of 57 males and 51 females. Table 1 shows the prevalence of TTV DNA in plasma and PBMC from the 108 individuals stratified by age. Nucleic acids were extracted from 50 μl of plasma by the High Pure Viral Nucleic Acid Kit (Boehringer Mannheim, Mannheim, Germany) and were dissolved in nuclease-free distilled water. Extracted nucleic acids corresponding to 25 μl of plasma served as the template for detection of TTV DNA by PCR. Nucleic acids were also extracted from PBMC equivalent to 2 ml of whole blood as described previously (13) and dissolved in 200 μl of Tris-HCl buffer (10 mM, pH 8.0) supplemented with 1 mM EDTA. A 10-μl portion thereof (equivalent to 100 μl of blood) was tested for TTV DNA by the two PCR methods.
TABLE 1.
PCR detection of TTV DNA in plasma and PBMC from healthy individuals
| Age (yr) | No. of patients | No. of patients possessing TTV DNAa
|
|||
|---|---|---|---|---|---|
| UTR PCR
|
N22 PCR
|
||||
| Plasma | PBMC | Plasma | PBMC | ||
| 16–19 | 26 | 26 (100) | 26 (100) | 4 (15) | 10 (38) |
| 20–29 | 29 | 26 (90) | 28 (97) | 3 (10) | 9 (31) |
| 30–39 | 21 | 20 (95) | 21 (100) | 4 (19) | 8 (38) |
| 40–49 | 19 | 18 (95) | 19 (100) | 6 (32) | 11 (53) |
| >50 | 13 | 13 (100) | 13 (100) | 3 (23) | 7 (54) |
| Total | 108 | 103 (95) | 107 (99) | 20 (19) | 45 (42) |
Numbers in parentheses are percentages of individuals in whom TTV DNA was detected.
UTR PCR, which detects TTV of essentially all genotypes, was carried out with nested primers by a slight modification of the method described previously (17). The first-round PCR was performed for 35 cycles with primers NG133 (sense, 5′-GTA AGT GCA CTT CCG AAT GGC TGA G-3′, representing nucleotides [nt] 91 to 115) and NG352 (antisense, 5′-GAG CCT TGC CCA TRG CCC GGC CAG-3′ [nt 229 to 252], R = A or G), and the second-round PCR was performed for 25 cycles with NG249 (sense, 5′-CTG AGT TTT CCA CGC CCG TCC GC-3′ [nt 111 to 133] mixed with an equal amount of the primer with the underlined four nucleotides replaced by ATGC) and NG351 (antisense, 5′-CCC ATR GCC CGG CCA GTC CCG AGC-3′ [nt 221 to 244]). The amplification product of the first-round PCR was 162 bp, and that of the second-round PCR was 134 bp. N22 PCR, which detects mainly genotypes 1 to 4, was performed with heminested primers as described previously (11, 14). The size of the amplification product of the first-round PCR was 286 bp, and that of the second-round PCR was 271 bp.
By UTR PCR, TTV DNA was found in plasma from 103 (95%) individuals and in PBMC from 107 (99%) individuals; only four individuals possessed TTV in PBMC without detectable free virions in plasma. There was only 1 (1%) individual among the 108 whose PBMC tested negative for TTV DNA. The frequency of TTV DNA detection by UTR PCR in either plasma or PBMC was high for all age groups. By N22 PCR, in contrast, prevalence rates were different between plasma and PBMC. TTV DNA was found in plasma from 20 of the 108 (19%) individuals and in PBMC from 45 (42%) individuals (P < 0.01 [chi-square test]). All of the 20 individuals whose plasma tested positive for TTV DNA also possessed TTV DNA in PBMC. TTV DNA detection by N22 PCR tended to be more frequent for the older individuals than for the younger individuals. TTV DNA was detected in plasma from the individuals aged ≥40 years about twice as frequently as in those <40 years old (9 of 32 [29%] versus 11 of 76 [15%]); the difference fell short of being significant, however (P = 0.09). The detection of TTV DNA in PBMC was significantly more frequent in individuals ≥40 years old than in individuals <40 years old (18 of 32 [56%] versus 27 of 76 [36%], P < 0.05). There were no differences in the detection of TTV DNA in plasma or PBMC between males and females.
Genotypes of TTV in plasma and PBMC from the 20 individuals whose plasma tested positive for TTV DNA by N22 PCR.
Using as a template the products of 286 bp in the first round of N22 PCR and in the presence of Perkin-Elmer AmpliTaq Gold (Roche Molecular Systems, Inc., Branchburg, N.J.), PCR was performed for 25 cycles (95°C for 30 s, with an additional 9 min in the first cycle; 58°C for 30 s; and 72°C for 40 s, with an additional 7 min in the last cycle) with type-specific sense and antisense primer pairs as described previously (12). These pairs were NG162-NG165 for genotype 1, NG198-NG174 for genotype 2, NG193-NG180 for genotype 3, and NG177-NG178 for genotype 4. Close to a single copy of TTV DNA of any genotype per tube was detected by the PCR. Semiquantitation of TTV of distinct genotypes was performed as follows. Nucleic acids extracted from plasma or PBMC were serially diluted 10-fold in distilled water containing 20 μg of glycogen (Boehringer Mannheim) per ml and tested for genotype 1, 2, 3, or 4 by PCR with N22 primers (NG059-NG063) in the first round and type-specific primers in the second round. The titer of TTV DNA of a certain genotype was expressed by the highest dilution (10n) testing positive.
Table 2 compares the four major TTV genotypes and TTV DNA titers between plasma and PBMC from the 20 (19%) individuals whose plasma and PBMC both tested positive for TTV DNA by N22 PCR. TTV of genotype 1 or 2 was detected in plasma and PBMC from 18 (90%) of them. A mixed infection with TTV strains of distinct genotypes in plasma was identified in six (30%) of them. At least one of the genotypes was genotype 1 in the six individuals with a mixed TTV infection; it was accompanied by genotype 2, 3, or 4. TTV genotypes detected in plasma were found invariably in PBMC from the same individuals in pairs. In addition, one or two TTV genotypes not present in plasma were detected in PBMC from eight (40%) individuals with TTV DNA titers of 101 or 102, except for two with a titer of 1. They all possessed genotype 3 or 4 or both in PBMC. These genotypes were not frequent in plasma, being detected in 5 (25%) of the 20 individuals. Genotype 3 or 4 or both occurred in PBMC from seven individuals who did not have TTV of these genotypes in plasma. Titers of TTV DNA of any genotype in PBMC (equivalent to 100 μl of blood) were equal to or 10 to 100 times higher than those in the corresponding plasma (25 μl).
TABLE 2.
TTV genotypes in paired plasma and PBMC samples
| Patient no. | Age (yr) and sexa | TTV genotype(s) (titer)
|
|
|---|---|---|---|
| Plasmab | PBMCc | ||
| 1 | 45, M | 1 (1) | 1 (101) |
| 2 | 32, M | 1 (102) | 1 (104), 3 (102) |
| 3 | 17, M | 1 (1) | 1 (101) |
| 4 | 27, F | 1 (101) | 1 (102) |
| 5 | 29, F | 1 (102) | 1 (102) |
| 6 | 32, F | 1 (102) | 1 (103) |
| 7 | 46, F | 1 (101) | 1 (101), 4 (102) |
| 8 | 43, F | 1 (101) | 1 (101), 3 (1) |
| 9 | 17, M | 2 (1) | 2 (101) |
| 10 | 45, M | 2 (101) | 2 (102) |
| 11 | 28, F | 2 (1) | 2 (101), 4 (101) |
| 12 | 18, F | 2 (1) | 2 (1), 3 (101), 4 (1) |
| 13 | 32, F | 4 (1) | 4 (1) |
| 14 | 45, F | 4 (1) | 4 (101) |
| 15 | 37, M | 1 (101), 2 (1) | 1 (102), 2 (1) |
| 16 | 52, M | 1 (103), 2 (101) | 1 (103), 2 (101), 3 (102) |
| 17 | 19, F | 1 (101), 3 (101) | 1 (101), 3 (102) |
| 18 | 53, F | 1 (1), 2 (1) | 1 (1), 2 (1), 3 (101) |
| 19 | 59, M | 1 (1), 3 (1) | 1 (1), 3 (101), 4 (1) |
| 20 | 49, M | 1 (102), 4 (1) | 1 (102), 4 (1) |
M, male; F, female.
TTV DNA titers in DNA copies per 25 μl of plasma are shown in parentheses.
TTV DNA titers in DNA copies in PBMC recovered from 100 μl of whole blood are shown in parentheses.
Genotypes of TTV in PBMC from the 25 individuals whose plasma was negative for TTV DNA by N22 PCR.
There were 25 (23%) individuals whose PBMC tested positive but whose plasma was negative for TTV DNA by N22 PCR. TTV genotypes were determined in PBMC from them, with the results shown in Table 3. Two distinct TTV genotypes were detected in PBMC from four of them, indicating a mixed infection. Genotype 1 or 2 was detected in PBMC from only 7 of the 21 (33%) individuals whose PBMC contained TTV of a single genotype. Genotype 3 or 4 was present in PBMC from the remaining 14 individuals. Of the 25 individuals with single or mixed TTV infection, TTV of genotype 3 or 4 or both occurred in 16 (64%) individuals, significantly more often (P < 0.01) than their occurrence in plasma from 5 of the 20 (25%) individuals who had TTV DNA in plasma detectable by N22 PCR (Table 3). Products from the second round of N22 PCR were inserted into pT7BlueT-Vector (Novagen, Inc., Madison, Wis.), and the clones obtained were sequenced by a method described elsewhere (17). The genotypes determined by PCR with type-specific primers were confirmed by the nucleotide sequences (Table 3). Titers of TTV DNA of any genotype in PBMC (equivalent to 100 μl of blood) were 102 in 3 individuals, 101 in 10 individuals, and 1 in the others.
TABLE 3.
Genotypes of TTV DNA in PBMC detectable by N22 PCR in the absence of TTV DNA in plasma
| Patient no. | Age (yr) and sexa | Genotype(s) determined by:
|
TTV isolate(s)c | |
|---|---|---|---|---|
| PCRb | Sequencing | |||
| 1 | 17, M | 1 (102) | 1 | TL01 (100) |
| 2 | 31, M | 1 (101) | 1 | TL02 (100) |
| 3 | 34, F | 1 (1) | 1 | TL03 (98.6) |
| 4 | 43, M | 1 (1) | 1 | TL04 (98.2) |
| 5 | 48, M | 1 (1) | 1 | TL05 (100) |
| 6 | 59, M | 1 (101) | 1 | TL06 (87.8) |
| 7 | 17, F | 2 (101) | 2 | TL07 (85.1) |
| 8 | 29, F | 3 (1) | 3 | TL08 (98.6) |
| 9 | 33, F | 3 (101) | 3 | TL09 (98.2) |
| 10 | 45, M | 3 (101) | 3 | TL10 (98.6) |
| 11 | 58, F | 3 (1) | 3 | TL11 (96.4) |
| 12 | 27, M | 3 (1) | 3 | TL12 (86.9) |
| 13 | 17, F | 3 (1) | 3 | TL13 (90.5) |
| 14 | 26, F | 3 (101) | 3 | TL14 (89.6) |
| 15 | 16, M | 4 (1) | 4 | TL15 (97.7) |
| 16 | 16, F | 4 (101) | 4 | TL16 (97.7) |
| 17 | 20, F | 4 (101) | 4 | TL17 (95.9) |
| 18 | 37, M | 4 (1) | 4 | TL18 (95.9) |
| 19 | 45, M | 4 (1) | 4 | TL19 (97.7) |
| 20 | 50, F | 4 (102) | 4 | TL20 (96.8) |
| 21 | 51, F | 4 (1) | 4 | TL21 (97.3) |
| 22 | 17, F | 1 (1), 2 (101) | 1, 2 | TL22-I (99.1), TL22-II (86.5) |
| 23 | 24, F | 1 (1), 2 (1) | 1, 2 | TL23-I (87.8), TL23-II (83.3) |
| 24 | 22, M | 2 (102), 4 (1) | 2, 4 | TL24-I (85.1), TL24-II (98.2) |
| 25 | 49, M | 3 (101), 4 (1) | 3, 4 | TL25-I (87.4), TL25-II (97.7) |
M, male; F, female.
TTV DNA titers in DNA copies in PBMC recovered from 100 μl of whole blood are shown in parentheses.
Discussion.
Detection of TTV DNA by UTR PCR and N22 PCR in 108 healthy individuals confirmed a very high prevalence of TTV infection in the healthy general population in Japan (17, 22). Of the 108 individuals tested, 103 (95%) possessed TTV DNA in plasma detectable by UTR PCR. The prevalence of TTV DNA detectable by UTR PCR was high in individuals over the ages 16 to 69, while that detectable by N22 PCR was more frequent in the older than in the younger individuals. The frequency of TTV DNA in PBMC was much higher than that in plasma, especially as determined by N22 PCR (19 versus 42%). Hence, certain genotypes of TTV appear to persist in PBMC after they are cleared from serum. For cytomegalovirus (CMV), CMV DNA was detected in 16 of 29 (55%) individuals who did not have evidence of infection detectable by antibodies to CMV (6). Individuals infected with TTV of genotype 1 clear the infection as they develop antibodies to TTV (25). They would, however, be able to harbor TTV of genotype 1 in PBMC, because it is secluded from circulating antibodies. This view needs to be evaluated by testing for genotype-specific antibodies in individuals who have TTV DNA of genotypes in PBMC not found in plasma.
There were remarkable differences in the distribution of TTV genotypes between PBMC and plasma. TTV genotypes not present in plasma were detected in 8 of the 20 (40%) individuals with TTV DNA detectable by N22 PCR in both PBMC and plasma, while genotypes detected in plasma were invariably found in PBMC. Genotype 3 or 4 or both were frequent in PBMC and were detected in 12 of the 20 (60%) individuals; they occurred in 8 of the 9 individuals who had the TTV genotypes in PBMC but not in plasma. By sharp contrast, genotype 1 or 2 prevailed in plasma and occurred in 18 of the 20 (90%) individuals. In the other 25 individuals with TTV DNA detectable by N22 PCR in PBMC unaccompanied by that in plasma, genotype 3 or 4 or both were prevalent and were detected in 16 (64%) individuals. Furthermore, mixed infection with TTV of two or more genotypes was more common in PBMC than in plasma. It was detected in 55% of PBMC, compared with 30% in plasma from the 20 individuals, and in 16% of PBMC from the 25 individuals. Mixed TTV infection has been reported to be present in sera from patients with hemophilia and those on maintenance hemodialysis (2, 23), and transfusions with blood or blood products and compromised immune response are implicated in it. The reported frequency of mixed TTV infection would be much higher if genotypes of TTV DNA were determined not only for sera but also for PBMC from these patients.
Observed differences in the distribution of TTV genotypes between PBMC and plasma might reflect the persistence of TTV infection in PBMC after the host clears it from serum. Furthermore, they might reflect a genotype-dependent infection of PBMC with TTV. There is a possibility that certain genotypes of TTV, such as 3 and 4, would have a predilection for hematopoietic cells, while those of the other genotypes have affinity with hepatocytes; replication of TTV in these two tissues has been indicated (16, 18). Such a tissue-dependent infection has been reported for different subtypes of human immunodeficiency virus type 1 (20, 21, 24).
Double-stranded TTV DNAs in replicative-intermediate forms are detected in bone marrow cells but not in PBMC (16). Hence, TTV would not be able to replicate in PBMC, leaving the reason for the presence of TTV in PBMC uncertain. There would be little, if any, passive absorption of TTV on PBMC, which were extensively washed by the method used. The detection of TTV genotypes in PBMC that were not present in the corresponding plasma lends supports to this view. High titers of TTV DNA of certain genotypes in PBMC may be taken as evidence of the accumulation of TTV in PBMC in infected individuals. Although TTV does not replicate in PBMC (16), it may well be infectious when introduced into other individuals by transfusion, and even in the same individuals when genotype-specific antibodies wane with time or are decreased by immunosuppressive drugs. Granulocyte-rich white cells are a major source of transfusion-transmitted CMV infection and are implicated in a third of the recipients of allogeneic marrow transplantation who acquire infection from seronegative donors (3). Concealed in a Trojan horse, TTV in PBMC would also serve as a reservoir of TTV for the transmission in some clinical and epidemiological settings.
Nucleotide sequence accession numbers.
The nucleotide sequence data in this paper have been deposited in the DDBJ/EMBL/GenBank nucleotide sequence databases under accession no. AB027199 to AB027227.
Footnotes
Corresponding author. Mailing address: Minamikawachi-Machi, Tochigi-Ken 329-0498, Japan. Phone: 81-285-58-7404. Fax: 81-285-44-1557. E-mail: immundiv@jichi.ac.jp.
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