Ttv dna virus

However, within a year after birth, the prevalence reaches the same level for children born to both TTV-positive and TTV-negative mothers even without breast-feeding. The non-coding region surrounding a short nt GC-rich stretch and occupying approximately one-third of the genome is considered to contain the putative replication origin.

A protein translated from the 3. The nature of the proteins translated by the other two mRNAs are still putative. To date, although T cells are considered to be the main site of TTV replication [23,24], other cell types may allow viral replication [21]. Additionally, among the body fluids examined, some studies have reported that TTV DNA is highly present in the saliva [25—28].

Therefore, saliva has gained an increased interest as a non-invasive screening approach to assess the TTV DNA viral load [28]. TTV DNA has been recently investigated in the saliva and compared with paired plasma samples obtained during allogeneic hematopoietic stem cell transplantation [29]. In order to provide clinical information in SOT recipients, this study aimed to investigate the TTV viral load in saliva and paired plasma samples in patients treated with renal transplantation.

Exclusion criteria were described in a previously published study by our group as follows: being submitted to dual kidney or multi-organ transplantation, undergoing immunosuppressive therapy prior to inclusion, being HIV-seropositive or presenting cognitive impairment affecting the ability to understand the informed consent form. Moreover, patients dropping out or having inconclusive blood or saliva samples for viral detection were also excluded from the study [30].

Clinical and demographic characteristics of the patients were collected during an interview. The samples saliva and blood were collected in three different experimental periods as follows: the first within 24 hours before renal transplantation, the second between 15 and 20 days and the third between 45 and 60 days after the surgery. Mouthwash samples were also collected from the patients, who were comfortably seated in a bright and well-ventilated room.

Blood samples were collected by nurses at the same moment of the saliva collection. All the participants signed an informed consent form. All samples were found to be suitable for DNA amplification.

The results were analysed with the SPSS software, version The Kruskall-Wallis test was used to observe whether viral loads in saliva and plasma were different between the immunosuppressive schemes used. Saliva and plasma samples were collected at three experimental times: 24 hours before transplantation, 15—20 days after transplantation and 45—60 days after transplantation.

In total, saliva and plasma samples were collected. Twenty patients The mean dialysis time was Most transplant organs came from living donors The most commonly used immunosuppressive regimen was a combination of tacrolimus, sodium mycophenolate and prednisone TTV DNA status in the paired saliva and plasma samples collected from the 71 patients before and after renal transplantation is shown in.

It was observed that the concordance was A higher viral load in plasma was observed for a drug regimen consisting of tacrolimus, sodium mycophenolate and prednisone compared to other regimens. Association of TTV viral load in saliva and plasma with immunosuppressive regimen used in the phase of maximum immunosuppression i. The comparison of TTV DNA status at different experimental times before and after transplantation in paired saliva and plasma samples is shown in and Figure 2.

Multiple comparative tests were also performed and showed that the DNA copy number of TTV in saliva differed significantly between the second and third collections, whereas in plasma it differed statistically in the third collection compared to the first and second one.

Viral loads in saliva and plasma were obtained by using total samples examined a , viral load in saliva and plasma obtained by using only the samples that were negative before transplantation time b and viral load in saliva and plasma obtained by using only the samples that were positive before transplantation time c is reported.

Additionally, it was of interest to examine the difference of the TTV DNA load in saliva and plasma coupled samples before and after 15—20 and 45—60 days renal transplantation. Then, the TTV DNA viral load was scheduled subdividing the total samples in the group of samples obtained from patients with a negative viral load Figure 2 b and in the group of samples obtained from patients with a positive viral load before transplantation Figure 2 c.

In this context, in presence of similar TTV viral load status before transplantation, it was observed that high differences of TTV DNA load in plasma samples mean of 4.

In this study, the TTV DNA status in paired saliva and plasma samples was investigated in 71 patients before and after 15—20 and 45—60 days renal transplantation. In saliva, particularly, the DNA copy number of TTV differed significantly between the second and third collections, whereas in plasma it differed significantly in the third collection compared to the first and second ones.

Additionally, plasma samples at 15—20 and 45—60 days after transplantation showed high increase of the TTV DNA viral load compared to the saliva samples at the same experimental times. In this context, the TTV viral load showed no normal distribution and showed high values in the plasma of patients on a drug regimen consisting of tacrolimus, sodium mycophenolate and prednisone compared to other regimens.

Several studies reported that monitoring of the TTV DNA load in blood may predict the risk of opportunistic infections and allograft rejection events in the SOT setting [22]. In particular, it has been shown that in SOT recipients e. Additionally, studies have investigated the kinetics of plasma TTV DNA after allogeneic hematopoietic stem cell transplantation, thus clearly demonstrating that the TTV DNA load decreases after conditioning therapy with an increased viral load and correlating the degree of T-cell immune reconstitution following engraftment [37—39].

Noteworthy, these data support the assumption that T-cells are the major site of TTV replication [23,37—41]. To date, there are studies on TTV DNA in saliva proposing that the oral cavity is another site for TTV viral replication or transmission route, thus potentially contributing to the total viral load [24—29].

Thus, recently the TTV DNA load in saliva after allogeneic hematopoietic stem cell transplantation was used to assess the potential utility of a non-invasive and rapid biological fluid collection thus replacing or complementing plasma sampling for predicting lymphocyte reconstitution after engraftment [29]. In the latter study, the TTV DNA loads were significantly higher in saliva than in plasma samples, which showed a direct correlation between TTV DNA loads in saliva and plasma and lymphocyte reconstitution after engraftment [29].

In our study, the TTV viral load in saliva was constantly found to be lower than in paired plasma samples, but with a similar increasing kinetics compared to that observed in plasma after transplantation. Also, a high increase of the TTV DNA viral load was observed in plasma samples at 15—20 and 45—60 days after transplantation compared to that obtained in paired saliva samples, especially in samples with positive viral load before the transplantation.

These observed differences between our study and previous ones could be related to the different clinical status of patients undergoing renal transplantation or allogeneic hematopoietic stem cell transplantation and to the drug regime used.

Also, since the TTV viral load in blood has been shown to have a wide variation, it should not be excluded that the methodology used in our study for DNA extractions from saliva and quantification of TTV DNA, could have contributed to the different results reported by other studies [25,26,29]. However, our study, performed in SOT patients in the absence of haematological disorders, confirmed that the TTV viral load is mainly associated to the viral replication in the lymphocytes replication-competent cells [23,37,38].

Also, this study reported that TTV viral shedding at early time after transplantation, in the saliva of patients with negative TTV DNA before transplantation, showed a viral load similar to that observed in paired plasma. Our study is hampered by several limitations, such as low number of samples analysed during the post-transplantation period, short-time period of examination after transplantation and lack of lymphocyte count assessment for evaluation of immune functionality.

Nevertheless, our data confirm that an easy and non-invasive sequential monitoring of the TTV DNA load in saliva samples can allow the assessment of the degree of immune functionality after transplant engraftment in combination with the monitoring of the plasma TTV DNA load in SOT patients. Therefore, further studies involving larger cohorts during a long time after transplantation should be conducted to confirm the potential of saliva sampling as an additional source to evaluate the TTV viral load in combination with plasma samples in SOT patients.

No potential conflict of interest was reported by the author s. Read article at publisher's site DOI : To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation. J Med Virol , 90 9 , 25 May Cited by: 1 article PMID: Viruses , 12 11 , 11 Nov Biol Blood Marrow Transplant , 26 4 , 09 Nov Cited by: 8 articles PMID: Transplant Rev Orlando , 33 3 , 04 Apr Cited by: 13 articles PMID: World J Gastroenterol , 26 15 , 01 Apr Contact us.

Europe PMC requires Javascript to function effectively. Recent Activity. Search life-sciences literature Over 39 million articles, preprints and more Search Advanced search. Search articles by 'Alexandre Mendes Batista'. Batista AM 1 ,. Caetano MW 1 ,. Stincarelli MA 2 ,. Search articles by 'Ana C Mamana'. DNA species of chromosomal origin, which emerged as a cloudy precipitate immediately after addition of ethanol, were removed.

The concentration of TTV in plasma is estimated to be 50 to 50, geometric mean, copies per ml Since sera and liver tissues from infected individuals contained TTV DNA in titers too low to be detected by Southern hybridization, the length and strandedness of the TTV genome were determined by the following procedure.

After electrophoresis, the gel was stained with ethidium bromide. TTV DNA samples extracted from paired serum and liver tissues from three viremic patients Cases 1 to 3 along with a molecular size marker bp DNA ladder [TaKaRa Shuzo] were subjected to electrophoresis, and areas corresponding to 1. The size of DNA was estimated by reference to slice numbers. The products were subjected to electrophoresis, and signals were compared for intensity.

Louis, Mo. Based on these findings, the gel corresponding to this size was cut into 20 slices at 2. The amplification was performed with or without digestion of the recovered DNAs from patients 1 to 3 by the restriction endonuclease Nde I TaKaRa Shuzo under conditions described previously The products of long-distance PCR with nested primers for amplification of the entire genome and those of inverted PCR are shown in the periphery.

ORF, open reading frame. B; upper panel Agarose gel electrophoresis of the amplification products of the long-distance PCR. The products of 3. An inverted PCR was performed, with the primers indicated in Fig. The polarity of two sequences was evaluated see regions a and b in Fig. One sequence, spanning nt to , was extended with the sense primer NG or the antisense primer NG, and the products were amplified with a primer pair NG and NG , which gave rise to a fragment of bp.

The other sequence, stretching from nt to , was extended with the sense primer NG or the antisense primer RD, and the products were amplified with another primer pair NG and NG to generate a fragment of bp.

The extension was also performed without primers to obtain mock products. An intensification of PCR signals, with prior extension by the used primer, verified the extension; it was attributed to the nature of the target strand that was complementary to the primer.

Using DNAs extracted from serum or liver tissues as templates, strand-specific primer extension was performed on two genomic regions a and b with sense or antisense primers specific for TTV. Nucleotides were numbered from position 1 nt 1 of the coding strand of the prototype TTV isolate of genotype 1a TA 21 , 22 , complementary to the viral strand, because TTV was found to have a minus-stranded DNA in the circulating virion.

B Amplification of extension products by PCR. Using extension products as templates, PCR was performed with primer pairs internal to those used for primer extension.

The extension was performed without primers to obtain mock products. The amplification products were subjected to electrophoresis, and signals were compared between the products with and without prior extension. DNAs recovered from paired serum and liver tissues from the three viremic individuals patients 1 to 3 were subjected to electrophoresis Fig.

One of them migrated at 2. Hence, these slower-migrating forms of TTV DNAs in liver tissues are in double-stranded, replicative intermediate forms. They were linearized to have a genomic size of 3.

Slower-migrating DNAs recovered from liver tissues were confirmed to have the nucleotide sequences specific for TTV by the following procedure. Slower-migrating TTV DNAs in liver tissues from patients 1 to 3 were recovered from gel slices slice 13, corresponding to 3. The slower-migrating DNA from patients 1 and 2 possessed sequences On the basis of these results, TTV DNAs in serum were single stranded while those in liver tissues existed in both single- and double-stranded forms.

These results were in agreement with the previous observation that TTV DNA in plasma is digested with mung bean nuclease that has a specificity for the single-stranded DNA A product of 3. The sequences were The connection between nt and was confirmed by determination of the bp sequence. The same results were obtained over two distinct regions a and b of the TTV genome. A weak signal was detected by PCR with region b primers in lanes with no primers. Hence, weak signals observed for the extension products with the sense primer on the single- and minus-stranded TTV DNA, as well as those for the extension products observed with the antisense primers on the single- and plus-stranded TTV DNA, were attributed to the amplification of these regions on the template.

The lower intensities of signals for region a than region b were due to the sizes of products of region a being less than half those of region b. The findings with single-stranded TTV DNA of plus or minus polarity attested to the credibility of extension procedures used to determine the polarity. When TTV DNAs from the serum of patient 1 were extended by these procedures, the amplification signal was intensified remarkably by prior extension with sense, but not antisense, primers for the two regions examined Fig.