Serum concentrations of TT virus and TT virus-like mini virus in patients developing AIDS

Changes in viraemia of two chronically replicating circoviruses, TT virus (TTV) and TTV-like mini virus (TLMV), were investigated in patients developing HIV-related immune deficiency. In a few cases, episodes of a considerable increase in viraemia were observed, but in most patients there were no discernible increases as immune deficiency progressed. The deterioration of immune function associated with AIDS did not by itself have any obvious effect on TTV and TLMV.

The immune dysfunction caused by HIV increases the chance of various other infectious agents proliferating, both the re-emergence of latent agents and de novo infections. Some agents seem more likely to take advantage of this opportunity than others, and it is not obvious whether this variation reflects differences in exposure, or the extent to which the various agents are affected by the particular section of the immune system disrupted by HIV. The human circoviruses TT virus (TTV) and TTV-like mini virus (TLMV) offer a chance to probe the effect of AIDS-related immune dysfunction on chronically replicating viruses.

It has previously been reported that AIDS, or a decrease in the CD4 cell count, correlates with an increase in TTV viraemia [^1-3], suggesting that the increase is a direct consequence of immune dysfunction. One report even suggested that TTV may act as a co-factor in the progression towards AIDS [¹]. These reports, however, have been based on group averages, rather than on following individual patients. In the present paper, serum titres of TTV and TLMV were measured in serial samples taken before and after the diagnosis of AIDS in order to get a better understanding of how these viruses respond to HIV-related immune deficiency.

TTV is capable of replicating continuously over several years, as evidenced from serial samples in which the same DNA sequence was found repeatedly [^4,5]. Moreover, recent, more sensitive, detection methods have suggested that TTV is present in at least 90% of the general population [^6,7]. Increased prevalences have been reported for HIV-positive individuals [^1,2], patients with hepatitis [⁸] or cancer [⁹], but in most of these reports all prevalences were well below 90%. The apparent differences may thus be partly due to the fact that an increase in viraemia will ease viral detection. If so, the differences in prevalence referred to above may be explained by assuming that various factors that disrupt health are likely to correlate with an elevated activity of TTV. It follows that the particular immune dysfunction caused by HIV is not necessarily the primary cause of any increase in circoviral viraemia observed in HIV patients. One purpose of the present investigation was to address this issue.

Individuals were selected from the Oslo HIV Cohort Study on the basis of the availability of ample serum samples taken before and after the diagnosis of AIDS, and the availability of laboratory results and clinical information. The diagnosis of AIDS made on the basis of the prevailing WHO recommendation at the time [¹⁰]. A total of 77 serum samples, from 12 individuals, taken in the period 1989-1995 were analysed. Various parameters were recorded at the time of sampling, the more important of these in this context are the CD4 and CD8 cell counts, the use of antiretroviral medicine (zidovudine), and clinical information including diagnosed co-infections.

Aliquots of 200 μl of serum, kept at -80°C, were used for the extraction of DNA with a QIAamp DNA Blood Mini Kit (Qiagen 40724 Hilden, Germany), according to the manufacturers' recommendations. The DNA was analysed using a TaqMan probe based real time polymerase chain reaction (PCR) as previously described [¹¹]. The primers were proved to be specific for TTV and TLMV, respectively, by analyses of sequenced PCR products [¹¹]. The C_T score was converted to the number of viral products by using a standard curve based on the amplification of dilutions of purified PCR products, obtained by primers other than those used in the real time PCR. TTV and TLMV analyses of samples from a single patient were performed in the same set-up, with three parallels of each sample and negative and positive controls included.

Some variations in titres were observed in all 12 HIV patients. Data for six of them are shown in Fig. 1. In two cases in particular, TTV and TLMV varied in a parallel fashion: the increase and subsequent decrease in viral load of case 1, and the extreme increase in titres observed towards the end in case 3. In case 6, TLMV increased subsequent to the diagnosis of AIDS, but TTV did not. In the remaining nine cases the diagnosis of AIDS had no obvious impact on TTV or TLMV titres, although the titres varied significantly over time. When comparing the average of TTV and TLMV titres before and after the AIDS diagnosis in all 12 patients, the amount of virus increased fourfold and twofold, respectively, but the differences were not significant (in the case of TTV, P = 0.07 was close to significance in a paired samples test). The CD4 cell count, or CD4 : CD8 cell ratio, are considered better indicators of remaining immune function than the somewhat arbitrary diagnosis of AIDS; however, these immune indicators did not offer any obvious additional explanatory power as to viral titres than did the AIDS diagnosis.

Fig. 1

Six of the patients were, part of the time, receiving antiretroviral medication in the form of zidovudine. There was no obvious correlation between this medication and TTV or TLMV titres, neither were there any obvious correlations as to the presence of secondary infections or other clinical conditions. In the three cases discussed above with considerable variations in circovirus viraemia, two genital herpes infections, one incidence of myelopathy, one Candida infection, and one incidence of dermatitis were recorded around the time of the AIDS diagnosis.

In order to investigate further the above-mentioned episodes of increased viraemia, the PCR products of selected samples were sequenced with a cycle sequencing kit, as previously described [¹²]. In case 1, the TTV sequence was unchanged from the second to the third sample (before and at the high titre), whereas the fifth sample revealed a double infection. In case 3, the TTV sequences of the fourth, fifth and seventh samples were identical, whereas the TLMV sequences of the sixth sample had a TA dinucleotide in place of an AT in the fourth sample. Considering the sequence heterogeneity of these viruses, this alteration seems more likely to be caused by mutation than by a novel infection. In case 6, however, the TLMV sequence of sample number 5 was only 88% identical to the third sample, suggesting superinfection.

The observed changes in TTV/TLMV titres could reflect co-infection with other agents, or some host factor possibly related to the HIV infection, or be a consequence of exposure to novel TTV/TLMV subtypes for which the immune defence was less prepared. In two of the cases (1 and 3), TTV and TLMV titres increased in a coordinated fashion, and in the absence of any appreciable change in DNA sequence; whereas in patient no. 6, in whom only the TLMV titre increased, the rise was accompanied by a change in sequence. This pattern suggests that in cases 1 and 3, host factors or co-infections were more likely to explain the elevated viraemia, whereas in case 6, the introduction of a novel TLMV could be the pertinent factor.

Although we did observe, in line with previous reports [^1-3], an overall trend in the direction of higher serum concentrations of TTV and TLMV as the HIV infection progressed; in most patients there were no appreciable changes. Moreover, in the three patients in whom distinct increases in circoviral activity were observed (cases 1, 3 and 6), only in one (case 3) was there a sustained augmentation of both viruses. HIV-related immune deficiency is thus apparently not sufficient to cause an increase in circovirus viraemia.

Both specific antibodies and interferon are apparently involved in clearing TTV particles from the blood [^13-15]. On the other hand, the previously reported inverse relationship between CD4 cell counts and TTV viraemia [^2,3], does not necessarily imply that the CD4 cell-dependent section of the immune system plays a role in fighting circoviruses. According to the present results, in a majority of patients even a substantial depletion of CD4 cells was not accompanied by elevated levels of TTV or TLMV. The more likely explanation seems to be that the HIV-induced depletion of CD4 cells occasionally induces some factor(s) that are responsible for the infrequent increase in circovirus viraemia. The elevated viraemia might be related to a suppression of particular sections of the immune system, such as phagocytic cells or interferon production, or other host factors could be responsible. Circoviral replication apparently depends on cell division [¹⁶], thus a stimulation of the replication of cells in which TTV and TLMV replicate, most likely liver cells [¹⁷] and bone marrow/mononuclear cells [^18,19], could boost viral activity.

Eva M. Moena

Jowita Slebodab

Bjørn Grindea

Acknowledgements

The authors would like to thank Sanela Numanovic for technical assistance, and Tom Øystein Jonassen for designing oligonucleotides, and for critical reading of the manuscript.