Presence of DNA of adeno-associated virus in subfertile couples, but no association with fertility factors.

BACKGROUND: Based on previous reports suggesting a role of adeno-associated virus (AAV) in miscarriage, the prevalence of AAV DNA in genital tracts of male and female partners of subfertile couples was determined to assess a potential association of AAV infection with clinically relevant parameters of male and female fertility. METHODS: A prospective study was performed in the outpatient infertility clinic of a university-based hospital. Semen samples and endocervical material obtained from 146 male and 134 female partners of asymptomatic subfertile couples were analyzed for the presence of AAV DNA (using nested PCR). Patients' medical histories and details of clinical examinations were recorded. Semen quality, including sperm functional capacity and the presence of antisperm antibodies (ASA) and seminal white blood cells (WBC), was assessed in aliquots of the same ejaculate. Detailed examinations of the cervical factor and other variables of female subfertility were performed. Both partners were screened for bacterial infection. RESULTS: The presence of AAV DNA in semen was not significantly related to semen quality, including sperm functional capacity or local ASA, nor was it coupled to the presence of AAV in the endocervical material of female partners. The presence of AAV DNA was not associated with the presence of other micro-organisms of the lower genital tract or with seminal WBC in men. AAV DNA in endocervical material was not related to a reduced quality of cervical mucus or to other female infertility factors. CONCLUSIONS: The presence of AAV DNA in semen samples or endocervical swabs showed no significant association with clinically relevant infertility factors. However, longitudinal studies may clarify previous suggestions of an influence of AAV infection on early pregnancy problems.

Lack of association of herpesviruses with brain tumors.

Gliomas are the most frequent primary brain tumors in humans. Many studies have been carried out on their etiology; however, the only confirmed risk factors are hereditary predisposing conditions and high dose of ionizing radiation. Recently, human cytomegalovirus (HCMV) gene products and nucleic acids were reported to be present in all of 27 glioma samples investigated in contrast to other brain tissues, and it was hypothesized that HCMV might play a role in glioma pathogenesis. To evaluate these findings, samples of 40 gliomas, 31 meningiomas, and 6 acoustic neurinomas (ACNs) were analyzed for the presence of HCMV macromolecules using polymerase chain reaction (PCR) and immunohistochemistry. Additionally, corresponding blood samples from 72 patients were analyzed for the presence of HCMV DNA to check for a possible contamination of tumor tissues with HCMV-infected blood cells. No HCMV DNA sequences were found, neither in brain tumor tissues nor in corresponding blood samples. Immunohistochemistry did not detect HCMV-specific proteins. Addressing a possible role of other herpesviruses as has been suggested in seroepidemiological studies, seroprevalence of antibodies to HCMV, herpes simplex virus (HSV), Epstein-Barr virus (EBV), and varicella-zoster virus (VZV) were determined by enzyme-linked immunosorbent assay (ELISA). Serological analyses of brain tumor patients showed no significant differences in the prevalences of antibodies to HCMV, HSV, EBV, or VZV compared to the general population. Thus, the data of the present study do not support the hypothesis of an association of herpesviruses with the development of primary brain tumors.

Oncolytic potential of rodent parvoviruses for cancer therapy in humans: a brief review.

Summary Rodent parvoviruses are promising candidates for oncolytic virotherapy of cancer in humans because of their oncotropism (preferential killing of transformed cells) in the absence of pathogenicity. Here, we give an overview concerning the possible application of parvovirus H-1 for cancer therapy, with specific emphasis on malignant brain tumours in humans.

Adeno-associated virus DNA in human gestational trophoblastic disease.

Previous studies had shown a correlation between infection with the human adeno-associated virus (AAV) and spontaneous abortion in early pregnancy. Furthermore, AAV DNA had been detected in cells of the human trophoblast lines, Jeg-3, JAr, and BeWo, in cells of the human amnion line, FL, and in trophoblasts from amnion fluids. Infectious AAV virions could be isolated from amnion fluids. To further analyse AAV infection during pregnancy, we tested material from Gestational Trophoblastic Disease for the presence of AAV DNA. With 63 tissue samples from patients from Brazil, including 49 hydatiform moles and 14 choriocarcinomas, nested PCR was performed to detect the presence of AAV DNA. In addition, 15 samples from spontaneous abortions were analysed. AAV DNA was found in 43 samples (28/49 hydatiform moles, 4/14 choriocarcinomas, 11/15 miscarriage material). These findings confirm AAV infection of embryo-derived tissue in humans and further suggest a role of AAV in miscarriage and trophoblastic disease.

Adeno-associated virus seropositivity and HPV-induced cervical cancer in Spain and Colombia.

Extensive experimental and limited epidemiologic data suggest that adeno-associated viruses (AAV) can have antioncogenic activity and may be protective factors for the development of cervical cancer. To examine the association between AAV-2 IgG antibodies and cervical neoplasia in Spain and Colombia, we tested for AAV-2 antibodies using an ELISA assay for 109 women with invasive cervical cancer, 100 population-based controls age-matched to the invasive cases, 77 women with carcinoma in situ (CIN III) and 100 clinic-based controls age-matched to the CIN III cases. Human papillomavirus (HPV) DNA was detected in cervical exfoliated cells by polymerase chain reaction using HPV-L1 and GP5+/6+ consensus primers. The prevalence of AAV-2 antibody titers >100 was significantly lower in invasive cervical cancer cases than control participants. When comparing women with invasive cancer with controls or with CIN III cases, a pattern of decreasing cervical cancer risk with increasing AAV-2 titers was observed. Elevated AAV antibody titers (>100) were inversely associated with invasive cervical cancer (OR 0.3; 95% CI 0.1-0.7), although results were not statistically significant after controlling for HPV (OR 0.4; 95% CI 0.1-1.6). In contrast, AAV-2 antibodies were not significantly associated with the risk of CIN III (OR 1.4; 95% CI 0.3-6.8). These results provide supportive evidence that AAV infection may be a protective factor for the development of invasive cervical cancer. Alternatively, the lower AAV-2 seroprevalence in invasive cervical cancer cases may be due to an immunosuppressive effect of cervical cancer on AAV antibody response. To investigate whether a direct viral interaction is occurring, future studies should aim to resolve at what frequency AAV is found in the genital tract and to clarify further whether AAV may infect the same HPV-positive cells in the cervix.

DNA of adeno-associated virus (AAV) in testicular tissue and in abnormal semen samples.

BACKGROUND: Human genital tissues, including spermatozoa, have been found to be frequently infected with the helper-virus dependent parvovirus, adeno-associated virus (AAV). METHODS: To assess the role of AAV infection in disorders of the male reproductive system, semen samples from 95 men (including 73 men attending a fertility programme) and testicular samples from patients with azoospermia (n = 38) or prostate cancer (n = 8) were analysed using polymerase chain reaction for the presence of AAV DNA. Semen quality was assessed according to World Health Organization guidelines and the grade of atrophy of testicular biopsies was determined histomorphologically. RESULTS: AAV DNA was detected in 38% (28/73) of ejaculates from men with abnormal semen analyses (oligoasthenozoospermia or asthenozoospermia) and in 4.6% of normal semen samples (1/22, P = 0.003). DNA from AAV helper-viruses (human papillomaviruses, cytomegalovirus) was detected at similar frequencies in normal and abnormal semen samples. In testes, AAV DNA was detected in 10 out of 38 biopsies from infertile men (26%), and in 2 out of 8 orchidectomy samples. CONCLUSION: The data show an increased incidence of AAV infection with abnormal semen analysis. Detection of AAV DNA in the testes might point to a role for AAV infection in male infertility, possibly by interfering with spermatozoa development.

Reduced prevalence of serum antibodies against adeno-associated virus type 2 in patients with adult T-cell leukaemia lymphoma.

Seroepidemiological studies have shown previously that cancer patients are less likely to have antibodies against the tumour suppressive adeno-associated virus (AAV) than control groups. To examine the influence of AAV infection on the development of adult T-cell leukaemia lymphoma (ATLL), an endemic disease in Southern Japan that is caused by infection with the human T-cell leukaemia virus type 1 (HTLV-I), the prevalence of serum antibodies to AAV type 2 (AAV-2) was tested in healthy HTLV-I carriers (n = 39) and patients with ATLL (n = 31). The results showed a significant difference in AAV-2 seropositivity between the two groups: Only 29% of the ATLL patients had IgG antibodies against AAV-2, whereas 84.6% of the healthy HTLV-I carriers were seropositive. Analysis of total serum IgG and antibodies against the Epstein-Barr virus (EBV) EBNA1 antigen showed that the lack of AAV antibodies in patients was not due to an ATLL-associated immune deficiency. The lower level of AAV-2 seropositivity in ATLL-patients may indicate that AAV-2 antibody-positive HTLV-I carriers might be less likely to develop ATLL or that loss of AAV-2 antibodies may parallel the development of disease.

Detection of adeno-associated virus in human semen: does viral infection play a role in the pathogenesis of male infertility?

OBJECTIVE: To evaluate the occurrence of adeno-associated virus (AAV) DNA and/or human papillomavirus (HPV) DNA in the semen of infertile men as a possible factor in the pathogenesis of male infertility. DESIGN: Descriptive pilot study. SETTING: University-based diagnostic and research laboratory. PATIENT(S): Semen specimens were collected from 30 men with diagnosed infertility and from 8 control subjects. INTERVENTION(S): Diagnostic spermiograms were made and the semen specimens were separated into seminal fluid, nonspermatozoal cells, and spermatozoa using a Ficoll gradient technique. MAIN OUTCOME MEASURE(S): The presence of AAV and HPV DNA in the different fractions of the ejaculates from the infertile men and the control subjects was detected by polymerase chain reaction. Semen quality was analyzed according to World Health Organization guidelines. RESULT(S): Adeno-associated virus DNA was detected in 30% (9/30) of the ejaculates from the infertile men. No AAV DNA was found in the ejaculates from the 8 control subjects. In 8 of 9 samples, AAV DNA could be found only in the spermatozoal fraction of the specimen. Seven of 9 semen specimens that contained viral DNA also demonstrated oligoasthenozoospermia. Both AAV and HPV DNA was found in the spermatozoal fraction of 3 of 30 specimens. CONCLUSION(S): The data demonstrate for the first time the occurrence of AAV infection in human semen. Sperm motility seems to be affected by the presence of AAV.

[Natural infection with adeno-associated viruses]. / Infection naturalle à virus adéno-associés.

Adeno-associated viruses (AAV) are parvoviruses which exhibit oncosuppressive properties as well as unique characteristics of integration. They have never been associated with human diseases. AAV are thus considered promising vectors for the corrective therapy of various gene defects. In this review, the (possible) consequences of AAV infection for human health, cancer development and recombinant AAV vectors are discussed with respect to recent results on the cellular and molecular targets of AAV infection in humans.

Evidence for infection of the human embryo with adeno-associated virus in pregnancy.

Previous reports have demonstrated the presence of DNA of the human helper virus-dependent adeno-associated parvovirus (AAV) in uterine tissue and curettage material from early miscarriage. To examine infection of embryonic tissue during pregnancy, amnion fluids were analysed for the presence of AAV. Using polymerase chain reaction, AAV DNA was detected in 64 out of 238 DNA samples extracted from amnion cells. DNA of helper viruses were found in 12% (papillomavirus) and 18% (cytomegalovirus) of the samples (double infections with AAV in eight and nine cases, respectively). Furthermore, infectious AAV virions were found in 13 out of 43 AAV DNA-containing samples. In mothers with AAV DNA-positive amnion fluids, premature amniorrhexis and premature labour occurred significantly more frequently (P < 0.001). Using an immunofluorescence assay, 24% of newborn sera (unrelated to the amnion fluid samples) were found to contain IgM antibodies to AAV, in most cases paralleled by IgM antibodies in the mother's sera. The data demonstrate that AAV infection can occur in utero at early and at late stages of pregnancy. The observed complications at delivery should encourage studies to clarify possible pathological consequences of AAV infection in pregnancy and a possible latent infection of the fetus.

Update on the prevalence of serum antibodies (IgG and IgM) to adeno-associated virus (AAV).

In view of presumed non-pathogenicity, tumor suppressive properties, and site-specific integration of the viral genome the human parvovirus, adeno-associated virus (AAV) has gained great interest as a gene transduction vector. Data on the seroprevalence of antibodies to AAV vary between reports, probably due to the different serological methods used. In order to understand better the immune response to AAV during natural infection, sera from different age groups and various geographical regions were compared for AAV antibodies using an ELISA. The data show that the prevalence of antibodies to AAV is similar in Europe (Germany, France, and Switzerland), Brazil, and Japan, indicating worldwide infection. It was confirmed that infection takes place during childhood. However, declining seropositivity thereafter and a second increase of seropositivity after 30 years of age suggests reinfection or reactivation of latent virus in particular as the prevalence of IgM antibodies in adults is relatively high. Furthermore, pregnant women were found to be significantly more frequently seropositive than non-pregnant controls, hinting at a reactivation of persistent AAV (up to 80% of women carry AAV in genital tissue) in specific hormonal conditions, e.g., pregnancy. Cross-reaction of serum antibodies with the different AAV types (defined by complement fixation) was observed by ELISA and neutralization tests confirming earlier results. The results suggest an unstable AAV antibody response allowing lifelong reinfection or reactivation of persisting virus possibly due to partial immunotolerance after an infection in utero, at delivery or during early infancy.

Functional analysis of different LMP1 proteins isolated from Epstein-Barr virus-positive carriers.

The Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and is implicated in the development of several human malignancies. Latent membrane protein 1 (LMP1), an EBV protein with known oncogenic properties, may be important in the pathogenesis of EBV-associated tumors, particularly nasopharyngeal carcinoma (NPC) and Hodgkin's disease (HD). Several reports suggested that sequence variations in the LMP1 gene may define a more aggressive, geographically restricted EBV-genotype. Most mutations in the LMP1 gene described are located within the C-terminus of the protein. However, the effect of these mutations on the biological function of the protein remains widely unknown. Therefore, this study aimed in investigating whether mutations detected in LMP1 genes isolated from different EBV-positive carriers have an effect on the biological function of the protein. For this purpose the LMP1 genes were amplified by nested PCR from DNA out of bone marrow and peripheral blood lymphocytes and sequenced. Three functional assays were performed in order to evaluate the biological activity of the different isolates: activation of the transcription factors NF-kappaB and AP-1 as well as the anchorage independent growth of LMP1 transfected ratl cells in soft agar. The results suggested that whereas differences in the activation of NF-kappaB through the various LMP1 isolates correlated tightly with their different expression levels, the outgrowth of transfected cells in soft agar did not and the transcription factor NF-kappaB therefore appeared not to be the major effector for the transformation of the rodent cell line ratl by LMP1. The various LMP1-isolates also differed in their capacity in activating the transcription factor AP-1. We found no correlation between the transforming ability of the LMPI isolates and activation of AP-1 suggesting that other so far uncharacterized domains also influence the transforming ability of the protein.

Enhanced sensitivity of small cell lung cancer cell lines to cisplatin and etoposide after infection with adeno-associated virus type 2.

In previous studies we have reported the sensitisation of human tumour cells to gamma irradiation and chemotherapeutic drugs upon infection with the human non-pathogenic adeno-associated virus type 2 (AAV-2) in vitro and in vivo. Treatment of small cell lung cancer (SCLC) is consistently hampered by relapses due to the selection of chemotherapy-resistant cell clones. Hence, we were interested to test whether selection of chemotherapy-resistant SCLC cells might be reduced or even prevented if chemotherapy is applied in combination with AAV-2 infection. In vitro proliferation assays indicated that the number of proliferating cells, after combined treatment with cisplatin and etoposide, can be significantly reduced by concomitant AAV-2 infection, as compared with treated but non-infected controls. H446 SCLC cells, which show resistance to etoposide/cisplatin chemotherapy (compared with a cell line which was never chemotherapeutically treated before, like NCI-H209) were significantly more sensitive after AAV-2 infection, suggesting that the therapeutic efficacy of chemotherapy in SCLC can be enhanced even if the cells are already relatively resistant to chemotherapy. Similarly, in vivo growth of tumours induced by inoculation of SCLC cells into immunocompromised nude mice was reduced more efficiently in AAV-2-infected animals compared with tumours in mice treated with chemotherapeutic drugs alone. These data extend and further support our previous reports on AAV functions which might be useful in improving the efficacy of chemotherapeutic drugs used in human cancer treatment.

Detection of infectious adeno-associated virus particles in human cervical biopsies.

Recently we reported that DNA of the human oncogenic papillomaviruses (HPV) and the tumor suppressive human helper virus-dependent parvoviruses, adeno-associated viruses type 2 (AAV-2), colocalize in cervical epithelium. To analyze whether infectious AAV particles are present in cervical tissue, we examined cervical biopsies from 36 patients with HPV-related lesions (squamous intraepithelial lesions) for the presence of AAV DNA and of infectious AAV. From each patient specimens from the lesion and from adjacent normal epithelium were analyzed. After PCR analysis AAV DNA-containing samples were purified by CsCl gradient centrifugation. The presence of AAV virions in CsCl gradients was analyzed and infectivity of AAV was determined. In addition, the biopsies were tested for the presence of HPV DNA. AAV DNA could be detected in biopsies from 23 of 36 patients. AAV particles were found in 11 AAV DNA-positive biopsies from 7 patients (lesions and/or normal tissue, respectively). AAV particles were found to be infectious virions in 10 of the 11 cases. These results demonstrate for the first time that infectious AAV can be isolated from human cervical biopsies, indicating a possible sexual transmission of AAV.

Discrimination between different types of human adeno-associated viruses in clinical samples by PCR.

Persistent infection of human tissues with the helper virus-dependent parvovirus, adeno-associated virus (AAV) was detected by polymerase chain reaction (PCR) using primer pairs detecting AAV types 2, 3 or 5. In order to develop PCR protocols which discriminate between the different serotypes of AAV, the DNA of AAV-5 was sequenced partially and compared with the published sequences of AAV-2 and -3. Type specific oligonucleotides and specific probes which allow the distinction between human AAV types by PCR are described.

Dose-dependent regression of HeLa cell-derived tumours in SCID mice after parvovirus H-1 infection.

Parvoviruses of rodents are endowed with oncosuppressive properties. In particular, parvoviral infections protect host animals from spontaneous and chemical- or virus-induced tumour initiation in laboratory animals. The present study was undertaken to substantiate the capacity of parvovirus H-1 to inhibit therapeutically the growth of established tumours originating from human carcinoma cells implanted in recipient mice. To this end, quickly growing s.c. carcinomas were established by injection of human cervical carcinoma cells (HeLa) into immunodeficient (SCID) mice. Tumour-bearing mice subsequently were inoculated with H-1 at various multiplicities of infection. H-1 virus infection led to regression of tumours, the onset and efficiency of which were dose-dependent.

Presence of integrated DNA sequences of adeno-associated virus type 2 in four cell lines of human embryonic origin.

The human helper virus-dependent parvovirus adeno-associated virus (AAV) has been found in human female genital tissues including material from first trimester miscarriage. In the latter case, AAV type 2 (AAV-2) DNA and viral proteins were detected mainly in the trophoblast cell layer of placenta. In this report, we present evidence that AAV DNA is also present in established human trophoblast cell lines (JEG-3, JAr, BeWo) and in the human amnion cell line FL. In cells of these lines, AAV-2 DNA could be detected both by PCR and Southern blot analysis. Restriction enzyme analysis indicated that AAV DNA was integrated into the host cell genome. Although the cell lines supported AAV replication when infected with AAV-2 and adenovirus type 2 (Ad2) as a helper virus, superinfection with Ad2 alone did not induce replication of AAV DNA, i.e. it failed to rescue AAV from its integrated state. This is probably due to rearrangements within the integrated AAV genome. The presence of AAV DNA in cells derived from human embryonic tissue corroborates the suggestion that human embryonic tissue may be one of the targets of AAV infection.

Human genital tissues containing DNA of adeno-associated virus lack DNA sequences of the helper viruses adenovirus, herpes simplex virus or cytomegalovirus but frequently contain human papillomavirus DNA.

The detection of DNA of the helper virus-dependent adeno-associated virus type 2 (AAV-2) in biopsies of material from spontaneous abortion and in tissue samples from the uterus raises the question of whether sequences of known helper viruses can be detected simultaneously within the same specimen despite the lack of histological evidence for the presence of lytic viruses. Therefore, we performed PCR analyses with primers detecting DNA sequences of viruses (adenovirus, herpes simplex virus and human cytomegalovirus) known for their helper activity in the replication of adeno-associated viruses. In addition, PCR was performed to detect DNA of human papillomaviruses (HPV), which were recently shown to be able to help AAV replication in vitro. In no cases were sequences of the known helper viruses found. However, HPV DNA was detected in approximately 60% of paraffin sections from uterus biopsies and cervical lesions containing AAV DNA and in approximately 70% of material from early miscarriage. This finding suggests that HPV may be a helper virus for AAV.