Enzo Cassai

Kaposi's Sarcoma–Associated Herpesvirus DNA Sequences in Prostate Tissue and Human Semen

BACKGROUND Sequences of novel herpesvirus, Kaposis sarcoma-associated herpesvirus (KSHV), have been indentified in Kaposis sarcoma tissue, but it is not known whether the virus is transmitted by sexual contact. METHODS Using the polymerase chain reaction (PCR), we searched for KSHV DNA sequences in ejaculates from 43 healthy men and tissue from the urogenital tract or prostate of 100 immunocompetent adults. RESULTS In an unblinded analysis, we identified KSHV DNA sequences in 2 of 20 tissue specimens from the urinary tract (10 percent; 15 men and 5 women), 3 of 46 specimens from the female genital tract (6.5 percent), 4 of 18 specimens from the glans or foreskin (22 percent), 7 of 16 specimens from the prostate (44 percent), and 30 or 33 ejaculates (91 percent). By contrast, such sequences were present in 1 of 18 samples of normal skin (5.5 percent) and 1 of 14 samples of peripheral-blood mononuclear cells (PBMCs; 7.1 percent). Ejaculates and PBMC samples from each of 10 study subjects were analyzed in a blinded, coded fashion, along with PBMCs and biopsy specimens of normal skin from 4 and 8 other patients, respectively. This analysis confirmed the presence of KSHV DNA sequences in semen. Viral DNA was not found in the sperm heads but was present in the fraction of the ejaculates that contained urothelial and other types of cells. Point mutations were found in PCR products amplified from both prostate tissue and sperm samples. CONCLUSIONS KSHV infects a large proportion of healthy adults and is probably transmitted by sexual contact.

Latent BK virus infection and Kaposi's sarcoma pathogenesis

We have analyzed by PCR skin lesions from classic, endemic and AIDS‐related Kaposis sarcoma (KS), as well as from KS‐derived cell lines, the presence of ubiquitous transforming viruses. BK virus (BKV), a transforming human papovavirus which has been associated with human tumors, was detected in 100% of KS skin lesions and 75% of KS cell lines. KS specimens contained a full‐length, intact BKV early region, but minor rearrangements were observed in some tumors. BKV was also detected with a high prevalence (57–67%) in genital tissues and sperm, thus fulfilling the role of a sexually transmitted agent in KS. The closely related JC virus (JCV), which has never been associated with human malignancies, was present in 11–20% of KS specimens and was detected with a low prevalence (0–21%) in genital tissues and sperm. Simian virus 40 (SV40) was not detected in any KS lesions. Herpes simplex virus (HSV) DNA sequences were detected in 20–25% of KS lesions. Malignant human papillomavirus (HPV) types 16 and 11 were detected in KS specimens with a similar prevalence of 11–83%, suggesting that the presence of HPV‐transforming sequences is not a specific trait of HPV interaction with KS tissue. Furthermore, JCV, SV40, HSV and HPV DNA sequences were not detected in KS cell lines, suggesting that these viruses are not associated to KS neoplastic cells in KS tissue. KS cell lines were also negative for DNA sequences of KS‐HV, the novel herpesvirus detected in primary KS lesions. The constant association of BKV DNA with KS lesions and KS cell lines suggests that BKV‐transforming functions may participate in the development of KS.

Herpes simplex virus DNA isolation from infected cells with a novel procedure

Abstract Herpes Simplex Virus type 1-infected cells were extracted in the presence of 0.25% Triton X-100-0.2 M NaCl. Viral DNA associated with proteins was found in the supernatant after low-speed centrifugation. Only viral DNA was recovered by this procedure, as shown by CsCl density analysis after deproteinization. Full-length viral DNA molecules were observed in the electron microscope.

Virologic and Immunologic Evidence Supporting an Association between HHV-6 and Hashimoto's Thyroiditis

Hashimotos thyroiditis (HT) is the most common of all thyroid diseases and is characterized by abundant lymphocyte infiltrate and thyroid impairment, caused by various cell- and antibody-mediated immune processes. Viral infections have been suggested as possible environmental triggers, but conclusive data are not available. We analyzed the presence and transcriptional state of human herpesvirus 6 (HHV-6) in thyroid fine needle aspirates (FNA) and peripheral blood mononuclear cells (PBMCs) from 34 HT patients and 28 controls, showing that HHV-6 DNA prevalence (82% vs. 10%, p≤0.001) and viral load were significantly increased in FNA from HT patients, and thyrocytes from HT FNA displayed a 100-fold higher HHV-6 DNA load compared to infiltrating lymphocytes. In addition, while HHV-6 was strictly latent in positive samples from controls, a low grade acute infection was detected in HT samples. HHV-6 variant characterization was carried out in 10 HT FNA samples, determining that all specimens harbored HHV-6 Variant A. The tropism of HHV-6 for thyroid cells was verified by infection of Nthy-ori3-1, a thyroid follicular epithelial cell line, showing that thyrocytes are permissive to HHV-6 replication, which induces de novo expression of HLA class II antigens. Furthermore, HHV-6-infected Nthy-ori3-1 cells become targets for NK-mediated killing, NK cells from HT patients show a significantly more efficient killing of HHV-6 infected thyroid cells than healthy controls, and HT patients have increased T-cell responses to HHV-6 U94 protein, associated to viral latency. These observations suggest a potential role for HHV-6 (possibly variant A) in the development or triggering of HT.

Human herpesvirus 6 infects the central nervous system of multiple sclerosis patients in the early stages of the disease

The presence and the replicative state of human herpesvirus 6 (HHV-6) were evaluated in clinical samples from multiple sclerosis (MS) patients at the first time of MS diagnosis. HHV-6 variant B was present in peripheral blood mononuclear cells of 5/32 (15%) patients, but persisted with a latent infection. Viral sequences were present also in cerebrospinal fluid (CSF), both free in the liquid (7/32, 22%) and latent in the cellular fraction (3/32, 9%), as shown by analysis of viral transcription. In these cases, variant A was detected. HHV-6 DNA sequences present in the CSF were associated to mature viral particles. In fact, in vitro infectious assays of CSF showed the presence of replication-competent virions. These results show that about 20% of MS patients have active foci of HHV-6 variant A infection in the early stages of the disease and suggest that viral replication takes place within the central nervous system.

Human papillomavirus type 16 DNA in genital tumours: a pathological and molecular analysis.

The presence of human papillomavirus type 16 (HPV16) DNA in 34 genital tract tumours of Italian female patients was investigated by Southern blot hybridization in high stringency conditions. HPV16 DNA was detected in 16 neoplasias, including cervical invasive and intraepithelial lesions as well as vulvar intraepithelial neoplasias and, to a lesser extent, vulvar invasive carcinomas. Appropriate control tissues included in the study were negative. The data suggest that integration of viral DNA had occurred in most tumours, both in invasive and in intraepithelial lesions. HPV16 variants or defective genomes, lacking the BamHI restriction site, were detected in three tumours.

Stable Transformation of Mouse, Rabbit and Monkey Cells and Abortive Transformation of Human Cells by BK Virus, a Human Papovavirus

Semi-permissive mouse, rabbit and monkey cells were stably transformed by BK virus (BKV). The specificity of transformation was demonstrated by the presence of BKV tumour (T) antigen in nuclei of transformed cells and by virus rescue with Sendai virus-mediated fusion or transfection. Two out of seven BKV-transformed cell lines were oncogenic. Permissive human cells were only abortively transformed by BKV, since morphologically modified cells persisted in culture for a few passages and eventually died.

Transcription Pattern of Human Herpesvirus 8 Open Reading Frame K3 in Primary Effusion Lymphoma and Kaposi's Sarcoma

ABSTRACT Human herpesvirus 8 (HHV-8) is found in immunoblastic B cells of patients with multicentric Castlemans disease (MCD) and, predominantly in a latent form, in primary effusion lymphoma (PEL) cells and Kaposis sarcoma (KS) spindle cells. Recent studies have shown that upon reactivation, HHV-8 expresses factors that downregulate major histocompatibility class I proteins and coactivation molecules and that may enable productively infected cells to escape cytotoxic T lymphocytes and natural killer cell responses. One of these viral factors is encoded by open reading frame (ORF) K3. Here we show that in PEL cells, ORF K3 is expressed through viral transcripts that are induced very early upon virus reactivation, including bicistronic RNA molecules containing coding sequences from viral ORFs K3 and 70. Specifically, we found that a bicistronic transcript was expressed in the absence of de novo protein synthesis, thereby identifying a novel HHV-8 immediate-early gene product. Several features of the RNA molecules encoding the K3 product, including multiple transcriptional start sites, multiple donor splicing sites, and potential alternative ATG usage, suggest that there exists a finely tuned modulation of ORF K3 expression. By contrast, ORF K3 transcripts are not detected in the majority of cells present in KS lesions that are latently infected by the virus, suggesting that there are other, as-yet-unknown mechanisms of immune evasion for infected KS spindle cells. Nevertheless, because HHV-8 viremia precedes the development of KS lesions and is associated with the recrudescence of MCD symptoms, the prompt expression of ORF K3 in productively infected circulating cells may be important for virus pathogenesis. Thus, molecules targeting host or viral factors that activate ORF K3 expression or inactivate the biological functions of the K3 product should be exploited for the prevention or treatment of HHV-8-associated diseases in at-risk individuals.

Detection of Antibodies Directed against Human Herpesvirus 6 U94/REP in Sera of Patients Affected by Multiple Sclerosis

ABSTRACT The association between human herpesvirus 6 (HHV-6) and multiple sclerosis (MS) is controversial. In fact, it is difficult to establish a causative role of HHV-6, due to the high prevalence of latently infected individuals in the healthy population. Therefore, the presence of virus sequences in tissue biopsy does not support a viral role, and serological assays do not show significant differences between MS patients and control populations. The only viral gene expressed during latency is U94/rep. Therefore, we have developed a serological assay for the detection of antibodies specifically directed against U94/REP protein. Different populations were analyzed by enzyme-linked immunosorbent assay, including healthy controls, MS patients, and subjects with diseases unrelated to HHV-6 infection, including other neurological diseases. The results show statistically significant differences (P > 0.01) between MS patients and control groups, both in antibody prevalence (87 and 43.9%, respectively) and in geometric mean titer (1:515 and 1:190, respectively). The detection of antibodies specific for HHV-6 U94/REP shows that the immune system is exposed to this antigen during natural infection. The higher prevalence and higher titers of antibodies to U94/REP suggest that MS patients and control groups might experience different exposures to HHV-6.

Human herpesvirus-8 (Kaposi's sarcoma-associated herpesvirus) ORF50 interacts synergistically with the tat gene product in transactivating the human immunodeficiency virus type 1 LTR

Human herpesvirus-8 (HHV-8) is a lymphotropic virus associated with several AIDS-related neoplasms. Two ORFs play a critical role in the regulation of virus replication: ORF50, encoding an immediate-early transcriptional activator, and ORF57, encoding a post-transcriptional regulator. We analysed their effects on the activation of the human immunodeficiency virus type 1 (HIV-1) LTR. ORF50 interacted synergically with tat, inducing a 10-fold enhancement of HIV-1 LTR transactivation. This effect occurred both in BCBL-1 cells, latently infected with HHV-8, and in HL3T1 cells, an epithelial cell line non-permissive to HHV-8 infection. Also, ORF57 enhanced tat-induced transactivation of HIV-1 LTR, but only in BCBL-1 cells, suggesting that its action was likely mediated by the induction of other viral functions. Finally, when both ORFs were expressed, the enhancement of transactivation induced by ORF50 was partially inhibited. The findings suggest that ORF57 can modulate ORF50 activity and that ORF50 may render biologically active small amounts of tat.