Anna Marta Degener

Polyomavirus persistence in lymphocytes: prevalence in lymphocytes from blood donors and healthy personnel of a blood transfusion centre.

BK and JC polyomaviruses (BKV and JCV) are widespread in humans and are thought to persist and reactivate under immune alterations. In addition to the kidney, lymphoid cells have been proposed as a site of latency. However, while this was shown to occur in immunocompromised patients, discordant data were published for healthy humans. To help to solve this issue, an extensive study (231 healthy subjects) was carried out on peripheral blood mononuclear cells (PBMC) from blood donors of two towns and from operators of a blood transfusion centre. To discriminate between past and recent infection, nested PCRs for BKV and JCV non-coding control region (NCCR) and VP1 DNA sequences were carried out. Twenty-two per cent of subjects had BKV NCCR, but only 7% also had BKV VP1, as detected by PCR assays of similar sensitivities; the latter positivity was found to decrease with age. In both towns, the BKV WW archetypal DDP strain, subtype I, was found. Only 0.9% of subjects contained JCV DNA, for both NCCR and VP1. Blood operators presented a statistically significant increased prevalence of BKV NCCR (3. 0-fold) and BKV VP1 (9.4-fold) sequences with respect to blood donors of comparable ages, suggesting the possibility of occupational risk of BKV (re)infection or reactivation. Since the possibility of amplifying BKV VP1 sequences from PBMC of healthy humans is lost with age, this suggests that PBMC are not a site of polyomavirus persistence in healthy individuals and that detection of BKV VP1 DNA in PBMC is probably indicative of recent infection or reactivation.

Transplacental transmission of human polyomavirus BK

The presence of BK virus (BKV) and JC virus (JCV) in autopsy materials (placenta, brain, and kidney) of aborted fetuses was investigated by PCR using two sets of primers, specific for the regulatory region (RR) and for the capsid protein VP1, respectively. The RR of BKV was detected in 12 samples of placenta and brain and in nine samples of kidney obtained from 15 fetuses. Out of the 12 positive cases, four placentas, one brain, and three kidney samples also showed the presence of BKV DNA in the VP1 region. Of 12 placentas from a control group with a normal pregnancy outcome, the RR of BKV was detected in six samples, four of which were also positive for the VP1 region. None of the samples from either group was positive for the RR of JCV. In two cases, the nucleotide sequence of the BK RR demonstrated that the viruses isolated from maternal and fetal tissues showed a high homology with one another and had a characteristic deletion of the R63 box compared to the archetype strain. The results indicate that BKV may be transmitted vertically. J. Med. Virol. 56:372–376, 1998.

Human Papillomaviruses and genital co-infections in gynaecological outpatients

BackgroundHigh grade HPV infections and persistence are the strongest risk factors for cervical cancer. Nevertheless other genital microorganisms may be involved in the progression of HPV associated lesions.MethodsCervical samples were collected to search for human Papillomavirus (HPV), bacteria and yeast infections in gynaecologic outpatients. HPV typing was carried out by PCR and sequencing on cervical brush specimens. Chlamydia trachomatis was identified by strand displacement amplification (SDA) and the other microorganisms were detected by conventional methods.ResultsIn this cross-sectional study on 857 enrolled outpatients, statistical analyses revealed a significant association of HPV with C. trachomatis and Ureaplasma urealyticum (at high density) detection, whereas no correlation was found between HPV infection and bacterial vaginosis, Streptococcus agalactiae, yeasts, Trichomonas vaginalis and U. urealyticum. Mycoplasma hominis was isolated only in a few cases both in HPV positive and negative women and no patient was infected with Neisseria gonorrhoeae.ConclusionAlthough bacterial vaginosis was not significantly associated with HPV, it was more common among the HPV positive women. A significant association between HPV and C. trachomatis was found and interestingly also with U. urealyticum but only at a high colonization rate. These data suggest that it may be important to screen for the simultaneous presence of different microorganisms which may have synergistic pathological effects.

Urothelial bladder carcinoma and viral infections: different association with human polyomaviruses and papillomaviruses.

Bladder cancer is the second most commonly occurring genitourinary cancer in adults. The interaction of different carcinogenic and co-carcinogenic agents are responsible for bladder urothelial carcinoma: alcohol and smoking habits, Schistosoma haematobium infection, exposition to chemicals, analgesic and antineoplastic drugs prolonged use. Recently also viral infections have been associated to this pathology. In this study the correlation between viral infections and bladder carcinoma has been evaluated. A group of 32 patients affected by primary bladder neoplasia has been analysed. A control group of 20 autoptic samples of healthy bladder was analysed. The DNA of the following viruses has been searched by Polymerase chain reaction (PCR): Adenovirus, Herpes simplex virus type 1 (HSV-1), Herpes simplex virus type 2 (HSV-2), Human Papillomaviruses (HPV), Polyomaviruses (BKV and JCV). In the examined population the association bladder carcinoma-HPV, found by others, has not been confirmed. The high percentage of human polyomaviruses present in the samples is a statistically significant data (p=0.0087) and allows to presume that BKV and JCV may play a role in the aetiology of bladder tumor. In particular the polyomavirus BK, which is found in significative percentage both in single infection (p=0.0036) and in co-infections with other viral species (p=0.035), may be an important co-factor in the pathogenesis of bladder carcinoma.

Human immunodeficiency virus infection in vitro activates naturally integrated human papillomavirus type 18 and induces synthesis of the L1 capsid protein.

Human papillomavirus (HPV) infections are prevalent in human immunodeficiency virus (HIV)-positive individuals. To highlight the effect of HIV on HPV expression, HPV-18-positive HIV-permissive HeLa-T4 cells were either infected with HIV-1 or treated with Tat or with the cytokines IL-1alpha, IL-1beta, IL-6 and TNF-alpha. The presence of HPV-18 E1 (early) and L1 (late) transcripts was then determined by dot-blot or Northern blot hybridization with E1 and L1 or with genomic HPV-18 DNA probes, respectively. Protein extracts from parallel cultures were challenged by Western blotting with an antiserum raised against an L1-beta-galactosidase hybrid protein. Results indicated that HeLa-T4 cells constitutively express E1 and L1 transcripts. When cells were infected with HIV, the amounts of E1 and L1 RNAs increased with time, followed by the de novo appearance of L1 protein. E1 and L1 transcripts were also increased, in a dose-dependent manner, by treatment of uninfected cultures with Tat or with IL-6, but were not affected by IL-1alpha, IL-1beta and TNF- alpha. Neither Tat nor IL-6 could induce L1 translation. These findings raise the hypothesis that the increase of HPV shedding and of HPV-associated diseases in HIV-infected individuals could be due in part to a direct or cytokine-mediated action of HIV, in addition to the HIV-induced immunodeficiency.

Detection of BK polyomavirus genotypes in healthy and HIV-positive children.

Urine samples from 211 community children (3–7 years age), from 33 HIV type-1 infected children and from 56 HIV- negative children were collected and analyzed for the presence of BK virus (BKV) DNA by PCR. PCR amplifications were carried out using primers specific for the BKV structural region VP1. We also investigated the distribution of BKV subtypes by a restriction fragment polymorphism assay (RFLP). We demonstrated BKV DNA in 3.8% of 211 community children with a higher prevalence of subtype I. In HIV-1 positive children we detected BKV DNA in 2 urine samples (6%) out of 33, both belonging to subtype I. The HIV-negative cluster did not show any positivity to BKV DNA. The results confirm a more frequent primary BKV infection in children of 3–5 years of age and a higher prevalence in hospitalized children affected by HIV-1. The most relevant finding was that among both the community and HIV-1 positive children the subtype I was the most frequently detected.

The human polyomavirus BK: Potential role in cancer.

In human cancer, a role has been suggested for the human polyomavirus BK, primarily associated with tubulointerstitial nephritis and ureteric stenosis in renal transplant recipients, and with hemorrhagic cystitis in bone marrow transplant (BMT) recipients. After the initial infection, primarily unapparent and without clinical signs, the virus disseminates and establishes a persistent infection in the urinary tract and lymphocytes. There is correlative evidence regarding potential role of polyomavirus BK in cancer. In fact, the BK virus (BKV) DNA (complete genome and/or subgenomic fragments containing the early region) is able to transform embryonic fibroblasts and cells cultured from kidney and brain of hamster, mouse, rat, rabbit, and monkey. Nevertheless, transformation of human cells by BKV is inefficient and often abortive. Evidence supporting a possible role for BKV in human cancer has accumulated slowly in recent years, after the advent of polymerase chain reaction (PCR). BKV is known to commonly establish persistent infections in people and to be excreted in the urine by individuals who are asymptomatic, complicating the evaluation of its potential role in development of human cancer. Therefore, there is no certain proof that human polyomavirus BK directly causes the cancer in humans or acts as a cofactor in the pathogenesis of some types of human cancer.

Rearrangement patterns of JC virus noncoding control region from different biological samples

The JC virus (JCV) is generally considered the etiological agent of progressive multifocal leukoencephalopathy (PML), a demyelinating brain illness, often associated with immunosuppression and significantly frequent in acquired immunodeficiency syndrome (AIDS) patients. The primary infection by JCV is usually asymptomatic and the virus can remain in a latent status in the kidney. As a consequence of immunological alterations of the host, the virus can show a genetic variability in the noncoding control region (NCCR) due to deletions, duplications, and insertions as compared with the archetype. The NCCR of the archetype strain can be divided into six regions, named boxes A to F. In this study, the authors evaluated the presence of the JCV genome in different biological samples, such as urine, peripheral blood mononuclear cells (PBMCs) and cerebral spinal fluid (CSF) by means of polymerase chain reaction (PCR). After sequencing of the PCR fragments, the NCCR structure of isolated JCV strains was analyzed in order to verify the presence of different viral variants. An analysis of the homology and of the multiple alignment of the obtained sequences in comparison with the archetype strain has been carried out. The results indicated the presence of different rearrangements among the analyzed samples. Whereas in the urine, the NCCR structure always appeared very similar to that of the archetype, in the PBMCs and CSF, the NCCR sequences showed specific and characteristic rearrangements as compared to the archetype. These different rearrangements could be correlated with the emerging of an NCCR organization more suitable for the development of PML.

Detection of human papillomavirus DNA, P53 and KI67 expression in penile carcinomas

Our study is aimed at evaluating the presence of p53 and Ki67 expression by immunohistochemistry in a series of 11 paraffin-embedded penile carcinomas. We also investigated the presence of Human Papillomavirus (HPV) DNA in these tumours and performed an accurate typing by DNA sequencing on positive samples. Immunohistochemistry (IHC) was performed with the anti-p53 and Ki67 mouse monoclonal antibodies. DNA extracted from small sections of each specimen was submitted to amplification with HPV specific general primers; PCR products of the proper length were purified and sequenced. IHC demonstrated nuclear accumulation of mutated p53 and Ki 67 expression in 10/11 tumour samples (90.9%). The prevalence of HPV DNA was 72.7%; the most prevalent type was HPV16. Sequencing analysis revealed the presence of HPV53 (12.5%), HPV18 (25%) and HPV16 (62.5%). Out of the p53 or Ki67 positive carcinomas the percentage of HPV positives was 80% and 70% respectively. Our results indicate that penile carcinoma is frequently associated to high risk HPV and with diffuse p53 and Ki67 expression.

Association of Human Papillomavirus Type 11 with Carcinoma of the Penis

Human papillomaviruses (HPVs) are epithelium-tropic viruses associated with several cutaneous, epithelial, and mucosal lesions. The oncogenic potential varies considerably among the more than 70 different genotypes so far identified. HPV 6 and 11 are generally found in benign genital condilomata or laryngeal papillomas, but they have been sporadically associated with genital malignancies. Polymerase chain reaction (PCR) primed by degenerated consensus oligonucleotides (from a late region of the HPV genome) allows one to amplify a broad spectrum of HPV, whereas the amplification with specific primers is restricted to a limited number of HPVs. Therefore, the restriction fragment length polymorphism assay permits one to identify the HPV type present in the PCR product. We report a case of an invasive verrucous carcinoma of the penis associated with HPV 11, a type previously considered noncarcinogenic.