Phylogeny and Classification of Human Papillomavirus (HPV)16 and HPV18 Variants Based on E6 and L1 genes in Tunisian Women with Cervical Lesions

Background: High-risk human papillomavirus (HPV) types are the main etiological factors for cervical cancer. HPV16 and HPV18 are generally the most common forms associated with development of high-grade cervical lesions. This study was undertaken to identify intratypic variants of HPV16 and HPV18 among women with cervical lesions in Tunisia. Materials and Methods: DNA was extracted from cervical samples collected from 49 women. using a PureLinkTM Genomic DNA mini Kit (Invitrogen). E6 and L1 open reading frames (ORF) were amplified by PCR and viral DNA amplicons were subjected to automated sequencing using Big Dye Terminators technology (Applied Biosystems). The obtained sequences were analyzed using an appropriate software program to allow phylogenetic trees to be generated. Results: HPV16 and HPV18 were detected in 15 and 5 cases, respectively. HPV16 E6 sequences clustered with the European German lineage (A2) whereas one isolate diverged differently in the L1 region and clustered with the African sub-lineage (B1). HPV 18 E6 sequences clustered with the European sub-lineage (A1) but L1 sequences clustered as a new clade which diverged from A1-A5. Conclusions: Our results suggest that the distribution of HPV16 and HPV18 sequences in women with cervical lesions in Tunisia is mainly related to European epidemiological conditions and point to the presence of recombinant HPV forms.

Detection of a new JCV strain of genotype A in a subpopulation of colorectal adenocarcinomas in Tunisia

The etiology of colorectal cancer (CRC) remains elusive in spite of major advances in knowledge of this disease and related risk factors. Several studies report the detection of human polyomavirus JC (JCV) in colorectal tumors and some suggest its association with CRC. Since many known human virus associations with cancer are linked to factors such as ethnic and geographical origin, it is interesting to search for the postulated association of JCV with CRC in different populations and regions. In this perspective, the present work was undertaken to assess the presence of JCV in CRC tumors in Tunisia. Fresh biopsies were obtained from both colorectal tumors and adjacent normal tissues of 47 CRC patients. Only tumors diagnosed as adenocarcinomas were included in the present study. Twenty patients with other gastroenterological disorders were taken as controls. DNA was extracted from fresh biopsies or formalin-fixed, paraffin-embedded tissue sections. A region of the viral T-Ag gene was amplified by PCR and the DNA amplicons were subjected to automated sequencing. JCV DNA was found in 22 (46%) of the adenocarcinomas but in none of the normal mucosa biopsies of either CRC or control patients. Sequence analysis indicated that the amplified DNA belonged to a new JCV variant of genotype A. The presence of JCV DNA was correlated with tumor location and grade. The data obtained suggest that JCV may be associated either with a subpopulation of colorectal tumors or with CRC in general, possibly through a hit and run mechanism.

NF-kappaB-mediated modulation of inducible nitric oxide synthase activity controls induction of the Epstein-Barr virus productive cycle by transforming growth factor beta 1.

Transforming growth factor beta 1 (TGF-ß1) signal transduction has been implicated in many second-messenger pathways, including the NF-κB pathway. We provide evidence of a novel TGF-ß1-mediated pathway that leads to extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, which in turn induces expression of an Epstein-Barr virus (EBV) protein, ZEBRA, that is responsible for the induction of the viral lytic cycle. This pathway includes two unexpected steps, both of which are required to control ERK 1/2 phosphorylation: first, a quick and transient activation of NF-κB, and second, downregulation of inducible nitric oxide synthase (iNOS) activity that requires the participation of NF-κB activity. Although necessary, NF-κB alone is not sufficient to produce downregulation of iNOS, suggesting that another uncharacterized event(s) is involved in this pathway. Dissection of the steps involved in the switch from the EBV latent cycle to the lytic cycle will be important to understand how virus-host relationships modulate the innate immune system.

Phosphatidylinositol 3-kinase/Akt pathway targets acetylation of Smad3 through Smad3/CREB-binding protein interaction: contribution to transforming growth factor beta1-induced Epstein-Barr virus reactivation.

Epstein-Barr virus, a ubiquitous human herpesvirus, is associated with the development of carcinomas and lymphomas. We previously showed that transforming growth factor beta1 (TGF-beta1) mediated the virus to enter the lytic cycle, which is triggered by expression of Z Epstein-Barr virus replication activator (ZEBRA), through the ERK 1/2 MAPK signaling pathway. We report here that Akt, activated downstream from ERK 1/2, was required for TGF-beta1-induced ZEBRA expression and enabled Smad3, a mediator of TGF-beta1 signaling, to be acetylated by direct interaction with the co-activator CREB-binding protein and then to regulate TGF-beta1-induced ZEBRA expression.

Activation of the lytic program of the Epstein-Barr virus in Burkitt's lymphoma cells leads to a two steps downregulation of expression of the proapoptotic protein BimEL, one of which is EBV-late-gene expression dependent.

The Epstein-Barr virus (EBV) generally latently infects its target cells with expression of genes conferring resistance to apoptosis. However, the modulation of apoptotic signals during lytic cycle remains poorly understood. We show here that resulting from viral reactivation in the EBV-positive Mutu-I and Akata Burkitt's lymphoma cell lines, a two steps proteasome-dependent downregulation of expression of the proapoptotic protein BimEL occurs. The first drop might be EBV-independent, is ERK 1/2 dependent, and BimEL is phosphorylated on Ser69. A second dramatic drop of the BimEL level observed during the lytic cycle is dependent of EBV-late-gene expression, ERK 1/2 independent, and no further phosphorylation of BimEL on Ser69 occurred. These results demonstrate for the first time, that the lytic cycle contributes to downregulation of BimEL and then could add to protection against apoptosis.

Parvovirus B19 infection in Tunisian patients with sickle-cell anemia and acute erythroblastopenia.

BACKGROUND: Human parvovirus B19 is the etiologic agent of erythema infectiosum in children. It is also associated with other clinical manifestations in different target groups. Patients with chronic hemolytic anemia are at high risk of developing acute erythroblastopenia following infection by the virus. They usually become highly viremic and pose an increased risk of virus transmission. Close monitoring of such high risk groups is required for epidemiologic surveillance and disease prevention activities. Here we report a molecular epidemiological study on B19 virus infection in Tunisian patients with chronic hemolytic anemia. METHODS: This study was conducted on 92 young chronic hemolytic anemia patients who attended the same ward at the National Bone Marrow Transplantation Center of Tunis and 46 controls from a different hospital. Screening for IgM and IgG anti-B19 antibodies was performed using commercially available enzyme immunoassays and B19 DNA was detected by nested PCR in the overlapping VP1/VP2 region. DNA was sequenced using dideoxy-terminator cycle sequencing technology. RESULTS: Anti-parvovirus B19 IgG antibodies were detected in 26 of 46 sickle-cell anemia patients, 18 of 46 beta-thalassemia and 7 of 46 controls. Anti-parvovirus B19 IgM antibodies were detected only in 4 of the sickle-cell anemia patients: two siblings and two unrelated who presented with acute erythroblastopenia at the time of blood collection for this study and had no history of past transfusion. B19 DNA was detected only in sera of these four patients and the corresponding 288 bp nested DNA amplicons were sequenced. The sequences obtained were all identical and phylogenetic analysis showed that they belonged to a new B19 virus strain of Genotype1. CONCLUSION: A new parvovirus B19 strain of genotype1 was detected in four Tunisian patients with sickle-cell anemia. Virus transmission appeared to be nosocomial and resulted in acute erythroblastopenia in the four patients. The possibility of independent transmission of this B19 variant to the patients is unlikely in light of the present epidemiological data. However this possibility cannot be ruled out because of the low genetic variability of the virus.

High-risk HPV types in Tunisia. A pilot study reveals an unexpectedly high prevalence of types 58 and 82 and lack of HPV 18 among female prostitutes.

"High-risk" HPVs (HR-HPV) have sharply different prevalences and there is evidence to suggest this may vary according to regions. Accurate description of HR-HPV circulation is a key feature for the rational design of prevention and screening campaigns. To gain insight into HR-HPV circulation in Tunisia, a pilot study was carried out on 64 healthy prostitutes working in the Tunis area. HPV detection and typing were carried out by MY09/MY11 PCR and restriction fragment length polymorphism (RFLP). A selected set of samples was also assayed by Gp5+/Gp6+ PCR and typed by direct sequencing. Out of 64 women, 28 were HPV positive. HPV-16 and HPV-58, both members of the genus Alpha-Papillomavirus, species 9, accounted for 10 and 7 cases with a prevalence rate of 38% and 27%, respectively. HPV-82, a member of species 5, ranked third with four cases (approximately 15%). Types 31, 33, 35; all members of species 9 were each detected once (approximately 4%) while neither HPV-18 nor related members of species 7 were detected. Type 72 and 83, both members of species 3, were the only low-risk types, each detected only once (4% each). Two samples could not be typed. The prevalence of HPV types appeared sharply different from that of neighboring areas. Should the existence of epidemiological pockets be confirmed by larger, more detailed studies, screening and vaccine campaigns will have to be designed carefully taking into account the actual epidemiological context of the target population.

Cytokine and immunoglobulin production by PWM-stimulated peripheral and tumor-infiltrating lymphocytes of undifferentiated nasopharyngeal carcinoma (NPC) patients.

BACKGROUND: Undifferentiated Nasopharyngeal Carcinoma (NPC) patients show a characteristic pattern of antibody responses to the Epstein-Barr virus (EBV) which is regularly associated with this tumor. However, no EBV-specific cytotoxic activity is detectable by the standard chromium-release assay at both peripheral and intratumoral levels. The mechanisms underlying this discrepancy between the humoral and cellular immune responses in NPC are still unknown, but might be related to an imbalance in immunoregulatory interleukin production. In this report, we investigated the ability of peripheral (PBL) and tumor- infiltrating (TIL) lymphocytes of undifferentiated NPC patients to produce in vitro three interleukins (IL-2, IL-6, IL-10) and three immunoglobulin isotypes (IgM, IgG, IgA). METHODS: Lymphocytes from 17 patients and 17 controls were cultured in the presence of Pokeweed mitogen (PWM) for 12 days and their culture supernatants were tested for interleukins and immunoglobulins by specific enzyme-linked immunosorbent assays (ELISA). Data were analysed using Student's t-test and probability values below 5% were considered significant. RESULTS: The data obtained indicated that TIL of NPC patients produced significantly more IL-2 (p = 0,0002), IL-10 (p = 0,020), IgM (p= 0,0003) and IgG (p < 0,0001) than their PBL. On the other hand, patients PBL produced significantly higher levels of IL-2 (p = 0,022), IL-10 (p = 0,016) and IgM (p = 0,004) than those of controls. No significant differences for IL-6 and IgA were observed. CONCLUSION: Taken together, our data reinforce the possibility of an imbalance in immunoregulatory interleukin production in NPC patients. An increased ability to produce cytokines such as IL-10 may underlie the discrepancy between humoral and cellular immune responses characteristic of NPC.