6B DNA Research Articles

Clinical Features of Complex Febrile Seizure Caused by Primary Human Herpesvirus 6B Infection

BackgroundIt is well known that febrile seizures are commonly occur in children with exanthem subitum. In this study, we compared the clinical features and backgrounds of patients with complex febrile seizures with and without primary human herpesvirus 6B infection. MethodsSixty-two patients were enrolled after experiencing their first febrile seizure. Primary human herpesvirus 6B infection was confirmed when human herpesvirus 6B DNA was detected and human herpesvirus 6B antibody was negative in serum obtained during the acute phase of infection. Patient age, gender, and features of seizures were evaluated between patients with and without human herpesvirus 6B infection. ResultsThirty patients with complex febrile seizure were diagnosed with primary human herpesvirus 6B infection. Those with primary human herpesvirus 6B infection (median, 13 months; range, seven to 39 months) were significantly younger than those without primary human herpesvirus 6B infection (median, 19 months; range, 10 to 59 months) (P = 0.001), and the proportion of males was significantly higher in patients without primary human herpesvirus 6B infection (male/female, 25/7) than in those with the infection (male/female, 14/16) (P = 0.017). An interval between fever onset and seizures of more than 24 hours was significantly more common in patients with primary human herpesvirus 6B infection (15 of the 30 patients) than in those without primary HHV-6B infection (two of 32 patients) (P < 0.001). ConclusionsA younger age at onset, a different gender ratio compared with febrile seizure due to other causes, and the length of interval between fever and seizures were features of complex febrile seizure associated human herpesvirus 6B infection. These findings may suggest a mechanism of complex febrile seizure onset different from that due to other causes.

CD4+ T Cells Coexpressing CD134 (OX40) Harbor Significantly Increased Levels of Human Herpesvirus 6B DNA Following Umbilical Cord Blood Transplantation.

Human herpesvirus 6B (HHV-6B) commonly reactivates after umbilical cord blood transplantation (UCBT) and is associated with delayed engraftment, fever, rash, and central nervous system dysfunction. Recently, CD134 (OX40) has been implicated as a potential viral entry receptor. We evaluated CD4+CD134+/neg-lo and CD8+CD134+/neg-lo cells at day 28 after UCBT in 20 subjects with previously documented HHV-6 reactivation and persistent viremia. Analysis of CD4+CD134+ cells as compared to CD4+CD134neg-lo cells showed 0.308 versus 0.129 copies of HHV-6B/cell (P = .0002). CD8+CD134+/neg-lo cells contained little to no HHV-6B copies. Following UCBT, CD4+CD134+ cells harbor significantly increased levels of HHV-6B, suggesting that CD134 (OX40) may facilitate viral entry.

Virus specific cell‐mediated immunity may play a role in controlling reactivated human herpesvirus 6B in patients under measles induced immunosuppression

For a better understanding of the cellular immune responses to reactivated HHV 6B the lymphoproliferative response to human herpesvirus 6B (HHV 6B) antigen was measured in three consecutive specimens obtained biweekly from 22 young children and infants suffering from acute measles, and in 19 influenza patients and nine healthy control subjects. HHV 6B DNA in peripheral blood mononuclear cells (PBMCs) was detected in 18 of 22 subjects with measles, but not in the influenza patients or the healthy population. A novel reactivation profile of HHV 6B was found in patients with measles in the milder form of immunosuppression than in patients with organ transplantation. HHV 6B specific lymphoproliferation activities increased correspondingly with reactivation of HHV 6B assessed by detecting HHV 6B DNA in PBMCs in patients with measles, but no significant change in either the antibody response to HHV 6B or DNAemia occurred in serial specimens obtained either from patients with influenza or healthy subjects. This novel form of HHV 6B reactivation without antibody response was observed in patients with measles. The dynamic fluctuations in lymphoproliferative responses in measles may represent the balance between HHV 6B reactivation and its suppression by the host immune system.

Synthesis and biological evaluation of a class of quinolone triazoles as potential antimicrobial agents and their interactions with calf thymus DNA

A novel series of quinolone triazoles were synthesized and characterized by IR, NMR, MS and HRMS spectra. All the newly prepared compounds were screened for their antimicrobial activities against seven bacteria and four fungi. Bioactive assay manifested that most of new compounds exhibited good or even stronger antibacterial and antifungal activities against the tested strains including multi-drug resistant MRSA in comparison with reference drugs Norfloxacin, Chloromycin and Fluconazole. The preliminary interactive investigations of compound 6b with calf thymus DNA by fluorescence and UV–vis spectroscopic methods revealed that compound 6b could effectively intercalate DNA to form compound 6b–DNA complex which might block DNA replication and thus exert its antimicrobial activities.

Studies on the establishment of a method to detect HPV 6b E7-specific antibodies of serum and cervical secretion from patients with condyloma acuminatum and its epidemiological significance

To establish a method for detection of the human papi11omavirus (HPV) 6b E7-specific antibodies in serum and cervical secretion from patients with condyloma acuminatum (CA). A full-length HPV 6b E7 gene was amplified by PCR from the CA tissue to construct the recombinant plasmid pET32a(+)/HPV 6b E7. The expression of prokaryotic protein was analyzed by SDS-PAGE and Western blot, then purified with Ni-NTA Agarose affinity column and used as an diagnostic antigen for establishing indirect ELISA method, to detect specific serum IgG and specific cervical secretion sIgA from 56 CA patients, 81 healthy control. Sera from 43 cervical cancer was served as control. HPV 6b DNA from 56 CA patients was identified by PCR. Data showed that the nucleotide homology of cloned sequence was 99.5%, compared to the standard sequences of HPV 6b E7 gene (GenBank accession number: NC001355). A high level expression of E7 fusion protein was obtained in prokaryotic expression system (40 µg/ml). Based on HPV 6b E7 fusion protein being used as coating antigen, results from ELISA showed that the absorbance rates (A) of serum IgG from CA, cervix cancer and healthy control groups were 1.82 ± 0.48, 1.36 ± 0.39 and 1.39 ± 0.27, respectively. The level of IgG antibody in the serum of CA group was significantly higher than that in cervix cancer group and healthy control (P < 0.05). The A values of cervical secretion sIgA in CA and healthy control groups were 0.63 ± 0.26 and 0.53 ± 0.06, respectively, while the level of sIgA antibody in the cervical secretion of CA group was also significantly higher than that in healthy controls (P < 0.05). The positive rate of HPV 6b E7 DNA in CA tissue was 78.6% (44/56) by PCR method. When compared the results measured by PCR, the HPV 6b E7-specific IgG and sIgA antibodies by ELISA used to detect the patients infected with HPV 6b infection, showed that the sensitivity rates were 68.2% (30/44) and 54.6% (24/44) respectively, and the specificity were all 100% (12/12). Based on the serum and cervical secretion specific HPV 6b E7 antibodies from patients with CA to diagnose HPV 6b infection, results showed medium sensitivity and high specificity, and could further be used to investigate the epidemiological characteristics of HPV 6b infection.

Presence of Epstein-Barr virus and human herpesvirus 6B DNA in multiple sclerosis patients: associations with disease activity

To assess the presence of Epstein-Barr virus (EBV) and human herpesvirus 6B (HHV-6B) DNA in saliva and plasma from multiple sclerosis (MS) patients enrolled in a randomized, double-blind, placebo-controlled valacyclovir treatment study. DNA was prepared following ultracentrifugation of saliva and plasma. EBV and HHV-6B DNAs were determined by real-time polymerase chain reaction. EBV and HHV-6B DNAs were detected in 41% and 65% of saliva samples, respectively. In patients treated with valacyclovir, the percentage of saliva samples with EBV was significantly reduced (9%; P = 0.000017), whereas the frequency of HHV-6B positive samples was unchanged (57%; P = 0.38). Longitudinal studies demonstrated a time-dependent reduction in the frequency of saliva samples containing EBV following valacyclovir treatment. In contrast, plasma contained EBV and HHV-6B DNAs in 17% and 25% of the samples, respectively, and these numbers were not significantly reduced following valacylovir treatment (13% and 16%, respectively), nor were they different from those of healthy controls (6% and 39%, respectively). Patients with high disease activity had a significantly higher frequency of EBV (P = 0.018) and HHV-6B (P = 0.023) positive samples than did patients with low disease activity. The presence of EBV and HHV-6B was strongly correlated in plasma (P < 0.00000001), but not in saliva (P = 0.41). MS patients express EBV and HHV-6B in both saliva and plasma, but only the expression of EBV in saliva is significantly reduced following valacyclovir treatment. Although EBV and HHV-6B DNAs can be detected in plasma from healthy individuals, the co-expression of both these viruses in MS patients is highly significant and further associated with clinical activity. The observations of viral DNA in plasma are consistent with an underlying immunologic defect in MS.

Asociación entre la integración del virus del papiloma humano y la pérdida de heterozigosidad del gen p53 en los carcinomas epidermoides de cabeza y cuello

AbstractHuman papillomavirus integration in cellular Dna and loss of heterozygosity of p53 gene are both related with tumour formation process by promoting genomic inestability that leads to dna abnormalities accumulation. In order to analyze the relationship between both events, 26 squamous cell carcinomas of the head and neck were studied. hpv 16 and 6b dna was detected by pcr in 8 cases (31%), whereas p53 loss of heterozygosity was present in 16 cases (61%). No correlation was found between both events and they were not related to clinical factors neither the prognosis. Consecuently, hpv integration and loss of heterozygosity of p53 seem to act independently in the genesis of these tumours.

The cellular DNA polymerase alpha-primase is required for papillomavirus DNA replication and associates with the viral E1 helicase.

Persistent infection by papillomaviruses involves the maintenance of viral DNA as a nuclear plasmid, the replication of which requires host DNA polymerases. The role of the cellular DNA polymerase alpha-primase holoenzyme was probed by using soluble extracts from rodent cells that replicate bovine papilloma virus 1 and human papilloma virus 6b DNA in the presence of the viral E1 helicase and the E2 transcription factor. Monoclonal antibodies directed against the catalytic 180-kDa subunit of polymerase alpha inhibit DNA synthesis in this system. Addition of purified human polymerase alpha-primase holoenzyme to neutralized extracts restores their DNA synthetic activity. The amino-terminal 424 amino acids of E1 forms a specific protein complex with the p180 polymerase subunit. Immune complexes can be isolated with antibodies directed against E1 that contain a DNA polymerase activity. Moreover, this polymerase activity can be neutralized by anti-polymerase alpha antibodies. Permissivity barriers were not encountered in this in vitro system, as bovine E1 can interface with the murine and human replication apparatus. Although the large tumor antigens encoded by simian virus 40 and polyoma share limited primary sequence homology with the papillomavirus E1 proteins, the organization of functional motifs at the level of primary protein structure is remarkably similar. In addition to their origin-specific DNA-binding activity, each of these helicases may function to help recruit the cellular polymerase alpha-primase complex to the viral replication origin.

In situ hybridization at light and electron microscopic levels: identification of human papillomavirus nucleic acids

A comparison of the sensitivities of biotinylated and 32P-labelled human papillomavirus type 6b DNA probes was made. Slot blot hybridization results showed that the sensitivity of biotinylated probes was consistent with that of 32P-labelled, that is, 0.1 pg of pBR 322 plasmid containing 8 kbp HPV cDNA. In situ hybridization using 35S-labelled probes was applied to tissue from condylomata acuminata. After autoradiography, many silver grains were seen concentrated over the superficial koilocytic nuclei with some grains present in the cytoplasm. Biotinylated probes were visualized by 4 different means, i.e., streptavidin alkaline phosphatase, streptavidin biotinylated horseradish peroxidase, monoclonal anti-biotin antibody with 15 nm colloidal gold and streptavidin 5 nm colloidal gold. Strong reaction products were localized in the superficial nuclei while the cytoplasm of koilocytes showed weak hybridization signal. Pre-embedding methods were carried out for electron microscopic studies in which numerous granular diaminobenzidine (DAB) products were present in the nuclear chromatin while viral particles themselves had much fewer DAB products. This suggested that hybridization occurred more efficiently to yet unassembled viral genomes than to matured virions. Post-embedding methods using 15 nm colloidal gold were performed and showed singly scattered or clustered gold particles in superficial koilocytic nuclei.

Human papillomavirus type 6b DNA required for initiation but not maintenance of transformation of C127 mouse cells

We describe the transformation of C127 mouse fibroblasts with human papillomavirus type 6b (HPV-6b) DNA, which is associated primarily with benign tumors of the human genital tract. The major transformed phenotype of the HPV-6b-transfected cells lines, which had been G418 selected, pooled, and maintained without subsequent selection, was tumorigenicity in nude mice. We found that, unlike that reported for other HPVs or papovaviruses, the transformed phenotype was expressed after a delay, in which the cells had undergone extensive culture passages (about 20 passages or 100 generations). Interestingly, the HPV-6b DNA had become reduced or nondetectable in copy number in the cells by the time the transformed phenotype was expressed and in most of the tumors induced by the cells in nude mice, indicating that high levels of HPV-6b DNA were not required for maintenance of the transformed phenotype. Clonal cell lines gave similar results. When continued G418 selection was used to maintain high-copy-number HPV-6b DNA, the cells were tumorigenic, indicating that high levels of HPV-6b DNA did not suppress tumorigenesis. These studies suggest that HPV-6b DNA initiates transformation of C127 cells but is dispensable for expression or maintenance of the transformed phenotype. Transformation by HPV-6b DNA in vitro may provide insights into the HPV type-specific association with benign versus malignant lesions in vivo and may elucidate some of the oncogenic processes involved in tumor progression.

Characterization of human papillomavirus type 6b DNA isolated from an invasive squamous carcinoma of the vulva

Human papillomavirus type 6 (HPV 6) DNA was detected in an episomal form in DNA extracted from an invasive squamous carcinoma of the vulva. The viral DNA, designated HPV6-T70, was molecularly cloned. Restriction analysis of the HPV6-T70 genome revealed an insertion of ≈35 by in the 5′ portion of the upstream regulatory region relative to the prototype HPV 6b genome, cloned from a benign genital wart ( E.-M. de Villiers, L. Gissmann, and H. zur Hausen, 1981, J. Virol. 40, 932–935). However, sequence analysis of the upstream regulatory region identified several alterations in the purine-thymidine-rich region spanning nucleotides 7292–7400. One insertion of 24 by at position 7323 represented an exact tandem duplication of nucleotides 7300–7323. A second insertion of 58 by at position 7350 had 84% positional identity to immediately adjacent HPV 6 sequences (nucleotides 7303–7356) and was also similar to the 74-bp insertion found at position 7348 in HPV-6vc, cloned from an invasive verrucous carcinoma of the vulva ( R. F. Rando, D. E. Groff, J. G. Chirikjian, and W. D. Lancaster, 1986, J. Virol. 57, 353–356). A deletion of 49 by (nucleotides 7351–7399) immediately followed the 58-bp insertion. The cloned HPV6-T70 DNA induced the morphological transformation of NIH 3T3 cells.

The physical state of human papillomavirus type 16 DNA in benign and malignant genital tumours.

Cloned DNA from human papillomavirus (HPV) type 16 was subjected to restriction enzyme analysis. A genome size of 7.8 +/- 0.1 kb was determined and restriction maps were prepared. Fragments of HPV 16 DNA were nick-translated and hybridized with fragments of HPV 6b DNA. The two genomes appeared to be colinear. The physical state of HPV 16 DNA in genital tumours was analysed. In each of six benign tumours the viral DNA was detected exclusively as 8 kb circles. In four malignant tumours the viral DNA appeared to be integrated within the host genome but one cervical carcinoma and one case of Bowen's disease also contained oligomeric episomal molecules of viral DNA. One cervical carcinoma (WV 2965), containing only integrated viral DNA, was examined in detail. HPV 16 DNA was integrated as head-to-tail tandem repeats at more than one site. Three virus/cell junction fragments from this tumour were cloned. Two contained lengths of repetitive cellular DNA and one a length of apparently single copy cellular DNA.