Search

The key to developing a therapeutic vaccine for chronic hepadnavirus infection lies in the characteristics of the host-immune response which leads to clearance of acute infection. Groups of 28-day-old ducks which had been surgically bursectomized (n = 10) or thymectomized (n = 13) on the day of hatch or were untreated (n = 21) were inoculated with 10(9) viral genome equivalents (vge) DHBV, then bled twice a week, and euthanased 40 days later. Serum and liver were tested for DHBV DNA and total leukocytes and peripheral blood mononuclear cells (PBMCs) counted. Liver and spleen sections were either stained with hematoxylin and eosin, and graded for inflammation or stained with peroxidase-labeled anti-human CD3 antibody and examined for T lymphocyte distribution. PBMC counts were similar in all groups. DHBV infection combined with bursectomy increased significantly, while thymectomy decreased significantly the total leukocyte count. The spleen and liver bursectomy increased T lymphocyte number while B cells were decreased. Converse changes were observed in thymectomized ducks. Histological evidence of hepatitis was present in infected control and bursectomized ducks but not in the uninfected control or infected thymectomized ducks. In control animals, DHBV challenge caused viremia in 17 and persistent infection in 11 (56%). Fewer thymectomized ducks (3/13, 23%) and significantly more (100%) bursectomized ducks remained persistently infected (P < 0.001). Unexpectedly, bursectomy led to persistence of infection while clearance of infection occurred normally in thymectomized ducks despite decreased T lymphocyte numbers. This suggests that clearance requires T and B lymphocyte collaboration., ((c) 2006 Wiley-Liss, Inc.)

High rates of cervical cancer have been reported from parts of China and this may reflect a predominance of cervical infection with particularly aggressive human papillomavirus (HPV) variants. This PCR-based investigation of cervical tumours from Sichuan province in central China demonstrated an HPV positivity rate of 88%. HPV 16 was most common (21/34, 61%), followed by HPV 18 (3/34, 9%), while types 33, 45, 58 and 59 were each identified in one specimen. Sequencing of up to 1349 bases of the 21 HPV 16-positive isolates, encompassing the enhancer/promoter of the upstream regulatory region (URR) and the E6 and E7 genes, revealed distinct patterns of genomic stability and variability. An overall mutation rate of 5% was seen in the URR. One isolate had a large deletion of 436 bases in the enhancer; while varying combinations of 21 point mutations were identified in the remainder, impacting several YY1, NF1, TEF-1 and Oct-1 sites. More sequence variations were found in E6 compared to E7 (81% vs. 52% of isolates showing at least one mutation), some of which resulted in changes to the translated amino acids. Since the E6/E7 genes encode the oncogenic proteins essential for malignant transformation, and as their expression is controlled by the URR, it is possible that some of the identified mutations altered the oncogenicity of the virus: either directly by changing amino acid sequences of the E6 or E7 oncoproteins, or indirectly through alterations to transcription factor binding sites in the URR., (Copyright 2000 Wiley-Liss, Inc.)

Duck hepatitis B virus (DHBV) has been a useful model for hepadnavirus infection. There have been few studies on immunity to DHBV and none describing the cell-mediated immune response by acute and chronically infected ducks. A duck hepatitis B antigen-specific blastogenesis assay was used to measure DHBV antigen-specific responses of duck peripheral blood (PBMC) and splenic mononuclear cells (SMCs) from uninfected control ducks, ducks acutely or chronically infected with DHBV, and ducks immune to DHBV. A comparison of the group mean responses by PBMC to DHBV surface antigen (DHBsAg) found that the immune group was significantly different to the other three groups (controls or unexposed, P < 0.0001; acutely infected, P< 0.01; chronically infected, P < 0.01). The responses to DHBsAg by PBMC of the acute group (P< 0.01) were significantly different also to that of the unexposed group. For DHBV core antigen (DHBcAg), significant differences in the responses were found between immune ducks and unexposed (P < 0.0005) and acutely infected (P < 0.05) groups. The SMC showed a significant difference between unexposed ducks and immune ducks (P< 0.05) in the group mean responses to DHBsAg. The responses to DHBcAg were significantly different between the immune group and the acute (P < 0.01) and unexposed (P < 0.01) groups. The group mean of unexposed ducks was also significantly different to that of acutely infected ducks (P < 0.01). This study indicates that the cellular immune response in immune animals differs from acutely and chronically infected ducks. Further studies of these differences may provide some explanations for the differing outcomes of DHBV infection.

Natural variation in hepatitis B virus (HBV) was studied in asymptomatic carriers originating from countries of high endemicity. HBV DNA was detected by dot blot and/or polymerase chain reaction (PCR) in 34 of the 184 members of 22 new immigrant families who agreed to be tested after one of their children had been found to have current or past hepatitis B in a school survey. PCR products from both the S and distal-X pre-C regions were sequenced. One vaccinated child had the classical "escape" mutation at amino acid position 126 in the S-gene and two other children and two adults had other substitutions at amino acid positions 133, 120, 165, and 159. Mutations were more frequent in the distal-X pre-C region and included two pre-C mutants and 13 other amino acid substitutions. The strains originating in the various countries were placed in almost identical groups by phylogenetic analysis using each amplicon, and determination of subtype by antigenic analysis gave the same result as sequencing. The S-data allowed recognition of three dominant strains within genotype B, while the distal-X pre-C data provided better discrimination between family groups. No change was found when the sequence of samples obtained for the study was compared with those collected from 14 of the children two years earlier. There was some evidence of horizontal spread in addition to vertical transmission. Reports of mutations of HBV in patients with severe or unusual clinical features should be interpreted with caution until the prevalence of the mutant in asymptomatic carriers has been determined.

The polymerase chain reaction (PCR) was used to investigate the prevalence and distribution of human papillomavirus (HPV)-16 DNA in paraffin sections of all pelvic lymph nodes removed from 14 patients with Stage Ib-cervical cancer at the time of resection of their primary tumours. The results were compared with those obtained from 8 women with no known history of cervical abnormality. In all, 22 cervical biopsies and 40 I lymph nodes (296 paraffin blocks) were examined. Nine of the 14 cervical cancer patients had primary tumours that were positive for HPV-16 DNA: only 3 of these had lymph nodes with histological evidence of metastasis, and HPV 16 DNA was detected in each of the corresponding paraffin blocks. HPV 16 DNA was also detected in varying proportions (8%-92%) of the histologically-negative lymph nodes from these women. There was no correlation between the HPV DNA-positive lymph nodes and their proximity to the primary tumour. HPV-16 DNA was not identified in any of the lymph nodes from the 5 women whose cancers were not HPV-16-related, or in those of women with no evidence of cervical abnormality. This preliminary survey suggests that HPV DNA is frequently transported from HPV-16-related cervical tumours to regional lymph nodes. However, its practical significance will not be clear until sufficient time has elapsed for correlation of the results with the clinical outcome.

An archival survey of 98 cervical cancer specimens dating from the 1920s to the 1980s was undertaken to determine whether changes had occurred in the prevalence of human papilloma-virus (HPV) DNA. HPV DNA was detected in paraffin sections of cancers fixed in 10% formalin by in situ hybridisation (ISH) using HPV 6, 11, 16, and 18 32P-labelled DNA probes under conditions of high stringency; and by the polymerase chain reaction (PCR) using 20-mer oligonucleotide primers to amplify 109 bases of the E6 region of HPV 16. In 30 instances results obtained from Southern blot hybridisations which had been carried out on specimens of fresh tissue from the same cancers collected during the 1980s were available for comparison. The rates of HPV DNA detection in cervical cancers ranged from 83% (by Southern or PCR) and 70% (by ISH) on specimens from the 1980s, to 50% and 63% (by ISH and PCR, respectively) on specimens from the 1920s. HPV 16 was by far the most common type, being identified by Southern or ISH in approximately 92% of HPV DNA-positive specimens. No significant change in the prevalence of HPV DNA, or of HPV types, in cervical cancers was found over the 65 year period examined.

The therapeutic efficacy of antiviral agents for postexposure prophylaxis to hepadnavirus infection has been studied using acyclovir and foscarnet in the duck hepatitis B virus (DHBV) model. A total of 112 Pekin-Aylesbury ducks were inoculated with DHBV at 11 days post-hatch. Three days later, groups of these birds were injected intraperitoneally twice daily for 10 days with acyclovir (25 mg/kg) or foscarnet (250 mg/kg) or phosphate-buffered saline. Serum samples were taken before, during, and up to 4 weeks post-treatment and were analysed for DHBV DNA by dot hybridization. Liver tissue obtained at sacrifice was examined for viral DNA and for histological changes. At completion of treatment with acyclovir, 21 of 22 ducks were not viremic, compared with 6 of 26 control birds (P less than 0.001). Four weeks after withdrawal of acyclovir, 12 of 20 ducks remained nonviremic, compared with 2 of 23 controls (P less than 0.01). In liver tissue, viral DNA was detected in 10 of 19 treated ducks, compared with 21/24 controls (P less than 0.01). Histological changes of hepatitis were present in more of the control birds than in the treated group. The results with foscarnet treatment were similar, although a smaller inoculum of DHBV was used and fewer control birds became infected. The administration of antiviral agents soon after exposure prevented productive infection in approximately 50% of birds. Therefore, the use of a safe antiviral agent such as acyclovir, which can be given orally, should be considered in post-exposure prophylaxis against human hepatitis B virus (HBV) infection.

Specific varieties of human papillomavirus (HPV) infecting the anogenital region were detected in clinical samples by use of a filter hybridization technique suitable for rapid screening of cervical and anal scrapes. In this way possibly benign types (HPV6 and HPV11) could be differentiated from types thought to be capable of malignant transformation (HPV 16 and HPV 18). Cervical or anal canal cells were applied directly to nylon filters and fixed by u.v. irradiation before hybridization with mixed viral DNA probes under both low- and high-stringency conditions. In addition, probe for the human Alu-repeated DNA sequence was used to assess the relative amount of total nucleic acids in each sample applied to the filter. HPV DNA was detected in 3 of 19 cervical scrapes from patients with no past or present history of wart virus infection or cervical dysplasia. Within a positive study group totalling 71 patients, HPV (6/11 or 16/18) was detected in cervical scrapes from 24% of 41 patients who did not have visible genital dysplasia, 30% of 27 patients with visible genital dysplasia or cervical intraepithelial neoplasia (CIN) I, and in 1 of 3 patients with past CIN II/III. In addition, HPV6/11 or 16/18 DNA was detected in anal scrapes from 3 of 6 male patients and in 85% of genital biopsies. A notably high proportion (4/6) of vaginal condylomata were positive with both the HPV6/11 and the HPV16/18 mixed viral DNA probes. Of the biopsies prepared for histopathology and positive for HPV DNA, the HPV group-specific antigen could be detected in only 60%.

The immune response to duck hepatitis B virus (DHBV) had not been elucidated. An assay was therefore established to detect the presence of antibody to DHB surface antigen (anti-DHBs) in serum of experimentally inoculated and naturally infected ducks. Anti-DHBs in serum was detected by indirect RIA from the percentage inhibition of binding of rabbit anti-DHBs to purified DHBsAg. Specificity was confirmed by positive and negative controls, infected and noninfected sera, and a mouse monoclonal antibody to DHB core antigen (anti-DHBc). Serum and liver samples were tested for DHBV DNA by dot-blot hybridization assay. Adult ducks repeatedly inoculated with DHBV remained non-viraemic but developed anti-DHBs. This antibody activity neutralized the infectivity of DHBV, which was experimentally inoculated into 1-day-old ducklings. In naturally infected flocks anti-DHBs was detected in a proportion of noninfected adult ducks as well as 1-day-old hatchlings. Anti-DHBs activity in hatchlings neutralized the infectivity of experimentally inoculated DHBV. Pekin ducks can therefore mount a neutralizing antibody response to DHBV, and immunity may be transferred in ovo from dam to off-spring.

It is now widely accepted that HPV types 16, 18, 31, and 33 are associated with the development of high grade intraepithelial neoplasia and malignant lesions in the cervix. On this basis, the identification of HPV types in cervical scrape samples has been advocated as a supplement to cytological screening tests. However, little is known of the distribution of the virus at different sites in the lower female genital tract or of how this distribution may change during the natural course of HPV infection. In this survey, HPV DNA dot hybridizations and, in some instances, Southern blot hybridizations with mixed HPV 6/11 and 16/18 probes were undertaken to detect HPV DNA in cervical scrapes and biopsies of the cervix, vagina, and vulva. A total of 92 women attending a Sydney hospital were screened: 59 of these patients had cervical disease, either invasive cervical carcinoma (CaCx) or cervical intraepithelial neoplasia (CIN), grades I-III. A group of 33 women who lacked evidence of cervical abnormalities served as controls. HPV DNA, predominantly type 16/18, was detected in the cervical biopsies of 96% of the CaCx patients, 80% of the CIN III patients, and 65% of the CIN I-II patients. In contrast only 9% of the cervical biopsies from the control group contained detectable HPV 6, 11, 16, or 18 DNA. A high proportion of the women with cervical abnormalities had evidence of concurrent vaginal and/or vulval papillomavirus involvement. The significance of these findings for routine screening and subsequent management of patients with HPV-associated cervical disease is discussed.