Historical cottontail rabbit papillomavirus studies raised early indications of a mammalian DNA oncogenic virus. Today, molecular cloning recognises numerous animal and human papillomaviruses (HPVs) and the development of in vitro transformation assays has escalated oncological research in HPVs. Currently, their detection and typing in tissues is usually by Southern blotting, in-situ hybridization and polymerase chain reaction methods. The complete papillomavirus virion constitutes a protein coat (capsid) surrounding a circular, double-stranded DNA organised into coding and non-coding regions. 8 early (E1-E8) open reading frames (ORFs) and 2 late (L1, L2) ORFs have been identified in the coding region of all papillomaviruses. The early ORFs encode proteins which interact with the host genome to produce new viral DNA while late ORFs are activated only after viral DNA replication and encode for viral capsid proteins. All papillomaviruses are obligatory intranuclear organisms with specific tropism for keratinocytes. Three possible courses of events can follow papillomaviruses entry into cells: (1) viral DNA are maintained as intranuclear, extrachromosomal, circular DNA episomes, which replicates synchronously with the host cell, establishing a latent infection; (2) conversion from latent into productive infection with assembly of complete infective virions; (3) integration of viral DNA into host cellular genome, a phenomenon seen in HPV infections associated with malignant transformation. Human papillomaviruses (HPVs) essentially induce skin and mucosal epithelial lesions. Various skin warts are well known to be HPV-associated (HPVs 1, 2, 3, 7 and 10). Besides HPVs 3 and 10, HPVs 5, 8, 17 and 20 have been recovered from Epidermodysplasia verruciformis lesions. Anogenital condyloma acuminatum, strongly linked with HPVs 6 and 11 are probably sexually transmitted. The same HPVs, demonstrable in recurrent juvenile laryngeal papillomas, are probably transmitted by passage through an infected birth canal. HPVs described in uterine cervical lesions are generally categorized into those associated with high (16, 18), intermediate (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) and low (6, 11, 26, 40, 42, 43, 44, 53, 54, 55, 62, 66) risk of cervical squamous carcinoma. Cervical adenocarcinoma, clear cell carcinoma and small cell neuroendocrine carcinoma have also been linked to HPVs, especially HPV18. Other lesions reported to be HPV-associated are: papillomas, dysplasia and carcinomas in the nasal cavity (HPV 6, 11, 57); squamous papilloma, condyloma acuminatum, and verruca vulgaris of the oral cavity (HPV 6, 11), oral focal epithelial hyperplasia (HPV 13, 32); warty lip lesions (HPV 2): and conjunctival papillomas (HPV 6, 11).
The Epstein-Barr virus (EBV) has been implicated as a contributing factor in the development of Hodgkin's disease. Western cases of Hodgkin's disease have shown the presence of EBV in Hodgkin and Reed-Sternberg cells in approximately 50%. We studied a total of 100 consecutive cases of Hodgkin's disease from Malaysia, with the aim to elucidate its association with EBV in a multi-ethnic Asian population. Of 34 patients (34%) less than 15 years of age (childhood), 25 had classical Hodgkin's disease (eight nodular sclerosis, 16 mixed cellularity, one lymphocyte depleted) and nine had lymphocyte predominance Hodgkin's disease. Of the 66 from patients aged 15 years and above, 33 had nodular sclerosis, 24 mixed cellularity, two lymphocyte depleted, one unclassifiable and six lymphocyte predominance Hodgkin's disease. The ethnic distribution of classical Hodgkin's disease was: Malay 23, Chinese 32 and Indian 30 (Malay:Chinese:Indian = 1:1.4:1.3), and the ethnic distribution in the 15 cases of lymphocyte predominance Hodgkin's disease was: Malay four, Chinese 10 and Indian one. Taking into account the ethnic distribution of the general population and of hospital admissions, there appears to be a significant predilection of classical Hodgkin's disease cases in ethnic Indian compared to non-Indian patients (chi-squared test, 0.025 > P > 0.01). Eighty-one cases were tested for the presence of EBV by in situ hybridization for EBV encoded RNA, and 57 cases by immunostaining for EBV latent membrane protein 1. In the younger age group, all except one of the 15 cases (nine mixed cellularity, six nodular sclerosis) showed the presence of EBV (93%). In the older age group, EBV was detected (52%) in the following proportion: 6/27 nodular sclerosis, 19/22 mixed cellularity, 1/2 lymphocyte depleted, 1/1 unclassifiable. None of the 14 cases of lymphocyte predominance Hodgkin's disease showed the presence of EBV in the Hodgkin and Reed-Sternberg cells. The findings suggest a strong association of EBV with Hodgkin's disease in Malaysians (41/67, 61%), in particular childhood cases (93%). In adults, the association with EBV is significantly higher in the mixed cellularity subtype (86%) compared with the nodular sclerosis subtype (22%).
Epstein-Barr virus (EBV) has consistently been detected in the tumour cells of nasopharyngeal carcinoma and lymphoepithelial-like carcinoma of the salivary glands, and have occasionally been found in similar tumours at other sites. Moreover, recent studies from various parts of the world including the Orient have shown about 10% of gastric carcinomas to be EBV-associated. We studied 50 gastric carcinomas from Malaysia to investigate its association with EBV in the Malaysian population. They comprised 37 intestinal and 13 diffuse type carcinomas from 32 male and 18 female patients, age range from 29 to 86 years with an ethnic distribution of Malay: Chinese: Indian with the ratio of 4: 27: 19. EBV gene and gene-expression were examined in sections of formalin-fixed, paraffin-embedded tissue using commercially available probes for detecting EBV encoded RNAs (EBERs) by in situ hybridization and monoclonal antibodies to EBV latent membrane protein-1 (LMP-1) by standard immunohistochemistry. Five of 50 gastric carcinomas showed EBER intranuclear positivity in all tumour cells but no cases expressed LMP-1. The EBV-associated cases were classified as intestinal type in 4 and diffuse type in one case and all were histologically unremarkable. EBV-positive tumours were found in 3 Chinese and 2 Indian patients with none in the small Malay group. Four EBV-positive tumours were in male patients, with age-range of 65 to 86 years. We conclude that our findings of about 10% of Malaysian gastric carcinomas being EBV-associated is in line with the results from other parts of the world and from other ethnic groups.
As a simian species, the langurs are not known to harbor simian retroviruses, except for one report on a simian Type D endogenous retrovirus from the spectacled langur (Trachypithecus obscurus) from Malaysia. The present report describes for the first time natural infection of the common Hanuman langur (Semnopithecus entellus) from India by a novel simian retrovirus (SRV). The new SRV is phylogenetically related to but distinct from the three molecularly characterized serotypes, SRV 1-3, of the five known serotypes of SRVs, based on sequence analyses from the 3'orf and env regions of the viral genome. The novel SRV isolated from the Indian Hanuman langur is provisionally named SRV-6.
Isolation of single cells permits analysis of DNA or RNA from individual cells among heterogeneous populations. This technique is particularly useful in the study of classical Hodgkin's lymphoma (cHL) due to the scarcity of H/RS tumor cells among large numbers of reactive leukocytes. In a previous study, we found a high frequency of dual LMP-1 variant (concurrent presence of deleted and nondeleted variants) in cHL from whole-tissue sections. For the present study, we applied a single-cell isolation technique to determine the LMP-1 oncogene variant in EBV-associated H/RS cells. Five cases of EBV-infected cHL, containing nondeleted (n=1), deleted (n=1) and dual infection (n=3) based on whole-tissue section analysis, were selected for study. Paraffin-embedded tissue sections were stained with antibody to LMP-1 and positively stained H/RS cells isolated using a semiautomated micromanipulator. Each isolated single cell was subjected to PCR for amplification of the LMP-1 gene flanking the 30 bp deletion region and Xho1 restriction site. Cases with either nondeleted variant or the deleted variant showed similar LMP-1 variant expression in isolated single H/RS cells. However, 1 of the 3 cases with dual variants showed only the deleted variant in H/RS cells. The other 2 cases showed mixed patterns of deleted, nondeleted and dual LMP-1 variants in isolated single H/RS cells. All cases showed loss of the Xho1 restriction site, with the exception of the case with nondeleted LMP-1. Results of single-H/RS cell analysis of the Xho1 restriction site concur with those of whole-tissue section amplification. A mixed pattern of LMP-1 variants was observed in isolated H/RS cells, and it is speculated that this is due to the accumulation of mutation and deletion events.
Archival oral tissues comprising 51 squamous cell carcinomas, 18 non-malignant lesions and 7 normal mucosa samples were investigated for human herpesvirus-6 (HHV-6)-encoded antigens and HHV-6 DNA. The virus-specific antigens were detected by an immunohistochemical method using monoclonal antibodies. Two further techniques used for HHV-6 DNA detection included the polymerase chain reaction (PCR) with virus-specific primers and in situ hybridization using digoxigenin-labelled oligonucleotides specific for HHV-6A and HHV-6B genotypes. A high proportion (79-80%) of the squamous cell carcinomas were positive for HHV-6 with the various detection methods. In cases of lichen planus and leukoplakia a high prevalence rate (67-100%) was noted with in situ hybridization and immunohistochemical techniques but a lower proportion (22-33%) was detected with the PCR method. All 7 normal tissues tested were negative for HHV-6. The HHV-6 variant B was found in 60% of the oral carcinoma tissues analysed. The study demonstrates the frequent presence of HHV-6 in neoplastic and non-malignant lesions of the oral cavity. While the role of HHV-6 in oral mucosal tissues remains to be determined, the in vitro tumorigenic potential of the virus suggests a possible role in the etiopathogenesis of oral lesions.
PURPOSE: The purpose of this study was to evaluate the role of HPV and p53 polymorphisms in oral squamous cell carcinomas (OSCC) affecting Malaysian population.
METHODS: We analysed frozen samples from 105 OSCC as well as 105 oral specimens derived from healthy individuals. PCR assays targeting two regions of the virus were used. PCR amplification for the analysis of p53 codon 72 arginine/proline alleles was carried out in a separate reaction.
RESULTS: HPV DNA was detected in 51.4% OSCC samples, while 24.8% controls were found to be HPV positive. HPV was found to be significantly associated with OSCC (P
LMP-1, an Epstein-Barr viral (EBV) latency protein, is considered a viral oncogene because of its ability to transform rodent fibroblasts in vivo and render them tumorigenic in nude mice. In human B cells, EBV LMP-1 induces DNA synthesis and abrogates apoptosis. LMP-1 is expressed in EBV-transformed lymphoblastoid cell lines, nasopharyngeal carcinoma (NPC), a subset of Hodgkin's disease (HD), and in EBV-associated lymphoproliferative disorders (EBV-LPDs). Recently, focused deletions near the 3' end of the LMP-1 gene (del-LMP-1, amino acids 346-355), in a region functionally related to the half-life to the LMP-1 protein, have been reported frequently in human immunodeficiency virus (HIV)-associated HD (100%) and EBV+ Malaysian and Danish peripheral T-cell lymphomas (100%, 61% respectively), but less frequently in cases of HD not associated with HIV (28%, 33%) and infectious mononucleosis (33%). To further investigate the potential relationship of del-LMP-1 to EBV-LPDs associated with immunosuppression or immunodeficiency, we studied 39 EBV-associated lymphoproliferations (10 benign, 29 malignant) from four distinct clinical settings: posttransplant (4 malignant, 1 reactive); HIV+ (18 malignant, 2 reactive); nonimmunodeficiency malignant lymphoma (ML) (7 cases); and sporadic EBV infection with lymphoid hyperplasia (7 cases). The presence of EBV within lymphoid cells was confirmed by EBV EBER1 RNA in situ hybridization or by polymerase chain reaction (PCR) analysis. EBV strain type and LMP-1 deletion status were determined by PCR. EBV strain types segregated into two distinct distributions: HIV+ (9 A; 11 B) and non-HIV (19 A, 0 B), consistent with previous reports. Overall, del-LMP-1 were found in 1 of 5 (20%) Burkitt lymphomas (BL); 17 of 24 (71%) aggressive non-Hodgkin's lymphoma (agg-NHL), and 2 of 10 (20%) reactive lymphoid proliferations. Of the agg-NHLs, del-LMP-1 were present in 4 of 4 PT-ML (100%); 10 of 15 HIV+ ML (67%); and 3 of 5 nonimmunodeficiency malignant lymphoma (ML, 60%). A total of 2 of 7 (28%) sporadic EBV-associated lymphoid hyperplasias contained a del-LMP-1. All del-LMP-1 were identical by DNA sequence analysis. No correlation was identified between the presence of del-LMP-1 and the EBV strain type observed. The high incidence of del-LMP-1 observed in agg-NHLs (71%), in contrast to the relatively low incidence observed in reactive lymphoid proliferations (28%), suggests that the deleted form may be preferentially selected in lymphomatous processes. All posttransplant agg-NHLs contained a del-LMP-1, and a similar frequency of del-LMP-1 was observed in both HIV-associated ML (66%) and nonimmunodeficiency ML (60%), suggesting that impairment of immune function alone is not a requirement for the expansion of malignant cells infected by EBV stains containing the deleted LMP-1 gene.