Complete genome sequence of a HPV31 isolate from laryngeal squamous cell carcinoma and biological consequences for p97 promoter activity.

Human papillomavirus type 31, although detected less frequently than HPV types 16 and 18, is associated with head and neck squamous cell carcinomas. Previous studies suggest that polymorphisms in the long control region (LCR) may alter the oncogenic potential of the virus. This study reports the first complete genome of a South African HPV31 isolate from a laryngeal squamous cell carcinoma. Sequence variations relative to the HPV31 prototype sequence were identified. The pBlue-Topo® vector, a reporter gene system was used to investigate the possible influence of these variations on the LCR promoter activity in vitro. Using mutagenesis to create two different fragments, ß-galactosidase assays were used to monitor the effect of nucleotide variations on the p97 promoter. Increased ß-galactosidase expression was observed in mutants when compared to the South African HPV31 LCR isolate. Enhanced transcriptional activity was observed with the mutant that possessed a single nucleotide change within the YY1 transcription factor binding site. In conclusion, sequence variation within the LCR of HPV31 isolates may have a functional effect on viral p97 promoter activity.

Whole-Genome Sequence and Comparative Analysis of Human Papillomavirus Type 18 Isolated from a Nasopharyngeal Carcinoma from South Africa.

We report the complete genome sequence of human papillomavirus type 18 isolated from a nasopharyngeal carcinoma in South Africa.

A simple and rapid approach to prepare Sindbis and West Nile viral RNA controls for differentiation between positive samples and laboratory contamination.

Reverse transcription-polymerase chain reaction (RT-PCR) is frequently used for surveillance and diagnosis of arboviruses and emerging viruses. A disadvantage of RT-PCR assays, especially nested assays, is the potential for false-positive results caused by laboratory contamination from either positive controls or positive samples. Positive reactors usually require sequence determination for confirmation which delay timeous reporting of a result. Thus, the aim of the study was to use a simple technique to prepare a positive control allowing true positives to be differentiated from laboratory contamination based on size differentiation for conventional PCR, or melt temperatures for real time assays. A flavivirus positive control and an alphavirus positive control were prepared for two RT-PCR assays that we are currently using for arbovirus surveillance in South Africa. Primers targeting a region of the partial genes of interest cloned in pGEM®T-easy were modified at the 5' ends with non-viral nucleotides. The resulting amplicons were circularised, resulting in pGEM®T-easy constructs with 51 and 65 non-viral bases inserted into the partial flaviviral and alphaviral genes respectively and used as template for transcribing RNA. Sequence analysis was used to confirm the manipulation of the partial genes. Using virus specific primer pairs, viral RNA could be readily differentiated from the modified positive controls either by size differentiation, or melt temperature in a SYBR®Green real time RT-PCR. This study demonstrates how simple recombinant technology can be used to produce a positive control that has application in the laboratory for surveillance studies or as a diagnostic tool using synthetic genes to abrogate the requirement for handling infectious virus.

Human papillomavirus in head and neck squamous cell carcinomas in a South African cohort.

BACKGROUND: Most tumours of the head and neck are attributable to smoking and alcohol use, but an increasing proportion of head and neck tumours are caused by human papillomaviruses (HPVs). The aim of this study was to use in house molecular assays to detect and genotype HPV in biopsies from patients with histologically confirmed head and neck squamous cell carcinomas. In addition, the results were compared with p16 immunohistochemistry staining, which has been described as a potential marker for HPV infection. METHODS: Biopsies of squamous cell carcinomas of the oropharynx, nasopharynx, larynx and hypopharynx from 112 South African patients were screened using three PCR assays targeting the L1 and E6 regions of HPV and p16 immunohistochemical staining. RESULTS AND CONCLUSION: HPV was identified in 7 (6.3%) tumours, while 22 (19.6%) had positive p16 immunohistochemical staining. There was concordance between the results obtained using the three PCR assays. There was substantial agreement between the results of molecular tests and p16 immunohistochemistry for hypopharyngeal carcinomas, but only fair agreement for laryngeal and oropharyngeal carcinomas.