Next-generation sequencing of cervical DNA detects human papillomavirus types not detected by commercial kits.

BACKGROUND: Human papillomavirus (HPV) is the aetiological agent for cervical cancer and genital warts. Concurrent HPV and HIV infection in the South African population is high. HIV positive (+) women are often infected with multiple, rare and undetermined HPV types. Data on HPV incidence and genotype distribution are based on commercial HPV detection kits, but these kits may not detect all HPV types in HIV + women. The objectives of this study were to (i) identify the HPV types not detected by commercial genotyping kits present in a cervical specimen from an HIV positive South African woman using next generation sequencing, and (ii) determine if these types were prevalent in a cohort of HIV-infected South African women. METHODS: Total DNA was isolated from 109 cervical specimens from South African HIV + women. A specimen within this cohort representing a complex multiple HPV infection, with 12 HPV genotypes detected by the Roche Linear Array HPV genotyping (LA) kit, was selected for next generation sequencing analysis. All HPV types present in this cervical specimen were identified by Illumina sequencing of the extracted DNA following rolling circle amplification. The prevalence of the HPV types identified by sequencing, but not included in the Roche LA, was then determined in the 109 HIV positive South African women by type-specific PCR. RESULTS: Illumina sequencing identified a total of 16 HPV genotypes in the selected specimen, with four genotypes (HPV-30, 74, 86 and 90) not included in the commercial kit. The prevalence's of HPV-30, 74, 86 and 90 in 109 HIV positive South African women were found to be 14.6%, 12.8%, 4.6% and 8.3% respectively. CONCLUSIONS: Our results indicate that there are HPV types, with substantial prevalence, in HIV positive women not being detected in molecular epidemiology studies using commercial kits. The significance of these types in relation to cervical disease remains to be investigated.

Genetic variability within the coat protein gene of Grapevine fanleaf virus isolates from South Africa and the evaluation of RT-PCR, DAS-ELISA and ImmunoStrips as virus diagnostic assays.

Grapevine fanleaf virus (GFLV) is responsible for severe fanleaf degeneration in grapevines of all major wine producing regions of the world, including South Africa. In order to successfully control the spread of the virus, specific and reliable diagnostic assays are necessary. The genetic variability of 12 GFLV isolates recovered from naturally infected grapevine plants in the Western Cape region of South Africa were characterised. These samples were subjected to RNA extraction, RT-PCR analysis and sequencing of the coat protein gene (2CCP). Sequence identities between different GFLV isolates from South Africa were between 86-99% and 94-99% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis based on the 2CCP gene sequences showed that the South African isolates form two distinct clades or sub-populations. The specificity and sensitivity of three diagnostic techniques (rapid-direct-one-tube-RT-PCR, DAS-ELISA and ImmunoStrips) for the detection of GFLV were analysed to determine the appropriate diagnostic assay for virus infection. Rapid-direct-one-tube-RT-PCR was found to be the most reliable technique for detection. This is the first report on sequence analysis of full-length 2CCP gene cDNA clones of GFLV isolates from South Africa.

Deep sequencing analysis of viruses infecting grapevines: Virome of a vineyard.

Double stranded RNA, isolated from 44 pooled randomly selected vines from a diseased South African vineyard, has been used in a deep sequencing analysis to build a census of the viral population. The dsRNA was sequenced in an unbiased manner using the sequencing-by-synthesis technology offered by the Illumina Genome Analyzer II and yielded 837 megabases of metagenomic sequence data. Four known viral pathogens were identified. It was found that Grapevine leafroll-associated virus 3 (GLRaV-3) is the most prevalent species, constituting 59% of the total reads, followed by Grapevine rupestris stem pitting-associated virus and Grapevine virus A. Grapevine virus E, a virus not previously reported in South African vineyards, was identified in the census. Viruses not previously identified in grapevine were also detected. The second most prevalent virus detected was a member of the Chrysoviridae family similar to Penicillium chrysogenum virus. Sequences aligning to two other mycoviruses were also detected.

Mapping of the 5' terminal nucleotides of Grapevine leafroll-associated virus 3 sgRNAs.

Grapevine leafroll-associated virus 3 (GLRaV-3) is a positive sense, single stranded RNA virus that has a detrimental effect on wine and table grapes worldwide. Previous studies have shown that GLRaV-3, like other closteroviruses produces subgenomic (sg) RNAs during replication and that these sgRNAs are deployed for the expression of the ORFs on the 3' half of the genome. In this study a dsRNA blot confirmed the presence of three, 3' co-terminal sgRNAs species [sgRNA(ORF3/4), sgRNA(ORF5) and sgRNA(ORF6)] in GLRaV-3-infected plant material when using a probe directed at the coat protein. The specific 5' terminal nucleotides for these sgRNAs as well as four additional sgRNAs [sgRNA(ORF7), sgRNA(ORF8), sgRNA(ORF9) and sgRNA(ORF10-12)] were determined by RLM-RACE for GLRaV-3 isolate GP18.