Ignacio G. Bravo

Bead-Based Multiplex Genotyping of Human Papillomaviruses

ABSTRACT Typing of human papillomaviruses (HPV) by DNA hybridization procedures, such as reverse line blot (RLB) assay, is sensitive and well validated. However, the application of these assays to high-throughput analyses is limited. Here, we describe the development of multiplex human papillomavirus genotyping (MPG), a quantitative and sensitive high-throughput procedure for the identification of multiple high- and low-risk genital HPV genotypes in a single reaction. MPG is based on the amplification of HPV DNA by a general primer PCR (GP5+/6+) and the subsequent detection of the products with type-specific oligonucleotide probes coupled to fluorescence-labeled polystyrene beads (Luminex suspension array technology). Up to 100 different HPV types can be detected simultaneously with MPG, and the method is fast and labor saving. We detected all 22 HPV types examined with high specificity and reproducibility (the median interplate coefficient of variation was below 10%). Detection limits for the different HPV types varied between 100 and 800 pg of PCR products. We compared the performance of MPG to an established RLB assay on GP5+/6+-PCR products derived from 94 clinical samples. The evaluation showed an excellent agreement (kappa = 0.922) but also indicated a higher sensitivity of MPG. In conclusion, MPG appears to be highly suitable for large-scale epidemiological studies and vaccination trials as well as for routine diagnostic purposes.

Worldwide human papillomavirus genotype attribution in over 2000 cases of intraepithelial and invasive lesions of the vulva

BACKGROUND Human papillomavirus (HPV) contribution in vulvar intraepithelial lesions (VIN) and invasive vulvar cancer (IVC) is not clearly established. This study provides novel data on HPV markers in a large series of VIN and IVC lesions. METHODS Histologically confirmed VIN and IVC from 39 countries were assembled at the Catalan Institute of Oncology (ICO). HPV-DNA detection was done by polymerase chain reaction using SPF-10 broad-spectrum primers and genotyping by reverse hybridisation line probe assay (LiPA25) (version 1). IVC cases were tested for p16(INK4a) by immunohistochemistry (CINtec histology kit, ROCHE). An IVC was considered HPV driven if both HPV-DNA and p16(INK4a) overexpression were observed simultaneously. Data analyses included algorithms allocating multiple infections to calculate type-specific contribution and logistic regression models to estimate adjusted prevalence (AP) and its 95% confidence intervals (CI). RESULTS Of 2296 cases, 587 were VIN and 1709 IVC. HPV-DNA was detected in 86.7% and 28.6% of the cases respectively. Amongst IVC cases, 25.1% were both HPV-DNA and p16(INK4a) positive. IVC cases were largely keratinising squamous cell carcinoma (KSCC) (N=1234). Overall prevalence of HPV related IVC cases was highest in younger women for any histological subtype. SCC with warty or basaloid features (SCC_WB) (N=326) were more likely to be HPV and p16(INK4a) positive (AP=69.5%, CI=63.6-74.8) versus KSCC (AP=11.5%, CI=9.7-13.5). HPV 16 was the commonest type (72.5%) followed by HPV 33 (6.5%) and HPV 18 (4.6%). Enrichment from VIN to IVC was significantly high for HPV 45 (8.5-fold). CONCLUSION Combined data from HPV-DNA and p16(INK4a) testing are likely to represent a closer estimate of the real fraction of IVC induced by HPV. Our results indicate that HPV contribution in invasive vulvar cancer has probably been overestimated. HPV 16 remains the major player worldwide.

CAIcal: A combined set of tools to assess codon usage adaptation

BackgroundThe Codon Adaptation Index (CAI) was first developed to measure the synonymous codon usage bias for a DNA or RNA sequence. The CAI quantifies the similarity between the synonymous codon usage of a gene and the synonymous codon frequency of a reference set.ResultsWe describe here CAIcal, a web-server available at http://genomes.urv.es/CAIcal that includes a complete set of utilities related with the CAI. The server provides useful important features, such as the calculation and graphical representation of the CAI along either an individual sequence or a protein multiple sequence alignment translated to DNA. The automated calculation of CAI and its expected value is also included as one of the CAIcal tools. The software is also free to be downloaded as a standalone application for local use.ConclusionThe CAIcal server provides a complete set of tools to assess codon usage adaptation and to help in genome annotation.ReviewersThis article was reviewed by Purificación López-García, Dan Graur, Rob Knight and Shamil Sunyaev.

HPV Involvement in Head and Neck Cancers: Comprehensive Assessment of Biomarkers in 3680 Patients

BACKGROUND We conducted a large international study to estimate fractions of head and neck cancers (HNCs) attributable to human papillomavirus (HPV-AFs) using six HPV-related biomarkers of viral detection, transcription, and cellular transformation. METHODS Formalin-fixed, paraffin-embedded cancer tissues of the oral cavity (OC), pharynx, and larynx were collected from pathology archives in 29 countries. All samples were subject to histopathological evaluation, DNA quality control, and HPV-DNA detection. Samples containing HPV-DNA were further subject to HPV E6*I mRNA detection and to p16(INK4a), pRb, p53, and Cyclin D1 immunohistochemistry. Final estimates of HPV-AFs were based on HPV-DNA, HPV E6*I mRNA, and/or p16(INK4a) results. RESULTS A total of 3680 samples yielded valid results: 1374 pharyngeal, 1264 OC, and 1042 laryngeal cancers. HPV-AF estimates based on positivity for HPV-DNA, and for either HPV E6*I mRNA or p16(INK4a), were 22.4%, 4.4%, and 3.5% for cancers of the oropharynx, OC, and larynx, respectively, and 18.5%, 3.0%, and 1.5% when requiring simultaneous positivity for all three markers. HPV16 was largely the most common type. Estimates of HPV-AF in the oropharynx were highest in South America, Central and Eastern Europe, and Northern Europe, and lowest in Southern Europe. Women showed higher HPV-AFs than men for cancers of the oropharynx in Europe and for the larynx in Central-South America. CONCLUSIONS HPV contribution to HNCs is substantial but highly heterogeneous by cancer site, region, and sex. This study, the largest exploring HPV attribution in HNCs, confirms the important role of HPVs in oropharyngeal cancer and drastically downplays the previously reported involvement of HPVs in the other HNCs.

Human papillomavirus DNA prevalence and type distribution in anal carcinomas worldwide

Knowledge about human papillomaviruses (HPV) types involved in anal cancers in some world regions is scanty. Here, we describe the HPV DNA prevalence and type distribution in a series of invasive anal cancers and anal intraepithelial neoplasias (AIN) grades 2/3 from 24 countries. We analyzed 43 AIN 2/3 cases and 496 anal cancers diagnosed from 1986 to 2011. After histopathological evaluation of formalin‐fixed paraffin‐embedded samples, HPV DNA detection and genotyping was performed using SPF‐10/DEIA/LiPA25 system (version 1). A subset of 116 cancers was further tested for p16INK4a expression, a cellular surrogate marker for HPV‐associated transformation. Prevalence ratios were estimated using multivariate Poisson regression with robust variance in the anal cancer data set. HPV DNA was detected in 88.3% of anal cancers (95% confidence interval [CI]: 85.1–91.0%) and in 95.3% of AIN 2/3 (95% CI: 84.2–99.4%). Among cancers, the highest prevalence was observed in warty–basaloid subtype of squamous cell carcinomas, in younger patients and in North American geographical region. There were no statistically significant differences in prevalence by gender. HPV16 was the most frequent HPV type detected in both cancers (80.7%) and AIN 2/3 lesions (75.4%). HPV18 was the second most common type in invasive cancers (3.6%). p16INK4a overexpression was found in 95% of HPV DNA‐positive anal cancers. In view of the results of HPV DNA and high proportion of p16INK4a overexpression, infection by HPV is most likely to be a necessary cause for anal cancers in both men and women. The large contribution of HPV16 reinforces the potential impact of HPV vaccines in the prevention of these lesions.

Seroprevalence of 34 Human Papillomavirus Types in the German General Population

The natural history of infections with many human papillomavirus (HPV) types is poorly understood. Here, we describe for the first time the age- and sex-dependent antibody prevalence for 29 cutaneous and five mucosal HPV types from 15 species within five phylogenetic genera (alpha, beta, gamma, mu, nu) in a general population. Sera from 1,797 German adults and children (758 males and 1,039 females) between 1 and 82 years (median 37 years) were analysed for antibodies to the major capsid protein L1 by Luminex-based multiplex serology. The first substantial HPV antibody reactions observed already in children and young adults are those to cutaneous types of the genera nu (HPV 41) and mu (HPV 1, 63). The antibody prevalence to mucosal high-risk types, most prominently HPV 16, was elevated after puberty in women but not in men and peaked between 25 and 34 years. Antibodies to beta and gamma papillomaviruses (PV) were rare in children and increased homogeneously with age, with prevalence peaks at 40 and 60 years in women and 50 and 70 years in men. Antibodies to cutaneous alpha PV showed a heterogeneous age distribution. In summary, these data suggest three major seroprevalence patterns for HPV of phylogenetically distinct genera: antibodies to mu and nu skin PV appear early in life, those to mucosal alpha PV in women after puberty, and antibodies to beta as well as to gamma skin PV accumulate later in life.

Functions, Structure, and Read-Through Alternative Splicing of Feline APOBEC3 Genes

BackgroundOver the past years a variety of host restriction genes have been identified in human and mammals that modulate retrovirus infectivity, replication, assembly, and/or cross-species transmission. Among these host-encoded restriction factors, the APOBEC3 (A3; apolipoprotein B mRNA-editing catalytic polypeptide 3) proteins are potent inhibitors of retroviruses and retrotransposons. While primates encode seven of these genes (A3A to A3H), rodents carry only a single A3 gene.ResultsHere we identified and characterized several A3 genes in the genome of domestic cat (Felis catus) by analyzing the genomic A3 locus. The cat genome presents one A3H gene and three very similar A3C genes (a-c), probably generated after two consecutive gene duplications. In addition to these four one-domain A3 proteins, a fifth A3, designated A3CH, is expressed by read-through alternative splicing. Specific feline A3 proteins selectively inactivated only defined genera of feline retroviruses: Bet-deficient feline foamy virus was mainly inactivated by feA3Ca, feA3Cb, and feA3Cc, while feA3H and feA3CH were only weakly active. The infectivity of Vif-deficient feline immunodeficiency virus and feline leukemia virus was reduced only by feA3H and feA3CH, but not by any of the feA3Cs. Within Felidae, A3C sequences show significant adaptive selection, but unexpectedly, the A3H sequences present more sites that are under purifying selection.ConclusionOur data support a complex evolutionary history of expansion, divergence, selection and individual extinction of antiviral A3 genes that parallels the early evolution of Placentalia, becoming more intricate in taxa in which the arms race between host and retroviruses is harsher.

E-CAI: a novel server to estimate an expected value of Codon Adaptation Index (eCAI)

BackgroundThe Codon Adaptation Index (CAI) is a measure of the synonymous codon usage bias for a DNA or RNA sequence. It quantifies the similarity between the synonymous codon usage of a gene and the synonymous codon frequency of a reference set. Extreme values in the nucleotide or in the amino acid composition have a large impact on differential preference for synonymous codons. It is thence essential to define the limits for the expected value of CAI on the basis of sequence composition in order to properly interpret the CAI and provide statistical support to CAI analyses. Though several freely available programs calculate the CAI for a given DNA sequence, none of them corrects for compositional biases or provides confidence intervals for CAI values.ResultsThe E-CAI server, available at http://genomes.urv.es/CAIcal/E-CAI, is a web-application that calculates an expected value of CAI for a set of query sequences by generating random sequences with G+C and amino acid content similar to those of the input. An executable file, a tutorial, a Frequently Asked Questions (FAQ) section and several examples are also available. To exemplify the use of the E-CAI server, we have analysed the codon adaptation of human mitochondrial genes that codify a subunit of the mitochondrial respiratory chain (excluding those genes that lack a prokaryotic orthologue) and are encoded in the nuclear genome. It is assumed that these genes were transferred from the proto-mitochondrial to the nuclear genome and that its codon usage was then ameliorated.ConclusionThe E-CAI server provides a direct threshold value for discerning whether the differences in CAI are statistically significant or whether they are merely artifacts that arise from internal biases in the G+C composition and/or amino acid composition of the query sequences.

Mucosal Human Papillomaviruses Encode Four Different E5 Proteins Whose Chemistry and Phylogeny Correlate with Malignant or Benign Growth

ABSTRACT We performed a phylogenetic study of the E2-L2 region of human mucosal papillomaviruses (PVs) and of the proteins therein encoded. Hitherto, proteins codified in this region were known as E5 proteins. We show that many of these proteins could be spurious translations, according to phylogenetic and chemical coherence criteria between similar protein sequences. We show that there are four separate families of E5 proteins, with different characteristics of phylogeny, chemistry, and rate of evolution. For the sake of clarity, we propose a change in the present nomenclature. E5α is present in groups A5, A6, A7, A9, and A11, PVs highly associated with malignant carcinomas of the cervix and penis. E5β is present in groups A2, A3, A4, and A12, i.e., viruses associated with certain warts. E5γ is present in group A10, and E5δ is encoded in groups A1, A8, and A10, which are associated with benign transformations. The phylogenetic relationships between mucosal human PVs are the same when considering the oncoproteins E6 and E7 and the E5 proteins and differ from the phylogeny estimated for the structural proteins L1 and L2. Besides, the protein divergence rate is higher in early proteins than in late proteins, increasing in the order L1 < L2 < E6 ≈ E7 < E5. Moreover, the same proteins have diverged more rapidly in viruses associated with malignant transformations than in viruses associated with benign transformations. The E5 proteins display, therefore, evolutionary characteristics similar to those of the E6 and E7 oncoproteins. This could reflect a differential involvement of the E5 types in the transformation processes.

Quantifying the phylodynamic forces driving papillomavirus evolution

The associations between pathogens and their hosts are complex and can result from a variety of evolutionary processes including codivergence, lateral transfer, or duplication. Papillomaviruses (PVs) are double-stranded DNA viruses ubiquitously present in mammals and are a suitable target for rigorous statistical tests of potential virus-host codivergence. We analyze the evolutionary dynamics of PV diversification by comparing robust phylogenies of PVs and their respective hosts using different statistical approaches to assess topological and branch-length congruence. Mammalian PVs segregated into four diverse major clades that overlapped to varying degrees in terms of their mammalian host lineages. The hypothesis that PVs and hosts evolved independently was globally rejected (P = 0.0001), although only 90 of 207 virus-host associations (43%) were significant in individual tests. Virus-host codivergence accounted roughly for one-third of the evolutionary events required to reconcile PV-host evolutionary histories. When virus-host associations were analyzed locally within each of the four viral clades, numerous independent topological congruencies were identified that were incompatible with respect to the global trees. These results support an evolutionary scenario in which early PV radiation was followed by independent codivergence between viruses within each of the major clades and their hosts. Moreover, heterogeneous groups of closely related PVs infecting non-related hosts suggest several interspecies transmission events. Our results argue thus for the importance of alternative events in PV evolution, in contrast to the prevailing opinion that these viruses show a high degree of host specificity and codivergence.