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  • SDSN - Greece
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  • UK Research and Innovation
  • Pathogen diversity, host specificity and virulence

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    Authors: Forth, Jan H.; Forth, Leonie F.; Lycett, Samantha; Bell-Sakyi, Lesley; +8 Authors

    Additional file 5: Supplementary Appendix. Phylogenetic and molecular clock analyses of ASFLI-elements from different tick genomes and ASFV using different clock-rates and substitution models implemented in BEAST. Supplementary Appendix. Figure A1-A5, Tables A1-A7. FigA1- Phylogenetic tree of ASFV and ASFLI-elements. FigA2 – Phylogenetic tree of NCLDV including ASFV and ASFLI-elements. FigA3 – Time-scaled tree for partial EP1242L sequences from tick samples and reference ASFV (non-integrated). FigA4 - Time-scaled tree for partial EP1242L sequences from tick samples only (5e-7 substitutions per site per year). FigA5 - Time-scaled tree for partial EP1242L sequences from tick samples (1e-8 substitutions per site and year). Table A1 - Summary of EP1242L fragments derived from ticks and the tick cell line OME/CTVM21 (OME21) used in the analysis. Table A2 - Indels in the tick sample sequences relative to the start of EP1242L in ASFV|KM111295|Kenya|Ken06/Bus|2006. Table A3 - Estimated root height and overall mean clock rate for strict clocks with fixed priors. Table A4 - Estimated root height and overall (averaged over all branches) mean clock rate for relaxed clocks with fixed priors of the rate of the relaxed clock (some variation). Table A5 - Estimated root height and overall (averaged over all branches) mean clock rate for relaxed clocks with normal or log-normal priors on the rate of the relaxed clock (most variation). Table A6 - Estimated root height and overall (averaged over all branches) mean clock rate for strict clocks with log-normal priors on the rate of the strict clock for the five ASFV-like sequences from tick samples only. Table A7 - Marginal likelihood estimation using Path Sampling and Stepping Stone Sampling, showing that the log-normal clock rate prior with mean = 5e-7 is best, but not significantly better than the other clock rates.

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    Authors: Forth, Jan H.; Forth, Leonie F.; Lycett, Samantha; Bell-Sakyi, Lesley; +8 Authors

    Additional file 20: Figure S7. BLAST-analysis for identification of ASFLI-element containing contigs and annotation. 66,745 SPAdes assembled contigs were blasted (BLASTn, NCBI, v2.6.0+) against a customised database comprising all sequences with the NCBI taxonomy ID 10497 (African swine fever virus) (as of 16 January 2018). Hits were filtered using a cut off e-value of 1x10-4 and a minimum alignment length of 150 bp, resulting in 34 contigs. These were then blasted against the complete NCBI database (The non-redundant nucleotide collection) to reliably identify and annotate ASFV-like sequences and areas of the host genome using default parameters. BLASTp search of >500 bp ORFs was performed against the “Non-redundant protein sequences” database using default parameters.

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    Authors: Forth, Jan H.; Forth, Leonie F.; Lycett, Samantha; Bell-Sakyi, Lesley; +8 Authors

    Additional file 7: Table S6. BLASTn results of SPAdes-assembled contigs from Ornithodoro porcinus France and Kenya17 containing ASFLI-elements. Shown are hits with the lowest e-value for ASFV-genes as obtained by Blastn against the entire NCBI database.

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    Authors: Forth, Jan H.; Forth, Leonie F.; Lycett, Samantha; Bell-Sakyi, Lesley; +8 Authors

    Additional file 17: Table S11. O. porcinus piRNA ping-pong signature analysed by PingPongPro v1.0 with default parameters and -b option (creates additional browser track files, which are suitable for display in common genome browsers). The closer the Score-value (1 minus the FDR-value: estimated fraction of signatures that have the same combination of properties, but that are not true ping-pong signatures) which is calculated from adenine bias at pos. 10 of the piRNA, stack height - e.g. the number of reads that make up the stack and independence of local coverage is to 1, the more likely is a true ping-pong signature.

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    Authors: Lu, Lu; Brierley, Liam; Robertson, Gail; Zhang, Feifei; +5 Authors

    AbstractTo have epidemic potential, a pathogen must be able to spread in human populations, but of human-infective RNA viruses only a minority can do so. We investigated the evolution of human transmissibility through parallel analyses of 1755 virus genome sequences from 39 RNA virus genera. We identified 57 lineages containing human-transmissible species and estimated that at least 74% of these lineages have evolved directly from non-human viruses in other mammals or birds, a public health threat recently designated “Disease X”. Human-transmissible viruses rarely evolve from virus lineages that can infect but not transmit between humans. This result cautions against focussing surveillance and mitigation efforts narrowly on currently known human-infective virus lineages and supports calls for a better understanding of RNA virus diversity in non-human hosts.

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    Authors: Forth, Jan H.; Forth, Leonie F.; Lycett, Samantha; Bell-Sakyi, Lesley; +8 Authors

    Additional file 9: Table S7. SPAdes-assembled contigs from deep sequencing data from libraries AGL001 and MPA001, as generated from museum-stored ticks. Data from libraries from museum-stored tick were mapped against ASFLI-element-containing databases of Ornithoidoros moubata (AGL001) and Ornithodoros porcinus (MPA001) using Bowtie2 (v.2.3.4.3) with default parameters. Subsequently, mapped reads were assembled using SPAdes and aligned to the corresponding ASFLI- element-contigs using MAFFT (v. 7.388) in Geneious.

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    Authors: Forth, Jan H.; Forth, Leonie F.; Lycett, Samantha; Bell-Sakyi, Lesley; +8 Authors

    Additional file 18: Figure S6. The reconstructed ASFV-like A104R protein is highly similar to its ASFV homologue (A) A rabbit antiserum raised against the reconstructed A104 gene recognised a flag-tagged and an untagged version of A104R protein (lanes A104-Flag and A104, respectively), but showed no specific reaction with extracts of tick cell lines OME/CTVM21, OME/CTVM22, OME/CTVM24, and OME/CTVM27. In extracts of WSL-HP cells infected with ASFV Kenya 1033, the serum reacted with a single band of 12 kDa which is similar to the calculated molecular weight of ASFV A104R (11.6 kDa). (B) The Coomassie stained gel confirms equal loading with proteins.

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    Authors: J. H. Forth; Leonie Forth; Samantha Lycett; Lesley Bell-Sakyi; +8 Authors

    BACKGROUND: African swine fever virus (ASFV) is a most devastating pathogen affecting swine. In 2007, ASFV was introduced into Eastern Europe where it continuously circulates and recently reached Western Europe and Asia, leading to a socio-economic crisis of global proportion. In Africa, where ASFV was first described in 1921, it is transmitted between warthogs and soft ticks of the genus Ornithodoros in a so-called sylvatic cycle. However, analyses into this virus’ evolution are aggravated by the absence of any closely related viruses. Even ancient endogenous viral elements, viral sequences integrated into a host’s genome many thousand years ago that have proven extremely valuable to analyse virus evolution, remain to be identified. Therefore, the evolution of ASFV, the only known DNA virus transmitted by arthropods, remains a mystery. - RESULTS: For the identification of ASFV-like sequences, we sequenced DNA from different recent Ornithodoros tick species, e.g. O. moubata and O. porcinus, O. moubata tick cells and also 100-year-old O. moubata and O. porcinus ticks using high-throughput sequencing. We used BLAST analyses for the identification of ASFV-like sequences and further analysed the data through phylogenetic reconstruction and molecular clock analyses. In addition, we performed tick infection experiments as well as additional small RNA sequencing of O. moubata and O. porcinus soft ticks. - CONCLUSION: Here, we show that soft ticks of the Ornithodoros moubata group, the natural arthropod vector of ASFV, harbour African swine fever virus-like integrated (ASFLI) elements corresponding to up to 10% (over 20 kb) of the ASFV genome. Through orthologous dating and molecular clock analyses, we provide data suggesting that integration could have occurred over 1.47 million years ago. Furthermore, we provide data showing ASFLI-element specific siRNA and piRNA in ticks and tick cells allowing for speculations on a possible role of ASFLI-elements in RNA interference-based protection against ASFV in ticks. We suggest that these elements, shaped through many years of co-evolution, could be part of an evolutionary virus-vector ‘arms race’, a finding that has not only high impact on our understanding of the co-evolution of viruses with their hosts but also provides a glimpse into the evolution of ASFV. Background Results - Evidence of ASFLI-elements in the O. moubata tick cell genome - Phylogenetic analysis shows ASFLI-elements are close relatives of ASFV sequences - ASFLI-elements are present in recently sampled O.moubata, O. porcinus and approx. 100-year-old O.moubata and O. porcinus field-collected ticks from Africa - Phylogenetic reconstruction using full-length mitochondrial genomes of soft ticks reveals a possible integration of an ASFLI-element might have occurred over 1.46–1.47 million years ago (mya) - Molecular clock analyses using ASFLI-elements from different Ornithodoros species provide an estimate for a time to the most recent common ancestor consistent with orthologous dating - Ornithodoros tick species and tick cell lines show differences in the infectability with various ASFV genotype isolates - RNA sequencing demonstrates ASFLI-element-specific mRNA—small-interfering and piwi-interacting RNAs in tick cells - The reconstructed ASFLI-A104R protein is highly similar to its ASFV homologue but is not expressed in tick cell lines Discussion Conclusion Methods - Virus strains - Tick rearing, tick infection and tick cell cultures - Nucleic acid extraction - Oligonucleotide design - PCR - qPCR - RT-qPCR - Sanger sequencing - Next-generation sequencing - Amplicon sequencing for assembly validation - Data analysis - Phylogenetic analysis - Clock rate estimates and Bayesian time-scaled trees - Protein expression and purification in E. coli and rabbit immunisation - Transfection - SDS-PAGE and immunoblotting - Statistical analysis

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    Authors: Forth, Jan H.; Forth, Leonie F.; Lycett, Samantha; Bell-Sakyi, Lesley; +8 Authors

    Additional file 13: Figure S4. siRNA/piRNA mapping against ASFV-Kenya1950 (GT X), Warthog (G IV) and two ASFLI-elements. siRNA (22 nt) and piRNA (28-29 nt) fractions from Ornithodoros porcinus were individually mapped against available ASFV whole-genome sequences (the ones showing the most mapped reads, ASFV-Kenya1950 (GT X), ASFV Wathog (GT IV) are shown) and two exemplary O. porcinus ASFLI-element containing contigs (NODE955 and NODE1089) using Bowtie 1 (1.1.2) in Geneious. Marked (arrows) are the most abundant small RNA molecules relating to the ASFV genes and ASFLI-elements.

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    Authors: Forth, Jan H.; Forth, Leonie F.; Lycett, Samantha; Bell-Sakyi, Lesley; +8 Authors

    Additional file 10: Table S8. qRT-PCR results of Ornithodoros ticks experimentally infected with different ASFV-genotype isolate. Shown are ASFV-P72 transcript-specific Cq-values of third nymphal stage ticks fed with defibrinated pig blood, containing either 1 x 104 HAU/ml or 1 x 106 HAU/ml ASFV-ken.rie1 (GT X) (A-D), 1 x 105 HAU/ml ASFV-Ken06.bus (GT IX) (E-F) or 1 x 104 HAU/ml ASFV-Sardinia (GT I) (G-H). Due to the limited number of field ticks available and feeding under artificial conditions, fifteen Ornithodoros porcinus ticks were collected in each of three experiments and ten in two experiments while for the laboratory-reared Ornithodoros moubata, twenty-five individuals were collected in each of three experiments and fifteen in one experiment. All samples were stored at – 80 °C until RNA-extraction and ASFV transcript-specific qRT-PCR analysis as described in the ‘Material and methods’ section.

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    Authors: Forth, Jan H.; Forth, Leonie F.; Lycett, Samantha; Bell-Sakyi, Lesley; +8 Authors

    Additional file 5: Supplementary Appendix. Phylogenetic and molecular clock analyses of ASFLI-elements from different tick genomes and ASFV using different clock-rates and substitution models implemented in BEAST. Supplementary Appendix. Figure A1-A5, Tables A1-A7. FigA1- Phylogenetic tree of ASFV and ASFLI-elements. FigA2 – Phylogenetic tree of NCLDV including ASFV and ASFLI-elements. FigA3 – Time-scaled tree for partial EP1242L sequences from tick samples and reference ASFV (non-integrated). FigA4 - Time-scaled tree for partial EP1242L sequences from tick samples only (5e-7 substitutions per site per year). FigA5 - Time-scaled tree for partial EP1242L sequences from tick samples (1e-8 substitutions per site and year). Table A1 - Summary of EP1242L fragments derived from ticks and the tick cell line OME/CTVM21 (OME21) used in the analysis. Table A2 - Indels in the tick sample sequences relative to the start of EP1242L in ASFV|KM111295|Kenya|Ken06/Bus|2006. Table A3 - Estimated root height and overall mean clock rate for strict clocks with fixed priors. Table A4 - Estimated root height and overall (averaged over all branches) mean clock rate for relaxed clocks with fixed priors of the rate of the relaxed clock (some variation). Table A5 - Estimated root height and overall (averaged over all branches) mean clock rate for relaxed clocks with normal or log-normal priors on the rate of the relaxed clock (most variation). Table A6 - Estimated root height and overall (averaged over all branches) mean clock rate for strict clocks with log-normal priors on the rate of the strict clock for the five ASFV-like sequences from tick samples only. Table A7 - Marginal likelihood estimation using Path Sampling and Stepping Stone Sampling, showing that the log-normal clock rate prior with mean = 5e-7 is best, but not significantly better than the other clock rates.

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    Authors: Forth, Jan H.; Forth, Leonie F.; Lycett, Samantha; Bell-Sakyi, Lesley; +8 Authors

    Additional file 20: Figure S7. BLAST-analysis for identification of ASFLI-element containing contigs and annotation. 66,745 SPAdes assembled contigs were blasted (BLASTn, NCBI, v2.6.0+) against a customised database comprising all sequences with the NCBI taxonomy ID 10497 (African swine fever virus) (as of 16 January 2018). Hits were filtered using a cut off e-value of 1x10-4 and a minimum alignment length of 150 bp, resulting in 34 contigs. These were then blasted against the complete NCBI database (The non-redundant nucleotide collection) to reliably identify and annotate ASFV-like sequences and areas of the host genome using default parameters. BLASTp search of >500 bp ORFs was performed against the “Non-redundant protein sequences” database using default parameters.

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    Authors: Forth, Jan H.; Forth, Leonie F.; Lycett, Samantha; Bell-Sakyi, Lesley; +8 Authors

    Additional file 7: Table S6. BLASTn results of SPAdes-assembled contigs from Ornithodoro porcinus France and Kenya17 containing ASFLI-elements. Shown are hits with the lowest e-value for ASFV-genes as obtained by Blastn against the entire NCBI database.

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    Authors: Forth, Jan H.; Forth, Leonie F.; Lycett, Samantha; Bell-Sakyi, Lesley; +8 Authors