乳腺癌熬过五年就不复发?专家:七年也是小高峰_治疗

2.9. IonTorrent Sequencing A total of 100 ng of DNA was subjected to enzymatic fragmentation using the reagents supplied in the NEBNext Fast DNA Fragmentation & L...

圣安东尼奥(EGMN)——根据圣安东尼奥乳腺癌研讨会上报告的一项德国大规模观察性研究结果，临床医生倾向于过高估计乳腺癌患者对芳香酶抑制剂辅助治疗的依从性。

德国吉森与马尔堡大学医院的PeymanHadji博士报告，在入组COMPACT(临床治疗中的依从性与关节痛)研究的2,313例绝经后乳腺癌患者中，80.5%声称在接受辅助内分泌治疗的第1年内服用了所有或几乎所有的阿那曲唑片剂。而负责这些患者的肿瘤医生中，则有93.2%认为其患者服用了所有或几乎所有的阿那曲唑片剂。

在COMPACT研究的受试者中，11.9%报告称在接受芳香酶抑制剂治疗之前的4周内已有关节痛，另有17%的患者报告称在开始阿那曲唑治疗后新发关节痛。

COMPACT研究仍在进行中，Hadji博士称将会评估临床实践中芳香酶抑制剂相关性关节痛的发生率和严重度，并将确定关节痛相关性治疗不依从的比例，以及比较不同治疗方案的疗效与费用。

大会主席ThomasJ.Smith博士指出，这些初步数据反映了一个重要信息：一些乳腺癌患者遇到困难却不想麻烦医生，因此医生必须询问每例乳腺癌患者是否在服用这些药物时遇到困难。另外值得注意的是，约有12%的乳腺癌患者在治疗前已有关节痛，还有近1/5的患者因服用芳香酶抑制剂而新发关节痛。

Smith博士还强调，目前尚缺乏高质量的随机试验数据用于指导芳香酶抑制剂相关性关节痛的治疗。Hadji博士也持相同观点，并表示虽然已在使用非甾体抗炎药(NSAID)方面积累了大量经验，却缺乏系统性。他还引述了一项显示针灸有效的双盲研究(J.Clin.Oncol.2010;28:1154-60)，认为其结果“非常理想”，希望肿瘤科医生能更勇于接受针灸。

(PDF) A Simple Method for Sample Preparation to Facilitate Efficient Whole-Genome Sequencing of African Swine Fever Virus
ArticlePDF AvailableA Simple Method for Sample Preparation to Facilitate Efficient Whole-Genome Sequencing of African Swine Fever VirusDecember 2019Viruses 11(12):1129DOI:10.3390/v11121129Authors:

Ferenc OlaszHungarian Academy of Sciences

István MészárosVeterinary Medical Research Institute

Szilvia MartonHungarian Academy of Sciences

Győző KajánCentre for Agricultural Research, Hungarian Academy of SciencesShow all 10 authorsHide

Download full-text PDFRead full-textDownload full-text PDFRead full-textDownload citation Copy link Link copied Read full-text Download citation Copy link Link copiedCitations (11)References (41)Figures (2)Abstract and FiguresIn the recent years, African swine fever has become the biggest animal health threat to the swine industry. To facilitate quick genetic analysis of its causative agent, the African swine fever virus (ASFV), we developed a simple and efficient method for next generation sequencing of the viral DNA. Execution of the protocol does not demand complicated virus purification steps, enrichment of the virus by ultracentrifugation or of the viral DNA by ASFV-specific PCRs, and minimizes the use of Sanger sequencing. Efficient DNA-se treatment, monitoring of sample preparation by qPCR, and whole genome amplification are the key elements of the method. Through detailed description of sequencing of the first Hungarian ASFV isolate (ASFV_HU_2018), we specify the sensitive steps and supply key reference numbers to assist reproducibility and to facilitate the successful use of the method for other ASFV researchers.

Qualifying data for four sequenced samples.… 

Nucleotide differences between ASFV_HU_2018 and two recent ASFV isolates.… Figures - available via license: Creative Commons Attribution 4.0 InternationalContent may be subject to copyright.

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virusesArticleA Simple Method for Sample Preparation to FacilitateEfficient Whole-Genome Sequencing of AfricanSwine Fever VirusFerenc Olasz 1, *, István Mészáros 1, Szilvia Marton 1, Gy˝oz˝o L. Kaján1, Vivien Tamás1,Gabriella Locsmándi 2, Tibor Magyar 1,Ádám Bálint 2, Krisztián Bányai 1and Zoltán Zádori 11Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungária krt. 21, 1143 Budapest,Hungary; meszaros.istvan@agrar.mta.hu (I.M.); marton.szilvia@agrar.mta.hu (S.M.);kajan.gyozo@agrar.mta.hu (G.L.K.); tamas.vivien@agrar.mta.hu (V.T.); magyar.tibor@agrar.mta.hu (T.M.);banyai.krisztian@agrar.mta.hu (K.B.); zadori.zoltan@agrar.mta.hu (Z.Z.)2Veterinary Diagnostic Directorate, National Food Chain Safety Office, Tábornok u. 2, 1149 Budapest,Hungary; LocsmandiG@nebih.gov.hu (G.L.); balintad@nebih.gov.hu (Á.B.)*Correspondence: olasz.ferenc@agrar.mta.hu; Tel.: +36-1-467-4060Received: 12 November 2019; Accepted: 4 December 2019; Published: 6 December 2019Abstract:In the recent years, African swine fever has become the biggest animal health threat to theswine industry. To facilitate quick genetic analysis of its causative agent, the African swine fevervirus (ASFV), we developed a simple and efficient method for next generation sequencing of the viralDNA. Execution of the protocol does not demand complicated virus purification steps, enrichmentof the virus by ultracentrifugation or of the viral DNA by ASFV-specific PCRs, and minimizes theuse of Sanger sequencing. Efficient DNA-se treatment, monitoring of sample preparation by qPCR,and whole genome amplification are the key elements of the method. Through detailed descriptionof sequencing of the first Hungarian ASFV isolate (ASFV_HU_2018), we specify the sensitive stepsand supply key reference numbers to assist reproducibility and to facilitate the successful use of themethod for other ASFV researchers.Keywords:African swine fever virus; ASFV; whole genome sequencing; whole genome amplification;NGS; Illumina; Hungarian ASFV strain; DNAse treatment; whole genome amplification1. IntroductionAfrican swine fever virus (ASF) is a devastating disease affecting Sus scrofa; it infects bothdomesticated pigs and wild boars. The ASF virus (ASFV) was most probably transmitted from itsnatural hosts, warthogs (Phacochoerus spp.), bushpigs (Potamochoerus spp.) and soft ticks of the genusOrnithodoros to domestic pigs (Sus scrofa domesticus) in southeast Africa [1,2]. The virus is endemic inthe Sub-Saharan region, where viral reservoir is maintained by a sylvatic cycle between the soft ticksand its natural hosts [3,4]. In countries with temperate climate, mainly direct contact between domesticpigs and wild boars, and indirect contact facilitated by human activity sustain the infectious cycle [4,5].First transmission of ASFV to domestic pigs was reported in East Africa in 1921 [3]. The virus wasintroduced into Europe in several successive waves; genotype I virus spread to the Southern Europein the 1950’s and 1960’s, while the well documented emergence of genotype II ASFV occurred inGeorgia in 2007 [6]. Since 2017 the virus has continuously spread westward in Eastern Europe, reachingHungary and Belgium, then in 2018 it arrived to China, and by now it became arguably the biggesteconomic and animal health threat to the swine industry of the world [7,8].ASFV is an enveloped virus with a large (170−190 kilo base pair) double stranded, covalentlyclosed, linear DNA genome, which contains around 200 open reading frames. The genome organizationViruses 2019,11, 1129; doi:10.3390/v11121129 www.mdpi.com/journal/viruses
Viruses 2019,11, 1129 2 of 15of the virus is reminiscent that of the poxviruses: it consists of a more conserved central area (approx.125 kb) flanked by two variable regions (38-47 kb and 13–16 kb, respectively) at the ends of thegenome [6,9,10].The ASFV has a high genetic and antigenic diversity. Determined by the p72 protein (B646L), sofar 24 genotypes have been identified, while based on hemadsorption inhibition at least 8 serotypes arerecognized [11,12]. All genotypes occur in Africa, from which genotype I of West African origin causedoutbreaks in the European, Caribbean, and South and Central American regions before 2007. However,the pandemic that originated from Georgia was caused by the emergence of a genotype II virus ofEast African origin [13]. Comparison of the complete sequences of the original Georgia 2007/1 genomeand the Polish ASFV/Pol/2015/Podlaskie genome isolated eight years later revealed only 95 nucleotidedifferences scattered in the 190 000 bp genome, suggesting a relatively slowin vivoevolution of thisgenotype II ASFV in pigs [14].Vaccine development is hampered by the lack of detailed knowledge about viral virulence andimmunological factors influencing the outcome of infection of and immune response to ASFV. Currentlythere are no available continuous cell lines supporting the replication of ASFV field isolates withoutmajor genetic makeovers. Adaptation of ASFV to established cell lines usually leads to genomedestabilization and loss of ability to replicate in macrophagesin vitroandin vivo. However, ASFV canbe isolated and replicated in primary macrophages without obvious genetic alterations [15,16].So far, only 69 near complete genome sequences (including unverified ones and sequences frompatents) have been deposited into the GenBank (status 31.10.2019) [17] despite the obvious animalhealth significance of the virus. Attempts to produce additional complete sequences of biologicallyrelevant ASFV strains are frequently thwarted by technical problems, such as difficulty in achievingsequencing grade ASFV DNA and applying the most suitable next generation sequencing (NGS)method for their production. To facilitate much needed epidemiological investigations, advanceresearch and vaccine development, it would be expedient to have a simple and reproducible methodfor full genome sequencing of the ASFV.In this paper we present a protocol that was successfully used to sequence the first Hungarianisolate of ASFV and in our opinion fulfills the aforementioned criteria.2. Materials and Methods2.1. PAM Preparation and CulturePorcine alveolar macrophages (PAMs) were prepared according to the OIE Manuals [18] andwere stored in RPMI-1640 medium containing 30% bovine serum and 10% DMSO at−72◦C. PAMswere cultured in PAM culturing media (RPMI-1640 medium supplemented with 10% (v/v) fetal bovineserum, 100 U/mL penicillin, 100µg/mL streptomycin, and 2 mM L-glutamine (Sigma-Aldrich, SaintLouis, MO, USA) at 37 ◦C and in 5% CO2.2.2. Virus Isolation and Immunofluorescence DetectionTissue homogenates of an ASFV-infected carcass were prepared with Tissue Lyser (QIAGEN,Hilden, Germany) in PAM culturing medium sterile-filtered with 0.2µm Acrodisc syringe filter (PallCorporation, NY, USA) and serially diluted in half-log steps. 2×104PAM cells (100µL) were plated in96-well plates, incubated for 16 h at 37◦C, and infected with 10µL of the diluted tissue homogenates.After three days of incubation, the supernatant was removed and stored at−72◦C, while cells werepermeabilized by 1% Triton-X and fixed in 3% formaldehyde solution. Infected cells were visualizedby anti-ASFV polyclonal sera and Goat Anti-Swine IgG (H+L) CF488A (Biotium, Fremont, CA, USA)secondary antibody. Positive cells were detected under an Axio Observer D1 inverted fluorescencemicroscope (Carl Zeiss Ag. Oberkochen, Germany).
Viruses 2019,11, 1129 3 of 152.3. Viral Stock PreparationPAM cells (105cells in 1ml PAM culturing media) were infected with 30µL high-titre ASFV-infectedPAM cell supernatant in several wells of a 24-well plate and incubated at 37◦C. At 72 h post infection(hpi), the supernatant was removed, aliquoted, and stored at −72 ◦C.2.4. DNAse TreatmentThe media of ASFV-infected cells were collected at 72 hpi and centrifuged at 13 000 g for 3 min toget rid of cellular debris. Subsequently, to 100µL of the supernatant 100µL DNase I solution containing20µL 10×FastDigest buffer (Thermo Fischer, Waltham, MA, USA), 40 mM MgCl2and 1.5µL of theDNase I (50 U/µL) (Thermo Fischer Scientific, Waltham, MA, USA) was added. The samples wereincubated at 37 ◦C for 1 h, after which 10 µL 0.5 M EDTA was added to stop the reaction.2.5. DNA PurificationViral DNA was purified with the High Pure Viral Nucleic Acid Kit (Roche, Basel Switzerland)following the manufacturer’s recommendations. In brief, 200µL binding buffer and 50µL proteinase Kwere added to 200µL sample, mixed, and incubated at 72◦C for 10 min. An additional 100µL bindingbuffer was added to the samples, mixed and transferred to the nucleic acid-binding membranes. Aftercentrifugation at 8000×gfor 1 min, the membranes were washed first with 500µL inhibitor removalbuffer and twice with 450 µL wash buffer. The DNA was eluted by 50 µL elution buffer.2.6. Quantitative PCRASFV specific dual quantitative PCR (qPCR) was executed by Virotype ASFV PCR Kit (Qiagen,Hilden, Germany) according to the manufacturer’s recommendation.2.7. Aspecific DNA AmplificationThe viral DNA was amplified using the REPLI-g Mini Kit (Qiagen, Hilden, Germany), followingthe manufacturer’s protocol. First, 5µL denaturing buffer was added to 5µL viral DNA sample andincubated at room temperature for 3 min. After that 10µL neutralizing buffer and 30µL master mix(containing 29µL REPLI-g Reaction Buffer and 1µL REPLI-g Mini DNA polymerase) were mixedwith the denatured sample. The tubes were incubated at 30◦C for 16 h, then the polymerase wasinactivated by heating up to 65 ◦C for 3 min.2.8. Amplified DNA Clean UpREPLI-g samples were purified using the NucleoSpin Gel and PCR clean-up Kit (Macherey-NagelDüren, Germany). Briefly, 200µL NTI buffer was added to 50µL of the sample. After mixing,the solution was loaded to the spin column and centrifuged at 11,000×gfor 1 min. The column waswashed first with 500, then with 200µL NT3 buffer. The remnant of the wash buffer was removedby centrifugation at 11,000×gfor 1 min. The DNA was then eluted in 20µL elution buffer, and itsconcentration was measured with NanoDrop 2000 (Thermo Fischer Scientific, Waltham, MA, USA).2.9. IonTorrent SequencingA total of 100 ng of DNA was subjected to enzymatic fragmentation using the reagents suppliedin the NEBNext Fast DNA Fragmentation Library Prep Set for Ion Torrent kit (New England BioLabs,Hitchin, United Kingdom) according to the manufacturer’s instructions with slight modifications.In brief, 8µL of DNA was mixed with 1µL of NEBNext DNA Fragmentation Reaction buffer, 0.5µLMgCl2(using a 10 mM stock), and 0.75µL NEBNext DNA Fragmentation Master Mix. The mixturewas incubated at 25◦C for 20 min, then at 70◦C for 10 min. The adaptor ligation was performedusing reagents from the same kit, whereas barcoded adaptors were retrieved from the Ion XpressBarcode Adapters (Thermo Fischer Scientific, Waltham, MA, USA). Reaction components were used at
Viruses 2019,11, 1129 4 of 15a reduced volume: 2µL T4 DNA Ligase Buffer for Ion Torrent, 2µL barcode adapter mixture, 0.5µLBstDNA Polymerase and 2µL T4 DNA Ligase were combined with the fragmentation reaction mixtureand nuclease-free water to obtain a final volume of 20µL. Adapter ligation was performed at 25◦C for15 min, terminated at 65◦C for 5 min. After cooling on ice slurry, 2.5µL of Stop Buffer was added tothe mixture. The barcoded library DNA samples were purified using the Gel/PCR DNA fragmentsextraction kit (Geneaid Biotech, Ltd., Taipei, Taiwan) according to the manufacturer’s instructions.The eluted DNA libraries were then run on 2% E-Gel SizeSelect II Agarose (Invitrogen, Carlsbad, CA,USA). Products between 300 and 350 bp were directly used in the PCR mixture of the NEBNext FastDNA Fragmentation Library Prep Set for Ion Torrent kit (New England BioLabs, Hitchin, UnitedKingdom) without further purification.Library amplification was made in a total volume of 50µL (the reaction mixture consisted of15µL sample, 7.5µL H2O, 25µL enzyme mix, and 2.5µL primer), the heat profile included an initialdenaturation at 98◦C for 30 s, followed by 12 amplification cycles (98◦C for 10 s, 58◦C for 30 s, 72◦Cfor 30 s) and terminated at 72◦C for 5 min. The products were purified using the Gel/PCR DNAfragments extraction kit (Geneaid). The library DNA was eluted in nuclease-free water and quantifiedfluorometrically on Qubit 2.0 equipment using the Qubit dsDNA BR assay kit (Invitrogen, Carlsbad,CA, USA). Subsequently, the library DNA was diluted to 10 to 14 pM, then clonally amplified byemulsion PCR. This step was carried out according to the manufacturer’s instructions using the IonPGM Hi-Q View OT2 Kit on an Ion OneTouch 2 instrument. Enrichment of the templated beads (onan Ion OneTouch ES machine) and further steps for pre-sequencing setup were performed accordingto the 200-bp protocol of the manufacturer. The sequencing protocol recommended for the Ion PGMsequencing kit on a 316 chip was strictly followed.2.10. Illumina SequencingIllumina®Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA) and NexteraXT Index Kit v2 Set A (Illumina, San Diego, CA, USA) were used to prepare Illumina specific libraries.DNA samples were diluted to 0.2 ng/µL in nuclease-free water (Promega, Madison, WI, USA) in afinal volume of 2.5µL. For the tagmentation reaction, 5µL Tagment DNA (TD) buffer with 2.5µLAmpliconTagment Mix (ATM) were used. Next, the samples were incubated at 55◦C for 6 min, usingthe GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA). The samples were thenallowed to cool to 10◦C before the immediate addition of 2.5µL of the Neutralize Tagment (NT) buffer.Neutralization was performed for 5 min at room temperature. A total of 7.5µL of the Nextera PCRMaster Mix (NPM) was combined with i5 and i7 index primers (2.5µL of each primer per well) andadded to the tagmented DNA sample. The index primers were incorporated into library DNA via12 PCR cycles (each cycle consisted of the following steps: 95◦C for 10 s, 55◦C for 30 s, followed by72 ◦C for 30 s). Following the PCR cycles, the samples were held at 72 ◦C for 5 min and then at 10 ◦C.The PCR products were purified using Gel/PCR DNA Fragments Extraction Kit (Geneaid BiotechLtd., Taipei, Taiwan). The concentration of the purified DNA samples was quantified with Qubit 2.0equipment using Qubit dsDNA HS Assay Kit (Thermo Fischer Scientific, Waltham, MA, USA). LibraryDNAs were pooled and denatured. Denatured library pool at a final concentration of 1.5 pM wasloaded onto a NextSeq 500/550 Mid Output flowcell and sequenced using an Illumina®NextSeq 500sequencer (Illumina, San Diego, CA, USA).2.11. Mapping and AssemblySequence reads obtained by NGS were trimmed by using Geneious Prime 2019.0.3 (BiomattersLtd., Auckland New Zealand). The reads were mapped against the pig genome (Sscrofa11.1,GenBank assembly accession: GCF_000003025.6) to determine and eliminate host DNA contaminations.The purified reads were assembled to the viral genome by mapping these to the sequence ASFVBelgium 2018/1 [19] strains as reference. The sensitivity of mapping was set to medium with three
Viruses 2019,11, 1129 5 of 15iteration. The graphic representation of coverage and gene predictions were made using GeneiousPrime 2019.0.3.2.12. Sanger SequencingThree regions were amplified using Primestar GXL kit with GC buffer (Takara Bio Inc., Japan),in 25µL final volume with 1µL of purified ASFV DNA according to the manufacturer’s instructions.The amplification was performed using the following PCR program: 98◦C 30 s, 30×(98◦C 10 s, 60◦C15 s, 68◦C 1 min), 68◦C 2 min. The primer pairs used for the amplification of the different fragmentsare listed in Table 1. The same primers were used for Sanger sequencing by BaseClear B.V. (Leiden,Netherlands). Sequences of the amplicons were well defined from both directions upstream of thepoly C/G on each fragment, while downstream of the C/G tracts sequence slippage was detected ineach case. The size of the poly C/G tracts and the actual sequence around them were determined bycomparing and analyzing the reads of the opposing strands.Table 1. Primers used to amplify three poly C/G regions.Regions Primers14067–14379 seqASF_14234-FCTGAGATAGCCAAATCAAAATAC seqASF_14234-RCGATTGTAAACTGTATAGTTAATCG15551–15809 seqASFV_15670-FCAAAGCAGCCTGTATATGCAATACC seqASFV_15670-RCAATCATTCTATTGTAAACTGTAGAG19845–20072 seqASFV_20022-FTAGTACATCAATGTTGTAAGTTTG seqASFV_20022-RCTATCTAAACGTGCTTCTATGAATTC2.13. Phylogenetic AnalysesTwo phylogenetic analyses were conducted: first, ASFV complete genomes were compared, thengenotype I and II strains were picked from this analysis together with an outgroup strain for new treeinference. The transposed 50genomic end of strain Estonia 2014 (LS478113) [20] was trimmed fromthe genome to ease aligning. For phylogenetic tree inference, multiple alignments were conductedusing MAFFT [21] with the legacy gap penalty, and phylogenetic calculations were performed usingRAxML-NG v0.9.0 [22] based on alignments edited in trimAl v1.3 [23]. Evolutionary model selectionwas performed using ModelTest-NG v0.1.5 [24] and the generalized time reversible (GTR) model hadthe highest probability combined with discrete Gamma rate categories (+G) and the proportion ofinvariant sites (+I) [25]. The robustness of the trees was determined with a non-parametric bootstrapcalculation using 1000 repeats. Phylogenetic trees were visualized using MEGA 7 [26], bootstrap valuesare given as percentages if they reached 75%.3. Results and DiscussionASFV_HU_2018 was isolated from homogenized organ samples of a wild boar carcass on PAMcells. The isolated virus was assigned to be sequenced to determine its genetic makeup and to gainmolecular epidemiological data that might help identify its origin. However, following publishedNGS-based general viral metagenome sequencing protocols we were unable to assemble the completeASFV genome sequence. The main reason of the failure was that viral sequences represented only aminor fraction ( 0.5%) of the total NGS reads [27]Similar problems were reported at the sequencing of other ASFV isolates and different methodicalapproaches were pursued to overcome the difficulties [28–32]. These include animal infections [33],
Viruses 2019,11, 1129 6 of 15the complicated purification of the virus from animal blood [14,33], the enrichment of the virus byultracentrifugation or that of the viral DNA by ASFV-specific PCR in the samples, and the excessiveuse of Sanger sequencing [14,29,30].The majority of the “ASFV sequencing papers” that made their virus/host genome read ratioavailable reported very low percentage (0.5–1.0%) [27,34] of viral reads that resulted in low coverageand consequently, the frequent use of specific PCR-Sanger sequencing (SPSS) to patch up ambiguous,uncovered or poorly covered genome stretches. Regrettably, large-scale use of SPSS can substantiallyincrease the time and money spent to achieve the complete sequence of a near 200 Kbp virus.Wen and coworkers [32], gave up altogether the random “shotgun” approach of NGS and applieda whopping 86 overlapping specific PCRs (and sequenced the amplicons by NGS) to complete anASFV genome after having very low percentage viral reads in their samples. This extreme examplemakes obvious that the full advantage of NGS for ASFV sequencing can only be exploited if a highpercentage of the sample DNA derives from the viral sequence.Having considered the aforementioned facts, our goal was to develop a simple and reliableNGS-based ASFV sequencing protocol, in which animal housing or ultracentrifugation are notinvolved, ASFV-specific PCRs can be minimized and can be executed in most of veterinary BSL3laboratories using standard equipment. To achieve this, we concentrated on enriching the ASFVgenome and decreasing the contaminating host genome in the input DNA samples using the simplesttools available to us.3.1. Maximizing Viral DNA ContentTo maximize viral titer, ASFV was propagated in PAM cells, and the dynamics of infected cells wasmonitored by immunofluorescence at 24, 48, and 72 hpi. The infection rate and attached cell numberat 24 and 48 hpi varied greatly (0.5–20% and 15–60%, respectively), even when the same cell andvirus lots were used in parallel infections. However, quasi independently of the 24 hpi infection rateusually 90% of the cells lysed at 72 hpi and ~90% of the remaining attached cells proved to be infected(Figure 1). These observations indicated that the supernatants of the infected cells had to be collectedat 72 hpi. They were centrifuged to get rid of contaminating cells and cellular debris and used for totalDNA extraction. For DNA quantification, the Virotype ASFV PCR Kit (Qiagen, Hilden, Germany)was chosen as it allowed the simultaneous detection of the ASFV DNA and the contaminating swinegenome by dual PCR. Ctvalues (~22 and ~29 for ASFV and pig DNA, respectively) indicated highmolar ratio of the ASFV genome in the average samples. However, our ultimate goal was to get a highsequence read ratio between the virus and the host and this can be only achieved by a high mass ratiobetween the two genomes in the NGS input sample. Knowing the Ctvalues and size of the host andthe ASFV genomes allowed us to roughly calculate the mass ratio of the two genomes. For example,assuming equally efficient amplifications and taking into consideration the size difference of the host(2.5×109bp of the haploid genome) and the ASFV (2×105bp) genomes, at least ~13.6 cycle difference(2.5×109/2×105=1.25×104=213.6) would be expected in a sample which contains equal mass ofviral and host DNA. However, this assessment certainly underestimates the Ctdifference measured insuch a sample, as the kit is optimized to reduce the effectiveness of the host-specific PCR, to increasethe sensitivity of the viral PCR [35]. Nonetheless, following the rationale presented above, the massratio of the viral and the contaminating host DNA can be calculated with the formula (2×105/2y)/(2.5×109/2z)×100 where y and z are Ctvalues measured in the two channels of the dual PCR, respectively.In our specific case (2×105/221.6)/(2.5×109/229.1)×100 =~1.44%. Thus, relatively high Ctnumbers(~29) measured in the second channel lagging behind with only 7–8 Ctvalues of the virus-specificchannel in the reality indicated very high swine genome contents ( 98.56%).
Viruses 2019,11, 1129 7 of 15Viruses 2019, 11, x FOR PEER REVIEW 7 of 15 Figure 1. The effect of African swine fever virus (ASFV) infection on primary macrophages at 72 h p.i. Cells were mock infected (A) or infected (B) with MOI of 3 of ASFV. The nuclei of the cells were visualized by Hoechst 33342 reagent (blue), infected cells (red) were detected by ASFV positive sera and CF488 labelled anti-pig secondary antibodies. Pictures were colored by computer. 3.2. Minimizing Contaminating Host Genome DNA DNAse treatment and ultracentrifugation are widely used to get rid of contaminating host DNA from ASFV samples [14,29,30,34]. Since ultracentrifuges are expensive and far from being standard appliances in veterinary BSL3 laboratories, we concentrated on increasing the effectiveness of the DNAse treatment and monitoring its result. Bovine serum-supplemented culture medium with cell lysate content is a complex protein solution with a potential to inhibit DNAse I activity through bivalent cation and DNA binding [36,37]. To ensure effective DNAse I treatment, the culture medium was diluted two-fold in a nuclease buffer containing added MgCl2. After 1-h incubation with 75U DNAse-I, the reaction was stopped with EDTA and viral nucleic acid was purified. In most cases, minimal increase (Ct +1-2) was detected between the Ct values of untreated and DNAse-treated samples indicating that a substantial part (25–50%) of viral DNA in the supernatant packed into DNAse-I resistant virions. The amount of host DNA on the other hand decreased markedly, however, the extent of reduction varied from sample to sample as indicated by the broad range of increases in the Ct value (4 to 13) in the second channel (Figure 2). These observations indicate the complexity and unpredictable nature of the virus-containing cell lysate and highlight the importance of the qPCR monitoring for the selection of the appropriate samples. Figure 1.The effect of African swine fever virus (ASFV) infection on primary macrophages at 72 hp.i. Cells were mock infected (A) or infected (B) with MOI of 3 of ASFV. The nuclei of the cells werevisualized by Hoechst 33342 reagent (blue), infected cells (red) were detected by ASFV positive seraand CF488 labelled anti-pig secondary antibodies. Pictures were colored by computer.3.2. Minimizing Contaminating Host Genome DNADNAse treatment and ultracentrifugation are widely used to get rid of contaminating host DNAfrom ASFV samples [14,29,30,34]. Since ultracentrifuges are expensive and far from being standardappliances in veterinary BSL3 laboratories, we concentrated on increasing the effectiveness of theDNAse treatment and monitoring its result.Bovine serum-supplemented culture medium with cell lysate content is a complex protein solutionwith a potential to inhibit DNAse I activity through bivalent cation and DNA binding [36,37]. To ensureeffective DNAse I treatment, the culture medium was diluted two-fold in a nuclease buffer containingadded MgCl2. After 1-h incubation with 75U DNAse-I, the reaction was stopped with EDTA and viralnucleic acid was purified. In most cases, minimal increase (Ct+1-2) was detected between the Ctvalues of untreated and DNAse-treated samples indicating that a substantial part (25–50%) of viralDNA in the supernatant packed into DNAse-I resistant virions. The amount of host DNA on the otherhand decreased markedly, however, the extent of reduction varied from sample to sample as indicatedby the broad range of increases in the Ctvalue (4 to 13) in the second channel (Figure 2). Theseobservations indicate the complexity and unpredictable nature of the virus-containing cell lysate andhighlight the importance of the qPCR monitoring for the selection of the appropriate samples.The addition of EDTA to inactivate the DNAse before DNA purification also seemed to be crucial.Omission of this step resulted in complete loss of the viral DNA in the following DNA purification step(data not shown), which suggests that inactivation of high concentrations of DNAse I is not a rapidprocess in the binding buffer of the applied purification kit (surprisingly, supplier does not indicateEDTA content) [38].
Viruses 2019,11, 1129 8 of 15Viruses 2019, 11, x FOR PEER REVIEW 8 of 15 Figure 2. Host and viral DNA content of differently treated ASFV samples. Quantitative dual PCR was executed by Virotype ASFV PCR Kit. Ct values of individual and averaged samples represented by colored circles and grey box respectively. Averages were calculated from numbers of samples indicated on X axis. Gray circles indicate minimum and maximum values. Ct values higher than 40 (undetectable host DNA) are represented by 41. Whole genome amplification (WGA) was executed by REPLI-g Mini Kit. The addition of EDTA to inactivate the DNAse before DNA purification also seemed to be crucial. Omission of this step resulted in complete loss of the viral DNA in the following DNA purification step (data not shown), which suggests that inactivation of high concentrations of DNAse I is not a rapid process in the binding buffer of the applied purification kit (surprisingly, supplier does not indicate EDTA content) [38]. 3.3. Nonspecific Amplification of the Viral DNA To increase the absolute amount of DNA for the following NGS sample preparation protocols, whole genome amplification (WGA) was performed by using the REPLI-g Mini Kit. WGA was expected to sustain the original high viral/host DNA ratio by randomly and evenly multiplying host and viral DNA. Purified DNA samples with the highest viral content (viral Ct ~23, host Ct 40) were chosen for the reaction. After the WGA reaction qPCR revealed a ~1000-fold increase in the viral DNA content in the reaction tube (Ct ~23 vs. Ct ~14), while the host DNA remained undetectable (Figure 2). DNA from the WGA reaction was purified with the NucleoSpin Gel and PCR clean-up kit and the purification resulted in 0.3–0.8 μg DNA/reaction that were used for Illumina and Ion Torrent NGS. 3.4. NGS Sequencing of the ASFV Genome In the last few years, Illumina and Ion Torrent systems became the most frequently used NGS platforms for whole genome sequencing of microorganisms. To find the most effective solution for ASFV sequencing [39], we compared the two methods by running two ASFV samples on each platform. Figure 2.Host and viral DNA content of differently treated ASFV samples. Quantitative dual PCR wasexecuted by Virotype ASFV PCR Kit. Ctvalues of individual and averaged samples represented bycolored circles and grey box respectively. Averages were calculated from numbers of samples indicatedon X axis. Gray circles indicate minimum and maximum values. Ctvalues higher than 40 (undetectablehost DNA) are represented by 41. Whole genome amplification (WGA) was executed by REPLI-gMini Kit.3.3. Nonspecific Amplification of the Viral DNATo increase the absolute amount of DNA for the following NGS sample preparation protocols,whole genome amplification (WGA) was performed by using the REPLI-g Mini Kit. WGA was expectedto sustain the original high viral/host DNA ratio by randomly and evenly multiplying host and viralDNA. Purified DNA samples with the highest viral content (viral Ct~23, host Ct 40) were chosen forthe reaction. After the WGA reaction qPCR revealed a ~1000-fold increase in the viral DNA content inthe reaction tube (Ct~23 vs. Ct~14), while the host DNA remained undetectable (Figure 2). DNA fromthe WGA reaction was purified with the NucleoSpin Gel and PCR clean-up kit and the purificationresulted in 0.3–0.8 µg DNA/reaction that were used for Illumina and Ion Torrent NGS.3.4. NGS Sequencing of the ASFV GenomeIn the last few years, Illumina and Ion Torrent systems became the most frequently used NGSplatforms for whole genome sequencing of microorganisms. To find the most effective solution forASFV sequencing [39], we compared the two methods by running two ASFV samples on each platform.Analysis of the sequence data revealed that the number of viral reads exceeded the reads ofthe contaminating nucleic acids in all four samples (Table 2). This finding verified that the DNAse Itreatment together with the applied monitoring procedure is in fact able to warrant that the majority ofthe DNA in the ASFV samples originate from the viral genome.
Viruses 2019,11, 1129 9 of 15Table 2. Qualifying data for four sequenced samples.Method Samples ofASFV_HUN_2018Viral Reads ofthe Total (%)Number ofViral ReadsMeanCoverage Std. Dev.Ion PGM System S43 90.4 179,325 197 129.1Ion PGM System S41 87.7 152,865 158 106.5NextSeq Illumina S1 50 6,835,057 2557 1624.9NextSeq Illumina S20 77 7,115,377 2692 1897.6Genomes were assembled by mapping the viral reads to the sequence of ASFV Belgium 2018/1 [19]strains as reference by using the Geneious Prime 2019.0.3 program. Numerous (~131) undefinednucleotides, mainly in the form of single and double nucleotide insertions/deletions (indels) werefound in homopolymer tracts of the assembled genome of the Ion Torrent platform-sequenced samples(minimum coverage 35) compared to the reference sequence. The high number of ambiguous readsmade the usefulness of the Ion Torrent platform questionable for ASFV genome sequencing and it wasomitted from further investigations.The number of undefined nucleotides were much less frequent in the Illumina platform output.After processing the data of one sample (~7 million viral reads), they were restricted to the three“longest” homopolymer regions (14224–14236 (13C), 15665–15680 (16C), 19991–20001 (11G)) containingmore than 10 C/G nucleotides. The sequence ambiguities of these regions could not even be resolvedby processing all viral reads of the two Illumina samples (around 14 million viral reads). Thus,to determine the accurate sequence of the ASFV_HU_2018 (Accession number: MN715134), these threeregions had to be sequenced by the Sanger method. The use of these SPSS indeed allowed completingthe full sequence of the isolate.Short of these three poly C/G tracts, the Illumina reads gave an even coverage for most of thegenome in both samples (Mean coverage: 2692.4 with Std Dev: 1897.6 and 2557 with Std Dev: 1624.9)except for the terminal regions where viral reads were underrepresented.Handling the data of only one channel (~1.8 million reads) still supplied very good coverage onmost of the genome and resulted in five short nucleotide stretches (spanning altogether ~440 nucleotides)with unsatisfying coverage ( 10) within the two terminal regions (1–3200 and 188,800–190601) thatimpeded the determination of the exact sequence (Figure 3). Although processing the data of two(3.6 million reads) or three (5.4 million reads) channels decreased the extension of the shortage of reads,it still left nucleotides unsatisfyingly ( 10) covered in these 5 regions (Figure 3). The lower coverage ofthe termini most probably comes from the reduced amplification of these regions by the REPLI-g MiniKit. The termini of the virus are covalently closed and contain inverted repeats (ITR) that facilitate thequick rehybridization of the template strands. Thus, the ITRs can impede the annealing of the randomprimers to these regions that leads to below average amplification and the underrepresentation ofthese regions in the sequence reads.In any case, it seems that the generation of around 7 million reads and 3 SPSS are needed toassemble the complete sequence of an ASFV isolate with our protocol if we want to minimize thenumber of SPSS. The significant decrease of the NGS reads necessitate the increase of SPSS in theterminal regions.
Viruses 2019,11, 1129 10 of 15Viruses 2019, 11, x FOR PEER REVIEW 10 of 15 Figure 3. Graphical visualization of the nucleotide coverage values of the ASFV genome in sample S20. Horizontal blue bars represent regions with unsatisfying ( 10) coverage in the terminal regions. Horizontal green bar labels the region with the three unresolvable C/G tracts. Numbers on the vertical scale indicate minimum and maximum coverage values. A, data from a single channel; B, data from two channels; C data from four channels. 3.5. Analysis of the Sequence Comparison of the ASFV_HU_2018 sequence to the ASFV Belgium 2018/1 sequence resulted in only 22 nucleotide mismatches at 15 sites; the majority of these (9 sites) being mutations in non-coding regions. Approximately the same number of differences (19 mismatches at 14 sites) was found between the ASFV_HU_2018 and the China/2018/AnhuiXCGQ isolate [32] (Table 3). This relatively low number of differences provide indirect evidence for the good quality of sequencing in the different labs. Table 3. Nucleotide differences between ASFV_HU_2018 and two recent ASFV isolates. Differences Between ASFV Belgium 2018/1 and ASFV_HUN_2018 Type Mutation Position Localisation Description of Differences Indel deletC 1384. non-coding region Indel deletT 2956. non-coding region Indel deletA 12570. ASFV G ACD 00190 CDS The ASFV_HUN_2018 contains the “common version” of gene. The adenine insertion is unique in the ASFV Belgium 2018/1. Indel delet4C 15670. MGF 110-13L The length of this cytosine rich region is variable among isolates. Indel delet2G 17845. non-coding region Indel delet3G 20001. ASFV G ACD 00350 CDS The length of this guanine rich region is variable among isolates. Indel deletG 21799. non-coding region Point mutation T- C 26419. MGF 360-10L N- S, This nucleotide position is variable among the isolates. Figure 3.Graphical visualization of the nucleotide coverage values of the ASFV genome in sampleS20. Horizontal blue bars represent regions with unsatisfying ( 10) coverage in the terminal regions.Horizontal green bar labels the region with the three unresolvable C/G tracts. Numbers on the verticalscale indicate minimum and maximum coverage values. (A), data from a single channel; (B), data fromtwo channels; (C) data from four channels.3.5. Analysis of the SequenceComparison of the ASFV_HU_2018 sequence to the ASFV Belgium 2018/1 sequence resulted inonly 22 nucleotide mismatches at 15 sites; the majority of these (9 sites) being mutations in non-codingregions. Approximately the same number of differences (19 mismatches at 14 sites) was found betweenthe ASFV_HU_2018 and the China/2018/AnhuiXCGQ isolate [32] (Table 3). This relatively low numberof differences provide indirect evidence for the good quality of sequencing in the different labs.Table 3. Nucleotide differences between ASFV_HU_2018 and two recent ASFV isolates.Differences between ASFV Belgium 2018/1 and ASFV_HUN_2018Type Mutation Position Localisation Description of DifferencesIndel deletC 1384. non-coding regionIndel deletT 2956. non-coding regionIndel deletA 12570. ASFV G ACD 00190 CDSThe ASFV_HUN_2018 contains the “common version”of gene. The adenine insertion is unique in the ASFVBelgium 2018/1.Indel delet4C 15670. MGF 110-13LThe length of this cytosine rich region is variable amongisolates.Indel delet2G 17845. non-coding regionIndel delet3G 20001. ASFV G ACD 00350 CDSThe length of this guanine rich region is variable amongisolates.Indel deletG 21799. non-coding regionPoint mutation T- C 26419. MGF 360-10L N- S, This nucleotide position is variable among theisolates.Indel insT 27422. non-coding regionIndel insT 73257. non-coding regionPoint mutation G- A 88348. C315RV- IThe “common version” of gene contains the codon ofvaline. The isoleucine is unique in the ASFV_HUN_2018.
Viruses 2019,11, 1129 11 of 15Table 3. Cont.Indel insG 103310. non-coding regionPoint mutation T- C 109659. B263R This synonym nucleotide change is uniquein the ASFV_HUN_2018.Point mutation A- G 145065. D117LL- PThe “common version” of gene contains codon ofproline. This amino acid change is uniquein the ASFV Belgium 2018/1.Quasispecies W (A/T) 190462. non-coding region Coverage: 318Adenine 58%; Thymine 42%Quasispecies S (C/G) 190470. non-coding region Coverage: 298Cytosine: 41%, Guanine 59%Differences between China/2018/AnhuiXCGQ and ASFV_HUN_2018Type Mutation Position Localisation Description of DifferencesIndel insA 1063. non-coding regionIndel insC 1392. non-coding regionIndel ins2C 14235. MGF 110-14LThe length of this cytosine rich region is variable amongisolates.Indel ins4G 17631. non-coding regionIndel deletG 17845. non-coding regionIndel delet2G 20001. ASFV G ACD 00350 CDSThe length of this guanine rich region is variable amongisolates.Point mutation G- A 88348. C315RV- IThe “common version” of gene contains the codon ofvaline. This amino acide is unique in theASFV_HUN_2018.Point mutation T- C 109659. B263R This synonym nucleotide change is unique in theASFV_HUN_2018.Point mutation A- G 129413. O174LS- PThis amino acide change is unique in theChina/2018/AnhuiXCGQPoint mutation A- G 129517. O174LF- SThis amino acide change is unique in theChina/2018/AnhuiXCGQPoint mutation A- G 129542 O174LS- PThis amino acide change is unique in theChina/2018/AnhuiXCGQIndel insA 190122. DP60RThe length of this cytosine rich region is variable amongisolates.Quasispecies W (A/T) 190462. non-coding region Coverage: 318Adenine 58%; Thymine 42%Quasispecies S (C/G) 190470. non-coding region Coverage: 298Cytosine: 41%, Guanine 59%It is appealing that the majority of the otherwise small differences are located in non-coding regions(9 and 7 sites, respectively). Considering that around 85% of the ASFV genome codes proteins andassuming random mutation distribution, the majority of the mutations should be localized in the codingregions (unless there is no purifying selection). This may suggest such a selection pressure on the viralproteins that sustains not only their protein but also their nucleotide sequences. Phylogenetic analysis ofthe ASFV complete genomes also support high genetic stability of the genotype II viruses. Interestingly,it reveals considerably less evolutionary distance among genotype II viruses than genotype I viruses(Figure 4). For example, the two most divergent genotype I viruses (Mkuzi 1979 [AY261362] andBA71V [U18466]) show ~90% nucleic acid identity (without the large deletions in BA71V) while thetwo most different genotype II ASFVs (2008/1 [MH910495] and Estonia 2014 [LS478113]) are almost99.9% identical. This raises the question of whether genotype II viruses evolve at a lower speed in pigsthan type I viruses do. However, the different evolutionary time of the two genotypes in pigs and theimproving reliability of the sequencing technology since the emergence of ASFV makes the questiondifficult to answer and it requires further research.
Viruses 2019,11, 1129 12 of 15Viruses 2019, 11, x FOR PEER REVIEW 12 of 15 It is appealing that the majority of the otherwise small differences are located in non-coding regions (9 and 7 sites, respectively). Considering that around 85% of the ASFV genome codes proteins and assuming random mutation distribution, the majority of the mutations should be localized in the coding regions (unless there is no purifying selection). This may suggest such a selection pressure on the viral proteins that sustains not only their protein but also their nucleotide sequences. Phylogenetic analysis of the ASFV complete genomes also support high genetic stability of the genotype II viruses. Interestingly, it reveals considerably less evolutionary distance among genotype II viruses than genotype I viruses (Figure 4). For example, the two most divergent genotype I viruses (Mkuzi 1979 [AY261362] and BA71V [U18466]) show ~90% nucleic acid identity (without the large deletions in BA71V) while the two most different genotype II ASFVs (2008/1 [MH910495] and Estonia 2014 [LS478113]) are almost 99.9% identical. This raises the question of whether genotype II viruses evolve at a lower speed in pigs than type I viruses do. However, the different evolutionary time of the two genotypes in pigs and the improving reliability of the sequencing technology since the emergence of ASFV makes the question difficult to answer and it requires further research. Figure 4. Phylogenetic analyses of complete ASFV genome sequences. The sequenced ASFV HU 2018 strain is highlighted using bold letters. Strains are represented using their NCBI Nucleotide accession numbers and their strain names. Genotypes I and II are marked. A. Phylogenetic analysis of different ASFV genotypes. The tree was rooted on the midpoint. B. Phylogenetic analysis of genotype I and II ASFV strains. The tree was rooted using strain Warthog as outgroup. C. The subtree of genotype II strains shown separately from the phylogenetic analysis of genotype I and II ASFV strains. 4. Conclusions We developed an ASFV sequencing protocol that gives a simple and effective solution to the common problem of viral DNA shortage and host DNA contamination in ASFV samples. Proper application of the nuclease treatment, whole genome amplification by random PCR, and most importantly, constant monitoring and evaluation of the effectiveness of different phases of sample preparation allowed us to take full advantage of NGS for ASFV genome sequencing. Via sequencing of the first Hungarian ASFV isolate, we highlighted sensitive steps and supplied key reference Figure 4.Phylogenetic analyses of complete ASFV genome sequences. The sequenced ASFV HU 2018strain is highlighted using bold letters. Strains are represented using their NCBI Nucleotide accessionnumbers and their strain names. Genotypes I and II are marked. (A). Phylogenetic analysis of differentASFV genotypes. The tree was rooted on the midpoint. (B). Phylogenetic analysis of genotype I and IIASFV strains. The tree was rooted using strain Warthog as outgroup. (C). The subtree of genotype IIstrains shown separately from the phylogenetic analysis of genotype I and II ASFV strains.4. ConclusionsWe developed an ASFV sequencing protocol that gives a simple and effective solution to thecommon problem of viral DNA shortage and host DNA contamination in ASFV samples. Properapplication of the nuclease treatment, whole genome amplification by random PCR, and mostimportantly, constant monitoring and evaluation of the effectiveness of different phases of samplepreparation allowed us to take full advantage of NGS for ASFV genome sequencing. Via sequencing ofthe first Hungarian ASFV isolate, we highlighted sensitive steps and supplied key reference numbersto assist reproducibility and to facilitate the successful use of this protocol for other ASFV researchers(Figure 5).
Viruses 2019,11, 1129 13 of 15Viruses 2019, 11, x FOR PEER REVIEW 13 of 15 numbers to assist reproducibility and to facilitate the successful use of this protocol for other ASFV researchers (Figure 5). Figure 5. Workflow of the ASFV sequencing protocol. Major steps of the process are depicted together with key values for quality control to ensure successful reproduction. vCt, viral genome Ct (cycle threshold) value; pCt, pig genome Ct value. Author Contributions: Methodology: F.O., I.M., S.M., T.M, G.L.; software: F.O., G.L.K., I.M., V.T.; validation: V.T., G.L., T.M., G.L.K.; data curation: I.M., F.O., S.M.; writing—original draft preparation: Z.Z., F.O., I.M.; writing—review and editing: Á.B., K.B., Z.Z., G.L.K.; supervision: Z.Z.; funding acquisition: Z.Z., T.M., Á.B., K.B.. Funding: This research was funded by the National Research, Development and Innovation Office, grant number NKFIH K119381 and by H2020-EU.3.2.1.1. VACDIVA ID:862874. The research of G. L. K. is supported by the OMA Foundation (grant 101öu6), and he is also the recipient of the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. Conflicts of Interest: The authors declare no conflict of interest References Figure 5.Workflow of the ASFV sequencing protocol. Major steps of the process are depicted togetherwith key values for quality control to ensure successful reproduction. vCt, viral genome Ct(cyclethreshold) value; pCt, pig genome Ctvalue.Author Contributions:Methodology: F.O., I.M., S.M., T.M, G.L.; software: F.O., G.L.K., I.M., V.T.; validation:V.T., G.L., T.M., G.L.K.; data curation: I.M., F.O., S.M.; writing—original draft preparation: Z.Z., F.O., I.M.;writing—review and editing:Á.B., K.B., Z.Z., G.L.K.; supervision: Z.Z.; funding acquisition: Z.Z., T.M.,Á.B., K.B.Funding:This research was funded by the National Research, Development and Innovation Office, grant numberNKFIH K119381 and by H2020-EU.3.2.1.1. VACDIVA ID:862874. The research of G.L.K. is supported by the OMAFoundation (grant 101öu6), and he is also the recipient of the János Bolyai Research Scholarship of the HungarianAcademy of Sciences.Conflicts of Interest: The authors declare no conflict of interest.
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Citations (11)References (41)... PAM cells were grown in RPMI 1640-containing L-glutamine (Lonza, Basel, Switzerland) medium supplemented with 10% fetal bovine serum (Euro Clone, Pero, Italy), 1% Na-pyruvate (Lonza), 1% non-essential amino acid solution (Lonza), and 1% antibiotic-antimycotic solution (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C in 5% CO2 in air gas phase. The infectious titer of serially diluted viral stock was calculated using an immunofluorescence (IF) assay as described earlier [22]. PAMs were cultivated in 6-well plates at a density of 3.3 × 10 5 cells and infected at a multiplicity of infection (MOI) of 10 plaque-forming units per cell at 4 h after cell seeding. ...... PAM cells were grown in RPMI 1640-containing L-glutamine (Lonza, Basel, Switzerland) medium supplemented with 10% fetal bovine serum (Euro Clone, Pero, Italy), 1% Na-pyruvate (Lonza), 1% non-essential amino acid solution (Lonza), and 1% antibiotic-antimycotic solution (Thermo Fisher Scientific, Waltham, MA, USA) at 37 • C in 5% CO 2 in air gas phase. The infectious titer of serially diluted viral stock was calculated using an immunofluorescence (IF) assay as described earlier [22]. PAMs were cultivated in 6-well plates at a density of 3.3 × 10 5 cells and infected at a multiplicity of infection (MOI) of 10 plaque-forming units per cell at 4 h after cell seeding. ...... The genome size of the various ASFV strains can significantly vary [38]. We used ASFV_HU_2018 isolate [22], a very close relative of the highly virulent strain Georgia 2007/1 [39] and compared it to the attenuated Vero cell adapted strain Ba71V used for the most detailed ASFV transcriptome analysis available so far [12]. The homologous regions of the two viruses show~98% similarity. ...Combined Short and Long-Read Sequencing Reveals a Complex Transcriptomic Architecture of African Swine Fever VirusArticleFull-text availableMar 2021

Gabor Torma

Dóra Tombácz

Zsolt Csabai

Zsolt BoldogkőiAfrican swine fever virus (ASFV) is a large DNA virus belonging to the Asfarviridae family. Despite its agricultural importance, little is known about the fundamental molecular mechanisms of this pathogen. Short-read sequencing (SRS) can produce a huge amount of high-precision sequencing reads for transcriptomic profiling, but it is inefficient for comprehensively annotating transcriptomes. Long-read sequencing (LRS) can overcome some of SRS’s limitations, but it also has drawbacks, such as low-coverage and high error rate. The limitations of the two approaches can be surmounted by the combined use of these techniques. In this study, we used Illumina SRS and Oxford Nanopore Technologies LRS platforms with multiple library preparation methods (amplified and direct cDNA sequencings and native RNA sequencing) for constructing the ASFV transcriptomic atlas. This work identified many novel transcripts and transcript isoforms and annotated the precise termini of previously described RNAs. This study identified a novel species of ASFV transcripts, the replication origin-associated RNAs. Additionally, we discovered several nested genes embedded into larger canonical genes. In contrast to the current view that the ASFV transcripts are monocistronic, we detected a significant extent of polycistronism. A multifaceted meshwork of transcriptional overlaps was also discovered.ViewShow abstract... Many parameters, in particular mean coverage, also deal with efficiency of viral DNA purification and quality of the sample. An important source of the low coverage seen in next-generation sequencing (NGS) of ASFV genomes is mainly related to the problem of eukaryotic DNA contamination, which has been approached by different strategies including animal infections, purification from animal blood, non-specific DNA amplification or probe-mediated viral DNA enrichment designed with known ASFV-sequences [10,12,15,16]. ASFV is the etiological agent of African swine fever [17][18][19], a serious disease affecting both wild boar and domestic pigs, which lastly emerged from East Africa to the Caucasus in 2007, where it spread to affect 28 countries in Europe, Oceania and Asia, including China [20,21]. ...... This method presents obvious economic and animal welfare concerns. There are some other techniques for enrichment of viral vs. cellular DNA, either by nonspecific amplification of DNA [16] or by removal of methylated DNA [56]. Viral DNA capture using specific probes based on known genotype-specific ASFV sequences has also been used as a method to obtain pure viral DNA [12], which in the case of mixed viral populations would still select only the DNA sequences displaying the specific DNA sequences bound by the probes, thus missing any other information present in the sample. ...Identification and Isolation of Two Different Subpopulations Within African Swine Fever Virus Arm/07 StockArticleFull-text availableOct 2020

Daniel Pérez-Núñez

Eva Castillo Rosa

Gonzalo Vigara Astillero

Yolanda RevillaNo efficient vaccines exist against African swine fever virus (ASFV), which causes a serious disease in wild boars and domestic pigs that produces great industrial and ecological concerns worldwide. An extensive genetic characterization of the original ASFV stocks used to produce live attenuated vaccine (LAV) prototypes is needed for vaccine biosecurity and control. Here, we sequenced for the first time the Arm/07 stock which was obtained from an infected pig during the Armenia outbreak in 2007, using an improved viral dsDNA purification method together with high coverage analysis. There was unexpected viral heterogeneity within the stock, with two genetically distinct ASFV subpopulations. The first, represented by the Arm/07/CBM/c2 clone, displayed high sequence identity to the updated genotype II Georgia 2007/1, whereas the second (exemplified by clone Arm/07/CBM/c4) displayed a hemadsorbing phenotype and grouped within genotype I based on a central region conserved among all members of this group. Intriguingly, Arm/07/CBM/c4 contained a unique EP402R sequence, produced by a single mutation in the N-terminal region. Importantly, Arm/07/CBM/c4 showed in vitro features of attenuated strains regarding innate immune response pathway. Both Arm/07/CBM/c2 and c4 represent well-characterized viral clones, useful for different molecular and virus-host interaction studies, including virulence studies and vaccine development.ViewShow abstract... ASFV mRNAs have 5 ′ cap structures and 3 ′ poly(A) tails added by the viral capping enzyme complex and the poly(A) polymerase, respectively (Salas et al., 1986). ASFV replicates relatively well in porcine primary alveolar macrophages (PAMs) in vitro, although the sensitivity of naïve PAM culture to ASFV infection varies batch by batch (Olasz et al., 2019). ...... The highly virulent Hungarian ASFV isolate ASFV_HU_2018 (ID Number: MN715134) was used for infection. The infectious titer of the serially diluted viral stock was calculated in PAMs using an immunofluorescence (IF) assay as described previously (Olasz et al., 2019). All work with the infectious virus was conducted at the biosafety level 3 (BSL3) laboratory of the Institute for Veterinary Medical Research, Center for Agricultural Research following all current EU regulations (European Commission, 2020). ...Short and Long-Read Sequencing Survey of the Dynamic Transcriptomes of African Swine Fever Virus and the Host CellsArticleFull-text availableJul 2020

Ferenc Olasz

Dóra Tombácz

Gabor Torma

Zsolt BoldogkőiView... Nucleotide sequences were determined by next-generation sequencing on an Ion Torrent PGM (Life Technologies/Thermo Fisher Scientific, Waltham, MA, USA) platform and/or on an Illumina ® NextSeq 500 sequencer (Illumina, San Diego, CA, USA) following the protocols described previously [16,17]. ...Recombination Events Shape the Genomic Evolution of Infectious Bronchitis Virus in EuropeArticleFull-text availableMar 2021Krisztina Bali

Adám Bálint

Attila Farsang, Dr

Krisztian BanyaiInfectious bronchitis of chicken is a high morbidity and mortality viral disease affecting the poultry industry worldwide; therefore, a better understanding of this pathogen is of utmost importance. The primary aim of this study was to obtain a deeper insight into the genomic diversity of field infectious bronchitis virus (IBV) strains using phylogenetic and recombination analysis. We sequenced the genome of 20 randomly selected strains from seven European countries. After sequencing, we created a genome sequence data set that contained 36 European origin field isolates and 33 vaccine strains. When analyzing these 69 IBV genome sequences, we identified 215 recombination events highlighting that some strains had multiple recombination breaking points. Recombination hot spots were identified mostly in the regions coding for non-structural proteins, and multiple recombination hot spots were identified in the nsp2, nsp3, nsp8, and nsp12 coding regions. Recombination occurred among different IBV genotypes and involved both field and vaccine IBV strains. Ninety percent of field strains and nearly half of vaccine strains showed evidence of recombination. Despite the low number and the scattered geographical and temporal origin of whole-genome sequence data collected from European Gammacoronaviruses, this study underlines the importance of recombination as a major evolutionary mechanism of IBVs.ViewShow abstract... Supernatants of tissue culture showing cytopathic changes were centrifuged at 10,000 × g for 5 min, and 200 [6]. The 150-cycle sequencing protocol was carried out to obtain 150-nt-long single reads. ...Genome sequencing of a novel variant of fowl adenovirus B reveals mosaicism in the pattern of homologous recombination eventsArticleFull-text availableMay 2021Arch Virol

Zalán G HomonnaySzilvia JakabKrisztina Bali

Krisztian BanyaiWe determined the genomic sequence of a Ukrainian strain of fowl adenovirus B (FAdV-B). The isolate (D2453/1) shared 97.2% to 98.4% nucleotide sequence identity with other viruses belonging to the species Fowl aviadenovirus B. Marked genetic divergence was seen in the hexon, fiber, and ORF19 genes, and phylogenetic analysis suggested that recombination events had occurred in these regions. Our analysis revealed mosaicism in the recombination patterns, a finding that has also been described in the genomes of strains of FAdV-D and FAdV-E. The shared recombination breakpoints, affecting the same genomic regions in viruses belonging to different species, suggest that similar selection mechanisms are acting on the key neutralization antigens and epitopes in viruses of different FAdV species.ViewShow abstract... The complete genomes of all 60 ASF viruses (available in GenBank) were successfully sequenced in this study (Table-1, data with identical sequences were removed) [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]. In addition, all available (by March 2020) ASFV genomes were downloaded and included in all analyses (see alignment in the supplementary material). ...Whole-genome-based phylogeny of African swine fever virusArticleFull-text availableOct 2020Vet. World

Levon AslanyanHranush AvagyanZaven KaralyanAim: A genome-scale phylogenetic analysis was used to infer the evolutionary dynamics of Asfarviridae - African swine fever virus (ASFV) - and better define its genetic diversity. Materials and methods: All complete ASFV genomes from NCBI s resource as of March 2020 were used. The phylogenetic analysis used maximum likelihood and neighbor-joining methods. The evolutionary models detection was done with the help of the package of programs MEGA-X. Algorithms were used to build phylogenetic trees for type B DNA polymerases of ASFV (n=34) and HcDNAV (n=2), as an external group. Results: An expedient categorization of the Asfarviridae family uses five clades. Genotype 1 (except for LIV 5/40 virus isolate) as well genotype 7 are assigned to the alpha clade; genotype 2 to the beta clade; genotypes 8, 9, and 10 to the gamma clade; genotype 5 to the delta clade; and genotypes 3, 4, and 20, as well as genotype 22 and the LIV 5/40 isolate to the epsilon clade. Branch lengths on the phylogenetic tree are proportional to genetic distance along the branch. Branches at the phylogenetic tree of Asfarviridae are much shorter than branches for Baculoviridae. Shorter branches in ASFVs population suggest that Asfarviridae evolved relatively recently and remain more closely related. Conclusion: We suggest applying more robust standards using whole genomes to ensure the correct classification of ASFV and maintain phylogeny as a useful tool.ViewShow abstract... Another tandem repeat sequence variation located between MGF 505-9R/10R genes has also been described as a molecular marker [24]. Nowadays, wide use of NGS caused it to become more affordable, therefore the technique is commonly applied to ASFV genomic sequencing, especially in light of its considerable size of almost 200 kb [2][3][4][5][6]8,[25][26][27]. Its usefulness for variation detection is invaluable; nonetheless, it is inadequate in terms of population studies. ...The Spillover of African Swine Fever in Western Poland Revealed Its Estimated Origin on the Basis of O174L, K145R, MGF 505-5R and IGR I73R/I329L Genomic SequencesArticleFull-text availableSep 2020

Natalia Mazur-Panasiuk

Marek Walczak

Małgorzata Juszkiewicz

Grzegorz WozniakowskiThe African swine fever epidemic occurred in Poland at the beginning of 2014 and, up to date, the disease has been spreading mainly in the eastern part of the country. Unexpectedly, in November 2019 an infected wild boar case was confirmed in Lubuskie voivodship in western Poland. During the following weeks, several dozen African swine fever virus (ASFV)-positive animals were notified in the neighboring area, causing severe concern regarding further spread of the disease to the mostly pig-dense region in Poland, namely, Wielkopolskie voivodship. Moreover, almost a year after, several infected wild boar cases were confirmed for the first time in Germany, just beyond the Polish border, sending out a shock wave through the global pig market. The whole genome sequence of ASFV, isolated from the first case of ASF in western Poland, and three selected viruses from other affected areas, revealed the tandem repeat and single nucleotide polymorphism (SNP) variations in reference to the Georgia 2007/1 strain. These data, supported by the conventional sequencing of selected genomic regions from a total of 154 virus samples isolated between 2017 and 2020 in Poland, shed a new light on pathogen epidemiology. The sequence variations within the O174L gene detected in this study showed that cases identified in western Poland might be originating from the so-called southern Warsaw cluster. Moreover, the viruses originating from the northern Warsaw cluster do not possess single nucleotide polymorphism (SNP) mutations within the K145R and MGF 505-5R genes, which are specific to all of the other Polish ASFV strains. These results led to a conclusion of their distinct origin. Supporting these results, the nucleotide sequencing of I73R/I329L intergenic region revealed its new, previously undescribed variant, called IGR IV, with an additional three tandem repeats of 10 nucleotides in comparison to the reference sequence of the Georgia 2007/1 strain.ViewShow abstract... at room temperature and permeabilized by 1 % Triton-X. Indirect FAT was carried out as described previously (Olasz et al., 2019) using anti-ASFV polyclonal pig sera and Goat Anti-Swine IgG (H + L) CF658 (Biotium, Fremont, CA, USA) secondary antibody. Positive cells were observed under an Axio Observer D1 inverted fluorescence microscope (Carl Zeiss Ag. ...Detection of African swine fever virus in cell culture and wild boar tissues using a commercially available monoclonal antibodyArticleMay 2020J VIROL METHODS

Szeredi, L.Erika Bakcsa

Zoltán Zádori

Károly ErdélyiViewDevelopment and clinical application of a novel CRISPR-Cas12a based assay for the detection of African swine fever virusArticleFull-text availableSep 2020BMC MICROBIOLXiaoying WangSheng HeNa Zhao

Chunhe GuoBackground: As no treatment or effective vaccine for African swine fever virus (ASFV) is currently available, a rapid, highly sensitive diagnostic is urgently needed to curb the spread of ASFV. Results: Here we designed a novel CRISPR-Cas12a based assay for ASFV detection. To detect different ASFV genotypes, 19 crRNAs were designed to target the conserved p72 gene in ASFV, and several crRNAs with high activity were identified that could be used as alternatives in the event of new ASFV variants. The results showed that the sensitivity of the CRISPR-Cas12a based assay is about ten times higher than either the commercial quantitative PCR (qPCR) kit or the OIE-recommended qPCR. CRISPR-Cas12a based assay could also detect ASFV specifically without cross-reactivity with other important viruses in pigs and various virus genotypes. We also found that longer incubation times increased the detection limits, which could be applied to improve assay outcomes in the detection of weakly positive samples and new ASFV variants. In addition, both the CRISPR-Cas12a based assay and commercial qPCR showed very good consistency. Conclusions: In summary, the CRISPR-Cas12a based assay offers a feasible approach and a new diagnostic technique for the diagnosis of ASFV, particularly in resource-poor settings.ViewShow abstractTranscriptome view of a killer: African swine fever virusArticleFull-text availableJul 2020BIOCHEM SOC T

Gwenny Cackett

Michal SýkoraFinn WernerAfrican swine fever virus (ASFV) represents a severe threat to global agriculture with the world s domestic pig population reduced by a quarter following recent outbreaks in Europe and Asia. Like other nucleocytoplasmic large DNA viruses, ASFV encodes a transcription apparatus including a eukaryote-like RNA polymerase along with a combination of virus-specific, and host-related transcription factors homologous to the TATA-binding protein (TBP) and TFIIB. Despite its high impact, the molecular basis and temporal regulation of ASFV transcription is not well understood. Our lab recently applied deep sequencing approaches to characterise the viral transcriptome and gene expression during early and late ASFV infection. We have characterised the viral promoter elements and termination signatures, by mapping the RNA-5′ and RNA-3′ termini at single nucleotide resolution. In this review, we discuss the emerging field of ASFV transcripts, transcription, and transcriptomics.ViewShow abstractShow moreA Deep-Sequencing Workflow for the Fast and Efficient Generation of High-Quality African Swine Fever Virus Whole-Genome SequencesArticleFull-text availableSep 2019

Jan Hendrik Forth

Leonie ForthJacqueline King

Anne PohlmannAfrican swine fever (ASF) is a severe disease of suids caused by African swine fever virus (ASFV). Its dsDNA genome (170–194 kbp) is scattered with homopolymers and repeats as well as inverted-terminal-repeats (ITR), which hamper whole-genome sequencing. To date, only a few genome sequences have been published and only for some are data on sequence quality available enabling in-depth investigations. Especially in Europe and Asia, where ASFV has continuously spread since its introduction into Georgia in 2007, a very low genetic variability of the circulating ASFV-strains was reported. Therefore, only whole-genome sequences can serve as a basis for detailed virus comparisons. Here, we report an effective workflow, combining target enrichment, Illumina and Nanopore sequencing for ASFV whole-genome sequencing. Following this approach, we generated an improved high-quality ASFV Georgia 2007/1 whole-genome sequence leading to the correction of 71 sequencing errors and the addition of 956 and 231 bp at the respective ITRs. This genome, derived from the primary outbreak in 2007, can now serve as a reference for future whole-genome analyses of related ASFV strains and molecular approaches. Using both workflow and the reference genome, we generated the first ASFV-whole-genome sequence from Moldova, expanding the sequence knowledge from Eastern Europe.ViewShow abstractModelTest-NG: A New and Scalable Tool for the Selection of DNA and Protein Evolutionary ModelsArticleFull-text availableAug 2019Mol Biol Evol

Diego Darriba

David Posada

Alexey M Kozlov

Tomáš FlouriModelTest-NG is a re-implementation from scratch of jModelTest and ProtTest, two popular tools for selecting the best-fit nucleotide and amino acid substitution models, respectively. ModelTest-NG is one to two orders of magnitude faster than jModelTest and ProtTest but equally accurate, and introduces several new features, such as ascertainment bias correction, mixture, and free-rate models, or the automatic processing of single partitions. ModelTest-NG is available under a GNU GPL3 license at https://github.com/ddarriba/modeltest.ViewShow abstractAnatomy, Bony Pelvis and Lower Limb, Foot VeinsArticleFull-text availableJun 2019

Bradley A Lezak

Matthew VaracalloLezak B, Varacallo M. Anatomy, Bony Pelvis and Lower Limb, Foot Veins. [Updated 2019 Jun 6]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK542295/ViewShow abstractComparative Analysis of Whole-Genome Sequence of African Swine Fever Virus Belgium 2018/1ArticleFull-text availableJun 2019Emerg Infect Dis

Jan Hendrik Forth

Marylene Tignon

Brigitte CayMartin BeerWe analyzed the whole-genome sequence of African swine fever virus Belgium 2018/1. The strain fits into the European genotype II ( 99.98% identity). The high-coverage sequence revealed 15 differences compared with an improved African swine fever virus Georgia 2007/1 sequence. However, in the absence of genetic markers, no spatial or temporal correlations could be defined.ViewShow abstractRAxML-NG: A fast, scalable, and user-friendly tool for maximum likelihood phylogenetic inferenceArticleFull-text availableMay 2019BIOINFORMATICS

Alexey M Kozlov

Diego Darriba

Tomáš Flouri

Alexandros StamatakisMotivation: Phylogenies are important for fundamental biological research, but also have numerous applications in biotechnology, agriculture, and medicine. Finding the optimal tree under the popular maximum likelihood (ML) criterion is known to be NP-hard. Thus, highly optimized and scalable codes are needed to analyze constantly growing empirical datasets. Results: We present RAxML-NG, a from scratch re-implementation of the established greedy tree search algorithm of RAxML/ExaML. RAxML-NG offers improved accuracy, flexibility, speed, scalability, and usability compared to RAxML/ExaML. On taxon-rich datasets, RAxML-NG typically finds higher-scoring trees than IQTree, an increasingly popular recent tool for ML-based phylogenetic inference (although IQ-Tree shows better stability). Finally, RAxML-NG introduces several new features, such as the detection of terraces in tree space and a the recently introduced transfer bootstrap support metric. Availability: The code is available under GNU GPL at https://github.com/amkozlov/raxml-ng. RAxML-NG web service (maintained by Vital-IT) is available at https://raxml-ng.vital-it.ch/. Supplementary information: Supplementary data are available at Bioinformatics online.ViewShow abstractThe first complete genomic sequences of African swine fever virus isolated in PolandArticleFull-text availableMar 2019

Natalia Mazur-Panasiuk

Grzegorz Wozniakowski

Krzysztof NiemczukAfrican swine fever (ASF) is a contagious, notifiable viral disease, which is considered a significant threat not only for European, but also for worldwide pork production, since recently the virus emerged within numerous Chinese pig herds. The disease was introduced in Poland in 2014 and up to the end of 2018, 213 outbreaks in pigs and 3347 cases in wild boars have been reported. The presented study describes the whole genome sequencing of seven Polish isolates, collected between 2016 and 2017, using next generation sequencing (NGS) technology. The complete, genomic sequences of these isolates were compared against five other closely related ASFV genomes, annotated in the NCBI database. The obtained sequences were from 189.393 to 189.405 bp long and encoded 187–190 open reading frames (ORFs). The isolates were grouped within genotype II and showed 99.941 to 99.956% nucleotide identity to the Georgia 2007/1 reference strain.ViewShow abstractGenome sequences derived from pig and dried blood pig feed samples provide important insights into the transmission of African swine fever virus in China in 2018ArticleFull-text availableFeb 2019Xuexia Wen

Xi-Jun HeXiang Zhang

Zhigao buViewRelationships between magnesium and protein concentrations in serum.ArticleFeb 1985CLIN CHEM

Martin KrollR J ElinWe determined concentrations of magnesium, total protein, albumin, and globulin in more than 74 000 serum specimens from patients and noted a direct linear relationship between the concentration of magnesium in serum and the concentrations of total protein, albumin, and globulin in serum. Albumin and magnesium concentrations are linearly related at high and low albumin concentrations; within the reference interval, however, the magnesium concentration is independent of the albumin concentration. Linear regression analysis suggests that 25% of the total serum magnesium is bound to albumin and 8% to globulins.ViewShow abstractThe biology of African swine fever: Literature reviewArticleJan 2019MAGY ALLATORVOSOK

István Mészáros

Ferenc Olasz

Vivien Tamás

Zoltán ZádoriStarting in 2007, the African swine fever (ASF) advanced seemingly unstoppably from the Caucasus region toward the western part of Europe, and in 2018 it reached Hungary. In the lack of vaccine, the spread of ASF constitutes the biggest economical and animal health threat to the Hungarian and worldwide swine industry. The African swine fever virus (ASFV) is the causative agent of the disease. ASFV is an enveloped virus with a large (170-190 kilo base pair) double stranded DNA genome that contains around 200 open reading frames. The virus is the sole member of the Asfaviridae family. ASFV has high genetic and antigenic variability, so far 23 genotypes and at least 8 serotypes were identified. It is the only known DNA arbovirus, its natural hosts are soft ticks belonging to the genus Ornithodoros, and African wild pig species (common warthog (Phacocheorus africanus) and bushpig (Potamochoerus larvatus)). In domestic pigs (Sus scrofa) the virus replicates mainly in macrophages. ASFV utilizes both clathrin-mediated endocytosis and macropinocytosis to enter the macrophages and it replicates in the cytoplasm. At least fourteen ASFV protein was shown to contain strong immunodeterminant epitops and some of them are able to induce at least partially neutralizing antibodies. It seems that cellular immunity, natural killer cells and CD8+ lymphocytes play crucial role in the induction of protective immunity against ASFV. In this paper the authors present the most essential biological knowledge about the ASFV and review its entry, replication and immunology in more details.ViewShow abstractGenome comparison of African swine fever virus China/2018/AnhuiXCGQ strain and related European p72 Genotype II strainsArticleJan 2019TRANSBOUND EMERG DISJingyue BaoQinghua WangPeng Lin

Zhiliang WangAfrican swine fever was introduced into China in August, 2018 and led to high mortality in domestic pigs. We reported the genome characterization of the China/2018/AnhuiXCGQ strain mainly based on next‐generation sequencing and comparison with related European p72 Genotype II strains. The genome was 189,393 bp long, encoding 181 open reading frames. Pair‐wise genome sequence comparison revealed 54‐107 variation sites between China/2018/AnhuiXCGQ and the other genotype II virulent strains, contributing to the change of expression or alteration of amino acid residues in 10‐38 genes. China/2018/AnhuiXCGQ strain shared the highest similarity with POL/2015/Podlaskie strain. Phylogenetic analysis based on 125 kb long conserved central region revealed that the China/2018/AnhuiXCGQ strain and 4 European genotype II strains grouped into 3 clusters. This study expanded our knowledge on the genetic diversity and evolution of ASFV and provided valuable information for diagnosis improvement and vaccine development. This article is protected by copyright. All rights reserved.ViewShow abstractShow moreAdvertisementRecommendationsDiscover moreProjectCharacterisation of the PRRSV 7ap

Ferenc Olasz

Béla Dénes

Adám Bálint[...]

Zoltán ZádoriView projectProjectTyping of Hungarian fowl adenovirus strains

Győző KajánView projectProjectFIP vaccine and diagnostic tool development

Attila Farsang, Dr

Adám Bálint

Sándor Belák[...]

Szeredi, L.View projectArticleThe biology of African swine fever: Literature reviewJanuary 2019 · Magyar Allatorvosok Lapja

István Mészáros

Ferenc Olasz

Vivien Tamás[...]

Zoltán ZádoriStarting in 2007, the African swine fever (ASF) advanced seemingly unstoppably from the Caucasus region toward the western part of Europe, and in 2018 it reached Hungary. In the lack of vaccine, the spread of ASF constitutes the biggest economical and animal health threat to the Hungarian and worldwide swine industry. The African swine fever virus (ASFV) is the causative agent of the disease. ... [Show full abstract] ASFV is an enveloped virus with a large (170-190 kilo base pair) double stranded DNA genome that contains around 200 open reading frames. The virus is the sole member of the Asfaviridae family. ASFV has high genetic and antigenic variability, so far 23 genotypes and at least 8 serotypes were identified. It is the only known DNA arbovirus, its natural hosts are soft ticks belonging to the genus Ornithodoros, and African wild pig species (common warthog (Phacocheorus africanus) and bushpig (Potamochoerus larvatus)). In domestic pigs (Sus scrofa) the virus replicates mainly in macrophages. ASFV utilizes both clathrin-mediated endocytosis and macropinocytosis to enter the macrophages and it replicates in the cytoplasm. At least fourteen ASFV protein was shown to contain strong immunodeterminant epitops and some of them are able to induce at least partially neutralizing antibodies. It seems that cellular immunity, natural killer cells and CD8+ lymphocytes play crucial role in the induction of protective immunity against ASFV. In this paper the authors present the most essential biological knowledge about the ASFV and review its entry, replication and immunology in more details.Read moreArticleFull-text availableA Deep-Sequencing Workflow for the Fast and Efficient Generation of High-Quality African Swine Fever...September 2019 · Viruses

Jan Hendrik Forth

Leonie ForthJacqueline King[...]

Anne PohlmannAfrican swine fever (ASF) is a severe disease of suids caused by African swine fever virus (ASFV). Its dsDNA genome (170–194 kbp) is scattered with homopolymers and repeats as well as inverted-terminal-repeats (ITR), which hamper whole-genome sequencing. To date, only a few genome sequences have been published and only for some are data on sequence quality available enabling in-depth ... [Show full abstract] investigations. Especially in Europe and Asia, where ASFV has continuously spread since its introduction into Georgia in 2007, a very low genetic variability of the circulating ASFV-strains was reported. Therefore, only whole-genome sequences can serve as a basis for detailed virus comparisons. Here, we report an effective workflow, combining target enrichment, Illumina and Nanopore sequencing for ASFV whole-genome sequencing. Following this approach, we generated an improved high-quality ASFV Georgia 2007/1 whole-genome sequence leading to the correction of 71 sequencing errors and the addition of 956 and 231 bp at the respective ITRs. This genome, derived from the primary outbreak in 2007, can now serve as a reference for future whole-genome analyses of related ASFV strains and molecular approaches. Using both workflow and the reference genome, we generated the first ASFV-whole-genome sequence from Moldova, expanding the sequence knowledge from Eastern Europe.View full-textArticleFull-text availableThe evolution of African swine fever virus in Sardinia (1978 to 2014) as revealed by whole genome se...March 2020 · Transboundary and Emerging Diseases

Claudia Torresi

Mariangela S. Fiori

Luigi Bertolotti[...]

Fredrik GranbergAfrican swine fever (ASF) is a highly contagious and lethal viral disease of pigs and wild boars, which is enzootic in many African countries and on the Italian island of Sardinia, where it has been present since 1978. Previous genetic analyses of Sardinian ASF virus (ASFV) isolates have revealed that they all belong to p72 genotype I, with only minor sequence variations. However, these studies ... [Show full abstract] examined only a few selected genes. To distinguish between these closely related isolates and better investigate ASFV evolution in Sardinia, we sequenced the complete genomes of 12 Sardinian ASFV isolates collected between 1978 and 2012, and compared them with 47/Ss/2008 and 26544/OG10. Most of the observed changes occurred in a time‐dependent manner; however, their biological significance remains unclear. As a whole, our results demonstrate the remarkable genetic stability of these strains, supporting a single source introduction of the virus.View full-textArticleFull-text availableThe Spillover of African Swine Fever in Western Poland Revealed Its Estimated Origin on the Basis of...September 2020 · Viruses

Natalia Mazur-Panasiuk

Marek Walczak

Małgorzata Juszkiewicz

Grzegorz WozniakowskiThe African swine fever epidemic occurred in Poland at the beginning of 2014 and, up to date, the disease has been spreading mainly in the eastern part of the country. Unexpectedly, in November 2019 an infected wild boar case was confirmed in Lubuskie voivodship in western Poland. During the following weeks, several dozen African swine fever virus (ASFV)-positive animals were notified in the ... [Show full abstract] neighboring area, causing severe concern regarding further spread of the disease to the mostly pig-dense region in Poland, namely, Wielkopolskie voivodship. Moreover, almost a year after, several infected wild boar cases were confirmed for the first time in Germany, just beyond the Polish border, sending out a shock wave through the global pig market. The whole genome sequence of ASFV, isolated from the first case of ASF in western Poland, and three selected viruses from other affected areas, revealed the tandem repeat and single nucleotide polymorphism (SNP) variations in reference to the Georgia 2007/1 strain. These data, supported by the conventional sequencing of selected genomic regions from a total of 154 virus samples isolated between 2017 and 2020 in Poland, shed a new light on pathogen epidemiology. The sequence variations within the O174L gene detected in this study showed that cases identified in western Poland might be originating from the so-called southern Warsaw cluster. Moreover, the viruses originating from the northern Warsaw cluster do not possess single nucleotide polymorphism (SNP) mutations within the K145R and MGF 505-5R genes, which are specific to all of the other Polish ASFV strains. These results led to a conclusion of their distinct origin. Supporting these results, the nucleotide sequencing of I73R/I329L intergenic region revealed its new, previously undescribed variant, called IGR IV, with an additional three tandem repeats of 10 nucleotides in comparison to the reference sequence of the Georgia 2007/1 strain.View full-textArticleFull-text availableAfrican swine fever whole-genome sequencing—Quantity wanted but quality neededAugust 2020 · PLoS Pathogens

Jan Hendrik Forth

Leonie ForthMartin Beer[...]

Sandra BlomeThe pandemic spread of African swine fever virus (ASFV) genotype II (GTII) has led to a global crisis. Since the circulating strains are almost identical, time and money have been mis-invested in whole-genome sequencing the last years. New methods, harmonised protocols for sample selection, sequencing, and bioinformatics are therefore urgently needed. View full-textDiscover the world s researchJoin ResearchGate to find the people and research you need to help your work.Join for free

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