A robust, cost-effective and widely applicable whole-genome sequencing protocol for capripoxviruses

Mathijs, Élisabeth; Haegeman, Andy; Clercq, Kris De; Borm, Steven Van; Vandenbussche, Frank

doi:10.1016/j.jviromet.2022.114464

Cited by 7 publications

(8 citation statements)

References 56 publications

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“…DNA extraction, LSDV genome amplification, massive parallel sequencing, and genome assembly were performed as previously described ( 38 ). Briefly, DNA was extracted from skin sample homogenates using a Puregene extraction kit (Qiagen), followed by PCR amplification of the genome by long-range PCR (amplicons of approximately 7,500 bp).…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, DNA was extracted from skin sample homogenates using a Puregene extraction kit (Qiagen), followed by PCR amplification of the genome by long-range PCR (amplicons of approximately 7,500 bp). Amplicons were equimolarly pooled per half genome and sequenced using a Nextera XT DNA Library Preparation Kit (Illumina) and a MiSeq Reagent Kit version 3, 2 × 300 bp (Illumina) as previously described ( 38 ). The sequencing run was performed at the Neuromics Support Facility–VIB Genomics Core (UAntwerp, Belgium).…”

Section: Methodsmentioning

confidence: 99%

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Complete genome reconstruction of the global and European regional dispersal history of the lumpy skin disease virus

Van Borm,

Dellicour,

Martin

et al. 2023

J Virol

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Lumpy skin disease virus (LSDV) causes a disease of economic importance affecting cattle. Its global epidemiology is complex due to the combination of vector-borne and anthropogenic spread, the circulation of vaccine-like recombinants, and the use of vaccines. The slow molecular evolution of its DNA genome limits the utility of genetic variation for accurate tracing based on evolutionary analyses, but this limitation has not yet been formally assessed. Furthermore, until present, whole genome sequencing in affected areas has remained patchy. This study combines the first fine-grained sampling of LSDV whole genomes from a time-constrained (2015–2017) southeastern European (SEE) LSDV outbreak, which we analyze along with curated public genomes to investigate the global and regional viral dispersal dynamics. First, haplotype networks visualizing the limited genetic variability associated with the SEE LSDV outbreak show intense intermixing between countries. We also assess at which spatial scale a correlation between genetic and geographic distances can be detected for LSDV. On a global scale, we show the importance of accounting for recombination events that can impact phylogenetic and phylogeographic reconstructions. Following the assessment of the temporal signal in the recombination-free alignment, our time-scaled continuous phylogeographic analysis of Kenya-like and recent wild-type viruses confirms the origin and global dissemination history of LSDV. Our analyses highlight the importance of careful selection and application of phylodynamic approaches to DNA viruses, as well as the importance of whole genome sampling in endemic and outbreak areas to improve our understanding of the evolution, epidemiology, and transmission dynamics of DNA viruses. IMPORTANCE Lumpy skin disease virus (LSDV) has a complex epidemiology involving multiple strains, recombination, and vaccination. Its DNA genome provides limited genetic variation to trace outbreaks in space and time. Sequencing of LSDV whole genomes has also been patchy at global and regional scales. Here, we provide the first fine-grained whole genome sequence sampling of a constrained LSDV outbreak (southeastern Europe, 2015–2017), which we analyze along with global publicly available genomes. We formally evaluate the past occurrence of recombination events as well as the temporal signal that is required for calibrating molecular clock models and subsequently conduct a time-calibrated spatially explicit phylogeographic reconstruction. Our study further illustrates the importance of accounting for recombination events before reconstructing global and regional dynamics of DNA viruses. More LSDV whole genomes from endemic areas are needed to obtain a comprehensive understanding of global LSDV dispersal dynamics.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Complete genome reconstruction of the global and European regional dispersal history of the lumpy skin disease virus

Van Borm,

Dellicour,

Martin

et al. 2023

J Virol

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“…In order to achieve uniform coverage of the entire genome, an enrichment step was performed prior to sequencing using an in-house long-range PCR that covered the entire genome with 23 overlapping amplicons of approximately 7.5 kb in length [ 43 ]. Briefly, PCRs were performed in a mix containing 1 M betaine, 0.5 μM of both forward and reverse primers, 0.4 mM CleanAmp dNTPs (TriLink Biotechnologies, San Diego, CA, USA) and 1 U of Q5 High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

“…The trimmed reads were de novo assembled using SPAdes v.3.15.2 [ 48 ] with optimised k values and a subsample of 40,000 paired-end reads as described previously [ 43 ]. The quality of the de novo assemblies was assessed and compared using Quast v.5.1.0 [ 49 ].…”

Section: Methodsmentioning

confidence: 99%

Recombinant LSDV Strains in Asia: Vaccine Spillover or Natural Emergence?

Vandenbussche

Mathijs

Philips

et al. 2022

Viruses

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From 2017 to 2019, several vaccine-like recombinant strains of lumpy skin disease virus (LSDV) were discovered in Kazakhstan and neighbouring regions of Russia and China. Shortly before their emergence, the authorities in Kazakhstan launched a mass vaccination campaign with the Neethling-based Lumpivax vaccine. Since none of the other countries in the affected region had used a homologous LSDV vaccine, it was soon suspected that the Lumpivax vaccine was the cause of these unusual LSDV strains. In this study, we performed a genome-wide molecular analysis to investigate the composition of two Lumpivax vaccine batches and to establish a possible link between the vaccine and the recent outbreaks. Although labelled as a pure Neethling-based LSDV vaccine, the Lumpivax vaccine appears to be a complex mixture of multiple CaPVs. Using an iterative enrichment/assembly strategy, we obtained the complete genomes of a Neethling-like LSDV vaccine strain, a KSGP-like LSDV vaccine strain and a Sudan-like GTPV strain. The same analysis also revealed the presence of several recombinant LSDV strains that were (almost) identical to the recently described vaccine-like LSDV strains. Based on their InDel/SNP signatures, the vaccine-like recombinant strains can be divided into four groups. Each group has a distinct breakpoint pattern resulting from multiple recombination events, with the number of genetic exchanges ranging from 126 to 146. The enormous divergence of the recombinant strains suggests that they arose during seed production. The recent emergence of vaccine-like LSDV strains in large parts of Asia is, therefore, most likely the result of a spillover from animals vaccinated with the Lumpivax vaccine.

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“…This is the first report of complete genome sequencing of SPPVs directly from clinical samples, although similar approaches have been described for LSDV and parapoxviruses (van Schalkwyk et al., 2021; Günther et al., 2017). Similar non‐culturing approaches employing either virus enrichment in vitro or amplicon pool preparation have been described for capripoxvirus genome sequencing (Mathijs et al., 2022). As mentioned previously, it is essential to analyse the complete SPPV genomes of isolates circulating in Russia and neighbouring countries and compare them to sequences from other affected countries.…”

Section: Resultsmentioning

confidence: 99%

Genetic characterization of sheep pox virus strains from outbreaks in Central Russia in 2018–2019

Krotova

Shalina

Mazloum

et al. 2022

Transbounding Emerging Dis

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This study investigates the phylogenomic relatedness between sheep pox viruses (SPPVs) circulating in Central Russia in 2018-2019 with the NISKHI vaccine strain used in the country, based on their complete genome sequences. The sheep pox outbreaks occurred 1 year apart in the adjacent regions of Tula and Moscow. Full genome sequences were generated by sequencing DNA directly obtained from Trizol-extracted scabs, using the DNBSEQ-400 platform (MGI Tech, China). Phylogenetic analysis indicated that the SPPV isolates from Russia clusters with previously published sequences from Srinagar in the Kashmir province of India in 2000 (SPPV-Srinagar strain) as well as SPPV A strain from Kazakhstan in 2000. The aforementioned cluster belonged to a sister clade containing the NISKHI vaccine strain, thus indicating that the recent outbreaks were not genetically linked to the widely used vaccine.

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A robust, cost-effective and widely applicable whole-genome sequencing protocol for capripoxviruses

Cited by 7 publications

References 56 publications

Complete genome reconstruction of the global and European regional dispersal history of the lumpy skin disease virus

Complete genome reconstruction of the global and European regional dispersal history of the lumpy skin disease virus

Recombinant LSDV Strains in Asia: Vaccine Spillover or Natural Emergence?

Genetic characterization of sheep pox virus strains from outbreaks in Central Russia in 2018–2019

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