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bioRxiv · 10.1101/444141

Use of Next Generation Sequencing to study two Cowpoxvirus outbreaks

Antwerpen, M.·Georgi, E.·Nikolic, A.·Zoeller, G.·Wohlsein, P.·Baumgaertner, W.·Peyrefitte, C.·Charell, R.·Meyer, H.
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Abstract

BackgroundBetween 2008 and 2011 about 40 cases of human cowpox were reported from Germany and France. Infections had been acquired via close contact to infected, young pet rats. Sequencing of the hemagglutinin gene of various cowpox virus (CPXV) isolates resulted in an identical and unique sequence in each case pointing to a common source. In a second CPXV outbreak in cats in a small animal clinic in Germany in 2015, four out of five hospitalized cats showed identical hemagglutinin sequences and thus, a hospital-acquired transmission was assumed.\n\nMethodsHomogenates of lesion material from rats, cats and humans were cultivated in cell culture. The genomes of 4 virus isolates, 9 CPXVs from our strain collections and from DNA of 3 paraffin-embedded lesion materials were determined by Next Generation Sequencing (NGS). For phylogenetic analyses a MAFFT-alignment was generated. A distance matrix based on concatenated SNPs was calculated and plotted as dendrogram using Unweighted Pair Group Method with Arithmetic mean (UPGMA) for visualization.\n\nResultsAligning of about 200.000 nucleotides of 8 virus isolates associated with the pet rat outbreak revealed complete identity of six genomes, the remainder two genomes differed in as little as 3 SNPs. When comparing this dataset with four already published CPXV genomes also associated with the pet rat outbreak, again a maximum difference of 3 SNPs was found. The outbreak which lasted from 2008 till 2011 was indeed caused by a single strain which has maintained an extremely high level of clonality over 4 years.\n\nAligning genomic sequences from 4 cases of feline cowpox revealed 3 identical sequences and one sequence which differed in 65 nucleotides. Although identical hemagglutinin sequences had been obtained from four hospitalized cats, genomic sequencing proved that a hospital-acquired transmission had occurred in only three cats.\n\nDiscussionAnalyzing the rather short sequence of the hemagglutinin gene is not sufficient to conduct molecular trace back analyses. Instead, whole genome sequencing is the method of choice which can even be applied to paraffin-embedded specimens.\n\nFunding StatementThis publication was supported by the European Virus Archive goes Global (EVAg) project that has received funding from the European Unions Horizon 2020 research and innovation program under grant agreement No 653316.\n\nThis study was also supported in part by the European Unions Horizon 2020 research and innovation program under grant agreement No 643476 (COMPARE).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Antwerpen, M., Georgi, E., Nikolic, A., Zoeller, G., Wohlsein, P., Baumgaertner, W., Peyrefitte, C., Charell, R., Meyer, H.. 2018-10-24. Use of Next Generation Sequencing to study two Cowpoxvirus outbreaks. https://doi.org/10.1101/444141

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