Use of Next Generation Sequencing to study two cowpox virus outbreaks

Markus H Antwerpen; Enrico Georgi; Alexandra Nikolic; Gudrun Zoeller; Peter Wohlsein; Wolfgang Baumgärtner; Christophe Peyrefitte; Remi Charrel; Hermann Meyer

doi:10.7717/peerj.6561

Use of Next Generation Sequencing to study two cowpox virus outbreaks

PeerJ. 2019 Mar 1:7:e6561. doi: 10.7717/peerj.6561. eCollection 2019.

Authors

Markus H Antwerpen¹, Enrico Georgi¹, Alexandra Nikolic¹, Gudrun Zoeller¹, Peter Wohlsein², Wolfgang Baumgärtner³, Christophe Peyrefitte⁴, Remi Charrel⁴, Hermann Meyer¹

Affiliations

¹ Bundeswehr Institute of Microbiology, Munich, Germany.
² Small Animal Clinic, University of Veterinary Medicine, Hanover, Germany.
³ Department of Pathology, University of Veterinary Medicine, Hanover, Germany.
⁴ Unité des Virus Emergents, Aix Marseille Université, Marseille, France.

Abstract

Background: Between 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. An identical and unique sequence of the hemagglutinin gene was found in various cowpox virus (CPXV) isolates pointing to a common source of infection. 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 had been assumed. Next-Generation Sequencing was performed in order to re-investigate the outbreaks, as epidemiological data could not confirm all cases.

Methods: Homogenates of lesion material from rats, cats and humans were cultivated in cell culture. The genomes of four virus isolates, nine CPXVs from our strain collections and from DNA of three 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.

Results: Aligning 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. Aligning genomic sequences from four 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.

Conclusion: Analyzing 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.

Keywords: Cowpox virus; Germany; Hospital; NGS; Outbreak.

Grants and funding

This publication was supported by the European Virus Archive goes Global (EVAg) project that has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 653316. This study was also supported in part by the European Union’s Horizon 2020 research and innovation program under grant agreement No 643476 (COMPARE). There was no additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.