Molecular diagnostics of aleutian mink disease virus: applied use of next generation sequencing and phylogenetics

Emma Elisabeth Hagberg

    Research output: Book/ReportPh.D. thesis

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    Abstract

    Aleutian Mink Disease virus (AMDV) is a parvovirus causing Aleutian Mink Disease (AMD), often referred to as plasmacytosis. It is a systemic infection affecting mink of all ages, and is globally the most important pathogen impacting mink farming. In Denmark AMDV has since 1999 been monitored by a national control program, which is based on serological screening of all animals and encourages infected farms to stamp out. Historically there has been no consensus about which genomic region of the virus to analyse e.g. in relation to surveillance, and most previous studies in this regard, have been based either on partial or entire genes, or on pure epidemiological data. Thus, when initiating this project, little was known about AMDV’s total genomic diversity and how the virus was spread between farms.

    Recent advances in the field of molecular diagnostics have made high throughput tools such as next generation sequencing cheaper and more easily available. Whole genome sequencing and advanced phylogenetic analyses have successfully been applied to describe the molecular evolution and transmission patterns for viruses such as Foot and Mouth Disease Virus (FMDV), Ebola, and avian influenza virus, however not previously for AMDV. The overall aim with this thesis was to investigate if next generation sequencing and phylogenetic analyses of full length isolates could improve our understanding of the total genomic diversity and evolution of AMDV. Additionally, we wanted to evaluate if this knowledge could contribute to the elucidation of AMDV transmission between farms and improve molecular diagnostics. During the first phase of this project a method for performing whole genome sequencing of AMDV was developed. This protocol enabled the sequencing of a large number of in vivo infectious AMDV isolates and provided the necessary dataset to act as foundation for the remaining analyses in the thesis. The first original paper (Manuscript 1) describes this protocol.

    Manuscript 2 is a proof-of-concept study which demonstrated the advantage of using the whole genome sequence approach, compared to the in Denmark traditionally used partial NS1 gene sequencing, for the elucidation of transmission pathways between farms. The study was performed on samples from a small local AMDV outbreak, and clearly illustrated that the phylogenies based on partial NS1 gene sequencing were uninformative and could not be used for determining transmission pathways, even in the light of supporting epidemiological data. The whole-genome approach on the other hand, confirmed the epidemiological hypothesis about the direction of spread.

    In Manuscript 3, the methodologies from Manuscript 1 and 2 were applied to generate the to-date most comprehensive phylogenetic and genetic analysis of full-length AMDV isolates, composed of more than 200 field strains. The study shed light on the diversity and evolutionary behaviour of two distinct AMDV strains, in addition to providing the first robust evolutionary rate-estimates. Altogether, the work presented in this thesis provides a contribution to the molecular diagnostics of AMDV, enables us better to understand the virus’ evolutionary behaviour in the context of mink farming, and is anticipated to be of value for more accurately tracing back in time the emergence of future outbreaks.
    Original languageEnglish
    PublisherTechnical University of Denmark
    Number of pages180
    Publication statusPublished - 2017

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