Outbreak tracking of Aleutian mink disease virus (AMDV) using partial NS1 gene sequencing

Abstract

BackgroundAleutian Mink Disease (AMD) is an infectious disease of mink (Neovison vison) and globally a major cause of economic losses in mink farming. The disease is caused by Aleutian Mink Disease Virus (AMDV) that belongs to the genus Amdoparvovirus within the Parvoviridae family. Several strains have been described with varying virulence and the severity of infection also depends on the host’s genotype and immune status. Clinical signs include respiratory distress in kits and unthriftiness and low quality of the pelts. The infection can also be subclinical.Systematic control of AMDV in Danish mink farms was voluntarily initiated in 1976. Over recent decades the disease was mainly restricted to the very northern part of the country (Northern Jutland), with only sporadic outbreaks outside this region. Most of the viruses from this region have remained very closely related at the nucleotide level for decades. However, in 2015, several outbreaks of AMDV occurred at mink farms throughout Denmark, and the sources of these outbreaks were not known.MethodsPartial NS1 gene sequencing, phylogenetic analyses data were utilized along with epidemiological to determine the origin of the outbreaks.ResultsThe phylogenetic analyses of partial NS1 gene sequences revealed that the outbreaks were caused by two different clusters of viruses that were clearly different from the strains found in Northern Jutland. These clusters had restricted geographical distribution, and the variation within the clusters was remarkably low. The outbreaks on Zealand were epidemiologically linked and a close sequence match was found to two virus sequences from Sweden. The other cluster of outbreaks restricted to Jutland and Funen were linked to three feed producers (FP) but secondary transmissions between farms in the same geographical area could not be excluded.ConclusionThis study confirmed that partial NS1 sequencing can be used in outbreak tracking to determine major viral clusters of AMDV. Using this method, two new distinct AMDV clusters with low intra-cluster sequence diversity were identified, and epidemiological data helped to reveal possible ways of viral introduction into the affected herds.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-017-0786-5) contains supplementary material, which is available to authorized users.

Global phylogenetic analysis of contemporary aleutian mink disease viruses (AMDVs)

“…Total DNA was extracted using the QIAmp®DNA Mini Kit with the Standard version 1 protocol automated on the QIAcube (Qiagen, Copenhagen, Denmark). PCR and Sanger sequencing was carried out as previously described [ 11 ]. The sequences names were tagged “f” or “w” to indicate if the mink was farmed or wild respectively, and included the country and region of sampling, and the sampling date as follows: AMDV_mink-f/w_Country_unique identifier_sampling date.…”

“…The partial NS1 sequencing of viruses from this outbreak revealed that the outbreaks were caused by two distinct viral strains, which clearly deviated from all viruses involved in previously known outbreaks in Denmark [ 11 ]. Furthermore, there was only limited nucleotide sequence homology to any AMDV sequences in GenBank.…”

“…Furthermore, there was only limited nucleotide sequence homology to any AMDV sequences in GenBank. Specifically, the partial NS1 sequences showed the presence of two new virus clusters; one named the Zealand cluster, as the viruses were all from farms located on Zealand, and the other named the “Holstebro” cluster containing viruses isolated from farms in both Jutland and Funen and with index case from the town Holstebro [ 11 ]. In order to investigate the possible origin of these new Danish outbreaks we collected AMDV strains from other countries with commercial mink production.…”

Duplex PCR for Detection of Aleutian Disease Virus from Biological and Environmental Samples

“…Phylogenetic analysis was carried out in MEGA6. A phylogenetic tree was obtained by the Maximum Likelihood method with 1000 bootstrap replications for the sequence coding NS1 protein as this fragment has been used to analysis of relationships between isolates and tracing of outbreaks [13,14].…”

“…Electrophoretic separation of PCR products: M -100 bp marker, reaction results for samples isolated from blood tested as individual samples (wells 1-4), pooled blood(5)(6)(7)(8) and spleens(9)(10)(11)(12)(13)(14)(15)(16) …”