Detection of mink astrovirus in Poland and further phylogenetic comparison with other European and Canadian astroviruses

Abstract

Mink astrovirus infection remains a poorly understood disease entity, and the aetiological agent itself causes disease with a heterogeneous course, including gastrointestinal and neurological symptoms. This paper presents cases of astrovirus infection in mink from continental Europe. RNA was isolated from the brains and intestines of animals showing symptoms typical of shaking mink syndrome (n = 6). RT-PCR was used to amplify astrovirus genetic material, and the reaction products were separated on a 1% agarose gel. The specificity of the reaction was confirmed by sequencing fragment coding RdRP protein (length of sequencing product 170 bp) from all samples. The presence of astrovirus RNA was detected in each of the samples tested. Sequencing and bioinformatic analysis indicated the presence of the same variant of the virus in all samples. Comparison of the variant with the sequences available in bioinformatics databases confirmed that the Polish isolates form a separate clade, closely related to Danish isolates. The dissimilarity of the Polish variant to those isolated in other countries ranged from 2.4% (in relation to Danish isolates) to 7.1% (in relation to Canadian isolates). Phylogenetic relationships between variants appear to be associated with the geographic distances between them. To our knowledge, this work describes the first results on the molecular epidemiology of MAstV in continental Europe. The detection of MAstV in Central Europe indicates the need for further research to broaden our understanding of the molecular epidemiology of MAstV in Europe.

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Vol:.(1234567890)
Virus Genes (2021) 57:258–265
https://doi.org/10.1007/s11262-021-01834-z
1 3
ORIGINAL PAPER
Detection ofmink astrovirus inPoland andfurther phylogenetic
comparison withother European andCanadian astroviruses
AndrzejJakubczak1 · MarekKowalczyk2· IlonaMazurkiewicz1· MarcinKondracki1
Received: 21 October 2020 / Accepted: 26 March 2021 / Published online: 15 April 2021
© The Author(s) 2021
Abstract
Mink astrovirus infection remains a poorly understood disease entity, and the aetiological agent itself causes disease with a
heterogeneous course, including gastrointestinal and neurological symptoms. This paper presents cases of astrovirus infection
in mink from continental Europe. RNA was isolated from the brains and intestines of animals showing symptoms typical
of shaking mink syndrome (n = 6). RT-PCR was used to amplify astrovirus genetic material, and the reaction products were
separated on a 1% agarose gel. The specificity of the reaction was confirmed by sequencing fragment coding RdRP protein
(length of sequencing product 170bp) from all samples. The presence of astrovirus RNA was detected in each of the sam-
ples tested. Sequencing and bioinformatic analysis indicated the presence of the same variant of the virus in all samples.
Comparison of the variant with the sequences available in bioinformatics databases confirmed that the Polish isolates form
a separate clade, closely related to Danish isolates. The dissimilarity of the Polish variant to those isolated in other coun-
tries ranged from 2.4% (in relation to Danish isolates) to 7.1% (in relation to Canadian isolates). Phylogenetic relationships
between variants appear to be associated with the geographic distances between them. To our knowledge, this work describes
the first results on the molecular epidemiology of MAstV in continental Europe. The detection of MAstV in Central Europe
indicates the need for further research to broaden our understanding of the molecular epidemiology of MAstV in Europe.
Keywords Mink astrovirus· Molecular diagnostics· Molecular polymorphism· Phylogenetics· Shaking mink syndrome
Introduction
Astroviruses are pathogens that infect a wide range of hosts
belonging to various species. Two genera are distinguished
within the family Astroviridae—Mamastrovirus and Avas-
trovirus, which include astroviruses that infect mammals and
birds, respectively. The virus has been detected in represent-
atives of mammals inhabiting both terrestrial environments
(e.g. pigs and cattle) and aquatic environments (dolphins), as
well as in birds and fish [1, 2]. A pathogen from this group
was diagnosed for the first time in children with diarrhea in
the mid-1970s. Today, alongside rotaviruses, it is one of the
main causes of viral gastrointestinal infections [3]. Astro-
viruses cause gastrointestinal diseases in humans (HastVs
1–8), sheep (OAstV), cattle (BoAstV), mink (MiAstV), pigs
(PoAstV), cats (FeAstV), dogs (CaAstV) and marine mam-
mals. In turkeys (TAstVs) and chickens (CAstV), they cause
nephritis and gastrointestinal diseases [3, 4]. Some strains of
astroviruses in humans and animals, such as mink, cattle and
sheep, can bypass the gastrointestinal tract, showing tropism
for nervous tissue. They then cause infections of organs of
the central nervous system (CNS), especially the brain [4].
Mink astrovirus (MiAstV) is known to play a major role in
mink pre-weaning diarrhea, and some of viruses detected
in mink faeces such as rotavirus C and hepatitis E virus
(HEV) are considered as zoonotic agents. These viruses are
not monitored in commercial mink, and the role of these
viral infections in mink health is not well understood [5].
The genetic material of astroviruses is single-stranded
RNA with positive polarization. The 6.8 to 7.9kb genome
contains a 5′UTR untranslated region followed by three
Edited by Zhen F. Fu.
* Andrzej Jakubczak
andrzej.jakubczak@up.lublin.pl
1 Institute ofBiological Basis ofAnimal Production, Faculty
ofAnimal Sciences andBioeconomy, University ofLife
Sciences inLublin, Lublin, Poland
2 Institute ofQuality Assessment andProcessing ofAnimal
Products, Faculty ofAnimal Sciences andBioeconomy,
University ofLife Sciences inLublin, Lublin, Poland
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259Virus Genes (2021) 57:258–265
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open reading frames—ORF1a, ORF1b and ORF2, a 3′UTR
region, and a poly(A)tail [3, 4, 6]. ORF1a and ORF1b code
for non-structural protein precursors, while ORF2 codes for
a structural protein precursor [4, 7]. ORF1 encodes protease
and RNA-dependent polymerase [3]. The subgenomic RNA
of the astrovirus, derived from ORF2, encodes a single, large
structural capsid protein (CP). Depending on the strain of the
virus, this capsid polyprotein precursor contains from about
775 to 785 amino acid residues, and also has a molecular
weight of 87–90 kilodaltons (kDa) [4]. The CP is an external
structural barrier that not only surrounds the nucleic acids,
but also interacts with the host, influencing cell tropism and
mediating entry into the cell. Furthermore, it is an antigen
that induces an immune response in the host [8].
Phylogenetic and genomic analyses indicate high homol-
ogy between astroviruses infecting humans and those iso-
lated from mink. There is evidence indicating the zoonotic
potential of astroviruses. After observing the occurrence of
diseases in people living near infected farms, Quan etal.
suggested that the pathogen may flow between mink and
humans [9]. The zoonotic potential of astroviruses is also
suggested by the results of Meliopoulos etal. [10], who con-
firmed the presence of antibodies against turkey astrovirus
in humans.
The aetiological factor for astrovirus infections in mink
is the MAstV-1 virus. Mink astrovirus infection is a disease
with a heterogeneous course and a diverse clinical picture.
When the pathogen colonizes the nervous system, shaking
mink syndrome (SMS) develops [11, 12]. Other clinical pic-
tures of the disease, often treated as one disease entity due
to the similarity of the symptoms, are pre-weaning diarrhea
[13] and wet mink syndrome (WMS).
In the case of WMS, viraemia results in increased activ-
ity of the apocrine glands, especially in the neck and tail
area, where a sticky, greasy secretion appears, to which the
disease owes its name. Affected mink kits display diarrhea
with concomitant excessive secretions from the cervical
apocrine glands, and exudate on the skin surface, the tail,
and the claws. Moreover, dehydration may ultimately lead
to the death of the affected kits [14]. The secretions may
cause deterioration in the quality of the fur, which takes
on a wavy structure. As the disease develops, alopecia may
occur at the site of excessive secretion of apocrine glands
[4]. The syndrome includes a characteristic symptom of
astrovirus infections, i.e. diarrhea, lasting up to 10days, usu-
ally foamy and yellowish, and often with an admixture of
undigested milk [13]. Animal faeces are infectious material
through which the virus can spread. In many cases, diarrhea
and fever result in dehydration and an overall decrease in
immunity, which is conducive to complications caused by
bacterial co-infections. The clinical picture may also include
behavioural changes in mink; sick individuals often make
sounds that resemble meowing. Pre-weaning diarrhea is a
syndrome affecting farm-raised neonatal mink kits. Apart
from diarrhea it causes greasy skin exudation, dehydration,
and distressed behavior. Moreover, dehydration may ulti-
mately lead to the death of the affected kits [15].
In view of the relatively little-known aetiology and epide-
miology of mink astrovirus infection, as well as the lack of
research on this subject in continental Europe, the aim of the
study is to examine molecular variation in MAstV on farms
in Poland and the relationship between the variants obtained
and previously isolated variants.
Materials andmethods
The study covered two farms in north-western Poland with
more than 10,000 mink. Symptoms typical of the neurologi-
cal form of astrovirus infection—shaking mink syndrome—
were observed in animals on these farms, such as tremors,
an unsteady gait, and awkward movements. Cases of wet
mink syndrome were also noted on the farms. The study
material comprised brains (n = 6) and intestines (n = 6) of
six mink that had died from the disease (3 mink from each
of the tested farm). The age of the tested animals ranged
from 4 to 7weeks. Tissues were collected and fixed in RNA
later reagent.
Viral RNA isolation
Isolation was carried out in two independent replicates using
the RNeasy Mini Kit (Qiagen) and the Total RNA Mini kit
(A&A Biotechnology). The tissues were suspended in lysis
buffers: 600μl RLT buffer was used in the case of the RNe-
asy Mini Kit, while the tissues were suspended in 800μl
fenozol for isolation with the Total RNA Mini Kit. Then the
samples were homogenized in a Tissue Lyser for 40s at a
frequency of 20Hz. The remainder of the isolation proce-
dure was carried out according to the recommendations of
the kit manufacturers. Genetic material was eluted in 50μl
of nuclease-free water.
Reverse transcription andPCR
Reverse transcription was performed using the QuantiTect
Reverse Transcription Kit (Qiagen). The gDNA Wipeout
Buffer reagent was used to remove DNA residue. Reverse
transcription was carried out according to the manufacturer’s
protocol and included a reaction with oligo-dT primers and
random hexameters to obtain total cDNA, with a recom-
mended incubation time of 42°C for 15min and 3min of
enzyme inactivation at 95°C.
PCR was carried out for all samples isolated from the
intestines and brains using both of the kits and transcribed
into cDNA (n = 24: 6 intestinal samples and 6 brain samples
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260 Virus Genes (2021) 57:258–265
1 3
for the Qiagen kit and 6 intestinal samples and 6 brain sam-
ples for the A&A Biotechnology kit). The primers used were
MA17 (reverse, 5′ GAG GAG TTT CAG ACA GAT G 3′) and
MA15 (forward 5′ CAA ATG CCT GGA AGA ACA C 3′),
proposed by Mittelholzer etal. [7]. Primers targeted a part
of RNA-dependent RNA-polymerase region (RdRp). The
reaction mixture contained 3μl DNA and 1 U Taq poly-
merase (AmpliTaq Gold 360 DNA Polymerase, Applied
Biosystems) in the manufacturer’s buffer, adjusted to a final
concentration of 2.5mM MgCl2; 0.8mM of each dNTP; and
1.2mM of each primer—25 μL total volume. The reaction
took place under the following conditions: 95°C for 10min,
40 cycles of 95°C for 45s, 54°C for 45s, 72°C for 45s,
and 72°C for 10min in a Labcycler Thermocycler (Senso-
Quest). The reaction products were separated on a 1% aga-
rose gel with ethidium bromide at 80V. Visualization and
archiving of the gel was carried out in Scion Image software.
Sequencing andbioinformatics analysis
The samples were purified with an EPPiC Fast kit (A&A
Biotechnology) and subjected to sequencing with the same
primers as in the standard reaction, using the BigDye Ter-
minator v3.1 Cycle Sequencing Kit (Applied Biosystems).
Sequencing PCR products were purified using the Extermi-
nator kit (A&A Biotechnology). The purified samples were
suspended in formamide, denatured, and then separated
on an ABI PRISM 3100 Avant Genetic Analyzer (Applied
Biosystems).
Sequencing results were assembled into contigs in DNA
Baser software to obtain fragments of 170bp. Specificity
was confirmed using the Blast application, and sequences
were compared with the NCBI bioinformatics database.
Sequences obtained during the analyses were compared
with sequences from the GenBank database in MEGA7 soft-
ware. The similarity between isolates was determined using
BioEdit software. Analysis of polymorphisms and phyloge-
netic analysis were carried out in MEGA7. The evolutionary
history was inferred using the neighbor-joining (NJ) method.
The percentage of replicate trees in which the associated
taxa are clustered together in the bootstrap test (1000 repli-
cates) are shown next to the branches [16].
Results
The RT-PCR method confirmed the presence of the genetic
material of the virus in both brain and intestinal samples
isolated using both kits. In each of the tested samples the
product of 178bp was obtained. The specificity of the reac-
tion was confirmed by sequencing all PCR products. After
sequencing the fragment of 170bp was obtained and used
in further bioinformatic analysis. The results show 100%
similarity for the tested fragment between isolates from the
two farms.
The similarity of the nucleotide sequence of RdRP gene
of the mink astrovirus variants from Polish farms with Dan-
ish, Swedish and Canadian variants deposited in the NCBI
database was assessed as well (Table1). The Polish isolates
showed the highest similarity to the variants isolated from
Danish farms, ranging from 96.4 to 97.6% (97.2% on aver-
age). Lower similarity was noted in comparison to Swedish
isolates, ranging from 94.1 to 96.4% (95.64% on average).
The greatest variation in relation to Polish isolates was found
for the MAstV variants from Canada, differing in 12 nucleo-
tides, which translated into a more than 7% difference within
the analysed fragment. Bioinformatics analysis indicated a
unique G3674A polymorphism in the Polish isolates, which
has thus far not been detected in other sequences deposited
in databases.
All polymorphisms between Danish and Polish isolates
are transitions, while in the case of Swedish farms there
were two transversions—T3514A and T3616A, both in the
case of an isolate deposited under number GU985458.1,
isolated from an individual with SMS (shaking mink syn-
drome). There was also a transversion for one isolate from
Canada—C3514A.
Among the polymorphisms examined, two were non-syn-
onymous and were associated with differences in the amino
acid sequence. In the Polish isolates, adenine was present at
position 3515, as in the case of most isolates from databases.
A difference in this position occurs only in the sequence
from Danish isolate deposed in NCBI database in 2003
under number AY196095.1, with guanine at position 3515.
The polymorphism involves a change at amino acid position
301—methionine (present in most of the tested sequences)
to valine (appearing only in sequence AY196095.1). The
other amino acid change (L317P) occurs in the sequence
deposited under number AY196100.1, where thymine is
found in nucleotide position 3564 (the codon encodes leu-
cine), and cytosine in the remaining sequences (the codon
encodes proline).
Phylogenetic analysis was performed to determine the
phylogenetic relationships between Polish isolates and
isolates from Denmark, Sweden and Canada (Fig.1). Five
main groups were distinguished, three of which form one
relatively closely related clade. The first group (the Danish
group) was formed by Danish isolates, whose percentage
similarity within the group is over 99% (Table2).
The bootstrap value obtained for the node is 88. The same
group includes clades grouping together the Polish isolates
and some of the Swedish isolates. Polish isolates, like the
Danish ones, were characterized by high homogeneity,
which was manifested by the presence of the same genetic
variant in all samples. Isolates from Sweden were signifi-
cantly less homogeneous. Based on phylogenetic analysis,
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261Virus Genes (2021) 57:258–265
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two heterogeneous groups were distinguished—Swed-
ish I—which included two closely related clades (98.2%
similarity) and Swedish II, which included, among others,
the GU985458.1 isolate, causing shaking mink syndrome
(SMS). This variant differed from representatives of the
Swedish I group by over 6% (differences from 6.5 to 7.1%).
The second isolate within the Swedish II group was depos-
ited under number AY196104.1. In relation to the Swedish
Table 1 Polymorphic nucleotides differentiating Polish isolates from variants from the NCBI database
Differences between database isolates and isolates from Polish farms are given (First letter—nucleotide or amino acid position in the database
variant, number—polymorphism location, second letter—nucleotide or amino acid in the Polish isolate. Bolded polymorphisms—nonsynony-
mous substytutuions
Country Accession number Polish iso-
late [%]
Polymorphic nucleotides
Denmark AY196095.1 96.4 G3515A > V301M, G3574A*, T3598C, G3604A, T3628C, C3631T
AY196096.1 97.0 G3574A*, T3598C, G3616A, T3628C, C3631T
AY196097.1 97.6 G3574A*, T3598C, T3628C, C3631T
AY196098.1 97.6 G3574A*, T3598C, T3628C, C3631T
AY196099.1 97.6 G3574A*, T3598C, T3628C, C3631T
AY196100.1 97.0 T3564C > L317P, G3574A*, T3598C, T3628C, C3631T
Sweden AY196101.1 95.8 C3508T, T3529C, C3547T, A3550G, G3574A*, T3598C, C3631T
AY196102.1 96.4 C3508T, C3547T, A3550G, G3574A*, T3583C, C3631T
AY196103.1 95.8 C3508T, T3529C, C3547T, A3550G, G3574A*, T3598C, C3631T
AY196104.1 95.2 T3514A, A3523G, T3529C, C3547T, G3559A, G3574A*, T3577C, T3583C
AY196105.1 95.8 C3508T, T3529C, C3547T, A3550G, G3574A*, T3598C, C3631T
NC_004579.1 96.4 C3508T, C3547T, A3550G, G3574A*, T3583C, C3631T
GU985458.1 94.1 T3514A, T3529C, C3547T, G3559A, G3574A*, T3577C, G3604A, C3613T, T3616A, T3628C
Canada MH282878.1 92.9 C3514A, C3520T, C3532T, C3547T, G3559A, G3574A*, T3583C, A3586G, T3589C, T3595C,
T3598C, C3607T
MH282880.1 92.9 T3514A, C3520T, C3532T, C3547T, G3559A, G3574A*, T3583C, A3586G, T3589C, T3595C,
T3598C, C3607T
Fig. 1 Phylogenetic analysis of the Polish MAstv isolate in relation to sequences deposited in the NCBI database. The tree was constructed using
the NJ method with a bootstrap value of 1000. Analysis based on 170bp fragment coding RdRp (RNA-dependent RNA polymerase) gene
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262 Virus Genes (2021) 57:258–265
1 3
I group it showed 94.7–95.2% similarity. The similarity
between the two sequences assigned to the Swedish II group
was 96.4%.
A separate group was a homogeneous clade consisting of
Canadian isolates (99.4% similarity within the group), which
showed significant differences in relation to variants of the
virus isolated in Europe. Compared to representatives of the
Danish group, the differences ranged from 7.1 to 8.3%. A
similar level of similarity was found in relation to most of
the Swedish isolates (average differences of 7–8%), except
for isolate AY196104.1, which showed over 94% similar-
ity to one of the Canadian isolates. Expanded phylogenetic
analysis, including astroviruses infecting representatives of
other species, confirmed that the mink astrovirus is the most
closely related to astroviruses infecting humans (Fig.2).
Discussion
Mink astrovirus infection remains a poorly characterized
disease entity. In 2002, Englund etal. investigated the rela-
tionship between the presence of astrovirus in the mink
intestines and faeces and the occurrence of pre-weaning
diarrhea in mink. The researchers confirmed the presence of
the pathogen in both types of material and indicated a pos-
sible causal relationship between the astrovirus and the onset
of disease [13]. The results obtained by Englund etal. were
based on histopathological examination and observations of
viral particles under an electron microscope. Therefore, it is
difficult to state conclusively whether the pathogens present
in the set of samples examined by that team included MAstV
or another member of the Astroviridae family.
In our own research, the presence of MAstV genetic
material was confirmed in both the brain and the intestines.
However, in contrast to Englund’s study [13], we observed a
different set of symptoms, much more similar to the shaking
mink syndrome described by Blomström etal. 2010 [12].
An increasing body of research indicates that astroviruses
may be responsible for the development of disease entities
with a diverse clinical picture. Most reports confirm gastro-
intestinal symptoms resulting from replication of the virus
in the intestines [17, 18], or in the case of avian astroviruses
in the liver as well [19, 20]. An increasing number of stud-
ies confirm the link between astroviruses and neurological
symptoms detected in pigs [21], cattle [22], or mink [12]. In
our research as well, the symptoms pointed to the neurologi-
cal form of the disease, and the presence of MAstV genetic
material was also confirmed in the brain of the animals.
Table 2 Comparison of the
RdRp (RNA-dependent RNA
polymerase) gene sequence
between variants available in
the NCBI database (similarity
was expressed as %)
Sweden Denmar
kC
anada
Country
Accession
number
Poland
AY196101.1
AY196102.1
AY196103.1
AY196104.1
AY196105.1
NC_004579.1
GU985458.1
AY196095.1
AY196096.1
AY196097.1
AY196098.1
AY196099.1
AY196100.1
MH282878.1
MH282880.1
Sweden
AY196101.1 95.8ID 98.2100 94.7100 98.2 93.5 95.8 96.4 97.0 97.0 97.0 96.4 92.3 92.3
AY196102.196.498.2 ID 98.295.298.2 100 92.9 95.2 95.8 96.4 96.4 96.4 95.8 92.9 92.9
AY196103.195.810098.2 ID 94.7100 98.2 93.5 95.8 96.4 97.0 97.0 97.0 96.4 92.3 92.3
AY196104.1 95.294.7 95.294.7 ID 94.7 95.2 96.4 92.9 93.5 94.1 94.1 94.1 93.5 93.5 94.1
AY196105.1 95.810098.2100 94.7 ID 98.2 93.5 95.8 96.4 97.0 97.0 97.0 96.4 92.3 92.3
NC_004579.196.498.2 100 98.295.298.2 ID 92.9 95.2 95.8 96.4 96.4 96.4 95.8 92.9 92.9
GU985458.194.193.5 92.993.596.493.5 92.9 ID 94.1 94.1 94.1 94.1 94.1 93.5 91.1 91.7
Denmark
AY196095.1 96.495.8 95.295.892.995.8 95.2 94.1 ID 98.2 98.8 98.8 98.8 98.2 91.7 91.7
AY196096.197.096.4 95.896.493.596.4 95.8 94.1 98.2 ID 99.4 99.4 99.4 98.8 92.3 92.3
AY196097.197.697.0 96.497.094.197.0 96.4 94.1 98.8 99.4 ID 100100 99.4 92.9 92.9
AY196098.197.697.0 96.497.094.197.0 96.4 94.1 98.8 99.4 100 ID 100 99.4 92.9 92.9
AY196099.1 97.697.0 96.497.094.197.0 96.4 94.1 98.8 99.4 100100 ID 99.4 92.9 92.9
AY196100.197.096.4 95.896.493.596.4 95.8 93.5 98.2 98.8 99.4 99.4 99.4 ID 92.3 92.3
Canada
MH282878.1 92.992.3 92.992.393.592.392.991.191.792.392.992.992.992.3 ID 99.4
MH282880.1 92.992.3 92.992.394.192.392.991.091.792.392.992.992.992.399.4 ID
Green colour—similarity within Swedish isolates, Blue colour—similarity within Danish isolates, Yellow
colour—similarity within Canadian isolates
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263Virus Genes (2021) 57:258–265
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Fig. 2 Analysis of phylogenetic relationships between MAstV and
astroviruses infecting humans, bats, pigs, rodents, dolphins, cattle,
dogs, cats, and sea lions. The analysis was carried out using the NJ
method, with a bootstrap value of 1000. Analysis based on 170bp
fragment coding RdRp (RNA-dependent RNA polymerase) gene
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264 Virus Genes (2021) 57:258–265
1 3
The results confirm the diagnostic effectiveness of the
primers proposed by Mittelholzer etal. [7]. In addition,
the presence of genetic material of the astrovirus was con-
firmed for the first time in continental Europe. Analysis of
the sequenced fragment indicates the presence of the same
variant of the virus in all samples tested, obtained from two
separate farms. Similarly, Mittelholzer etal. [7] reported
high similarity in a study conducted on Danish and Swedish
farms, which showed 96.7–100% similarity of the isolates.
The researchers observed high similarity of the virus isolates
within each of the countries studied, but at the same time
variation between them. Analysis of polymorphisms by Mit-
telholzer enabled clear differentiation between Danish and
Swedish strains [7].
The high similarity observed by the Mittelholzer team
and in our research may be due to both the relatively short
length of the analysed sequence and the conserved nature of
the fragment coding RdRp, which can be used for diagnos-
tic purposes. However, molecular characterization remains
a very important element in the study of diseases caused
by astroviruses, including understanding of their underly-
ing cause and the mechanisms of their onset and develop-
ment. The usefulness of this type of analysis is confirmed by
research conducted on mink with diarrhea symptoms from
Chinese farms. Sun etal. confirmed the presence of genetic
material of astroviruses, but interestingly, none of them was
a representative of MAstV, and additional bioinformatic
analysis indicated the possibility of mink infection with
astrovirus from birds [23]. The researchers formulated an
interesting hypothesis regarding transmission of the patho-
gen via feed obtained from infected birds and the possibil-
ity of interspecies infection. The possibility of interspecies
transmission is also indicated by Quan [9], who detected
encephalitis in a boy with X-linked agammaglobulinaemia.
The authors suggest that one of the potential causes was
infection from mink from a nearby farm, but due to the com-
plicated history of the disease, as well as immunosuppres-
sion in the patient, conclusive determination of the source
of infection is not possible.
The phylogenetic analysis carried out in our study points
to a relationship between the genetic variant of the virus and
the country where the samples were isolated, which also
confirms the observations of Mitlleholzer [7] for Swedish
and Danish isolates. The differences detected may indicate
multiple and independent MAstV infections originating in
different primary outbreaks.
In addition to the study by Mitlleholzer etal. [7] men-
tioned above, MAstV phylogenetics has been studied by
Blomström [12], who obtained a tree similar to the one in
the present study. Isolate GU985458.1 presented by the
researchers formed a separate clade in relation to most
of the isolates associated with the occurrence of pre-
weaning diarrhea, which could indicate the presence of
polymorphisms influencing the tropism of the pathogen
for nerve tissue. However, the results of the present study
group the Polish isolate obtained from individuals with
the neurological form of the disease together with iso-
lates associated with pre-weaning diarrhea. Therefore,
the ORF1b region containing the RdRp gene seems not
to affect tropism or the clinical picture of the infection,
especially as the polymorphisms between GU985458.1
and most sequences are synonymous.
Bearing in mind the potential for cross-species infec-
tions caused by astroviruses, as well as their widespread
dissemination, it seems reasonable to analyse the RdRp
fragment to confirm infection and as a fragment ena-
bling preliminary assessment of the diversity of isolates
obtained in relation to the global pool of the virus.
Conclusions
Molecular diagnostics and epidemiology are increasingly
used as a tool to understand the spread and evolution of
infectious agents. The subject of mink astrovirus infec-
tion remains poorly understood, as confirmed by both the
small number of sequences deposited in bioinformatics
databases and the small number of studies in the PubMed
database.
The study confirmed the presence of MAstV astrovi-
rus genetic material in the mink brain and intestines with
a clinical picture indicative of shaking mink syndrome.
To our knowledge, this work describes the first results of
molecular epidemiology of MAstV in continental Europe.
Previous analyses have concerned Scandinavia (Sweden and
Denmark), Canada, and China. The detection of MAstV in
Central Europe indicates the need for further research that
will not only enable a better understanding of the aetiology
of such disease entities as pre-weaning diarrhea and shak-
ing mink syndrome, but also broaden current knowledge of
MAstV molecular epidemiology in Europe.
Author contributions AJ and MK conceived and planned the experi-
ments, methodology, AJ, MK—laboratory and bioinformatic analy-
sis, AJ, MK and IM—preparing of manuscript, AJ, MK, IM and M
Kon. All authors have read and agreed to the published version of the
manuscript.
Declarations
Conflict of interest The authors declare that there is no conflict of in-
terest.
Ethical approval Approval for these tests was obtained from the 2nd
Local Ethics Committee for Experiments on Animals at the University
of Life Sciences in Lublin (approval no. 83/2009).
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