Download Genetic sequencing and analysis of the infectious pancreatic

BSc Thesis
Genetic sequencing and analysis of the
infectious pancreatic necrosis virus VP2
coding region from Faroese isolates
Áki Vang & Unn Vagnsdóttir Johannesen
NVDRit
2013:08
Heiti / Title
Genetic sequencing and analysis of the
infectious pancreatic necrosis virus VP2 coding
region from Faroese isolates
Arvafrøðilg raðing og greining av smittandi
briskertilsvevnaðardeyða virusinum VP2
koduøkinum frá føroyskum reinalum
Høvundar / Authors
Vegleiðari / Supervisor
Ábyrgdarvegleiðari / Responsible Supervisor
Ritslag / Report Type
Latið inn / Submitted
NVDRit
©
ISSN
Útgevari / Publisher
Bústaður / Address
    @
Áki Vang & Unn Vagnsdóttir Johannesen
Debes H. Christiansen, Heilsufrøðiliga Starvsstovan
Svein-Ole Mikalsen, Fróðskaparsetur Føroya
BSc Thesis, Biology
BSc ritgerð, lívfrøði
21. juni 2013
2013:08
Náttúruvísindadeildin og høvundarnir 2013
1601-9741
Náttúruvísindadeildin, Fróðskaparsetur Føroya
Nóatún 3, FO 100 Tórshavn, Føroyar (Faroe Islands)
+298 352 550

+298 352 551

[email protected]
Vang and Johannesen, 2013
Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from
Faroese isolates
Table of Contents
Samandráttur.................................................................................................................. 2
Abstract.......................................................................................................................... 2
1 Introduction................................................................................................................. 3
1.1 Infectious pancreatic necrosis virus.....................................................................3
1.1.1 Structure and genetics of virus.....................................................................3
1.1.2 Pathogenesis and pathology.........................................................................6
1.2 History of IPN outbreaks.....................................................................................6
1.3 Phylogeny of birnaviruses................................................................................... 7
2 Aims of this study......................................................................................................10
3 Materials and methods...............................................................................................11
3.1 Sample collection...............................................................................................11
3.2 Isolation of viral RNA....................................................................................... 11
3.3 One step RT-PCR...............................................................................................11
3.4 DNA sequencing................................................................................................13
3.5 Sequence analysis.............................................................................................. 13
3.6 Phylogenetic analysis........................................................................................ 14
4 Results....................................................................................................................... 15
4.1 Generated cDNA sequences.............................................................................. 15
4.2 BLAST analysis.................................................................................................16
4.3 Deduced amino acid sequences......................................................................... 17
4.4 Phylogenetic relationships.................................................................................20
4.4.1 Evolutionary trees...................................................................................... 20
4.4.2 Distance matrix.......................................................................................... 21
5 Discussion................................................................................................................. 22
5.1 Phylogeny for Faroese isolates.......................................................................... 22
5.2 Phylogenic comparison with foreign isolates.................................................... 22
5.3 Amino acid sequence, protein structure and virulence motifs...........................23
6 Conclusion.................................................................................................................29
7 Cited literature...........................................................................................................30
8 Acknowledgments .................................................................................................... 35
9 Appendix................................................................................................................... 36
9.1 A1.......................................................................................................................36
9.2 A2.......................................................................................................................41
9.3 A3.......................................................................................................................44
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Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
Samandráttur
Smittandi briskertilsvevnaðardeyði (en: IPN) er sjúka, sum ávirkar ymsar fiskar og marin ryggleys
djór. Sjúkan kemst av smittandi briskertilsvevnaðardeyða virusinum (en: IPNV). IPN verður hildin
at hava ein tann stórsta týdning fíggjarliga fyri alivinnuna, og sjúkan er sera skaðilig fyri laks. IPNV
er limur í familjuni Birnaviridae og slektini Aquabirnavirus. Virusið inniheldur tveir streingir av
RNAase-mótstøðuførum RNA, nevndir segment A og B. Segment B kodar fyri einum proteini
(virus protein 1, VP1), meðan Segmant A kodar fyri fýra proteinum (VP2-VP5).
Í hesari kanning hava vit raða stóran part av VP2 kodandi økinum hjá fleiri føroyskum
reinalum. Raðini vórðu samanborin við útlendsk reinali, við raðum frá GenBank. Hetta vísti, at
Føroysk og útlendsk reinali eru 98-99% samlík. Útgreining av amino sýru raðnum meðan hugt var
eftir kendum sjúkukveikingarmeinskmotivum, bendir á at føroysk reinali eru sum heild ikki
sjúkukveikingarmeinsk, sum kann greiða frá, hví vit einans kenna til fá útbrot av IPN í føroyskum
sjógvi. Samanbering av føroyskum reinalum, við fjartskyld reinali vísur, at tey føroysku og
tættskyldu útlendsku reinalini kunnu bólkast í ein genobólk.
Abstract
Infectious pancreatic necrosis disease (IPN) affects various fish and marine invertebrates. It is
caused by the pancreatic necrosis virus (IPNV). IPN is considered one of the most economically
important diseases affecting the aquafarming industry, as it is detrimental to salmoid fishes. IPNV
is a member of the family Birnaviridae, and the genus Aquabirnavirus. IPNV's genetic material
consist of two strands of RNase resistant RNA, called segments A and B. Segment B encodes for a
single protein (virus protein 1, VP1), while segment A encodes for four proteins (VP2 to VP5).
In this study, we have sequenced a major part of the VP2 coding region on RNA segment A,
from Faroese isolates. The sequences have been compared with related foreign isolates with
sequences available from GenBank. The Faroese isolates and most related foreign isolates are 9899% identical. Studying the deduced amino acid sequences and looking at known virulence motifs
suggest, that Faroese isolates are largely avirulent, which explains the lack of marine outbreaks in
the Faroe Islands. Using previously established genogroups, we have placed the Faroese and most
related foreign isolates in one single genogroup.
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Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
1 Introduction
1.1 Infectious pancreatic necrosis virus
Infectious pancreatic necrosis is a disease that affects several species of fish, and is caused by the
virus named after it. Since the discovery of the disease, it has come to be known as one of the most
commercially important diseases affecting the aquafarming industry, and has been detected in many
countries around the world.
1.1.1 Structure and genetics of virus
The infectious pancreatic necrosis virus (IPNV) is a double-stranded RNA virus, containing two
RNA molecules that are RNAase-resistant (Dobos 1976; Macdonald and Yamamoto 1977). It
belongs to the virus family Birnaviridae (Brown, 1986) and is the type species of genus
Aquabirnavirus (Fauquet et al. 2005). As is the case with all aquabirnaviruses, the virion is
comprised of an icosahedron that is not enveloped by a membrane. The virion is circa 60 nm in
diameter (Leong et al. 2000). The structure of the virion is illustrated in Fig 1.1. and the molecular
weight and functions of the virus proteins are shown in Table 1.1.
Segment A contains a large open reading frame (ORF) which codes for the 106 kDa
polyprotein NH2-preVP2-NS protease-VP3-COOH. This protein is cleaved by a virus-encoded
serine-lysine protease, VP4 (Duncan and Dobos, 1986), to produce virus protein 3 (VP3) and the
precursor to VP2. (There seems to be some discussion on the naming of the VP4. Sometimes it is
called NS protease, and sometimes it is simply called VP4, so the polyprotein could also be named
NH2-preVP2-VP4-VP3-COOH) (Wu et al, 1998). During the maturation of the virus, this precursor
is again cleaved to produce VP2. It is this virus protein that we will be examining. VP3 is an
internal capsid protein (Dobos, 1995).
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Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
Table 1.1. The molecular weight (Mw) and the functions of the five IPNV virus proteins. (Skjesol, 2009)
Fig. 1.1: (Left) Three dimensional structure of the IPNV virion. (Right) Schematic drawing showing the
positions of the three virus proteins and the two RNA segments within the virus particle (Anonymous, 2005).
Genome segment A does also contain a different ORF, which encodes a small 17 kDa
polypeptide, VP5, that overlaps the 5'-end of the larger reading frame, corresponding to the amino
teminal of polyprotein (Magyar and Dobos, 1994). This gene has been shown to have an antiapoptotic function (Hong et al., 2002). Genomic segment A's nucleotide sequence has been
determined for five separate IPNV strains: The Jasper strain (Duncan and Dobos, 1986), the N1
strain (Havarstein et al. 1990), the DRT strain (Chung et al., 1994, GenBank accession no.
D265527), the West Buxton strain (Yao and Vakharia, 1998) and the Sp strain (Mason, 1992).
VP1 is the product of genomic segment B, and this is an internal protein, which acts as a
RNA-dependent RNA polymerase. VP1 is present in the virus particle both as an internal protein
and as the genome linked protein VPg (Calvert, et al., 1991). It has also been shown that both
genomic segments contain non-coding regions, which may be important for polymerase recognition
and genome packing (Duncan et al., 1991). A schematic illustration of both genomic segments of
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Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
the Jasper strain of IPNV is shown in Fig. 1.2. This structure of the genome map also describes the
infectious bursal disease virus (IBDV) (Kilbenge, et al. 1988), which could mean that such structure
is characteristic of Birnaviruses.
In conclusion, the unique characteristics of Birnaviruses are: Genome segment A contains
two gene sequences, the polyprotein is cleaved by a virus coded protease and VPg is attached to the
5'-end of the polyprotein coding region (Dobos, 1995). For a review of the genetic structure of the
IPNV see Dobos (1995).
Fig 1.2. A schematic illustration of IPNV genome segments A and B, of the Jasper strain. (A) The two top bars show
the ORF's of segment A: The 17 kDa ORF and the ORF which contains the pVP2, NS and VP3. The borders between
pVP2, NS and VP3 show the cleavage sites for the NS-proteases. The numbers above the figure show the location of inline methionine residues, denoted “met” on the right. The denotions “aa” and “nt” mark the locations of important
amino acids and key nucleotides respectively. VPg shows the genome linked protein that is attached to the 5' end of the
RNA strand. Black arrowheads show potential N-glycosylation sites. Y/L shows the dipeptide Tyr-Leu located at
positions 491 and 725, corresponding to the cleavage sites. (B) Genome segment B. The bar shows the VP1 ORF, and
VPg is the genome linked form of VP1. Important amino acids and key nucleotides are shown as above. The black bars
show the GTP-binding region, three polymerase motifs (I, II and III), secondary structures (beta sheet and alpha helix)
and the carboxy terminal region (+) (Dobos, 1995).
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Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
1.1.2 Pathogenesis and pathology
Studies have shown that the infection of fish cells by IPNV is initiated when virus particles are
internalized by means of endocytosis (Couve, et al. 1992). Most enveloped viruses need an acidic
environment in the endosome to be able to enter the cytosol, however this is not the case for IPNV
(Espinoza and Kuznar, 1997).
The virus replication can occur in many types of cells at temperatures below 24 oC (Wolf and
Mann, 1980), and takes place in the cytosol. A study showed that after 16-20 hours of viral
replication, cells of brook trout (Salvelinus fontinalis) showed classical symptoms of a cytopathic
effect (CPE) (Malsberger and Cerini, 1963).
The clinical features of infectious pancreatic necrosis in salmon are similar to those which
McKnight and Roberts observed in 1976 in rainbow trout, these being that fry are found in the
surface water film or at outflows at the farms. These fry, which would also have acquired a darker
color, would be making shimmering movements, lying on the side hyperventilating or whirling.
Young fry are severely affected by necrosis of the pancreatic acinar cells, with minor lipid necrosis.
The liver is also generally shows severe necrosis (Roberts and Pearson, 2005).
Fish that have completed the first feeding cycle, and have a robust growth may not be as
gravely affected, but losses can amount to 70%, although losses in older fingerlings usually are
about 10-20%. Symptoms of the diseased fingerlings are the same as with the fry, but may also
include hanging head up, or lying in the water. The transfer of young salmoid fishes to salt water
can be difficult, and make the salmon susceptible to stress and infection, but fish that have survived
an IPN infection will usually not be affected by the transfer (Roberts and Pearson, 2005).
1.2 History of IPN outbreaks
The IPN virus was the first virus to be isolated from fish (Olsen, 2001). IPN was detected as a
severe contagious disease that effected salmon fry in the fresh water stage. The mortality could be
up to 100% (Roberts and Pearson, 2005). The IPN disease was first described in United States in
1941 under the name acute catarrhal enteritis (McGonigle, 1941) because of its histopathological
findings and only changed name to infectious pancreatic necrosis in mid 1950’s (Wood et al 1955).
The virus is highly contagious and was thought only to affect salmonid species. The etiology later
showed that the IPN virus affects fresh water and marine salmonid species as well as some non6
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Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
salmonid fish species, molluscs, and crustacean (Hill 1982; Hill and Way 1995).
The first IPN virus isolates in Europe were described in France in 1965, in Denmark 1968
(Vestergård-Jørgensen and Bregnballe, 1969), in Sweden 1969, in Scotland 1971, in Norway 1975
(Håstein og Krogsrud, 1976). There was a rapid increase of IPN outbreaks in Norway from 1982
(Krogsrud et al. 1989). P/F Fiskaaling stopped importing roe from Norway in 1984 because of the
rapid increase of IPN outbreaks (ARSF).
The first Faroese IPNV isolate was detected in the spring of 1986, and in 1987 the IPN virus
was found in some smolt farms and most of the aquafarms (Strøm 1987). The IPN is isolated in
many other European countries. IPNV was also isolated in Japan in 1971 and Chile in 1981
(McAllister and Reyes, 1984). IPN has become one of the biggest economic burdens in the
aquaculture (Hill 1982; Hill and Way 1995).
In 2001 alone IPN cost the Faroe Islands fish farming industry around 100 million DDK.
There are no exact numbers on the total cost of the industry but it is significant (Joensen, 2002). In
Norway IPN has been the source to a massive economic loss. In 1997 this was considered to be
around 60 million US dollars per year, in the preceding years (Christie, 1997). The IPN outbreaks
in Norway have three-folded from 2001 to 2002, and the disease is considered to be the most
economically important disease facing the Norwegian fish farming industry (Sandvik, 2002).
1.3 Phylogeny of birnaviruses
As stated earlier, the IPNV is a member of the Birniviridae, and we have also mentioned some
general characteristics of this virus family above. IPNV is a member of the genus Aquabirnavirus,
and this group is the largest and most diverse in the virus family, and includes viruses from both
fish and invertebrates (Wolf, 1988).
Research in the relationships of Birnaviruses have shown that a majority of the virus species
are related antigenically, regardless of geographic location or host species. These related viruses
form the Serogroup A, which is the predominant serogroup worldwide (Caswell-Reno, 1989).
Serogroup A contains nine cross-reacting serotypes: A2 (type strain West Buxton), A2 (type strain
Sp), A3 (type strain Ab), A4 (type strain He), A5 (type strain Te), A6 (type strain Canada 1), A7
(type strain Canada 2), A8 (type strain Canada 3) and A9 (Type strain Jasper-ATCC VR 1325)
(Blake et al., 2001).
It had been shown, by comparing deduced amino acid sequences from a 310 bp cDNA
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Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
fragment at the junction of the C-terminus of the pVP2 and NS coding regions of 17 isolates, that
Serogroup A consists of only three major genogroups (Heppell et al., 1993). A study by Blake et al.
(2001) showed the relationships of birnaviruses based on the VP2 coding region of genome segment
A. The results of this study demonstrate that a large number of viruses from around the world can
be grouped into six genogroups. These genogroups also correspond to geographic origin. This
study, which looked at the whole of the VP2 ORF, thus refined the results obtained by Heppell et al.
These six genogroups are shown in Fig. 1.3.
Serogroup B, on the other hand, is only represented by few antigenically unrelated aquatic
birnaviruses, which comprise only of one serotype (Hill and Way, 1995).
Fig. 1.3: Cladogram representing the phylogenic relationships of auqatic birnaviruses, based on amino acid sequences
of VP2. The branch length represents the distance between sequence pairs, and the numbers show the bootstrap values
(Blake et al., 2001).
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Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
Genus
Virus species
Host
Aquabirnavirus
IPNV, Tellina virus 2 (TV-2),
European eel virus (EEV),
Oyster virus (OV), Yellowtail
ascites virus (YTAV)
Fish, crustaceans and molluscs
Table 1.2: The virus species within the genus Aquabirnavirus and their host species (Fauquet, 2005).
Fig. 1.3 illustrates the relationship between IPNV and some of the other viruses in genus
Aquabirnavirus, which are listed in Table 1.2. Note that viruses belonging to IPNV are found mixed
with other Aquabirnaviruses.
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Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
2 Aims of this study
The aim of this study is to determine the genetic sequences of viruses obtained from samples from
smolt farms from the Faroe Islands. We will also compare these sequences to each other, and
foreign sequences found in GenBank. This should give an indication to what genogroup and
serotype is present in Faroese smolt farms.
Comparison of Faroese IPNV strains with foreign strains will also show if these strains can
be considered identical, or if there is a novel strain found, and it could also be of interest in inferring
where an infection is likely to have come from. As it is possible to deduce the amino acid sequence
of the VP2 protein from the DNA sequence, we will also be examining the amino acid sequences,
which will show us if any of the Faroese isolates have any known virulence motifs.
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Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
3 Materials and methods
3.1 Sample collection
14 virus samples were collected from seven smolt farms from around the Faroe Islands. The
samples were taken from smolt kidneys, heart and pancreas. The farms were given designations,
FAR01-FAR07, to protect their anonymity.
3.2 Isolation of viral RNA
Isolation of viral RNA from tissue samples was carried out using the Qiagen RNeasy kit. The tissue
samples were first cut to sizes of about 10-30 mg. These were placed in a microtube with 500µl
Buffer RLT, which was placed in a steel sphere an cooled. When frozen, the sample was thawed and
homogenized in a Retsch Mixer Mill MM 30 at 30 Hz for 4 min. The sample is turned and
homogenized for 4 min. once more. The homogenate is centrifuged 16100 x G for 5 min. 350 µl
70% ethanol and 350 µl of the homogenized solution is added to a new tube, and mixed well by
pipetting. 700 µl of the lysate is added to a Rneasy mini column and centrifuged at 9400 x G for 15
sec. The flow through is discarded. 700 µl of RW1 is added to the column and centrifuged at 9400 x
G for 15 sec. The flow through is discarded once more. 500 µl RPE is added, and centrifuged at
9400 x G for 15 sec., and the flow through is discarded. 500 µl RPE is added once more, and
cenrtifuged at the same strength, but for 2 min. Afterwards the flow through and tube is discarded,
and the column is placed in a new tube. This tube is centrifuged at 9400 x G for 1 min. and the tube
is discarded. Now, the column is placed in a 1.5 ml tube and 100 µl RNase free distilled water is
added, and incubated for 1 min. The tube is then centrifuged at 9400 x G for 1 min.
3.3 One step RT-PCR
The viral RNA sampled from the smolt farms was treated with reverse-transcription PCR using the
Qiagen One Step RT-PCR kit, which produces cDNA. The primers used for amplification were
IPNvp2-2F (5'-GAGTCACAGTCCTGAATC-3') and IPNvp2-2R (5'TCGGACAAGAACTCCCGAG-'3), as described by Santi et al. 2004. These primers should give a
product of 1100 bp.
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Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
Fig 3.1: Schematic showing the placement of the primers on the target sequence. The forward primer is shown
in green, and the reverse primer is shown in blue.
Solutions for the PCR reaction were as follows, in each tube: RNA sample (2.00 µl), RTPCR Buffer (4.00 µl), dNTP Mix (0.80 µl), 10 µM stock solution forward primer (1.20 µl), 10 µM
reverse primer (1.20 µl), One Step Enzyme Mix (0.80 µl) and distilled water (10.00 µl). This
mixture will give a total volume of 20 µl per tube. The reverse-transcriptase polymerase chain
reaction was carried out in an AB 2720 Thermal Cycler at 50 oC for 30 mins.
The program then directly continued with a polymerase chain reaction carried out with a
schedule of 95oC for 30 seconds and then 40 cycles of 94oC for 30 sec., 60oC for 30 sec. and 72oC
for 90 sec. Electrophoresis of the samples was performed using DNA chips in an Agilent 2100
Bioanalyzer, to conclude if there was ample product of the correct length.
The RT-PCR products were purified using the JetQuick purification spin kit: 80 µl of H1
buffer were added to each PCR tube. This mixture was then transferred to a JetQuick spin column,
placed in a 2 ml microtube. All the columns were then centrifuged at 12,000 x G for 1 min. 500 µl
H2 buffer solution was then added to each spin column and centrifuged at 12,000 x G for 2 min. If
the spin column was not completely dry at this stage, the centrifugation was repeated.
The dry JetQuick spin column was now subsequently placed in a 1.5 ml microtube, and 50
µl of 70oC distilled water was placed on the filter in the column. Centrifugation at 12,000 x G for 2
min. was then completeted. The purified PCR product could now be found in the 1.5 ml microtube.
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Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
3.4 DNA sequencing
Sequencing of the PCR product was performed with the BigDye Terminator v 1.1 Cycle Sequencing
kit from Applied Biosystems. The reaction solution for the cycle sequencing was as follows:
BigDye Terminator v. 1.1 Ready Reaction Mix (1.00 µl), BigDyeTerm.v1.1/3.1 Seq.Buffer x5 (2.00
µl), 3.30 µM primer (0.48 µl), distilled water (5.52 µl) and a template (1.00 µl). The reaction
volume totaled 10 µl, and was mixed twice for each template, once for the forward primer and once
for the reverse primer.
The cycle sequencing was carried out in the AB 2720 Thermal Cycler with a program
starting at 94oC for 2 min. followed by a 25 cycles of 96oC for 30 sec., 50oC for 5 sec. and 60oC for
4 min. The program ended with 20oC set indefinitely.
The sequence products were purified by adding 1 µl of sodium acetate and 25 µl 98%
ethanol to the 10 µl PCR solution. This mixture rested for 15 min. and was then centrifuged at
20,000 x G for 15 min. All the fluid was then discarded, and 80 µl of 70% ethanol was added to the
empty tubes. The ethanol was then discarded, and the tubes were dried at 90 oC for no more than 30
seconds. 10 µl Hi-Di formamide was then added to each tube. The sequence products were then
denatured at 90oC for two minutes, after which they were cooled in plastic freeze blocks for two
minutes. Now, the sequence products were ready to be analyzed in the 3100 Sequencer from
Applied Biosystems Inc. DNA sequencing worked for all viral samples except FAR02.
3.5 Sequence analysis
The generated sequences were edited using the Sequencing Analysis Software from Applied
Biosystems Inc. The subsequent alignment of the sequences was carried out, using ClustalW2 from
EBI (Larkin et al., 2007; Goujon, et al., 2010).
Comparison of the Faroese strains with foreign strains was carried out using the Basic Local
Alignment Search Tool (BLAST) from NCBI (Zhang et al., 2000; Morgulis et al., 2008). Using
BLAST, it is possible to enter a query DNA or amino acid sequence into the search engine, and then
see which sequences available in the database are most similar to the query sequence. The three first
isolates from the BLAST search for each strain were picked to be used for later comparison, as can
be seen in Table 4.1.
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Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
3.6 Phylogenetic analysis
To be able to infer genetic grouping and phylogenetic relationships of the isolated viruses, we
computed several analyses using the MEGA5 software (Tamura et al., 2011). For analyzing the
relationships among the Faroese isolates, and between the Faroese and foreign isolates, we
constructed phylogenetic trees, using the Maximum-Likelihood method based on the Tamura-Nei
model (Tamura and Nei, 1993). We also calculated the pairwise distances among the Faroese
isolates, which show the number of base substitutions per site. This analysis was performed using
the Maximum-Likelihood model (Tamura et al., 2004). These distance matrices can be seen in the
appendix, A3.
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Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
4 Results
4.1 Generated cDNA sequences
The smolt farms have all given a designation, FAR01-07, and the sequences from each location
will be known as one of these numbers. All obtained sequences can be seen in the appendix A1. All
of the sequences represent the VP2 coding region of the genome segment A, which encodes for the
outer capsid proteins.
The sequences were aligned using ClustalW, and can be seen in the appendix, A2. The
alignment shows, that the nucleotide sequences differed at 17 nucleotide sites. From the editing and
alignment we ended up with seven sequences that were 982 bp long, and one that was 976 bp long.
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Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
4.2 BLAST analysis
We used the BLAST software to find out which previously published virus sequences available in
GenBank were most similar to those we isolated from Faroese samples. No parameters were
changed when using BLAST, and we performed the search using the database designated 'other'.
It should be noted, that the search using BLAST resulted in several other 97%-99% matches,
which were not included in this study.
Closest hits in GenBank
Faroese
isolate
GenBank
acc. no.
Isolate
Host
Country
Authors
Max identity
FAR01
HQ457190.1
N-136
Atlantic
salmon
Norway
Mutoloki
and Evensen
(2011)
98%
FAR01
AY379742.1
NVI-016
Bluegill
Norway
Santi et al.
(2004)
98%
FAR01
AJ489229.1
88R
Oyster
Spain
Cutrín et al.
(2004)
98%
FAR03
AJ489229.1
88R
Oyster
Spain
Cutrín et al.
(2004)
99%
FAR03
HQ457169.1
C-1.3
Atlantic
salmon
Chile
Mutoloki
and Evensen
(2011)
99%
FAR03
HQ457171.1
C-1
Atlantic
salmon
Chile
Mutoloki
and Evensen
(2011)
99%
FAR04
HQ457193.1
NVH-108
Atlantic
salmon
Norway
Mutoloki
and Evensen
(2011)
99%
FAR04
HQ457186.1
NVH-105
Atlantic
salmon
Norway
Mutoloki
and Evensen
(2011)
99%
FAR04
HQ457169.1
C-1.3
Atlantic
salmon
Chile
Mutoloki
and Evensen
(2011)
99%
FAR05
HQ457190.1
N-136
Atlantic
salmon
Norway
Mutoloki
and Evensen
(2011)
99%
FAR05
AY379742.1
NVI-016
Bluegill
Norway?
Santi et al.
99%
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Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
(2004)
FAR05
AJ489229.1
88R
Oyster
Spain
Cutrín et al.
(2004)
99%
FAR06-1
AJ489229.1
88R
Oyster
Spain
Cutrín et al.
(2004)
99%
FAR06-1
HQ457169.1
C-1.3
Atlantic
salmon
Chile
Mutoloki
and Evensen
(2011)
99%
FAR06-1
HQ457171.1
C-1
Atlantic
salmon
Chile
Mutoloki
and Evensen
(2011)
99%
FAR06-2
AJ489229.1
88R
Oyster
Spain
Cutrín et al.
(2004)
99%
FAR06-2
HQ457169.1
C-1.3
Atlantic
salmon
Chile
Mutoloki
and Evensen
(2011)
99%
FAR06-2
HQ457171.1
C-1
Atlantic
salmon
Chile
Mutoloki
and Evensen
(2011)
99%
FAR07-1
AJ489229.1
88R
Oyster
Spain
Cutrín et al.
(2004)
99%
FAR07-1
HQ457169.1
C-1.3
Atlantic
salmon
Chile
Mutoloki
and Evensen
(2011)
99%
FAR07-1
HQ457171.1
C-1
Atlantic
salmon
Chile
Mutoloki
and Evensen
(2011)
99%
FAR07-2
HQ457190.1
N-136
Atlantic
salmon
Norway
Mutoloki
and Evensen
(2011)
98%
FAR07-2
AY379742.1
NVI-016
Bluegill
Norway
Santi et al.
(2004)
98%
FAR07-2
AJ489229.1
88R
Oyster
Spain
Cutrín et al.
(2004)
98%
Table 4.1: Three foreign isolates are shown for each Faroese isolate, with GenBank accession number, isolate name,
host organism, country of origin, authors responsible for isolation the virus and the max identity score of the isolate.
The foreign isolates chosen, were those with the highest maximum identity.
4.3 Deduced amino acid sequences
The amino acid sequence was deduced using the MEGA5 software. Notice that to achieve the
17
Vang and Johannesen, 2013
Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
proper reading frame, the first guanine residue was deleted from the sequence for FAR01, FAR04,
FAR05, FAR06-1, FAR06-2, FAR07-1 and FAR07-2. Variations in amino acids among the different
isolates are shown in Table 4.2. One important thing to note, is that we have arbitrarily related all
isolates, local and foreign, to the FAR01. This should not be understood as if FAR01 is the sequence
most related to the ancestral sequence.
When discussing the amino acid sequence changes, we will be grouping them into three
groups. At this point we will only be discussing the Faroese proteins:
•
Group 1 (G1): No change in amino acid property or size of side chain (or slight size
change). These changes are found in positions 187: FAR03, FAR05, FAR06-1, FAR06-2
and FAR07-1 (S"T), in pos. 245: FAR03, FAR05, FAR06-1, FAR06-2 and FAR07-1
(G"S), in pos. 248: Same proteins as above (E"D), in pos. 253: All except FAR01 (D"E),
in pos. 282: FAR03, FAR05, FAR06-1, FAR06-2 and FAR07-1 (N"S).
•
Group 2 (G2): No change in amino acid property but size of side-chain changes. On pos.
314: FAR04 (I"F) and in pos. 216: FAR06-1 (V"A)
•
Group 3 (G3): Change in amino acid property. These changes are found in pos. 217: FAR04
(P"T), in pos. 249: FAR03, FAR05, FAR06-1, FAR06-2 and FAR07-1 (R"Q), in pos. 252:
FAR04 (D"V) and in pos. 315: All except FAR01 (D"N).
18
Vang and Johannesen, 2013
Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
FAR S
01
187
FAR
03
I
199
V
216
P
217
T
221
T
G
245
A
247
S
FAR
04
E
248
R
249
D
Q
T
D
252
V
D
253
N
282
E
S
E
I
314
D
315
A
319
V
323
N
F
N
FAR
05
T
S
D
Q
E
S
N
FAR
06-1
T
S
D
Q
E
S
N
FAR
06-2
T
S
D
Q
E
S
N
FAR
07-1
T
S
D
Q
E
S
N
A
A
318
T
FAR
07-2
N136
S
Q
N
E
D
N
T
E
F
NVI016
S
Q
N
E
D
N
T
E
F
S
Q
N
E
N
T
88R
T
C-1
A
S
Q
V
E
N
T
C1.3
A
S
Q
V
E
N
T
NV
H108
T
NV
H105
T
A
S
T
Q
V
E
N
T
S
T
Q
V
E
N
T
Table 4.2: The positions of amino acid mutations are shown. The top label denotes an amino acid, and its location in the
FAR01 sequence. A variation in amino acids, in other isolates is shown by denoting that particular amino acid. All
positions are in relation to the whole of the VP2 protein. GenBank accession numbers for foreign isolates can be seen in
Table 4.1.
19
Vang and Johannesen, 2013
Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
4.4 Phylogenetic relationships
4.4.1 Evolutionary trees
In order to understand the phylogenetic relationship of the Faroese isolates, we constructed a
phylogenetic tree using the Maximum-Likelihood method based on the Tamura-Nei model. Fig 4.1
shows the phylogeny of the isolated Faroese strains.
Fig. 4.1: Phylogenetic tree showing the evolutionary history of the Faroese IPNV isolates, based on VP2 nucleoties.
The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model. The
bootstratp value after 1000 replications is shown next to the branches. The scale indicates 0.001 substitutions per
nucleotide position, with branch lengths measured in the number of substitutions per site. The analysis involved 8
nucleotide sequences. Positions containing gaps and missing data were eliminated, otherwise all nucleotide positions
were included. There were a total of 976 positions in the final dataset.
To understand the relationship between the Faroese isolates, and those isolates, which were
found to be closely related, using the BLAST analysis, we also constructed a phylogenetic tree
including those foreign isolates. This tree was calculated using the same method as above, and is
illustrated in Fig. 4.2.
20
Vang and Johannesen, 2013
Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
Fig. 4.2: Phylogenetic tree showing the evolutionary history of the Faroese and related foreign isolates, based on VP2
nucleotide sequences. The evolutionary history was inferred by using the Maximum Likelihood method based on the
Tamura-Nei model. The bootstrap value after 1000 replications. is shown next to the branches. The scale indicates 0.002
substitutions per nucleotide position. The analysis involved 15 nucleotide sequences. All positions containing gaps and
missing data were eliminated, otherwise all nucleotide positions were included. There were a total of 976 positions in
the final dataset.
4.4.2 Distance matrix
The degree to which viral isolates differ from one another, can be calculated to a percentage. To
visualize this, distance matrices can be very useful to show both how viruses differ and how they
are similar. We computed a distance matrix comprising of the Faroese isolates using the MEGA5
software, which uses the Maximum Composite Likelihood model. This matrix shows the percentage
of which the isolates are similar and dissimilar, and is shown in the Appendix, A3, Table 9.1.
A distance matrix incorporating the foreign strains was also calculated, to show the
divergence and identity between the Faroese isolates and the foreign isolates. This matrix is shown
in the Appendix, A3, Table 9.2.
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Vang and Johannesen, 2013
Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
5 Discussion
5.1 Phylogeny for Faroese isolates
Looking at the phylogenetic tree shown in Fig. 4.1, the Faroese isolates cluster into two separate
groups. The first group incorporating FAR03, FAR06-1, FAR06-2, FAR07-1 and FAR05. The
second group includes FAR04, FAR01 and FAR07-2.
The phylogenetic trees shown are calculated using the nucleotide sequence. We chose to use
the nucleotide sequence instead of the amino acid sequence, because the Faroese isolates are so
closely related that a change in nucleotide sequence may not constitute a change in the amino acid
sequence. Indeed FAR01 and FAR07-2 have exactly the same amino acid sequence, while the
nucleotide sequence differs. The first thing of note is that two isolates, sampled in the same location
FAR07 are grouped separately. This should not be surprising, as there may well be several strains of
IPNV at each location. There is also a possibility that some samples have included two separate
viral isolates, but this could be cause for further investigation. Looking at the tree, it is valid to
speculate, if FAR05 can be considered a progenitor the the rest of the isolates in the first group.
Even though the Faroese strains can be clearly separated into two groups, they are very similar. If
we inspect the distance matrix shown in Table 9.1 we see that all of the Faroese isolates have an
identity percentage above 99%. Mutoloki and Evensen (2011) considered IPNV isolates, with a
similarity of 97-99% to be identical. If we were to use the same criteria, all of the Faroese isolates
must be considered identical.
5.2 Phylogenic comparison with foreign isolates
Considering Table 4.1 we see, that all of the foreign isolates included in this study have a maximum
identity of 98-98%. If we again were to use the criteria from Mutoloki and Evensen (2011), we
would conclude that these foreign isolates were identical to the Faroese. When inspecting Fig. 4.2
we can see that none of the foreign isolates are grouped with the Faroese isolates. They are divided
into two groups. The foreign and Faroese isolates do have a high degree of similarity, as can be seen
in the distance matrices in the Appendix, A3, Table 9.1 and Table 9.2. We can therefore suggest that
both local and foreign isolates are descended from one virus.
If we look closer at the foreign branch of the tree, we see that the second split, does not have
a bootstrap value shown, which means that the bootstrap value is below 50. This means that the
22
Vang and Johannesen, 2013
Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
grouping at this point has low statistical reliability which suggests that 88R could have been
grouped at the same level as N-136 and NVI-016.
The third branching in the tree has a bootstrap value of 56, which means it is just above the
threshold, which suggests that 88R more closely related to the progenitor than C-1 and C-1.3.
Considering that the host organism for 88R is oysters (Cutrín et al., 2004), and the Chilean isolates
originate from salmon (Mutoloki and Evensen, 2011), this shows that IPNV has a great range of
host diversity.
Grouping of the Chilean isolates from NVH-105 and NVH-108 has the greatest confidence,
with a bootstrap value of 90. These results reinforce the conclusion in the original study (Mutoloki
and Evensen, 2011), which considered the Chilean and Norwegian isolates to be part of the same
genogroup. The study did not include 88R and NVI-016, which originates from bluegills (Santi et
al., 2004). We can however, with some certainty state that all of these foreign isolates belong to the
same genogroup, which would also include the Faroese isolates.
5.3 Amino acid sequence, protein structure and virulence motifs
Considering these variations in amino acid sequence, the variable positions can be placed on a
ribbon representation model of the VP2 protein, to better understand the influence these mutations
may have on protein function.
23
Vang and Johannesen, 2013
Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
Fig. 5.1: Ribbon representation of the IPNV VP2 molecule. The base domain is shown in green, the shell domain is
shown in blue and the protruding domain is shown in orange. The black arrows point to the locations where the amino
acid mutations are located, and with their positions indicated in the boxes. The overview is based on the sequences
mentioned in Table 4.2, and the numbers indicate the residue positions. Adapted from (Coulibaly, et al., 2010)
The Faroese sequences only include a part of the VP2 sequence so we can't compare the
sequence with the whole structure of the VP2 as shown on Fig.5.1. The amino acid sequence that is
aligned FAR01, which is shown in Fig. 5.2, is from Protein Data Bank and has the accession code
3IDE. By comparing Fig. 5.1 and Fig. 5.2, we can visualize the three-dimensional structure of the
VP2 protein. The protein sequence is part of the whole projection domain, most of the base domain,
a very small part of the shell domain and the whole AA’ domain.
24
Vang and Johannesen, 2013
Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
FAR01
VP2
-----------------------------------------------------------MNTNKATATYLKSIMLPETGPASIPDDITERHILKQETSSYNLEVSESGSGVLVCFPGAP 60
FAR01
VP2
-----------------------------------------------------------GSRIGAHYRWNANQTGLEFDQWLETSQDLKKAFNYGRLISRKYDIQSSTLPAGLYALNGT 120
FAR01
VP2
----------------------------------------------------PYVRLEDE 8
LNAATFEGSLSEVESLTYNSLMSLTTNPQDKVNNQLVTKGVTVLNLPTGFDKPYVRLEDE 180
********
FAR01
VP2
TPQGLQSMNGAKMRCTAAIAPRRYEIDLPSQRLPPVPATGTLTTLYEGNADIVNSTTVTG 68
TPQGLQSMNGAKMRCTAAIAPRRYEIDLPSQRLPPVPATGTLTTLYEGNADIVNSTTVTG 240
************************************************************
FAR01
VP2
DINFGLAERPADDTKFDFQLDFMGLDNDVPVVTVVSSVLATNDNYRGVSAKMTQSIPTEN 128
DINFSLAEQPADETKFDFQLDFMGLDNDVPVVTVVSSVLATNDNYRGVSAKMTQSIPTEN 300
****.***:***:***********************************************
FAR01
VP2
ITKPITRVKLSYKIDQQAAIGNVATLGTMGPASVSFSSGNGNVPGVLRPITLVAYEKMTP 188
ITKPITRVKLSYKINQQTAIGNVATLGTMGPASVSFSSGNGNVPGVLRPITLVAYEKMTP 360
**************:**:******************************************
FAR01
VP2
LSILTVAGVSNYELIPNPELLKNMVTRYGKYDPEGLNYAKMILSHREELDIRTVWRTEEY 248
LSILTVAGVSNYELIPNPELLKNMVTRYGKYDPEGLNYAKMILSHREELDIRTVWRTEEY 420
************************************************************
FAR01
VP2
KERTRVFNEITDFSSDLPTSKAWGWRDIVRGIRKVAAPVLSTLFPMAAPLIGMADQFIGD 308
KERTRVFNEITDFSSDLPTSKA-------------------------------------- 442
***********************
FAR01
VP2
LTKTNAAGGRYHSMAAGGR 327
-------------------
Fig 5.2: Alignment of FAR01 and IPNV VP2 sequence from Protein Data Bank, accession code 3IDE. The colors
represent the various protein domains. Red: β-sheets of the projection-domain (P) of the VP2; green: α-helix of the
base-domain (B) of the VP2; dark blue: β-sheets in the shell-domain (S) of the VP2; light blue: α-helix in the shelldomain (S) and pink: the is the AA’ flop. The yellow are the differences from our VP2 protein and the VP2 from the
Protein Data Bank. The nucleotide numbers for FAR01 are in relation to the sequence length, and not the length of the
whole protein. Adapted from (Coulibaly et al., 2010).
There are four variable regions in the amino acid sequence, before discussing further, we
will reiterate, that all changes are in relation to FAR01, but FAR01 should not be considered an
ancestral strain, as it is chosen arbitrarily.
The first region is pos. 187-199 and it contains two changes, a G1 change and a G3 change.
The second variable region is pos. 216-221. This region contains one G2 change and six cases of a
G3 change collected over three positions (216, 217, 221). Pos. 245-253 contains 30 G1 changes and
22 G3 changes collected over six positions (245, 247, 248, 249, 252, 253). The last variable region,
pos. 314-323 includes three G2 changes and 23 G3 changes collected over five positions (314, 315,
318, 319, 323). According to Fig. 5.1, the first region is found on the AA flop, the second variable
region can be found on loops on the protruding domain of the VP2 protein. Pos. 314-323 are located
25
Vang and Johannesen, 2013
Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
on beta sheets. Considering these results, we can see that FAR03, FAR05, FAR06-1, FAR06-2 and
FAR07-1 follow the mutation pattern of the foreign proteins closely, and FAR04 follows to a lesser
degree. From this we can conclude that the first Faroese phylogenetic group is more similar to the
foreign isolates, while FAR04 is slightly less similar.
This could indicate that these variable regions are important for the proteins ability to attach
to and enter cells, or escaping the detection and attack by immune system. Research by Santi et al.
(2004) has shown that pos. 217, 221 and 247, with the virulence motif T 217A221T247 have a high
virulence. The study concluded that pos. 217 has particular importance for variation in IPNV
virulence and that a threonine on pos. 217 was associated with a high degree of virulence. A change
from alanine to threonine on a loop region, could be considered to be important, because the change
is from a hydrophobic residue to a polar residue. Considering the change takes place on a loop, we
could speculate that when a hydrophobic residue is present on the loop, the loop configuration turns
inwards towards the protein, and when a polar residue is present, the loop turns outwards from the
protein. This position could therefore be speculated to have importance for attachment to cells. A
later study by Song et al. (2005) concluded that both pos. 217 and 221 are important for virulence
with the motif T217A221 resulting in a high virulence. The motif P 217A221 results in moderate
virulence and strains with a threonine on pos. 221 are almost avirulent. Table 5.1 shows the amino
acids of Faroese isolates on the three positions associated with virulence.
Isolate
217
221
247
FAR01
Pro
Thr
Ala
FAR03
Pro
Thr
Ala
FAR04
Thr
Thr
Ala
FAR05
Pro
Thr
Ala
FAR06-1
Pro
Thr
Ala
FAR06-2
Pro
Thr
Ala
FAR07-1
Pro
Thr
Ala
FAR-2
Pro
Thr
Ala
Table 5.1: Virulence motifs of Faroese isolates. Amino acids on pos. 217, 221 and 247 are shown.
Taking these virulence motifs into consideration, this could explain why very few IPNV
outbreaks have been detected in aquafarms in the Faroe Islands as opposed to Norway (Debes H.
Christiansen, pers. Comm.). The Norwegian isolates NVH-105 has the T 217A221 virulence motif, and
26
Vang and Johannesen, 2013
Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
must be considered virulent, while C-1 and C-1.3 have the P 217A221 and are moderately virulent.
It is interesting that most of the Faroese and foreign proteins have a P 217T221 motif, and it could
therefore be suspected, that the ancestral strain was not highly virulent. It should be noted, that
Santi et al. (2004) also concluded that the length of the NS protein was related to virulence. If an
isolate encodes a truncated NS protein, it is highly virulent. In our study, we did not include the
VP4 gene, which codes for the NS protein, however this should be an interesting topic for further
inquiry.
Fig. 5.3: Phylogenetic tree showing the evolutionary history of the Faroese isolates, the closest foreign isolates and
more distantly related isolates, based on VP2 nucleotide sequences. The evolutionary history was inferred by using the
Maximum Likelihood method based on the Tamura-Nei model. The bootstrap value is shown next to the branches. The
scale indicates 0.05 substitutions per nucleotide site. The bootstrap value after 1000 replications is shown next to the
branches, The analysis involved 22 nucleotide sequences. All positions containing gaps and missing data were
eliminated, otherwise all nucleotide sequences included. There were a total of 973 positions in the final dataset.
Genogrouping according to Blake et al. (2001).
To illustrate the relationship between Faroese isolates and more distantly related foreign isolates, we
created another phylogenetic tree, shown in Fig. 5.3. For this analysis we included WB (Genogroup
27
Vang and Johannesen, 2013
Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
1), Canada 2 (Genogroup 2), Ab (Genogroup 3), Canada 1 (Genogroup 4), OV2 (Genogroup 5), He
(Genogroup 6) and Buhl (Genogroup 1) strains. This phylogenetic tree illustrates that Faroese
isolates are related to these strains by some distance. Comparing Fig. 1.3 and Fig. 5.3, and using the
same genogrouping as Blake et al. (2001), we can infer that Faroese isolates, and the most closely
related foreign isolates belong to Genogroup 5.
28
Vang and Johannesen, 2013
Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
6 Conclusion
The findings of this study, demonstrate that the various Faroese IPNV strains have a very high
degree of similarity (ca. 99%), and that foreign isolates most closely related to these viruses can be
found in Norway, Spain and Chile, showing great geographic variability of closely related IPNV
strains. The Spanish strain's host organism was an oyster, so IPNV does also have a large host
diversity.
The amino acid sequences deduced from generated cDNA sequences, showed a mutation
pattern involving pos. 187-199, 216-221, 245-282 and 314-323. High virulence motifs involve a
threonine on pos. 217 and and alanine 221, while the Faroese sequences contain a proline on pos.
217 and a threonine on pos. 221. This suggests that the Faroese isolates are of low virulence, which
explains why IPNV outbreaks in the Faroe Islands have mainly occurred in smolt farms.
Comparing these Faroese isolates with more distantly related isolates, and using previously
established genogroups, we conclude that the Faroese and closely related foreign isolates belong to
one genogroup.
29
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Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
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Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
8 Acknowledgments
This study was carried at the Faroese Food and Veterinary Agency, Department of Pathology, who
also funded the study. We would like to thank our advisors, Prof. Svein-Ole Mikalsen from the
University of the Faroe Islands, and Debes H. Christiansen from the Faroese Food and Veterinary
Agency for their help and advice during the process of researching the topic and during the
laboratory work. A special thanks goes to Marita Næs for her help and patience in the laboratory.
Lastly, we would also like to thank all of the staff at the Faroese Food and Veterinary Agency for
their hospitality.
35
9. Appendix
The sections:
“9.1 A1 Edited DNA sequences”
“9.2 A2 Aligned DNA sequences:”
(Page 36 to 43) are not available in this web-version of the bachelor thesis.
If you are interested in these sequences, please contact:
Debes Christiansen [email protected]
at the “Faroese Food- and veterinary authority (Heilsufrøðiliga starvsstovan)”
Vang and Johannesen, 2013
Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
9.3 A3
Distance matrices
FAR01
FAR01
FAR03
FAR04
FAR05
FAR06-1
FAR06-2
FAR07-1
FAR07-2
99.988
99.990
99.990
99.988
99.986
99.988
99.999
99.988
99.998
~100
99.999
~100
99.989
99.987
99.988
99.986
99.988
99.991
99.998
99.997
99.998
99.991
99.999
~100
99.989
99.999
99.988
FAR03
0.012
FAR04
0.010
0.012
FAR05
0.010
0.002
0.013
FAR06-1
0.012
~0
0.012
0.002
FAR06-2
0.014
0.001
0.014
0.003
0.001
FAR07-1
0.012
~0
0.012
0.002
~0
0.001
FAR07-2
0.001
0.011
0.009
0.009
0.011
0.012
99.989
0.011
Table 9.1: Distance matrix calculated using the Maximum Composite Likelihood model, based on the nucleotide
sequences. The lower matrix shows the percent divergence, and the upper matrix shows the percent identity.
44
Vang and Johannesen, 2013
Genetic sequencing and analysis of the infectious pancreatic necrosis virus VP2 coding region from Faroese isolates
FAR
01
FAR
01
FAR
03
FAR
04
FAR
05
FAR
06-1
FAR
06-2
FAR
07-1
FAR
07-2
N136
NVI016
88R
C-1
C-1.3 NVH- NVH108 105
99.988 99.980 99.980 99.988 99.987 99.988 99.999 99.982 99.982 99.982 99.981 99.981 99.979 99.979
FAR
03
0.012
FAR
04
0.010
0.012
FAR
05
0.010
0.002
0.012
FAR
06-1
0.012
~0
0.012
0.002
FAR
06-2
0.013
0.001
0.013
0.003
0.001
FAR
07-1
0.012
~0
0.012
0.002
~0
0.001
FAR
07-2
0.001
0.011
0.009
0.009
0.011
0.012
0.011
N136
0.018
0.014
0.014
0.014
0.014
0.015
0.014
0.017
NVI016
0.018
0.014
0.014
0.014
0.014
0.015
0.014
0.017
~0
88R
0.018
0.011
0.013
0.013
0.011
0.012
0.011
0.017
0.006
0.006
C-1
0.019
0.012
0.012
0.015
0.012
0.013
0.012
0.018
0.009
0.009
0.005
C-1.3
0.019
0.012
0.012
0.015
0.012
0.013
0.012
0.018
0.009
0.009
0.005
~0
NVH108
0.021
0.015
0.012
0.017
0.015
0.016
0.015
0.020
0.011
0.011
0.007
0.004
0.004
NVH105
0.021
0.015
0.012
0.017
0.015
0.016
0.015
0.020
0.011
0.011
0.007
0.002
0.002
99.988 99.998
~100
99.999
~100
99.989 99.986 99.986 99.989 99.988 99.988 99.985 99.985
99.988 99.988 99.987 99.988 99.991 99.986 99.986 99.987 99.988 99.988 99.988 99.988
99.998 99.997 99.998 99.991 99.986 99.986 99.987 99.985 99.985 99.983 99.983
99.999
~100
99.989 99.986 99.986 99.989 99.988 99.988 99.985 99.985
99.999 99.988 99.985 99.985 99.988 99.987 99.987 99.984 99.984
99.989 99.986 99.986 99.989 99.988 99.988 99.985 99.985
99.983 99.983 99.983 99.982 99.982 99.980 99.980
~100
99.994 99.991 99.991 99.989 99.989
99.994 99.991 99.991 99.989 99.989
99.995 99.995 99.993 99.993
~100
99.996 99.998
99.996 99.998
99.998
0.002
Table 9.2: Distance matrix including the foreign isolates. The matrix is calculated using the Maximum Composite
Likelihood model, based on the nucleotide sequences. The lower matrix shows the percent divergence, and the upper
matrix shows the percent identity.
45