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Ultra-deep sequencing of VHSV isolates contributes to understanding the role of viral quasispecies



The high mutation rate of RNA viruses enables the generation of a genetically diverse viral population, termed a quasispecies, within a single infected host. This high in-host genetic diversity enables an RNA virus to adapt to a diverse array of selective pressures such as host immune response and switching between host species. The negative-sense, single-stranded RNA virus, viral haemorrhagic septicaemia virus (VHSV), was originally considered an epidemic virus of cultured rainbow trout in Europe, but was later proved to be endemic among a range of marine fish species in the Northern hemisphere. To better understand the nature of a virus quasispecies related to the evolutionary potential of VHSV, a deep-sequencing protocol specific to VHSV was established and applied to 4 VHSV isolates, 2 originating from rainbow trout and 2 from Atlantic herring. Each isolate was subjected to Illumina paired end shotgun sequencing after PCR amplification and the 11.1 kb genome was successfully sequenced with an average coverage of 0.5-1.9 × 106 sequenced copies. Differences in single nucleotide polymorphism (SNP) frequency were detected both within and between isolates, possibly related to their stage of adaptation to host species and host immune reactions. The N, M, P and Nv genes appeared nearly fixed, while genetic variation in the G and L genes demonstrated presence of diverse genetic populations particularly in two isolates. The results demonstrate that deep sequencing and analysis methodologies can be useful for future in vivo host adaption studies of VHSV.
Schönherz et al. Vet Res (2016) 47:10
DOI 10.1186/s13567-015-0298-5
Ultra-deep sequencing ofVHSV isolates
contributes tounderstanding the role ofviral
Anna A. Schönherz1, Niels Lorenzen2, Bernt Guldbrandtsen1, Bart Buitenhuis1 and Katja Einer‑Jensen3*
The high mutation rate of RNA viruses enables the generation of a genetically diverse viral population, termed a qua‑
sispecies, within a single infected host. This high in‑host genetic diversity enables an RNA virus to adapt to a diverse
array of selective pressures such as host immune response and switching between host species. The negative‑sense,
single‑stranded RNA virus, viral haemorrhagic septicaemia virus (VHSV), was originally considered an epidemic virus
of cultured rainbow trout in Europe, but was later proved to be endemic among a range of marine fish species in the
Northern hemisphere. To better understand the nature of a virus quasispecies related to the evolutionary potential
of VHSV, a deep‑sequencing protocol specific to VHSV was established and applied to 4 VHSV isolates, 2 originating
from rainbow trout and 2 from Atlantic herring. Each isolate was subjected to Illumina paired end shotgun sequenc‑
ing after PCR amplification and the 11.1 kb genome was successfully sequenced with an average coverage of
0.5–1.9 × 106 sequenced copies. Differences in single nucleotide polymorphism (SNP) frequency were detected both
within and between isolates, possibly related to their stage of adaptation to host species and host immune reactions.
The N, M, P and Nv genes appeared nearly fixed, while genetic variation in the G and L genes demonstrated presence
of diverse genetic populations particularly in two isolates. The results demonstrate that deep sequencing and analysis
methodologies can be useful for future in vivo host adaption studies of VHSV.
© 2016 Schönherz et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
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publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Viral haemorrhagic septicaemia virus (VHSV) is an RNA
virus endemic to marine and freshwater fish species. It
represents one of the most important viral pathogens
in salmonid fish in continental Europe where it heavily
affects cultured rainbow trout, causing a severe systemic
disease with mortality rates as high as 90% [1] and thus
resulting in extensive economical loses to the aquaculture
industry [2, 3].
VHSV is a single-stranded RNA virus of negative polar-
ity that belongs to the genus Novirhabdovirus, within the
family Rhabdoviridae. Genetic analyses show that the
virus clusters into four major phylogenetic clades classi-
fied as genotypes I–IV with further subdivision of geno-
types I and IV. Genotypes are correlated to geographic
regions rather than host species with genotype I–III cir-
culating in Europe and genotype IV circulating in North
America and Asia [47]. Although these phylogenetic
analyses cannot define host species specificity, they do
demonstrate that isolates of VHSV from rainbow trout
are principally members of genotypes Ia, Ic and Id, and
the isolates adapted to marine host species are members
of genotypes Ib, Ie and II–IV. e genetic differentiation
of rainbow trout and marine adapted isolates is also phe-
notypically manifested in different pathogenicity patterns
towards the two host groups [8]. Rainbow trout adapted
isolates are highly pathogenic to rainbow trout but show
very low or no pathogenicity in marine fish species and
vice versa.
Moreover, it has been revealed that rainbow trout
adapted isolates have evolved from the marine environ-
ment and are the result of cross-species transmission
followed by subsequent host adaptation [5, 9, 10]. Cross-
species transmission from marine hosts to cultured
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Page 2 of 12
Schönherz et al. Vet Res (2016) 47:10
rainbow trout has occurred several times. Recently,
cross-species transmission events have been reported in
rainbow trout cultured in marine waters of Finland [11,
12], Norway [13] and Sweden [14, 15].
e repeated emergence of VHSV into cultured rain-
bow trout illustrates the high evolutionary potential of
the virus. is is a feature common to all RNA viruses
[16, 17]. RNA viruses have high replication rates, large
population sizes, and exceptionally high mutation rates
commonly ranging from 103 to 105 mutations per
nucleotide, per replication [1618]. e high mutation
rates result from an error-prone RNA polymerase lacking
proofreading abilities [18, 19]. As a consequence, RNA
virus populations sustain high genetic diversity, most
likely resulting in the formation of a quasispecies. A qua-
sispecies is composed of a dominant nucleotide sequence
and a spectrum of low frequency variants arising from
mutations during virus replication [18]. While appear-
ing a wasteful strategy, this feature enables an RNA virus
to rapidly adapt to environmental changes or new hosts.
However, very little is known about viral population
structures and dynamics, especially the low-frequency
mutant spectrum.
Until recently, viral evolutionary dynamics were inves-
tigated based on consensus genome sequencing, ignoring
that viral samples actually represent complex, heteroge-
neous populations, of non-identical genome sequences.
Consensus sequencing, however, only identifies the dom-
inant or major viral sequence present in a sample but is
uninformative about the mutant spectrum of minority
variants present in the population [20, 21]. Consequently,
consensus genome sequences provide an incomplete
picture of the within- and between-host viral evolu-
tionary diversity during cross-species transmission and
In contrast Next-Generation Sequencing (NGS) tech-
niques provide a rapid and cost-effective analysis of viral
population diversity at an unprecedented level of detail
[20, 2224]. e great depth of resolution and high-
throughput nature of NGS platforms allows investigation
of viral evolution at the within- and between-host scale.
e main focus of this study was to develop a VHSV
specific NGS protocol applicable across viral genotypes
with the aim of understanding the evolutionary potential
of VHSV through characterization of its viral quasispe-
cies. An overlapping long range PCR amplification pro-
tocol specific to VHSV was developed targeting isolates
of genotype I–III. e nucleotide sequences of four cell
culture passaged VHSV isolates were analysed using the
Illumina HiSeq sequencing platform and coverage rates
between 0.5 and 1.9×106 were achieved allowing char-
acterization of the viral population diversity in all four
isolates of VHSV.
Materials andmethods
Virus isolates andviral propagation
Deep sequencing of viral populations was conducted
using four VHSV isolates (Table1): two isolates origi-
nating from freshwater cultured rainbow trout (DK-
3592b, DK-9895174), and two isolates originating from
Atlantic herring (4p168, 1p49). Both rainbow trout iso-
lates belong to genotype I whereas the two marine iso-
lates belong to genotypes II (1p49) and III (4p168). All
four viral stocks were propagated in bluegill fry cells
(BF-2; [25]) as described earlier [26]. When complete
cytopathic effect (CPE) was observed, cell medium
was harvested, filtered through a 0.2-µm Minisart fil-
ter (Bie & Bernsten) and the filtrate was centrifuged
at low speed (5000 RPM) for 30min at 4°C to remove
cell debris. Subsequently, the supernatant was recov-
ered and subjected to ultracentrifugation at 86000×g
for 2h at 4°C to pellet viral particles. e pellet was
harvested and stored at 80°C or directly subjected to
RNA extraction.
RNA extraction
Total RNA was extracted from replicate samples for each
isolate using the RNeasy Mini kit (Qiagen) following
manufacturer’s recommendations for extraction of RNA
from cell culture. Total RNA from each replicate was
eluted in 30 µL nuclease-free water that was treated with
DEPC (Qiagen) and finally pooled. Two microliters were
used to quantify the concentration of RNA; the remain-
der was stored at 80°C. e concentration of extracted
RNA was determined using a spectrophotometer (Nan-
oDrop, ermo Scientific) and the final concentration
of the pooled samples was in range of 16–40ng/µL per
Table 1 Data related tothe four viral haemorrhagic septicaemia virus isolates used inthis study.
Isolate Genotype Host Geographic origin Year ofisolation Original sample type References Cell culture passage
DK‑3592b Ia Rainbow trout Denmark 1985 Tissue pool, fish pool [28, 39, 40] 8 Pass BF2
DK‑9895174 Ia Rainbow trout Denmark 1998 Tissue pool, fish pool [5] 5 Pass BF2
1p49 II Atlantic herring Baltic Sea 1996 Tissue pool, fish pool [8] 1 Pass EPC, 5 pass BF2
4p168 III Atlantic herring Skagerrak 1997 Tissue pool, fish pool [41] 8 Pass BF2
Page 3 of 12
Schönherz et al. Vet Res (2016) 47:10
Reverse transcription
Reverse transcription (RT) of the full-length VHSV
genome was performed using the SuperScript III First-
Strand Synthesis System for RT-PCR (Invitrogen) and
a VHSV genome specific primer (Table2). RT was per-
formed following manufacturer’s recommendations.
Briefly, 1µL cDNA primer (0.01mM) and 1µL dNTPs
(10 mM) were added to 8 µL total RNA, incubated at
65°C for 5min and placed on ice. Subsequently, 10µL
cDNA synthesis mix (2µL 10× buffer, 4µL MgCl2, 2µL
DTT, 1µL RNase OUT, 1µL SuperScript III reverse tran-
scriptase) were added and incubated at 50°C for 50min,
85°C for 5min, and placed on ice. Finally, 1µL RNase H
was added followed by incubation at 37°C for 20min to
remove the original viral RNA from the new synthesized
cDNA. In total, 20µL of full VHSV genome length cDNA
was synthesized and either stored at 80°C or immedi-
ately subjected to PCR amplification.
Polymerase chain reaction (PCR) andDNA purication
PCR amplification of the full length VHSV genome was
performed using the Platinum® Taq DNA Polymer-
ase High Fidelity kit (Invitrogen) and single primer set
amplifying a 11,014bp region covering all 6 open read-
ing frames, all intergenic regions and partial regions of
the leader and trailer sequence (sense primer VHSV_
Frag1I_nt18_+s: 5-GAG TTA TGT TAC ARG GGA
CAG G-3; antisense primer VHSV_Frag4I_nt11032_-s:
fication was performed for all four isolates but full-length
genome amplification could only be established for 3 of
the isolates (DK-3592b, DK-9895174, 1p49) and with
unwanted smaller fragments (Figure 1). To maximize
coverage depth, the genome was divided into four ampli-
cons that were numbered sequentially as amplicon 1–4
starting from the 5 end of the genome with amplicons
ranging from 2797 to 3709bp in length and overlapping
with the adjacent amplicons by 274–790bp (Table2).
Primers were designed to target conserved regions of the
VHSV genome irrespective of host origin and genotype
(Table 2). PCR amplification was performed for each
fragment and isolate separately using the Platinum® Taq
DNA Polymerase High Fidelity kit (Invitrogen) and the
corresponding primer sets. Amplification was conducted
in a total volume of 50µL in a MX Pro-Mx3005P ther-
mocycler. Reactions contained 2µL cDNA, 5µL 10x high
Fidelity PCR buffer, 1µL dNTPs (10mM), 2µL MgSO4
(50mM), 0.2 µL Platinum4 Taq High Fidelity Polymer-
ase, 1µL sense primer (0,01mM), 1µL antisense primer
(0.01 mM), and 37 µL nuclease free water. Amplicons
were produced using the following cycling program:
94°C for 1min, followed by 25 cycles of 94°C for 30s,
58°C for 30s, and 68°C for 4min, with a final step of
68°C for 5min. Individual PCR products were visualized
using agarose gel electrophoresis running 6µL on a 1%
agarose gel. A total of 30µL of the remaining amplified
DNA was purified using the QIAquick PCR Purification
kit (Qiagen) following manufacturer’s recommendations.
Purified DNA was eluted in 50 µL EB buffer (10 mM
Tris·Cl, pH 8.5). DNA concentration was determined by
fluorescence detection using a Qubit® Fluorometer (Inv-
itrogen) and the Quant-iT dsDNA BR Assay kit (Life
Technologies). Subsequently, amplicons of the same iso-
late were adjusted to equivalent concentrations, com-
bined to one sample and stored at 20°C.
Library preparation andsequencing
e paired end library preparation, as well as Illumina
HiSeq (2×100) sequencing of the four samples, was per-
formed on contract basis by Beckman Coulter Genomics.
Briefly, library construction was performed using sheared
DNA as input to the SPRI-TE system and LC cartridges
(Beckman Coulter, Inc.). TruSeq PE indexes (Illumina)
were added by ligation, the libraries were amplified by
PCR, and purified with AMPure XP beads (Beckman
Coulter, Inc.). e libraries were clustered on Illumina
Table 2 Primers used forRT-PCR amplication.
Names include +s or s, which reects the positive or negative sense orientation, respectively.
* Primer used for reverse transcription of the genomes.
Name Sequence Amplicon (length inbp) Overlapping region
VHSV_Frag1_nt2815_s CGATTGTAGYAGTCCTTCGC Amplicon 1 (2797 bp) Amplicon 1–2 = 274 bp
VHSV_Frag2_nt6250_s CGTAGGTAGGAACCCTGTC Amplicon 2 (3709 bp) Amplicon 2–3 = 790 bp
VHSV_Frag3_nt8287_s TGTGTCCGCCAAATGGTGTA Amplicon 3 (2827 bp) Amplicon 3–4 = 431 bp
VHSV_Frag4I_nt11032_s TCTCCAAATGGAAAGAAGGACT Amplicon 4 (3176 bp)
Page 4 of 12
Schönherz et al. Vet Res (2016) 47:10
High-Output v3 Flowcells and sequenced using a HiSeq
model 2500 sequencer (Illumina). Beckman Coulter
Genomics performed the primary software analysis using
the Illumina instrument application Real-Time Analy-
sis software, and hereby converted raw images into base
calls. Demultiplexing was then performed resulting in
FASTQ-formatted read-groups using the CASAVA soft-
ware package.
Bioinformatics analysis
e demultiplexed FASTQ files provided by Beckman
Coulter Genomics were analysed in a single workflow
using the specified tools (subsequently marked with
“”) which all are available in the CLC Genomics Work-
bench V7.5.1. e paired reads were trimmed using
the “Trim sequence” tool 4 times for removal gen-
eral adapters, PCR adaptors, PCR primers, and finally
ambiguous nucleotides (maximum number of ambi-
guities = 2) as well as nucleotides with low quality
scores (limit=0.05). e virus isolates DK-3592b and
DK-9895174 were mapped against a reference sequence
of DK-3592b (NCBI Accession number KC778774),
while the isolates 1p49 and 4p168 obtained from her-
ring were mapped against a VHSV isolate from sea-
reared rainbow trout (strain FA281107; NCBI Accession
number No EU481506). e following default param-
eters were used during mapping of reads to their refer-
ence sequence: mismatch cost=2; insertion cost= 3;
deletion cost = 3; length fraction = 0.5; similarity
fraction = 0.8. e mapped reads were subsequently
realigned and explored using the “InDel and Struc-
tural Variation Detection” tool. e hereby-generated
guidance variant track was subsequently used dur-
ing an additional realignment of the reads. Consensus
sequence and alignment of these against their respec-
tive reference sequences were performed using the
“Extract Consensus sequence” and “Create Alignment”
tools, respectively.
Figure1 Generation of the four overlapping amplicons (1–4) to cover the entire viral genomes of each isolate. The VHSV isolates, are
here abbreviated a DK‑3592b; b DK‑9895174; c 1p49; d 4p168. The individual RT‑PCR reactions are shown in A; B re‑amplified Amplicon 3 of sample
b*; C amplification of the whole genome RT‑PCR reaction; D pooled amplicon samples (1:1:1:1) for each of the VHSV samples used for library prepa‑
Page 5 of 12
Schönherz et al. Vet Res (2016) 47:10
Mapping efficiency and coverage of the individual sam-
ples were determined using the tools “Create Detailed
Mapping Report” and “Create Statistics for Target
Regions”, respectively.
Finally, single- and multiple nucleotide variants and
short insertions and deletions were called using the “Low
Frequency Variant Detector” tool where broken pairs and
non-specific read matches were ignored. e minimum
coverage, count and frequency were set to 1000, 200
and 1%, respectively, while the required significance was
0.01% and the relative read direction filter significance
was set to 1019.
To reduce the number of false positives due to PCR
errors, quality filtering was performed using the fol-
lowing conservative thresholds: equality in forward and
reverse reads at least 0.35, average quality at least 30,
and statistical testing of read position and read direc-
tion probability should exceed 1012. Since marginal
variants could still be related to cell culture passaging,
a final frequency-filtering criterion was applied includ-
ing polymorphic sites with intermediate allele frequency
(5–95%) but excluding polymorphic sites with minor allel
frequency below 5%. For each of these filtering steps, the
functional consequence was determined by translating
the encoded CDS’s using the “Amino Acid Changes” tool.
Statistical analysis
Patterns of genetic variation within and between isolates
were investigated using the statistical program R (version
3.1.0 R Core Team, 2014). All analyses conducted were
based on variant calls that passed quality control criteria.
Only polymorphisms with allele frequencies between 5
and 95% were retained for analysis. e frequency filter
was applied to ensure that variants investigated repre-
sented polymorphisms of the original isolate instead of
variants introduced during cell culture, RT-PCR ampli-
fication or sequencing and to ensure that those variants
represented genetic variation present within the original
viral population.
To identify whether genetic variation differed between
genes or isolates, a generalized linear model compari-
son analysis was conducted assuming that the number
of substitutions, including both synonymous- and non-
synonymous substitutions, as the response variable fol-
lowed a Poisson distribution. e explanatory variables
were gene (
: 6 levels representing genes encoding the
nucleoprotein (N), phosphoprotein (P), matrix protein
(M), glycoprotein (G), non-structural protein (NV), and
the RNA-dependent RNA polymerase (L)), and isolate (
4 levels representing the isolates DK-3592b, DK-9895174,
1p49, 4p168), as well as a regression coefficient taking
into account the effect of the length of the gene in nucle-
otides. e full linear model was:
is the parameter of the Poisson distribution, gi
is the effect of gene i, sj is the effect of isolate j, log (li)
is the logarithm of the length of gene i. e coefficient
of the logarithm of the gene length was fixed to 1. ree
models were investigated: (1) the full model including all
explanatory variables; (2) a reduced model without gene
as explanatory variable; and (3) a reduced model with-
out isolate as explanatory variable. e generalized linear
models were fitted using the “glm” function from the R
package “stats. Models were compared using the “anova
function in R.
To identify contrasts of genetic variation between
genes, genes were divided into two groups: (1) genes
with low genetic variation (N, P, M, NV); (2) genes with
high genetic variation (G, L). e generalized linear
model comparison analysis was repeated, but with gene
grouped into 2 levels (high genetic variation, low genetic
variation), isolate (4 levels) and gene length included
as explanatory variables, investigating whether genetic
variation in the G and L gene is higher compared to the
remaining genes. A Bonferroni correction was applied to
account for all possible groupings of 6 genes. e total
number of possible groupings is given by the sixth Bell
number minus 2, B6= 203, that is correcting for 201
simultaneous (implicit) tests. e two cases that are sub-
tracted are the case of each gene having a separate effect
and the case of all genes having the same effect. When
looking at contrasts among genes, this is the most con-
servative choice.
To identify contrasts of genetic variation between iso-
lates, isolates were divided into two groups: (1) isolates
with low genetic variation (DK-3592b, DK-9895174,
4p168); (2) isolates with high genetic variation (1p49).
e generalized linear model comparison analysis was
repeated, but with isolate grouped into 2 levels (high
genetic variation, low genetic variation), gene (6 levels)
and gene length included as explanatory variables, inves-
tigating whether genetic variation in 1p49 is higher com-
pared to the reaming isolates. e Bonferroni correction
was adjusted to correct for all possible groupings of 4 iso-
lates (B4=15).
Amplicon amplication
Based on agarose gel electrophoresis, amplicons of
expected size were synthesized by RT-PCR. However,
small amounts of shorter amplicons were also observed
(Figure 1A). Attempts to extract the DNA bands with
the expected amplicon size resulted in too low a con-
centration of purified DNA. Instead, a 1:1:1:1 amplicon
mixture was made from purified, but not gel-extracted
Page 6 of 12
Schönherz et al. Vet Res (2016) 47:10
samples, and therefore included some amplicons that
were shorter than desired (Figure 1D). e obtained
coverage data (Figure 2) showed unexpected increase
in coverage (up to 20×) at the first nucleotide upstream
of primer 3 end and at the sequences in the overlap-
ping regions of amplicon 1 and 2, amplicon 2 and 3,
and amplicon 3 and 4, respectively. is finding was
unexpected since the performed PCR amplifications
were performed in individual tubes, and not as multi-
plex reactions. Whether a minor cross contamination of
primer pairs may have occurred either during synthesis
at the producer or during handling of the primers in the
lab remains to be determined.
Coverage andmapping ecacy
e number of generated raw paired-end reads for DK-
3592b, DK-9895174, 1p49 and 4p168 was 266.4× 106,
127.6×106, 76.4 ×106 and 224.4 × 106, respectively.
Trimming removed between 3.2 and 6% of the reads
resulting in 260.6 × 106, 120.2× 106, 71.8 × 106 and
212.9×106 reads, respectively.
Based on a BLAST search two different reference
genomes were identified. Isolate DK-3592b (genotype I)
was identified as an appropriate reference genome for
isolates DK-3592b and DK-9895174 (both genotype I),
whereas isolate FA281107 (genotype III) was identified as
the best reference genome for isolates 1p49 (genotype II)
and 4p168 (genotype III).
Mapping efficacy of isolate DK-3592b, DK-9895174,
4p168 to their respective reference genomes was above
97%) and contained comparable fractions of paired reads
(above 88%). e fourth isolate (1p49) was approximately
10% lower with respect to the number of generated reads
as well as the mapping efficiency (Table3).
e average coverage was very high for all four isolates
but with some variations. e average coverage depths of
isolate 1p49 (5.28×105) was approximately 72% that of
the isolate with the highest coverage (isolate DK-3592b
with 1.9×106 coverage). However, the coverage across
the genomes appears more even for 1p49 than for the
other 3 samples due to less PCR contamination/artefacts
as discussed above (Figure2).
Alignment of mapped consensus sequences to their
respective reference genome revealed 99.93, 99.03 and
98.34% sequence identity for isolates DK-3592b, 4p168,
and DK-9895174, respectively, but only 90.09% sequence
identity for isolate 1p49.
Variant detection
In the analysis performed, we mainly focus on polymor-
phisms with frequencies between 5 and 95% (Table4),
which likely reflect polymorphisms established in vivo
(before cell culture propagation). Variants at population
frequencies below 1% were assumed to represent poly-
morphisms established invivo, variants that were estab-
lished during cell culture, as well as artefacts of RT-PCR
amplification and NGS sequencing [27]. e experimen-
tal design could not provide a method to distinguish
between the different sources of observed polymor-
phisms; therefore, a relatively conservative frequency fil-
ter was applied, excluding variants below a 5% frequency
Following quality- and frequency filtering, the highest
genetic variation at intermediate allele frequencies was
detected for isolate 1p49 with 100 variants, followed by
DK-3592b with 10 variants, DK-9895174 with 9 variants,
and 4p168 with 3 variants (Table4). Variants recorded
included the minor and the major allele detected for each
Figure2 Coverage of mapped reads across the whole VHSV genomes for all four isolates. The four overlapping amplicons (1–4) and CDS
regions are visualized according to the genome numbers of Acc No KC778774. The obtained mapping coverage for each nucleotide position of the
individual virus isolates is shown using a logarithmic scale.
Page 7 of 12
Schönherz et al. Vet Res (2016) 47:10
polymorphic side that passed quality control require-
ments. us, called variants do not represent the number
of polymorphic sites detected, but rather the total allelic
variation. A total of 89, 8, 7 and 2 polymorphic sites with
intermediate allele frequencies were detected for 1p49,
DK-9895174, DK-3592b and 4p168, respectively. Poly-
morphic sites were located in the N gene (DK-3592b), the
G gene (all isolates), the NV gene (1p49), and the L gene
(DK-3592b, 1p49). No genetic variation at intermediate
allele frequencies was detected in the M and P gene, or in
the intergenic regions. In Figure3, positions of polymor-
phic sites across the genome are shown representing the
allele frequency of the dominant variant (50–95% allele
frequency). e frequency of dominant alleles was shown
as some variants of minor allele frequency did not pass
the quality requirements and thus were not recorded.
Based on Table4 and Figure3, the G and L genes show
the highest level of genetic variation. Furthermore, iso-
late 1p49 shows higher genetic variation than the other
three isolates. Patterns of genetic variation within and
between isolates were confirmed by generalized linear
model comparison analysis. Comparing the full model
with the two model reductions revealed a significant dif-
ference between the full- and reduced models without
gene as explanatory variable (p value =9.955e10) and
between the full- and reduced models without isolate as
explanatory variable (p value=2.2e16). Hence, both the
genes as well as the isolates have an effect on the num-
ber of substitutions observed and the local substitution
rate because gene length was included in each model as
a regression coefficient. Comparison between genes of
high genetic variation to genes with low genetic variation
revealed that the average number of substitutions in the
G and L gene was significantly higher compared to other
genes (p value=0.002) (Figure4). In addition, compari-
son between isolates of high genetic variation with iso-
lates of low genetic variation revealed that the average
number of substitutions in isolate 1p49 was significantly
higher compared to the other isolates (p value=2.6e15)
(Figure 4). In terms of amino acid substitutions,
DK-9895174 and 1p49 displayed the highest variability.
is was even more evident when comparing numbers of
substitutions after quality filtration only (Table4).
In general, resequencing projects rarely exceeds a read
depth of 100×, which barely exceeds the depth needed
in order to correct for sequencing errors. In this study,
we established a protocol which provided an average cov-
erage of the four analysed VHSV genomes of between
0.5 and 1.9 million reads, while low coverage areas were
observed only in the initial part of the leader region, as
well as in the trailer region.
e results clearly demonstrated that the four isolates
of VHSV were composed of a genetically diverse virus
population, the first requirement for the formation of
a quasispecies. Intra-host genetic variation has been
demonstrated previously for other rhabdoviruses such
as rabies virus [22] but to our knowledge, this study is
the first to describe the intra-host genetic diversity in a
novirhabdovirus using ultra deep sequencing.
Statistical comparison of local substitution rates across
genes revealed that the G and L genes comprised the
majority of the genetic variation and showed signifi-
cantly different substitution rates compared to the N, P,
M and NV genes. ese findings are likely related to the
essential functions of these proteins: e G protein is
exposed on the surface of the virus particle and involved
in receptor recognition as well as cell entry. At the same
time, G is the target of neutralizing antibodies. Selection
pressure on G thus includes both the need for ability to
adapt to variation in host cell surface molecules as well
as ability to escape from the host adaptive immune sys-
tem [28]. e L gene is responsible for genome replica-
tion (transcription and translation), hence the observed
higher genetic variation may indicate the need to be able
Table 3 Read mapping oftrimmed reads againsta reference genome froma VHSV isolate ofsame host adaption environ-
Referencegenome DK-3592b (Acc No KC778774) FA281107 (Acc No EU481506)
Virus isolate DK-3592b DK-9895174 1p49 4p168
No ofreads In % No ofreads In % No ofreads In % No ofreads In %
Mapped reads 257 431 536 98.8 117 684 334 97.8 63 880 245 89.0 209 126 393 98.2
Not mapped reads 3 179 450 1.2 2 611 384 2.2 7 936 247 11.0 3 799 011 1.8
Reads in pairs 236 329 444 90.7 106 731 672 88.7 56 208 078 78.2 197 135 466 92.6
Broken paired reads 21 102 092 8.1 10 952 662 9.1 7 672 167 10.7 11 990 927 5.6
Total reads 260 610 986 100.0 120 295 718 100.0 71 816 492 100.0 212 925 404 100.0
Average coverage 1 871 006 959 252 528 148 1 729 031
Page 8 of 12
Schönherz et al. Vet Res (2016) 47:10
Table 4 Variant detection incoding andnone coding regions ofthe four analyzed VHSV genomes.
The table summarizes the distribution of nucleotide (Nt) and amino acid (Aa) variants before and after lteringprocedures. Two ltering procedures were applied based on read quality (Q) and variant frequency at
polymorphic sites (F). Read quality ltering was applied using the following parameters: equality in forward and reverse reads should be at least 0.35, average quality at least 30, while statistical testing of read position- as
well as read direction probability should exceed 1012 . Filtering on variant frequency was conducted to distinguish true variants from errors introduced during sequence preparation and sequencing. Only polymorphic
sites with variants with intermediate allel frequencies (5–95%) were incluede whereas polymorphic sites with minor allele frequencies below 5% were excluded from analysis.
a Coverage of trailer was below 80×.
Reference genome DK-3592b (Acc No KC778774) FA281107 (Acc No EU481506)
Isolate DK-3592b DK-9895174 1p49 4p168
Filtering Nt Aa Nt, Q Aa, Q Nt, QF Aa, QF Nt Aa Nt, Q Aa, Q Nt, QF Aa, QF Nt Aa Nt, Q Aa, Q Nt, QF Aa, QF Nt Aa Nt, Q Aa, Q Nt, QF Aa, QF
Leader 4 0 0 – 7 0 0 – 12 0 0 – 0 0 0
N 7 1 5 0 2 0 15 3 12 1 0 0 200 33 94 15 0 0 14 8 10 7 0 0
Inter N‑P 3 1 0 3 2 0 11 6 0 0 0 0
P 2 1 3 1 0 0 17 4 17 3 0 0 72 11 48 7 0 0 5 2 5 2 0 0
Inter P‑M 2 0 0 3 1 0 26 8 0 6 5 0
M 2 1 0 0 0 0 13 5 6 2 0 0 88 18 29 5 0 0 9 3 6 1 0 0
Inter M‑G 0 0 0 1 1 0 20 11 0 5 4 0
G 22 9 17 4 5 1 60 27 48 22 9 4 286 45 161 16 7 1 32 7 15 3 2 1
Inter G‑Nv 6 0 0 5 3 0 17 13 0 0 5 0
Nv 20 9 12 1 0 0 23 12 22 11 0 0 65 33 37 18 2 0 11 4 5 3 1 0
Inter Nv‑L 4 0 0 6 5 0 11 8 0 2 1 0
L 15 1 8 0 3 0 85 10 65 5 0 0 869 73 464 32 91 4 51 12 38 5 0 0
Trailera0 – 0 0 0 – 0 0 0 – 0 0 0 – 0 0
Total 87 22 46 6 10 1 238 61 182 44 9 4 1677 213 879 93 100 5 135 36 94 21 3 1
Page 9 of 12
Schönherz et al. Vet Res (2016) 47:10
Figure3 Detected polymorphic positions with intermediate allele frequency (5–95%) representing the allele frequency of the domi-
nant allele (frequency between 50 and 95%). Colours of vertical lines indicate different genes: pink N, yellow P, red M, green G, light blue NV, blue
L, dotted vertical lines intergenic regions. Horizontal line represents the 95% frequency threshold. Red stars at top of vertical lines indicate amino acid
change in the dominant allele.
Figure4 Box plot of number of detected substitutions with intermediate allele frequencies (5–95%). Number of substitutions is
represented as a count of substitution events recorded for polymorphic sites with intermediate allele frequencies (5–95%). Accord‑
ingly, substitution events resulting into alleles with frequencies below 5% were ignored. A The substitution counts for the individual gene across all
four isolates for each of the six genes (four counts per gene). B Substitution counts for the individual isolate across all six genes for each of the four
isolates (six counts per isolate).
Page 10 of 12
Schönherz et al. Vet Res (2016) 47:10
to adapt to replication conditions in different cell types
or host species. Recently, a single amino acid substitu-
tion (I1012F) in the L protein was shown to be associated
with a change in the invitro virulence of an isolate of
VHSV obtained from marine fish. Following the substitu-
tion, this isolate was able to replicate in primary cultures
of rainbow trout gill epithelial cells. While the parental
isolate was avirulent to rainbow trout, virulence of the
recombinant variant invivo was not analysed [29]. e
present study included two isolates originating from wild
Atlantic herring (1p49 and 4p168) that are known to be
avirulent for rainbow trout [8, 30]. Sequences of these
isolates were mapped against those of an isolate of VHSV
originating from the marine environment. However, this
reference isolate (FA281107) originated from sea-reared
rainbow trout and represents the first VHSV isolate of
genotype III pathogenic to rainbow trout [13]. In both
herring isolates, isoleucine (I) was detected at amino acid
position 1012 in the L protein within the list of quality-
filtered variants at a frequency higher than 99%, while the
marine reference isolate known to be pathogenic to rain-
bow trout showed a phenylalanine (F) at this position. To
further investigate this aspect, we aligned the 23 VHSV L
protein sequences currently available at NCBI, and found
a general correlation with the presence of the I1012F
substitution when the host is rainbow trout (Figure5).
ere were, however, four exceptions which included the
virulent SVA-1033 isolate and two plaque clones of the
same isolate and a plaque clone of another mixed isolate
(SVA-14). Based on typing using monoclonal antibodies,
the Swedish isolates SVA-14 and SVA-1033 contains a
mixed virus population (N. Lorenzen unpublished data)
so the available Sanger consensus sequences might there-
fore be misleading, and the reason why plaque-cloning
attempts have been performed. Our findings show the
same trend as the invitro findings reported by Kim etal.
[29], although it remains to be proven e.g. by reverse
genetics that the single substitution I1012F facilitates
an increase in the invivo virulence of a marine strain of
VHSV for rainbow trout. Statistical comparison of local
substitution rates across isolates reveal that the 1p49 iso-
late indeed behaved differently compared to the other
three isolates. e high number of nucleotide substitu-
tions might indicate a low and unstable state within the
fitness landscape. While it cannot be excluded that the
higher stability of 4p168 isolate could be due to passage
of the latter in one cell line only (BF2) compared to two
for 1p49 (BF2 and EPC), the difference might also reflect
different host/environmental conditions at the geo-
graphical sites of virus isolation. Both marine isolates
were obtained from tissue pools of herring. However,
while isolate 4p168 was from Skagerrak, a region with
stable high salinity, limited fish species diversity and low
prevalence of VHSV occurring in only a few species, iso-
late 1p49 was from the Baltic Sea, characterized by high
fluctuations in salinity and with rather high prevalence of
VHSV in a range of different fish species [31]. In terms
of the two freshwater VHSV isolates, both derived from
serious disease outbreaks in freshwater-farmed rain-
bow trout, the observed differences in variability might
also be related to host conditions. Although isolate DK-
3592B displayed slightly higher nucleotide variability fol-
lowing frequency filtering, this was not reflected at the
amino acid level, where isolate DK-9895174 displayed the
Figure5 Alignment of all L protein sequences available in GenBank. Amino acid position 1012 is highlighted by the vertical box.
Page 11 of 12
Schönherz et al. Vet Res (2016) 47:10
highest variability. When looking at the data after qual-
ity filtering but before frequency filtering, the higher
variability of DK-9895174 became even more prominent
(Table4). While isolate DK-3592b was derived back in
1985, where almost all Danish VHSV isolates belonged to
a single serotype (based on a plaque neutralization test),
isolate DK-9895174 was isolated 14 years later, when
serotyping data revealed a higher frequency of isolates
not neutralized by reference antibodies raised against the
original VHSV F1 isolate [32, 33]. To confirm whether
our NGS data reflects viral quasispecies related adapta-
tions to selective pressures set by the host availability or
immune defence as discussed above will require more
extensive analyses.
Errors may occur at any of the many steps involved in
deep sequencing of the evolving pathogen population,
including RNA extraction; reverse transcription; PCR
amplification of target regions; library preparation and
sequencing; read quality control and filtering and map-
ping. In a deep sequencing analysis of a mixture of HIV
clones, the estimated PCR chimera rate was 1.9% [34],
while the average error rates when using the Illumina
platform have been estimated at between 0.31 and 1.66%
[35]. Estimation of the actual accumulated error rate in
the performed experimental setting is not possible at this
point, a number of internal controls (e.g. sample repli-
cates) is needed for this. Instead we filtered using con-
servative quality parameters as well as ignored variants at
frequencies below 5%.
In the present study, 0.5–1.9 million×coverage was
obtained. However, during the process of data analy-
sis, we realized that a number of control samples are
needed in order to be able to determine that mutations
detected at extremely low frequencies (under 1%) repre-
sent true polymorphisms. Such controls could consist of
plaque-cloned viral samples and control plasmids. ese
would provide the baseline for the error rate due to the
methods used. Once determined, the established deep-
sequencing methodology would prove very powerful for
analysis of tissue samples from viral adaptation studies.
With respect to the actual sample source, the use of an
enrichment approach, either by hybridization [36, 37]
or through enriching for virus-specific RNA by deplet-
ing host genomic DNA and rRNA, might be relevant to
explore as well [38]. Nevertheless, since all virus isolates
had been propagated invitro, our results may not fully
reflect how VHSV acts as a quasispecies in vivo. is
will need further deep sequencing analyses of VHSV
genomes directly derived from infected fish.
e overall aim of this study was to better understand
the potential for evolution and host adaption of VHSV
by applying a deep-sequencing approach. Based on the
multiple and unique results obtained during this study,
we find that the established deep sequencing and applied
bioinformatics- and statistical analysis methods are valu-
able, and will probably be even more when used in future
invivo host adaption studies of VHSV.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AAS prepared the RNA samples, generated the overlapping RT‑PCR amplifica‑
tions for library preparation and sequencing, performed the statistical analysis
and wrote significant parts of the manuscript, NL contributed to planning of
experiment and discussion of the results, BG assisted with the statistical analy‑
sis and discussion of results, BB contributed to the planning of the experiment
and discussion of the results, KEJ contributed to the planning of the experi‑
ment, performed the bioinformatic analysis and wrote significant parts of the
manuscript. All authors contributed to the manuscript. All authors read and
approved the final manuscript.
The authors thank Niels Jørgen Olesen (Technical University of Denmark) for
supplying the VHSV isolates, and Hanne Buchholz and Lisbeth Troels ( Techni‑
cal University of Denmark) for their excellent technical assistance. This work
was supported by Grants from The Danish Agency for Science, Technology
and Innovation (Grant Nos 09‑066097 and 09‑065033/FTP) and EU‑FP7 (PATH‑
SEEK, project reference 304875, and TargetFish, project reference 7311993).
Author details
1 Department of Molecular Biology and Genetics, Center for Quantitative
Genetics and Genomics, Aarhus University, Blichers Allé 20, P.O. Box 50,
8830 Tjele, Denmark. 2 Department of Animal Science, Aarhus University,
Blichers Allé 20, P.O. Box 50, 8830 Tjele, Denmark. 3 QIAGEN AAR, 8000 Århus,
Received: 2 February 2015 Accepted: 1 June 2015
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... However, heavily infected fish can carry the virus without showing any clinical signs (Cornwell, Eckerlin, et al., 2012;Groocock et al., 2012). Viral haemorrhagic septicaemia virus, like most RNA viruses, shows high evolutionary potential because of its high replication rate and mutation rate of 10 −3 -10 −5 mutations per nucleotide, per replication (Schönherz, Lorenzen, Guldbrandtsen, Buitenhuis, & Einer-Jensen, 2016 (Elsayed et al., 2006). Viral haemorrhagic septicaemia virus IVb spread quickly throughout multiple Great Lakes fish species. ...
... Reverse transcription was completed following the protocol described by Schönherz et al. (2016) and the instructions in the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). ...
... The 20 μl of VHSV genome cDNA was synthesized from the RNA extracted with the MagMax-96 viral RNA isolation kit as described above and stored at −80°C. The PCR was performed using a slight modification of the protocol used by Schönherz et al. (2016) and described by Getchell et al. (2017). Instead of using the described cycling programme, a touchdown programme was used to increase the stringency of the amplification. ...
Eleven viral haemorrhagic septicaemia virus (VHSV) genotype IVb isolates were sequenced, and their genetic variation explored to determine the source of a VHS outbreak on the eastern shore of Cayuga Lake. An active fish kill of round gobies (Neogobius melanostomus, Pallas) was intensively sampled at King Ferry, NY and nearby Long Point State Park in May 2017. Gross lesions observed on 67 moribund round gobies and two rock bass (Ambloplites rupestris, Rafinesque) included moderately haemorrhagic internal organs and erythematous areas on the head, flank, and fins. RT‐qPCR tests for VHSV were positive for all 69 fish. Viral isolation on epithelioma papulosum cyprinid cells showed cytopathic effect characteristic of VHSV for six round goby samples from King Ferry. The complete nucleotide sequence of the VHSV IVb genomes of five Cayuga Lake round goby isolates were derived on an Illumina platform along with 2017 VHSV IVb isolates from round gobies collected from the following: Lake Erie near Dunkirk, NY; the St. Lawrence River near Clayton and Cape Vincent, NY; and Lake St. Lawrence near Massena, NY. The phylogenetic tree created from these aligned sequences and four other complete VHSV IVb genomes shows Cayuga Lake isolates are closely related to the Lake Erie isolates.
... cDNA was synthesized from total RNA extracted from the tissue samples using SuperScript IV (Invitrogen), following manufacturer's instructions. Genomic cDNA was amplified in four segments using primers from Schönherz [59], substituting VHSV_Frag1I_nt18_+s with a more specific primer (5'GAGAGCTGCAGCACTTCACCG C3'), and 1 μl cDNA in 25 μl polymerase chain reactions (PCRs) with One Taq DNA polymerase (New England Biolabs). Amplicons were examined under UV light on 1% agarose gels stained with ethidium bromide. ...
... Additional PCRs were conducted to amplify the front 700 nucleotides (NTs) and end 400 NTs of the genome, with 45 s extension time, in order to complete the whole genomes. The front segment utilized VHSV_Frag1I_nt18_+s (5'GAGTTATGTTACARGGGACAGG3') [59] and anti-sense 5'TGACCGAGATGGCAGATC3', and end primers were designed by us based on the VHSV-IVb original reference genome (GenBank: GQ385941) (End sense: 5'CCCAG ATGCTATCACCGAGAA3', End anti-sense: 5'ACAAAGAATCCGAGGCAGGAG3'). Those cleaned products were Sanger-sequenced at Cornell DNA Services (Ithaca, NY), and checked and aligned by us. ...
Full-text available
A unique and highly virulent subgenogroup (-IVb) of Piscine novirhabdovirus, also known as Viral Hemorrhagic Septicemia Virus (VHSV), suddenly appeared in the Laurentian Great Lakes, causing large mortality outbreaks in 2005 and 2006, and affecting >32 freshwater fish species. Periods of apparent dormancy have punctuated smaller and more geographically restricted outbreaks in 2007, 2008, and 2017. In this study, we conduct the largest whole genome sequencing analysis of VHSV-IVb to date, evaluating its evolutionary changes from 48 isolates in relation to immunogenicity in cell culture. Our investigation compares genomic and genetic variation, selection, and rates of sequence changes in VHSV-IVb, in relation to other VHSV genogroups (VHSV-I, VHSV-II, VHSV-III, and VHSV-IVa) and with other Novirhabdoviruses. Results show that the VHSV-IVb isolates we sequenced contain 253 SNPs (2.3% of the total 11,158 nucleotides) across their entire genomes, with 85 (33.6%) of them being non-synonymous. The most substitutions occurred in the non-coding region (NCDS; 4.3%), followed by the Nv-(3.8%), and M-(2.8%) genes. Proportionally more M-gene substitutions encoded amino acid changes (52.9%), followed by the Nv-(50.0%), G-(48.6%), N-(35.7%) and L-(23.1%) genes. Among VHSV genogroups and sub-genogroups, VHSV-IVa from the northeastern Pacific Ocean has shown the fastest substitution rate (2.01x10-3), followed by VHSV-IVb (6.64x10-5) and by the VHSV-I,-II and-III genogroups from Europe (4.09x10-5). A 2016 gizzard shad (Dorosoma cepedianum) from Lake Erie possessed the most divergent VHSV-IVb sequence. The in vitro immunogenicity analysis of that sample displayed reduced virulence (as did the other samples from 2016), in comparison to the original VHSV-IVb isolate (which had been traced back to 2003, as an PLOS ONE
... 19 MinION technology offers important cost-and time-saving advantages over Sanger and Illumina systems, and thus could become instrumental for aquatic viral genotype and subtype characterization and monitoring in the future. 17,18,19,[27][28][29][30][31][32][33][34][35][36][37][38][39][40] In recent years, the application of viroinformatics (i.e., an amalgamation of virology with bioinformatics) has provided new ways to formulate scientific questions, thereby strengthening the progress of viral pathogenesis research. NGS applications involve the management of large datasets, requiring highly-specialized bioinformatics software and pipelines to process and analyze sequencing data using parallel computing (i.e., supercomputers). ...
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In this short review, we highlight the importance of combining cell culture and next-generation sequencing for the study of viruses in aquaculture. Moreover, we summarize some key examples of previously published studies that have implemented this approach and discuss the advantages of nanopore sequencing and other long-read sequencing technologies. With the rapid advances of genomic research, selection of the best tool to carry out analyses, it’s a computational challenge, but the potential for their applications is enormous. Therefore, this mini-review highlights important NGS bioinformatic tools and recent studies in cell culture for the study of aquatic virology.
... To date, there are several successful applications of next-generation sequencing (NGS), coupled with standard virus isolation technique, for discovery and characterizing genomes of novel piscine viral pathogens such as tilapia lake virus (TiLV) responsible for die-off in farmed tilapia in Israel (Bacharach et al., 2016), piscine reovirus and piscine totivirus causing HSMI (heart and skeletal muscle inflammation) and cardiomyopathy syndrome in salmon, respectively (Haugland et al., 2011;Palacios et al., 2010), and Lates calcarifer Birnavirus (LCBV) in farmed barramundi in Singapore (Chen et al., 2019). The rapid recovery of already-known virus genomes from infected samples can be a foremost benefit of NGS, which was successfully implemented to determine genome variation in cyprinid herpesvirus (CyHV)-2 (Davison et al., 2013), CyHV-3 (Hammoumi et al., 2016) and viral haemorrhagic septicaemia virus (Schonherz, Lorenzen, Guldbrandtsen, Buitenhuis, & Einer-Jensen, 2016) in goldfish, carp and rainbow trout, respectively, in a single sequencing run. ...
Scale drop disease virus (SDDV) is a novel viral pathogen considered to be distributed in farmed barramundi (Lates calcarifer) in South‐East Asia. Despite the severity of the disease, only limited genomic information related to SDDV is available. In this study, samples of SDDV‐infected fish collected in 2019 were used. The microbiome of brain tissue was investigated using Illumina HiSeq DNA sequencing. Taxonomic analysis showed that SDDV was the main pathogen contained in the affected barramundi. De novo metagenome assembly recovered the SDDV genome, named isolate TH2019, 131 kb in length, and comprised of 135 ORFs. Comparison between this genome and the Singaporean SDDV reference genome revealed that the nucleotide identity within the aligned region was 99.97%. Missense, frameshift, insertion and deletion mutations were identified in 26 ORFs. Deletion of four deduced amino acid sequence in ORF_030L, identical to the SDDV isolate previously identified in Thailand, would be a potential biomarker for future strain classification. Interestingly, the genome of SDDV TH2019 harboured a unique 7,695‐bp‐long genomic region containing six hypothetical protein‐encoded genes. Collectively, this study demonstrated that the SDDV genome can be sequenced directly, although with limited coverage depth, using metagenomic analysis of barramundi sample with severe infection.
... As reviewed in Quiñones-Mateu et al. (2014) [42], this approach is being increasingly used in diagnostic laboratories, given the significance of VAF in human clinical virology [43]. It would be of great interest to implement this practice on a routine basis also for animal viruses, where the selection at viral population level of signatures implicated in immune evasion and host jump are particularly relevant [44][45][46][47][48]. ...
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Background: Next generation sequencing (NGS) is becoming widely used among diagnostics and research laboratories, and nowadays it is applied to a variety of disciplines, including veterinary virology. The NGS workflow comprises several steps, namely sample processing, library preparation, sequencing and primary/secondary/tertiary bioinformatics (BI) analyses. The latter is constituted by a complex process extremely difficult to standardize, due to the variety of tools and metrics available. Thus, it is of the utmost importance to assess the comparability of results obtained through different methods and in different laboratories. To achieve this goal, we have organized a proficiency test focused on the bioinformatics components for the generation of complete genome sequences of salmonid rhabdoviruses. Methods: Three partners, that performed virus sequencing using different commercial library preparation kits and NGS platforms, gathered together and shared with each other 75 raw datasets which were analyzed separately by the participants to produce a consensus sequence according to their own bioinformatics pipeline. Results were then compared to highlight discrepancies, and a subset of inconsistencies were investigated more in detail. Results: In total, we observed 526 discrepancies, of which 39.5% were located at genome termini, 14.1% at intergenic regions and 46.4% at coding regions. Among these, 10 SNPs and 99 indels caused changes in the protein products. Overall reproducibility was 99.94%. Based on the analysis of a subset of inconsistencies investigated more in-depth, manual curation appeared the most critical step affecting sequence comparability, suggesting that the harmonization of this phase is crucial to obtain comparable results. The analysis of a calibrator sample allowed assessing BI accuracy, being 99.983%. Conclusions: We demonstrated the applicability and the usefulness of BI proficiency testing to assure the quality of NGS data, and recommend a wider implementation of such exercises to guarantee sequence data uniformity among different virology laboratories.
... Diverse approaches have been used in the evolutionary analysis and phylogenetic studies of salmonids viruses, such as IHNV, IPNV, and VHSV, using deep sequence data or evolutionary dynamics methods [11,[21][22][23]. Our study aimed to analyze the sequences of the Korean IHNV isolates and compare them with those of IHNV strains from other countries, especially other Asian isolates. ...
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Infectious hematopoietic necrosis virus (IHNV), one of the most important pathogenic fish viruses, affects trout fisheries and causes considerable economic losses. Currently, in Korea, more studies on IHNV infection are being reported. However, relatively less data is available on Korean isolates than on those from other countries. Few studies have focused on gene sequence analyses of IHNV glycoprotein (G) gene and almost none have focused on other gene fragments. Therefore, considering the dearth of adequate phylogenetic and genomic studies on Korean IHNV strains because of the lack of data, our study aimed to provide sufficient relevant data by sequencing the complete genome of the IHNV strain SNU1, which was recently isolated from a Korean rainbow trout farm. Moreover, we focused on expanding the perspectives on the phylogenesis of IHNV isolates from Korea and other Asian countries. IHNV was isolated from pooled hematopoietic tissue samples using Epithelioma papulosum cyprinid (EPC) cells, and phylogenetic analysis and genome study were conducted using complete G, N, and nonvirion (NV) gene sequences. Our main achievements were the development of a phylogenetic analytical method based on the NV gene and complete genome sequence analysis of the IHNV strain SNU1, which was compared with other Asian isolate sequences.
... VHS has been identified with genotypes I-III predominantly occurring in Europe and genotype IV in North America and Asia (Snow et al., 1999;Nishizawa et al., 2002;Kim et al., 2003;Einer-Jensen et al., 2004;Lumsden et al., 2007). Genotypes are correlated to geographic regions rather than host species but do not have any correlation to serotyping with neutralizing (G protein-specific) antibodies (Kahns et al., 2012;Einer-Jensen et al., 2014;Schönherz et al., 2016). Genotypes Ia, Ic and Id isolates from rainbow trout and genotypes Ib, Ie and II-IV isolates from marine species (Skall et al., 2005;Gadd et al., 2011). ...
During a fish disease outbreaks in 2015-2016 suspected to viral hemorrhagic septicemia virus (VHSV), moribund rainbow trout fries were obtained from fish farms. Samples of kidney, heart and spleen tissues were taken for examination. The full coding region of the G gene was amplified by RT-PCR reaction. Genotyping was carried out by phylogenetic analysis with reference sequences of four VHS genogroup. A phylogenetic tree was constructed with the maximum likelihood method and the Kumara 2-parameter substitution model by using MEGA software version 6.06. A maximum likelihood phylogenetic analysis reveals that all the Iranian VHS strains belonged to clade Ia-2 of genotype Ia. The intra-sequence comparison of Iranian VHS strains ranged from 99.1% to 100% (average was 99.55%). In this study, two strains (Chaharmahal and Bakhtiari (1) and Chaharmahal and Bakhtiari (3) showed 100% identity but 0.1% differences with Isfahan strain. The Iranian strains belonging to European genogroup are related (99% identity) to VHSV from recent Italian strains (VHSV/O.mykiss/I/TN/133/Apr10).
Teleost fish are indispensable model organisms for comparative immunology research that should lead to an improved understanding of the general principles of vertebrate immune system design. Although numerous studies on fish immunology have been conducted, knowledge about the cell types that orchestrate piscine immune systems remains limited. Here, we generated a comprehensive atlas of immune cell types in zebrafish spleen on the basis of single-cell transcriptome profiling. We identified 11 major categories from splenic leukocyte preparations, including neutrophils, natural killer cells, macrophages/myeloid cells, T cells, B cells, hematopoietic stem and progenitor cells, mast cells, remnants of endothelial cells, erythroid cells, erythroid progenitors, and a new type of serpin-secreting cells. Notably, we derived 54 potential subsets from these 11 categories. These subsets showed differential responses to spring viremia of carp virus (SVCV) infection, implying that they have diverse roles in antiviral immunity. Additionally, we landscaped the populations with the induced expression of interferons and other virus-responsive genes. We found that trained immunity can be effectively induced in the neutrophil and M1-macrophage subsets by vaccinating zebrafish with inactivated SVCV. Our findings illustrated the complexity and heterogeneity of the fish immune system, which will help establish a new paradigm for the improved understanding of fish immunology.
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Piscine novirhabdovirus = Viral Hemorrhagic Septicemia Virus (VHSV) first appeared in the Laurentian Great Lakes with large outbreaks from 2005 to 2006, as a new and novel RNA rhabdovirus subgenogroup (IVb) that killed >30 fish species. Interlude periods punctuated smaller more localized outbreaks in 2007, 2010, and 2017, although some fishes tested positive in the intervals. There have not been reports of outbreaks or positives from 2018, 2019, or 2020. Here, we employ a combined population genetics and phylogenetic approach to evaluate spatial and temporal evolutionary trajectory on its G‐gene sequence variation, in comparison with whole‐genome sequences (11,083 bp) from a subset of 44 individual isolates (including 40 newly sequenced ones). Our results show that IVb (N = 184 individual fish isolates) diversified into 36 G‐gene haplotypes from 2003 to 2017, stemming from two originals (“a” and “b”). G‐gene haplotypes “a” and “b” differed by just one synonymous single‐nucleotide polymorphism (SNP) substitution, remained the most abundant until 2011, then disappeared. Group “a” descendants (14 haplotypes) remained most prevalent in the Upper and Central Great Lakes, with eight (51%) having nonsynonymous substitutions. Group “b” descendants primarily have occurred in the Lower Great Lakes, including 22 haplotypes, of which 15 (68%) contained nonsynonymous changes. Evolutionary patterns of the whole‐genome sequences (which had 34 haplotypes among 44 isolates) appear congruent with those from the G‐gene. Virus populations significantly diverged among the Upper, Central, and Lower Great Lakes, diversifying over time. Spatial divergence was apparent in the overall patterns of nucleotide substitutions, while amino acid changes increased temporally. VHSV‐IVb thus significantly differentiated across its less than two decades in the Great Lakes, accompanied by declining outbreaks and virulence. Continuing diversification likely allowed the virus to persist at low levels in resident fish populations, and may facilitate its potential for further and future spread to new habitats and nonacclimated hosts. This study analyses the evolutionary trajectory of a novel virus over its 17‐year history, examining the dynamics of its host relationships and its patterns across geographic space and evolutionary time. We sequence its entire genome and discern that the virus has significantly changed, with its original genotypes disappearing and being replaced in a quasispecies trajectory. It also has become less virulent, appearing to adapt for long‐term persistence at low levels in the host populations.
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Viral Hemorrhagic Septicemia Virus (VHSV) (Piscine novirhabdovirus) appeared in the Laurentian Great Lakes in 2005, constituting a unique and highly virulent genogroup (IVb), which killed >32 fish species in large 2005 and 2006. Periods of apparent dormancy punctuated smaller outbreaks in 2007, 2008, and 2017. We conducted the first whole genome analysis of IVb, evaluating its evolutionary changes using 46 isolates, in reference to immunogenicity in cell culture, and the genomes of other VHS genogroups (I–IVa) and other Novirhabdoviruses. IVb isolates had 253 genomic nucleotide substitutions (2.3% of the total 11,158 nucleotides), with 85 (16.6%) being non-synonymous. The greatest number of substitutions occurred in the non-coding region (NCDS; 4.3%) followed by the Nv- (3.8%), and M- (2.8%) genes. The M-gene possessed the greatest proportions of amino acid changes (52.9%), followed by the Nv- (50.0%), G- (48.6%), N- (35.7%) and L- (23.1%) genes. Among VHS genogroups, IVa from the northeastern Pacific exhibited the fastest substitution rate (2.01x10-3), followed by IVb (6.64x10-5), and I/III from Europe (4.09x10-5). A 2016 gizzard shad isolate from Lake Erie was the most divergent IVb isolate (38 NT, 15.0%, 15 AA), yet exhibited reduced virulence with in vitro immunogenicity analyses, as did other 2016 isolates, in comparison to the first IVb isolate (2003). The 2016 isolates exhibited a lower impact on innate antiviral responses, suggesting phenotypic effects. Results suggest continued sequence change and lower virulence over the history of IVb, which may facilitate its long-term persistence in fish host populations.
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Chlamydia trachomatis is a pathogen of worldwide importance, causing more than 100 million cases of sexually transmitted infections annually. Whole-genome sequencing is a powerful high resolution tool that can be used to generate accurate data on bacterial population structure, phylogeography and mutations associated with antimicrobial resistance. The objective of this study was to perform whole-genome enrichment and sequencing of C. trachomatis directly from clinical samples. C. trachomatis positive samples comprising seven vaginal swabs and three urine samples were sequenced without prior in vitro culture in addition to nine cultured C. trachomatis samples, representing different serovars. A custom capture RNA bait set, that captures all known diversity amongst C. trachomatis genomes, was used in a whole-genome enrichment step during library preparation to enrich for C. trachomatis DNA. All samples were sequenced on the MiSeq platform. Full length C. trachomatis genomes (>95-100% coverage of a reference genome) were successfully generated for eight of ten clinical samples and for all cultured samples. The proportion of reads mapping to C. trachomatis and the mean read depth across each genome were strongly linked to the number of bacterial copies within the original sample. Phylogenetic analysis confirmed the known population structure and the data showed potential for identification of minority variants and mutations associated with antimicrobial resistance. The sensitivity of the method was >10-fold higher than other reported methodologies. The combination of whole-genome enrichment and deep sequencing has proven to be a non-mutagenic approach, capturing all known variation found within C. trachomatis genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of C. trachomatis directly from clinical samples and has the potential to be adapted to other pathogens with a similar clonal nature.
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Unlabelled: Viral hemorrhagic septicemia virus (VHSV) is separated into four different genotypes (I to IV) with different sublineages (K. Einer-Jensen, P. Ahrens, R. Forsberg, and N. Lorenzen, J. Gen. Virol. 85:1167-1179, 2004; K. Einer-Jensen, J. Winton, and N. Lorenzen, Vet. Microbiol. 106:167-178, 2005). European marine VHSV strains (of genotypes I to III) are, in general, nonpathogenic or have very low pathogenicity to rainbow trout after a waterborne challenge, and here we also show that genotype IVa is nonpathogenic to trout. Despite several attempts, it has not been possible to link genomic variation to in vivo virulence. In vitro virulence to gill epithelial cells (GECs) has been used as a proxy for in vivo virulence, and here we extend these studies further with the purpose of identifying residues associated with in vitro virulence. Genotype Ia (DK-3592B) and III (NO/650/07) isolates, which are pathogenic to rainbow trout (O. B. Dale, I. Orpetveit, T. M. Lyngstad, S. Kahns, H. F. Skall, N. J. Olesen, and B. H. Dannevig, Dis. Aquat. Organ. 85:93-103, 2009), were compared to two marine strains that are nonpathogenic to trout, genotypes Ib (strain 1p8 [H. F. Mortensen, O. E. Heuer, N. Lorenzen, L. Otte, and N. J. Olesen, Virus Res. 63:95-106, 1999]) and IVa (JF-09). DK-3592 and NO/650/07 were pathogenic to GECs, while marine strains 1p8 and JF-09 were nonpathogenic to GECs. Eight conserved amino acid substitutions contrasting high- and low-virulence strains were identified, and reverse genetics was used in a gain-of-virulence approach based on the JF-09 backbone. Mutations were introduced into the G, NV, and L genes, and seven different virus clones were obtained. For the first time, we show that a single amino acid mutation in conserved region IV of the L protein, I1012F, rendered the virus able to replicate and induce a cytopathic effect in trout GECs. The other six mutated variants remained nonpathogenic. Importance: This is the first study to clearly link in vitro virulence of viral hemorrhagic septicemia virus (VHSV) with an amino acid residue in the L protein, a site located in conserved region IV of the L protein. In vitro virulence is documented by induction of cytopathic effects and viability studies of gill epithelial cells, and the observed cellular responses to infection are associated with increased viral replication levels. There are no previous studies addressing the importance of the L protein or the RNA-dependent RNA polymerase for virus virulence in vitro or in vivo. Therefore, the findings reported here should broaden the search for pathogenicity traits in novirhabdoviruses, and there is a possibility that the polymerase participates in defining the host species virulence of various VHSV strains.
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One of the hurdles to understanding the role of viral quasispecies in RNA virus cross-species transmission (CST) events is the need to analyze a densely sampled outbreak using deep sequencing in order to measure the amount of mutation occurring on a small time scale. In 2009, the California Department of Public Health reported a dramatic increase (350) in the number of gray foxes infected with a rabies virus variant for which striped skunks serve as a reservoir host in Humboldt County. To better understand the evolution of rabies, deep-sequencing was applied to 40 unpassaged rabies virus samples from the Humboldt outbreak. For each sample, approximately 11 kb of the 12 kb genome was amplified and sequenced using the Illumina platform. Average coverage was 17,448 and this allowed characterization of the rabies virus population present in each sample at unprecedented depths. Phylogenetic analysis of the consensus sequence data demonstrated that samples clustered according to date (1995 vs. 2009) and geographic location (northern vs. southern). A single amino acid change in the G protein distinguished a subset of northern foxes from a haplotype present in both foxes and skunks, suggesting this mutation may have played a role in the observed increased transmission among foxes in this region. Deep-sequencing data indicated that many genetic changes associated with the CST event occurred prior to 2009 since several nonsynonymous mutations that were present in the consensus sequences of skunk and fox rabies samples obtained from 20032010 were present at the sub-consensus level (as rare variants in the viral population) in skunk and fox samples from 1995. These results suggest that analysis of rare variants within a viral population may yield clues to ancestral genomes and identify rare variants that have the potential to be selected for if environment conditions change.
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The serological variation among 127 isolates of viral hemorrhagic septicaemia virus (VHSV) was examined by plaque neutralization tests (PNT) using a panel of 4 neutralizing monoclonal antibodies (MAbs) and 1 neutralizing rabbit antiserum (PAb). Three distinct neutralization patterns were observed. All isolates in the largest group (I) were neutralized at high titres by the MAbs and the PAb. This group included isolates representing the previously described 'Serotypes' 1 and 2. The isolates in Group II were efficiently neutralized by 1 of the MAbs and the PAb but not, unless at low titres, by the remaining 3 MAbs. Isolates represented by previously described 'serotype' 3 were in this group. The isolates in Group III were not neutralized at all by any of the MAbs and not, or only moderately, neutralized by the PAb. Isolates with this reaction pattern have become the most frequent ones from VHS outbreaks in Denmark in recent years. Pronounced crossreaction between Groups I, II & III in Western blotting using polyclonal rabbit antibodies combined with the ability of 1 antiserum to neutralize 120 out of the 127 isolates indicate that the polyclonal antibodies used define only 1 serotype of VHSV. Groups I to III should be regarded as subtypes. The present panel of antibodies did not allow a distinction between pathogenic and nonpathogenic isolates. No relationship was observed between host species and the reaction pattern as all 3 reaction patterns were present among the 5 isolates originating from fish species other than rainbow trout Oncorhynchus mykiss.
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With the advent of Next Generation Sequencing (NGS) technologies, the ability to generate large amounts of sequence data has revolutionized the genomics field. Most RNA viruses have relatively small genomes in comparison to other organisms and as such, would appear to be an obvious success story for the use of NGS technologies. However, due to the relatively low abundance of viral RNA in relation to host RNA, RNA viruses have proved relatively difficult to sequence using NGS technologies. Here we detail a simple, robust methodology, without the use of ultra-centrifugation, filtration or viral enrichment protocols, to prepare RNA from diagnostic clinical tissue samples, cell monolayers and tissue culture supernatant, for subsequent sequencing on the Roche 454 platform. As representative RNA viruses, full genome sequence was successfully obtained from known lyssaviruses belonging to recognized species and a novel lyssavirus species using these protocols and assembling the reads using de novo algorithms. Furthermore, genome sequences were generated from considerably less than 200 ng RNA, indicating that manufacturers' minimum template guidance is conservative. In addition to obtaining genome consensus sequence, a high proportion of SNPs (Single Nucleotide Polymorphisms) were identified in the majority of samples analyzed. The approaches reported clearly facilitate successful full genome lyssavirus sequencing and can be universally applied to discovering and obtaining consensus genome sequences of RNA viruses from a variety of sources.
A sanitation programme for stamping-out viral haemorrhagic septicaemia (VHS) was implemented in Denmark in 1965. The programme has resulted in a dramatic reduction in the number of infected rainbow trout farms, from ~400 to 26. The programme is carried out on a voluntary basis at the expense of the involved fish farm owners. The fact that financial support for the eradication of VHS and IHN may be made available from the European Union may enhance the efforts towards further eradication of the disease in Europe. The finding of a VHS virus (VHSV)-like virus in the marine environment is a matter of major concern, but improved diagnostic procedures may enable discrimination between the highly pathogenic freshwater strains and the apparently less virulent marine strains.
Rabbit antisera against viral hemorrhagic septicemia virus (VHSV) produced by two immunization procedures were compared for neutralization and immunochemical properties against homologous and heterologous strains. The VHSV isolate used as the immunogen was a member of a serogroup not neutralized by previously available antisera. The results from this study suggested that frequent intravenous (IV) injections of rabbits with viral antigens were superior to adjuvant-mediated, combined subcutaneous and intraperitoneal (SC/IP) injections for the production of neutralizing antisera. All IV injected rabbits produced high neutralization titers against the homologous VHSV isolate but not against an isolate from a different serogroup. The SC/IP injected rabbits had no significant neutralization titers against either the homologous VHSV strain or two isolates of a heterologous VHSV strain. Sera from all injected rabbits reacted in indirect immunofluorescence (IF) assays with either strain; however, the SC/IP injected rabbits produced higher titers against the heterologous VHSV strain by ELISA (enzyme-linked immunosorbent assay). By Western blotting, neutralizing antisera primarily stained the viral glycoprotein (G) whereas the nonneutralizing sera stained all the vital structural proteins equally well. Our results demonstrate that immunization procedures to produce antisera against VHSV in rabbits determine whether the resultant antibodies will have primarily neutralizing or binding capabilities.