Porcine cytomegalovirus detection by nanopore-based metagenomic sequencing in a Hungarian pig farm

Abstract

The rapid diagnosis of infectious diseases has an essential impact on their control, treatment, and recovery. Oxford Nanopore Technologies (ONT) sequencing opens up a new dimension in applying clinical metagenomics. In a large-scale pig farm in Hungary, four fattening and one piglet nasal swab pooled samples were sequenced using ONT for metagenomic analysis. Long reads covering 53.69% of the porcine cytomegalovirus genome were obtained in the piglet sample. The 650 bp long read matching the glycoprotein B gene of the virus is sequentially most similar to Japanese, Chinese and Spanish isolates.

Full text

Porcine cytomegalovirus detection by
nanopore-based metagenomic sequencing in a
Hungarian pig farm
Adrienn Gr´
eta T ´
oth1, Regina Fiam2,3,´
Agnes Becsei2, S´
andor Spis´
ak4, Istv´
an Csabai2,
L´
aszl ´
o Makrai5, Tam´
as Reibling1, and Norbert Solymosi*1
1Centre for Bioinformatics, University of Veterinary Medicine, 1078 Budapest, Hungary
2Department of Physics of Complex Systems, E¨
otv¨
os Lor´
and University, 1117 Budapest, Hungary
3Saint Ignatius Jesuit College of Excellence, 1085 Budapest, Hungary
4Institute of Enzymology, Research Centre for Natural Sciences, 1117 Budapest, Hungary
5
Department of Microbiology and Infectious Diseases, University of Veterinary Medicine, 1143 Budapest, Hungary
*solymosi.norbert@gmail.com
ABSTRACT
The rapid diagnosis of infectious diseases has an essential impact on their control, treatment and recovery. Oxford Nanopore
Technologies (ONT) sequencing opens up a new dimension in applying clinical metagenomics. In a large-scale pig farm in
Hungary, four fattening and one piglet nasal swab pooled samples were sequenced using ONT for metagenomic analysis. Long
reads covering 53.69% of the porcine cytomegalovirus genome were obtained in the piglet sample. The 650 bp long read
matching the glycoprotein B gene of the virus is sequentially most similar to Japanese, Chinese and Spanish isolates.
Introduction
In both human and animal health, the rapid diagnosis of diseases, including infectious diseases, has a crucial impact on their
treatment and recovery. Oxford Nanopore Technologies (ONT) sequencing opens up a new dimension in the application of
clinical metagenomics
1
in veterinary medicine
2
. This third-generation sequencing technique can rapidly provide information on
specific samples’ microbial components in a few hours to some days. While the preceding and parallel NGS (next-generation
sequencing) methodologies provide higher sequence detection reliability, the sequencing time does not allow rapid microbial
diagnostics in practice.
The results presented here are from a study aimed at gaining experience in the clinical metagenomic applicability of ONT
in veterinary medicine. Here, we present only the main virological result of veterinary relevance: the detection of porcine
cytomegalovirus (PCMV) sequences. The infection occurs in almost all pig populations, but clinical disease is rare, except in
young piglets, where it can be fatal.
3
However, since xenotransplantation from PCMV-infected pigs affects the recovery and
survival of human patients, screening these donor animals has become an important issue.4–6
Although the virus has already been identified by PCR in Hungary
7
, a phylogenetic comparison of its sequence has not yet
been performed. In addition to ONT-based viral detection, the similarity of its genome sequence to the available genomes is
studied in this work.
Materials and Methods
Nasal swab samples were collected from 5-week-old piglets of the same stable and from 16 (sample id: 3, 4) and 19-week-old
(id: 1, 2) fattening pigs of two-two boxes of two stables from a Hungarian large-scale swine farm located near the town of
Szekszárd on 21 November 2022. After sample collection, the nasal swabs were transported on ice and stored at -20 °C before
the laboratory procedures. Porcine nasal swabs were pooled in nuclease-free molecular biology water as follows. Each five
fattening pig samples deriving from the same stable and box, and two piglet pools of four-four piglet samples were created.
DNA extraction and metagenomics library preparation
DNA extraction was performed with QIAamp Fast DNA Stool Mini Kit from Qiagen. The concentrations of the extracted DNA
solutions were evaluated with an Invitrogen Qubit 4 Fluorometer using the Qubit dsDNA HS (High Sensitivity) Assay Kit. The
concentrations of the 2 piglet samples were insufficient for library preparation. Thus, the two extracted piglet-deriving DNA
solutions were pooled and concentrated with a vacuum concentrator. Consequently, the library preparation was conducted
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on DNA deriving from four fattening pig nasal swab samples and one piglet nasal swab sample. The metagenomic long-read
library was prepared by the Ligation Sequencing Kit (SQK-LSK110) combined with the PCR-free Native Barcoding Expansion
1-12 (EXP-NBD104) from ONT. The sequencing was implemented with a MinION Mk1C sequencer using an R9.4.1. flow cell
from ONT.
Bioinformatic analysis
From the generated FAST5 files, one fast (configuration file:
dna_r9.4.1_450bps_fast_mk1c.cfg
) and one high-
accuracy (configuration file:
dna_r9.4.1_450bps_hac_mk1c.cfg
) base calling was performed by ONT’s Guppy
basecaller (v6.4.2,
https://nanoporetech.com/community
). The further analytical steps were done using the
two-way called sequences parallel. The raw reads were adapter trimmed and quality-based filtered by Porechop (v0.2.4,
https://github.com/rrwick/Porechop
) and Nanofilt (v2.6.0)
8
, respectively. The resulting reads were taxonomi-
cally classified using Kraken2
9
with the NCBI non-redundant nucleotide database
10
. Evaluating the taxon hits, the cleaned reads
were mapped to the reference genome of Suid betaherpesvirus 2 (KF017583.1) by minimap2.
11,12
The sequence matching the
glycoprotein B gene (
gB
) was used in the phylogenetic analysis. For that purpose, we used the available (19/12/2022) sequences
with complete or partial
gB
CDSs. By blastn (BLAST v2.13.0+)
13
with default settings, pairwise alignments were performed to
identify the matching range and strand of the subject sequences. By the MUSCLE aligner (v3.8.1551)
14
, multiple sequence
alignment was performed on the cropped subject and the query sequences. To construct a maximum-likelihood tree
15
, the func-
tion pml, optim.pml (
model=’JC’
,
optNNi=TRUE
,
optBf=TRUE
,
optQ=TRUE
,
optInv=TRUE
,
optGamma=TRUE
,
optEdge=TRUE
) from the phangorn (v2.10) package was applied.
16
All data processing and visualization were performed in
the R environment.17
Results
In total, 5.94 and 5.97 gigabases of data were generated during the 72 hours of sequencing, according to fast and high-accuracy
basecalling. The descriptive statistics of the sequences generated by the two basecalling procedures are summarized in Figure 1.
The high-accuracy basecalling generated slightly more nucleotides and longer reads, while the number of reads was equal.
Gigabase
Median read length
Read count (M)
fattening 1 fattening 2 fattening 3 fattening 4 piglet unclassified
0.0
0.5
1.0
1.5
0
250
500
750
1000
0.0
0.5
1.0
1.5
Barcode
Basecall
fast
hac
Figure 1. Descriptive statistics of raw reads. The high-accuracy (hac) basecalling, compared to the fast one, generated slightly
more nucleotides and longer reads, while the number of reads was equal.
In the sample fattening 1, 2, 3, 4, the matched read number on the Suid betaherpesvirus 2 reference genome was 2, 5, 8, 1,
respectively. None of the four fattening samples had a read that matched
gB
. Of the unclassified (without barcode) reads, 25
matched the genome, and no hit was on gene
gB
. In the piglet sample, 315 reads aligned to the reference genome, covering
53.69% of it.
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One sequence overlaps the
gB
gene and covers the region between nucleotides 49507 and 50172 of the representative
reference genome. Where the identity was 631/671 (94%) and 641/665 (96%) with gaps 32/671 (4%) and 18/665 (2%) for
the fast and high-accuracy basecalled sequence, respectively. The high-accuracy called sequence similarity with other
gB
sequences is shown by cladogram in Fig 2. Based on sequence similarity, the multiple sequence alignment of the closest strains
is shown in Fig 3. The deletions identified in our sample are not found in the other
gB
genes. In addition to the deletions, only
two positions (insertions at 87 and 139) of the sequential variations are shown in Fig 3, where no other
gB
gene modifications
were found.
AF394056.1
FJ870561.1
FJ844360.1
KX575702.1
KX575699.1
KX575694.1
KX575686.1
KX575684.1
KX575682.1
KX575680.1
KX575679.1
KX575678.1
KX575676.1
KX575675.1
KX575674.1
KX575672.1
KX575671.1
KX575670.1
KX575668.1
KX575667.1
KX575666.1
KX575701.1
KC342285.1
KC342275.1
KC342269.1
KC342268.1
KC342284.1
KC342274.1
KC342283.1
KC342279.1
KC342282.1
KC342280.1
KC342286.1
KC342267.1
KC342288.1
KC342287.1
KC342281.1
KC342278.1
KC342276.1
KC342273.1
KC342272.1
KC342270.1
JN701021.1
KC342266.1
KC342271.1
KX575691.1
KX575688.1
strain_RT
KX575698.1
KX575693.1
AF268040.2
KX575690.1
KX575689.1
LC064808.1
KF017583.1
AF268041.2
FJ870563.1
KX575692.1
KX575708.1
KX575707.1
KX575706.1
KX575705.1
KX575704.1
KX575703.1
KX575700.1
KX575695.1
KX575687.1
KX575685.1
KX575683.1
KX575681.1
KX575677.1
KX575673.1
KX575669.1
KX575697.1
KX575665.1
KX575696.1
EF460488.1
HQ686080.1
FJ595497.1
FJ870562.1
FJ870564.1
AB771707.1
KC342277.1
KC342289.1
AB771708.1
AF268039.2
AF394057.1
HQ686081.1
AB771706.1
Origin
China
Hungary
Japan
South Korea
Spain
United Kingdom
NA
Figure 2. Cladogram based on gB gene sequence similarity. The strain_RT is the sequence obtained by high-accuracy
basecalling.
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strain_RT_fast TCTGTTCATTAAATACCTCTTTAGAAGTTCCATTAAAAATGGCTGATATCTCCAACCGATTCACCTGTACCGAGTATANAATTATN87
strain_RT_hac TCTGTTCATTAAATACCTCTTTAGAAGTTCCATTAAAAAATGGTGATATCTNCAACCGATTCACCTGTACCGAGTATAAAATTATC87
AF268040.2 TCTGTTCATTAAATACCTCTTTAGAAGTTCCATTAAAAAATGGTGATATCTNCAACCGATTCACCCGTACCGAGTATAAAATTATN87
FJ870563.1 TCTGTTCATTAAATACCTCTTTAGAAGTTCCATTAAAAAATGGTGATATCTNCAACCGATTCACCTGTACCGAGTATAAAATTATN87
LC064808.1 TCTGTTCATTAAATACTTCTTTAGAAGTTCCATTAAAAAATGGTGATATCTNCAACCGATTCACCTGTACCGAGTATAAAATTATN87
KF017583.1 TCTGTTCATTAAATACTTCTTTAGAAGTTCCATTAAAAAATGGTGATATCTNCAACCGATTCACCTGTACCGAGTATAAAATTATN87
AF268041.2 TCTGTTCATTAAATACTTCTTTAGAAGTTCCATTAAAAAATGGTGATATCTNCAACCGATTCACCTGTACCGAGTATAAAATTATN87
strain_RT_fast CATATGGGTAATCAGATTTACCAATAGTATCAGTTACTATGCAATTAATAGAAAGATGAGCTTTTATATAACCAATGGGTTCCATA 173
strain_RT_hac CATATGGGTAATCAGATTTACCAATAGTATCAGTTACTATGCAATTAATAGAAAGATGAGCTTTTATATAACCAATGGGTTCCATA 173
AF268040.2 CATATGGGTAATCAGATTTACCCATAGTATCAGTTATTATGCAATTAATAGNAAGATGAGCTTTTATATAACCAATGGGTTCCATA 173
FJ870563.1 CATATGGGTAATCAGATTTACCCATAGTATCAGTTATTATGCAATTAATAGNAAGATGAGCTTTTATATAACCAGTGGGTTCCATA 173
LC064808.1 CATATGGGTAATCAGATTTACCCATAGTATCAGTTACTATGCAATTAATAGNAAGATGAGCTTTTATATAACCAATGGGTTCCATA 173
KF017583.1 CATATGGGTAATCAGATTTACCCATAGTATCAGTTACTATGCAATTAATAGNAAGATGAGCTTTTATATAACCAATGGGTTCCATA 173
AF268041.2 CATATGGGTAATCAGATTTACCCATAGTATCAGTTACTATGCAATTAATAGNAAGATGAGCTTTTATATAACCAATGGGTTCCATA 173
strain_RT_fast TGTGAACTGAAAATCAGGCGTGGATATGTAACGAGTATTAATAGTAGATCCAAATTTCAAACGATATATTTGTATTGTNNNNTTTG 259
strain_RT_hac TGTGAACTGAAAATCAGGCGTGGATATGTAACGAGTATTAATAGTAGATCCAAATTTCAAACGATATATNNNNNTTGTATTGTTTG 259
AF268040.2 TGTGAACTGAAAATCAGGCGTGGATATGTAACGAGTATTAATAGTAGATCCAAATTTCAAACGATATAATCTCATTGTATTGTTTG 259
FJ870563.1 TGTGAACTGAAAATCAGGCGTGGATATGTAACGAGTATTAATAGTAGATCCAAATTTCAAACGATATAATCTCATTGTATTGTTTG 259
LC064808.1 TGTGAACTGAAAATCAGGCGTGGATATGTAACGAGTATTAATAGTAGATCCAAATTTCAAACGATATAATCTCATTGTATTGTTTG 259
KF017583.1 TGTGAACTGAAAATCAGGCGTGGATATGTAACGAGTATTAATAGTAGATCCAAATTTCAAACGATATAATCTCATTGTATTGTTTG 259
AF268041.2 TGTGAACTGAAAATCAGGCGTGGATATGTAACGAGTATTAATAGTAGATCCAAATTTCAAACGATATAATCTCATTGTATTGTTTG 259
strain_RT_fast TATTATCATCTTTATGATATACCCGATAATTTATTCCCTGGTTTCTAATCTCGGCAGCGGAAAAAACATTGACCATTCAAATTTAT 345
strain_RT_hac TATTATCATCTTTATGATATACCCGATAATTTATTCCCTGGTTTCTAATCTCGGCAGCGGNAAAAACATTGACCATTCAAATTTAT 345
AF268040.2 TATTATCATCTTTATGATATACACGATAATTTATTCCCTGGTTTCTAATCTCGGCAGCGGNAAAAACATTGACCATTTAAATTTAT 345
FJ870563.1 TATTATCATCTTTATGATATACCCGATAATTTATTCCCTGGTTTCTAATCTCGGCAGCGGNAAAAACATTGACCATTCAAATTTAT 345
LC064808.1 TATTATCATCTTTATGATATACCCGATAATTTATTCCCTGGTTTCTAATCTCGGCAGCGGNAAAAACATTGACCATTCAAATTTAT 345
KF017583.1 TATTATCATCTTTATGATATACCCGATAATTTATTCCCTGGTTTCTAATCTCGGCAGCGGNAAAAACATTGACCATTCAAATTTAT 345
AF268041.2 TATTATCATCTTTATGATATACCCGATAATTTATTCCCTGGTTTCTAATCTCGGCAGCGGNAAAAACATTGACCATTCAAATTTAT 345
strain_RT_fast ATATCCAGCTTCATCTACAGGAACGGGCACTTTATATGAAGACCTATCAACGAGATAGATGACATGAACGTCTCTATACGTCGTTT 431
strain_RT_hac ATATCCAGCTTCATCTACAGGAACGGGCACTTTATATGAAGACCTATCAACGAGATAGATGACATGAACGTCTCTATACGTCGTTT 431
AF268040.2 ATATCCAGCTTCATCTACAGGAACGGGCACTTTATATGAAGACCTATCAACAAGATAGATGACATGAACGTCTCTATACGTCGTTT 431
FJ870563.1 ATATCCAGCTTCATCTACAGGAACGGGCACTTTATATGAAGACCTATCAACAAGATAGATGACATGAACGTCTCTATACGTCGTTT 431
LC064808.1 ATATCCAGCTTCATCTACAGGAACGGGCACTTTATATGAAGACCTATCAACGAGATAGATGACATGAACGTCTCTATACGTCGTTT 431
KF017583.1 ATATCCAGCTTCATCTACAGGAACGGGCACTTTATATGAAGACCTATCAACAAGATAGATGACATGAACGTCTCTATACGTCGTTT 431
AF268041.2 ATATCCAGCTTCATCTACAGGAACGGGCACTTTATATGAAGACCTATCAACAAGATAGATGACATGAACGTCTCTATACGTCGTTT 431
strain_RT_fast GAAACGACAATTCCTTGGTATACGTCTAACAAAAAAAAATTATTGCGGAACTATANTTTTCTTAAACAATAACAAAATACCTTCCA 517
strain_RT_hac GAAACGACAATTCCTTGGTATACATCCTAACAAAAAAAGTNNGTGCGGAACTATATTTTTCTTAAACAATAACNAAATACCTTNCA 517
AF268040.2 GAAACGACAATTCCTTGGTATACGTCCTAACAAAAAAAGTNNGTGCGGAACTATATTTTTCTTAAACAATAACAAAATACCTTNCA 517
FJ870563.1 GAAACGACAATTCCTTGGTATACGTCCTAACAAAAAAAGTNNGTGCGGAACTATATTTTTCTTAAACAATAACAAAATACCTTNCA 517
LC064808.1 GAAACGACAATTCCTTGGTATACGTCCTAACAAAAAAAGTNNGTGCGGAACTATATTTTTCTTAAACAATAACAAAATACCTTNCA 517
KF017583.1 GAAACGACAATTCCTTGGTATACGTCCTAACAAAAAAAGTNNGTGCGGAACTATATTTTTCTTAAACAATAACAAAATACCTTNCA 517
AF268041.2 GAAACGACAATTCCTTGGTATACGTCCTAACAAAAAAAGTNNGTGCGGAACTATATTTTTCTTAAACAATAACAAAATACCTTNCA 517
strain_RT_fast GAATACTGANNNNNNNNNNNTTACTTATTACAAGTGATATAATTATNAAATCTATATAGATCTGTTCCAACGACACATATTGCATA 603
strain_RT_hac GAATACTGAAGNNNNNNNNNATACTTATTACAAGTGATATAATTATNAAATCTATATAGATCTGTTCCAACGGCNCATATTGCATA 603
AF268040.2 GAATACTGAGTCTCCGTATCATACTTATTACAAGTGATATAATTATCAAATCTATATAGATCTGTTCCAACGGCNCATATTGCATA 603
FJ870563.1 GAATACTGAGTCTCCGTATCATACTTATTACAAGTGATATAATTATCAAATCTATATAGATCTGTTCCAACGGCNCATATTGCATA 603
LC064808.1 GAATACTGAGTCTCCGTATCATACTTATTACAAGTGATATAATTATCAAATCTATATAGATCTGTTCCAACGGCNCATATTGCATA 603
KF017583.1 GAATACTGAGTCTCCGTATCATACTTATTACAAGTGATATAATTATCAAATCTATATAGATCTGTTCCAACGGCNCATATTGCATA 603
AF268041.2 GAATACTGAGTCTCCGTATCATACTTATTACAAGTGATATAATTATCAAATCTATATAGATCTGTTCCAACGGCNCATATTGCATA 603
strain_RT_fast CACGAAAAGGATANTTTTCATCATCGCTTGCTTCAGTGTATTCCTGTGAAGAATTTCCGGTAATGTNNN 672
strain_RT_hac CACGAAAAGGATATTTTTCATCATCGCTTGCTTCAGTGTATTCCTGTGAAAAATTTCCGGTAATGTTCA 672
AF268040.2 CACGAAAAGGATATTTTTCATCATCGCTTGCTTCAGTGTATTCCTGTGAAAAATTTCCGGTAATGTTCA 672
FJ870563.1 CACGAAAAGGATATTTTTCATCATCGCTTGCTTCAGTGTATTCCTGTGAAAAATTTCCGGTAATGTTCA 672
LC064808.1 CACGAAAAGGATATTTTTCATCATCGCTTGCTTCAGTGTATTCCTGTGAAAAATTTCCGGTAATGTTCA 672
KF017583.1 CACGAAAAGGATATTTTTCATCATCGCTTGCTTCAGTGTATTCCTGCGAAAAATTTCCGGTAATGTTCA 672
AF268041.2 CACGAAAAGGATATTTTTCATCATCGCTTGCTTCAGTGTATTCCTGTGAAAAATTTCCGGTAATGTTCA 672
Figure 3. Multiple sequence alignments with the most similar strains. The sequence matching gene gB basecalled with fast
and high-accuracy configurations is represented by label strain_RT_fast and strain_RT_hac, respectively. Strain
FJ870563.1 and KF017583.1 (representative reference genome) strains originated from China, AF268041.2 and
LC064808.1 from Japan, while the AF268040.2 one from Spain. The unique insertions are highlighted by blue.
Discussion
By an ONT-based metagenomic study, we identified sequences of PCMV, including a 650 bp long one that matched the
gB
gene. It is the second report of the virus presence in Hungary but the first with a comparable genomic sequence.
High-accuracy basecalling resulted in fewer polymorphisms and shorter deletions compared to fast basecalling. Although
we cannot know with certainty the exact sequence of the virus in our sample, we suppose that polymorphisms identified by
the high-accuracy approach may be reliable. Not only do the deletions represent a difference from the reference genome, but
none of the
gB
genes has similar ones. A closer look at the pattern of the reference genome at the beginning of the deletions in
Figure 3reveals a sequence
TCTC
. This may be a short tandem repeat (STR or microsatellite), which Delahaye and Nicolas
18
associate with the appearance of deletions originating from the ONT-basecall. Unfortunately, only one read in our samples
matched the
gB
gene. Perhaps if we had sequenced the samples more deeply and had more overlapping reads, these deletions
could have been filled in.
The vast majority of PCMV sequences were found in the piglet sample, which the age-specificity of infection and disease
can explain. A persistent but mild problem on the farm for several years is the presence of sneezing and rhinitis in piglets up
to 6 weeks of age. Several pathogens have been identified in the past to investigate the background of this problem, but their
treatment has not resulted in a solution. The small number of reads in the fattening pigs may be a consequence of barcode
cross-talk and are, in fact, derived from the piglet sample.
19
However, it is also possible that minimal levels of the virus are
present in the fatteners. This is supported by our metagenomic analysis of nasal swabs from fattening pigs on the same farm,
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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using Illumina sequencing in 2019, in which two pooled samples were analyzed. One had 205, and the other 118 reads matching
the PCMV reference genome, but no reads for the gB gene.
The experience of our study tells us that ONT-metagenomics can be a promising tool for rapidly detecting pathogens in
farm animals. However, instead of using multiplex sequencing as we have done, we should consider using smaller, single-use
flow cells to sequence samples individually, avoiding the barcode cross-talks.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Availability of data and material
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
Competing interests
The authors declare that they have no competing interests.
Funding
The research was supported by the European Union’s Horizon 2020 research and innovation program under Grant Agreement
No. 874735 (VEO).
Author contributions statement
NS takes responsibility for the data’s integrity and the data analysis’s accuracy. AGT, NS, and TR conceived the concept
of the study. AGT, LM, NS, RF, and TR performed sample collection. AGT, ÁB, RF, and SS did the DNA extraction and
metagenomics library preparation. NS participated in the bioinformatic analysis. AGT and NS participated in the drafting of
the manuscript. AGT, IC, and NS completed the manuscript’s critical revision for important intellectual content. All authors
read and approved the final manuscript.
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