Identification of a Novel Hepacivirus in Domestic Cattle from
Germany
Christine Baechlein,a,b Nicole Fischer,c,d Adam Grundhoff,d,e Malik Alawi,e,f Daniela Indenbirken,e Alexander Postel,a
Anna Lena Baron,a Jennifer Offinger,g Kathrin Becker,h Andreas Beineke,h Juergen Rehage,i Paul Bechera,b
ABSTRACT
Hepatitis C virus (HCV) continues to represent one of the most significant threats to human health. In recent years, HCV-related
sequences have been found in bats, rodents, horses, and dogs, indicating a widespread distribution of hepaciviruses among animals. By applying unbiased high-throughput sequencing, a novel virus of the genus Hepacivirus was discovered in a bovine serum sample. De novo assembly yielded a nearly full-length genome coding for a polyprotein of 2,779 amino acids. Phylogenetic
analysis confirmed that the virus represents a novel species within the genus Hepacivirus. Viral RNA screening determined that
1.6% (n ⴝ 5) of 320 individual animals and 3.2% (n ⴝ 5) of 158 investigated cattle herds in Germany were positive for bovine
hepacivirus. Repeated reverse transcription-PCR (RT-PCR) analyses of animals from one dairy herd proved that a substantial
percentage of cows were infected, with some of them being viremic for over 6 months. Clinical and postmortem examination
revealed no signs of disease, including liver damage. Interestingly, quantitative RT-PCR from different organs and tissues, together with the presence of an miR-122 binding site in the viral genome, strongly suggests a liver tropism for bovine hepacivirus,
making this novel virus a promising animal model for HCV infections in humans.
IMPORTANCE
Livestock animals act as important sources for emerging pathogens. In particular, their large herd size and the existence of multiple ways of direct and food-borne infection routes emphasize their role as virus reservoirs. Apart from the search for novel viruses, detailed characterization of these pathogens is indispensable in the context of risk analysis. Here, we describe the identification of a novel HCV-like virus in cattle. In addition, determination of the prevalence and of the course of infection in cattle
herds provides valuable insights into the biology of this novel virus. The results presented here form a basis for future studies
targeting viral pathogenesis of bovine hepaciviruses and their potential to establish zoonotic infections.
I
t is estimated that over 185 million people are infected with
hepatitis C virus (HCV) worldwide (1). HCV represents the type
species of the genus Hepacivirus within the family Flaviviridae,
which also includes the genera Flavivirus, Pestivirus, and Pegivirus.
Although treatment options will be significantly expanded in
coming years, most patients living in developing countries will not
profit from novel drug therapies, and protective vaccines are still
not available to date (2, 3). The development of several rodent
models has only partially overcome the challenges to studying
HCV in vivo (4). Alternatively, a relative of HCV, the GB virus B
(GBV-B), is considered a surrogate HCV infection model (5, 6). In
recent years, it became apparent that hepaciviruses are more widespread than originally suspected: the identification of HCV-like
sequences in wild and domestic animals broadened the spectrum
of alternative animal models. In 2011, an HCV-like virus was
found in dogs (7). However, serology and PCR-based studies revealed that the natural reservoirs are not dogs but horses. Subsequently, these sequences were designated nonprimate hepaciviruses (NPHV) (8, 9). Moreover, a great diversity of hepacivirus
sequences was detected in rodents and bats from Europe, Africa,
Asia, and Central America (10, 11). In addition to HCV-like sequences, other viral genomes assigned to the proposed genus Pegivirus were identified, expanding the family Flaviviridae (11–16).
July 2015 Volume 89 Number 14
Some of these viruses have been discovered through unbiased
high-throughput sequencing methods (next-generation sequencing [NGS]) (11, 14, 16). Recent examples from human as well as
veterinary diagnostics nicely document the benefit of NGS approaches in the discovery of previously unknown viruses associated with specific diseases (17, 18). In addition, these techniques
allow the detection of pathogens that have not yet been described
Received 27 February 2015 Accepted 17 April 2015
Accepted manuscript posted online 29 April 2015
Citation Baechlein C, Fischer N, Grundhoff A, Alawi M, Indenbirken D, Postel A,
Baron AL, Offinger J, Becker K, Beineke A, Rehage J, Becher P. 2015. Identification
of a novel hepacivirus in domestic cattle from Germany. J Virol 89:7007–7015.
doi:10.1128/JVI.00534-15.
Editor: J.-H. J. Ou
Address correspondence to Paul Becher, [email protected].
C.B. and N.F. contributed equally to this article.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/JVI.00534-15.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JVI.00534-15
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Institute of Virology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Hannover, Germanya; German Center for Infection Research, Partner
Site Hannover-Braunschweig, Hannover, Germanyb; Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg,
Germanyc; German Center for Infection Research, Partner Site Hamburg-Lübeck-Borstel, Hamburg, Germanyd; Heinrich Pette Institute, Leibniz Institute for Experimental
Virology, Research Group Virus Genomics, Hamburg, Germanye; Bioinformatics Core, University Medical Center Hamburg-Eppendorf, Hamburg, Germanyf; Veterinary
Practice, Legau, Germanyg; Department of Pathology, University of Veterinary Medicine Hannover, Hannover, Germanyh; Clinic for Cattle, University of Veterinary Medicine
Hannover, Hannover, Germanyi
Baechlein et al.
identification of bovine hepacivirus B1. (B) Serum samples included in BovHepV RT-PCR screening. (C) Schematic overview of sampling sites: 158 herds from
six federal states of Germany were included. Red dots indicate the origin of BovHepV-positive herds. (D) Summary of serum sampling in two dairy herds.
in clinically healthy animals. Successful spillover to human individuals with subsequent adaptation to the new host involves a
multistep process, and zoonotic transmission dynamics call for
careful modeling (19). Nevertheless, transmission rates of pathogens correlate with population densities, favoring livestock animals to act as sources for emerging viruses (20, 21). Therefore, it is
of major importance to search for possible virus reservoirs and to
describe the risks arising for public health. By applying such an
approach to livestock animals, we identified a thus far unknown
hepacivirus in a serum sample from cattle. The major aims of this
study were to characterize the genomic and biological properties
of the virus, including its distribution among cattle in Germany,
duration of viremia, course of infection, clinical and pathological
signs in infected animals, and viral tropism. Our results pave the
way for future studies addressing the zoonotic potential of bovine
hepacivirus (BovHepV) infections and the establishment of a
novel infection model for HCV.
MATERIALS AND METHODS
Sample material. Blood samples were either derived directly from dairy
farms or obtained from animals submitted to the Clinic for Cattle (University of Veterinary Medicine Hannover). None of the animals was infected experimentally. A serum pool composed of sera from seven individual animals originating from seven distinct dairy herds was analyzed by
high-throughput sequencing. A prevalence study included 320 individual
bovine serum samples from 158 different herds, with a sample size varying
between 1 and 10 serum samples per herd. These samples were taken
between 2012 and 2014 and originated from six German federal states.
Details concerning sampling are given in Fig. 1. Whole blood samples
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were centrifuged at 860 ⫻ g for 10 min. Serum was collected manually and
stored at ⫺80°C.
Liver enzymes. Serum levels of aspartate-aminotransferase (AST)
(GOT [ASAT]-liquid UV kit, standardized to the International Federation of Clinical Chemistry [IFCC]; Mti Diagnostics GmbH, Idstein, Germany), ␥-glutamyl transferase (␥-GT) (Gamma GT, standardized to the
IFCC; Dialab GmbH, Wiener Neudorf, Austria), glutamate dehydrogenase (GLDH) (GLDH Cobas; Roche Diagnostics GmbH, Mannheim,
Germany), and total bilirubin (Labor⫹Technik Lehmann GmbH, Berlin,
Germany) were measured photometrically using an automated analysis
system (ABX Pentra400; Horiba ABX Diagnostics).
RNA isolation. RNA was isolated from 140 ␮l of serum (QIAamp
Viral RNA minikit; Qiagen, Hilden, Germany) as recommended by the
manufacturer. Total RNA from tissue samples was extracted with the help
of QIAzol and RNeasy minikits (Qiagen, Hilden, Germany). RNA was
eluted in RNase-free water and stored at ⫺80°C.
Library preparation and high-throughput sequencing. Illumina libraries from 15 ng of total RNA, as measured by Qubit (Invitrogen) after
DNase treatment, were generated using a modified protocol of a
ScriptSeq, version 2, RNA-Seq (high-throughput sequencing of RNA
transcripts) kit (Epicentre Biotechnologies) (17). Briefly, RNA was subjected to size fragmentation, followed by cDNA synthesis and addition of
a terminally tagged oligonucleotide. Di-tagged cDNA was purified with
Agencourt AMPure XP beads, followed by amplification (15 cycles). The
fragment size distribution of the library was analyzed on a BioAnalyzer
High Sensitivity LabChip. Libraries diluted at 2 nM were multiplex sequenced on an Illumina MiSeq (2 ⫻ 250-bp, paired-end sequencing run;
2.3 to 6.3 million reads/sample).
Bioinformatic analysis. Analysis of RNA transcripts was conducted as
recently described (17, 22). Reads were aligned to cDNA sequences of the
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FIG 1 Summary of bovine serum samples taken during the study. (A) Pooling of serum samples and subsequent high-throughput sequencing led to the
Hepacivirus Infections of Domestic Cattle
July 2015 Volume 89 Number 14
proteases NS2 and NS3 were extrapolated by manually comparing the
polyprotein sequence with previously reported sequences (5, 8, 10, 11,
29). For phylogenetic analyses, the viral sequences were truncated to the
coding regions, and the evolutionary history was inferred by using the
maximum-likelihood method based on the general time reversible model
realized by MEGA, version 6 (30). Bootstrap analysis was performed with
1,000 iterations. A sequence divergence scan over the entire open reading
frame (ORF) was accomplished by the program Sequence Distance implemented in the SSE package (31).
Pathology. At necropsy of animal 463, different samples were collected for histology. Tissues were fixed in 10% formalin, embedded in
paraffin, sectioned at a thickness of 5 ␮m, and stained with hematoxylin
and eosin for histological examination.
Statistics. Results of AST, ␥-GT, GLDH, and total bilirubin were
tested for statistical differences of the means between BovHepV-positive
and -negative cows by use of a t test (IBM SPSS Statistics). The level of
significance was set to a P value of ⬍0.05.
Nucleotide sequence accession numbers. Sequences of the bovine
hepaciviruses identified in this study have been deposited in the GenBank
under accession numbers KP641123 to KP641127.
RESULTS
Identification of a novel hepacivirus in bovine serum. Pooled
bovine serum samples of seven animals from different herds were
screened for the presence of known and putative novel viruses by
unbiased high-throughput RNA sequencing (17, 22) (Fig. 1). De
novo assembly and iterative mapping of sequencing reads obtained by Illumina MiSeq short-read sequencing recovered seven
contigs (between 448 and 870 bp in size) distantly related to sequences of the family Flaviviridae (see Tables S1 and S2 in the
supplemental material). There was no evidence for the presence of
any other viral sequences in these samples. The individual animal
contributing the flaviviral sequences, animal B1, was identified by
PCR with primers NS3_310bp_fwd and NS3_310bp_rev. RNA
isolated from serum of animal B1 was subsequently subjected to a
second MiSeq NGS analysis (see Table S3). From this second analysis 8.64% of all nonhost reads mapped to a single contig of 8,841
nt (minimal coverage, 1,096 over 99% of the contig) which was
readily classified as a novel hepacivirus due to blastn and blastx
matches. The contig contained one single open reading frame
(ORF) of 8,340 nucleotides which encodes a polyprotein of 2,779
amino acids (aa). A blastp search against the NCBI nr database
revealed highly significant homology to hepacivirus polyproteins
(E value of ⬍1e⫺180; 36% sequence identity and 51% sequence
similarity shared with the most closely related database entry).
Alignments were contiguous and extended over nearly the entire
length of the polyprotein (query coverage, 97%). Results from
pairwise alignments between individual mature hepaciviral proteins expected to be processed from the polyprotein and mature
proteins of other hepacivirus species are summarized in Table 1.
All coding segments exhibited significant homology to known
hepacivirus proteins, thus strongly suggesting that the assembled
8,841-nt contig represents an authentic viral genome. The most
highly conserved regions were those encoding NS3 and NS5B,
with 52.4% amino acid identity and 75.4% similarity in the NS3
protein and 45.2% amino acid identity and 72.1% similarity in the
NS5B region (Table 1). The overall pattern and degree of sequence
diversity suggest that the sequence recovered by next-generation
sequencing is a novel hepacivirus which was termed bovine hepacivirus (BovHepV).
Prevalence and genetic diversity of BovHepV in Germany.
To gain an insight into the prevalence of bovine hepacivirus in
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human reference assembly (Ensembl GRCh38) using Bowtie2 (version
2.2.3) (23). Reads yielding significant alignments were considered originating from the host and excluded from further analysis. Trinity (version
r20140717) was employed for assembly. To estimate contig abundance,
reads were aligned to assembled contigs using Bowtie2. Putative PCR
duplicates were excluded from abundance estimation. For taxonomic
classification, all contigs of at least 399 nucleotides (nt) were iteratively
aligned to the NCBI nt and nr (nucleotide and nonredundant, respectively) databases using megablast, blastn, and blastx tools of the BLAST⫹
package (version 2.2.30) and a lowest-common-ancestor (LCA) algorithm.
Variant calling. Reads were aligned to the corresponding reference
assemblies using Bowtie2 (version 2.2.3) (23). SAMtools (version 0.1.18)
(24) was employed to remove putative PCR duplicates. Alignments of
reads belonging to the same reference assemblies were merged. Variants
were called with FreeBayes (version 0.9.18-1-g4233a23) (25). Putative
variants were filtered for quality (threshold 20) and positions at which at
least one sample supported both the reference and an alternative sequence
with at least five reads. Alignments in the vicinity of called variants were
visually assessed using the Integrative Genomics Viewer (version 2.3.40)
(26).
Reverse transcription-PCR (RT-PCR). cDNA was synthesized using
4.0 ␮l of RNA, random primers, and Moloney murine leukemia virus reverse
transcriptase (Life Technologies, Carlsbad, CA, USA). Hepacivirus-specific
RNA was detected by using two sets of primers: NS3_310bp_fwd (5=-CCGC
AAGGGCTATAGTGTGT-3=) and NS3_310bp_rev (5=-GGCGGTGGCAA
GCAAAAATA-3=) spanning a 310-bp fragment of the NS3 coding region.
PCRs were carried out using hot-start PCR master mix (Thermo Fisher Scientific, Vilnius, Lithuania) under the following conditions: 95°C for 15 min,
followed by 40 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, with a
final step at 72°C for 7 min. Primers targeting highly conserved regions of the
NS3 coding region (Hepaci_NS3_fwd, 5=-TTGTGCTTGCCACSGCYACYC
C-3=; Hepaci_NS3_rev, 5=-TCRAAGTTGCCRGTGTACCCKGT-3=) gave
rise to a 318-bp-spanning amplicon. These primers were designed following
alignments of nucleotide sequences corresponding to the NS3 region of several hepacivirus sequences, including HCV (GenBank accession number
M62321), GBV-B (GenBank AF179612), NPHV (GenBank JQ434008), and
rodent (GenBank NC_021153) and bat (KC796091 and GenBank
KC796074) hepaciviruses. Here, the following temperature profile was chosen: 95°C for 15 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, and
72°C for 30 s, with a final step at 72°C for 7 min.
Quantitative RT-PCR. For quantification of viral RNA, an RNA standard was constructed. A fragment of the NS3 coding region was amplified
with the primer pair NS3_310bp_fwd/rev and inserted into pCR2.1 (Life
Technologies, Carlsbad, CA, USA). BamHI-digested DNA was in vitro transcribed using T7 polymerase. Remaining DNA was cleared with Turbo DNase
(both enzymes from a MEGAscript kit; Ambion, Inc., Austin, TX, USA), and
RNA was purified (MEGAclear kit, Ambion, Inc., Austin, TX, USA). RNA
concentrations were determined using a NanoDrop 2000 UV-visible light
(UV-Vis) spectrophotometer, and a log10 dilution series was used to calculate
genome equivalents. TaqMan RT-PCR was applied under the following conditions: 50°C for 30 min and 95°C for 2 min, followed by 40 cycles of 95°C for
15 s, 54°C for 30 s, and 68°C for 30 s, using a SuperScript III One-Step RTPCR System (Life Technologies, Carlsbad, CA, USA). The sequences of the
primers and probe were the following: BovHepV_fwd1, 5=-GCTCGGCTTA
CATACTCTAC-3=; BovHepV_rev1, 5=-GAATGGTAGTGGAATCGGTG3=; BovHepV_probe1, 5=-FAM-TTTACCATCCGCCAAAAATCTGCCATA
A-BHQ1-3= (where FAM is 6-carboxyfluorescein and BHQ1 is Black Hole
quencher 1). All samples were analyzed in duplicates using three independent
RNA preparations.
Sequence analysis. Nucleotide sequences of viruses classified to the
genus Hepacivirus were downloaded from GenBank, and corresponding
amino acid sequences were aligned using the Clustal W multiple alignment tool as implemented in BioEdit (27). Cleavage sites for cellular signal
peptidases were predicted in silico (28), whereas cleavage sites for the viral
Baechlein et al.
TABLE 1 Sequence identity and similarity of mature BovHepV proteins
compared to those of other hepaciviruses
Value for the indicated virus in relation to
BovHepV (%)a
GBV-B
RoHepV
HCV1a
NPHV
BatHepV
Amino acid sequence
identity
Core
E1
E2
P7
NS2
NS3
NS4A
NS4B
NS5A
NS5B
35.2
32
30.8
32.8
25.8
45.2
28.6
37.9
28.1
45.2
31.1
29.5
30.3
41.7
28.4
42
37.5
28.2
34
43.8
33.3
27.6
22.2
30
27.9
41.3
34.3
28.2
28.7
32.6
32.9
26
23.6
24.5
25.2
43
25
26.7
34
35.9
39.7
28.5
26.7
27.3
29.3
52.4
31
36.4
30
41.6
Amino acid sequence
similarity
Core
E1
E2
P7
NS2
NS3
NS4A
NS4B
NS5A
NS5B
59.1
64
55.3
62.3
59.1
71.6
54.8
68.3
53.7
72.1
57
61.1
57
66.7
60.3
68.7
66.7
69.6
67.9
70.5
53.8
58.9
43.3
60
57.1
69.3
60
69.4
58.1
63.7
56.6
60.8
43.6
53.1
55.8
71.4
59.6
66.1
66
66.4
61.6
56.1
56.2
67.3
62.6
75.4
47.6
69.2
56.5
70.3
a
Nonintersecting local alignments of the BovHepV-encoded proteins with the protein
sequences indicated were performed using the online tool LALIGN, version 36.3.6
(August 2014). GenBank accession numbers of the sequences included are as follows:
BovHepV, KP641123; GBV-B, AF179612; rodent hepacivirus (RoHepV), KC411807;
HCV1a, M62321; NPHV, JQ434008; bat hepacivirus (BatHepV), KC796077. Amino
acid sequence identity and similarity are indicated as percentages and as follows: plain
text, 20 to 40%; underlining, 40 to 60%; gray shading, 60 to 80%.
Germany, 320 individual bovine serum samples originating
from 158 herds were screened by RT-PCR using primers
Hepaci_NS3_fwd and Hepaci_NS3_rev. By this means, four viremic animals originating from distinct herds were identified (serum samples 209, 379, 438, and 463) (Fig. 1). Thus, including the
initially identified BovHepV-positive animal B1, the prevalence
accounted for 3.2% on the herd level and for 1.6% on the individual level. Nearly complete genome sequences from the four additional BovHepV variants were obtained by high-throughput sequencing (see Table S5 in the supplemental material). Gaps were
TABLE 2 Comparison of predicted hepacivirus polyprotein cleavage sites
Cleavage site sequence at:a
Virus
C/E1
E1/E2
E2/p#
p#/NS2
NS2/NS3
NS3/NS4A
NS4A/NS4B
NS4B/NS5A
NS5A/NS5B
HCV 1a
NPHV
GBV-B
Bat hepacivirus PDB-112
Rodent hepacivirus
BovHepV B1
ASA/YQV
GE./SV.
C.G/AR.
.ES/VPA
VEP/KPL
V.G/.RH
VDA/ETH
.SC/DSD
TSG/NPI
AA./MPV
SV./APV
.E./T.T
AEA/ALE
. . ./Y.S
.SG/YPL
.WG/WPA
SY./QPP
.T./..L
AYA/LDT
.W./F.N
.S./F..
.Q./ASL
VE./FSS
VT./..F
RLL/API
. . ./S..
AIT/..F
ERN/..M
Q.S/S.V
APC/S..
VVT/STW
TQ./NA.
.N./.GT
YSA/GGL
SW./GGL
LDV/WGA
EEC/SQH
. . ./FD
FS./. . .
. . ./M.T
. . ./ALD
. . ./WGF
TPC/SGS
QN./DFT
DD./GLI
AE./D.M
E../TD.
V../GFN
VCC/SMS
ES./.L.
FS./.M.
SKM/.R.
ET./TY.
KE./.Y.
a
GenBank accession numbers for the sequences are as follows: HCV 1a, M62321; NPHV, JQ434008; GBV-B, AF179612; bat hepacivirus, KC796077; rodent hepacivirus, KC411807.
Cleavage is indicated by a slash. p#, p7 or p13.
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Parameter and
protein
filled by RT-PCR and nucleotide sequencing of the obtained PCR
products. The 5= terminal sequences of BovHepV were confirmed
by applying a 5= RACE System for Rapid Amplification of cDNA
Ends (Life Technologies, Carlsbad, CA, USA), whereas the 3= terminal sequences of BovHepV B1 and 463 were determined by
5=/3= RACE (Roche Diagnostics, Mannheim, Germany). All five
sequences were submitted to the GenBank (accession numbers
KP641123 to KP641127). The BovHepV sequences identified in
this study showed low to moderate genome sequence diversity on
the nucleotide level, ranging from 7 to 9% (see Table S5). Calculation of pairwise amino acid distances indicated low divergence
among the five BovHepV sequences from this study but large distances to other hepaciviruses including HCV and NPHV (see Fig.
S1 in the supplemental material).
Genome characterization of BovHepV. The genome of
BovHepV B1 consists of 8,881 nucleotides and contains one large
ORF encoding a polyprotein of 2,779 aa. This polyprotein comprises considerably fewer amino acid residues than that of HCV 1a
(3,011 aa) or NPHV (2,942 to 2,946 aa) but is more similar in
length to the polyproteins of GBV-B (2,864 aa) and rodent hepacivirus strains (SAR-3/RSA/2008, 2,781 aa; SAR-46/RSA/2008,
2,781 aa). Putative cleavage sites specific for signal peptidases
(processing of E1, E2, and p7/p13) and cleavage sites essential for
processing of the nonstructural proteins NS2 to NS5B were identified. Some of them were shown to be well conserved among the
hepacivirus sequences analyzed here (Table 2). Thus, BovHepV
exhibits the well-known genomic organization typical for hepaciviruses: NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5BCOOH. The ORF is flanked by 5= and 3= untranslated regions
(UTRs) consisting of 294 and 247 nucleotides, respectively. The 5=
UTR of the BovHepV B1 is characterized by secondary structures
of a previously described hepaciviral internal ribosomal entry site.
As shown in Fig. S2 in the supplemental material, two miR-122
target sites can be identified in BovHepV B1, with one site located
in the 5= UTR.
The BovHepV B1 sequence was analyzed for genomic subpopulations identified by single nucleotide polymorphisms at specific nucleotide positions. Using a frequency cutoff of minimally
10%, 10 positions within the polyprotein coding sequence showing variant nucleotides not resulting in changes of either cleavage
sites or amino acids essential for cleavage activity of NS2 or NS3
were identified (see Table S4 in the supplemental material).
Phylogenetic analysis of bovine hepaciviruses. A phylogenetic tree built upon the whole open reading frame proved the
affiliation of BovHepV to the genus Hepacivirus. BovHepV is
only distantly related to HCV, NPHV, and other hepaciviruses.
Hepacivirus Infections of Domestic Cattle
complete coding sequences of hepaciviruses. Bootstrap analysis was performed with 1,000 replicates (numbers next to the branches are percentages). Bootstrap
values below 70% are not shown. The tree was rooted to hepatitis GB virus A. Sequences downloaded from GenBank are cited with their accession numbers.
Sequences are identified as follows: blue triangles, HCV; green squares, NPHV; gray inverted triangles, bat hepacivirus; purple circles, rodent hepacivirus; teal
diamond, GBV-B; red open circles, BovHepV.
Moreover, BovHepV branches very deeply next to GBV-B, bat
hepacivirus, and rodent hepacivirus sequences, with a large genetic distance among them as well (Fig. 2). Also, phylogenetic
reconstruction clearly demonstrated a narrow cluster of the five
BovHepV sequences obtained in this study forming a separate
phylogenetic branch of hepaciviruses.
Acute and chronic BovHepV infections in two single dairy
herds in Germany. The course of BovHepV infections was investigated in two dairy herds by RT-PCR. In dairy herd 1, at 6 months
after the first sampling which led to the identification of BovHepV
from animal B1, 14 adult animals (35.0%) from the 40 cows comprising the herd had viral RNA in their blood. Three months later,
11 out of 35 (31.4%) animals tested positive, whereas again 3
months later, 6 out of 34 (17.6%) investigated serum samples
contained viral RNA. Five animals stayed viremic throughout this
period, whereas others tested negative at one or two sampling time
points (Table 3). In dairy herd 2, 12 of 40 randomly selected dairy
cows (30.0%) were virus positive. Sera from 10 calves tested
negative, including the sample from an offspring calf of one
July 2015 Volume 89 Number 14
BovHepV-positive cow. Serum concentrations of liver enzymes
together with total bilirubin determined for 12 virus-positive and
28 virus-negative cows from herd 2 revealed no significant differences between BovHepV-positive and -negative animals (Fig. 3).
Postmortem analysis and determination of viral genome
loads in different organs and tissues. For one BovHepV-infected
cow (463), the effect of the infection was investigated in more
detail. This animal had been delivered to the Clinic for Cattle and
tested positive during screening for BovHepV. The animal was
euthanized and submitted to postmortem analyses. Macroscopic
examination revealed focal tension lipidosis of the liver. Subsequent histological investigation showed focal lipidosis (tension
lipidosis) and mild diffuse centrilobular lipidosis, while degenerative or inflammatory changes suggestive of viral infection were
not observed. Viral genome load in different tissues of animal 463
was investigated by quantitative RT-PCR. Highest loads of
BovHepV RNA (2.92 ⫻ 105 genome equivalents per mg) were
detected in the liver. In comparison, the serum contained 8.94 ⫻
104 viral genome copies per microliter. Significantly lower num-
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FIG 2 Phylogenetic analysis of hepaciviruses from human and various animal species including cattle. A maximum-likelihood tree is presented based on the
Baechlein et al.
TABLE 3 Course of infection in dairy herd 1
Infection status by sampling datea
August 2013
February 2014
May 2014
August 2014
1
2
3
4
5
6
7
8
9
10
*
*
*
*
*
*
*
*
*
*
Neg
Pos
Neg
Pos
Pos
Pos
Neg
Neg
Neg
Neg
Neg
Pos
Neg
Pos
Neg
Pos
Neg
*
Neg
Neg
Neg
Neg
Neg
Pos
Neg
Pos
Neg
*
Neg
Neg
11
12
13
14
15
16
17
18
19
20
*
*
*
*
Pos
*
*
*
*
*
Neg
Neg
Neg
Pos
Neg
Pos
Pos
Neg
Neg
Pos
Neg
Neg
Neg
Neg
Pos
Neg
Pos
Neg
Neg
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
21
22
23
24
25
26
27
28
29
30
*
*
*
*
*
*
*
Neg
*
*
Neg
Neg
Neg
Pos
Neg
Neg
Neg
Pos
Neg
Neg
Neg
Neg
Neg
Pos
*
Neg
*
Neg
Neg
*
Neg
Neg
pos
Neg
*
Neg
*
Neg
Neg
*
31
32
33
34
35
36
37
38
39
40
*
Neg
*
*
*
Neg
*
*
*
*
Pos
Pos
Neg
Neg
Neg
Pos
Neg
Neg
Pos
Neg
Pos
Pos
*
Neg
Neg
Pos
Neg
Neg
Pos
Neg
Pos
Neg
*
Neg
Neg
Pos
Neg
Neg
Neg
*
a
Positive results are highlighted. *, not tested.
bers of BovHepV genome equivalents (ⱕ4.15 ⫻ 103) or no viral
RNA was detected in the other organs and tissues analyzed. The
viral genome load in the liver lymph node was significantly higher
(5.09 ⫻ 103) than that in other mesenterial lymph nodes (Fig. 4).
DISCUSSION
After the discovery of hepatitis C virus, human HCV and the related primate GBV-B first described in 1995 represented the only
known members of the newly formed genus Hepacivirus for more
than 2 decades (12, 32, 33). The recent detection of hepacivirus
sequences in different animal species indicated the widespread
presence of hepaciviruses. Here, a thus far unknown hepacivirus
was identified from cattle and was named bovine hepacivirus
(BovHepV). Estimation of evolutionary distances and phyloge-
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Animal
netic analysis showed that the five BovHepV sequences identified
from different herds are closely related to each other but display
large genetic distances from other previously reported hepaciviruses.
Among domestic animals, dogs and horses have been the only
species reported so far to carry hepaciviruses. The first HCV-related virus identified in nonprimate animal species was described
by Kapoor et al. (7), who detected HCV-related sequences in nasal
swabs from dogs. However, subsequent studies identified nearly
identical sequences in horses but failed to retrieve similar or additional sequences from dogs, suggesting that the initial study
might, in fact, have detected a horse virus that was transmitted to
a dog (8, 9, 34). NPHV has been shown to be widespread among
horses since approximately 2 to 7% of investigated animals
showed signs of viremia (8, 9, 34, 35). Similarly, the results of the
present study show that 1.6% of 320 investigated cattle sera collected from 158 different holdings contained BovHepV RNA genomes. Each of the five virus-positive animals originated from a
distinct herd, leading to the assumption that BovHepV is widely
disseminated in Germany.
In two of the test-positive herds, the virus prevalence on the
individual level accounted for 30 to 35%. Serial investigations in
herd 1 involving serum samples taken at an interval of 3 months
imply that BovHepV is able to establish chronic infections in cattle
as five animals were proven to carry viral RNA in their blood at
every time point of sampling over more than half a year. This is
similar to NPHV since a very recent study proved that horses carry
the virus for over 6 months (35). Taken together, these data indicated that both bovine and equine nonprimate hepaciviruses
share one of the main characteristics with HCV, which is the
chronic outcome of in vivo infections (reviewed in reference 36).
Until now, one could only speculate how the virus was introduced into the herds or how the virus was transmitted between
different animals. Mass vaccination without changing needles between individual animals represents a serious risk to passing on
infectious agents. Also, semen has to be considered a potential
source of infection. Analysis of samples from a limited number of
BovHepV-positive animals available so far provided no evidence
for viral excretion via milk, feces, urine, or nasal discharge (data
not shown). However, investigations of samples from a larger
number of BovHepV-positive animals are required for a final conclusion concerning virus excretion and routes of transmission.
In only a few cases, animal hepaciviruses have been linked to
clinical disease. The canine hepacivirus (CHV) was initially found
in dogs with respiratory illness and was also detected occasionally
in liver samples (7). However, later studies failed to link idiopathic
hepatitis in dogs with CHV infection (34, 37). Elevated concentrations of liver-specific enzymes without impairment of liver
function in NPHV-infected horses have been recently reported
(35). In contrast, only one single recently published case report
associated NPHV infection in a horse with liver disease (38). In the
present study, a potential effect of BovHepV infection on the liver
was investigated in two different ways. Serum concentrations of
liver enzymes of adult cows were determined; however, no significant difference was seen in comparisons of infected and noninfected animals. Instead, levels of enzymatic activity notably exceeding threshold values (AST, ⬍100 U/liter; ␥-GT, ⬍33U/liter;
GLDH, ⬍14 U/liter) were measured in both groups. This observation can certainly be attributed to a strongly increased metabolic rate in milking dairy cows. This issue could be resolved by
Hepacivirus Infections of Domestic Cattle
cows. AST, aspartate-aminotransferase; ␥-GT, ␥-glutamyl transferase; GLDH, glutamate dehydrogenase.
analyzing metabolic parameters in a virus-positive cohort of beef
cattle. One BovHepV-positive cow was submitted to postmortem
analyses, but liver injury attributable to virus infections was not
observed. However, it is not known how long this animal had been
infected. Given the possibility of an acute infection, the absence of
pathological signs due to virus replication in the liver is not surprising. To clarify the possible impact of BovHepV on animal
health, experimental infections or screening approaches involving
liver material from slaughtered cattle will have to be conducted.
Independent of the induction of clinical disease, a putative
tissue tropism of BovHepV as a hepatotropic virus such as HCV is
of significant interest. According to the results of our analysis, the
highest concentration of viral RNA (⬎105 genome equivalents/
mg) was found in the liver. It appears reasonable to assume that
this result is directly linked to efficient viral replication in the liver,
which is further supported by the presence of a bona fide miR-122
binding site within the 5= UTR of the BovHepV genome. Among
vertebrates, the sequence of mature miR-122 is fully conserved
(39, 40). Furthermore, miR-122 constitutes the most abundantly
expressed microRNA in the bovine liver (41), suggesting a possible role in supporting hepacivirus replication. In contrast, the
considerably lower numbers, about 103 or fewer genome equivalents/mg, in other organs and tissues strongly supplied with blood
are most likely due to the high viral genome loads detected in the
serum. Considering human-to-animal contacts including exposure to blood and tissues as well as food-borne infections, cattle
represent one of the most relevant animal species for spillover
infections from animals to humans. In particular, farmers, veterinarians, and slaughterhouse workers are frequently exposed to
BovHepV present at high concentration in the blood and liver of
cattle. Therefore, future studies on BovHepV will focus on pathogenicity, clarification of the zoonotic potential, and possible transmission routes to humans. In summary, a thus far unknown
HCV-like virus in domestic cattle was identified via unbiased RNA
sequencing. The observation of chronic infections in cattle together with the presence of high viral genome loads in the liver of
FIG 4 BovHepV genome equivalents per milligram of tissue and microliter of serum determined by quantitative RT-PCR. Mean values and standard deviations
of three independent experiments are shown. Samples were taken during necropsy from BovHepV-positive animal 463.
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FIG 3 Box-plots of serum concentrations of liver enzymes and of total bilirubin determined for BovHepV-positive (n ⫽ 12) and BovHepV-negative (n ⫽ 28)
Baechlein et al.
infected animals makes BovHepV a promising model for HCV.
Moreover, it will be of particular interest to investigate whether
BovHepV infections affect animal and human health.
ACKNOWLEDGMENTS
The project is funded by the German Center for Infection Research/Thematic Translational Unit, Emerging Infections.
We thank Ester Barthel, Hossein Naghilouy Hidaji, and Franziska
Peest for excellent technical assistance.
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