JVI Accepts, published online ahead of print on 29 October 2014 J. Virol. doi:10.1128/JVI.02718-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Co-circulation of two distinct genetic and antigenic lineages of proposed influenza D virus
2
in cattle
3
Running Title: Influenza D virus in cattle
4
Authors: Emily A. Collina,b,c*#, Zizhang Sheng d, Yuekun Lang c, Wenjun Ma c, Ben M.
5
Hausea,c*#, Feng Li b
6
a
7
b
8
South Dakota, USA
9
c
Newport Laboratories Inc., Worthington, Minnesota, USA Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings,
Veterinary Diagnostic Laboratory and Department of Diagnostic Medicine and Pathobiology,
10
Kansas State University, Manhattan, Kansas, USA
11
d
12
York, USA
13
*Present Address: Veterinary Diagnostic Laboratory, Kansas State University, Manhattan,
14
Kansas
15
#Correspondence: Emily Collin, Veterinary Diagnostic Laboratory, Kansas State University,
16
L213 Mosier Hall, Manhattan, Kansas, (785)-532-4466, [email protected], Ben Hause,
17
Veterinary Diagnostic Laboratory, Kansas State University, L227 Mosier Hall, Manhattan,
18
Kansas, (785) 532-4278, [email protected]
19
Keywords: influenza, bovine, hemagglutination inhibition, hemagglutinin esterase fusion,
20
bovine respiratory disease
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New
1
1
Abstract: Viruses with approximately 50% homology to human influenza C virus (ICV) have
2
recently been isolated from swine and cattle. The overall low homology to ICV, lack of antibody
3
cross reactivity to ICV in hemagglutination inhibition (HI) and agar gel immunodiffusion assays
4
and inability to productively reassort with ICV led to the proposal that these viruses represented
5
a new genus of influenza, influenzavirus D (IDV). To further our understanding of the
6
epidemiology of IDV, real time reverse transcription PCR was performed on a set of 208
7
samples from bovines with respiratory disease. Ten samples (4.8%) were positive and six
8
viruses were successfully isolated in vitro. Phylogenetic analysis of full genome sequences of
9
these six new viruses and four previously reported viruses revealed two distinct co-circulating
10
lineages represented by D/swine/Oklahoma/1334/2011 (D/OK) and
11
D/bovine/Oklahoma/660/2013 (D/660) which frequently reassorted with one another. Antigenic
12
analysis using the HI assay and lineage-representative D/OK and D/660 antiserum found up to an
13
approximate 10-fold loss in cross reactivity against heterologous clade antiserum. One isolate,
14
D/bovine/Kansas/3-13/2011 (D/3-13), clustered with the D/660 lineage, but also had high HI
15
titers to heterologous (D/OK) clade antiserum. Molecular modeling of the hemagglutinin
16
esterase fusion protein of D/3-13 identified a mutation at position 212 as a possible antigenic
17
determinant responsible for the discrepant HI results. These results suggest that IDV is common
18
in bovines with respiratory disease and at least two genetic and antigenically distinct clades co-
19
circulate.
20 21 22
2
1
Importance:
2
A novel bovine influenza virus was recently identified. Detailed genetic and antigenic studies
3
led to the proposal that this virus represents a new genus of influenza, influenzavirus D (IDV).
4
Here we show that IDV is common in clinical samples of bovine respiratory disease complex
5
(BRDC), with prevalence similar to other established BRDC etiological agents. These results
6
are in good agreement with near ubiquitous seroprevalence of IDV previously found.
7
Phylogenetic analysis of complete genome sequences found evidence for two distinct co-
8
circulating lineages of IDV which freely reassort. Significant antigenic differences were
9
observed between the two lineages that generally agreed with the surface glycoprotein
10
hemagglutinin esterase phylogeny. These results, in addition to the ability of IDV to infect and
11
transmit in multiple mammalian species, warrant additional studies to determine the pathogenic
12
potential of IDV.
13 14 15 16 17 18 19 20
3
1
Introduction:
2
Influenza viruses are single-stranded, negative-sense (-), segmented RNA viruses belonging to
3
the family Orthomyxoviridae (1). Influenza A (IAV) and influenza B (IBV) both contain eight
4
genomic segments, including two surface glycoproteins, the hemagglutinin (HA) and
5
neuraminidase (NA), whereas influenza C (ICV) only has seven segments with one surface
6
glycoprotein, the hemagglutinin-esterase-fusion (HEF) protein (2,3). While the vast genetic
7
diversity of IAV is found in waterfowl, only limited subtypes infect mammals. IBV and ICV are
8
found principally in humans and rarely infect other species. IBV is a component of seasonal
9
influenza epidemics with clinically significant disease while ICV infects most humans during
10
childhood and typically results in mild respiratory symptoms and fever (1,4-6).
11
In 2011, an influenza virus with moderate homology to ICV was isolated from swine in
12
Oklahoma (D/OK) exhibiting influenza-like symptoms. Sequence analysis showed
13
approximately 50% homology to human ICVs (7). D/OK did not cross react with antibodies
14
against human ICV in hemagglutination inhibition (HI) and agar gel immunodiffusion (AGID)
15
assays. Limited seroprevalence in swine and humans to D/OK (9.5% and 1.3%, respectively)
16
suggested that an alternate species was the reservoir of this novel virus (7). HI assays on bovine
17
sera found seven out of eight herds with titers greater than 40 to both D/OK and the bovine
18
D/660 strain. Eighteen percent of bovine respiratory disease samples were positive by RT-PCR
19
assay targeting the PB1 gene of D/OK. Virus isolation, genome sequencing and phylogenetic
20
analysis showed that D/OK and three bovine isolates were closely related and did not reassort
21
with human ICV (8). Likewise, in vitro reassortment experiments between D/OK and human
22
ICV failed to identify viable reassortant viruses. Reassortment of viral segments can yield viable
23
progeny within the same genera but not across genera of influenza (2,9,10). Taken together, 4
1
these results led to the proposal to classify D/OK-like viruses as a new genus of influenza virus,
2
influenzavirus D (IDV), with bovines as the potential reservoir (8).
3
While the current three genera of influenza, influenza A, B, and C, all share similar genetic
4
ancestry, they have diverged over time (2). ICV undergo reassortment frequently in nature,
5
which results in greater genetic diversity of the viruses (3,6,11,12). ICV is a product of multiple
6
lineage evolution, a result of co-circulating strains in the human population (6,10,13,14). As
7
influenza B and C viruses have further diverged from IAV, significant mutations resulted in the
8
lack of viable reassortant viruses between influenza B and C, and they both are thought to be
9
evolutionary stable (10,15). The discovery of IDV warrants new research into its evolutionary
10
history as well as its epidemiology and ecology.
11
Bovine respiratory disease complex (BRDC) is the most economically significant disease of the
12
beef industry with losses due to morbidity, mortality, treatment costs and reduced carcass value
13
(16,17). Established viral etiological agents include bovine viral diarrhea virus (BVDV), bovine
14
herpes virus (BHV-1), bovine respiratory syncytial virus (BRSV), and parainfluenza virus (PI3).
15
In the past several years there has been increasing evidence that bovine respiratory coronavirus
16
also contributes to BRDC in feedlot cattle (17,18). The finding of IDV in cattle warrants further
17
investigation into its possible role as a BRDC etiological agent.
18
To further investigate the epidemiology of this proposed new genus, a large sample set of BRDC
19
cases were screened by quantitative real time reverse transcription PCR (qRT-PCR) to determine
20
the molecular epidemiology of IDV in association with other bovine respiratory disease viral
21
agents. Phylogenetic analysis of full genome sequences, along with hemagglutination inhibition
22
(HI) assay was performed to characterize the genetic and antigenic diversity of IDV.
5
1
Materials and Methods:
2
Molecular screening of bovine viruses. Clinical samples from bovine respiratory disease
3
submissions to the Kansas State University Veterinary Diagnostic Lab (n=208) were screened by
4
a BRDC PCR panel which detects BVDV, BHV-1, BRSV, PI3, and BCV. The 208 samples,
5
consisting of nasal and pharyngeal swabs and lung tissue, originated from twelve states primarily
6
in the Midwest: Nebraska (n=48), Kansas (n=116), Colorado (n=6), Missouri (n=1), Mississippi
7
(n=7), Texas (n=4), Oklahoma (n=2), Idaho (n=2), Montana (n=6), Oregon (n=4), Washington
8
(n=11), and Virginia (n=1). Additionally, samples were analyzed for IDV using quantitative real
9
time reverse transcription PCR (qRT-PCR) (7).
10
Cells and Viruses. Human rectal tumor cells (HRT-18G) (ATCC CRL-11663) were maintained
11
in Dulbecco’s modified Eagles medium (DMEM) supplemented with 5% fetal bovine serum
12
(FBS). Swine testicle cells (ST) (ATCC CRL-1746) were maintained in the same manner. Both
13
cell lines were grown at 37°C with ±5% CO 2 before infection with viruses. IDV were isolated at
14
Newport Laboratories from PCR-positive field samples. D/swine/Oklahoma/1334/2011 (D/OK)
15
virus was previously isolated from swine exhibiting influenza-like symptoms (7).
16
D/bovine/Minnesota/628/2013 (D/628), D/bovine/Minnesota/729/2013 (D/729), and
17
D/bovine/Oklahoma/660/2013 (D/660) were previously isolated from bovine respiratory disease
18
samples submitted for diagnostic testing (8).
19
Virus Isolation. Virus isolation was performed on IDV qRT-PCR positive bovine samples
20
using confluent monolayers of HRT-18G cells in DMEM without FBS with 0.5 µg/mL trypsin,
21
at 37°C with ±5% CO 2. Viral growth was confirmed by qRT-PCR and the hemagglutination
22
assay (HA) using 0.5% washed turkey red blood cells, as IDV is non-cytopathic on HRT-18G
6
1
cells. Following one passage on HRT-18G cells, viruses were passaged to ST cells in DMEM
2
without trypsin. Growth was confirmed by qRT-PCR, HA, and cytopathic effects (CPE) after
3
each passage. PCR-positive samples failing to show CPE or increase in qRT-PCR and HA titers
4
were passaged three times before they were called negative.
5
Full-genome sequencing and analysis. The MegaMax viral RNA kit was used to isolate the
6
viral RNA. Full-genome sequencing was performed as previously described (8). In brief,
7
sequencing libraries were prepared from full-genome amplicons using the NEBNext Fast DNA
8
kit for Ion Torrent. D/OK (accession numbers NC_922305 to NC_922311) was used as the
9
template to assemble the contigs using the SegMan NGen module from DNAStar. Phylogenetic
10
analysis was performed using MEGA6.0 software using the Maximum Likelihood algorithm
11
with 1000 bootstrap replicates to verify tree topology.
12
Serology. Rabbit polyclonal antisera generated using D/660 and D/OK were used in the
13
hemagglutination inhibition (HI) assay. All viruses were assayed in triplicate. Colostrum
14
deprived/cesarean derived (CDCD, Struve Laboratories) sera was used as a negative control.
15
The HI test was performed according to the WHO protocol, using turkey red blood cells (19).
16
Mean HI titers and standard deviation were calculated from triplicate data. Heterologous mean
17
HI titers were normalized to mean homologous HI titers (mean heterologous titers / mean
18
homologous titers). Relative HI titers were reported to account for differences in homologous HI
19
titers between D/OK and D/660.
20
HEF Structure Modeling
21
The HEF amino acid sequence of D/3-13 HEF was queried against all sequences in the PDB
22
structure database using BLAST (20). The HEF structure from ICV C/Johannesburg/1/66 (PDB 7
1
ID: 1FLC) (21), which showed the highest sequence identity to D/3-13 HEF (53% sequence
2
identity), was chosen as template. The sequences of D/3-13 and ICV HEF were then aligned
3
using Muscle (22). The model of 3-13 HEF was built using Modeller (23). The quality of the
4
structure was checked using Verify 3D (17). For demonstration purpose, the sialic acid binding
5
template was shown on the D/3-13 HEF structure.
6 7
Results
8
Molecular Epidemiology: Ten of the 208 (4.8%) samples submitted for BRDC diagnostic
9
testing were positive for IDV by qRT-PCR with Ct values ranging from 21-29. These samples
10
consisted of nine nasal swabs and one swab of unknown type. In addition, other viruses
11
associated with BRDC were detected by qRT-PCR, with 36% (n=75), 7% (n=15), 5% (n=11),
12
3% (n=7), and
1
Co-circulation of two distinct genetic and antigenic lineages of proposed influenza D virus
2
in cattle
3
Running Title: Influenza D virus in cattle
4
Authors: Emily A. Collina,b,c*#, Zizhang Sheng d, Yuekun Lang c, Wenjun Ma c, Ben M.
5
Hausea,c*#, Feng Li b
6
a
7
b
8
South Dakota, USA
9
c
Newport Laboratories Inc., Worthington, Minnesota, USA Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings,
Veterinary Diagnostic Laboratory and Department of Diagnostic Medicine and Pathobiology,
10
Kansas State University, Manhattan, Kansas, USA
11
d
12
York, USA
13
*Present Address: Veterinary Diagnostic Laboratory, Kansas State University, Manhattan,
14
Kansas
15
#Correspondence: Emily Collin, Veterinary Diagnostic Laboratory, Kansas State University,
16
L213 Mosier Hall, Manhattan, Kansas, (785)-532-4466, [email protected], Ben Hause,
17
Veterinary Diagnostic Laboratory, Kansas State University, L227 Mosier Hall, Manhattan,
18
Kansas, (785) 532-4278, [email protected]
19
Keywords: influenza, bovine, hemagglutination inhibition, hemagglutinin esterase fusion,
20
bovine respiratory disease
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New
1
1
Abstract: Viruses with approximately 50% homology to human influenza C virus (ICV) have
2
recently been isolated from swine and cattle. The overall low homology to ICV, lack of antibody
3
cross reactivity to ICV in hemagglutination inhibition (HI) and agar gel immunodiffusion assays
4
and inability to productively reassort with ICV led to the proposal that these viruses represented
5
a new genus of influenza, influenzavirus D (IDV). To further our understanding of the
6
epidemiology of IDV, real time reverse transcription PCR was performed on a set of 208
7
samples from bovines with respiratory disease. Ten samples (4.8%) were positive and six
8
viruses were successfully isolated in vitro. Phylogenetic analysis of full genome sequences of
9
these six new viruses and four previously reported viruses revealed two distinct co-circulating
10
lineages represented by D/swine/Oklahoma/1334/2011 (D/OK) and
11
D/bovine/Oklahoma/660/2013 (D/660) which frequently reassorted with one another. Antigenic
12
analysis using the HI assay and lineage-representative D/OK and D/660 antiserum found up to an
13
approximate 10-fold loss in cross reactivity against heterologous clade antiserum. One isolate,
14
D/bovine/Kansas/3-13/2011 (D/3-13), clustered with the D/660 lineage, but also had high HI
15
titers to heterologous (D/OK) clade antiserum. Molecular modeling of the hemagglutinin
16
esterase fusion protein of D/3-13 identified a mutation at position 212 as a possible antigenic
17
determinant responsible for the discrepant HI results. These results suggest that IDV is common
18
in bovines with respiratory disease and at least two genetic and antigenically distinct clades co-
19
circulate.
20 21 22
2
1
Importance:
2
A novel bovine influenza virus was recently identified. Detailed genetic and antigenic studies
3
led to the proposal that this virus represents a new genus of influenza, influenzavirus D (IDV).
4
Here we show that IDV is common in clinical samples of bovine respiratory disease complex
5
(BRDC), with prevalence similar to other established BRDC etiological agents. These results
6
are in good agreement with near ubiquitous seroprevalence of IDV previously found.
7
Phylogenetic analysis of complete genome sequences found evidence for two distinct co-
8
circulating lineages of IDV which freely reassort. Significant antigenic differences were
9
observed between the two lineages that generally agreed with the surface glycoprotein
10
hemagglutinin esterase phylogeny. These results, in addition to the ability of IDV to infect and
11
transmit in multiple mammalian species, warrant additional studies to determine the pathogenic
12
potential of IDV.
13 14 15 16 17 18 19 20
3
1
Introduction:
2
Influenza viruses are single-stranded, negative-sense (-), segmented RNA viruses belonging to
3
the family Orthomyxoviridae (1). Influenza A (IAV) and influenza B (IBV) both contain eight
4
genomic segments, including two surface glycoproteins, the hemagglutinin (HA) and
5
neuraminidase (NA), whereas influenza C (ICV) only has seven segments with one surface
6
glycoprotein, the hemagglutinin-esterase-fusion (HEF) protein (2,3). While the vast genetic
7
diversity of IAV is found in waterfowl, only limited subtypes infect mammals. IBV and ICV are
8
found principally in humans and rarely infect other species. IBV is a component of seasonal
9
influenza epidemics with clinically significant disease while ICV infects most humans during
10
childhood and typically results in mild respiratory symptoms and fever (1,4-6).
11
In 2011, an influenza virus with moderate homology to ICV was isolated from swine in
12
Oklahoma (D/OK) exhibiting influenza-like symptoms. Sequence analysis showed
13
approximately 50% homology to human ICVs (7). D/OK did not cross react with antibodies
14
against human ICV in hemagglutination inhibition (HI) and agar gel immunodiffusion (AGID)
15
assays. Limited seroprevalence in swine and humans to D/OK (9.5% and 1.3%, respectively)
16
suggested that an alternate species was the reservoir of this novel virus (7). HI assays on bovine
17
sera found seven out of eight herds with titers greater than 40 to both D/OK and the bovine
18
D/660 strain. Eighteen percent of bovine respiratory disease samples were positive by RT-PCR
19
assay targeting the PB1 gene of D/OK. Virus isolation, genome sequencing and phylogenetic
20
analysis showed that D/OK and three bovine isolates were closely related and did not reassort
21
with human ICV (8). Likewise, in vitro reassortment experiments between D/OK and human
22
ICV failed to identify viable reassortant viruses. Reassortment of viral segments can yield viable
23
progeny within the same genera but not across genera of influenza (2,9,10). Taken together, 4
1
these results led to the proposal to classify D/OK-like viruses as a new genus of influenza virus,
2
influenzavirus D (IDV), with bovines as the potential reservoir (8).
3
While the current three genera of influenza, influenza A, B, and C, all share similar genetic
4
ancestry, they have diverged over time (2). ICV undergo reassortment frequently in nature,
5
which results in greater genetic diversity of the viruses (3,6,11,12). ICV is a product of multiple
6
lineage evolution, a result of co-circulating strains in the human population (6,10,13,14). As
7
influenza B and C viruses have further diverged from IAV, significant mutations resulted in the
8
lack of viable reassortant viruses between influenza B and C, and they both are thought to be
9
evolutionary stable (10,15). The discovery of IDV warrants new research into its evolutionary
10
history as well as its epidemiology and ecology.
11
Bovine respiratory disease complex (BRDC) is the most economically significant disease of the
12
beef industry with losses due to morbidity, mortality, treatment costs and reduced carcass value
13
(16,17). Established viral etiological agents include bovine viral diarrhea virus (BVDV), bovine
14
herpes virus (BHV-1), bovine respiratory syncytial virus (BRSV), and parainfluenza virus (PI3).
15
In the past several years there has been increasing evidence that bovine respiratory coronavirus
16
also contributes to BRDC in feedlot cattle (17,18). The finding of IDV in cattle warrants further
17
investigation into its possible role as a BRDC etiological agent.
18
To further investigate the epidemiology of this proposed new genus, a large sample set of BRDC
19
cases were screened by quantitative real time reverse transcription PCR (qRT-PCR) to determine
20
the molecular epidemiology of IDV in association with other bovine respiratory disease viral
21
agents. Phylogenetic analysis of full genome sequences, along with hemagglutination inhibition
22
(HI) assay was performed to characterize the genetic and antigenic diversity of IDV.
5
1
Materials and Methods:
2
Molecular screening of bovine viruses. Clinical samples from bovine respiratory disease
3
submissions to the Kansas State University Veterinary Diagnostic Lab (n=208) were screened by
4
a BRDC PCR panel which detects BVDV, BHV-1, BRSV, PI3, and BCV. The 208 samples,
5
consisting of nasal and pharyngeal swabs and lung tissue, originated from twelve states primarily
6
in the Midwest: Nebraska (n=48), Kansas (n=116), Colorado (n=6), Missouri (n=1), Mississippi
7
(n=7), Texas (n=4), Oklahoma (n=2), Idaho (n=2), Montana (n=6), Oregon (n=4), Washington
8
(n=11), and Virginia (n=1). Additionally, samples were analyzed for IDV using quantitative real
9
time reverse transcription PCR (qRT-PCR) (7).
10
Cells and Viruses. Human rectal tumor cells (HRT-18G) (ATCC CRL-11663) were maintained
11
in Dulbecco’s modified Eagles medium (DMEM) supplemented with 5% fetal bovine serum
12
(FBS). Swine testicle cells (ST) (ATCC CRL-1746) were maintained in the same manner. Both
13
cell lines were grown at 37°C with ±5% CO 2 before infection with viruses. IDV were isolated at
14
Newport Laboratories from PCR-positive field samples. D/swine/Oklahoma/1334/2011 (D/OK)
15
virus was previously isolated from swine exhibiting influenza-like symptoms (7).
16
D/bovine/Minnesota/628/2013 (D/628), D/bovine/Minnesota/729/2013 (D/729), and
17
D/bovine/Oklahoma/660/2013 (D/660) were previously isolated from bovine respiratory disease
18
samples submitted for diagnostic testing (8).
19
Virus Isolation. Virus isolation was performed on IDV qRT-PCR positive bovine samples
20
using confluent monolayers of HRT-18G cells in DMEM without FBS with 0.5 µg/mL trypsin,
21
at 37°C with ±5% CO 2. Viral growth was confirmed by qRT-PCR and the hemagglutination
22
assay (HA) using 0.5% washed turkey red blood cells, as IDV is non-cytopathic on HRT-18G
6
1
cells. Following one passage on HRT-18G cells, viruses were passaged to ST cells in DMEM
2
without trypsin. Growth was confirmed by qRT-PCR, HA, and cytopathic effects (CPE) after
3
each passage. PCR-positive samples failing to show CPE or increase in qRT-PCR and HA titers
4
were passaged three times before they were called negative.
5
Full-genome sequencing and analysis. The MegaMax viral RNA kit was used to isolate the
6
viral RNA. Full-genome sequencing was performed as previously described (8). In brief,
7
sequencing libraries were prepared from full-genome amplicons using the NEBNext Fast DNA
8
kit for Ion Torrent. D/OK (accession numbers NC_922305 to NC_922311) was used as the
9
template to assemble the contigs using the SegMan NGen module from DNAStar. Phylogenetic
10
analysis was performed using MEGA6.0 software using the Maximum Likelihood algorithm
11
with 1000 bootstrap replicates to verify tree topology.
12
Serology. Rabbit polyclonal antisera generated using D/660 and D/OK were used in the
13
hemagglutination inhibition (HI) assay. All viruses were assayed in triplicate. Colostrum
14
deprived/cesarean derived (CDCD, Struve Laboratories) sera was used as a negative control.
15
The HI test was performed according to the WHO protocol, using turkey red blood cells (19).
16
Mean HI titers and standard deviation were calculated from triplicate data. Heterologous mean
17
HI titers were normalized to mean homologous HI titers (mean heterologous titers / mean
18
homologous titers). Relative HI titers were reported to account for differences in homologous HI
19
titers between D/OK and D/660.
20
HEF Structure Modeling
21
The HEF amino acid sequence of D/3-13 HEF was queried against all sequences in the PDB
22
structure database using BLAST (20). The HEF structure from ICV C/Johannesburg/1/66 (PDB 7
1
ID: 1FLC) (21), which showed the highest sequence identity to D/3-13 HEF (53% sequence
2
identity), was chosen as template. The sequences of D/3-13 and ICV HEF were then aligned
3
using Muscle (22). The model of 3-13 HEF was built using Modeller (23). The quality of the
4
structure was checked using Verify 3D (17). For demonstration purpose, the sialic acid binding
5
template was shown on the D/3-13 HEF structure.
6 7
Results
8
Molecular Epidemiology: Ten of the 208 (4.8%) samples submitted for BRDC diagnostic
9
testing were positive for IDV by qRT-PCR with Ct values ranging from 21-29. These samples
10
consisted of nine nasal swabs and one swab of unknown type. In addition, other viruses
11
associated with BRDC were detected by qRT-PCR, with 36% (n=75), 7% (n=15), 5% (n=11),
12
3% (n=7), and
