Induction of type I interferons by a novel porcine reproductive and respiratory syndrome virus isolate.

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is known to interfere with the signaling of type I interferons (IFNs). Here we found PRRSV A2MC2 induced type I IFNs in cultured cells. A2MC2 replication in MARC-145 cells resulted in the synthesis of IFN-α2 protein, transcript elevation of the IFN-stimulated genes ISG15 and ISG56, and the proteins of the signal transducer and activator of transcription 2 (STAT2) and ISG56. A2MC2 infection of primary porcine pulmonary alveolar macrophages (PAMs) also led to the elevation of the two proteins, but had little cytopathic effect. Furthermore, A2MC2 infection of MARC-145 or PAM cells had no detectable inhibitory effect on the ability of IFN-α to induce an antiviral response. Sequencing analysis indicated that A2MC2 was closely related to VR-2332 and Ingelvac PRRS MLV with an identity of 99.8% at the nucleotide level. The identification of this IFN-inducing PRRSV isolate may be beneficial for vaccine development against PRRS.

Full text

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Induction of type I interferons by a novel porcine reproductive and
respiratory syndrome virus isolate
Yuchen Nana,1, Rong Wanga,1, Meiyan Shena,2, Kay S. Faabergc, Siba K. Samalb, Yan-Jin Zhanga,n
aMolecular Virology Laboratory, VA-MD Regional College of Veterinary Medicine and Maryland Pathogen Research Institute, University of Maryland, 8075 Greenmead Drive,
College Park, MD 20742, USA
bVirology Laboratory, VA-MD Regional College of Veterinary Medicine and Maryland Pathogen Research Institute, University of Maryland, College Park, MD 20742, USA
cVirus and Prion Research Unit, National Animal Disease Center, ARS, USDA, Ames, IA 50010, USA
a r t i c l e i n f o
Article history:
Received 11 March 2012
Returned to author for revisions
4 May 2012
Accepted 14 May 2012
Keywords:
Interferon induction
Porcine reproductive and respiratory
syndrome virus (PRRSV)
IFN
IFN signaling
a b s t r a c t
Porcine reproductive and respiratory syndrome virus (PRRSV) is known to interfere with the signaling
of type I interferons (IFNs). Here we found PRRSV A2MC2 induced type I IFNs in cultured cells. A2MC2
replication in MARC-145 cells resulted in the synthesis of IFN-a2 protein, transcript elevation of the
IFN-stimulated genes ISG15 and ISG56, and the proteins of the signal transducer and activator of
transcription 2 (STAT2) and ISG56. A2MC2 infection of primary porcine pulmonary alveolar macro-
phages (PAMs) also led to the elevation of the two proteins, but had little cytopathic effect.
Furthermore, A2MC2 infection of MARC-145 or PAM cells had no detectable inhibitory effect on the
ability of IFN-a to induce an antiviral response. Sequencing analysis indicated that A2MC2 was closely
related to VR-2332 and Ingelvac PRRS MLV with an identity of 99.8% at the nucleotide level. The
identification of this IFN-inducing PRRSV isolate may be beneficial for vaccine development
against PRRS.
& 2012 Elsevier Inc. All rights reserved.
Introduction
Porcine reproductive and respiratory syndrome virus (PRRSV)
is a positive-sense single-stranded RNA virus belonging to the
family Arteriviridae (Faaberg et al., 2011; Meulenberg, 2000). It
causes an economically important disease, resulting in an esti-
mated $560 million loss per year to the swine industry in the
United States (Neumann et al., 2005). PRRSV is known to inhibit
the synthesis of type I interferons (IFNs) in infected pigs (Albina
et al., 1998; Buddaert et al., 1998). IFNs are not detectable in the
lungs of pigs, in which PRRSV actively replicates. PRRSV-infected
pigs develop delayed and low titer neutralizing antibodies
(Labarque et al., 2000) and weak cell-mediated immune responses
(Xiao et al., 2004). Suppression of innate immunity may be an
important contributing factor to PRRSV modulation of host
immune responses.
PRRSV can be propagated in vitro in an epithelial-derived
monkey kidney cell line, MARC-145 (Kim et al., 1993), and in
primary cultures of porcine pulmonary alveolar macrophages
(PAMs). PAMs are the main target cells for PRRSV during its acute
infection of pigs (Rossow et al., 1995). PRRSV infection of PAM
and MARC-145 cells in vitro leads to a very low expression of
interferon-a (IFN-a) for viral strains studied to date (Albina et al.,
1998; Lee et al., 2004; Miller et al., 2004).
Type I IFNs, such as IFN-a and -b, are critical to innate
immunity against viral infection and contribute to the modulation
of adaptive immunity (Takaoka and Yanai, 2006). The innate
immune system is activated after cellular pattern recognition
receptors (PRR) sense pathogen associated molecular patterns
(PAMPs) of invading pathogens. Host PRRs for RNA viruses
include Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs).
Activation of the TLR or RLR pathways eventually leads to the
secretion of type I IFNs. The binding of type I IFNs to their
receptors activates the Janus kinase (JAK)-signal transducer and
activator of transcription (STAT) pathway, which induces expres-
sion of IFN-stimulated genes (ISGs) and results in the establish-
ment of an antiviral state (Darnell et al., 1994; Schindler and
Darnell, 1995; Stark et al., 1998).
Some PRRSV strains suppress IFN-b expression in MARC-145
cells and PRRSV non-structural proteins (nsp) 1, 2, 4, and 11 inhibit
IFN induction when over-expressed (Beura et al., 2010; Chen et al.,
2010; Kim et al. 2010). PRRSV can also inhibit IFN downstream
signaling and expression of ISGs in both MARC-145 and PAM cells
(Patel et al., 2010). The nuclear translocation of STAT1/STAT2/IRF9
heterotrimers was blocked in PRRSV-infected cells, while the IFN-
induced phosphorylation of STAT1 and STAT2 was not affected.
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/yviro
Virology
0042-6822/$-see front matter & 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.virol.2012.05.015
nCorresponding author. Tel.: þ1 301 314 6596; fax: þ1 301 314 6855.
E-mail address: zhangyj@umd.edu (Y.-J. Zhang).
1These authors have equal contribution.
2Present address: Shandong Vocational College of Veterinary Medicine and
Animal Science, Weifang, Shandong, China.
Please cite this article as: Nan, Y., et al., Induction of type I interferons by a novel porcine reproductive and respiratory syndrome
virus isolate. Virology (2012), http://dx.doi.org/10.1016/j.virol.2012.05.015
Virology ] (]]]]) ]]]–]]]

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Many efforts to control PRRS have been attempted, but few are
successful because of the antigenic and genomic diversity among
PRRSV isolates, and persistence of the virus in infected herds.
Attenuated live virus vaccines have been commercially available
for years; however, outbreaks of PRRS resulting from viral strains
nearly identical in sequence to the vaccine strain have been
reported (Botner et al., 1997; Opriessnig et al., 2002). Outbreaks
of atypical or acute PRRS in vaccinated pigs have raised serious
concern about the efficacy and safety of the current vaccines
(Madsen et al., 1998; Meng, 2000).
In the present study, we discovered a PRRSV isolate that
induced production of type I IFNs in both MARC-145 and PAM
cells and appeared to have an undetectable inhibitory effect on
the ability of IFN-a to induce an antiviral response. Its infection of
PAMs led to an undetectable cytopathic effect. Characterization of
this unique isolate will be beneficial to the study of PRRSV-cell
interactions and the development of improved vaccines.
Results
Detection of antiviral activity in cell culture supernatant from
A2MC2-infected MARC-145 cells
In studying PRRSV interference of IFN signaling, we discovered
one PRRSV cell culture isolate that did not inhibit IFN signaling
but induced antiviral activity in MARC-145 cells. After plaque
purification ofthis isolatethree
named A2MC2.
Vero cells are not susceptible to PRRSV infection and were
used as an indicator cell line for our studies. NDV-GFP is sensitive
to type I IFNs, so pre-treatment of Vero cells with IFN-a inhibited
NDV-GFP replication and was included as an assay control.
Treatment of Vero cells with dilutions of A2MC2-infected
MARC-145 cell culture supernatant reduced the number of
NDV-GFP-positive cells (Fig. 1A), which indicated that the NDV
times,oneplaquewas
replication was inhibited. This result suggested the existence of
type I IFNs in the culture supernatant of the A2MC2-infected cells.
To further confirm that the antiviral activity was due to
interferons, Western blot analysis was conducted to assess the
protein levels of interferon-stimulated gene 56 (ISG56) and
STAT2, in Vero cells. Blotting results showed that both ISG56
and STAT2 were elevated after treatment with the cell culture
supernatant from A2MC2-infected MARC-145 cells (Fig. 1B). The
levels of the proteins were similar to those of cells treated with
1000 U/ml IFN-a. These results suggested that A2MC2 induced
synthesis of type I IFNs in MARC-145 cells.
PRRSV is known to inhibit production of type I IFNs. To confirm
that A2MC2 is a genuine PRRSV isolate, an inhibition assay was
performed by using antisense peptide-conjugated phosphorodia-
midate morpholino oligomer (PPMO), 5UP1, which inhibits repli-
cation of PRRSV in MARC-145 cells in a sequence-specific manner
(Zhang et al., 2006). A scrambled control PPMO CP1 was included
as a negative control. An indirect immunofluorescence assay
showed that 5UP1 blocked A2MC2 replication in MARC-145 cells
(Fig. 1C), while CP1 had no effect. The lysate of A2MC2-infected
MARC-145 cells was used for Western blot analysis with pig
antiserum against PRRSV. Lysate samples of VR-2385- and MLV-
infected cells were included as controls. Blotting results showed
that A2MC2-infected cells had a band profile similar to MLV,
though the bands were at a weaker intensity (Fig. 1D). The
difference in band pattern between VR-2385 and MLV is likely
because there is a deletion in nsp2 of VR-2385 (Ni et al., 2011).
These results confirmed that A2MC2 was a strain of PRRSV.
Genotyping of this isolate was then performed.
Genotyping of PRRSV A2MC2 strain
RT-PCR was conducted for the whole A2MC2 RNA genome.
Sequencing of the cDNA was done and sequence analysis showed
that it closely resembles Ingelvac PRRS MLV (GenBank ID:
AF066183) and VR-2332 (GenBank ID: U87392), strains of
A2 Sup: 1:2 1:4 1:8 1:16
A2 Sup: 1:32 1:64
IFN
Mock
A2 Mock IFN Mock
STAT2
ISG56
Tubulin
17
26
43
56
130
kDa
PRRSV
Tubulin
A2MC2: + + + -
PPMO: - 5UP1 CP1 -
N
DAPI
VR MLV A2 Mock
72
95
Fig. 1. Detection of antiviral activity in cell culture supernatant from A2MC2-infected MARC-145 cells. (A) Inhibition of NDV-GFP replication in Vero cells. Vero cells were
treated with dilutions of cell culture supernatant of A2MC2-infected MARC-145 cells. The Vero cells were inoculated with NDV-GFP 12 h after the treatment, and observed
under fluorescence microscopy at 24 h post-infection. Treatment of the cells with IFN-a at a final concentration of 1000 U/ml was included as a positive control. (B)
Elevation of STAT2 and ISG56 proteins in Vero cells after treatment with the supernatant from A2MC2-infected MARC-145 cells detected by Western blot analysis. Vero
cells treated with IFN-a and mock-treated were included as positive and negative controls, respectively. Blotting with b-tubulin antibody was done to normalize protein
loading. (C) Inhibition of A2MC2 replication in MARC-145 cells by PRRSV-specific peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) 5UP1. A
scrambled control PPMO CP1 was included as a negative control. An indirect immunofluorescence assay with PRRSV N-specific monoclonal antibody was conducted. The
bottom panel of images shows the nuclear DNA stained with 40,6-19 diamidino-2-phenylindole (DAPI). (D) Detection of PRRSV proteins in whole cell lysate of A2MC2-
infected cells (A2 lane) by Western blotting with pig antiserum. Cell lysate samples from PRRSV VR-2385-infected (VR lane) or MLV-infected cells were included as positive
controls. Molecular weight markers are illustrated on the left.
Y. Nan et al. / Virology ] (]]]]) ]]]–]]]
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Please cite this article as: Nan, Y., et al., Induction of type I interferons by a novel porcine reproductive and respiratory syndrome
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Page 3

genotype 2 PRRSV, at identity of 99.8%. There were a total of 28
nucleotide (nt) variations when compared to VR-2332, resulting
in 14 amino acid changes (Supplementary Table 1). The nucleo-
tide variations are scattered from nt 4681 to the end of the
genome (Fig. 2). The first 4680 nucleotides are identical to VR-
2332. There were a total of 34 nucleotide variations when
compared to strain MLV, resulting in 19 different amino acids.
Compared to both VR-2332 and MLV, A2MC2 has 15 unique
nucleotides scattered from nt 4681 to the end of the genome
(Fig. 2). Ten of the unique changes are between nt 4681 and nt
10037 of the A2MC2 genome. The sequence from nt 11667 to
14420 of A2MC2 is the same as VR-2332 except 4 unique
nucleotide variations. The sequence from nt 14421 to the end of
the A2MC2 genome is the same as MLV except 1 unique nucleo-
tide variation.
At the amino acid level, the A2MC2 differences when com-
pared to VR-2332 were located in nsp3, nsp7, nsp8, nsp10, nsp11,
nsp12, GP3, and M; and the variations from MLV sequence were
located in nsp1b, nsp2, nsp8, nsp10, nsp11, nsp12, GP2, GP3, GP5,
and M (Supplementary Table 1). Six unique amino acid changes
occurred in A2MC2 when compared to VR-2332 and MLV:
threonine to serine in nsp8, serine to alanine and proline to
leucine in nsp10, serine to glycine in nsp12, methionine to valine,
and isoleucine to valine in GP3 (Table 1). Nsp10 is a RNA helicase
that unwinds dsRNA (Bautista et al., 2002), while the functions of
nsp8 and nsp12 are unknown. GP3 is a glycoprotein found in
PRRSV virions as a minor structural component (de Lima et al.,
2009). The genomic sequence of A2MC2 when compared to both
that of VR-2332 and MLV indicated that A2MC2 was possibly a
chimera derived from these two strains.
Growth properties of A2MC2 in MARC-145 and PAM cells
To determine the growth properties of A2MC2 in MARC-145
cells, a multi-step growth curve analysis was conducted. The cells
were inoculated at a MOI of 0.01, 0.1 and 1 TCID50 per cell,
respectively. Cell culture supernatant samples were collected
daily for five days after the inoculation and titrated for virus
yield. The cells inoculated with 0.01 TCID50 per cell had the
highest virus yield, 106.67TCID50per ml on day 3, while the cells
inoculated with 1 TCID50per cell had the lowest yield, lower than
103(Fig. 3A). The virus yields of cells with 0.01 TCID50increased
from 103on day 1 to 106.67on day 3, and remained at 106on day
5. The virus yield of cells with 1 TCID50per cell decreased from
103.5on day 1 to 102.5on day 5. The virus yields of cells with 0.01
TCID50on day 3, 4 and 5 were significantly higher than those from
cells with 0.01 and 1 TCID50.
To further characterize the growth properties of A2MC2,
plaque assays were conducted in comparison with VR-2385 and
MLV strains. MARC-145 cells were inoculated with A2MC2, VR-
2385, and MLV, respectively. Plaques were observed on 4 dpi after
neutral red staining. The A2MC2-infected cells resulted in a small
plaque morphology at around less than 1 mm in diameter, similar
to VR-2385, while cells infected with MLV revealed a plaque
morphology at around 5 mm in diameter, which was at least
5 times larger than those of A2MC2 (Fig. 3B). This result indicated
that A2MC2 replication in MARC-145 cells was different from that
of MLV.
To test if A2MC2 caused cytopathic effects (CPE) after infection
of PAM cells, as it does in MARC-145 cells, we inoculated primary
PAMs with PRRSV at the MOI of 0.05 TCID50per cell and observed
the cells at 20 hpi under bright field microscopy. A2MC2 infection
of PAMs caused no observable CPE, while VR-2385 and NVSL led
to cell death (Fig. 3C). A2MC2-infected cells appeared similar to
MLV-infected or uninfected PAM cells in morphology. A cell
viability assay was conducted to assess the relative viability level
between treatments. A2MC2-infected PAM cells showed a similar
viability rate as was seen in uninfected cells, as did MLV infection,
while VR-2385significantly reduced
(Fig. 3D). This result was consistent with the CPE observed under
bright field microscopy.
To determine virus yield in PAMs, cell culture supernatant was
collected at 24 hpi and titrated in MARC-145 cells by IFA. The
virus yields of A2MC2, MLV, VR-2385, and NVSL were 103.8, 103.6,
105.2, and 104.4TCID50per ml, respectively (Fig. 3E). The results
showed that viral yields of A2MC2 and MLV were significantly
lower than VR-2385 (Po0.05), but did not vary much from the
viral yield of NVSL.
viabilityto0.14-fold
A2MC2 replication induces strong expression of STAT2 and ISG56 in
MARC-145 cells
To determine if A2MC2 replication induces IFN-stimulated
genes in MARC-145 cells, the cells were infected with the virus
at 1 TCID50per cell and harvested at 24 h post-infection (hpi).
Western blot analysis showed that the levels of STAT2 and ISG56
in MARC-145 cells were remarkably elevated after A2MC2 infec-
tion (Fig. 4A). Treatment of A2MC2-infected cells with PPMO
5UP1 abolished the elevation, which indicated that the inhibition
of A2MC2 removed the stimulation. UV-inactivated A2MC2 failed
VR-2332
MLV
A2MC2
1
4680
10037
11666
14420 15411
Fig. 2. Illustration of sequence variation of A2MC2 in comparison to VR-2332 and MLV. The top line indicates the genomic sequence of VR-2332 and the numbers above
the line indicate nucleotide positions in the genome. The nucleotide variations of MLV in comparison with VR-2332 are indicated by narrow vertical bars. The nucleotide
variations of A2MC2 in comparison with VR-2332 are indicated by both narrow and wide vertical bars, among which the narrow vertical bars indicates they are the same
as MLV and the wide bars indicates they are unique for A2MC2 sequence.
Table 1
Nucleotide variations in A2MC2 sequence leading to unique amino acid changes
compared with both MLV and VR-2332a.
Positionb
Nucleotidec
Amino acidd
Proteine
A2MC2MLVVR-2332 A2MC2MLVVR-2332
7621
9627
9655
12012
12972
12975
T
G
T
G
G
G
A
T
C
A
A
A
A
T
C
A
A
A
S
A
L
G
V
V
T
S
P
S
M
I
T
S
P
S
M
I
nsp8
nsp10
nsp10
nsp12
GP3
GP3
aGenBank accession numbers: VR-2332 (GenBank ID: U87392), MLV (Gen-
Bank ID: AF066183), and A2MC2 (GenBank ID: JQ087873).
bNucleotide positions are indicated on left column based on VR-2332
sequence.
cNucleotides at the indicated positions are listed.
dAmino acids derived from the codon of indicated nucleotides are listed.
eProteins corresponding to the amino acids derived from the codon of
indicated nucleotide positions are listed on the right column.
Y. Nan et al. / Virology ] (]]]]) ]]]–]]]
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Please cite this article as: Nan, Y., et al., Induction of type I interferons by a novel porcine reproductive and respiratory syndrome
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Page 4

to induce the elevation of these two proteins, indicating that the
elevation was A2MC2 replication-dependent.
Real-time RT-PCR was conducted to detect the transcripts of
IFN-b, ISG15, and ISG56 in MARC-145 cells. Compared to mock-
treated control wells, A2MC2 infection at 1 TCID50 per cell
induced 422-, 73-, and 509-fold RNA elevations of IFN-b, ISG15,
and ISG56, respectively (Fig. 4B). The IFN-b transcript in A2MC2-
infected cells was 105-fold higher than that of IFN-a-treated
PRRSV-negative cells. The average levels of ISG15 and ISG56
transcripts in A2MC2-infected cells without external IFN-a were
1.68- and 1.6-fold, respectively, lower than those in IFN-a-treated
PRRSV-negative cells. The differences of ISG15 and ISG56 between
the A2MC2-infected and the IFN-a-treated uninfected cells were
statistically insignificant. Addition of IFN-a to A2MC2-infected cells
did not lead to a significant increase in expression of these three
genes, compared to A2MC2-infected cells without external IFN-a.
As different MOI led to variable virus yields in MARC-145 cells,
the protein levels of STAT2 and ISG56 in the cells after infection
with different MOIs were assessed. Western blotting showed that
the greater the MOI that was used to infect MARC-145 cells, the
higher the level of STAT2 and ISG56 at 24 hpi detected (Fig. 4C).
By 48 hpi, the cells with 0.1 TCID50had similar levels of these two
proteins to 1 TCID50. By 72 hpi, the cells with 1 TCID50had the
lowest level of these two proteins. The results indicated that the
high MOI inoculum was able to induce early synthesis of the two
proteins, while a low MOI led to delayed induction.
Comparison with other PRRSV strains on IFN production in MARC-
145 cells
To compare A2MC2 with other PRRSV strains in regards to IFN
induction, MARC-145 cells were inoculated with A2MC2, VR-
2385, NVSL, MLV, and VR-2332 at 1 TCID50per cell, separately.
These strains were selected because they are frequently used in
this laboratory, because VR-2385 (Halbur et al., 1995; Meng et al.,
1994), VR-2332 (Benfield et al., 1992), and NVSL (Kwon et al.,
2006) are PRRSV strains of varying virulence in experimentally
infected pigs, and Ingelvac PRRS MLV is a licensed modified live
vaccine strain. Cell culture supernatant samples were collected at
24 hpi and used to treat Vero cells for the IFN bioassay.
A2MC2
VR-2385
MLV
Mock
0
1
2
3
4
5
6
7
Days post-infection
Virus yield (Log10/ml)
1.0 MOI
0.1 MOI
0.01 MOI
1
2
345
**
**
**
0
0.2
0.4
0.6
0.8
1
1.2
Relative fold
PRRSV: - A2 MLV NV VR
Virus yield (Log10/ml)
PRRSV: A2 MLV VR NV
0
1
2
3
4
5
6
**
*
MLVNVSLA2MC2 VR-2385 Mock
Fig. 3. Growth properties of A2MC2 in MARC-145 and PAM cells. (A) Multi-step growth curve of A2MC2 in MARC-145 cells. The cells were inoculated with A2MC2 virus at
a multiplicity of infection of 0.01, 0.1 or 1 TCID50per cell. Virus yields at different time points after inoculation were titrated by an immunofluorescence assay. Error bars
represent variation of three repeated experiments. Significant differences in virus yields between cells with 0.01 TCID50and the rest two other groups are denoted by ‘‘**’’,
which indicate P-value ofo0.01. (B) Plaque assay completed in MARC-145 cells. The cells were infected with diluted A2MC2, VR-2385 or MLV and overlaid with agarose. A
plate of mock-infected cells was included as a negative control. Plaques were revealed at 4 dpi and photographed for comparison. (C) Cytopathic effect in PRRSV-infected
PAMs. PAM cells were inoculated with PRRSV and at 20 hpi, observed using bright field microscopy. Mock-infected cells were included for comparison. PRRSV VR-2385 and
NVSL led to cell death and lysis, while A2MC2 and MLV had little cytopathic effect. (D) Cell viability assay of PAMs. PRRSV-infected PAMs were assayed at 20 hpi with
CellTiter-Glo kit (Promega). Relative fold cell viability in comparison with uninfected PAMs were plotted. Only VR-2385-infected cells had significantly lower viability
(denoted by ‘‘**’’, indicating Po0.01) viability than uninfected PAMs. A2: A2MC2, NV: NVSL, VR: VR-2385. (E) Virus yield titrated using MARC-145 cells. Cell culture
supernatant samples from PRRSV-infected PAMs at 24 hpi were titrated in MARC-145 cells by IFA. Median tissue culture infectious dose per ml is shown. Error bars
represent variation of three repeated experiments. The virus yields of A2MC2 and MLV were significantly lower (denoted by ‘‘*’’, indicating Po0.05) than VR-2385.
Y. Nan et al. / Virology ] (]]]]) ]]]–]]]
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Please cite this article as: Nan, Y., et al., Induction of type I interferons by a novel porcine reproductive and respiratory syndrome
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Page 5

Supernatants from A2MC2-infected cells inhibited NDV-GFP
replication in Vero cells, while supernatant samples from
MARC-145 cells infected with VR-2385, VR-2332, NVSL or MLV
had no effect on NDV-GFP propagation in Vero cells (Fig. 5A).
Real-time RT-PCR analysis showed that A2MC2 induced an
820-fold elevation of IFN-b transcripts in MARC-145 cells, sig-
nificantly higher than that induced by VR-2385, VR-2332, NVSL,
or MLV (Fig. 5B). Western blot analysis showed that VR-2385, VR-
2332, NVSL, and MLV infection had no effect on STAT2 and ISG56
protein level, while A2MC2 infection led to a higher amount of the
two proteins (Fig. 5C).
ELISA was done to detect IFN level in culture supernatants of
MARC-145 cells infected with A2MC2, VR-2385, VR-2332, or MLV,
respectively. Due to paucity of ELISA kits for type I IFNs of
monkeys, only the level of monkey IFN-a2 was quantified. The
level of IFN-a2 in culture supernatant of A2MC2-infected cells
was 46.6 pg/ml and significantly higher than the supernatants of
MARC-145 cells infected with VR-2385, VR-2332 or MLV (Fig. 5D).
These results indicated that A2MC2 induced synthesis of type I
IFNs in MARC-145 cells, while the other four PRRSV strains
inhibited IFN induction.
Kinetics of IFN-b expression in A2MC2-infected MARC-145 cells
To further examine the expression of IFN-b in A2MC2-infected
MARC-145 cells, the cells were harvested at 2, 4, 6, 8, 10, 12, and
24 hpi for RNA isolation and real-time RT-PCR. The IFN-b mRNA
increased from 2-fold at 2 hpi to 474-fold at 24 hpi (Fig. 6A). The
large increase of IFN-b transcripts started at 8 hpi. This result
indicates that IFN-b expression increased concurrently with
A2MC2 replication. Viral RNAs at these time points were detected
by real-time RT-PCR. The results showed that the viral RNAs
detected at 8, 10, 12 and 24 hpi were 3.2-, 2.3-, 4.2-, and 2.5-fold,
respectively, higher than 2 hpi (Fig. 6B). The relatively small
increase in the viral RNA level is consistent with the result of
the multi-step growth curve showing limited virus replication in
cells inoculated with 1 TCID50per cell.
The data above showed that A2MC2 induced expression of
type I IFNs. We were interested in the status of JAK-STAT
signaling pathway in A2MC2-infected cells. STAT1 and STAT2
proteins are key players in JAK-STAT signaling, a pathway acti-
vated by type I IFNs (Darnell et al., 1994; Schindler and Darnell,
1995; Stark et al., 1998). Phosphorylation of STAT1 and STAT2 is
an early step in the pathway after IFNs bind to their receptors. To
determine if A2MC2-induced IFNs resulted in the activation of
these two proteins, we tested the phosphorylation status of STAT1
and STAT2 in MARC-145 cells at 0, 9, 16 and 24 hpi. The selection
of 9 hpi was based on the speculation that after increase of IFN-b
transcript at 8 hpi, phosphorylation of STAT1 and STAT2 would be
detected. The inclusion of 16 and 24 hpi was based on the
speculation that along with the increase of IFN-b expression,
phosphorylation of STAT1 and STAT2 would continue. The result
showed that the levels of phosphorylated STAT1 at tyrosine 701
(STAT1-Y701) and STAT2 at tyrosine 690 (STAT2-Y690) were
greatly increased at 9 hpi (Fig. 6C), indicating A2MC2-induced
IFNs led to the activation of STAT1 and STAT2. Moreover, the total
STAT2 and ISG56 were detectable at 9 hpi and increased at 16 and
24 hpi (Fig. 6C). This result suggested that A2MC2-induced IFNs
resulted in the activation of JAK–STAT signaling, which then led to
the increased expression of STAT2 and ISG56.
A2MC2 induces elevated expression of ISGs in primary porcine
alveolar macrophages
PAMs are the main target cells for PRRSV infection in vivo. To
determine the effect of A2MC2 on IFN synthesis in PAM cells,
PAMs were infected with A2MC2 and harvested for Western blot
analyses at 20 hpi. Infections of PAMs with VR-2385 and MLV
were included as controls. Compared to uninfected cells, A2MC2
infection resulted in the elevation of STAT2 and IFI56 (equivalent
to ISG56 in primates), while VR-2385 led to no change of these
two proteins, and MLV led to a slight elevation of STAT2 (Fig. 7A). To
test the effect of these virus strains on IFN signaling, IFN-a was
added to PAMs at 12 hpi. PAMs infected with A2MC2 and MLV had
elevated STAT2 and IFI56 to a similar level of uninfected cells treated
A2MC2:
IFN:
PPMO:
STAT2
ISG56
Tubulin
Induction folds
A2MC2:
IFN:
MOI: 1.0
STAT2
ISG56
Tubulin
STAT2
ISG56
Tubulin
STAT2
ISG56
Tubulin
24 hpi
48 hpi
72 hpi
0
200
400
600
800
1000
IFN-β
ISG15
ISG56
*
*
0.1 0.01 -
UV
-
5UP1 CP1
- + +
+ - +
-
-
--
-
-
--
--
-
+
++++
+
Fig. 4. A2MC2 replication induces elevated expression of IFN-stimulated genes in MARC-145 cells. (A) Elevation of STAT2 and ISG56 detected by Western blotting. The
cells were infected with A2MC2 or UV-inactivated virus at 1 TCID50per cell, followed by treatment with PPMO 5UP1 to inhibit A2MC2 replication, and at 24 hpi, treated
with or without IFN-a. Cell lysate from uninfected cells was included as a control. (B) Elevation of IFN-b, ISG15 and ISG56 expression detected by real-time PCR. Treatment
of the cells with IFN-a was included as a control. Relative induction folds in comparison with mock-treated cells are shown. Error bars represent variation between
repeated experiments. Significant differences between A2MC2-infected cells and the uninfected cells are denoted by ‘‘*’’, which indicates a P-value ofo0.05. (C) Kinetics of
STAT2 and ISG56 expression in MARC-145 cells infected with different MOIs of A2MC2. The cells were harvested at 24, 48 and 72 hpi for Western blot amalyses. Samples of
uninfected cell lysates were included as controls.
Y. Nan et al. / Virology ] (]]]]) ]]]–]]]
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Please cite this article as: Nan, Y., et al., Induction of type I interferons by a novel porcine reproductive and respiratory syndrome
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Page 6

with external IFN-a, while VR-2385-infected cells had no increase in
STAT2 and IFI56 levels (Fig. 7A). This result indicated that A2MC2
and MLV had an undetectable effect on IFN downstream signaling
while VR-2385 inhibited the IFN activation in PAMs.
The IFN bioassay was conducted to assess IFNs in culture
supernatantofA2MC2-infectedPAMs. CRL2843cellsare
immortalized porcine alveolar macrophages that are not suscep-
tible to PRRSV infection (Weingartl et al., 2002). The cells were
treated with dilutions of the supernatant from A2MC2-infected
PAMs and, on the next day, inoculated with NDV-GFP. The
supernatant dilutions up to 1:8 inhibited NDV-GFP replication,
compared with mock-infected cells (Fig. 7B). This result indicated
Mock A2MC2 VR-2385 VR-2332
IFN MLV NVSL Mock
PRRSV:
IFN:
STAT2
ISG56
Tubulin
A2VRMLV NVSL
IFN-β mRNA (folds)
0
200
400
600
800
1000
VR1
VR VR1 A2
- + - - -
0
10
20
30
40
50
60
A2VR MLV
VR1
IFN-α2 level (pg/ml)
**
**
- - - - - + -
- VR NV MLV A2 - -- -
Fig. 5. Comparison of A2MC2 to other PRRSV strains in IFN production using MARC-145 cells. (A) IFN bioassay in Vero cells. Cell culture supernatants from MARC-145 cells
infected with an MOI of 1 TCID50each of PRRSV strains A2MC2, VR-2385, VR-2332, MLV, or NVSL, respectively, were collected at 36 hpi. Vero cells were treated with 1:4
dilution of the respective supernatants for 12 h, and then infected with NDV-GFP. Fluorescence microscopy was conducted at 24 hpi. Treatment with IFN-a was included as
a positive control. (B) IFN-b expression in MARC-145 cells detected by real-time RT-PCR. The cells were infected with PRRSV and harvested for detection of IFN-b
transcript. Relative fold of induction in comparison with uninfected cells are shown. Error bars represent variation of three repeated experiments. Significant difference
between A2MC2 and the rest of the samples is denoted by ‘‘**’’, which indicates Po0.01. A2: A2MC2; VR: VR-2385; VR1: VR-2332. (C) STAT2 and ISG56 protein level in
MARC-145 cells detected by Western blotting. Treatment of uninfected cells with IFN-a was included as a positive control. A2: A2MC2; VR: VR-2385; NV: NVSL; VR1: VR-
2332. (D). IFN-a2 level in culture supernatants of MARC-145 cells infected with A2MC2, VR-2385, VR-2332, and MLV, respectively. ELISA analyses were conducted to
quantify the IFN-a2 levels and concentrations were calculated on the basis of a standard curve. The significant difference between A2MC2 and the rest of the samples is
denoted by ‘‘**’’, which indicates Po0.01.
0
100
200
300
400
500
600
Time (h):
Time (h):
STAT1-Y701
STAT2-Y690
STAT2
ISG56
Tubulin
Viral RNA (folds)
Time (h): 2
46810 1224 24681012 24
0
1
2
3
4
5
IFN-β mRNA (folds)
24 1690
Fig. 6. Time-course kinetics of IFN-b expression and activation of the JAK–STAT signaling pathway in A2MC2-infected MARC-145 cells. (A). Time-course kinetics of IFN-b
expression. The cells were infected with A2MC2 at 1 TCID50per cell and harvested at 2, 4, 6, 8, 10, 12, and 24 hpi for real-time RT-PCR detection of IFN-b transcript. Relative
fold of induction in comparison with uninfected cells are shown. Error bars represent variation between repeated experiments. (B) Viral RNA levels detected by real-time
RT-PCR. Relative fold of viral RNA in comparison with that detected at 2 hpi are shown. (C) Activation of the JAK-STAT signaling pathway. The cells were infected with
A2MC2 at an MOI of 1 TCID50per cell and harvested at 0, 9, 16, and 24 hpi for Western blot analysis of phosphorylated STAT1 (STAT1-Y701) and STAT2 (STAT2-Y690),
whole STAT2, and ISG56.
Y. Nan et al. / Virology ] (]]]]) ]]]–]]]
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Please cite this article as: Nan, Y., et al., Induction of type I interferons by a novel porcine reproductive and respiratory syndrome
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Page 7

that the culture supernatant of A2MC2-infected PAMs contained
interferons.
To determine if the induction of IFNs by A2MC2 is not limited
to PAMs from one piglet, PAMs from three other piglets were
inoculated with A2MC2 and incubated for 20 h. Western blot
analysis showed that A2MC2 infection induced the elevation of
STAT2 in PAM cells from all three other piglets (Fig. 7C). The level
of STAT2 in A2MC2-infected PAMs was similar to that in PAMs
treated with external IFN-a.
Discussion
Many PRRSV strains are known to inhibit the production of
type I IFNs in cultured cells and in infected pigs. A2MC2 induces
the synthesis of interferons in MARC-145 and primary PAM cells,
and will be useful in studying PRRSV interference with host
innate immune responses. In this study, the induction of IFNs
by A2MC2 was examined from several aspects. First, the culture
supernatant from A2MC2-infected MARC-145 cells protected
Vero cells from NDV infection. Vero cells are defective in IFN
production and not susceptible to PRRSV. Pretreatment of the
cells with dilutions of A2MC2-derived supernatant induced an
antiviral response in Vero cells that inhibited the replication of
NDV. The result was corroborated by elevation of STAT2 and
ISG56, two genes stimulated by IFN signaling in Vero cells.
Second, the elevated expression of STAT2 and ISG56 was
shown in A2MC2-infected MARC-145 cells. Virus replication was
essential for the elevation of the two proteins because UV-
inactivated virus and PPMO inhibition of PRRSV replication did
not lead to elevation of these host proteins. The results suggested
that viral replication in the cytoplasm stimulated PRRs, leading to
IFN synthesis. The elevation of the transcripts of ISG15 and ISG56
further confirmed the observation. This result also ruled out the
possibility of contamination by other swine pathogens because
PPMO-mediated inhibition of A2MC2 led to an absence of IFN
induction. Other common swine pathogens, such as porcine
respiratory coronavirus (PRCV) or swine influenza virus (SwIV),
can induce synthesis of a high level bioactive IFN-a (Van Reeth
et al., 1999). Interestingly, A2MC2 infection at different MOIs
induced variable levels of ISG expression. The higher the MOI, the
earlier A2MC2 induced ISG elevation. The lower the MOI, the later
the virus-induced ISG expression and the lower level of induction.
This result provided an explanation as to why an MOI of 1 TCID50
per cell led to a lower virus yield than MOIs of 0.1 and 0.01
TCID50. It indicates that the inoculum of 0.01 TCID50 per cell
resulted in limited initial virus replication, leading to a weaker
stimulation on cellular PRRs, which allowed the virus to complete
its replication cycles. On the other hand, the inoculum at 1 TCID50
per cell stimulated a more robust response in regards to PRRs and
IFN synthesis, which in turn inhibited virus spread to neighboring
cells or continued replication.
Third, several other PRRSV strains including the vaccine strain
MLV were shown to inhibit IFN production. IFN-a2 was detected
in culture supernatant from A2MC2-infected cells, but not in the
samples from cells infected with VR-2332, VR-2385, or MLV. It
was possible that there were other subtypes of type I IFNs in the
supernatant from A2MC2-infected cells, as IFN-b mRNA level
significantly increased. We further tested IFN production in
A2MC2 infection of PAM cells. The STAT2 and IFI56 were elevated
in A2MC2-infected cells, but not in VR-2385-infected cells. The
A2MC2 induction of IFNs was pig-independent as PAMs isolated
from three other pigs had similar elevations of STAT2 after
A2MC2 infection. Infection of PAMs with MLV also led to a slight
elevation in STAT2 level, which indicates that MLV might induce
STAT2 albeit at much lower levels than A2MC2.
Analysis of the cDNA sequence of the A2MC2 genome showed
that it was highly homologous to both VR-2332 and MLV. This
result indicated that A2MC2 might be a chimera of VR-2332 and
MLV strains. Based on sequence analysis, we reasoned that the
first 4.6 kb and the fragment from nt 11966 to 14420 were
derived from VR-2332; fragments of nt 10697 to 11666 and nt
14421 to the end of the genome were possibly of MLV origin; and
the fragment of nt 4681 to 10037 was derived from either one of
them, but with mutations as 10 unique changes are located in this
fragment. Compared to both VR-2332 and MLV, A2MC2 possessed
6 unique amino acids distributed in nsp8, nsp10, and nsp12, and
GP3. Based on this sequence comparison, future experiments on
virus mutagenesis using reverse genetic technology will be
conducted to pinpoint the A2MC2 sequences that are involved
in the loss of inhibition of IFN production.
Both A2MC2 and MLV have an undetectable effect on the
ability of IFN-a to induce an antiviral response, as their infection
of PAM cells did not affect expression of STAT2 and IFI56
activated by external IFN-a. This result is consistent with a
previous report that virulent strain VR-2385 inhibits IFN signaling
while MLV does not (Patel et al., 2010). The plaque morphology of
PRRSV:
IFN:
STAT2
IFI56
Tubulin
1:2
A2 Sup:
1:8
1:4
Pig 1
+
STAT2
Tubulin
Pig 2
+
Pig 3
+
A2:
IFN:
IFN1:16
-
-
-
---
-
+
-
--
-
+
-
--
-
+
-
--
-
+
+++
A2MLV VR A2 MLV VR
Fig. 7. A2MC2 induces expression of IFN-stimulated genes in primary porcine
pulmonary alveolar macrophages (PAMs). (A) STAT2 and IFI56 detected by
Western blotting. PAMs were infected with PRRSV VR-2385, A2MC2, and MLV,
and at 12 hpi, treated with or without IFN-a. The cells were harvested at 20 hpi for
Western blotting. Cell lysate samples from uninfected PAMs with or without IFN
treatment were included as controls. (B) IFN bioassay in CRL2843 cells. Super-
natant from A2MC2-infected PAMs was diluted and added to the CRL2843 cells
12 h before NDV-GFP inoculation. The cells were observed 24 h after NDV-GFP
inoculation. Treatment of the cells with swine IFN-a at a final concentration of
1000 U/ml was included as a positive control. (C) A2MC2 induces elevation of
STAT2 in PAM cells from different piglets. PAMs from three piglets were
inoculated with A2MC2 at 0.05 TCID50per cell, respectively, and incubated for
20 h. Cell lysate samples from IFN-a-treated PAM cells were included as positive
controls. Cell lysate samples from non-infected cells were included as negative
controls in the Western blot analyses.
Y. Nan et al. / Virology ] (]]]]) ]]]–]]]
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Please cite this article as: Nan, Y., et al., Induction of type I interferons by a novel porcine reproductive and respiratory syndrome
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Page 8

A2MC2 was much smaller than that of MLV, which indicated that
MLV replicated with faster kinetics than A2MC2 in MARC-145
cells. In addition, A2MC2 infection did not lead to any observable
cytopathic effect in PAM cells, and A2MC2-infected cells were of
similar viability as mock-infected or MLV-infected cells. These
features indicated that like MLV, A2MC2 might be less suitable to
replicate in PAMs.
The well-characterized cellular PRRs for IFN production during
virus infection include the TLR and RLR pathways. Activation of
either the TLR or the RLR pathway leads to the phosphorylation of
interferon regulatory factor 3 (IRF-3), followed by its nuclear
translocation and transcriptional activation of type I IFNs. We
tested if A2MC2 replication led to IRF-3 phosphorylation using
polyinosinic-polycytidylic acid (poly(I:C)), a synthetic analog of
double-stranded RNA (dsRNA), as a positive control. The phos-
phorylated IRF-3 was detected in the poly(I:C)-treated cells but
undetectable in cells infected with A2MC2 (data not shown). The
result that A2MC2 infection led to increase of IFN-b transcript
from 8 hpi to less than 1000-fold at 24 hpi (Fig. 6) indicated that
A2MC2 replication might lead to a low level of IRF-3 signaling for
an extended period. The reason for this conjecture is that poly(I:C)
treatment led to over a 10,000-fold induction of IFN-b transcript
with a peak at 6 h after treatment (data not shown). The
continuous detection of phosphorylation of both STAT1 and
STAT2 in A2MC2-infected MARC-145 cells from 9 to 24 hpi
supports this speculation, while stimulation with external IFN-a
leads to transient increase of phosphorylation of STAT1 that was
reduced below the detection level after 2.5 h treatment (Patel
et al., 2010).
In conclusion, we have discovered a strain of PRRSV, A2MC2,
that induced IFN production in both MARC-145 and PAM cells
while other tested PRRSV strains inhibited IFN induction. Speci-
fically, A2MC2 induced type I IFNs and led to an elevation of IFN-
stimulated genes. Full genome sequence analysis showed that
A2MC2 was closely related to VR-2332 and MLV. The identifica-
tion of A2MC2 as an IFN-inducer may be beneficial for studying
PRRSV interference in IFN signaling, and developing improved
vaccines for protective immunity against PRRS.
Materials and methods
Cells and viruses
MARC-145 (Kim et al., 1993) and Vero cells (ATCC CCL-81)
were grown in Dulbecco’s Modified Eagle’s Medium (DMEM)
supplemented with 10% fetal bovine serum (FBS). Immortalized
porcine macrophages (CRL2843) were cultured in RPMI1640
medium supplemented with 10% FBS. Primary PAM cells were
prepared from bronchoalveolar lavage of 4-week-old PRRSV-
negative piglets. The preparation and subsequent culture of PAMs
in RPMI1640 culture medium were conducted, as previously
described (Patel et al., 2008). PRRSV strains A2MC2, VR-2385,
NVSL 97-7895, and Ingelvac PRRS MLV were used to inoculate
MARC-145 cells at 1 multiplicity of infection (MOI). Virus titers
were determined in MARC-145 cells for the median tissue culture
infectious dose (TCID50), as previously described (Zhang et al.,
2006). Avirulent LaSota Newcastle disease virus carrying the gene
of green fluorescence protein (NDV-GFP) was propagated in Vero
cells, as previously described (Kim and Samal, 2010).
Virus inactivation was conducted with a UV cross-linker
(Spectrolinker XL-1500, Agilent Technologies, Santa Clara, CA) at
1200 mJ/cm2for two 10-min pulses at 1-min interval. The
inactivation was confirmed by the absence of virus replication
in MARC-145 cells at 72 h post-infection (hpi) as assessed by
immunofluorescence assay (IFA).
For interferon stimulation, universal type I IFN-a (R&D Sys-
tems, Minneapolis, MN) was added to the cultured cells at a final
concentration of 1000 U/ml. The cells were harvested at indicated
time points for further analysis.
Interferon bioassay
Vero cells were seeded into cell culture plates, incubated
overnight, and, on the next day, treated with culture supernatant
from PRRSV-infected MARC-145 cells. The cells were infected
with LaSota NDV-GFP 12 h after the treatment. Fluorescence
microscopy was conducted 24 h after infection to observe GFP-
positive cells.
Immunofluorescence assay (IFA)
An IFA was carried out as previously reported with an
N-specific monoclonal antibody EF11 to detect PRRSV N-proteins
in MARC-145 cells on coverglass slips (Zhang et al., 1998). Specific
reactions between EF11 and the N-protein were detected with
goat anti-mouse IgG-fluorescein isothiocyanate (FITC) conjugate
(Sigma, St. Louis, MO). The coverglass was mounted onto slides
using SlowFade Gold antifade reagent containing 406-diamidino-
2-phenylindole (DAPI) (Life Technologies Corporation, Carlsbad,
CA) and observed under fluorescent microscopy.
Western blot analysis
Cell lysate samples were analyzed by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot
analysis as described previously (Zhang et al., 2007). Briefly,
separated proteins from SDS-PAGE were transferred onto a
nitrocellulose membrane and probed with antibodies against
STAT2 (Santa Cruz Biotechnology, Santa Cruz, CA), b-tubulin
(Sigma), phospho-STAT2 (STAT2-Y690) (Santa Cruz Biotechnol-
ogy), phospho-STAT1 (STAT1-Y701) (Millipore, Billerica, MA), and
ISG56 (Thermo Fisher Scientific, Rockford, IL). The chemilumines-
cent signal was recorded digitally by Quantity One Program,
Version 4.6, in a ChemiDoc XRS imaging system (Bio-Rad Labora-
tories, Hercules, CA). Pig antiserum against PRRSV NVSL strain
(NVSL, Ames, IA) was used to detect PRRSV proteins in lysate of
PRRSV-infected cells (Patel et al., 2010).
RNA isolation, reverse transcription, and real-time PCR
Total RNA was isolated from MARC-145 and PAM cells with
TRIzolsReagent (Life Technologies) following the manufacturer’s
instructions. Reverse transcription and real-time PCR were con-
ducted as previously described (Patel et al., 2008, 2009). Tran-
scripts of ribosomal protein L32 (RPL32) were also amplified from
the samples of PAM and MARC-145 cells and used to normalize
the total input RNA. Primers used in this study to conduct reverse
transcription and real-time RT-PCR were previously described
(Patel et al., 2010). Relative transcript levels were quantified by
the 2?DDCTmethod (Livak and Schmittgen, 2001) and shown as a
relative fold of change in comparison with mock-treated control.
Cell viability assay
Viability of PAMs was determined with CellTiter-Glo Cell
Viability Assay (Promega). Briefly, CellTiter-Glo reagent was
added to cells in a 96-well plate and the luminescence signal
was measured with VICTOR3TMMultilabel Counter (Perkin-Elmer
Life and Analytical Sciences, Wellesley, MA). Relative percentages
of luminescence intensity were calculated by comparison to
controls.
Y. Nan et al. / Virology ] (]]]]) ]]]–]]]
8
Please cite this article as: Nan, Y., et al., Induction of type I interferons by a novel porcine reproductive and respiratory syndrome
virus isolate. Virology (2012), http://dx.doi.org/10.1016/j.virol.2012.05.015

Page 9

Plaque assay
Plaque assays were done with modifications from a previously
described protocol (O’Reilly et al., 1992). MARC-145 cells were
seeded into 35 mm culture dishes and incubated overnight.
PRRSV was diluted in a ten-fold series and added to the cells.
The virus inoculum was removed 2 h after inoculation and
replaced with 0.5% agarose overlay containing complete growth
medium. The cells were stained with another layer of agarose
overlay containing neutral red at 50 mg/ml 4 days after inocula-
tion. Plaques were observed after overnight incubation.
Quantifying IFN level by ELISA
Culture supernatant samples of MARC-145 cells infected with
A2MC2, VR-2385, VR-2332, or MLV at a MOI of 1 TCID50per cell
were collected at 24 hpi. Detection of IFN-a2 in each sample was
done by using VeriKineTMCynomolgus/Rhesus Interferon-Alpha
Serum ELISA kit (PBL InterferonSource, Piscataway, NJ) according
to the manufacturer’s instruction. The concentration of IFN-a2 in
the samples was calculated on the basis of a standard curve
prepared from supplied IFN-a2 in the kit.
Sequencing
A2MC2 genomic RNA was isolated from cell culture super-
natant with TRIzol LS reagent (Life Technologies). Reverse tran-
scription of theviral RNAwith
TCAATTCAGGCCTAAAGTTG-30) and P6-7-R (50-CGCCCTAATTGAA-
TAGGTGACTT-30) was done with Maxima reverse transcriptase
(Thermo Fisher Scientific). PCR amplification was done with
Phusion high-fidelity DNA polymerase (New England Biolab,
Ipswich, MA). 50-RACE (rapid amplification of cDNA ends) and
30-RACE of the A2MC2 genome were done as previously described
(Sambrook and Russell, 2001). Sequencing of the PCR products
was performed with ABI Prism 3130 Genetic Analyzer (Life
Technologies). Sequence assembly and analysis was done with
LaserGene Core Suite (DNASTAR Inc., Madison, WI). The GenBank
accession number of the cDNA sequence of the A2MC2 genome is
JQ087873.
primers32nsp12R1(50-
Statistical analysis
Differences in indicators between treatment samples, such as
cellular RNA level between the groups in the presence or absence
of PRRSV infection, were assessed by the Student t-test. A two-
tailed P-value of less than 0.05 was considered significant.
Acknowledgments
We are grateful to Dr. Joseph F. Urban at Human Nutrition
Research Center, USDA, Beltsville, MD for his gift of the lung
lavage of piglets. We thank Sa Xiao at University of Maryland,
College Park, MD for his advice and discussion in sequencing of
A2MC2 cDNA. Y. Nan and R. Wang were partially supported by
China Scholarship Council. M. Shen was partially supported by
Shandong Bureau of Education, China. This project was supported
by National Pork Board, USA.
Appendix A. Supporting information
Supplementary data associated with this article can be found
in the online version at doi:10.1016/j.virol.2012.05.015.
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Please cite this article as: Nan, Y., et al., Induction of type I interferons by a novel porcine reproductive and respiratory syndrome
virus isolate. Virology (2012), http://dx.doi.org/10.1016/j.virol.2012.05.015