Research in Veterinary Science 93 (2012) 1060–1065

Contents lists available at SciVerse ScienceDirect

Research in Veterinary Science

journal homepage: www.elsevier .com/locate / rvsc

Inhibitory effects of recombinant porcine interferon-a on high- andlow-virulence porcine reproductive and respiratory syndrome viruses

Shijuan Dong a,b, Yanbo Yin c, Shiyuan Shen a,b, Yanyan Guo c, Minjie Gao d, Wanhua Zhang a,Yumin Zhu a,b, Ruisong Yu a,b, Zhongping Shi d, Zhen Li a,b,⇑a Institute of Animal Science and Veterinary Medicine, Shanghai Academy of Agricultural Sciences, Shanghai 201106, Chinab Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai 201106, Chinac College of Animal Science and Veterinary Medicine, Qingdao Agricultural University, Qingdao, Shandong 266109, Chinad Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, Jiangsu 214122, China

a r t i c l e i n f o

Article history:Received 21 April 2011Accepted 6 December 2011

Keywords:Interferon-aPorcine respiratory and reproductionsyndromeVirusChallengeLymphocyteImmunology

0034-5288/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.rvsc.2011.12.006

⇑ Corresponding author at: Institute of Animal ScieShanghai Academy of Agricultural Sciences, Shangha62206391; fax: +86 21 62207858.

E-mail address: zhenli60@163.com (Z. Li).

a b s t r a c t

The inhibitory effects of recombinant porcine interferon alpha (rPoIFN-a) on the propagation of low-virulence PRRSV (lvPRRSV) in MARC-145 cells, and on the progress and severity of high virulence PRRSV(hvPRRSV)-induced infections in pigs, were determined. Pre-treatment of MARC-145 cells with increasingconcentrations of rPoIFN-a prior to infection with lvPRRSV decreased the observed cytopathic effects(CPEs) in a concentration-dependent manner. Viral propagation and antibody response were temporarilydelayed in swine treated with rPoIFN-a either at the same time as the hvPRRSV challenge was adminis-tered or post-challenge. Exposure of challenged animals to rPoIFN-a after the onset of disease symptomsalleviated associated hyperthermia. Variations in lymphocyte subsets indicated that rPoIFN-a treatmentmight alleviate damage to the immune system or enhance propagation of host cytotoxic T-lymphocyteswhen the treatment was applied simultaneously with the virus or 1 dpc, respectively.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Porcine reproductive and respiratory syndrome (PRRS) iscaused by PRRS virus (PRRSV), a single-stranded positive-senseRNA virus belonging to the family Arteriviridae in the orderNidovirales. Since 2006, when highly virulent PRRSV (hvPRRSV)first appeared in China, PRRS has been regarded as a major threatto the Chinese pig industry. Highly virulent PRRSV contains a dis-continuous deletion of 30 amino acids in the second nonstructuralprotein (NSP2) (Tian et al., 2007) and, although these deletions donot appear to be related to virulence (Zhou et al., 2009), they doprovide a useful marker for highly virulent strains of the virus.Highly virulent PRRSV causes an acute infection with high morbid-ity and mortality rates, and disease prevention is difficult due tothe lack of an effective vaccine.

PRRSV infections were found to compromise the host’s immunesystem, possibly due to the inhibition of adequate cytokine expres-sion (Albina et al., 1998a; Thanawongnuwech et al., 2001;Murtaugh et al., 2002; Miller et al., 2004). Production of type I

ll rights reserved.

nce and Veterinary Medicine,i 201106, China. Tel.: +86 21

interferons (IFNs) was affected (Albina et al., 1998b; Buddaertet al., 1998) which, in turn, delayed the production of IFN-c, theonset of cellular immunity and the formation of neutralizing anti-bodies (Mateu and Diaz, 2008). Further support for this hypothesiswas provided by research showing that swine IFNs had broad-spectrum antivirus activity and exhibited immunoregulatory func-tions both in vivo and in vitro (Buddaert et al., 1998). For example,porcine IFN-c (PoIFN-c) protected swine from infection with foot-and-mouth disease virus (FMDV) or delayed the appearance ofclinical symptoms (Yao et al., 2008), while swine IFN-b protectedboth MARC-145 cells and porcine alveolar macrophages (PAMs)from PRRSV infection (Overend et al., 2007). Furthermore, IFN-ainhibited PRRSV replication in cultured cells, and elevated IFN-ain animal tissues substantially attenuated subsequent PRRSV repli-cation (Albina et al., 1998b; Buddaert et al., 1998).

All the data above suggested that it might be practical to treathvPRRSV-infected pigs with porcine IFN-a (PoIFN-a) and we haveundertaken a two-part investigation to test this hypothesis. Thisinvestigation consisted of a cell-based study to determine if recom-binant PoIFN-a (rPoIFN-a) exerted an inhibitory effect on low vir-ulence PRRSV (lvPRRSV) [a weakly virulent strain, manipulation ofwhich did not require a biological safety level-3 (BSL-3) labora-tory], and an animal-based study to determine if rPoIFN-a pro-vided any protection to pigs challenged with hvPRRSV.

S. Dong et al. / Research in Veterinary Science 93 (2012) 1060–1065 1061

2. Materials and methods

2.1. Virus, rPoIFN-a and cell populations

Recombinant PoIFN-a (rPoIFN-a) was expressed in Pichia pasto-ris KM71H cells grown in a bench-scale bioreactor (Yu et al., 2010).Yeast cells were removed by centrifugation (15,000g, 0.5 h, 4 �C),the supernatant was dialyzed for 24 h against two changes of0.2 mM Na phosphate buffer solution, and the dialyzed solutionwas then lyophilized in the presence of stabilizing additives (Donget al., 2010). The antiviral activity of rPoIFN-a (5 � 105 IU/mL) wasmeasured using the WISH–VSV assay system by quantifying theextent to which pretreatment of WISH cells with rPoIFN-a inhib-ited the cytopathic effect (CPE) caused by subsequent exposureof the cells to vesicular stomatitis virus. The CPE was determinedmicroscopically without staining.

The hvPPRSV strain used in animal challenge experiments,strain JXA1, was isolated in Jiangxi, China in 2006 (Tian et al.,2007) and, in common with other hvPRRSV strains, has discontin-uous deletions of 30 amino acids in NSP2. Low virulence porcinereproductive and respiratory syndrome virus (lvPRRSV) was theNorth American genotype VR2332 vaccine strain (BoehringerMannheim Corporation, Germany).

MARC-145 cells were grown in Dulbecco’s Modified Eagle Med-ium – High Glucose (DMEM; Gibco, Carlsbad, CA, USA) in the pres-ence of 10% fetal calf serum (Gibco).

2.2. Cellular test for anti-lvPRRSV activity of rPoIFN-a

rPoIFN-a antiviral activity against lvPRRSV was determinedusing MARC-145 cells, suspensions of which were seeded into sev-enteen 96 well plates (6 � 104 cells/well) comprising 16 testgroups and one control group. After the cells had grown to forma monolayer, culture medium was removed and the cells of the testgroups were incubated with twofold serial dilutions of rPoIFN-a.After further incubation for 20 h at 37 �C, 100 lL of 10-fold serialdilutions of lvPRRSV were added to both test and control groupsand the CPE was monitored microscopically without staining inevery case. The 50% tissue culture infective dose (TCID50) forlvPRRSV was calculated according to the Reed–Muench method(Reed and Muench, 1938).

2.3. Anti-hvPRRSV activity of rPoIFN-a in vivo

Twenty-five 4-week-old pigs that tested negative for bothPRRSV RNA and antiserum were divided randomly into five groupsand housed in level II animal biocontainment facilities located atthe Aolan Biotech Company (Qingdao, Shandong, China). Treat-ments administered to the different groups are shown in Table 1.Control group C1 was not treated with rPoIFN-a or challenged withhvPRRSV, while control group C2 was challenged with hvPRRSVbut not exposed to rPoIFN-a. Test groups (T groups) were differen-tiated according to when they were exposed to rPoIFN-a. Pigs in T1group were treated by rPoIFN-a on the same day of challenge, T2

Table 1Experimental treatments.

Group Treatments

Challenge with hvPRRSV (day 0)

Control 1 (C1) NoControl 2 (C2) YesT1 YesT2 YesT3 Yes

group was treated one day after challenge, and T3 group was trea-ted when symptoms began to appear. For experimental purposes,the time of challenge was designated as day zero. Pigs were chal-lenged with hvPRRSV at a dosage of 1.40 � 107 TCID50 by intramus-cular injection, selected on the basis of published data (e.g.Hermann et al., 2005) demonstrating that this route is effectivein establishing homogeneous infections within groups of animals,in the right side of the neck. Recombinant PoIFN-a was adminis-tered on three consecutive days by intramuscular injection in theleft side of the neck. Blood samples were collected at 0, 2, 4, 6, 8,10, 13, 16, 20, 24 days post-challenge (dpc). All experiments wereconducted according to approved procedures relating to animalexperimentation carried out in registered Chinese institutions. Inthis context, an ethical endpoint based on clinical symptoms (fol-lowed by culling) would normally have been adopted as an alter-native to death, but published data describing viable options arelimited. Consequently, in our experiments, either complete recov-ery or death of the challenged animals was adopted as theendpoint.

2.4. Clinical monitoring

After challenge, pigs were visually inspected daily for generaland clinical symptoms, including fever, loss of appetite, respiratoryabnormalities and skin rash, and rectal temperatures and bodyweights were recorded. Clinical respiratory scores (ranging from0 to 3) were calculated as described previously (Martelli et al.,2009).

2.5. Detection of PRRSV antibodies

PRRSV antibodies were detected using an enzyme-linkedimmunosorbent assay (ELISA) kit (IDEXX Laboratories Inc.,Westbrook, ME, USA) according to the manufacturer’s instructions.

2.6. Detection of neutralizing antibodies

Serum samples taken at 16, 20 and 24 dpc were analyzed forPRRSV neutralizing antibodies as follows: 0.025 mL hvPRRSV sus-pension diluted to 200 TCID50 was mixed with the same volumeof serum sample diluted fourfold. Mixtures were incubated for1 h at 37 �C and then inoculated onto a monolayer of MARC-145cells growing in separate wells of a well-plate. The CPE producedin each well was examined after 120 h and the titer of neutralizingantibody in each serum sample was expressed as ND50 (the highestdilution that neutralized 50% of the virus activity), calculated usingthe Reed–Muench formula (Reed and Muench, 1938).

2.7. Detection of PRRSV RNA

Highly virulent PRRSV RNA levels in serum samples were as-sayed using quantitative real-time RT-PCR. Viral RNA was ex-tracted from serum samples using TRIzol LS (Invitrogen, Carlsbad,CA, USA) according to the manufacturer’s protocol. After

Exposure to rPoIFN-a rPoIFN-a dose (IU)

No exposure –No exposure –Day 0 3 � 106

Day 1 2 � 106

When symptoms appeared 2 � 106

1062 S. Dong et al. / Research in Veterinary Science 93 (2012) 1060–1065

suspension in 20 lL DEPC-treated water, RNA samples were sub-jected to real-time RT-PCR using a one-step multiplex qRT-PCRkit (AgPath-ID™ PRRSV Reagent Kit) and a ABI 7500 fast sequencedetection system (Applied Biosystems, Carlsbad, CA, USA).

2.8. Flow cytometry analysis of peripheral blood lymphocytes

Phenotypes of lymphocyte sub-populations were determinedusing flow cytometry. Blood samples (100 lL) collected 0, 2, 4, 6,8, 10, 13, 16, 24 dpc were heparinized and mixed with 2 lL of spe-cific antibody in a plastic tube. After incubating in the dark at roomtemperature for 30 min, 2 mL of hemolysin (FACS Lysing Solution –Becton, Dickinson, Franklin Lakes, NJ, USA) were added to the tubeto lyse the red blood cells. Samples were then centrifuged (400g,5 min) and the pellets were resuspended in 0.2 mL phosphate buf-fer saline (PBS), pH 7.4. Flow cytometry analysis was performedusing double staining with anti-CD4a-PE, anti-CD8-SPRD andanti-CD3e-FITC (Southern Biotech, Birmingham, AL, USA) andemploying a BD Vantage Cell Sorter Flow Cytometer (Becton,Dickinson).

2.9. Statistical analysis

Statistical analyses (one-way ANOVA) were performed usingSPSS software (SPSS11.5 for Windows). Data are expressed as meanvalues ± standard error (SEM). The approximate normality of distri-bution of all the values was confirmed by the Kolmogorov–Smirnovtest. The t-test was employed to determine the statistical signifi-cance (P < 0.05) of the differences between the means. Data relatingto viral RNA copies were converted to log10 to maintain a normaldistribution.

3. Results

3.1. Inhibition of PRRSV propagation in MARC-145 cells by rPoIFN-a

Recombinant PoIFN-a had a clear inhibitory effect on viruspropagation (Fig. 1) which gradually diminished with increasingdilution of the reagent. Corresponding increases in viral titers werealso observed with increasing dilution of the rPoIFN-a reagent, a215 dilution resulting in a 104 increase in viral titers.

3.2. Clinical symptoms in challenged pigs

Symptoms began to appear in challenged animals between 4and 5 dpc and included petechiae in the ears, discharges fromthe mouth and eyes, high fever (40–42 �C), depression, loss ofappetite, coughing, dyspnea and shivering. No differences were ob-served in the respiratory scores of animals in group C2 and othertrial groups. Pigs began to die from 6 dpc, and the mortality ratein challenged animals was 80–100% (Fig. 2). One pig survived in

Fig. 1. Effect of rPoIFN-a on lvPRRSV titers in MARC-145 cells. NC is the negativecontrol group that was infected with lvPRRSV but not pretreated with rPoIFN-a.

each of the T2 and T3 groups but, in the other challenged groups,all the animals died from complications.

Rectal temperatures of animals in C1 were 39.82 ± 0.29 �C,whereas the average rectal temperatures of test group animalsreached 41 �C at 8 dpc. Recombinant PoIFN-a treatment temporar-ily depressed the fever symptoms in T2 and T3 groups (Fig. 3).Temperatures were significantly lower (p < 0.05) in the T2 groupthan in the C2 animals following the three injections of rPoIFN-aand remained stable for four days thereafter. Temperatures inthe T3 group were close to 41 �C after challenge, but decreasedto significantly lower values compared with the C2 group(p < 0.05) after administration of rPoIFN-a. There was a slight in-crease in rectal temperatures when the animals in T1 were simul-taneously challenged and administered the interferon reagent.However, temperatures fell to significantly lower values comparedwith C2 animals (p < 0.05) 24 h later.

3.3. PRRSV RNA

No PRRSV RNA was detected in group C1 pigs but was recordedin all the animals of the challenged groups (Fig. 4) and reachedhighest levels between 5 and 6 dpc. At 2 dpc, the mean PRRSVRNA copy number for group C2 was significantly higher than thecorresponding values for groups T1 and T2 (p < 0.05). At 8 dpc,the mean PRRSV RNA copy number for T3 was significantly lowerthan for group C2 (p < 0.05).

3.4. Antibody response to PRRSV challenge

According to the recommended cut-off value (0.40) of the testkit, none of the serum samples taken from group C1 animals scoredpositive for PRRSV antibodies, but sera taken from challenged ani-mals at 7 dpc were all judged to be positive (Fig. 5). Antibody levelsin T1 and T2 animals were significantly lower compared withgroup C2 pigs at 8 dpc (p < 0.05).

3.5. Neutralizing antibodies

No neutralizing antibodies were detected in serum samples ta-ken from all the pigs in both the challenged and control groups.

3.6. Flow cytometry analysis of peripheral blood lymphocytes

Changes in the populations of CD4+, CD8+, CD3+CD8high andCD4+CD8+ cells are shown in Fig. 6. In the early stages followingchallenge, significant decreases in CD8+ and CD4+CD8+ T cell countswere observed in samples taken from most groups, but these val-ues then gradually increased in the blood of challenged animalsover the next 8 days. With the exception of group T1 samples,CD4+ cell counts exhibited a downward trend between 2 and4 dpc, but significant increases then occurred during the next6 days. An increase in CD3+CD8high cells was recorded in groupT2 samples at 10 dpc.

4. Discussion

Initial experiments demonstrated that the capacity of PRRSVvaccine strain, VR2332, to propagate in MARC-145 cells was signif-icantly decreased if the cells were pretreated with rPoIFN-a.Although these experiments would have been more meaningfulin terms of developing effective measures for controlling hvPRRSVin China if a highly virulent strain had been employed, this was notfeasible given the standard of bio-containment facilities available.Previous research has shown that pretreatment of PAMs with re-combinant porcine IFN-a1 significantly decreased PRRSV yields

Fig. 2. Survival patterns among pigs after challenged with hvPRRSV at 0 dpc. One pig each from groups T1 and T2 died on 0 dpc from stress-related reactions following viralchallenge. Pigs in group C2, which were challenged with hvPRRSV without rPoIFN-a, began to die from 5 dpc. Pigs in T1 group, which were challenged with hvPRRSV andgiven rPoIFN-a simultaneously on day 0 began to die from 9 dpc. No pigs in groups C2 and T1 survived beyond 11 dpc. Pigs in group T2, which were challenged with hvPRRSVon day 0 and given rPoIFN-a on day 1, began to die from 8 dpc. Group T3 pigs, which were given rPoIFN-a after the appearance of disease symptoms, began to die on 6 dpc.One pig each from groups T2 and T3 survived viral challenge.

Fig. 3. Rectal temperatures of pigs in control and test groups. Group T1 waschallenged with hvPRRSV and administered rPoIFN-a on day 0. Group T2 waschallenged with hvPRRSV on day 0 and administered 2 � 106 IU rPoIFN-a on day 1.Group T3 was given rPoIFN-a after the appearance of disease symptom. Group C2was challenged with hvPRRSV but not administered rPoIFN-a. Data are expressed asmean ± SD; (⁄) significant difference (⁄p < 0.05) compared with C2 group. The lowercase letter c on X-axis indicates the time of viral challenge.

Fig. 4. Highly virulent hvPRRSV genomic copy numbers in pig sera determinedusing real-time RT-PCR. Viral RNA copies were determined in pigs inoculated withhvPRRSV on day 0, converted to log10 and expressed as the mean ± SD. Statisticalanalysis was performed using a mixed linear model for repeated measures. (⁄)Indicates a significant difference (p < 0.05) compared with the C2 group which waschallenged with hvPRRSV but not administered rPoIFN-a.

S. Dong et al. / Research in Veterinary Science 93 (2012) 1060–1065 1063

and the number of antigen-expressing cells (Buddaert et al., 1998).Subsequently, porcine circovirus 2 (PCV2)-induced interferon-awas also reported to decrease PRRSV infection of PAMs (Changet al., 2005).

The findings above prompted us to test the effects of rPoIFN-aon pigs challenged with hvPRRSV which, from 2006/7, was preva-lent in many provinces of China, resulting in high swine mortalityrates and serious economic losses to the industry. As expected, theadopted hvPRRSV strain produced the typical symptoms and highmortality rates associated with hvPRRSV infections (Tian et al.,2007). Animals were exposed to rPoIFN-a at different dpc in an

attempt to identify a time point when the treatment diminishedor completely inhibited viral propagation within the host. How-ever, no obvious differences were evident in terms of the survivaltimes of animals comprising Groups T1, T2, T3 and C2, althougha tendency for increased survival times was noted among pigs inboth T2 and T3. In vivo sensitivity of PRRSV to IFN-a was also dem-onstrated in an earlier study, but the impact on survival timescould not be assessed because the viral strain used for challengewas too virulent for the host defense system or other commonlyadopted therapies to control (Buddaert et al., 1998). AlthoughrPoIFN-a treatment failed to prevent most of the challenged pigsfrom dying, careful analysis of the effects of the cytokine on theprogress of the interaction between the virus and the host stillallow conclusions to be drawn. In addition to possibly prolonging

Fig. 5. ELISA S/P ratios for PRRSV antibody in experimental pig groups afterinoculation with hvPRRSV. S/P ratios shown represent values that are higher thanthe cut-off value of 0.40, which is considered seropositive for PRRSV. After 7 dpc,serum antibody to PRRSV was detected in all of the challenged animals andcontinued to increase for a further 3 days. The antibody levels in group C2 animalswere significantly higher than groups T1 and T2 at 8 dpc. For the most part, datarelating to group C1 overlapped with the X-axis. Data are expressed as themean ± SD (p < 0.05). The lower case letter c along the X-axis indicates the time ofchallenge.

1064 S. Dong et al. / Research in Veterinary Science 93 (2012) 1060–1065

the survival of challenged animals, rPoIFN-a also alleviated diseasesymptoms and the extent of the viremia. Thus, rPoIFN-a treatmenttemporarily repressed fever symptoms (Fig. 3), and real-time PCRdata demonstrated that exposure to rPoIFN-a decreased the quan-tity of viral RNA in challenged animals (Fig. 4). Interferon-a-med-iated reduced disease signs in swine challenged with PRRSV,including a diminished febrile reaction, a lower percentage of lunginvolvement, and delayed viral replication, viremia and antibodyresponse, have been reported previously (Brockmeier et al., 2009).

Infection of PRRSV has been reported to induce a vigorous anti-PRRSV antibody response that was detectable by ELISA starting at7–9 dpc (Yoon et al., 1994; Lopez and Osorio, 2004). Our study re-vealed that anti-PRRSV antibody was detectable 7 dpc in chal-lenged animals although antibody titers were lower in pigstreated with rPoIFN-a than in group C2 animals. This was unex-pected since members of the interferon sub-family of cytokinesare considered to be immuno-enhancing agents (Biron, 1998;Bonjardim, 2005). However, this anomaly may be due to the de-pressed PRRSV propagation in the treatment groups resulting in aweaker pathogen stimulus to the host immune system.

Fig. 6. Percentage changes in CD4+, CD8+, CD3+CD8high and CD4+CD8+ cell counts during tC and D: variations in CD4+ and CD8+ cells, respectively. Values are expressed as means0 dpc..

We were unable to detect neutralizing antibodies (NAs) in chal-lenged pigs before 24 dpc, and the NA response usually appearedaround the third or fourth week post infection (PI) or even later.It has been suggested that the delayed development and low titersof neutralizing antibodies are due to a poor milieu for cytokinestimulation and/or the existence of a decoy epitope (Chang et al.,2008; Ostrowski et al., 2002). We had expected to detect NAs ear-lier than 4 weeks PI as a result of exposure to rPoIFN-a, but thistreatment did not appear to be play a critical role.

Although pigs developed both humoral and cell-mediated im-mune responses against PRRSV infection, our understanding ofprecisely which immune mechanisms are induced remains incom-plete (Darwich et al., 2010; Gomez-Laguna et al., 2009). Therefore,in order to determine the impact of hvPRRSV infection and inter-feron treatment on the cellular immunology of the host, we ana-lyzed the changes occurring in lymphocyte subsets during thefirst 10 dpc (Fig. 6). The negligible changes in the proportion ofCD4+ T cells in group T1 at 2 dpc implied that simultaneousrPoIFN-a treatment and PRRSV challenge might improve cell-med-iated immunity (Katze et al., 2002). Counts of CD3+CD8high cells,which generally considered to be cytotoxic T-lymphocytes (CTL)(Lamontagne et al., 2003; Gomez-Laguna et al., 2009; Tingstedtand Nielsen, 2004), increased significantly 10 dpc in group T2.

Although a single higher dosage of rPoIFN-a had been plannedfor animals in the T1 group, the dosage was administered in erroron three consecutive days. Consequently, for the purpose of analy-sis and interpretation, data relating to T1 was compared only withthat for the control group.

In summary, the antiviral function of rPoIFN-a has been demon-strated in terms of the inhibitory effect of the cytokine on lvPRRSVproliferation in MARC-145 cells. Furthermore, exposing swine torPoIFN-a prior to challenge with hvPRRSV alleviated fever symp-toms, reduced the virus burden, and prolonged host survival times.When applied 1 dpc, rPoIFN-a may also enhance CTL levels inhvPRRSV-challenged pigs, and improve cell-mediated immunitywhen exposure and viral challenge are carried out simultaneously.

he experimental period. Panel A: CD3+CD8high cells; Panel B: CD4+CD8+ cells; Panels± SD. (⁄) significant difference (p < 0.05) compared with the data in every group at

S. Dong et al. / Research in Veterinary Science 93 (2012) 1060–1065 1065

However, our data do not support a major independent role forrPoIFN-a in protecting swine from infection with hvPRRSV.

Acknowledgements

We thank Yong Zou for kindly providing lvPRRSV and theMARC-145 cells, and Dr. John Buswell for linguistic revision. Thisresearch was financed by a grant (No. 073919108) from theShanghai Science and Technology Committee, a grant from theNational High Technology Research and Development Program ofChina (863 Program) (No. 2011AA10A208) and a grant from theShanghai Key Laboratory of Agricultural Genetics and Breeding tosupport guest scientists.

References

Albina, E., Carrat, C., Charley, B., 1998a. Interferon-a response to swine arterivirus(PoAV), the porcine reproductive and respiratory syndrome virus. Journal ofInterferon and Cytokine Research 18, 485–490.

Albina, E., Piriou, L., Hutet, E., Cariolet, R., Hospitalier, R.L., 1998b. Immuneresponses in pigs infected with porcine reproductive and respiratorysyndrome virus (PRRSV). Veterinary Immunology and Immunopathology 61,49–66.

Biron, C.A., 1998. Role of early cytokines, including alpha and beta interferons (IFN-alpha/beta), in innate and adaptive immune responses to viral infections.Seminars in Immunology 10, 383–390.

Bonjardim, C.A., 2005. Interferons (IFNs) are key cytokines in both innate andadaptive antiviral immune responses and viruses counteract IFN action.Microbes and Infection 7, 569–578.

Brockmeier, S.L., Lager, K.M., Grubman, M.J., Brough, D.E., Ettyreddy, D., Sacco, R.E.,Gauger, P.C., Loving, C.L., Vorwald, A.C., Kehrli Jr., M.E., Lehmkuhc, H.D., 2009.Adenovirus-mediated expression of interferon-a delays viral replication andreduces disease signs in swine challenged with porcine reproductive andrespiratory syndrome virus. Viral Immunology 22, 173–180.

Buddaert, W., Van Reeth, K., Pensaert, M., 1998. In vivo and in vitro interferon (IFN)studies with the porcine reproductive and respiratory syndrome virus.Advances in Experimental Medicine and Biology 440, 461–467.

Chang, H.C., Peng, Y.T., Chang, H.L., Chaung, H.C., Chung, W.B., 2008. Phenotypic andfunctional modulation of bone marrow-derived dendritic cells by porcinereproductive and respiratory syndrome virus. Veterinary Microbiology 129,281–293.

Chang, H.W., Jeng, C.R., Liu, J.J., Lin, T.L., Chang, C.C., Chia, M.Y., Tsai, Y.C., Pang, V.F.,2005. Reduction of porcine reproductive and respiratory syndrome virus(PRRSV) infection in swine alveolar macrophages by porcine circovirus 2(PCV2) - induced interferon-alpha. Veterinary Microbiology 108, 167–177.

Darwich, L., Diaz, I., Mateu, E., 2010. Certainties, doubts and hypotheses in porcinereproductive and respiratory syndrome virus immunobiology. Virus Research154, 123–132.

Dong, S., Zhu, Y., Yu, R., Shen, S., Li, Z., 2010. Screening of freeze-drying stabilizationadditives for recombinant porcine interferon-a preparation. Chinese Journal ofVeterinary Drug 44, 37–40.

Gomez-Laguna, J., Salguero, F.J., De Marco, M.F., Pallares, F.J., Bernabe, A., Carrasco,L., 2009. Changes in lymphocyte subsets and cytokines during European porcinereproductive and respiratory syndrome: increased expression of IL-12 and IL-10and proliferation of CD4-CD8high. Viral Immunology 22, 261–271.

Hermann, J.R., Munoz-Zanzi, C.A., Roof, M.B., Burkhart, K., Zimmerman, J.J., 2005.Probability of porcine reproductive and respiratory syndrome (PRRS) virus

infection as a function of exposure route and dose. Veterinary Microbiology 110,7–16.

Katze, M.G., He, Y., Gale Jr., M., 2002. Viruses and interferon: a fight for supremacy.Nature Reviews Immunology 2, 675–687.

Lamontagne, L., Page, C., Larochelle, R., Magar, R., 2003. Porcine reproductive andrespiratory syndrome virus persistence in blood, spleen, lymph nodes, andtonsils of experimentally infected pigs depends on the level of CD8high T cells.Viral Immunology 16, 395–406.

Lopez, O.J., Osorio, F.A., 2004. Role of neutralizing antibodies in PRRSV protectiveimmunity. Veterinary Immunology and Immunopathology 102, 155–163.

Martelli, P., Gozio, S., Ferrari, L., Rosina, S., De Angelis, E., Quintavalla, C., Bottarelli,E., Borghetti, P., 2009. Efficacy of a modified live porcine reproductive andrespiratory syndrome virus (PRRSV) vaccine in pigs naturally exposed to aheterologous European (Italian cluster) field strain: clinical protection and cell-mediated immunity. Vaccine 27, 3788–3799.

Mateu, E., Diaz, I., 2008. The challenge of PRRS immunology. The Veterinary Journal177, 345–351.

Miller, L.C., Laegreid, W.W., Bono, J.L., Chitko-McKown, C.G., Fox, J.M., 2004.Interferon type I in porcine reproductive and respiratory syndrome virusinfected Marc-145 cells. Archives of Virology 149, 2453–2463.

Murtaugh, M.P., Xiao, Z., Zuckermann, F., 2002. Immunological responses of swineto porcine reproductive and respiratory syndrome virus infection. ViralImmunology 15, 533–547.

Ostrowski, M., Galeota, J.A., Jar, A.M., Platt, K.B., Osorio, F.A., Lopez, O.J., 2002.Identification of neutralizing and non-neutralizing epitopes in the porcinereproductive and respiratory syndrome virus GP5 ectodomain. The Journal ofVirology 76, 4241–4250.

Overend, C., Mitchell, R., Rompato, D.He.G., Grubman, M.J., Garmendia, A.E., 2007.Recombinant swine beta interferon protects swine alveolar macrophages andMarc-145 cells from infection with porcine reproductive and respiratorysyndrome virus. Journal of General Virology 88, 925–931.

Reed, J.L., Muench, H., 1938. A simple method of estimating fifty percent endpoint.American Journal of Hygiene 27, 493–497.

Thanawongnuwech, R., Young, T.F., Thacker, B.J., Thacker, E.L., 2001. Differentialproduction of proinflammatory cytokines: in vitro PRRSV and Mycoplasmahyopeumoniae co-infection model. Veterinary Immunology andImmunopathology 79, 115–127.

Tian, K., Yu, X., Zhao, T., Feng, Y., Cao, Z., Wang, C., Hu, Y., Chen, X., Hu, D., Tian, X., Liu, D.,Zhang, S., Deng, X., Ding, Y., Yang, L., Zhang, Y., Xiao, H., Qiao, M., Wang, B., Hou, L.,Wang, X., Yang, X., Kang, L., Sun, M., Jin, P., Wang, S., Kitamura, Y., Yan, J., Gao, G.,2007. Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRSin China and molecular dissection of the unique hallmark. PLoS ONE 2, e526.

Tingstedt, J.E., Nielsen, J., 2004. Cellular immune responses in the lungs of pigsinfected in utero with PRRSV: an immunohistochemical study. ViralImmunology 17, 558–564.

Yao, Q., Huang, Q., Cao, Y., Qian, P., Chen, H., 2008. Porcine interferon-gammaprotects swine from foot-and-mouth disease virus (FMDV). VeterinaryImmunology and Immunopathology 122, 309–311.

Yoon, I.J., Joo, H.S., Goyal, S.M., Molitor, T.W., 1994. A modified serum neutralizationtest for the detection of antibody to porcine reproductive and respiratorysyndrome virus in swine sera. Journal of Veterinary Diagnostic Investigation 6,289–292.

Yu, R., Dong, S., Zhu, Y., Jin, H., Gao, M., Duan, Z., Zheng, Z., Shi, Z., Li, Z., 2010.Effective and stable porcine interferon-a production by Pichia pastoris fed-batch cultivation with multi-variables clustering and analysis. BioprocessBiosystems Engineering 33, 473–483.

Zhou, L., Zhang, J., Zeng, J., Yin, S., Li, Y., Zheng, L., Guo, X., Ge, X., Yang, H., 2009. The30-amino-acid deletion in the nsp2 of highly pathogenic porcine reproductiveand respiratory syndrome virus emerging in China is not related to its virulence.Journal of Virology 83, 5156–5167.