| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Pathobiology and Veterinary Science, University of Connecticut, 61 North Eagleville Road, Storrs, CT, USA
2 College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
3 Plum Island Animal Disease Center, ARS, USDA, Greenport, NY, USA
Correspondence
A. E. Garmendia
Garmendi{at}Uconnvm.uconn.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) produced in HEK 293 cells infected with a recombinant, replication-defective human adenovirus 5 (Ad5) encoding the swIFN- gene was tested for antiviral activity against Porcine reproductive and respiratory syndrome virus (PRRSV). MARC-145 cells were incubated overnight with dilutions of supernatant fluids from HEK 293 cells infected with Ad5-swIFN- or with an Ad5 control virus (Ad5-Blue). Treated cells were infected with PRRSV; MARC-145 cells incubated with Ad5-Blue supernatants developed cytopathic effects (CPE), whereas those incubated with swIFN- showed no CPE. To confirm the antiviral activity of swIFN-, culture fluids from Ad5-swIFN--infected cells were affinity-purified on a Sepharose–anti-swIFN- matrix, and the resulting fractions exhibited antiviral activity upon infection with PRRSV. The antiviral effects were specific, as they were blocked by mAbs against swIFN-. Additional cultures of MARC-145 cells treated with swIFN--containing supernatants or affinity-purified swIFN- were infected with PRRSV and tested by real-time RT-PCR for viral RNA in culture supernatants at various times post-inoculation. These experiments confirmed the protective effects of swIFN-. swIFN- was also tested for antiviral activity on porcine alveolar macrophages (PAMs) obtained by bronchoalveolar lavage from PRRSV-negative swine. PAMs were treated with dilutions of swIFN- or Ad5-Blue culture fluids, infected with PRRSV and tested for viral RNA by real-time RT-PCR. The viral load data showed a dose-dependent protection in swIFN--treated PAMs, whereas no protection was evident from Ad5-Blue culture fluids. The data demonstrate that swIFN- protects both MARC-145 cells and PAMs from PRRSV infection. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Type I interferons (IFN-
/) are major players in such protection and are expressed through a highly regulated transcriptional process in response to most intracellular infectious agents, especially viruses (Derbyshire, 1989; Biron & Sen, 2001). However, many viruses, including Porcine reproductive and respiratory syndrome virus (PRRSV), induce variable levels of pro-inflammatory cytokines and IFNs (Van Reeth et al., 1999), yet also have developed mechanisms to subvert the host innate immune response (Albina et al., 1998; Buddaert et al., 1998).
PRRSV is considered one of the most important pathogens of swine and affects the industry adversely as a result of direct and indirect losses (reviewed by Rossow, 1998). PRRSV has become distributed widely throughout the world and substantial efforts are ongoing to design rational and effective control strategies.
Whilst IFN- constitutes an early host response to PRRSV (Chung et al., 2004), there is mounting evidence that the virus circumvents the host innate response, resulting in an inadequate induction of type I IFNs (Albina et al., 1998; Buddaert et al., 1998; reviewed by Murtaugh et al., 2002). As a consequence, there is a delayed production of IFN-
, cellular immunity and neutralizing antibodies (Royaee et al., 2004), resulting in delayed viral clearance (Batista et al., 2004; Murtaugh et al., 2002; Xiao et al., 2004). In a recent study, it was found that induction of type I IFN transcripts in cells by double-stranded RNA was inhibited significantly by exposure to PRRSV, suggesting that the virus may interfere with type I IFN at the transcriptional level (Miller et al., 2004). However, in another study, it was shown that a PRRSV field isolate enhanced poly(IC)-induced IFN- production strongly in porcine alveolar macrophages (PAMs), but this priming effect was inhibited by other PRRSV isolates (Lee et al., 2004). These authors suggested that inhibition of IFN- production occurs by inhibiting host protein synthesis. Clearly, additional work is needed to determine the role of viral proteins in controlling the host innate response. Considering that infection with PRRSV results in a poor type I IFN induction (IFN-), suppression of innate immunity may be a mechanism that could explain, at least in part, the peculiarities of the host response to PRRSV. However, there appears to be a high level of variability between PRRSV field isolates in their sensitivity and capacity to induce or suppress IFN- production (Lee et al., 2004). These differences may account for differences in virulence and pathogenicity of PRRSV strains.
The mechanisms of protective immunity against PRRSV are not understood completely, although there appears to be a consensus that both neutralizing antibody and cellular immunity are required for protection. Despite a high variability between animals, a strong cellular immunity, as measured by IFN- ELISPOTs, appeared to correlate with protection from PRRSV (Lowe et al., 2005). Another study showed that treatment with interleukin-12 (IL-12) resulted in lower virus titres in lungs of infected pigs, which also correlated with higher in vitro levels of IFN- and lower levels of IL-10 produced by PAMs from treated animals (Carter & Curiel, 2005). Furthermore, the application of either IL-12 or IFN- resulted in higher IFN- levels, although this did not necessarily result in a lower viraemia (Meier et al., 2004). However, in an earlier study, inoculation of pigs with porcine respiratory coronavirus, a potent inducer of IFN-, provided protection from a subsequent PRRSV infection, as shown by a significant reduction in virus titres in lungs (Buddaert et al., 1998). Moreover, a recent study demonstrated that PAMs infected dually with Porcine circovirus-2 (PCV-2) and PRRSV resulted in reduced cytopathic effects (CPE) compared with infection with only PRRSV (Chang et al., 2005). The infection with PCV-2 appeared to override the inhibition of IFN- induction by PRRSV, as higher levels of IFN- were detected in dually PCV-2/PRRSV-infected PAMs. Together, the data support an important role for type I IFNs in the initial innate response and the subsequent activation of adaptive immunity.
We have previously constructed a replication-defective human adenovirus type 5 (Ad5) vector containing the swine IFN- gene (Ad5-swIFN-) and demonstrated high levels of expression of biologically active IFN- protein in IBRS-2 (swine kidney) cells (Chinsangaram et al., 2003). Interestingly, the Ad5 vector coding for porcine IFN- protected pigs completely from challenge with Foot-and-mouth disease virus (FMDV) (Chinsangaram et al., 2003) and partially protected cattle from challenge with the same virus (Wu et al., 2003).
The present study was conducted to examine the ability of swIFN- to neutralize PRRSV in vitro as a step towards understanding its possible antiviral role in vivo. The protection of PAMs and MARC-145 cells from PRRSV infection by swIFN- is discussed.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
gene (Ad5-swIFN-) or containing the -galactosidase gene (Ad5-Blue) was propagated in HEK 293 cells essentially as described previously (Chinsangaram et al., 2003; Moraes et al., 2001).
Swine IFN-.
Ad5-swIFN- was used to generate swIFN- by infection of 293 cells. Culture supernatants from HEK 293 cells infected with Ad5-Blue served as the control in the assays. Briefly, Ad5-swIFN-- and Ad5-Blue-infected 293 cells were processed 48–72 h post-infection as follows: cells and culture supernatants were clarified twice by low-speed centrifugation (300 g) in a Beckman TH-4 rotor. The resulting supernatants were centrifuged at 28 000 r.p.m. for 2 h to pellet the virus. The supernatants were then acidified (pH 2.0) for 18 h at 4 °C and adjusted to pH 7.2 for use in the assays.
Antiviral swIFN- tests.
The antiviral activity of swIFN- against PRRSV was tested on MARC-145 cells or PAMs. In general, two criteria were used to measure the antiviral activity of swIFN-: the presence or absence of CPE as an end point and a real-time RT-PCR as a quantitative test to measure the concentration of viral RNA in culture supernatants at different times post-inoculation. Occasionally, culture supernatants were also assayed for the presence of virus by titration on MARC-145 cells. For the antiviral tests, the cells were primed for 18–20 h with dilutions of Ad5-swIFN- or control Ad5-Blue culture supernatants (control supernatants) processed as described above. The cells were then infected with PRRSV for 1.5–2.0 h, washed and replenished with Ad5-swIFN- or Ad5-Blue supernatant. For detection of CPE, the cells were incubated for up to 7 days post-inoculation. In some experiments, supernatants from cells treated as described above were collected for titration of virus in MARC-145 cells. A comparison of the sensitivity to IFN- was also performed with several strains of PRRSV. The specificity of the protection was determined by blocking swIFN- with mAbs generated as described below.
Real-time RT-PCR.
To quantify PRRSV RNA by real-time RT-PCR, Ad5-swIFN-- or control supernatant-primed MARC-145 cells or PAMs were inoculated with virus for 1.5–2.0 h at 37 °C and then washed four times with serum-free medium, at which point a sample of supernatant for each treatment was collected as time 0. The cells were then replenished with the appropriate supernatants and incubated for an additional 72–96 h, and culture supernatants were sampled for real-time RT-PCR analysis. Briefly, viral RNA was extracted from supernatants with TRIzol LS (Invitrogen) following the manufacturer's protocol. RNA pellets were suspended in 50 µl DEPC-treated water and a reverse transcription (RT) reaction was performed by utilizing an RT reaction kit (Applied Biosystems), 10 µl RNA and a PRRSV open reading frame 7 (ORF-7)-specific RT primer (ORF7 RT, 5'-TCGCCCTAAT-3'). Two ORF-7-specific primers were designed to amplify a 200 bp fragment in the conserved ORF-7 region by utilizing the DNAMan software (Real-time 7 F, 5'-AATAACAACGGCAAGCAGCA–3'; Real-time 7 R, 5'-GCACAGTATGATGCGTAGGC-3'). Real-time PCR was performed by using SYBRGreen PCR master mix, 0.25 µM each primer and 2.5 µl of the RT reaction, following the manufacturer's protocol (Applied Biosystems). Samples were heated for 10 min at 94 °C and a three-step cycle (30 s at 94 °C, 30 s at 64 °C and 30 s at 72 °C) was repeated 40 times. A standard curve was generated with purified viral RNA derived from caesium chloride gradient-purified PRRSV and utilized to determine the viral RNA concentration in test samples. The viral RNA was quantified spectrophotometrically, aliquotted and stored at –80 °C for further use. Aliquots of the viral RNA were tested periodically to ensure integrity and consistency.
Monoclonal antibodies.
A series of mAbs against swIFN- was produced in our laboratory. Briefly, BALB/c mice were immunized with an expression vector carrying the swine IFN- gene. Hybridomas were produced by using single spleen-cell suspensions from these mice and Sp2/0 myeloma cells by standard polyethylene glycol-mediated fusion. Clones were screened against Centricon-100-filtered, pH 2.0-treated and neutralized 293 cell-culture supernatants containing recombinant swIFN-. Positive clones were expanded further and cloned by limiting dilution. Upon cloning two or three times, hybridoma fluids were retested to ensure reactivity against swIFN- and the isotype was determined.
Affinity purification of swIFN-.
To purify swIFN-, affinity columns were developed with monoclonal anti-swIFN- antibodies. For this purpose, an anti-swIFN- mAb designated 10E9 (IgG2b), developed in our laboratory, was purified from hybridoma culture fluids on Sepharose–anti-mouse IgG or Sepharose–protein A columns. The purified anti-swIFN- mAb fractions were pooled and coupled to CNBr-activated Sepharose beads according to the instructions of the manufacturer (Pharmacia) to generate a Sepharose–anti-swIFN- matrix. The Sepharose–anti-swIFN- matrix was packed in a column and utilized to fractionate swIFN- from culture fluids of HEK 293 cells infected with Ad5-swIFN- (Moraes et al., 2001). The bound swIFN- was eluted with glycine/HCl buffer (pH 2.5) and adjusted to pH 7.2 by immediate dialysis against PBS (pH 7.2). The fractions were acidified (to pH 2.0) overnight and adjusted to pH 7.2. The protein concentration of the fractions was quantified by spectrophotometry at a wavelength of 280 nm, then the fractions were aliquotted and frozen at –70 °C for further use.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
blocks virus replication in MARC-145 cells produced in Ad5-swIFN--infected HEK 293 cells was initially examined in MARC-145 cells, a cell line used routinely to propagate PRRSV (Kim et al., 1993). Briefly, culture supernatants from HEK 293 cells infected with either Ad5-swIFN- or control Ad5-Blue, treated as described above, were incubated overnight with MARC-145 cells and then infected with PRRSV strain Mo8981. Cells lacking swIFN- and inoculated with virus (virus control) or cells without swIFN- or virus (cell control) were included. Cells treated with Ad5-swIFN- and infected with PRRSV did not develop CPE, whereas cells treated with control supernatants developed CPE (Table 1). Protection was evident up to a dilution of 1 : 160 for swIFN- when using 30 TCID50 per well, whereas CPE was recorded in cells even at the lowest dilution (1 : 5) of control supernatants at both high and low virus concentrations. These results are summarized in Table 1 and represent data from two or more similar tests using two separate preparations of Ad5-swIFN- and control Ad5-Blue supernatants. The protective effects were dose-dependent, as shown by the decrease of antiviral activity at higher dilutions of swIFN- versus the higher virus concentration (Table 1). Similar results were obtained against FMDV using culture supernatants obtained from Ad5-swIFN--infected IBRS-2 cells (a swine cell line) (Chinsangaram et al., 2003).
|
on PRRSV replication, we followed virus growth by real-time RT-PCR. MARC-145 cells treated with Ad5-swIFN- supernatants had approximately 50–150-fold lower viral RNA loads at 72 h post-infection, compared with virus control or cell control supernatant-treated cells, respectively (Table 2). Priming of MARC-145 cells with affinity-purified fractions of swIFN- reduced the replication of PRRSV significantly, as demonstrated by the lower viral RNA loads detected 72 h post-infection compared with the virus control. The antiviral activity was dose-dependent, as it was reduced at higher dilutions of the purified fractions (Table 3). The antiviral activity was higher in fractions 2 and 3, indicating that these fractions contained the highest concentration of swIFN- per unit of mass, as all of the affinity fractions were used at the same concentrations. This result reflects the specificity of the mAb and confirms the antiviral activity of swIFN- against PRRSV. The real-time RT-PCR results correlated with the presence or absence of CPE observed in swIFN--treated, PRRSV-infected MARC-145 cells.
|
|
or control supernatants followed by infection with PRRSV, were tested for viral RNA loads 24 and 48 h post-infection by real-time RT-PCR. PAMs treated with Ad5-swIFN- had viral RNA loads more than 200-fold lower than those detected in cells treated with control supernatants or the virus control at 24 h post-infection (Table 4). In contrast, the control supernatant-treated cells, which had visible CPE in earlier experiments, had a significant increase in virus RNA load 24 h post-infection (from 0 to 7.40 pg in 200 µl), comparable to that observed in the virus-infected control (from 0 to 9.60 pg in 200 µl) (Table 4). At 48 h, viral RNA loads in the virus control increased to 20.8 pg in 200 µl and, for unknown reasons, the viral load of control supernatant-treated cells was only 1.9 pg in 200 µl. In clear contrast, the Ad5-swIFN--treated cells had viral RNA loads lower than 0.2 pg in 200 µl at both time points, which were at least 13 times lower than that detected with the control supernatant at 48 h and up to 140 times lower than that detected with the virus control at 48 h.
|
blocked by mAb to swIFN- mAbs with swIFN- prior to priming and infection of MARC-145 cells with PRRSV isolate Mo25544 resulted in inhibition of the antiviral activity. This was evident by the presence of CPE in the antibody- and IFN-treated cells, but not in control cells treated with the same amount of IFN and no antibody, which were protected fully from PRRSV infection (data not shown). As expected, CPE was also evident in the virus control and in cells primed with control supernatants and infected with PRRSV. To confirm these results, Ad5-swIFN- or control supernatants were diluted serially and incubated with a constant amount of antibody before priming and infection of cells. The results of the latter experiment, summarized in Table 5 and Fig. 1, confirmed that the antiviral activity of swIFN- was abolished specifically and significantly by one of the anti-swIFN- mAbs (10E9, IgG2b) and partially by the other mAb (10F1, IgM). This was determined by the presence of CPE in the cells (Table 5) and significantly higher viral RNA loads in IFN- plus anti-IFN-primed cells, compared with cells primed with IFN reacted with isotype controls (Fig. 1). Thus, compared with the isotype controls, there was an increase in viral RNA of 41- and 8.5-fold with 10E9, and 2.2- and 3.1-fold with 10F1 when 20 or 10 units of swIFN- was used per well. The ratios were calculated after subtracting the viral RNA load detected with IFN alone.
|
|
varied among different isolates of PRRSV tested. Whilst two field isolates, Mo25544 and Mo8981, and a vaccine strain were clearly sensitive to swIFN-, another isolate, PDV130 9301 (NVSL), was less sensitive (Table 6). The swIFN--sensitive isolates did not induce CPE in cells treated with IFN, and virus replication was undetectable in MARC-145 cells. These isolates replicated and induced CPE in cells treated with control supernatant. In contrast, the IFN-resistant isolate, PDV130 9301 (NVSL), induced CPE comparable to that in the virus control in cells treated either with Ad5-swIFN- or Ad5-Blue control supernatants. Forty-eight hours post-inoculation, supernatants were collected and titrated for PRRSV in MARC-145 cells. Virus replication was not detected with the IFN-sensitive isolates, whilst the resistant isolate had titres of 102.5 TCID50 in 50 µl in supernatants from cells treated with swIFN- or control supernatants, respectively. It is not clear why isolate Mo25544 had some reduction in titre in control supernatant-treated cells compared with that of the untreated virus control.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
against PRRSV in vitro. This is based on a series of experiments conducted with both MARC-145 cells, a cell line that supports the growth of PRRSV, and PAMs, which are naturally susceptible to infection with PRRSV. Recombinant swIFN-, produced in 293 cells infected with Ad5-swIFN-, protected MARC-145 cells from infection with PRRSV, as demonstrated by the inhibition of CPE over the course of a 7 day post-infection period and by a decrease in viral RNA load as measured by real-time RT-PCR. Similarly, treatment of PAMs with swIFN- reduced the viral RNA load in PRRSV-infected cells dramatically. The inhibition was specific to swIFN-, as supernatants from Ad5-swIFN--infected 293 cells partially purified by affinity chromatography had enhanced antiviral effect, mAbs produced against swIFN- blocked its biological activity and supernatants from control Ad5-Blue-infected 293 cells had no activity. Real-time RT-PCR analysis confirmed that the presence or absence of CPE correlated with differences in the amount of viral RNA.
Whilst the actual mechanism of protection conferred by exogenous swine IFN- in either cell used in this study was not investigated, it is clear that it is capable of overriding PRRSV inhibition of type I IFN production reported in MARC-145 cells earlier (Miller et al., 2004; Lee et al., 2004). Several mechanisms are probably involved in conferring such protection, as suggested with type II IFN (Bautista & Molitor, 1999; Rowland et al., 2001). Our data suggest that the observed antiviral effects of swine IFN- are not species-specific, as these occurred not only in PAMs, but also in MARC-145 cells, a primate cell line. This is consistent with an earlier study in which MARC-145 cells were protected from PRRSV infection by treatment with human IFN- or IFN- (Rowland et al., 2001).
The protection of alveolar macrophages conferred by swIFN- in this study is significant and highly relevant, as these cells are the principal target cells for PRRSV (Rossow, 1998). Therefore, the in vitro antiviral effects shown here should be verified in vivo to determine the actual significance of swIFN- in protection of swine from PRRSV. PRRSV is no doubt an intriguing virus, due to its peculiar course of infection and development of a protective immune response. Evidence has accumulated showing that, upon infection with PRRSV, there is a poor induction of IFN-, which appears to affect the ensuing adaptive immune responses critically, including delayed IFN- and neutralizing-antibody production, ultimately leading to persistent infection (Albina et al., 1998; Buddaert et al., 1998; Royaee et al., 2004; Xiao et al., 2004).
Data from a number of studies on the effect and role of IFN- in swine associate type I IFNs, primarily IFN-, with an antiviral function (Lee et al., 2004). However, the sensitivity of PRRSV to IFN- varied among different American field isolates, as did the ability to induce or suppress IFN- production (Lee et al., 2004). Thus, the IFN--inducing or -suppressing phenotypes reflect virulence differences encountered in field isolates of PRRSV. Much less is known about the function and role of swIFN-. We have tested the effect of swIFN- against a few isolates of PRRSV and our data appear to be consistent with the variability in sensitivity of PRRSV isolates shown with IFN- (Lee et al., 2004). For example, the two isolates from Missouri, Mo25544 and M08981, that were sensitive to swIFN- in our study were also sensitive to IFN- (S. B. Kleiboeker, personal communication). However, at this time, the basis for this differential effect with swIFN- remains unknown. A more detailed molecular comparison between IFN-sensitive and -resistant PRRSV isolates is clearly needed to address this question, to identify what viral protein(s) is involved and to determine whether the differences are at the transcriptional or translational stages of IFN production (Lee et al., 2004). Recently Lee & Kleiboeker (2005) demonstrated that PRRSV infection activates nuclear factor 
B (NF-B), a transcription factor required for synthesis of type I IFN mRNAs. Therefore, the mechanism that PRRSV utilizes to limit type I IFN responses involves other steps in IFN gene activation. In addition, whether PRRSV isolates have a differential ability to induce IFN- remains unknown. The poor IFN- induction observed during infection with PRRSV (Albina et al., 1998; Lee et al., 2004) may be the result of an early inhibition of IFN- induction mediated by the virus.
Our data clearly show an antiviral activity of swIFN- in vitro. The actual role of swIFN- in vivo has not been elucidated directly, but is presumably associated intimately with phases I and II of the innate immune response to pathogens and promoting and strengthening the linkage between innate and adaptive immunity. To test this assumption, we plan to perform challenge experiments using IFN-sensitive and -resistant PRRSV isolates in animals primed by inoculation with Ad5-swIFN- and to measure the appropriate response end points, including protection. Overcoming the type I IFN suppression observed in PRRSV-infected animals (Albina et al., 1998; Buddaert et al., 1998), now widely recognized in the pathogenesis of PRRS, may be key in the development of a timely adaptive response and control of the infection.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Batista, L., Pijoan, C., Dee, S., Olin, M., Molitor, T., Joo, H. S., Xiao, Z. & Murtaugh, M. (2004). Virological and immunological responses to porcine reproductive and respiratory syndrome virus in a large population of gilts. Can J Vet Res 68, 267–273.[Medline]
Bautista, E. M. & Molitor, T. (1999). IFN gamma inhibits porcine reproductive and respiratory syndrome virus replication in macrophages. Arch Virol 144, 1191–1200.[CrossRef][Medline]
Biron, C. A. & Sen, G. C. (2001). Interferons and other cytokines. In Fields Virology, 4th edn, pp. 321–351. Edited by D. M. Knipe & P. M. Howley. Philadelphia, PA: Lippincott Williams & Wilkins.
Buddaert, W., Van Reeth, K. & Pensaert, M. (1998). In vivo and in vitro interferon (IFN) studies with the porcine reproductive and respiratory syndrome virus. Adv Exp Med Biol 440, 461–467.[Medline]
Carter, Q. L. & Curiel, R. E. (2005). Interleukin-12 (IL-12) ameliorates the effects of porcine respiratory and reproductive syndrome virus (PRRSV) infection. Vet Immunol Immunopathol 107, 105–118.[CrossRef][Medline]
Chang, H. W., Jeng, C. R., Li, J. J., Lin, T. L., Chang, C. C., Chia, M. Y., Tsai, Y. C. & Pang, V. F. (2005). Reduction of porcine reproductive respiratory syndrome virus (PRRSV) infection in swine alveolar macrophages by porcine circovirus 2 (PCV2)-induced interferon-alpha. Vet Microbiol 108, 167–177.[CrossRef][Medline]
Chinsangaram, J., Moraes, M. P., Koster, M. & Grubman, M. J. (2003). Novel viral disease control strategy: adenovirus expressing alpha interferon rapidly protects swine from foot-and-mouth disease. J Virol 77, 1621–1625.
Chung, H. K., Lee, J. H., Kim, S. H. & Chae, C. (2004). Expression of interferon-alpha and Mx1 protein in pigs acutely infected with porcine reproductive and respiratory syndrome virus (PRRSV). J Comp Pathol 130, 299–305.[CrossRef][Medline]
Derbyshire, J. B. (1989). The interferon sensitivity of selected porcine viruses. Can J Vet Res 53, 52–55.[Medline]
Kim, H. S., Kwang, J., Yoon, I. J., Joo, H. S. & Frey, M. L. (1993). Enhanced replication of porcine reproductive and respiratory syndrome (PRRS) virus in a homogenous subpopulation of MA-104 cell line. Arch Virol 133, 477–483.[CrossRef][Medline]
Lee, S. M. & Kleiboeker, S. B. (2005). Porcine arterivirus activates the NF-B pathway through IB degradation. Virology 342, 47–59.[CrossRef][Medline]
Lee, S. M., Schommer, S. K. & Kleibocker, S. B. (2004). Porcine reproductive and respiratory syndrome virus field isolates differ in in vitro interferon phenotypes. Vet Immunol Immunopathol 102, 217–231.[CrossRef][Medline]
Lowe, J. E., Husmann, R., Firkins, L. D., Zuckermann, F. A. & Goldberg, T. L. (2005). Correlation of cell-mediated immunity against porcine reproductive and respiratory syndrome virus with protection against reproductive failure in sows during outbreaks of porcine reproductive and respiratory syndrome in commercial herds. J Am Vet Med Assoc 226, 1707–1711.[CrossRef][Medline]
Meier, W. A., Husmann, R. J., Schnitzlein, W. N., Osorio, F. A., Lunney, J. K. & Zuckermann, F. A. (2004). Cytokines and synthetic double-stranded RNA augment the T helper 1 immune response of swine to porcine reproductive and respiratory syndrome virus. Vet Immunol Immunopathol 102, 299–314.[CrossRef][Medline]
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 virus-infected MARC-145 cells. Arch Virol 149, 2453–2463.[CrossRef][Medline]
Moraes, M. P., Mayr, G. A. & Grubman, M. J. (2001). pAd5-Blue: direct ligation system for engineering recombinant adenovirus constructs. Biotechniques 31, 1050–1056.[Medline]
Murtaugh, M. P., Xiao, Z. & Zuckermann, F. (2002). Immunological responses of swine to porcine reproductive and respiratory syndrome virus infection. Viral Immunol 15, 533–547.[CrossRef][Medline]
Rossow, K. D. (1998). Porcine reproductive and respiratory syndrome. Vet Pathol 35, 1–20.[Abstract]
Rowland, R. R. R., Robinson, B., Stefanick, J., Kim, T. S., Guanghua, L., Lawson, S. R. & Benfield, D. A. (2001). Inhibition of porcine reproductive and respiratory syndrome virus by interferon-gamma and recovery of virus replication with 2-aminopurine. Arch Virol 146, 539–555.[CrossRef][Medline]
Royaee, A. R., Husmann, R. J., Dawson, H. D., Calzada-Nova, G., Schmitzlein, W. M., Zuckermann, F. A. & Lunney, J. K. (2004). Deciphering the involvement of innate immune factors in the development of the host response to PRRSV vaccination. Vet Immunol Immunopathol 102, 199–216.[CrossRef][Medline]
Van Reeth, K., Labarque, G., Nauwynck, H. & Pensaert, M. (1999). Differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity. Res Vet Sci 67, 47–52.[CrossRef][Medline]
Wu, Q., Brum, M. C., Caron, L., Koster, M. & Grubman, M. J. (2003). Adenovirus-mediated type I interferon expression delays and reduces disease signs in cattle challenged with foot-and-mouth disease virus. J Interferon Cytokine Res 23, 359–368.[CrossRef][Medline]
Xiao, Z., Batista, L., Dee, S., Halbur, P. & Murtaugh, M. P. (2004). The level of virus-specific T-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of virus load. J Virol 78, 5923–5933.
Received 21 September 2006;
accepted 30 October 2006.
This article has been cited by other articles:
|
|
 |
|
  L. C. Miller, K. M. Lager, and M. E. Kehrli Jr. Role of Toll-Like Receptors in Activation of Porcine Alveolar Macrophages by Porcine Reproductive and Respiratory Syndrome Virus Clin. Vaccine Immunol., March 1, 2009; 16(3): 360 - 365. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Genini, P. L. Delputte, R. Malinverni, M. Cecere, A. Stella, H. J. Nauwynck, and E. Giuffra Genome-wide transcriptional response of primary alveolar macrophages following infection with porcine reproductive and respiratory syndrome virus J. Gen. Virol., October 1, 2008; 89(10): 2550 - 2564. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
