Immunological responses of swine to porcine reproductive and respiratory syndrome virus infection

The immunology of porcine reproductive and respiratory syndrome virus (PRRS) begins with an initial encounter of PRRSV with the pig. Regardless of the route of entry of PRRSV--via inhalation, intramuscular vaccination, insemination, or other routes--productive infection occurs predominately in alveolar macrophages of the lung. Thus, innate responses of the lung and the alveolar macrophage comprise the initial defense against PRRSV. The virus appears not to elicit innate interferon and cytokine responses characteristic of other strongly immunogenic viral pathogens, and its effects are consistent with induction of a weak adaptive immune response. Humoral and cell-mediated immunity is induced in due course, and results in clearance of virus from the circulation but not from lymphoid tissues, where the infection becomes persistent. Subsequent reexposure to PRRSV elicits an anamnestic response that is partially to completely protective. Within this unconventional picture of anti-PRRSV immunity lie a variety of unresolved issues, including the nature of protective immunity within individual pigs and among pigs in commercial populations, the efficacy of protective immunity against genetically different PRRSV isolates, the effects of developmental age, sex, genetics, and other host factors on the immune response to PRRSV, and the possible suppression of host immunity to other pathogens.     

Quasispecies variation of porcine reproductive and respiratory syndrome virus during natural infection

Porcine reproductive and respiratory syndrome virus (PRRSV) displays notorious genetic, antigenic, and clinical variability. Little is known, however, about the nature and extent of viral variation present within naturally infected animals. By amplifying and cloning the open reading frame 5 gene from tonsils of naturally infected swine, and by sequencing individual clones, we characterized viral diversity in nine animals from two farms. All animals harbored multiple PRRSV variants at both the nucleic and the amino acid levels. Structural variation and rates of synonymous and nonsynonymous nucleotide substitution were no different within known epitopes than elsewhere. Analysis of molecular variance indicated that differences between farms, among animals within farms, and within individual animals accounted for 92.94, 3.84, and 3.22% of the total viral genetic variability observed, respectively. PRRSV exists during natural infection as a quasispecies distribution of related genotypes. Positive natural selection for immune evasiveness does not appear to maintain this diversity.     

Mechanisms of suppression of interferon production by porcine reproductive and respiratory syndrome virus

The recently emerged porcine reproductive and respiratory syndrome virus (PRRSV) leads to one of the most economically significant infectious diseases of swine worldwide. The virus modulates the host innate and adaptive immunity to escape its immune response to facilitate the infection. Interferons (INFs) are principal antiviral cytokines, which represent components of the innate immunity and are regarded as a bridge between the innate and adaptive immunity. Currently, accumulating evidence indicates that the virus has developed various strategies to counteract the IFN production. Here, various mechanisms utilized by the virus to antagonize the IFN induction are reviewed.     

Identification of porcine alveolar macrophage glycoproteins involved in infection of porcine respiratory and reproductive syndrome virus

Summary.  The aim of this study was to identify the receptor(s) for PRRSV on porcine alveolar macrophages (PAMs) by producing monoclonal antibodies (MAbs) against these cells. Hybridoma supernatants were selected for their ability to block PRRSV infection. Four MAbs, 1-8D2, 9.4C7, 9.9F2, and 3-3H2 inhibited infection and recognised cell surface, PAM-specific antigens as shown by immunofluorescence and immunoperoxidase monolayer assay. These MAbs were then used to identify cellular proteins involved in PRRSV infection by radioimmunoprecipitation assays (RIPAs). MAbs 1-8D2 and 9.9F2 each recognised a 150 kDa-polypeptide doublet, while MAbs 9.4C7 and 3-3H2 both recognised a 220 kDa-polypeptide. Glycosidase treatment demonstrated all these polypeptides to be N-glycosylated. Thus, multiple glycoproteins appear to be involved in infection of PAMs by PRRSV. Received April 17, 2002; accepted July 25, 2002     

Heteroclite subgenomic RNAs are produced in porcine reproductive and respiratory syndrome virus infection

Porcine reproductive and respiratory syndrome virus (PRRSV) was shown to produce atypical subgenomic RNAs that contain open reading frame la nucleotides and are present under a wide variety of culture conditions, including high and low multiplicities of infection, in simian and porcine host cells, and during infection with cell-adapted and wild-type PRIRSV strains. Sequence analysis demonstrated that they are heterogeneous in 5-3' junction sequence and size and may code for different predicted fusion proteins. This is the first report of these novel RNA5 in arteriviruses and we have termed them heteroclite (meaning 'deviating from common forms or rules") subgenomic RNAs. The unique properties of these subgenomic RNAs include (a) apparent association with normal virus infection and stability during serial passage, (b) packaging of heteroclite RNAs into virus-like particles, (c) short, heterogeneous sequences which may mediate the generation of these RNAs, (d) a primary structure which consists of the two genomic termini with one large internal deletion, and (eJ little apparent interference with parental virus replication. These subgenomic RNA5 may be critical to, or a necessary side product of, viral replication. The expression of these novel RNA species support the template-switching model of similarity-assisted RNA recombination. In summary, PRRSV readily undergoes nonhomologous RNA recombination to generate heteroclite sub-genomic RNA5.     

Stress-activated protein kinases are involved in porcine reproductive and respiratory syndrome virus infection and modulate virus-induced cytokine production

The present study examined the role of the p38 MAPK and JNK pathways during PRRSV infection in immortalized porcine alveolar macrophage (PAM) cells. Infection with PRRSV was found to progressively activate p38 and JNK1/2 up to 36 h postinfection and then their phosphorylation levels dramatically decreased to baseline at 48 h postinfection. In contrast, UV-inactivated PRRSV failed to trigger phosphorylation of these SAPKs, indicating that the post-entry process is responsible for their activation. Independent treatment of cells with a selective p38 or JNK inhibitor markedly impaired PRRSV infection, resulting in significant reduction in synthesis of viral genomic and subgenomic RNAs, viral protein expression, and progeny virus production. Notably, cytokine production in PAM cells infected with PRRSV was shown to be altered by inhibiting these SAPKs. Altogether, our data suggest that the p38 and JNK signaling pathways play pivotal roles in PRRSV replication and may regulate immune responses during virus infection.     

Identification of new defective interfering RNA species associated with porcine reproductive and respiratory syndrome virus infection

In the present study, seven new defective interfering (DI) RNA species of porcine reproductive and respiratory syndrome virus (PRRSV) were identified. RT-PCR, Northern blot and sequence analyses indicated that these DI RNA specie have deletions of 8513-9176 nucleotides located between Nsp1/Nsp2 and Nsp10. Compared with the previous DI RNAs of PRRSV reported, they have three distinct characteristics: much smaller deletion sizes; different nucleotide repeats (2-12nt) used in the junction sites and in-frame deletions. The results further suggested that the similarity-assisted RNA recombination may be the main cause of generation of DI RNAs in PRRSV and probably in other arteriviruses.     

Thymocyte and peripheral blood T lymphocyte subpopulation changes in piglets following in utero infection with porcine reproductive and respiratory syndrome virus

Piglets infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV) are born severely immunocompromised. In this article we more closely examine the effects of in utero PRRSV infection on circulating and thymic T cell populations. Numbers of CD4+, CD8+, and dual-positive lymphocytes were quantitated in circulation and in the thymus during the 2 weeks following birth. At birth we found that the number of circulating lymphocytes was suppressed by 60%. Lymphocyte numbers were also suppressed by 42% at day 7, but by day 14 the number of lymphocytes had rebounded and was actually 47% greater than controls. At birth and day 7, a drop in the number of CD4+ cells could partially explain the suppression we observed, while the rebound in total lymphocyte numbers seen at day 14 was due to a nearly fourfold increase in the number of circulating CD8+ cells. As a result, the normal CD4+:CD8+ ratio of between 1.4 and 2.2 for neonatal pigs was reduced to 0.1-0.5. The thymuses of infected piglets were found to be 50% smaller than those of control pigs and were characterized by cortical involution and severe cortical depletion of thymocytes. Analysis of the population of thymocytes revealed that double-positive thymocytes were suppressed to a greater degree than either single positive subpopulation. In addition, we show that the number of thymocytes undergoing apoptosis was increased twofold in piglets infected with PRRSV. Taken together, these results help explain the dramatic immunosuppression observed in neonatal animals infected in utero with PRRSV.     

Gammadelta lymphocyte response to porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be one of the most important diseases facing swine industry today. Following PRRSV infection pigs develop both humoral and cell-mediated responses following PRRSV exposure; however, the relative importance in protection and clearance of the virus is not yet completely understood. Swine contain a large percentage of gammadelta T-lymphocytes in peripheral circulation capable of responding to various pathogens in both an innate and specific immune response. The objectives of this study were to determine whether gammadelta lymphocytes functionally respond to PRRSV upon initial exposure and re-exposure. Four month old PRRSV free gilts were intranasally inoculated with a field isolate MN-30100 then assessed at various time points post infection. On day 120, pigs were re-exposed with MN-30100 PRRSV strain and subsequently were bled on days 0, 7, and 14 post re-exposure. Lymphocyte subpopulations, antigen specific proliferation, and IFN-gamma production were evaluated throughout the study. Circulating gammadelta lymphocytes in PRRSV exposed animals expanded between days 14 to 70 (d14-d70, p = 0.016); following antigen stimulation, gammadelta lymphocyte proliferated by day 14 (d0-d14, p = 0.001) continuing through day 60. gammadelta lymphocytes produced IFN-gamma by day 14 pi continuing through day 50 (d0-d50, p = 0.004). Following re-exposure both gammadelta+ and CD4+ lymphocytes increased in IFN-gamma production. These results are not fully conclusive on the role of gammadelta lymphocytes against PRRSV; the data indicate that gammadelta lymphocytes specifically respond to PRRSV.     

Induction of autophagy enhances porcine reproductive and respiratory syndrome virus replication

Autophagy is an evolutionarily conserved lysosome-dependent degradation pathway that acts in the maintenance of cellular homeostasis and plays important functions in viral replication and pathogenesis. In this study, we investigated the role of autophagy in the replication of porcine reproductive and respiratory syndrome virus (PRRSV), an agent that has caused devastating losses in the international swine industry since the late 1980s. Using protein quantification and microscopy, we observed that PRRSV infection results in LC3-I/II conversion, an increased accumulation of punctate GFP-LC3-expressing cells, and a higher number of autophagosome-like double-membrane vesicles in the cytoplasm of host cells. Inhibition of autophagy using 3-methyladenine (3-MA) or small interfering RNAs targeting ATG7 and Beclin-1 led to a significant reduction in PRRSV titers and protein expression. Conversely, induction of autophagy by rapamycin resulted in increased viral replication. These results demonstrate that PRRSV infection induces autophagy which, in turn, enhances viral replication efficiency.     

IL-18 expression in pigs following infection with Mycoplasma hyopneumoniae.

Little is known about the detail of the immune response during infection of pigs with Mycoplasma hyopneumoniae (Mhp). To further understand this important porcine pathogen, we examined the interleukin-18 (IL- 18) response in experimentally infected piglets. We found that large amounts of IL-18 were produced in the bronchoalveolar lavage fluids (BALF) of pigs experimentally infected with Mhp. However, the concentration of interferon-gamma (IFN-gamma) in the same BALF was negatively correlated with that of IL-18. The antibody response against Mhp was found to be associated with the IL-18 concentration in the BALF. Immunohistochemical staining revealed that both IL-18 and IL-18 receptor alpha chain (IL-18Ralpha) were present in macrophages and plasma cells in the lungs of Mhp-infected pigs. Lung mononuclear cells isolated from pneumonic lesions secreted IL-18 and prostaglandin E(2) (PGE(2)) in vitro, and PGE(2) production was enhanced by stimulation with IL-18. These results indicate that IL-18 produced in the pig lung contributes to the development of innate and acquired immune responses against Mhp as a proinflammatory cytokine rather than as an IFN-gamma-inducing factor and may be involved in immunomodulation in pigs.     

Porcine reproductive and respiratory syndrome virus infects mature porcine dendritic cells and up-regulates interleukin-10 production

Porcine reproductive and respiratory syndrome virus (PRRSV) infects mature dendritic cells (mDCs) derived from porcine monocytes and matured with lipopolysaccharide. The infection of mDCs induced apoptosis, reduced the expression of CD80/86 and major histocompatibility complex class II molecules, and increased the expression of interleukin-10, thus suggesting that such mDC modulation results in the impairment of T-cell activation.     

Encephalomalacic lesions in pigs dually infected with porcine reproductive and respiratory syndrome virus and pseudorabies virus

Four pigs (group 1) were infected with an aerosol containing porcine reproductive and respiratory syndrome virus (PRRSV) followed 7 days later by pseudorabies virus (PRV). Three further pigs (group 2) received PRRSV alone, two (group 3) received PRV alone, and two (group 4) remained as uninfected controls. Despite the admittedly small numbers of animals, the experiment appeared to throw light on aspects of synergy. Thus, the group 1 pigs showed severe neurological signs characterized by ataxia and muscular tremors. Total cell numbers in the bronchoalveolar lavage fluid were increased in all PRRSV-infected pigs, and PRRSV antigen was detected in the alveolar macrophages. Total cell numbers in the cerebrospinal fluid of group 1 pigs were considerably greater than those demonstrated in group 3, but no PRV antigen was found. Pigs of groups 1 and 2 showed pulmonary lesions, characterized by interstitial pneumonia and PRRSV antigen immunolabelling. Non-suppurative encephalitis was found in five of the six pigs of groups 1 and 3. In particular, one group 1 animal had severe necrotizing encephalitis with intranuclear inclusion bodies and associated immunolabelling of PRV antigen. The other three group 1 pigs had prominent malacic lesions, with macrophages. These neuropathological findings strongly suggested that PRRSV infection in pigs enhances the severity of brain lesions caused PRV.     

Brain lesions in pigs dually infected with porcine reproductive and respiratory syndrome virus and pseudorabies virus

Four pigs were inoculated with an aerosol containing porcine reproductive and respiratory syndrome virus (PRRSV) followed 14 days later by inoculation with pseudorabies virus (PRV). The four dually infected pigs showed severe clinical signs, and one died on day 6 after infection with PRV. As demonstrated previously, the clinical disease was much more severe than that produced by either virus alone. All four dually infected pigs developed severe non-suppurative encephalitis, two had tonsillitis, two had necrotizing bronchiolitis, and one had lymphadenitis. The distribution of lesions corresponded closely with the detection of intranuclear inclusion bodies and PRV antigen. High numbers of TUNEL-positive cells detected in the thymus were associated with thymic atrophy.