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.     

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.     

Neutralizing antibody responses of pigs infected with natural GP5 N-glycan mutants of porcine reproductive and respiratory syndrome virus

In order to assess the effect of the N-glycans associated with the GP5 neutralization epitope of porcine reproductive and respiratory syndrome virus (PRRSV) on the neutralizing antibody (Ab) response of swine, groups of young pigs were infected with PRRSV strains differing in N-glycosylation pattern. The humoral immune response to strain VR-2332, harboring four potential N-glycan sites, was compared to that of two natural field isolates carrying mutations either abolishing the N-glycosylation site at position 44 (N44) or the two N-glycosylation sites in the hypervariable region upstream of the neutralization epitope (HV-1). The pigs were bled at intervals and their sera were assayed for neutralizing Abs by indirect and competition ELISAs using peptides containing the GP5 neutralization epitope, and selectively for infectivity neutralization of a number of PRRSV strains. In addition, viremia was monitored by quantitative RT-PCR, and anti-N-protein Ab formation was measured by HerdChek ELISA. The neutralizing Ab responses as measured by peptide ELISA varied greatly between individual pigs infected with each PRRSV strain. Some pigs generated high titers of peptide binding Abs between 7 and 28 days post infection (p.i.), whereas other pigs had not generated a response by 90 days p.i. However, the HV-1-infected pigs generated Abs to the neutralization epitope more rapidly and to a 5-10 times higher level than VR-2332-infected pigs, and the Abs neutralized the homologous HV-1 virus 10-20 times more efficiently than PRRSV strains VR-2332, N44, MN184, or SDSU73. In contrast, most N44-infected pigs generated neutralizing Abs only after 42 days p.i. and only to low levels. The results suggest that the deletions of the N-glycans or other amino acid substitutions in the GP5 ectodomains of the mutants affect the immunogenicity of the neutralization epitope and the specificity of the Abs raised to it but not the sensitivity of the virions to Ab neutralization.     

猪繁殖与呼吸综合征病毒感染仔猪淋巴细胞亚群的动态

目的：研究SPF仔猪感染猪繁殖与呼吸综合征病毒后对猪瘟苗的免疫应答受到抑制的机理。方法：利用流式细胞术检测了SPF仔猪感染PRRS病毒BJ-4后外周血淋巴细胞亚群的动态。结果：外周血CD3^ 、CD4^ 、CD8^ 和SLA-DR^ 表达细胞在感染早期比例下降;感染猪扁桃体的CD3^ 和CD4^ CD8^ 细胞亚群比例下降;肠系膜淋巴结的CD3^ 细胞亚群在感染后细胞在感染早期比例下降;感染猪扁桃体的CD3^ 和CD4^ CD8^ 细胞亚群比例下降;肠系膜淋巴结的CD3^ 细胞在感染后下降,但是SLA-DR^ 细胞亚群有逐渐升高的趋势。结论：仔猪感染PRRS病毒后淋巴细胞各亚群的比例下降可能会抑制机体对其它病原体的免疫反应,扁桃体的细胞比例变化有利于其它呼吸道病原体的混合感染或继发感染。     

Pathogenesis of Korean type 1 (European genotype) porcine reproductive and respiratory syndrome virus in experimentally infected pigs

The aim of this study was to elucidate the pathogenesis of experimental infection with Korean type 1 porcine reproductive and respiratory syndrome virus (PRRSV) by defining the virus distribution, sites of viral replication, viraemia and gross and microscopical lesions in conventional pigs studied for 28 days after intranasal inoculation. Mean rectal temperature was significantly higher in infected pigs than in negative control pigs at 2 days post inoculation (dpi) (P=0.004), 3 dpi (P<0.001), 4 dpi (P=0.003) and 5 dpi (P=0.034). The log(10)TCID(50)/ml of type 1 PRRSV increased significantly at 0-1 dpi (P=0.024) and 5-7 dpi (P=0.029), but decreased at 10-14 dpi (P=0.026) and 14-21 dpi (P=0.012) in infected pigs. Infected pigs developed multifocal, tan-mottled areas of lung tissue with irregular and indistinct borders. Microscopical lesions, when present, were multifocal, mild to moderate, generally most extensive at 5-7 dpi (P=0.036), and were nearly resolved at 28 dpi. Type 1 PRRSV nucleic acid and antigen were detected exclusively within the cytoplasm of macrophages and type I and II pneumocytes. The score for PRRSV-positive cells increased at 3-7 dpi (P<0.05) and decreased at 10-14 dpi (P=0.034) in infected pigs. Thus, respiratory disease was reproduced in conventional pigs by infection with Korean type 1 PRRSV.     

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.     

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.     

The evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with VR-2332.

GP5, the principal envelope glycoprotein of porcine reproductive and respiratory syndrome virus (PRRSV), contains a hypervariable region within the ectodomain which is responsible for generating diversity in field isolates. The purpose of this study was to gain insight into the possible origin of this diversity by following GP5 sequence changes in pigs exposed to PRRSV strain VR-2332 in utero. A region of the PRRS virus genome containing portions of ORF4 and ORF5 was amplified directly from tissues of infected pigs from birth to 132 days of age. We observed the emergence of a new PRRSV population, identified by a single nucleotide change in the ectodomain. The Asp to Asn change at amino acid 34 was also found as a minor component in pigs that expressed the wild-type sequence. The results from this study suggest that the variability in the ectodomain of ORF5 is the result of positive or negative selection, of which the mechanism remains to be determined.     

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.     

Temporary CD8+ T-cell depletion in pigs does not exacerbate infection with porcine reproductive and respiratory syndrome virus (PRRSV)

Several studies have demonstrated a consistent increase in the CD8+ T-cell subset of pigs following infection with porcine reproductive and respiratory virus (PRRSV). Consequently, it has been suggested that CD8+ T-cells may play an important role in protection against this infection. In order to test this hypothesis, we examined five 5-week-old pigs, which had been depleted for CD8+ T-cells by treatment with anti-CD8 mAb injections, starting 2 days before inoculation with PRRSV. Virus-inoculated and sham-inoculated age-matched pigs served as controls. Blood samples were collected continuously, together with organ material at necropsy, to study kinetics of leukocyte subpopulations, antibody production and virus persistence in individual pigs. Significant lower CD8+ T-cell counts on day 0, that is, before virus challenge, in the anti-CD8 mAb treated pigs compared to the control pigs, confirmed the depletion effect of specific mAb therapy. Almost complete depletion of cell subsets expressing the CD8+ antigen was obtained on day 2 and 5 post infection (PI) with nadir less than 1% of peripheral blood mononuclear cells (PBMC). One week PI, an increase in T-cell subsets was observed for both anti-CD8 mAb treated pigs and virus-inoculated control pigs. T-memory cells and cytotoxic T-cells reached levels comparative with the virus-inoculated control pigs on days 8 and 12 PI, respectively, whereas NKcells remained suppressed for the rest of the experimental period. An extraordinary increase of T-helper cells in the anti-CD8 mAb treated pigs with a significantly higher level than in the virus-inoculated and sham-inoculated control pigs, was observed from day 12 PI. In conclusion, it was established that CD8+ T-cell depletion in the early phase of PRRSV infection neither caused increased disease nor influenced the ability to clear virus in the treated pigs.     

Induction of T helper 3 regulatory cells by dendritic cells infected with porcine reproductive and respiratory syndrome virus

Delayed development of virus-specific immune response has been observed in pigs infected with the porcine reproductive and respiratory syndrome virus (PRRSV). Several studies support the hypothesis that the PRRSV is capable of modulating porcine immune system, but the mechanisms involved are yet to be defined. In this study, we evaluated the induction of T regulatory cells by PRRSV-infected dendritic cells (DCs). Our results showed that PRRSV-infected DCs significantly increased Foxp3⁺CD25⁺ T cells, an effect that was reversible by IFN-α treatment, and this outcome was reproducible using two distinct PRRSV strains. Analysis of the expressed cytokines suggested that the induction of Foxp3⁺CD25⁺ T cells is dependent on TGF-β but not IL-10. In addition, a significant up-regulation of Foxp3 mRNA, but not TBX21 or GATA3, was detected. Importantly, our results showed that the induced Foxp3⁺CD25⁺ T cells were able to suppress the proliferation of PHA-stimulated PBMCs. The T cells induced by the PRRSV-infected DCs fit the Foxp3⁺CD25⁺ T helper 3 (Th3) regulatory cell phenotype described in the literature. The induction of this cell phenotype depended, at least in part, on PRRSV viability because IFN-α treatment or virus inactivation reversed these effects. In conclusion, this data supports the hypothesis that the PRRSV succeeds to establish and replicate in porcine cells early post-infection, in part, by inducing Th3 regulatory cells as a mechanism of modulating the porcine immune system.     

Increased production of proinflammatory cytokines following infection with porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae

Induction of the proinflammatory cytokines interleukin-1 (IL-1) (alpha and beta), IL-6, IL-8, IL-10, IL-12, and tumor necrosis factor alpha (TNF-alpha) in pulmonary alveolar macrophages (PAMs) was assessed following experimental infection with porcine reproductive and respiratory syndrome virus (PRRSV) and/or Mycoplasma hyopneumoniae by using in vivo and in vitro models. The in vivo model consisted of pigs infected with PRRSV and/or M. hyopneumoniae and necropsied at 10, 28, or 42 days postinfection. Pigs infected with both pathogens had a greater percentage of macroscopic lung lesions, increased clinical disease, and slower viral clearance than pigs infected with either pathogen alone. The pigs infected with both PRRSV and M. hyopneumoniae had significantly increased levels of mRNA for many proinflammatory cytokines in PAMs collected by bronchoalveolar lavage (BAL) at all necropsy dates compared to those in uninfected control pigs. Increased levels of IL-1beta, IL-8, IL-10, and TNF-alpha proteins in BAL fluid, as measured by enzyme-linked immunosorbent assay, confirmed the increased cytokine induction induced by the pathogens. An in vitro model consisted of M. hyopneumoniae-inoculated tracheal ring explants cultured with PRRSV-infected PAMs. PAMs were harvested at 6 or 15 h postinfection with either or both pathogens. The in vitro study detected increased IL-10 and IL-12 mRNA levels in PAMs infected with PRRSV at all time periods. In addition, IL-10 protein levels were significantly elevated in the culture supernatants in the presence of M. hyopneumoniae-inoculated tracheal ring explants. The increased production of proinflammatory cytokines in vivo and in vitro associated with concurrent M. hyopneumoniae and PRRSV infection may play a role in the increased rates of pneumonia associated with PRRSV infection. The increased levels of IL-10 may be a possible mechanism that PRRSV and M. hyopneumoniae use to exacerbate the severity and duration of pneumonia induced by PRRSV and modulate the respiratory immune response.     

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.     

Distribution of a Korean strain of porcine reproductive and respiratory syndrome virus in experimentally infected pigs, as demonstrated immunohistochemically and by in-situ hybridization

In an experiment with 40 specific pathogen-free pigs aged 3 days, the distribution of a Korean isolate of porcine reproductive and respiratory syndrome virus (PRRSV) was assessed immunohistochemically and by in-situ hybridization for a period of 28 days after intranasal inoculation. The most consistent and intense labelling for PRRSV was in the lung, the virus persisting in pulmonary macrophages for at least 28 days. The middle lobe of the lung was the optimum site for the detection of PRRSV antigens and nucleic acids, and the interstitial macrophage was the main cell type in which PRRSV was identified. Other tissues and cells in which the virus was detected included macrophages and dendritic cells in the tonsil, lymph nodes, spleen and Peyer's patches, and macrophages in the hepatic sinusoids and adrenal gland. The experiment suggested that the pathogenesis of PRRSV infection may be summarized thus: initial entry of virus through tonsillar and pulmonary macrophages, followed within 3 days by viraemia and subsequent interstitial pneumonia.     

Real-time PCR for quantitation of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 in naturally-infected and challenged pigs

Real-time PCR assays were developed for quantitative detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2). The established real-time PCR for the quantitation of PRRSV cDNA and PCV2 DNA were found to be in the 9-log(10) linear dynamic range with excellent linearity and reliable reproducibility. Using these techniques, the distribution and quantitation of PRRSV and PCV2 in naturally infected and challenged pigs were investigated. The viral concentrations were expressed as the mean log(10) viral DNA or cDNA copy numbers per mg or ml of tested samples. For pigs infected naturally with both viruses, the lung, spleen, tonsil and lymphoid organs had the highest viral burdens with ranges from 5.73 to 8.38 and 5.65 to 6.91 for PRRSV and PCV2, respectively. The injection of formalin-inactivated Salmonella choleraesuis emulsified in complete Freund's adjuvant 1 week before and after the inoculation of both viruses resulted in PRRSV replication enhancement 2 weeks post-challenge. However, this facilitated the clearance of PRRSV 4 weeks post-challenge. Results from this study show that the established quantitative PCR could be a useful tool when applied to vaccine development and pathogenesis studies in the future.