Immunopathogenesis of lung lesions induced by the highly virulent Rosalía strain of PRRSV-1 circulating in Spain since 2020.

Global analysis of ubiquitome in PRRSV-infected pulmonary alveolar macrophages

To document shedding of porcine reproductive and respiratory syndrome (PRRS) virus in mammary gland secretions of experimentally inoculated sows, to evaluate effects of vaccination during gestation on virus shedding during the subsequent lactation, and to evaluate shedding of PRRS virus in milk of sows in commercial herds. 6 sows seronegative for PRRS virus were used for experiment 1, and 2 sows were retained for experiment 2. For experiment 3, 202 sows in commercial herds were used. In experiment 1, 2 sows were inoculated with PRRS virus, 2 sows were vaccinated with modified-live PRRS virus vaccine, and 2 sows served as control pigs. Mammary gland secretions were assayed for PRRS virus. In experiment 2, pregnant vaccinated sows from experiment 1 were vaccinated with another modified-live PRRS virus vaccine. Mammary gland secretions were assayed in the same manner as for experiment 1. For experiment 3, milk collected from 202 sows in commercial herds was assayed for PRRS virus. In experiment 1, PRRS virus was detected in mammary gland secretions of both vaccinated and 1 of 2 virus-inoculated sows. In experiment 2, virus was not detected in samples from either vaccinated sow. In experiment 3, all samples yielded negative results. Naïve sows inoculated late in gestation shed PRRS virus in mammary secretions. Previous vaccination appeared to prevent shedding during the subsequent lactation. Results for samples obtained from sows in commercial herds suggested that virus shedding in mammary gland secretions of such sows is uncommon.

Virulence and genotype-associated infectivity of interferon-treated macrophages by porcine reproductive and respiratory syndrome viruses

ORF1 but not ORF2 dependent differences are important for in vitro replication of PCV2 in porcine alveolar macrophages singularly or coinfected with PRRSV

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.

Porcine reproductive and respiratory syndrome virus (PRRSV) is highly limited to only cell subsets that express PRRSV receptors. Reproductive organ revealing the typical signs of PRRSV infection may be the critical site of problem syndromes. Persistent PRRSV producing re-infection via horizontal or vertical transmission could not be eradicated from herds. This research examined the possibility of porcine endometrium to be a PRRSV permissive cell�and serves as the primary cause of the persistent PRRSV. Cellular and immunological in response to PRRSV relevant to viral replication, shedding and re-circulation was assessed. The different outcomes between the different genotypes (type I vs. II), and routes of infection (apical vs. basolateral) were compared. Porcine glandular endometrial epithelial cells (PE) isolated from 4-6 months old PRRSV-free pre-puberty gilts (n=5 pigs) were cultured in standard medium DMEM with 5% fetal bovine serum until 90% confluent. Fresh isolated PRRSV type I or type II (at TCID100/2 ml) were inoculated to apical or basolateral membrane of PE for 1 hr. The occurrence of cytopathic effect (CPE) were observed daily. PRRSV-positive cells and cellular PRRSV mediator proteins, CD151, CD163, sialoadhesin (Sn), integrin and vimentin, were quantified by immunohistochemistry (IHC). Related cytokine secretion, CCL2, IL-1?, IL-6, IL-8, IFN-g and TNF-? was measured by enzyme linked immunosorbent assay (ELISA), at 0, 2, 4 and 6 day-post-infection (dpi). The mRNA expression of PRRSV mediator, toll-like receptors (TLRs) and cytokines were�evaluated by real-time RT-PCR. Effects of PRRSV re-infection in modulating all the responses�of primary infection were considered by repeating the infection at 4 dpi at the same PE. At early stage of infection, at 4dpi, CPE along and PRRSV proteins was observed in apical PRRSV infected PE, but was observed later in basolateral-infected PE. Infection with type II produced these infectivity effects rather than type I (p<0.05). Prior to infection,�mediator proteins CD151, Sn, integrin and vimentin�but not CD163 were expressed. Type I up-regulated CD151, CD163, Sn and integrin mRNA�higher than mock and type II (p<0.05). Changes of mediator proteins were observed differently during 4-6 dpi (p<0.05). Apical infection with type I up-regulated all mediators except Sn, whereas type II down-regulated Sn, integrin and vimentin. Basolateral type I and II infection down-regulated integrin and vimentin (p<0.05), but up-regulated CD151, CD163 and Sn (p<0.05). Up-regulation of TLR1/TLR3 and TLR10 were induced by primary infection with type I and II, respectively. All primary infection down-regulated TLR4 mRNA (p<0.05). Re-infection with PRRSV particularly type I up-regulated the level of TLR1, TLR2 and TLR4 expression (p<0.05). Down-regulation of TLR5 and TLR8 were later observed in primary or re-infected PE cells (p<0.05). Re-infection with type I or II completely decreased IL-6 mRNA, but not other genes. Primary PRRSV infection could not alter CCL2, IL1?, IL-8 and IFN-g�secreted by PE , but type I infection increased IL-6 secretion (p<0.05). Primary or re-infection with PRRSV type I or II dampened TNF-? secretion significantly (p<0.05). Noticeably, in the present study, supernatant collected from all PRRSV-infected cells contains PRRSV at TCID100/ml throughout the study. In summary, endometrial cells are susceptible to either apical and basolateral PRRSV infection, and long-lasting re-circulate PRRSV. Effects of primary infection may be mediated by TLRs or mediators. Modification in the synthesis of TLRs, PRRSV mediators and cytokines by PRRSV could be enhanced or suppressed depending on time course, genotype or route of infection. PRRSV type II has more virulence than type I but PRRSV type I produced more�to susceptibility for executive infection. Therefore, direct PRRSV infection to PE cells may play a role in PRRSV-induced reproductive failure and may be the cause of persistent PRRSV infection in sow.

Activation of pro- and anti-inflammatory responses in lung tissue injury during the acute phase of PRRSV-1 infection with the virulent strain Lena

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses in the swine industry. Frequent mutations and recombinations account for PRRSV immune evasion and the emergence of novel strains. In this study, we isolated and characterized two novel PRRSV-2 strains from Southwest China exhibiting distinct recombination patterns. They were designated SCABTC-202305 and SCABTC-202309. Phylogenetic results indicated that SCABTC-202305 was classified as lineage 8, and SCABTC-202309 was classified as lineage 1.8. Amino acid mutation analysis identified unique amino acid substitutions and deletions in ORF5 and Nsp2 genes. The results of the recombination analysis revealed that SCABTC-202305 is a recombinant with JXA1 as the major parental strain and NADC30 as the minor parental strain. At the same time, SCABTC-202309 is identified as a recombinant with NADC30 as the major parental strain and JXA1 as the minor parental strain. In this study, we infected piglets with SCABTC-202305, SCABTC-202309, or mock inoculum (control) to study the pathogenicity of these isolates. Although both isolated strains were pathogenic, SCABTC-202305-infected piglets exhibited more severe clinical signs and higher mortality, viral load, and antibody response than SCABTC-202309-infected piglets. SCABTC-202305 also caused more extensive lung lesions based on histopathology. Our findings suggest that the divergent pathogenicity observed between the two novel PRRSV isolates may be attributed to variations in the genetic information encoded by specific genomic regions. Elucidating the genetic determinants governing PRRSV virulence and transmissibility will inform efforts to control this devastating swine pathogen.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) is one of the most critical pathogens impacting the global swine industry. Frequent mutations and recombinations have made the control of PRRSV increasingly difficult. Following the NADC30-like PRRSV pandemic, recombination events involving PRRSV strains have further increased. We isolated two novel field PRRSV recombinant strains, SCABTC-202305 and SCABTC-202309, exhibiting different recombination patterns and compared their pathogenicity in animal experiments. The isolates caused higher viral loads, persistent fever, marked weight loss, moderate respiratory clinical signs, and severe histopathologic lung lesions in piglets. Elucidating correlations between recombinant regions and pathogenicity in these isolates can inform epidemiologic tracking of emerging strains and investigations into viral adaptive mechanisms underlying PRRSV immunity evasion. Our findings underscore the importance of continued genomic surveillance to curb this economically damaging pathogen.

Effect of porcine reproductive and respiratory syndrome virus (PRRSV) (isolate ATCC VR-2385) infection on bactericidal activity of porcine pulmonary intravascular macrophages (PIMs): in vitro comparisons with pulmonary alveolar macrophages (PAMs)

Comparison of the Pathogenesis of Single or Dual Infections with Type 1 and Type 2 Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) is the most economically significant disease affecting swine production in the United States (US). The etiologic agent of PRRS is an RNA virus named as PRRS virus (PRRSV). Substantial economic losses attributed to PRRS are due to reduced reproductive performance in sows and reduced growth rate and increased mortality in growing animals. In the US, PRRSV activity is routinely monitored by production systems and/or veterinary clinics. A group of swine production systems in the US voluntarily share their PRRSV breeding herd incidence to a program that gathers, aggregates, and reports the weekly incidence and prevalence among participants. As of February of 2020, most of the participants are from large production systems that represented 2.8 million sows. Currently, there is no information on PRRSV detection in the broader industry which totals of 6.46 million sows. Additionally, American Association of Swine Veterinarians (AASV) guidelines for PRRSV monitoring (from 2011) was based on the use of serum samples. However, there is a recent description of processing fluid as a population-based sample type to monitor PRRSV herd status more efficiently. The validation of this new sample type raises the need to better understand the practical applications of this sample type to monitor PRRSV. Considering (a) the high economic importance of PRRS to the US swine industry, (b) the current gap of information about PRRSV activity in most breeding herds, and in growing animals, and the (c) gap in knowledge about applications of processing fluids to monitor and surveil PRRSV, there is the critical need to develop additional tools for improvement in the monitoring and surveillance systems for PRRSV in the US swine industry. Therefore, the overall objectives of this dissertation were: a) to develop a capability to reliably and consistently track PRRSV detection over time, age group, geographical space, and specimen in the US swine industry; and b) to develop monitoring and surveillance systems to enable veterinarians to make science-driven decisions to support disease prevention, detection, and control strategies. To that end, a capability to monitor and report the detection of PRRSV RNA by RT-PCR at the four major US swine-centric veterinary diagnostic laboratories was built. A procedure to receive data, a process to compile to the same format, aggregate, report, and a capability for continuous automated updates was developed. This information was used to describe the macroepidemiological aspects of PRRSV RNA detection by RT-PCR in the US

BackgroundIn this study, six enzyme-linked immunosorbent assays (ELISA), intended for routine porcine reproductive and respiratory syndrome virus (PRRSV) herd monitoring, are tested for their ability to detect PRRSV specific antibodies in the serum of pigs after vaccination with an inactivated PRRSV type 1 vaccine and subsequent infection with a highly pathogenic (HP) PRRSV field strain. For this reason, ten piglets (group V) from a PRRSV negative herd were vaccinated twice at the age of 2 and 4 weeks with an inactivated PRRSV vaccine. Ten additional piglets (group N) from the same herd remained unvaccinated. Three weeks after second vaccination, each of the piglets received an intradermal application of an HP PRRSV field strain. Serum samples were taken before first vaccination as well as before and 3, 7, 10 and 14 days after HP PRRSV application. All serum samples were tested for PRRSV RNA by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) as well as for PRRSV antibodies with all six study ELISAs.ResultsAt the beginning of the study (before vaccination), all of the piglets were PRRSV antibody negative with all study ELISAs. They also tested negative for PRRSV RNA measured by RT-qPCR. From day 3 after HP PRRSV application until the end of the study, a viremia was detected by RT-qPCR in all of the piglets. On day 0 (day of HP PRRSV application), nine out of ten piglets of the pre-vaccinated group tested PRRSV antibody positive with one of the tested ELISAs, although with lower S/P values than after infection. On day 10 after HP PRRSV application, all study ELISAs except one had significantly higher S/P or OD values, respectively more positive samples, in group V than in group N.ConclusionsOnly one of the tested ELISAs was able to detect reliably PRRSV antibodies in pigs vaccinated with an inactivated PRRSV vaccine. With most of the tested ELISAs, higher S/P values respectively more positive samples after PRRSV infection were seen in the pre-vaccinated group than in the non-vaccinated.

BackgroundAirborne transmitted pathogens, such as porcine reproductive and respiratory syndrome virus (PRRSV), need to interact with host cells of the respiratory tract in order to be able to enter and disseminate in the host organism. Pulmonary alveolar macrophages (PAM) and MA104 derived monkey kidney MARC-145 cells are known to be permissive to PRRSV infection and replication and are the most studied cells in the literature. More recently, new cell lines developed to study PRRSV have been genetically modified to make them permissive to the virus. The SJPL cell line origin was initially reported to be epithelial cells of the respiratory tract of swine. Thus, the goal of this study was to determine if SJPL cells could support PRRSV infection and replication in vitro.ResultsThe SJPL cell growth was significantly slower than MARC-145 cell growth. The SJPL cells were found to express the CD151 protein but not the CD163 and neither the sialoadhesin PRRSV receptors. During the course of the present study, the SJPL cells have been reported to be of monkey origin. Nevertheless, SJPL cells were found to be permissive to PRRSV infection and replication even if the development of the cytopathic effect was delayed compared to PRRSV-infected MARC-145 cells. Following PRRSV replication, the amount of infectious viral particles produced in SJPL and MARC-145 infected cells was similar. The SJPL cells allowed the replication of several PRRSV North American strains and were almost efficient as MARC-145 cells for virus isolation. Interestingly, PRRSV is 8 to 16 times more sensitive to IFNα antiviral effect in SJPL cell in comparison to that in MARC-145 cells. PRRSV induced an increase in IFNβ mRNA and no up regulation of IFNα mRNA in both infected cell types. In addition, PRRSV induced an up regulation of IFNγ and TNF-α mRNAs only in infected MARC-145 cells.ConclusionsIn conclusion, the SJPL cells are permissive to PRRSV. In addition, they are phenotypically different from MARC-145 cells and are an additional tool that could be used to study PRRSV pathogenesis mechanisms in vitro.

Behaviour of pigs with viral and bacterial pneumonia

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus 2 (PCV2) are economically important pathogens in swine, and pigs with dual infections of PCV2 and PRRSV consistently have more severe clinical symptoms and interstitial pneumonia. However, the synergistic pathogenesis mechanism induced by PRRSV and PCV2 co-infection has not yet been illuminated. Therefore, the aim of this study was to characterize the kinetic changes of immune regulatory molecules, inflammatory factors and immune checkpoint molecules in porcine alveolar macrophages (PAMs) in individuals infected or co-infected with PRRSV and/or PCV2. The experiment was divided into six groups: a negative control group (mock, no infected virus), a group infected with PCV2 alone (PCV2), a group infected with PRRSV alone (PRRSV), a PCV2–PRRSV co-infected group (PCV2–PRRSV inoculated with PCV2, followed by PRRSV 12 h later), a PRRSV–PCV2 co-infected group (PRRSV–PCV2 inoculated with PRRSV, followed by PCV2 12 h later) and a PCV2 + PRRSV co-infected group (PCV2 + PRRSV, inoculated with PCV2 and PRRSV at the same time). Then, PAM samples from the different infection groups and the mock group were collected at 6, 12, 24, 36 and 48 h post-infection (hpi) to detect the viral loads of PCV2 and PRRSV and the relative quantification of immune regulatory molecules, inflammatory factors and immune checkpoint molecules. The results indicated that PCV2 and PRRSV co-infection, regardless of the order of infection, had no effect on promoting PCV2 replication, while PRRSV and PCV2 co-infection was able to promote PRRSV replication. The immune regulatory molecules (IFN-α and IFN-γ) were significantly down-regulated, while inflammatory factors (TNF-α, IL-1β, IL-10 and TGF-β) and immune checkpoint molecules (PD-1, LAG-3, CTLA-4 and TIM-3) were significantly up-regulated in the PRRSV and PCV2 co-infection groups, especially in PAMs with PCV2 inoculation first followed by PRRSV. The dynamic changes in the aforementioned immune molecules were associated with a high viral load, immunosuppression and cell exhaustion, which may explain, at least partially, the underlying mechanism of the enhanced pulmonary lesions by dual infection with PCV2 and PRRSV in PAMs.