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Linda J. Saif - One of the best experts on this subject based on the ideXlab platform.

  • decline of transmissible gastroenteritis virus and its complex evolutionary relationship with Porcine Respiratory Coronavirus in the united states
    Scientific Reports, 2019
    Co-Authors: Linda J. Saif, Fangzhou Chen, Todd P Knutson, Stephanie Rossow, Douglas Marthaler
    Abstract:

    The epidemiology and genetic diversity of transmissible gastroenteritis virus (TGEV) in the United States (US) was investigated by testing clinical cases for TGEV by real time RT-PCR between January 2008 and November 2016. Prevalence of TGEV ranged between 3.8–6.8% and peaked during cold months until March 2013, in which prevalence decreased to < 0.1%. Nineteen complete TGEV genomes and a single strain of Porcine Respiratory Coronavirus (PRCV) from the US were generated and compared to historical strains to investigate the evolution of these endemic Coronaviruses. Sixteen of our TGEV strains share 8 unique deletions and 119 distinct amino acid changes, which might greatly affect the biological characteristics of the variant TGEV, and resulted in a “variant” genotype of TGEV. The “variant” genotype shared similar unique deletions and amino acid changes with the recent PRCV strain identified in this study, suggesting a recombination event occurred between the ‘‘variant’’ TGEV and PRCV. Moreover, the results indicate the “variant” genotype is the dominant genotype circulating in the US. Therefore, this study provides insight into the occurrence, origin, genetic characteristics, and evolution of TGEV and PRCV circulating in the US.

  • Nasal Swabs of Infected Pigs Diagnosis of Transmissible Gastroenteritis Virus and Porcine Respiratory Coronavirus from Feces and Development of a Reverse Transcription-Nested Polymerase Chain Reaction Assay for Differential
    2013
    Co-Authors: Lomi Kim, K. Sestak, Kyeongok Chang, Anil V Parwani, Linda J. Saif
    Abstract:

    Transmissible gastroenteritis virus (TGEV), a Coronavirus, replicates in intestinal enterocytes and causes diarrhea in young pigs. Porcine Respiratory Coronavirus (PRCV), a spike (S) gene natural deletion mutant of TGEV, has a Respiratory tissue tropism and causes mild or subclinical Respiratory infections. Conventional antigen-based diagnostic tests fail to differentiate TGEV and PRCV, and a blocking ELISA test to serologically differentiate TGEV/PRCV-infected pigs is conducted on convalescent serum retrospectively after disease outbreaks. A reverse transcription (RT)-nested polymerase chain reaction (PCR) with primers targeted to the S gene deletion region to differentiate TGEV/PRCV was developed. The specificity of the RT-nested PCR was confirmed with reference and recent field strains of TGEV/PRCV, and its sensitivity was analyzed by testing nasal and fecal samples collected from pigs at various days postinoculation (DPI) with TGEV or PRCV. Specific PCR products for TGEV/PRCV were detected only with the homologous reference or field Coronaviruses and for 10–14 DPI of pigs with TGEV (feces) or PRCV (nasal samples). The RT-nested PCR assay was more sensitive than antigen-based assays on the basis of duration of virus detection in experimentally infected pigs and was directly applicable to nasal as well as fecal specimens from the field. Transmissible gastroenteritis virus (TGEV) is a member of the Coronaviridae family and is enveloped with a positive-stranded RNA genome.3,7 Porcine Respiratory Coronavirus (PRCV) represents a natural deletion mutant of TGEV that appeared in 1983–1984 in Europe and in 1988 in the US.3 Coronaviruses have 3 major structural proteins: the spike (S), the integral membrane glycoprotein, and the nucleocapsid protein.3 TGEV replicates primarily in small intestinal enterocytes, whereas PRCV replicates predominantly in the Respiratory tract.3,7 According to sequence comparisons of PRCV and TGEV, PRCV has a large deletion in the 59 region of the S gene and minor deletions in genes 3 and 3-1.3,11 These deletions are thought to influence the viral tissue tropism and virulence. The deletion size in the S gene ranges from 621 to 681 bp depending on the origin of the strain.11 Recently, strains of TGEV with reduced enteropathogenicity were reported in the field.6 A similar suspect TGEV outbreak of reduced virulence (mild diarrhea and intestinal lesions, slow disease spread among pigs) in nursery pigs from a swine herd in the US Midwest was investigated. Diagnosis of TGEV in these pigs was sporadic and inconsistent and presumably complicated by the presence of antibodies to PRCV confirmed by a blocking differential ELISA test on sera from a number of pigs in this herd (L. J. Saif and P. Lewis, unpublished). However, this latter test showed inconsistent results for TGEV/PRCV differentiation with serially collected samples from the same pigs within the herd (inconsistent individual immune status), and some pigs in the From the Food Animal Health Research Program, Department of Veterinary Preventive Medicine, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691 (Kim, Chang, Parwani, Saif), and the School of Veterinary Medicine, Tufts University, North Grafton, MA 01536-1895 (Sestak). Received for publication May 20, 1999. herd tested only PRCV positive, whereas others were TGEV positive (inconsistent herd immune status). These new TGEV strains may represent naturally occurring recombinants with reduced virulence between TGEV and PRCV strains, or the presence of PRCV antibodies in these herds may have complicated the diagnosis and modulated the severity of conventional TGEV infections. TGEV is a major cause of neonatal diarrhea and also causes enzootic diarrhea in older pigs.7 It costs the swine industry in the US nearly $200 million a year.7 PRCV causes infected swine to be diagnosed as TGEV positive in conventional serologic tests.8 Several investigators have described the use of molecular assays to detect and differentiate TGEV/PRCV strains including reverse transcription–polymerase chain reaction (RT-PCR),5 cDNA probes,12,13 in situ hybridization,10 and RT-PCR/restriction fragment length polymorphism.2 To differentiate TGEV/PRCV with reference virus strains from tissue culture, an RT-PCR assay was developed with primers targeted to the S gene deletion.5 These investigators used restriction endonuclease analysis to confirm the identity of their RT-PCR products. Use of the RT-nested PCR assay for detection and differentiation of TGEV/PRCV directly from nasal swabs or feces has not been reported. Therefore, the objective of this study was to develop and use RT-nested PCR assays to detect and differentiate TGEV/PRCV directly from fecal and nasal swab specimens from experimentally infected pigs and from field outbreak specimens. Four field samples were obtained from a midwest swine herd with sporadic diarrhea cases in nursery pigs. The BW 021898B sample consisted of intestinal contents from a nursery pig with mild diarrhea (clinically suspect for transmissible gastroenteritis). Three nasal swab samples (BW126, BW154, and BW155) were obtained from normal TGEVseronegative sentinel nursery pigs placed in contact with the diarrheic pigs in the same nursery. Swine testicular (ST) cells were used for virus isolation, growth, and cell culture 386 Brief communications Table 1. Reference and field TGEV and PRCV strains. Isolate Isolation date Location P no. (PP)* Source TGEV reference strains M5C Miller M6 Miller P115 Purdue 1965 1965 1952 Ohio Ohio Indiana 2 (2) 6 (2) 115 E. Bohl, OARDC,† Wooster, OH L. J. Saif, OARDC, Wooster, OH E. Bohl, OARDC, Wooster, OH PRCV reference strains ISU-1 ISU-3 199

  • Nitric oxide is elicited and inhibits viral replication in pigs infected with Porcine Respiratory Coronavirus but not Porcine reproductive and Respiratory syndrome virus.
    Veterinary immunology and immunopathology, 2010
    Co-Authors: Kwonil Jung, Gourapura J. Renukaradhya, Ashita Gurnani, Linda J. Saif
    Abstract:

    There is little information on the role of nitric oxide (NO) in innate immunity to Respiratory Coronavirus (CoV) infections. We examined NO levels by Greiss assay in bronchoalveolar lavage (BAL) of pigs infected with either Porcine Respiratory Coronavirus (PRCV) or Porcine reproductive and Respiratory syndrome virus (PRRSV), a member of Nidovirales, like CoV. The antiviral effects of NO on these two viruses were tested in an in vitro system using a NO donor, S-nitroso-N-acetylpenicillamine (SNAP). We detected a large increase in NO levels in BAL fluids of PRCV-infected pigs, but not in PRRSV-infected pigs. Pulmonary epithelial cell necrosis induced by PRCV coincided with increased NO. Moreover, NO levels in cell culture medium of PRRSV-infected alveolar macrophages (AMs) did not differ from that of mock-infected AMs. Antiviral assays showed that NO significantly inhibited PRCV replication in swine testicular (ST) cells, whereas PRRSV was not susceptible to NO based on the conditions tested. Our study suggests that unlike PRRSV which induces apoptosis in AMs, Respiratory CoVs such as PRCV that infect pulmonary epithelial cells and cause cytolysis, induce NO production in the Respiratory tract. Thus, NO may play a role in innate immunity to Respiratory CoV infections by inhibiting viral replication.

  • Porcine reproductive and Respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to Porcine Respiratory Coronavirus in pigs.
    Viral immunology, 2010
    Co-Authors: Gourapura J. Renukaradhya, Kwonil Jung, Konstantin P. Alekseev, Ying Fang, Linda J. Saif
    Abstract:

    We performed a comprehensive analysis of innate and adaptive immune responses in dual-virus infected pigs to understand whether a pre-existing immunomodulatory Respiratory viral infection affects the overall immunity to a subsequent Porcine Respiratory Coronavirus (PRCV) infection in pigs. Pigs were either mock-infected or infected with Porcine reproductive and Respiratory syndrome virus (PRRSV), a virus known to cause immunosuppressive Respiratory disease, and then pigs were co-infected with PRCV, which normally causes subclinical Respiratory infection. We collected samples for six independent experiments from 178 pigs that were also used for pathological studies. We detected a significant reduction in innate NK-cell-mediated cytotoxic function in PRRSV-infected pigs, which was synergistically further decreased in pigs co-infected with PRCV. Subsequently, in association with clinical signs we observed elevated levels of proinflammatory (IL-6), Th-1 (IL-12), and regulatory (IL-10 and TGF-β) cytokines. Increased frequencies of CD4CD8 double-positive T lymphocytes and myeloid cells, in addition to the elevated Th-1 and proinflammatory cytokines in dual-infected pigs, contributed to the severity of lung disease in pigs. The results of our study clarify how each virus modulates the host innate and adaptive immune responses, leading to inflammatory reactions and lung pathology. Thus measurements of cytokines and frequencies of immune cells may serve as indicators of the progression of Respiratory viral co-infections, and provide more definitive approaches for treatment.

  • Porcine reproductive and Respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with Porcine Respiratory Coronavirus: implications for Respiratory viral co-infections
    Journal of General Virology, 2009
    Co-Authors: Kwonil Jung, Konstantin P. Alekseev, Gourapura J. Renukaradhya, Ying Fang, Yuxin Tang, Linda J. Saif
    Abstract:

    The innate immune response is critical for host defence against Respiratory Coronaviruses (CoVs). This study demonstrated that an ongoing Respiratory virus infection compromises innate immune responses and affects the pathogenesis of a Respiratory CoV co-infection. An innate immunosuppressive Respiratory virus infection was established by infecting weaned pigs with Porcine reproductive and Respiratory syndrome virus (PRRSV); 10 days later, the pigs were exposed to Porcine Respiratory Coronavirus (PRCV). The PRRSV/PRCV dual-infected pigs had reduced weight gains, a higher incidence of fever and more severe pneumonia compared with either single infection. Significant suppression of innate immune responses [reduced alpha interferon (IFN-α) levels in the lungs and reduced blood natural killer cell cytotoxicity] by the ongoing PRRSV infection was observed in dual-infected pigs, which coincided with exacerbated pneumonia during early PRCV infection. The subsequent PRCV infection led to enhanced PRRSV replication in the lungs and a trend towards increased serum T-helper type 1 (Th1) (IFN-γ) but decreased Th2 [interleukin (IL)-4] responses, further exacerbating PRRSV pneumonia. Following PRCV infection, more severe PRRSV-related pulmonary alveolar macrophage (PAM) apoptosis occurred, as determined by an in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay, suggesting increased PRRSV replication in PAMs. Collectively, these observations suggest interactive effects between PRCV and PRRSV via early innate (IFN-α) and later adaptive Th1 (IFN-γ) and Th2 (IL-4) immune responses. These findings imply that an existing immunomodulating Respiratory viral co-infection may be a contributing factor to more severe pneumonia in Respiratory CoV disease. This study provides new insights into host–pathogen interactions related to co-infection by CoVs and other Respiratory viruses.

Maurice Pensaert - One of the best experts on this subject based on the ideXlab platform.

  • Effect of Porcine Respiratory Coronavirus infection on lipopolysaccharide recognition proteins and haptoglobin levels in the lungs.
    Microbes and infection, 2006
    Co-Authors: Steven Van Gucht, Maurice Pensaert, Eric Cox, Kalina R. Atanasova, Filip Barbé, Kristien Van Reeth
    Abstract:

    Porcine Respiratory Coronavirus (PRCV) potentiates Respiratory disease and proinflammatory cytokine production in the lungs upon intratracheal inoculation with lipopolysaccharide (LPS) at 1 day of infection. This study aimed to quantify LPS-binding protein (LBP), CD14 and haptoglobin in the lungs throughout a PRCV infection. LBP and CD14 recognize LPS and enhance its endotoxic activity, whereas haptoglobin dampens it. Gnotobiotic pigs were inoculated intratracheally with PRCV (n = 34) or saline (n = 5) and euthanized 1-15days post inoculation (DPI). Virus was detected in the lungs from 1 to 9DPI. Cell-associated CD14 in lung tissue increased up to 15 times throughout the infection, due to an increase in highly CD14+ monocyte-macrophages from 1 to 12DPI and CD14+ type 2 pneumocytes from 7 to 9DPI. LBP and soluble CD14 levels in bronchoalveolar lavage fluids were elevated from 1-12DPI, with up to 35- and 4-fold increases, respectively. Haptoglobin levels increased significantly (x4.5) at 7DPI. In addition, we found that PRCV could sensitize the lungs to LPS throughout the infection, but the response to LPS appeared less enhanced at the end of infection (7DPI). The marked increases in LBP, CD14 and haptoglobin were not correlated with the extent of the LPS response.

  • A potential role for tumour necrosis factor-α in synergy between Porcine Respiratory Coronavirus and bacterial lipopolysaccharide in the induction of Respiratory disease in pigs
    Journal of medical microbiology, 2000
    Co-Authors: Kristien Van Reeth, Hans Nauwynck, Maurice Pensaert
    Abstract:

    This study examined whether exposure of pigs to both Porcine Respiratory Coronavirus (PRCV) and bacterial lipopolysaccharide (LPS) can potentiate Respiratory disease and lung secretion of tumour necrosis factor-α (TNF-α) and interleukin-1 (IL-1). Caesarian-derived colostrum-deprived pigs were inoculated intratracheally with PRCV, with LPS from Escherichia coli O111:B4 (20 μg/kg), or with a combination of the two, and killed at set times after inoculation. Clinical signs, virus replication and (histo)pathological changes in the lungs, percentage of neutrophils and bioactive TNF-α and IL-1 in broncho-alveolar lavage (BAL) fluids were examined. The effects of separate virus or LPS inoculations were subclinical and failed to induce high and sustained cytokine levels. In a preliminary study, pigs were inoculated with PRCV and then with LPS 24 h later and killed sequentially. Severe Respiratory disease and significantly enhanced TNF-α titres (208–3601 U/ml versus 40–89 U/ml after LPS only) were seen during the first 12 h after LPS inoculation. IL-1 levels (106–1631 U/ml versus 28–654 U/ml after LPS only) were also increased, but persisted for longer after clinical recovery than TNF-α. In a second study, pigs were inoculated with PRCV and subsequently with LPS at various time intervals ranging from 0 to 24 h, and killed 5 h after inoculation with LPS. A time interval of at least 12 h between inoculations was necessary for prominent Respiratory signs to develop. Production of TNF-α, but not IL-1, was also dependent on the time interval between inoculations and was tightly correlated with disease. Lung neutrophil infiltration and pathological changes were comparable after combined PRCV-LPS and single LPS inoculations, and were not associated with disease. These data show that exposure to high endotoxin concentrations in swine buildings can precipitate Respiratory disease in PRCV-infected pigs, and that TNF-α is probably an important mediator of these effects. This is the first in-vivo demonstration of synergy between Respiratory viruses and LPS.

  • dual infections of feeder pigs with Porcine reproductive and Respiratory syndrome virus followed by Porcine Respiratory Coronavirus or swine influenza virus a clinical and virological study
    Veterinary Microbiology, 1996
    Co-Authors: Kristien Van Reeth, Hans Nauwynck, Maurice Pensaert
    Abstract:

    Abstract Dual infections of pigs with Porcine reproductive and Respiratory syndrome virus (PRRSV) followed by a second common Respiratory virus, either Porcine Respiratory Coronavirus (PRCV) or swine influenza virus (SIV), were studied. The aim was to determine if dual infections, as compared to single virus infections, result in enhanced clinical manifestations. It was also examined if PRRSV replication affects replication of PRCV or SIV in the Respiratory tract. Groups of conventional 10 week old pigs were inoculated with PRRSV-only (3 pigs), PRCV-only (4 pigs) or SIV-only (4 pigs). Dual inoculations with PRRSV-PRCV (4 pigs) and PRRSV-SIV (3 groups of 4, 4 and 5 pigs) were performed at a 3 day interval. A group of uninoculated control pigs (8 pigs) was included. The infection with PRRSV-only induced a transient fever (40.2°C) at 2 DPI, but no Respiratory signs. The PRCV-only infection remained subclinical. The SIV-only infection resulted in a one day fever (40.1°C) with moderate tachypnoea and dyspnoea. Mean weight gain in the virus-inoculated groups was retarded compared with the control group. The PRRSV-PRCV infection induced a 9 day lasting fever (peak 40.9°C) with tachypnoea, dyspnoea and productive coughing. The PRRSV-SIV infection resulted in fever and Respiratory signs in all 3 groups. Clinical signs, however, were more pronounced in group 1 than in groups 2 and 3. Pigs of group 1 showed fever during 10 days (peak 41.4°C), tachypnoea, marked dyspnoea with abdominal breathing, and a productive cough. Pigs of groups 2 and 3 had fever for 5 and 3 days (peaks 40.6 and 40.3°C) respectively and mild Respiratory disorders. Mean weight gain during 14 DPI of the 2nd virus was 5.9 kg in the PRRSV-PRCV group and 4.0, 6.8 and 6.7 kg in PRRSV-SIV groups 1, 2 and 3 respectively. Mean weight gain during the corresponding period in the PRRSV-only group was 8.6 kg. It was concluded that dual infections with viruses causes more severe disease and growth retardation than single PRRSV infection. PRCV excretion curves were similar in single and dual virus inoculated groups. Excretion of SIV was delayed by 2 days in the dual inoculated pigs. Thus, replication of the second virus is not (PRCV) or only slightly (SIV) affected by a prior infection with PRRSV.

  • an adenovirus recombinant expressing the spike glycoprotein of Porcine Respiratory Coronavirus is immunogenic in swine
    Journal of General Virology, 1996
    Co-Authors: Paul Callebaut, Luis Enjuanes, Maurice Pensaert
    Abstract:

    The full-length spike (S) gene of Porcine Respiratory Coronavirus (PRCV) was inserted into the genome of human adenovirus type 5 downstream of the early transcription region 3 promoter. The recombinant virus replicated in cultures of the swine testicle ST cell line and directed the synthesis of S antigen with a maximum yield of approximately 26 µg per 106 cells. The antigen was cell-associated except in the late phase of the infection, when a small amount (3.5 µg per 106 cells) was released. The cell-associated antigen consisted of polypeptides of molecular mass 160 kDa and 175 kDa, comigrating with the authentic precursor S′ and the mature S protein of PRCV, respectively. The extracellular recombinant antigen corresponded to the 175 kDa mature protein. Some recombinant S protein was exposed on the cell surface and was recognized by neutralization-mediating anti-S monoclonal antibodies. Piglets, inoculated oronasally with the recombinant adenovirus vector developed PRCV-neutralizing serum antibodies and were partially protected against PRCV challenge, demonstrating the potential of live adenovirus as vaccine vector.

  • Dual infections of feeder pigs with Porcine reproductive and Respiratory syndrome virus followed by Porcine Respiratory Coronavirus or swine influenza virus: a clinical and virological study.
    Veterinary microbiology, 1996
    Co-Authors: Kristien Van Reeth, Hans Nauwynck, Maurice Pensaert
    Abstract:

    Dual infections of pigs with Porcine reproductive and Respiratory syndrome virus (PRRSV) followed by a second common Respiratory virus, either Porcine Respiratory Coronavirus (PRCV) or swine influenza virus (SIV), were studied. The aim was to determine if dual infections, as compared to single virus infections, result in enhanced clinical manifestations. It was also examined if PRRSV replication affects replication of PRCV or SIV in the Respiratory tract. Groups of conventional 10 week old pigs were inoculated with PRRSV-only (3 pigs), PRCV-only (4 pigs) or SIV-only (4 pigs). Dual inoculations with PRRSV-PRCV (4 pigs) and PRRSV-SIV (3 groups of 4, 4 and 5 pigs) were performed at a 3 day interval. A group of uninoculated control pigs (8 pigs) was included. The infection with PRRSV-only induced a transient fever (40.2 degrees C) at 2 DPI, but no Respiratory signs. The PRCV-only infection remained subclinical. The SIV-only infection resulted in a one day fever (40.1 degrees C) with moderate tachypnoea and dyspnoea. Mean weight gain in the virus-inoculated groups was retarded compared with the control group. The PRRSV-PRCV infection induced a 9 day lasting fever (peak 40.9 degrees C) with tachypnoea, dyspnoea and productive coughing. The PRRSV-SIV infection resulted in fever and Respiratory signs in all 3 groups. Clinical signs, however, were more pronounced in group 1 than in groups 2 and 3. Pigs of group 1 showed fever during 10 days (peak 41.4 degrees C), tachypnoea, marked dyspnoea with abdominal breathing, and a productive cough. Pigs of groups 2 and 3 had fever for 5 and 3 days (peaks 40.6 and 40.3 degrees C) respectively and mild Respiratory disorders. Mean weight gain during 14 DPI of the 2nd virus was 5.9 kg in the PRRSV-PRCV group and 4.0, 6.8 and 6.7 kg in PRRSV-SIV groups 1, 2 and 3 respectively. Mean weight gain during the corresponding period in the PRRSV-only group was 8.6 kg. It was concluded that dual infections with viruses causes more severe disease and growth retardation than single PRRSV infection. PRCV excretion curves were similar in single and dual virus inoculated groups. Excretion of SIV was delayed by 2 days in the dual inoculated pigs. Thus, replication of the second virus is not (PRCV) or only slightly (SIV) affected by a prior infection with PRRSV.

Kwonil Jung - One of the best experts on this subject based on the ideXlab platform.

  • Nitric oxide is elicited and inhibits viral replication in pigs infected with Porcine Respiratory Coronavirus but not Porcine reproductive and Respiratory syndrome virus.
    Veterinary immunology and immunopathology, 2010
    Co-Authors: Kwonil Jung, Gourapura J. Renukaradhya, Ashita Gurnani, Linda J. Saif
    Abstract:

    There is little information on the role of nitric oxide (NO) in innate immunity to Respiratory Coronavirus (CoV) infections. We examined NO levels by Greiss assay in bronchoalveolar lavage (BAL) of pigs infected with either Porcine Respiratory Coronavirus (PRCV) or Porcine reproductive and Respiratory syndrome virus (PRRSV), a member of Nidovirales, like CoV. The antiviral effects of NO on these two viruses were tested in an in vitro system using a NO donor, S-nitroso-N-acetylpenicillamine (SNAP). We detected a large increase in NO levels in BAL fluids of PRCV-infected pigs, but not in PRRSV-infected pigs. Pulmonary epithelial cell necrosis induced by PRCV coincided with increased NO. Moreover, NO levels in cell culture medium of PRRSV-infected alveolar macrophages (AMs) did not differ from that of mock-infected AMs. Antiviral assays showed that NO significantly inhibited PRCV replication in swine testicular (ST) cells, whereas PRRSV was not susceptible to NO based on the conditions tested. Our study suggests that unlike PRRSV which induces apoptosis in AMs, Respiratory CoVs such as PRCV that infect pulmonary epithelial cells and cause cytolysis, induce NO production in the Respiratory tract. Thus, NO may play a role in innate immunity to Respiratory CoV infections by inhibiting viral replication.

  • Porcine reproductive and Respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to Porcine Respiratory Coronavirus in pigs.
    Viral immunology, 2010
    Co-Authors: Gourapura J. Renukaradhya, Kwonil Jung, Konstantin P. Alekseev, Ying Fang, Linda J. Saif
    Abstract:

    We performed a comprehensive analysis of innate and adaptive immune responses in dual-virus infected pigs to understand whether a pre-existing immunomodulatory Respiratory viral infection affects the overall immunity to a subsequent Porcine Respiratory Coronavirus (PRCV) infection in pigs. Pigs were either mock-infected or infected with Porcine reproductive and Respiratory syndrome virus (PRRSV), a virus known to cause immunosuppressive Respiratory disease, and then pigs were co-infected with PRCV, which normally causes subclinical Respiratory infection. We collected samples for six independent experiments from 178 pigs that were also used for pathological studies. We detected a significant reduction in innate NK-cell-mediated cytotoxic function in PRRSV-infected pigs, which was synergistically further decreased in pigs co-infected with PRCV. Subsequently, in association with clinical signs we observed elevated levels of proinflammatory (IL-6), Th-1 (IL-12), and regulatory (IL-10 and TGF-β) cytokines. Increased frequencies of CD4CD8 double-positive T lymphocytes and myeloid cells, in addition to the elevated Th-1 and proinflammatory cytokines in dual-infected pigs, contributed to the severity of lung disease in pigs. The results of our study clarify how each virus modulates the host innate and adaptive immune responses, leading to inflammatory reactions and lung pathology. Thus measurements of cytokines and frequencies of immune cells may serve as indicators of the progression of Respiratory viral co-infections, and provide more definitive approaches for treatment.

  • Porcine reproductive and Respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with Porcine Respiratory Coronavirus: implications for Respiratory viral co-infections
    Journal of General Virology, 2009
    Co-Authors: Kwonil Jung, Konstantin P. Alekseev, Gourapura J. Renukaradhya, Ying Fang, Yuxin Tang, Linda J. Saif
    Abstract:

    The innate immune response is critical for host defence against Respiratory Coronaviruses (CoVs). This study demonstrated that an ongoing Respiratory virus infection compromises innate immune responses and affects the pathogenesis of a Respiratory CoV co-infection. An innate immunosuppressive Respiratory virus infection was established by infecting weaned pigs with Porcine reproductive and Respiratory syndrome virus (PRRSV); 10 days later, the pigs were exposed to Porcine Respiratory Coronavirus (PRCV). The PRRSV/PRCV dual-infected pigs had reduced weight gains, a higher incidence of fever and more severe pneumonia compared with either single infection. Significant suppression of innate immune responses [reduced alpha interferon (IFN-α) levels in the lungs and reduced blood natural killer cell cytotoxicity] by the ongoing PRRSV infection was observed in dual-infected pigs, which coincided with exacerbated pneumonia during early PRCV infection. The subsequent PRCV infection led to enhanced PRRSV replication in the lungs and a trend towards increased serum T-helper type 1 (Th1) (IFN-γ) but decreased Th2 [interleukin (IL)-4] responses, further exacerbating PRRSV pneumonia. Following PRCV infection, more severe PRRSV-related pulmonary alveolar macrophage (PAM) apoptosis occurred, as determined by an in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay, suggesting increased PRRSV replication in PAMs. Collectively, these observations suggest interactive effects between PRCV and PRRSV via early innate (IFN-α) and later adaptive Th1 (IFN-γ) and Th2 (IL-4) immune responses. These findings imply that an existing immunomodulating Respiratory viral co-infection may be a contributing factor to more severe pneumonia in Respiratory CoV disease. This study provides new insights into host–pathogen interactions related to co-infection by CoVs and other Respiratory viruses.

  • Cytokine Responses in Porcine Respiratory Coronavirus-Infected Pigs Treated with Corticosteroids as a Model for Severe Acute Respiratory Syndrome
    Journal of virology, 2008
    Co-Authors: Xinsheng Zhang, Konstantin P. Alekseev, Kwonil Jung, Anastasia N. Vlasova, Nagesh Hadya, Linda J. Saif
    Abstract:

    The effectiveness and potential immunosuppressive effects of anti-inflammatory glucocorticoids in the lungs of severe acute Respiratory syndrome (SARS) patients are undefined. We treated Porcine Respiratory Coronavirus (PRCV)-infected conventional pigs with the corticosteroid dexamethasone (DEX) as a model for SARS. Innate and Th1 cytokines in bronchoalveolar lavage (BAL) and serum were elevated in PRCV-infected pigs compared to controls, but were decreased after DEX treatment in the PRCV-infected, DEX-treated (PRCV/DEX) pigs. Although decreased in BAL, Th2 cytokine levels were higher in serum after DEX treatment. Levels of the proinflammatory cytokine interleukin-6 in BAL and serum were decreased in PRCV/DEX pigs early but increased later compared to those in phosphate-buffered saline-treated, PRCV-infected pigs, corresponding to a similar trend for lung lesions. PRCV infection increased T-cell frequencies in BAL, but DEX treatment of PRCV-infected pigs reduced frequencies of T cells; interestingly B and SWC3a(+) (monocytes/macrophages/granulocytes) cell frequencies were increased. DEX reduced numbers of PRCV-stimulated Th1 gamma interferon-secreting cells in spleen, tracheobroncheolar lymph nodes, and blood. Our findings suggest that future glucocorticoid treatment of SARS patients should be reconsidered in the context of potential local immunosuppression of immune responses in lung and systemic Th1 cytokine-biased suppression.

  • altered pathogenesis of Porcine Respiratory Coronavirus in pigs due to immunosuppressive effects of dexamethasone implications for corticosteroid use in treatment of severe acute Respiratory syndrome Coronavirus
    Journal of Virology, 2007
    Co-Authors: Kwonil Jung, Xinsheng Zhang, Konstantin P. Alekseev, Anastasia N. Vlasova, Doosung Cheon, Linda J. Saif
    Abstract:

    The pathogenesis and optimal treatments for severe acute Respiratory syndrome (SARS) are unclear, although corticosteroids were used to reduce lung and systemic inflammation. Because the pulmonary pathology of Porcine Respiratory Coronavirus (PRCV) in pigs resembles SARS, we used PRCV as a model to clarify the effects of the corticosteroid dexamethasone (DEX) on Coronavirus (CoV)-induced pneumonia. Conventional weaned pigs (n = 130) in one of four groups (PRCV/phosphate-buffered saline [PBS] [n = 41], PRCV/DEX [n = 41], mock/PBS [n = 23], and mock/DEX [n = 25]) were inoculated intranasally and intratracheally with the ISU-1 strain of PRCV (1 x 10(7) PFU) or cell culture medium. DEX was administered (once daily, 2 mg/kg of body weight/day, intramuscularly) from postinoculation day (PID) 1 to 6. In PRCV/DEX pigs, significantly milder pneumonia, fewer PRCV-positive cells, and lower viral RNA titers were present in lungs early at PID 2; however, at PID 4, 10, and 21, severe bronchointerstitial pneumonia, significantly higher numbers of PRCV-positive cells, and higher viral RNA titers were observed compared to results for PRCV/PBS pigs. Significantly lower numbers of CD2(+), CD3(+), CD4(+), and CD8(+) T cells were also observed in lungs of PRCV/DEX pigs than in those of PRCV/PBS pigs at PID 8 and 10, coincident with fewer gamma interferon (IFN-gamma)-secreting cells in the tracheobronchial lymph nodes as determined by enzyme-linked immunospot assay. Our results confirm that DEX treatment alleviates PRCV pneumonia early (PID 2) in the infection but continued use through PID 6 exacerbates later stages of infection (PID 4, 10, and 21), possibly by decreasing cellular immune responses in the lungs (IFN-gamma-secreting T cells), thereby creating an environment for more-extensive viral replication. These data have potential implications for corticosteroid use with SARS-CoV patients and suggest a precaution against prolonged use based on their unproven efficacy in humans, including possible detrimental secondary effects.

Prem S. Paul - One of the best experts on this subject based on the ideXlab platform.

  • Pathogenicity of three isolates of Porcine Respiratory Coronavirus in the USA.
    The Veterinary record, 2003
    Co-Authors: P. G. Halbur, Francisco J. Pallarés, Tanja Opriessnig, E. M. Vaughn, Prem S. Paul
    Abstract:

    The pathogenicity of three isolates of Porcine Respiratory Coronavirus (AR310, LEPP and 1894) from the USA was assessed in specific pathogen-free pigs. Pigs inoculated with 1894 developed mild Respiratory disease and pigs inoculated with AR31o and LEPP developed moderate Respiratory disease from four to 10 days after they were inoculated, but all the pigs recovered fully by 14 days after inoculation. Gross and microscopic examination revealed mild (1894) to moderate (AR31o and LEPP) multifocal bronchointerstitial pneumonia from four to 10 days after inoculation. The lesions were characterised by necrotising bronchiolitis, septal infiltration with mononuclear cells, and a mixed alveolar exudate. No clinical signs or microscopic lesions were observed in control pigs that had not been inoculated.

  • An Overview of Immunological and Genetic Methods for Detecting Swine Coronaviruses, Transmissible Gastroenteritis Virus, and Porcine Respiratory Coronavirus in Tissues
    Advances in experimental medicine and biology, 1997
    Co-Authors: Theerapol Sirinarumitr, Patrick G. Halbur, Prem S. Paul, John P. Kluge
    Abstract:

    Transmissible gastroenteritis (TGE) is an enteric disease of swine caused by a Coronavirus, designated as transmissible gastroenteritis virus (TGEV). Commonly used methods for TGEV detection include viral isolation and detection of the viral antigen by indirect immunofluorescence (IFA), immunoperoxidase, and immunogold silver staining. Each of these techniques has some advantages and disadvantages. In general IFA and immunohistochemistry are preferred over viral isolation as TGEV isolation is not very reliable because not all field isolates replicate in cell cultures. The diagnosis of TGEV has become more complicated since the emergence of Porcine Respiratory Coronavirus (PRCV). PRCV is believed to be a TGEV mutant, and can not be easily differentiated from TGEV by immunological tests. Nucleic acid probes and polymerase chain reaction (PCR) have successfully been used to detect and differentiate these viruses. These techniques can detect viral nucleic acids in the specimen but do not provide information on the cell types infected by these viruses. Recently we have developed isotopic and nonisotopic in situ hybridization techniques (ISH) for the detection of these viral nucleic acids in formalin-fixed paraffin-embedded tissues. Furthermore, this procedure can differentiate between TGEV-and PRCV-infected cells. By ISH, TGEV is detected in the mature absorptive enterocytes of tissues infected by TGEV and the crypt epithelial cells are also infected but to a lesser extent. For PRCV, the main infected cells are epithelial cells of the bronchioles, type II pneumocytes, and alveolar and septal macrophages. ISH is an excellent tool for studying molecular pathogenesis of these two viruses especially when used in combination with immunohistochemistry.

  • in situ hybridization technique for the detection of swine enteric and Respiratory Coronaviruses transmissible gastroenteritis virus tgev and Porcine Respiratory Coronavirus prcv in formalin fixed paraffin embedded tissues
    Journal of Virological Methods, 1996
    Co-Authors: Theerapol Sirinarumitr, Prem S. Paul, John P. Kluge, Patrick G. Halbur
    Abstract:

    The in situ hybridization (ISH) technique was developed to detect the swine Coronaviruses, transmissible gastroenteritis virus (TGEV) and Porcine Respiratory Coronavirus (PRCV), in cell culture and tissue sections from TGEV-or PRCV-infected pigs. The 35S-labeled RNA probes were generated from two plasmids pPSP.FP1 and pPSP.FP2 containing part of the S gene of TGEV. The procedure was first standardized in cell cultures. The radiolabeled pPSP.FP2 probe detected both TGEV and PRCV in virus-inoculated cell cultures, whereas pPSP.FP1 probe detected TGEV but not PRCV. The probe was then used to detect TGEV or PRCV in tissues of pigs experimentally infected with TGEV or PRCV or naturally infected with TGEV. Again, the probes detected TGEV in intestines of experimentally and naturally infected pigs and PRCV in the lungs of experimentally infected pigs. TGEV RNA was detected mainly within the enterocytes at the tips of villi and, less often, within some crypt epithelial cells. PRCV was shown to replicate mainly in the bronchiolar epithelial cells and in lesser amount in type II pneumocytes, type I pneumocytes, alveolar macrophages and bronchial epithelial cells, respectively. ISH has potential applications as a diagnostic test for the detection and differentiation of TGEV and PRCV in tissues and in studies to gain a better understanding of the mechanism of pathogenesis of enteric and Respiratory Coronavirus infections.

  • Use of nonradioactive cDNA probes to differentiate Porcine Respiratory Coronavirus and transmissible gastroenteritis virus isolates
    Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians Inc, 1996
    Co-Authors: Eric M. Vaughn, Patrick G. Halbur, Prem S. Paul
    Abstract:

    Porcine Respiratory Coronavirus (PRCV), a member of the family Coronaviridae, is antigenically related to transmissible gastroenteritis virus (TGEV) of swine. PRCV, now thought to be a mutant of TGEV, was first isolated in 1984 from pigs in Belgium that were seropositive for TGEV but did not have a history of clinical transmissible gastroenteritis (TGE). Since the initial isolation of PRCV, it has been found that infections of swine in Europe with PRCV are widespread. PRCV has also been isolated in swine in the United States, 7,11,20,24 but its prevalence in herds within the United States is not known. There are several similarities between PRCV and TGEV. Both viruses have 3 major structural proteins: the surface spike (S) glycoprotein, the integral membrane glycoprotein, and an internal nucleoprotein. Nucleotide sequences of PRCV isolates thus far studied show that they are closely related to TGEV but that there are some striking differences. PRCV isolates have a characteristic deletion in the 5' end of the S gene when compared to TGEV, and PRCV has a different tissue tropism than TGEV. TGEV replicates in both the Respiratory and intestinal tissues and causes gastroenteritis, 14 whereas PRCV replicates to high titers in lung tissue of swine and with little or no replication in the intestinal tissues and no evidence of gastroenteritis and villous atrophy. PRCV is antigenically related to TGEV in that polyclonal sera which neutralize TGEV also neutralize PRCV. Thus, conventional serologic methods are not useful in determining if a swine herd with anti-TGEV antibodies has been infected with PRCV or TGEV. Anti-TGEV neutralizing monoclonal antibodies (MAbs) directed against the S glycoprotein readily neutralize PRCV; however, there are some nonneutralizing anti-TGEV MAbs directed against the S glycoprotein that can be used to distinguish between PRCV and TGEV isolates in a competitive binding assay. Of the European PRCV isolates that have had their nucleotide sequences published, all have a 672-nucleotide deletion in the 5' end of the S gene. The US PRCV isolates Ind/89 and ISU-1 have a 681-nucleotide deletion present in the 5' end of the S gene. Recently the PRCV isolates AR310 and LEPP have been shown to have a smaller S gene deletion of 621 nucleotides present. Additionally, the PRCV isolate IA1894 has recently been shown to have a 678-nucleotide deletion in the 5' end of its S gene. Hence, a cDNA probe that encompasses the region of the TGEV S gene that is characteristically deleted from PRCV isolates can be used

  • Sequence comparison of Porcine Respiratory Coronavirus isolates reveals heterogeneity in the S, 3, and 3-1 genes.
    Journal of virology, 1995
    Co-Authors: Eric M. Vaughn, Patrick G. Halbur, Prem S. Paul
    Abstract:

    Four new Porcine Respiratory Coronavirus (PRCV) isolates were genetically characterized. Subgenomic mRNA patterns and the nucleotide sequences of the 5' ends of the S genes, the open reading frame (ORF) 3/3a genes, and the ORF 3-1/3b genes of these PRCV isolates were determined and compared with those of other PRCV and transmissible gastroenteritis virus (TGEV) isolates. The S, ORF 3/3a, and ORF 3-1/3b genes are under intense study because of their possible roles in determining tissue tropism and virulence. Northern (RNA) blot analysis of subgenomic mRNAs revealed that mRNA 2, which encodes for the S gene, of the PRCV isolates migrated faster than the mRNA 2 of TGEV. The PRCV isolates AR310 and LEPP produced eight subgenomic mRNA species, the same number as produced by the virulent Miller strain of TGEV. However, the PRCV isolates IA1894 and ISU-1 produced only seven subgenomic mRNA species. All four of the PRCV isolates were found to have a large in-frame deletion in the 5' end of the S gene; however, the size and location of the deletion varied. Analysis of the ORF 3/3a gene nucleotide sequences from the four PRCV isolates also showed a high degree of variability in this area. The ORF 3 gene of the PRCV isolates AR310 and LEPP was preceded by a CTAAAC leader RNA-binding site, and the ORF 3 gene was predicted to yield a protein of 72 amino acids, the same size as that of the virulent Miller strain of TGEV. The PRCV isolates AR310 and LEPP are the first PRCV isolates found to have an intact ORF 3 gene. The ORF 3a gene of the PRCV isolate IA1894 was preceded by a CTAAAC leader RNA-binding site and was predicted to yield a truncated protein of 54 amino acids due to a 23-nucleotide deletion. The CTAAAC leader RNA-binding site and ATG start codon of ORF 3 gene of the PRCV isolate ISU-1 were removed because of a 168-nucleotide deletion. Analysis of the ORF 3-1/3b gene nucleotide sequences from the four PRCV nucleotides isolates also showed variability.

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  • Cytokine Responses in Porcine Respiratory Coronavirus-Infected Pigs Treated with Corticosteroids as a Model for Severe Acute Respiratory Syndrome
    Journal of virology, 2008
    Co-Authors: Xinsheng Zhang, Konstantin P. Alekseev, Kwonil Jung, Anastasia N. Vlasova, Nagesh Hadya, Linda J. Saif
    Abstract:

    The effectiveness and potential immunosuppressive effects of anti-inflammatory glucocorticoids in the lungs of severe acute Respiratory syndrome (SARS) patients are undefined. We treated Porcine Respiratory Coronavirus (PRCV)-infected conventional pigs with the corticosteroid dexamethasone (DEX) as a model for SARS. Innate and Th1 cytokines in bronchoalveolar lavage (BAL) and serum were elevated in PRCV-infected pigs compared to controls, but were decreased after DEX treatment in the PRCV-infected, DEX-treated (PRCV/DEX) pigs. Although decreased in BAL, Th2 cytokine levels were higher in serum after DEX treatment. Levels of the proinflammatory cytokine interleukin-6 in BAL and serum were decreased in PRCV/DEX pigs early but increased later compared to those in phosphate-buffered saline-treated, PRCV-infected pigs, corresponding to a similar trend for lung lesions. PRCV infection increased T-cell frequencies in BAL, but DEX treatment of PRCV-infected pigs reduced frequencies of T cells; interestingly B and SWC3a(+) (monocytes/macrophages/granulocytes) cell frequencies were increased. DEX reduced numbers of PRCV-stimulated Th1 gamma interferon-secreting cells in spleen, tracheobroncheolar lymph nodes, and blood. Our findings suggest that future glucocorticoid treatment of SARS patients should be reconsidered in the context of potential local immunosuppression of immune responses in lung and systemic Th1 cytokine-biased suppression.

  • altered pathogenesis of Porcine Respiratory Coronavirus in pigs due to immunosuppressive effects of dexamethasone implications for corticosteroid use in treatment of severe acute Respiratory syndrome Coronavirus
    Journal of Virology, 2007
    Co-Authors: Kwonil Jung, Xinsheng Zhang, Konstantin P. Alekseev, Anastasia N. Vlasova, Doosung Cheon, Linda J. Saif
    Abstract:

    The pathogenesis and optimal treatments for severe acute Respiratory syndrome (SARS) are unclear, although corticosteroids were used to reduce lung and systemic inflammation. Because the pulmonary pathology of Porcine Respiratory Coronavirus (PRCV) in pigs resembles SARS, we used PRCV as a model to clarify the effects of the corticosteroid dexamethasone (DEX) on Coronavirus (CoV)-induced pneumonia. Conventional weaned pigs (n = 130) in one of four groups (PRCV/phosphate-buffered saline [PBS] [n = 41], PRCV/DEX [n = 41], mock/PBS [n = 23], and mock/DEX [n = 25]) were inoculated intranasally and intratracheally with the ISU-1 strain of PRCV (1 x 10(7) PFU) or cell culture medium. DEX was administered (once daily, 2 mg/kg of body weight/day, intramuscularly) from postinoculation day (PID) 1 to 6. In PRCV/DEX pigs, significantly milder pneumonia, fewer PRCV-positive cells, and lower viral RNA titers were present in lungs early at PID 2; however, at PID 4, 10, and 21, severe bronchointerstitial pneumonia, significantly higher numbers of PRCV-positive cells, and higher viral RNA titers were observed compared to results for PRCV/PBS pigs. Significantly lower numbers of CD2(+), CD3(+), CD4(+), and CD8(+) T cells were also observed in lungs of PRCV/DEX pigs than in those of PRCV/PBS pigs at PID 8 and 10, coincident with fewer gamma interferon (IFN-gamma)-secreting cells in the tracheobronchial lymph nodes as determined by enzyme-linked immunospot assay. Our results confirm that DEX treatment alleviates PRCV pneumonia early (PID 2) in the infection but continued use through PID 6 exacerbates later stages of infection (PID 4, 10, and 21), possibly by decreasing cellular immune responses in the lungs (IFN-gamma-secreting T cells), thereby creating an environment for more-extensive viral replication. These data have potential implications for corticosteroid use with SARS-CoV patients and suggest a precaution against prolonged use based on their unproven efficacy in humans, including possible detrimental secondary effects.

  • complete genomic sequences a key residue in the spike protein and deletions in nonstructural protein 3b of us strains of the virulent and attenuated Coronaviruses transmissible gastroenteritis virus and Porcine Respiratory Coronavirus
    Virology, 2007
    Co-Authors: Xinsheng Zhang, Nagesh Hadya, Yuxin Tang, Mustafa Hasoksuz, David J Spiro, Rebecca A Halpin, Shiliang Wang, Sarah Stollar, Daniel Janies, Elodie Ghedin
    Abstract:

    Transmissible gastroenteritis virus (TGEV) isolates that have been adapted to passage in cell culture maintain their infectivity in vitro but may lose their pathogenicity in vivo. To better understand the genomic mechanisms for viral attenuation, we sequenced the complete genomes of two virulent TGEV strains and their attenuated counterparts: virulent TGEV Miller M6 and attenuated TGEV Miller M60 and virulent TGEV Purdue and attenuated TGEV Purdue P115, together with the ISU-1 strain of Porcine Respiratory Coronavirus (PRCV-ISU-1), a naturally occurring TGEV deletion mutant with an altered Respiratory tropism and reduced virulence. Pairwise comparison at both the nucleotide (nt) and amino acid (aa) levels between virulent and attenuated TGEV strains identified a common change in nt 1753 of the spike gene, resulting in a serine to alanine mutation at aa position 585 of the spike proteins of the attenuated TGEV strains. Alanine was also present in this protein in PRCV-ISU-1. Particularly noteworthy, the serine to alanine mutation resides in the region of the major antigenic site A/B (aa 506–706) that elicits neutralizing antibodies and within the domain mediating the cell surface receptor aminopeptidase N binding (aa 522–744). Comparison of the predicted polypeptide products of ORF3b showed significant deletions in the naturally attenuated PRCV-ISU-1 and TGEV Miller M60; these deletions occurred at a common break point, suggesting a related mechanism of recombination that may affect viral virulence or tropism. Sequence comparisons at both genomic and protein levels indicated that PRCV-ISU-1 had a closer relationship with TGEV Miller strains than Purdue strains. Phylogenetic analyses showed that virulence is an evolutionarily labile trait in TGEV and that TGEV strains as a group share a common ancestor with PRCV.