Browsing by Author "Czub, Markus"
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- ItemOpen AccessCharacterization of Neutralizing and Non-neutralizing Epitopes of Porcine Circovirus 2 Capsid Protein(2015-07-09) Nourozieh, Narges; Czub, MarkusVaccination is the most efficacious way to prevent Porcine circovirus 2 (PCV2)-associated diseases. In experimental pig studies, vaccine-induced neutralizing antibodies (NAs) appear to play a major role in protection from PCV2 infection. The immune response to PCV2 vaccination of farmed pigs has not been studied in detail. I hypothesize that NAs target conformational epitope(s) present on the surface of PCV2 particles. Highly purified PCV2 particles were found to expose only conformational but not linear epitopes on their surface. The screening 160 sera from farmed pigs showed that reactivity of sera with PCV2 particles correlated positively with the level of NA titer, suggesting that NAs recognize surface-exposed conformational epitope(s). This finding was supported by depleting antibodies reacting with linear epitopes of PCV2. No significant change was observed in the level of NA titer after antibody depletion. Altogether, these data suggest that NAs target conformational epitope(s) on the surface of PCV2 particles.
- ItemOpen AccessCharacterizing the pro-inflammatory cytokine response by dendritic cells upon exposure to PrP proteins(2013-09-24) Ma, Roger; Czub, MarkusDendritic cell (DC) interaction with infectious prions (PrPSc) represents an important part of prion pathogenesis. Previous studies have shown that DCs are likely among the first immune cells to interact with PrPSc after oral exposure. This study looks at the initial exposure of PrPSc on DC activation by measuring TNFα and IL-12 production. There was no significant production of TNFα or IL-12 by bone marrow derived DCs (BMDC) after exposure to PrPSc infected brain homogenate, however, BMDCs had lower capacity to produce IL-12 after secondary stimulation with LPS. Semi-purified PrPSc also did not activate BMDCs, however, the recombinant MoPrP 23-231 and MoPrP 90-231 elicited TNFα and IL-12 production. These results may not be surprising since PrPSc closely resembles cellular PrPC and therefore, DCs recognized PrP in brain homogenates as self-antigens. However, MoPrP 23-231 and MoPrP 90-231 were recognized as foreign antigens since the proteins were produced in bacteria.
- ItemOpen AccessComparative innate responses induced by Toll-like receptor (TLR)7 and 21 ligands against infectious bronchitis virus infection(2019-01-26) De Silva Senapathi, Yaseshwari Upasama; Abdul-Careem, Mohamed Faizal; Czub, Markus; Van Marle, GuidoToll-like receptor (TLR)7 and 21 ligands, resiquimod and cytosine-guanosine (CpG) oligonucleotides (ODNs) respectively have been used in ovo (pre-hatch) to enhance or prime an early immune response in chickens to provide protection against microbial pathogens. Yet, their protective efficacy against an infectious bronchitis virus (IBV) infection encountered post-hatch has not been evaluated. Thus, our objectives were to investigate the efficacy of resiquimod and CpG ODNs against a post-hatch IBV infection by delivering the ligands in ovo at embryo day (ED)18 and then, to determine possible mechanisms of protection. We found upregulation of interleukin (IL)-1β and interferon (IFN)-γ mRNA levels and considerable expansions of macrophage and cluster of differentiation (CD)8α+ T cell populations in lungs of chicken as early as day one post-hatch, following pre-hatch delivery of resiquimod. When the resiquimod pre-treated day-old chickens were infected with IBV, reduction in viral shedding via oral and fecal routes was observed at 3 days post-infection (dpi). Similarly, in CpG ODN pre-treated birds at 3 dpi, we found increased recruitment of macrophages, CD8α+ and CD4+ T lymphocytes in addition to up-regulation of IFN-γ and IL-1β mRNA concentrations in chicken lungs. However, only in ovo delivered CpG ODNs significantly reduced the morbidity and mortality associated with IBV infection. Overall, these studies bring us closer to understanding mechanisms behind CpG ODNs and resiquimod induced immune responses in chickens when used as stand-alone prophylactic agents in ovo.
- ItemOpen AccessCytotoxicity of orf3 proteins from porcine circoviruses(2010) Chaiyakul, Mark; Czub, Markus
- ItemOpen AccessDetection of Antibodies Against the Open Reading Frame Three Protein of Porcine Circovirus 2 in Pigs(2014-07-10) Hawkes, Aaron; Czub, MarkusThe swine pathogen porcine circovirus 2 (PCV2) is the primary etiological agent of a syndrome that is damaging to the swine industry. The PCV2 genome contains three major open reading frames (ORFs) and ORF3 appears to play a role in PCV2-related pathology and dissemination. No data have been published confirming that PCV2ORF3 protein is expressed in PCV2-infected swine. Here it is hypothesized that αPCV2ORF3 antibodies are present in the sera of most adult swine. Swine sera were screened for αPCV2ORF3 antibodies, the presence of which would imply PCV2ORF3 expression. About 90% of the tested sera from weaned swine appear to have at least some reactivity to rPCV2ORF3, implying that PCV2ORF3 is expressed in vivo. Serum reactivity to PCV2ORF3 did not correlate with PCV2 viral load or vaccination status. These results do not indicate or rule out any particular biological role for PCV2ORF3.
- ItemOpen AccessHepatitis B Virus (HBV) Infection in Peripheral Blood Mononuclear Cells of HBV Mono-infected and HBV/Human Immunodeficiency Virus Type-1 Co-infected Patients(2014-07-17) Lee, Zengina; Coffin, Carla; Czub, MarkusIt is unknown whether HIV-1 co-infection impacts HBV lymphotropism. We hypothesize that concomitant HIV-1 infection will affect HBV detection in PBMC subsets. We compared HBV genome detection in whole PBMC and CD4+ T, CD8+ T, CD14+ monocyte, CD19+ B and CD56+ NK cell subsets isolated from 14 HBV mono-infected (4/14 with a second sample collected after starting antivirals) and 6 HBV/HIV-1 co-infected patients on antivirals using nested PCR/nucleic hybridization and/or quantitative PCR assays. HBV DNA was detected in most target PBMC subsets regardless of HIV-1 co-infection and antiviral treatment; with the exception of the CD4+ T cell subset from HBV/HIV-1 + patients. All whole PBMC analyzed (13/13 HBV treatment naïve mono-infected, 4/4 follow-up cases on antivirals, and 3/3 HBV/HIV-1 co-infected) were HBV genome positive. The data suggests that HBV infection in CD4+ T cells is affected by concomitant HIV-1 infection.
- ItemOpen AccessHost responses in laying hens following infectious bronchitis vaccination: Comparison of two vaccination strategies(2021-06-03) Buharideen, Sabrina Marsha; Abdul Careem, Mohamed Faizal; Czub, Markus; Niu, Dongyan; Gedamu, LashitewThe infectious bronchitis virus (IBV) causes infectious bronchitis (IB) and causes nephritis and reproductive tract abnormalities depending on the infecting IBV strain. Vaccination for the control of IB has been practiced for decades. Although it has been shown that administration of inactivated vaccine following priming with live attenuated vaccines in pullets induces protection of laying hens against IB, the immunological basis of this protective response has not been investigated adequately. The first objective was to inactivate and formulate the IBV Massachusetts (Mass) variant that was isolated from a flock with shell-less egg syndrome (SES) as an in-house adjuvanted vaccine and test whether it induces an antibody-mediated immune response. Although we observed that the in-house adjuvanted inactivated IBV Mass variant vaccine induces a positive antibody-mediated immune response, it does not induce an antibody-mediated immune response similar to that of a commercial inactivated IBV Mass vaccine. The second objective of the study was to compare two vaccination strategies adopted by the Canadian poultry industry in terms of their ability to induce an adequate immune response in the IBV-impacted tissues in laying hens. Vaccination strategy 1 (multiple live attenuated vaccines) and vaccination strategy 2 (combination of live attenuated vaccines given multiple times and one inactivated vaccine) were given to pullets between 3 and 16 weeks of age. Serum anti-IBV antibodies, recruitment of T cell subsets, and interferon (IFN)-γ mRNA expression were measured at 10 weeks post-last vaccination, in selected tissues. We observed that vaccination strategy 2 induced higher serum anti-IBV antibody response and IFN-γ mRNA expression in the lungs, kidneys and reproductive tract. Both vaccination strategies 1 and 2 recruited CD4+ T cells in the lungs and isthmus and CD8+ T cells in all the examined tissues except the uterus. Serum collected from chickens vaccinated with vaccination strategy 2 was able to neutralize the IBV Mass variant that caused SES in Western Canadian layers, indicating the potential ability of vaccination strategy 2 to protect laying hens against this IBV variant. Overall, our findings indicate that administration of live attenuated vaccines followed by an inactivated vaccine, induces better host responses in laying hens.
- ItemOpen AccessInduction of Antiviral Response Against Avian Infectious Laryngotracheitis Virus Infection(2015-06-09) Thapa, Simrika; Careem, Faizal; Czub, MarkusToll-like receptors (TLRs) recognize pathogen associated molecular patterns (PAMPs). The PAMPs that act as ligands for TLRs prompt downstream signalling leading to antimicrobial effects. However, the details of antiviral responses of lipotechoic acid (LTA) and CpG DNA, which act as ligands for TLR-2 and -21 respectively, elicited against avian viruses are scarce. We investigated whether in ovo delivery of LTA and CpG DNA induces antiviral responses against infectious laryngotracheitis virus (ILTV) infection in chickens. We found that in ovo delivery of these two ligands reduces ILTV infections in lungs pre- and post-hatch. However, only CpG DNA could reduce mortality and morbidity due to ILTV infection encountered post-hatch. The expression of IL-1β mRNA and increase of macrophage numbers in lungs were found to be correlates of observed antiviral responses. Thus, LTA and CpG DNA can be candidate TLR ligands worthy of further investigation for the control of ILTV infection in chickens.
- ItemOpen AccessInteraction of Porcine circovirus 2 with the Swine Immune System(2017) Solis Worsfold, Cristina Marie; Czub, Markus; Yates, Robin; Jirik, Frank; Gilch, SabinePorcine circovirus 2 (PCV2) is a virus with a single-stranded, DNA circular genome that is ubiquitous in pig populations worldwide. PCV2 is the causative agent of the post-weaning multisystemic wasting syndrome (PMWS), a multifactorial disease that affects six to twelve-week-old pigs, and which is characterized by weight loss and immunosuppression. PCV2 vaccination has diminished the presentation of PMWS in the field, although PCV2 infection is not prevented. PCV2 infects lymphocytes and it depends on the host enzymes to replicate. Enhanced PCV2 infection rates are associated with increased mitotic activity, although the effect of PCV2 replication on lymphoid cell function is unknown. The main goal of this thesis was to understand the interactions of PCV2 with the swine immune system, by determining the effect of PCV2 on the antibody response in pigs under field conditions, and studying the impact of the primary PCV2 infection on lymphocyte activation, proliferation, and viability. As shown in Chapter 2, a PCV2 persistent infection was detected in farmed pigs of all age groups. Furthermore, a great variability in their neutralizing antibody titers was observed, regardless of their vaccination status. Chapter 3 provides details about the establishment of an in vitro cell model to study the effect of primary PCV2 infection on the immune cells, by using snatched-farrowed, porcine colostrum deprived (SF-pCD) PCV2-free pigs as blood donors of PCV2-naïve peripheral blood mononuclear cells (PBMCs). These cells were exposed to the polyclonal mitogens ionomycin/PMA and PCV2 infection. As shown in chapters 4 & 5, enhanced PCV2 infection rates in ionomycin/PMA-stimulated PBMCs, decreased proliferation in PCV2 infected cells, and high bystander cell death rates in PCV2-exposed PBMCs were observed. This thesis contributes to the current knowledge on PCV2 immunology by bringing insight into factors that contribute to viral pathogenesis and immune modulation and emphasizes the need of developing new vaccines that prevent PCV2 infection.
- ItemOpen AccessInvestigation into Equine Papillomavirus Type 2 Prevalence and Diagnosis(2018-09-28) Wachoski-Dark, Garrett Louis; Knight, Cameron G.; Klein, C.; Czub, Markus; Munday, John S.Equine papillomavirus type 2 (EcPV-2) has been consistently associated with most equine genital cancers and a subset of cancers at other locations. The prevalence of EcPV-2 in healthy horses has been estimated to be <20% for DNA presence, and 10-36% for anti-EcPV-2-L1 seroprevalence. However, no study has thus far examined the prevalence of EcPV-2 at the herd-level or over multiple timepoints. This thesis examined EcPV-2 prevalence in a predominantly closed herd with a known EcPV-2 infected horse over three timepoints. We found that DNA prevalence (0-4%) was equivalent to the general horse population, but that seroprevalence (51.5%) was higher. Additionally, no study has examined the distribution of EcPV-2 infection in situ, nor compared such a technique to PCR. We found that RNA ISH concorded with PCR in 80% of cases. We also recapitulated previous results concerning EcPV-2 distribution in situ as well as found EcPV-2 in numerous novel locations.
- ItemOpen AccessLytic Reactivation of Porcine Lymphotropic Herpesvirus 3(University of Calgary, 2018-09-24) Luu, Gia; Hundt, Jana; Rowell, Jared; Czub, MarkusEpstein-Barr virus (EBV) has infected more 90% of the world’s population, and is the cause of 2% of all neoplasms globally. In pigs, a closely related gammaherpes virus was identified called Porcine Lymphotrophic Herpesvirus 3 (PLHV3), that also causes lymphoproliferative disorders that resemble those caused by EBV. The purpose of this research is to generate a high titer viral stock of PLHV3 by shifting the viral life cycle from latency to lytic replication using baculovirus expression systems (BEVS) in latently infected cell lines. Infected lymphoblastic cell lines (LCL) will be infected with baculovirus vector carrying two PLHV3 immediate early genes that are crucial for lytic reactivation, BZLF1 and BRLF1. These immediate early genes will become expressed after the infection to produce recombinant proteins in high quantity, thus allowing viral reactivation. BZLF1, BRLF1 and an appropriate mammalian promoter will be cloned into the vector using PCR cloning. Acquiring a high titer viral stock will allow the generation of a chimeric PLHV3-EBV virus that could be used to establish a porcine model for studying EBV.
- ItemOpen AccessPorcine Circovirus Cap-Induced Apoptosis of Non-Infected PK15 Cells(2019-07-16) Rowell, Jared S.; Czub, Markus; Zheng, Xilong; Jirik, Frank Robert; Knight, Cameron G.; Corcoran, Jennifer A.Porcine Circovirus 2 (PCV2) is a pathogen of major importance for swine production around the world. Despite development of several vaccines and robust vaccination programs, PCV2 continues to persistently transmit within and between swine herds causing infections marked by immunosuppression via lymphocyte depletion. This study aims to improve understanding of porcine circovirus diseases (PCVD) pathogenesis caused by PCV capsid cytotoxicity which activates apoptosis in non-infected bystander cells. Putatively non-pathogenic Porcine Circovirus 1 (PCV1) was also included to help understand why there is a difference in pathogenicity between PCV1 and PCV2. Porcine Circovirus 3 (PCV3) has recently emerged worldwide as a possible etiological agent of clinical syndromes observed in swine that are similar to PCVD caused by PCV2. The capsid protein of PCV3 (PCV 3 Cap) was also included in this study to provide initial information regarding the cytotoxicity of this protein. Results were obtained through flow cytometric analysis of apoptosis using Annexin V-FITC and 7-AAD to measure apoptosis markers and cell death respectively. Flow cytometry results were reinforced by also utilizing a TUNEL assay in conjunction with confocal imaging to detect cells undergoing DNA fragmentation leading to apoptosis. Results show that monomeric PCV2 Cap, 1% formaldehyde inactivated and dialyzed PCV2, PCV1 virus-like particles (VLPs), PCV2 VLPs, and PCV3 VLPs are all able to activate apoptosis in porcine kidney (PK15) cells at a rate of ~%30 after a 24-hour exposure; this apoptotic similar to apoptosis caused by 50 µM etoposide, which is a strong chemical inducer of apoptosis. These results coupled with preliminary data obtained by pre-treating PK15 cells with caspase-8 or caspase-9 inhibitors suggest the involvement of caspase-8 and caspase-9 in PCV2 Cap induced apoptosis and lend weight to the idea of PCV2 Cap as a primary virulence factor during infections. In contrast, PCV1 Cap induced apoptosis does not appear to involve caspase-8 or caspase-9 indicating an alternate pathway to PK15 cell apoptosis, while PCV3 Cap appears to induce PK15 cell apoptosis with some involvement of caspase-9.
- ItemOpen AccessA Prion Protein Gene Polymorphism at Codon 138 Modulates Chronic Wasting Disease Pathogenesis(2021-08) Arifin, Maria Immaculata; Gilch, Sabine; Schätzl, Hermann; Czub, Markus; Jirik, Frank; Mathiason, Candace; Musiani, MarcoPrion diseases are fatal and infectious neurodegenerative diseases caused by prions. Chronic wasting disease (CWD) is a prion disease of cervids found in North America (NA), Scandinavia and South Korea. Although there are no reports of CWD in caribou (Rangifer tarandus spp.) in NA so far, previous findings show that reindeer (R. t. tarandus) are susceptible to CWD. Single amino acid substitutions (SAAS) within the cervid prion protein (PrP) sequence have been shown to prolong survival times and produce incomplete attack rates upon CWD infection. Prion protein SAAS have been found in caribou populations in NA, including a serine to asparagine substitution at codon 138 (S138N). Previous studies reported that animals harboring the N variant at this codon were either resistant or less susceptible to natural CWD prion exposure. Based on these reports, we hypothesized that the S138N PrP amino acid substitution modulates CWD pathogenesis. We report that the 138N allele frequency is rare among caribou in areas with high risk of contact with CWD-infected species, particularly in woodland caribou (R. t. caribou) herds in Saskatchewan and Alberta. We also report that the barren-ground caribou (R. t. groenlandicus) herds have higher frequencies of the 138N allele. We found that the S138N SAAS did not alter endogenous PrP properties, but rather impairs the prion conversion process. Transgenic knock-in (KI) mice expressing the 138NN PrP genotype did not develop clinical disease up to 700 days post-inoculation (dpi), whilst their wild-type deer (138SS) counter parts succumbed to CWD between ~450-580 dpi. The 138NN KI mice did, however, harbor prions capable of inducing conversion in an in vitro prion conversion assay. Remarkably, even upon intracerebral prion inoculation, seeding activity was first detected in the spleens of these KI mice. Our findings provide new insights into the role of PrP genotype in tissue tropism of prion replication. Caribou in NA are a Threatened species and an essential resource for Indigenous people. Thus, determining the mechanisms by which the 138N allele modulates CWD pathogenesis is important for future CWD management strategies, especially in areas where caribou are at a high risk of contracting the disease.
- ItemOpen AccessTiming recombinant prion protein conversion as a measure of prion activity in chronic wasting disease(2014-05-23) Gray, John Geoffrey; Czub, MarkusChronic Wasting Disease (CWD) is a fatal neurological disease affecting cervids caused by prions. Infected cervids shed the CWD prion in bodily fluids and excrement, contaminating the environment and creating an agricultural and ecological calamity. Preclinical antemortem CWD testing method is demanded by CWD risk management programs. In vitro PrP-conversion assays have been developed as potential tools for such an approach with increasing sensitivity for prion detection. However, no method has been routinely employed thus far. Timing recombinant-PrP conversion into amyloid fibrils, seeded by elk CWD prion, as a diagnostic method is presented herein. The assay, termed “RePLICA”, is at least as sensitive for detecting elk CWD in brain tissues as Tg(CerPrP-M132)1536+/- and Tg(CerPrP-E226)5037+/- mouse bioassay models. The assay performs within a period of 35 hours, is consistently reproducible, and functions on elk brain and tonsil tissues. There are indications RePLICA has the potential to titre CWD infectivity.