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Carolyn A Wilson - One of the best experts on this subject based on the ideXlab platform.

  • Three cysteine residues of SLC52A1, a receptor for the Porcine Endogenous Retrovirus-A (PERV-A), play a critical role in cell surface expression and infectivity.
    Virology, 2017
    Co-Authors: Winston Colon-moran, Takele Argaw, Carolyn A Wilson
    Abstract:

    Porcine Endogenous Retrovirus-A (PERV-A), a gammaRetrovirus, infects human cells in vitro, thus raising the potential risk of cross-species transmission in xenotransplantation. Two members of the solute carrier family 52 (SLC52A1 and SLC52A2) are PERV-A receptors. Site-directed mutagenesis of the cDNA encoding SLC52A1 identified that only one of two putative glycosylation signals is occupied by glycans. In addition, we showed that glycosylation of SLC52A1 is not necessary for PERV-A receptor function. We also identified that at a minimum, three cysteine residues are sufficient for SLC52A1 cell surface expression. Mutation of cysteine at position 365 and either of the two cysteine residues in the C-terminal tail at positions 442 or 446 reduced SLC52A1 surface expression and PERV-A infection suggesting that these residues may contribute to overall structural stability and receptor function. Understanding interactions between PERV-A and its cellular receptor may provide novel strategies to prevent zoonotic infection in the setting of xenotransplantation.

  • Susceptibility of Porcine Endogenous Retrovirus to anti‐retroviral inhibitors
    Xenotransplantation, 2016
    Co-Authors: Takele Argaw, Winston Colon-moran, Carolyn A Wilson
    Abstract:

    Background Porcine Endogenous Retrovirus (PERV) is an Endogenous Retrovirus that poses a risk of iatrogenic transmission in the context of pig-to-human xenotransplantation. The lack of a means to control PERV infection in the context of pig-to-human xenotransplantation is a major concern in the field. In this study, we set out to evaluate the ability of currently licensed anti-HIV drugs, and other types of anti-retroviral compounds, to inhibit PERV infection in vitro. Methods We used target cells stably expressing one of the known PERV viral receptors, an infectious molecular clone, PERV-A 14/220, and at least one drug from each class of anti-retroviral inhibitors as well as off-label drugs shown to have anti-viral activities. The susceptibility of PERV-A 14/220 LacZ to the anti-retroviral drugs was determined from infected cells by histochemical staining. Results We extend the results of previous studies by showing that, in addition to raltegravir, dolutegravir is found to have a potent inhibitory activity against PERV replication (IC50 8.634 ±0.336 and IC50 3.06 ± 0.844 nM, respectively). The anti-HIV drug zidovudine (AZT) showed considerable anti-PERV activity with IC50 of 1.923 ±0.691 μM as well. Conclusions The study results indicate that some of the licensed anti-retroviral drugs may be useful for controlling PERV infection. However, the efficacy at nanomolar concentrations put forward integrase inhibitors as a drug that has the potential to be useful in the event that xenotransplantation recipients have evidence of PERV transmission and replication.

  • susceptibility of Porcine Endogenous Retrovirus to anti retroviral inhibitors
    Xenotransplantation, 2016
    Co-Authors: Takele Argaw, Winston Colonmoran, Carolyn A Wilson
    Abstract:

    Background Porcine Endogenous Retrovirus (PERV) is an Endogenous Retrovirus that poses a risk of iatrogenic transmission in the context of pig-to-human xenotransplantation. The lack of a means to control PERV infection in the context of pig-to-human xenotransplantation is a major concern in the field. In this study, we set out to evaluate the ability of currently licensed anti-HIV drugs, and other types of anti-retroviral compounds, to inhibit PERV infection in vitro. Methods We used target cells stably expressing one of the known PERV viral receptors, an infectious molecular clone, PERV-A 14/220, and at least one drug from each class of anti-retroviral inhibitors as well as off-label drugs shown to have anti-viral activities. The susceptibility of PERV-A 14/220 LacZ to the anti-retroviral drugs was determined from infected cells by histochemical staining. Results We extend the results of previous studies by showing that, in addition to raltegravir, dolutegravir is found to have a potent inhibitory activity against PERV replication (IC50 8.634 ±0.336 and IC50 3.06 ± 0.844 nM, respectively). The anti-HIV drug zidovudine (AZT) showed considerable anti-PERV activity with IC50 of 1.923 ±0.691 μM as well. Conclusions The study results indicate that some of the licensed anti-retroviral drugs may be useful for controlling PERV infection. However, the efficacy at nanomolar concentrations put forward integrase inhibitors as a drug that has the potential to be useful in the event that xenotransplantation recipients have evidence of PERV transmission and replication.

  • Comparison of the convergent receptor utilization of a retargeted feline leukemia virus envelope with a naturally-occurring Porcine Endogenous Retrovirus A
    Virology, 2012
    Co-Authors: Peter M. Mazari, Carolyn A Wilson, Daniel R Salomon, Katherine T. Marcucci, Takele Argaw, Leonardo Valdivieso, Xia Zhang, Monica J. Roth
    Abstract:

    In vitro screening of randomized FeLV Envelope libraries identified the CP isolate, which enters cells through HuPAR-1, one of two human receptors utilized by Porcine Endogenous Retrovirus-A (PERV-A), a distantly related gammaRetrovirus. The CP and PERV-A Envs however, share little amino acid homology. Their receptor utilization was examined to define the common receptor usage of these disparate viral Envs. We demonstrate that the receptor usage of CP extends to HuPAR-2 but not to the Porcine receptor PoPAR, the cognate receptor for PERV-A. Reciprocal interference between virus expressing CP and PERV-A Envs was observed on human cells. Amino acid residues localized to within the putative second extracellular loop (ECL-2) of PAR-1 and PAR-2 are found to be critical for CP envelope function. Through a panel of receptor chimeras and point mutations, this area was also found to be responsible for the differential usage of the PoPAR receptor between CP and PERV-A.

  • Identification of two distinct structural regions in a human Porcine Endogenous Retrovirus receptor, HuPAR2, contributing to function for viral entry
    Retrovirology, 2009
    Co-Authors: Katherine T. Marcucci, Carolyn A Wilson, Takele Argaw, Daniel R Salomon
    Abstract:

    Background Of the three subclasses of Porcine Endogenous Retrovirus (PERV), PERV-A is able to infect human cells via one of two receptors, HuPAR1 or HuPAR2. Characterizing the structure-function relationships of the two HuPAR receptors in PERV-A binding and entry is important in understanding receptor-mediated gammaretroviral entry and contributes to evaluating the risk of zoonosis in xenotransplantation.

Yasuhiro Takeuchi - One of the best experts on this subject based on the ideXlab platform.

  • Suboptimal Porcine Endogenous Retrovirus Infection in Non-Human Primate Cells: Implication for Preclinical
    2013
    Co-Authors: Giada Mattiuzzo, Yasuhiro Takeuchi
    Abstract:

    Background: Porcine Endogenous Retrovirus (PERV) poses a potential risk of zoonotic infection in xenotransplantation. Preclinical transplantation trials using non-human primates (NHP) as recipients of Porcine xenografts present the opportunity to assess the zoonosis risk in vivo. However, PERV poorly infects NHP cells for unclear reasons and therefore NHP may represent a suboptimal animal model to assess the risk of PERV zoonoses. We investigated the mechanism responsible for the low efficiency of PERV-A infection in NHP cells. Principal Findings: Two steps, cell entry and exit, were inefficient for the replication of high-titer, human-tropic A/C recombinant PERV. A restriction factor, tetherin, is likely to be responsible for the block to matured virion release, supported by the correlation between the levels of inhibition and tetherin expression. In rhesus macaque, cynomolgus macaque and baboon the main receptor for PERV entry, PERV-A receptor 1 (PAR-1), was found to be genetically deficient: PAR-1 genes in these species encode serine at amino acid 109 in place of the leucine in human PAR-1. This genetic defect inevitably impacts in vivo sensitivity to PERV infection of these species. In contrast, African green monkey (AGM) PAR-1 is functional, but PERV infection is still poor. Although the mechanism is unclear, tunicamycin treatment, which removes N-glycosylated sugar chains, increases PERV infection, suggesting a possible role for the glycosylation of the receptors. Conclusions: Since cynomolgus macaque and baboon, species often used in pig-to-NHP xenotransplantation experiments

  • Potential zoonotic infection of Porcine Endogenous Retrovirus in xenotransplantation.
    Methods in molecular biology (Clifton N.J.), 2012
    Co-Authors: Giada Mattiuzzo, Yasuhiro Takeuchi, Linda Scobie
    Abstract:

    Porcine Endogenous Retrovirus (PERV) is considered the major biosafety issue in xenotransplantation. Several techniques have been employed for the analysis of the PERV status in the animal donor and for the assessment of PERV transmission/infection in the xenograft recipient. In this chapter, methods to assess the expression of PERV and the potential for PERV transmission from a donor animal are described in addition to the identification of relevant loci within the Porcine genome.PERV detection can be carried out using several techniques of which quantitative polymerase chain reaction (PCR) and RT-PCR are the most sensitive. However, other procedures can be employed such as detection of reverse transcriptase activity (i.e. viral replication) in the sample or immunostaining of the infected cells using an anti-PERV antibody. The PERV transmission assay has been described to identify the transmission phenotype of the pig donor, and subsequent risk from a donor. This assay can, therefore, direct the selection of the most suitable animal. Finally, it is important to determine the presence of critical PERV loci involved in transmission in the pig genome and compare between different animals. One of the methods for the analysis of these PERV integration sites is described.

  • Differential resistance to cell entry by Porcine Endogenous Retrovirus subgroup A in rodent species-4
    2011
    Co-Authors: Giada Mattiuzzo, Magda Matouskova, Yasuhiro Takeuchi
    Abstract:

    Copyright information:Taken from "Differential resistance to cell entry by Porcine Endogenous Retrovirus subgroup A in rodent species"http://www.retrovirology.com/content/4/1/93Retrovirology 2007;4():93-93.Published online 14 Dec 2007PMCID:PMC2241639. PCR products on genomic DNA were aligned using Clustal W software [48]. and have the same identical a.a. sequence in the ECL2 (boxed) of other murine species. RatPAR from different rat cell lines have identical a.a. sequences

  • Differential resistance to cell entry by Porcine Endogenous Retrovirus subgroup A in rodent species-3
    2011
    Co-Authors: Giada Mattiuzzo, Magda Matouskova, Yasuhiro Takeuchi
    Abstract:

    Copyright information:Taken from "Differential resistance to cell entry by Porcine Endogenous Retrovirus subgroup A in rodent species"http://www.retrovirology.com/content/4/1/93Retrovirology 2007;4():93-93.Published online 14 Dec 2007PMCID:PMC2241639.ic receptors huPAR-2 with L109P mutation and muPAR with P109L mutation (B) were incubated with 1 ml of medium (grey filled) or with the supernatant of 293T containing N-terminal c-myc tagged soluble PERV-A 14/220 SU ENV (bold line). The cells were then immunostained with an anti- human c-myc antibody and a PE conjugated anti-mouse IgG secondary antibody. The histograms show a representative result of at least three independent experiments

  • Differential resistance to cell entry by Porcine Endogenous Retrovirus subgroup A in rodent species-1
    2011
    Co-Authors: Giada Mattiuzzo, Magda Matouskova, Yasuhiro Takeuchi
    Abstract:

    Copyright information:Taken from "Differential resistance to cell entry by Porcine Endogenous Retrovirus subgroup A in rodent species"http://www.retrovirology.com/content/4/1/93Retrovirology 2007;4():93-93.Published online 14 Dec 2007PMCID:PMC2241639.s huPAR-1 (grey bar) and the mutant H1M g were introduced into QT6 cells by MLV-based retroviral vectors. 50–70% of the QT6 cell population showed PAR expression as confirmed by anti-HA staining. These cultures were infected with EGFP(PERV-A). Cells were harvested 48 hours later and PERV-A infection was measured by flow cytometry as percentage of EGFP-positive cells. Arrows indicate infection below detectable levels. Results are expressed as average ± SEM from three independent experiments

Daniel R Salomon - One of the best experts on this subject based on the ideXlab platform.

  • Comparison of the convergent receptor utilization of a retargeted feline leukemia virus envelope with a naturally-occurring Porcine Endogenous Retrovirus A
    Virology, 2012
    Co-Authors: Peter M. Mazari, Carolyn A Wilson, Daniel R Salomon, Katherine T. Marcucci, Takele Argaw, Leonardo Valdivieso, Xia Zhang, Monica J. Roth
    Abstract:

    In vitro screening of randomized FeLV Envelope libraries identified the CP isolate, which enters cells through HuPAR-1, one of two human receptors utilized by Porcine Endogenous Retrovirus-A (PERV-A), a distantly related gammaRetrovirus. The CP and PERV-A Envs however, share little amino acid homology. Their receptor utilization was examined to define the common receptor usage of these disparate viral Envs. We demonstrate that the receptor usage of CP extends to HuPAR-2 but not to the Porcine receptor PoPAR, the cognate receptor for PERV-A. Reciprocal interference between virus expressing CP and PERV-A Envs was observed on human cells. Amino acid residues localized to within the putative second extracellular loop (ECL-2) of PAR-1 and PAR-2 are found to be critical for CP envelope function. Through a panel of receptor chimeras and point mutations, this area was also found to be responsible for the differential usage of the PoPAR receptor between CP and PERV-A.

  • Identification of two distinct structural regions in a human Porcine Endogenous Retrovirus receptor, HuPAR2, contributing to function for viral entry
    Retrovirology, 2009
    Co-Authors: Katherine T. Marcucci, Carolyn A Wilson, Takele Argaw, Daniel R Salomon
    Abstract:

    Background Of the three subclasses of Porcine Endogenous Retrovirus (PERV), PERV-A is able to infect human cells via one of two receptors, HuPAR1 or HuPAR2. Characterizing the structure-function relationships of the two HuPAR receptors in PERV-A binding and entry is important in understanding receptor-mediated gammaretroviral entry and contributes to evaluating the risk of zoonosis in xenotransplantation.

  • Functional hierarchy of two L domains in Porcine Endogenous Retrovirus (PERV) that influence release and infectivity.
    Virology, 2008
    Co-Authors: Katherine T. Marcucci, Yuri Martina, Carolyn A Wilson, Frank Harrison, Daniel R Salomon
    Abstract:

    Abstract The Porcine Endogenous Retrovirus (PERV) Gag protein contains two late (L) domain motifs, PPPY and P(F/S)AP. Using viral release assays we demonstrate that PPPY is the dominant L domain involved in PERV release. PFAP represents a novel retroviral L domain variant and is defined by abnormal viral assembly phenotypes visualized by electron microscopy and attenuation of early PERV release as measured by viral genomes. PSAP is functionally dominant over PFAP in early PERV release. PSAP virions are 3.5-fold more infectious in vitro by TCID 50 and in vivo results in more RNA positive tissues and higher levels of proviral DNA using our human PERV-A receptor (HuPAR-2) transgenic mouse model [Martina, Y., Marcucci, K.T., Cherqui, S., Szabo, A., Drysdale, T., Srinivisan, U., Wilson, C.A., Patience, C., Salomon, D.R., 2006. Mice transgenic for a human Porcine Endogenous Retrovirus receptor are susceptible to productive viral infection. J. Virol. 80 (7), 3135–3146]. The functional hierarchies displayed by PERV L domains, demonstrates that L domain selection in viral evolution exists to promote efficient viral assembly, release and infectivity in the virus–host context.

  • Lack of cross-species transmission of Porcine Endogenous Retrovirus (PERV) to transplant recipients and abattoir workers in contact with pigs.
    Transplantation, 2007
    Co-Authors: Manuel Hermida-prieto, Daniel R Salomon, Nieves Doménech, Isabel Moscoso, T.m. Dı́az, Jennifer K. Ishii, Rafael Mañez
    Abstract:

    This study investigated the potential transmission of Porcine Endogenous Retrovirus (PERV) to solid-organ transplant recipients and abattoir workers in contact with pigs. Blood samples were obtained from volunteer healthy blood donors (Group A; n=33); pig-breeding farmers who had undergone a liver transplant (Group B; n=14); and pig abattoir workers (Group C; n=49). A second blood sample was obtained 1 year after the first sample from 10 of the abattoir workers (Group D). Tests included investigation for PERV-DNA, PERV-RNA, pig-specific mitochondrial DNA, a quantitative detection of PERV nucleic acids, and antibodies to PERV by two different Western Blots. All polymerase chain reaction and Western Blots assays were negative for PERV or antibodies to PERV. Therefore, the risks of cross-species transmission of PERV appear to be negligible for immunocompetent individuals and allotransplant recipients, even if they are in close and repeated contact with live pigs or pig tissues.

  • Mice Transgenic for a Human Porcine Endogenous Retrovirus Receptor Are Susceptible to Productive Viral Infection
    Journal of virology, 2006
    Co-Authors: Yuri Martina, Stephanie Cherqui, Carolyn A Wilson, Katherine T. Marcucci, Clive Patience, A. Szabo, T. Drysdale, U. Srinivisan, Daniel R Salomon
    Abstract:

    Porcine Endogenous Retrovirus (PERV) is considered one of the major risks in xenotransplantation. No valid animal model has been established to evaluate the risks associated with PERV transmission to human patients by pig tissue xenotransplantation or to study the potential pathogenesis associated with PERV infection. In previous work we isolated two genes encoding functional human PERV receptors and proved that introduction of these into mouse fibroblasts allowed the normally nonpermissive mouse cells to become productively infected (T. A. Ericsson, Y. Takeuchi, C. Templin, G. Quinn, S. F. Farhadian, J. C. Wood, B. A. Oldmixon, K. M. Suling, J. K. Ishii, Y. Kitagawa, T. Miyazawa, D. R. Salomon, R. A. Weiss, and C. Patience, Proc. Natl. Acad. Sci. USA 100:6759-6764, 2003). In the present study we created mice transgenic for human PERV-A receptor 2 (HuPAR-2). After inoculation of transgenic animals with infectious PERV supernatants, viral DNA and RNA were detected at multiple time points, indicating productive replication. This establishes the role of HuPAR-2 in PERV infection in vivo; in addition, these transgenic mice represent a new model for determining the risk of PERV transmission and potential pathogenesis. These mice also create a unique opportunity to study the immune response to PERV infection and test potential therapeutic or preventative modalities.

Joachim Denner - One of the best experts on this subject based on the ideXlab platform.

  • Human SAMHD1 restricts the xenotransplantation relevant Porcine Endogenous Retrovirus (PERV) in non-dividing cells
    The Journal of general virology, 2019
    Co-Authors: Hussein Al-shehabi, Joachim Denner, Uwe Fiebig, Juliane Kutzner, Torsten Schaller, Norbert Bannert, Henning Hofmann
    Abstract:

    The release of Porcine Endogenous Retrovirus (PERV) particles from pig cells is a potential risk factor during xenotransplantation by way of productively infecting the human transplant recipient. Potential countermeasures against PERV replication are restriction factors that block retroviral replication. SAMHD1 is a triphosphohydrolase that depletes the cellular pool of dNTPs in non-cycling cells starving retroviral reverse transcription. We investigated the antiviral activity of human SAMHD1 against PERV and found that SAMHD1 potently restricts its reverse transcription in human monocytes, monocyte-derived dendritic cells (MDDC), or macrophages (MDM) and in monocytic THP-1 cells. Degradation of SAMHD1 by SIVmac Vpx or CRISPR/Cas9 knock-out of SAMHD1 allowed for PERV reverse transcription. Addition of deoxynucleosides alleviated the SAMHD1-mediated restriction suggesting that SAMHD1-mediated degradation of dNTPs restricts PERV replication in these human immune cells. In conclusion, our findings highlight SAMHD1 as a potential barrier to PERV transmission from pig transplants to human recipients during xenotransplantation.

  • Tolerance and immune response to the Porcine Endogenous Retrovirus in German landrace pigs immunised with viral proteins.
    Virus Research, 2015
    Co-Authors: Joachim Denner, Björn Petersen, Heiner Niemann
    Abstract:

    Immunisation of goats, mice, rats, rabbits, guinea pigs, and hamsters with the recombinant ectodomain of the Porcine Endogenous Retrovirus (PERV) transmembrane envelope (TM) protein (p15E) induced binding and neutralising immune antibodies in all animals. In contrast, no antibodies were induced when pigs were immunised with p15E, indicating that pigs are tolerant to their Endogenous Retroviruses, at least to the TM protein. To answer the question of whether pigs are tolerant to other structural proteins of PERV, we immunised German landrace pigs with p15E, this time in conjunction with the surface envelope proteins gp70 and the core capsid Gag protein p27CA. To ensure that the pigs were immunocompetent and that immunisation was successful, all animals also received an injection of an unrelated protein, keyhole limpet hemocyanin (KLH). Whereas all animals produced antibodies against KLH, no animals produced antibodies against the viral envelope proteins, thus confirming previous results for p15E and extending them to the other envelope protein, gp70. However, the pigs did produce antibodies against p27CA, indicating that there is no tolerance to the core capsid protein of PERV.

  • Novel neutralising antibodies targeting the N-terminal helical region of the transmembrane envelope protein p15E of the Porcine Endogenous Retrovirus (PERV)
    Immunologic Research, 2014
    Co-Authors: Alexander Waechter, Joachim Denner
    Abstract:

    Previously, immunising different species with the transmembrane envelope protein p15E of the Porcine Endogenous Retrovirus (PERV), neutralising antibodies were induced which recognised epitopes in the fusion peptide proximal region (FPPR) and in the membrane-proximal external region (MPER). Only the MPER-specific antibodies were shown to neutralise and these antibodies targeted epitopes in the MPER similarly localised as the epitopes recognised by antibodies broadly neutralising HIV-1 such as 2F5 and 4E10. To study whether neutralising antibodies could be induced immunising with subunits of p15E, recombinant proteins corresponding to the N-terminal, the C-terminal helical region (NHR, CHR) and a p15E with a mutation in the Cys–Cys loop were produced. Whereas none of these antigens induced MPER-specific neutralising antibodies, the animals immunised with the FPPR/NHR subunit and the mutated p15E produced neutralising antibodies binding to the NHR. Therefore, for the first time, antibodies specific for the NHR and neutralising PERV were described.

  • Neutralization of Porcine Endogenous Retrovirus by antibodies against the membrane-proximal external region of the transmembrane envelope protein.
    Journal of General Virology, 2013
    Co-Authors: Alexander Waechter, Magdalena Eschricht, Joachim Denner
    Abstract:

    Immunization of different species including goats, rats, hamsters and guinea pigs with the recombinant ectodomain of the transmembrane envelope (TM) protein p15E of Porcine Endogenous Retrovirus (PERV) has been shown to result in the production of virus-neutralizing antibodies. The sera recognize two groups of epitopes, one located in the fusion peptide-proximal region (FPPR) and the second in the membrane-proximal external region (MPER) of p15E. Most interestingly, the epitopes in the MPER are similar to epitopes in the TM protein gp41 of human immunodeficiency virus type 1 (HIV-1) recognized by mAbs 2F5 and 4E10, which broadly neutralize HIV-1. To study which epitope and which antibody population are involved in the process of neutralization of PERV, this study generated a new antiserum in a goat using an elongated ectodomain of p15E. The immune serum neutralized PERV at a higher titre and recognized broader epitopes in the FPPR and MPER of p15E. For the first time, antibody subpopulations were isolated from this serum using affinity chromatography with immobilized proteins and peptides corresponding to the FPPR and MPER of p15E. Only the affinity-purified antibodies specifically binding the MPER neutralized PERV, indicating that, as in the case of HIV-1, the MPER is an important target of neutralizing activity.

Giada Mattiuzzo - One of the best experts on this subject based on the ideXlab platform.

  • Suboptimal Porcine Endogenous Retrovirus Infection in Non-Human Primate Cells: Implication for Preclinical
    2013
    Co-Authors: Giada Mattiuzzo, Yasuhiro Takeuchi
    Abstract:

    Background: Porcine Endogenous Retrovirus (PERV) poses a potential risk of zoonotic infection in xenotransplantation. Preclinical transplantation trials using non-human primates (NHP) as recipients of Porcine xenografts present the opportunity to assess the zoonosis risk in vivo. However, PERV poorly infects NHP cells for unclear reasons and therefore NHP may represent a suboptimal animal model to assess the risk of PERV zoonoses. We investigated the mechanism responsible for the low efficiency of PERV-A infection in NHP cells. Principal Findings: Two steps, cell entry and exit, were inefficient for the replication of high-titer, human-tropic A/C recombinant PERV. A restriction factor, tetherin, is likely to be responsible for the block to matured virion release, supported by the correlation between the levels of inhibition and tetherin expression. In rhesus macaque, cynomolgus macaque and baboon the main receptor for PERV entry, PERV-A receptor 1 (PAR-1), was found to be genetically deficient: PAR-1 genes in these species encode serine at amino acid 109 in place of the leucine in human PAR-1. This genetic defect inevitably impacts in vivo sensitivity to PERV infection of these species. In contrast, African green monkey (AGM) PAR-1 is functional, but PERV infection is still poor. Although the mechanism is unclear, tunicamycin treatment, which removes N-glycosylated sugar chains, increases PERV infection, suggesting a possible role for the glycosylation of the receptors. Conclusions: Since cynomolgus macaque and baboon, species often used in pig-to-NHP xenotransplantation experiments

  • Potential zoonotic infection of Porcine Endogenous Retrovirus in xenotransplantation.
    Methods in molecular biology (Clifton N.J.), 2012
    Co-Authors: Giada Mattiuzzo, Yasuhiro Takeuchi, Linda Scobie
    Abstract:

    Porcine Endogenous Retrovirus (PERV) is considered the major biosafety issue in xenotransplantation. Several techniques have been employed for the analysis of the PERV status in the animal donor and for the assessment of PERV transmission/infection in the xenograft recipient. In this chapter, methods to assess the expression of PERV and the potential for PERV transmission from a donor animal are described in addition to the identification of relevant loci within the Porcine genome.PERV detection can be carried out using several techniques of which quantitative polymerase chain reaction (PCR) and RT-PCR are the most sensitive. However, other procedures can be employed such as detection of reverse transcriptase activity (i.e. viral replication) in the sample or immunostaining of the infected cells using an anti-PERV antibody. The PERV transmission assay has been described to identify the transmission phenotype of the pig donor, and subsequent risk from a donor. This assay can, therefore, direct the selection of the most suitable animal. Finally, it is important to determine the presence of critical PERV loci involved in transmission in the pig genome and compare between different animals. One of the methods for the analysis of these PERV integration sites is described.

  • Differential resistance to cell entry by Porcine Endogenous Retrovirus subgroup A in rodent species-4
    2011
    Co-Authors: Giada Mattiuzzo, Magda Matouskova, Yasuhiro Takeuchi
    Abstract:

    Copyright information:Taken from "Differential resistance to cell entry by Porcine Endogenous Retrovirus subgroup A in rodent species"http://www.retrovirology.com/content/4/1/93Retrovirology 2007;4():93-93.Published online 14 Dec 2007PMCID:PMC2241639. PCR products on genomic DNA were aligned using Clustal W software [48]. and have the same identical a.a. sequence in the ECL2 (boxed) of other murine species. RatPAR from different rat cell lines have identical a.a. sequences

  • Differential resistance to cell entry by Porcine Endogenous Retrovirus subgroup A in rodent species-3
    2011
    Co-Authors: Giada Mattiuzzo, Magda Matouskova, Yasuhiro Takeuchi
    Abstract:

    Copyright information:Taken from "Differential resistance to cell entry by Porcine Endogenous Retrovirus subgroup A in rodent species"http://www.retrovirology.com/content/4/1/93Retrovirology 2007;4():93-93.Published online 14 Dec 2007PMCID:PMC2241639.ic receptors huPAR-2 with L109P mutation and muPAR with P109L mutation (B) were incubated with 1 ml of medium (grey filled) or with the supernatant of 293T containing N-terminal c-myc tagged soluble PERV-A 14/220 SU ENV (bold line). The cells were then immunostained with an anti- human c-myc antibody and a PE conjugated anti-mouse IgG secondary antibody. The histograms show a representative result of at least three independent experiments

  • Differential resistance to cell entry by Porcine Endogenous Retrovirus subgroup A in rodent species-1
    2011
    Co-Authors: Giada Mattiuzzo, Magda Matouskova, Yasuhiro Takeuchi
    Abstract:

    Copyright information:Taken from "Differential resistance to cell entry by Porcine Endogenous Retrovirus subgroup A in rodent species"http://www.retrovirology.com/content/4/1/93Retrovirology 2007;4():93-93.Published online 14 Dec 2007PMCID:PMC2241639.s huPAR-1 (grey bar) and the mutant H1M g were introduced into QT6 cells by MLV-based retroviral vectors. 50–70% of the QT6 cell population showed PAR expression as confirmed by anti-HA staining. These cultures were infected with EGFP(PERV-A). Cells were harvested 48 hours later and PERV-A infection was measured by flow cytometry as percentage of EGFP-positive cells. Arrows indicate infection below detectable levels. Results are expressed as average ± SEM from three independent experiments