New short-term heat inactivation method of cytomegalovirus (CMV) in breast milk: impact on CMV inactivation, CMV antibodies and enzyme activities

2019 ◽  
Vol 104 (6) ◽  
pp. F604-F608 ◽  
Author(s):  
Jens Maschmann ◽  
Denise Müller ◽  
Katrin Lazar ◽  
Rangmar Goelz ◽  
Klaus Hamprecht
Keyword(s):  
Breast Milk ◽  
Enzyme Activities ◽  
Primary Source ◽  
Wild Type Virus ◽  
Heat Inactivation ◽  
Target Cells ◽  
Igg Antibodies ◽  
Wild Type ◽  
Short Term ◽  
Milk Whey

ObjectivesBreast milk (BM) is the primary source of cytomegalovirus (CMV) transmission to premature infants with potentially harmful consequences. We therefore wanted to evaluate temperature and duration of short-term BM pasteurisation with respect to CMV inactivation, effect on CMV-IgG antibodies and BM enzyme activities.Methods116 artificially CMV-spiked BM and 15 wild-type virus-infected samples were subjected for 5 s to different temperatures (55°C–72°C). CMV-IE-1 expression in fibroblast nuclei was assessed using the milk whey fraction in short-term microculture. BM lipase and alkaline phosphatase (AP) activities and CMV binding using CMV-recomLine immunoblotting and neutralising antibodies using epithelial target cells were analysed before and after heating.ResultsA minimum of 5 s above 60°C was necessary for CMV inactivation in both CMV-AD-169 spiked and wild-type infected BM. Lipase was very heat sensitive (activities of 54% at 55°C, 5% at 60°C and 2% at 65°C). AP showed activities of 77%, 88% and 10%, respectively. CMV-p150 IgG antibodies were mostly preserved at 62°C for 5 s.ConclusionOur results show that short-term pasteurisation of BM at 62°C for 5 s might be efficient for CMV inactivation and reduces loss of enzyme activities, as well as CMV binding, and functional CMV antibodies.

scholarly journals Protease-Dependent Uncoating of a Complex Retrovirus

2005 ◽  
Vol 79 (14) ◽  
pp. 9244-9253 ◽  
Author(s):  
Jacqueline Lehmann-Che ◽  
Marie-Lou Giron ◽  
Olivier Delelis ◽  
Martin Löchelt ◽  
Patricia Bittoun ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Viral Genome ◽  
Productive Infection ◽  
Wild Type Virus ◽  
Target Cells ◽  
Wild Type ◽  
Microtubule Organizing Center ◽  
Viral Protease ◽  
Virus Life Cycle ◽  
Organizing Center

ABSTRACT Although retrovirus egress and budding have been partly unraveled, little is known about early stages of the replication cycle. In particular, retroviral uncoating, a process during which incoming retroviral cores are altered to allow the integration of the viral genome into host chromosomes, is poorly understood. To get insights into these early events of the retroviral cycle, we have used foamy complex retroviruses as a model. In this report, we show that a protease-defective foamy retrovirus is noninfectious, although it is still able to bud and enter target cells efficiently. Similarly, a retrovirus mutated in an essential viral protease-dependent cleavage site in the central part of Gag is noninfectious. Following entry, wild-type and mutant retroviruses are able to traffic along microtubules towards the microtubule-organizing center (MTOC). However, whereas nuclear import of Gag and of the viral genome was observed for the wild-type virus as early as 8 hours postinfection, incoming capsids and genome from mutant viruses remained at the MTOC. Interestingly, a specific viral protease-dependent Gag cleavage product was detected only for the wild-type retrovirus early after infection, demonstrating that cleavage of Gag by the viral protease at this stage of the virus life cycle is absolutely required for productive infection, an unprecedented observation among retroviruses.

scholarly journals Discordance between Bovine Leukemia Virus Tax Immortalization In Vitro and Oncogenicity In Vivo

2000 ◽  
Vol 74 (21) ◽  
pp. 9895-9902 ◽  
Author(s):  
Jean-Claude Twizere ◽  
Pierre Kerkhofs ◽  
Arsène Burny ◽  
Daniel Portetelle ◽  
Richard Kettmann ◽  
...  
Keyword(s):  
Viral Replication ◽  
Bovine Leukemia Virus ◽  
Leukemia Virus ◽  
Wild Type Virus ◽  
Target Cells ◽  
Phosphorylation Sites ◽  
Primary Cells ◽  
Wild Type

ABSTRACT Bovine leukemia virus (BLV) Tax protein, a transcriptional activator of viral expression, is essential for viral replication in vivo. Tax is believed to be involved in leukemogenesis because of its second function, immortalization of primary cells in vitro. These activities of Tax can be dissociated on the basis of point mutations within specific regions of the protein. For example, mutation of the phosphorylation sites at serines 106 and 293 abrogates immortalization potential in vitro but maintains transcriptional activity. This type of mutant is thus particularly useful for unraveling the role of Tax immortalization activity during leukemogenesis independently of viral replication. In this report, we describe the biological properties of BLV recombinant proviruses mutated in the Tax phosphorylation sites (BLVTax106+293). Titration of the proviral loads by semiquantitative PCR revealed that the BLV mutants propagated at wild-type levels in vivo. Furthermore, two animals (sheep 480 and 296) infected with BLVTax106+293 developed leukemia or lymphosarcoma after 16 and 36 months, respectively. These periods of time are within the normal range of latencies preceding the onset of pathogenesis induced by wild-type viruses. The phenotype of the mutant-infected cells was characteristic of a B lymphocyte (immunoglobulin M positive) expressing CD11b and CD5 (except at the final stage for the latter marker), a pattern that is typical of wild-type virus-infected target cells. Interestingly, the transformed B lymphocytes from sheep 480 also coexpressed the CD8 marker, a phenotype rarely observed in tumor biopsies from chronic lymphocytic leukemia patients. Finally, direct sequencing of the tax gene demonstrated that the leukemic cells did not harbor revertant proviruses. We conclude that viruses expressing a Tax mutant unable to transform primary cells in culture are still pathogenic in the sheep animal model. Our data thus provide a clear example of the discordant conclusions that can be drawn from in vitro immortalization assays and in vivo experiments. These observations could be of interest for other systems, such as the related human T-cell leukemia virus type 1, which currently lack animal models allowing the study of the leukemogenic process.

scholarly journals Class I MHC can present an endogenous peptide to cytotoxic T lymphocytes.

1989 ◽  
Vol 170 (3) ◽  
pp. 1033-1038 ◽  
Author(s):  
J L Whitton ◽  
M B Oldstone
Keyword(s):  
Cytotoxic T Lymphocytes ◽  
Class I ◽  
Wild Type Virus ◽  
Short Peptides ◽  
Target Cells ◽  
Viral Sequence ◽  
Wild Type ◽  
Class I Mhc ◽  
Ctl Epitope ◽  
A Cell

Since class I MHC glycoproteins may function by "screening and selecting" degraded proteins, we wished to determine whether very short peptides made within a cell were detected and bound by MHC, and presented for T cell perusal. We show that a 22 amino acid viral sequence containing a Db-restricted nonameric CTL epitope is sufficient to direct CTL recognition/lysis of H2b target cells. The mechanism of epitope presentation is by the "natural" endogenous route, and appears to direct lysis as effectively as wild-type virus infection, in which the epitope is part of a 236 residue glycoprotein.

scholarly journals The study of antibody responses to influenza neuraminidase using a lentiviral pseudotype based ELLA

2017 ◽  
Author(s):  
Fabrizio Biuso ◽  
George Carnell ◽  
Emanuele Montomoli ◽  
Nigel Temperton
Keyword(s):  
Antibody Response ◽  
Specific Antibody ◽  
Virus Entry ◽  
Antibody Responses ◽  
Surface Glycoprotein ◽  
Wild Type Virus ◽  
Target Cells ◽  
Type Virus ◽  
Wild Type ◽  
Reassortant Viruses

AbstractInfluenza pseudotypes represent an alternative to wild type virus for serological assays. To date, pseudotypes (PV) have predominantly been used as surrogates for wild type viruses in microneutralisation assays, where the surface glycoprotein of interest and a reporter gene (such as Luciferase) are used to assess if virus entry into target cells could be inhibited by serum antibodies. The influenza neuraminidase (NA) has the ability to bud and release new virions with or without the contribution of Haemagglutinin (HA). Influenza pseudotypes expressing NA alone, or with HA, were produced to evaluate the antibody response against NA using the enzyme-linked lectin assay (ELLA). The expression of an avian HA with human NAs has enabled the detection of specific antibody reponses against the human circulating subtypes of NA. Within this study a PV-based ELLA assay has been investigated with a pilot panel of sera prepared for an international CONSISE study. Preliminary results have confirmed that the assay is sensitive and could potentially represent a valid alternative to the classical ELLA assay, which requires the employment of reassortant viruses.

scholarly journals Moderate Influence of Human APOBEC3F on HIV-1 Replication in Primary Lymphocytes

2010 ◽  
Vol 84 (18) ◽  
pp. 9613-9617 ◽  
Author(s):  
Lubbertus C. F. Mulder ◽  
Marcel Ooms ◽  
Susan Majdak ◽  
Jordan Smedresman ◽  
Caitlin Linscheid ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Human Lymphocytes ◽  
Wild Type Virus ◽  
Target Cells ◽  
Wild Type ◽  
Peripheral Blood Mononuclear ◽  
Relative Contribution ◽  
Individual Contributions ◽  
Comparable Activity ◽  
Hiv 1

ABSTRACT Multiple APOBEC3 proteins are expressed in HIV-1 target cells, but their individual contributions to viral suppression when expressed at endogenous levels remain largely unknown. We used an HIV NL4-3 mutant that selectively counteracts APOBEC3G (A3G) but not APOBEC3F (A3F) to dissect the relative contribution of A3F to the inhibition of HIV-1 replication in primary human lymphocytes (peripheral blood mononuclear cells [PBMCs]). This HIV Vif mutant replicated similarly to wild-type virus in PBMCs, suggesting that the effect of A3F on HIV restriction in these cells is limited. The different A3F variants found in PMBC donors displayed either comparable activity or less activity than wild-type A3F. Lastly, the endogenous A3F mRNA and protein expression levels in PBMCs were considerably lower than those of A3G. Our results suggest that A3F neutralization is dispensable for HIV-1 replication in primary human T-lymphocytes.

scholarly journals Human Cytomegalovirus Reactivation During Lactation: Impact of Antibody Kinetics and Neutralization in Blood and Breast Milk

Nutrients ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 338 ◽  
Author(s):  
Katrin Lazar ◽  
Tabea Rabe ◽  
Rangmar Goelz ◽  
Klaus Hamprecht
Keyword(s):  
Breast Milk ◽  
Human Cytomegalovirus ◽  
Igg Antibodies ◽  
Binding Assays ◽  
Virus Shedding ◽  
Milk Whey ◽  
Viral Loads ◽  
Cytomegalovirus Reactivation ◽  
Specific Igg ◽  
Functional Antibodies

Human cytomegalovirus (HCMV) is shed into breast milk in nearly every seropositive woman during lactation. This reactivation shows mostly a self-limited, unimodal course. The dynamics and functional role of HCMV-specific-IgG in breast milk and in plasma during reactivation are unknown. Milk whey viral loads were monitored with real-time PCR in 18 HCMV-seropositive mothers over two months postpartum. HCMV-antibody binding assays (ECLIA) and antigen-specific immunoblotting were performed from plasma and corresponding milk samples. Epithelial-cell-specific neutralization was used to analyze functional antibodies in plasma- and whey-pools. Viral loads in milk whey showed unimodal courses in 15 of 18 mothers with peak viral loads around one month postpartum. HCMV-specific-IgG-antibodies increased significantly in plasma and milk whey during reactivation. The mean levels of plasma IgG were about 275-fold higher than in whey. Only antibodies against tegument protein p150 were continuously expressed in both compartments. Anti-glycoprotein-B1 IgG-antibodies were variably expressed in whey, but continuously in plasma. Neutralization assays showed 40-fold higher NT-50 values in plasma compared to whey at two months postpartum. During reactivation, HCMV-specific-IgG reactivities and neutralizing capacities are much lower in whey than in plasma. Therefore, their specific role in the decrease and discontinuation of virus-shedding in milk remains unclear.

scholarly journals Porcine Circovirus 2 Uses Heparan Sulfate and Chondroitin Sulfate B Glycosaminoglycans as Receptors for Its Attachment to Host Cells

2006 ◽  
Vol 80 (7) ◽  
pp. 3487-3494 ◽  
Author(s):  
Gerald Misinzo ◽  
Peter L. Delputte ◽  
Peter Meerts ◽  
David J. Lefebvre ◽  
Hans J. Nauwynck
Keyword(s):  
Heparan Sulfate ◽  
Chondroitin Sulfate ◽  
Pcv2 Infection ◽  
Porcine Circovirus ◽  
Wild Type Virus ◽  
Target Cells ◽  
Wild Type ◽  
Porcine Circovirus 2 ◽  
Pcv2 Replication

ABSTRACT Monocyte/macrophage lineage cells are target cells in vivo for porcine circovirus 2 (PCV2) replication. The porcine monocytic cell line 3D4/31 supports PCV2 replication in vitro, and attachment and internalization kinetics of PCV2 have been established in these cells. However, PCV2 receptors remain unknown. Glycosaminoglycans (GAG) are used by several viruses as receptors. The present study examined the role of GAG in attachment and infection of PCV2. Heparin, heparan sulfate (HS), chondroitin sulfate B (CS-B), but not CS-A, and keratan sulfate reduced PCV2 infection when these GAG were incubated with PCV2 prior to and during inoculation of 3D4/31 cells. Enzymatic removal of HS and CS-B prior to PCV2 inoculation of 3D4/31 cells significantly reduced PCV2 infection. Similarly, when PCV2 virus-like particles (VLP) were allowed to bind onto 3D4/31 cells in the presence of heparin and CS-B, attachment was strongly reduced. Titration of field isolates and low- and high-passage laboratory strains of PCV2 in the presence of heparin significantly reduced PCV2 titers, showing that the capacity of PCV2 to bind GAG was not acquired during in vitro cultivation but is an intrinsic feature of wild-type virus. When Chinese hamster ovary (CHO) cells were inoculated with PCV2, relative percentages of PCV2-infected cells were 27% ± 8% for HS-deficient and 12% ± 10% for GAG-deficient cells compared to wild-type cells (100%). Furthermore, it was shown using heparin-Sepharose chromatography that both PCV2 and PCV2 VLP directly interacted with heparin. Together, these results show that HS and CS-B are attachment receptors for PCV2.

scholarly journals Glycan Engagement Dictates Hydrocephalus Induction by Serotype 1 Reovirus

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Jennifer Stencel-Baerenwald ◽  
Kerstin Reiss ◽  
Bärbel S. Blaum ◽  
Daniel Colvin ◽  
Xiao-Nan Li ◽  
...  
Keyword(s):  
Cell Surface ◽  
Viral Disease ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Ependymal Cells ◽  
Target Cells ◽  
Type Virus ◽  
Wild Type ◽  
Newborn Mice ◽  
Serotype 1

ABSTRACTReceptors expressed on the host cell surface adhere viruses to target cells and serve as determinants of viral tropism. Several viruses bind cell surface glycans to facilitate entry, but the contribution of specific glycan moieties to viral disease is incompletely understood. Reovirus provides a tractable experimental model for studies of viral neuropathogenesis. In newborn mice, serotype 1 (T1) reovirus causes hydrocephalus, whereas serotype 3 (T3) reovirus causes encephalitis. T1 and T3 reoviruses engage distinct glycans, suggesting that glycan-binding capacity contributes to these differences in pathogenesis. Using structure-guided mutagenesis, we engineered a mutant T1 reovirus incapable of binding the T1 reovirus-specific glycan receptor, GM2. The mutant virus induced substantially less hydrocephalus than wild-type virus, an effect phenocopied by wild-type virus infection of GM2-deficient mice. In comparison to wild-type virus, yields of mutant virus were diminished in cultured ependymal cells, the cell type that lines the brain ventricles. These findings suggest that GM2 engagement targets reovirus to ependymal cells in mice and illuminate the function of glycan engagement in reovirus serotype-dependent disease.IMPORTANCEReceptor utilization strongly influences viral disease, often dictating host range and target cell selection. Different reovirus serotypes bind to different glycans, but a precise function for these molecules in pathogenesis is unknown. We used type 1 (T1) reovirus deficient in binding the GM2 glycan and mice lacking GM2 to pinpoint a role for glycan engagement in hydrocephalus caused by T1 reovirus. This work indicates that engagement of a specific glycan can lead to infection of specific cells in the host and consequent disease at that site. Since reovirus is being developed as a vaccine vector and oncolytic agent, understanding reovirus-glycan interactions may allow manipulation of reovirus glycan-binding properties for therapeutic applications.

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