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1.
Forensic Sci Int Genet ; 48: 102353, 2020 09.
Article in English | MEDLINE | ID: mdl-32668397

ABSTRACT

The imprints left by persistent DNA viruses in the tissues can testify to the changes driving virus evolution as well as provide clues on the provenance of modern and ancient humans. However, the history hidden in skeletal remains is practically unknown, as only parvovirus B19 and hepatitis B virus DNA have been detected in hard tissues so far. Here, we investigated the DNA prevalences of 38 viruses in femoral bone of recently deceased individuals. To this end, we used quantitative PCRs and a custom viral targeted enrichment followed by next-generation sequencing. The data was analyzed with a tailor-made bioinformatics pipeline. Our findings revealed bone to be a much richer source of persistent DNA viruses than earlier perceived, discovering ten additional ones, including several members of the herpes- and polyomavirus families, as well as human papillomavirus 31 and torque teno virus. Remarkably, many of the viruses found have oncogenic potential and/or may reactivate in the elderly and immunosuppressed individuals. Thus, their persistence warrants careful evaluation of their clinical significance and impact on bone biology. Our findings open new frontiers for the study of virus evolution from ancient relics as well as provide new tools for the investigation of human skeletal remains in forensic and archaeological contexts.


Subject(s)
DNA, Viral/analysis , Femur/chemistry , Forensic Genetics , Adult , Aged , Aged, 80 and over , Female , Femur/virology , Genotype , High-Throughput Nucleotide Sequencing , Humans , Male , Middle Aged , Parvovirus B19, Human/genetics , Polymerase Chain Reaction , Sequence Analysis, DNA
2.
Vet J ; 198(2): 376-81, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24135548

ABSTRACT

Transplacental viral infection of the fetus can result in abnormal trabecular and cortical bone modeling in long bones through impaired bone resorption and formation. Although such infections are frequently associated with neonatal fractures in humans and animals, their effect on the biomechanical properties of the developing skeleton remain poorly understood. The goal of this study was to determine the effects of transplacental bovine viral diarrhea virus (BVDV) infection on the biomechanical properties of fetal femora. Pregnant heifers were inoculated intranasally with non-cytopathic BVDV or media alone on day 75 of gestation to produce persistently infected (PI) and control fetuses, respectively, which were then removed on days 192 and 245 of gestation. Histomorphometry, compositional analysis and 'four-point bending until failure' were performed on fetal femora. Altered cortical geometry largely accounted for differences in calculated elastic modulus (PI vs. control, and day 192 vs. day 245) and ultimate stress (day 192 vs. day 245). Fetal infection with BVDV did not significantly impair inherent biomechanical properties of bone but rather resulted in decreased periosteal apposition rates, manifested as smaller femoral mid-diaphyseal diameters. There were no differences between PI and control fetuses in cortical thickness ratio, ash density or calcium/phosphorous content; however, cortical thickness ratio decreased with fetal age. Thus even when cortical thickness ratios are similar, differences in mid-diaphyseal diameter affect the error associated with the calculation of stress and strain by classical beam theory equations.


Subject(s)
Bovine Virus Diarrhea-Mucosal Disease/transmission , Diarrhea Viruses, Bovine Viral/physiology , Femur/virology , Fetus/embryology , Hemorrhagic Syndrome, Bovine/transmission , Infectious Disease Transmission, Vertical/veterinary , Pregnancy Complications, Infectious/veterinary , Uterus/virology , Animals , Biomechanical Phenomena , Bovine Virus Diarrhea-Mucosal Disease/physiopathology , Bovine Virus Diarrhea-Mucosal Disease/virology , Cattle , Female , Femur/anatomy & histology , Femur/physiology , Hemorrhagic Syndrome, Bovine/physiopathology , Hemorrhagic Syndrome, Bovine/virology , Pregnancy , Pregnancy Complications, Infectious/physiopathology , Pregnancy Complications, Infectious/virology
3.
Methods Mol Biol ; 737: 367-90, 2011.
Article in English | MEDLINE | ID: mdl-21590405

ABSTRACT

In vivo targeted gene delivery to hematopoietic stem cells (HSCs) would mean a big step forward in the field of gene therapy. This would imply that the risk of cell differentiation and loss of homing/-engraftment is reduced, as there is no need for purification of the target cell. In vivo gene delivery also bypasses the issue that no precise markers that permit the isolation of a primitive hHSC exist up to now. Indeed, in vivo gene transfer could target all HSCs in their stem-cell niche, including those cells that are "missed" by the purification criteria. Moreover, for the majority of diseases, there is a requirement of a minimal number of gene-corrected cells to be reinfused to allow an efficient long-term engraftment. This requisite might become a limiting factor when treating children with inherited disorders, due to the low number of bone marrow (BM) CD34(+) HSCs that can actually be isolated. These problems could be overcome by using efficient in vivo HSC-specific lentiviral vectors (LVs). Additionally, vectors for in vivo HSC transduction must be specific for the target cell, to avoid vector spreading while enhancing transduction efficiency. Of importance, a major barrier in LV transduction of HSCs is that 75% of HSCs are residing in the G0 phase of the cell cycle and are not very permissive for classical VSV-G-LV transduction. Therefore, we engineered "early-activating-cytokine (SCF or/and TPO)" displaying LVs that allowed a slight and transient stimulation of hCD34(+) cells resulting in efficient lentiviral gene transfer while preserving the "stemness" of the targeted HSCs. The selective transduction of HSCs by these vectors was demonstrated by their capacity to promote selective transduction of CD34(+) cells in in vitro-derived, long-term culture-initiating cell colonies and long-term NOD/SCID repopulating cells. A second generation of these "early-acting-cytokine"-displaying lentiviral vectors has now been developed that is fit for targeted in vivo gene delivery to hCD34(+) cells. In the method presented here, we describe the in vivo gene delivery into hCD34(+) cells by intramarrow injection of these new vectors into humanized BALB/c Rag2( null )/IL2rgc ( null ) (BALB/c RAGA) mice.


Subject(s)
Antigens, CD34/metabolism , Gene Transfer Techniques , Genetic Therapy/methods , Transduction, Genetic/methods , Animals , Cell Line , Clinical Trials as Topic , Cloning, Molecular/methods , Cytokines/genetics , Cytokines/metabolism , Femur/virology , Genetic Engineering , Genetic Vectors/biosynthesis , Genetic Vectors/isolation & purification , Genetic Vectors/therapeutic use , Hematopoietic Stem Cells/virology , Humans , Injections/methods , Lentivirus/genetics , Lentivirus/metabolism , Mice , Mice, Inbred BALB C , Mice, Transgenic , Recombinant Fusion Proteins/metabolism , Virion/genetics , Virion/metabolism
4.
Transplant Proc ; 37(6): 2799-801, 2005.
Article in English | MEDLINE | ID: mdl-16182813

ABSTRACT

We report the microbiological contamination rate of sterilely procured 3953 tissue allografts obtained during 22 years of activity for musculoskeletal reconstruction from 1982 to 2003. From 1987 to 2000, allograft retrievals were performed in 191 cadaveric donors and in 323 living donors. In the former group 30 retrievals (15.7%) were excluded based on laboratory criteria. Among living donors 108 femoral heads (33.4%) were also excluded by the same criteria. The microbiological contamination rate of sterilely procured allografts in the operating room was 8.3% for cadaveric donors and 18.2% for living donors. A questionable positive serology for HIV antigen was registered in two non-heart-beating donors. Hepatitis C virus antibodies were positive in two other non-heart-beating donors. Hepatitis B virus serological markers were positive or questionable in more than 11 non-heart-beating donors. In living donors 20 femoral heads were excluded (6.1%) due a positive or questionable hepatitis B virus serology. One femoral head donor showed a positive HTLV-I antibody and another one a positive syphilis serology. No positive serology cases for the HIV antibodies were found. No cases were registered of transmission of viral diseases from the donor to the recipient. Our extremely rigorous criteria led to the exclusion of a considerable number of both donors and allografts.


Subject(s)
Bone Banks , Tissue Banks , Transplantation, Homologous/statistics & numerical data , Cadaver , Femur/microbiology , Femur/virology , HTLV-I Antibodies/analysis , Hospitals, University , Humans , Living Donors/statistics & numerical data , Portugal , Retrospective Studies , Syphilis/diagnosis , Tissue Donors/statistics & numerical data , Transplantation, Homologous/adverse effects
5.
J Gene Med ; 7(6): 782-91, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15693034

ABSTRACT

With the aim of simplifying recombinant-adeno-associated virus (rAAV) delivery in muscle, a new femoral intra-arterial technique was designed and tested in rodents (rats and mice). Two serotypes, several promoters and transgenes (reporter or therapeutic) were tested using this administration route. The new route is both easy to perform and efficient. Its usefulness as a tool to assess gene delivery constructs in the muscle was established in the context of recombinant AAV serotypes 1 and 2, and with the ubiquitous CMV and two muscle-specific (C5-12 and CK6) promoters. Both serum monitoring of a secreted protein (murine alkaline phosphatase: muSEAP) and slide staining were used to compare the different constructs. Significantly different patterns of expression in kinetics of expression (muSEAP) and homogeneity of fiber transduction (staining) were evidenced with the different promoters tested, and compared with intra-muscular expression patterns. Detailed studies of differential transduction in leg and thigh muscles showed equivalent efficacy, except in rectus femoris, and to a lesser extent in soleus. In light of these results and prior data, intra-arterially mediated gene transfer mechanism is discussed.


Subject(s)
Dependovirus/genetics , Femur/virology , Gene Transfer Techniques , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Recombinant Proteins , Alkaline Phosphatase/metabolism , Animals , Cytomegalovirus/genetics , Dependovirus/classification , Femur/blood supply , Femur/pathology , Gene Expression , Genes, Reporter , Injections, Intra-Arterial , Kinetics , Lower Extremity/blood supply , Lower Extremity/pathology , Lower Extremity/virology , Luciferases/metabolism , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Muscle, Skeletal/enzymology , Promoter Regions, Genetic , Rats , Rats, Sprague-Dawley , Serotyping , Transduction, Genetic , Transgenes
6.
Biologicals ; 31(1): 75-82, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12623062

ABSTRACT

Several virus inactivation procedures like heat treatment, gamma irradiation and chemical sterilization are used to increase the safety of bone tissue transplants. In this study we present data on the virus-inactivating effect of heat disinfection on human femoral heads, using the Marburg bone bank system 'Lobator sd-2'. Three enveloped viruses (human immunodeficiency virus type 2 [HIV-2], bovine viral diarrhoea virus as a model for Hepatitis C virus [HCV], and the herpesvirus pseudorabies virus), and three non-enveloped viruses (hepatitis A virus, poliomyelitis virus, and bovine parvovirus) were investigated. In a model system the central part of human femoral heads was contaminated with the respective cell-free virus suspension, establishing a direct contact between virus and native bone tissue. The core temperature in the femoral heads during the sterilization process was determined in additional model experiments. A temperature of 82.5 degrees C, given by the manufacturer as the effective temperature for virus inactivation, was maintained for at least 15 min in decartilaged femoral heads with a diameter of < or = 56 mm. Heat treatment using the Lobator sd-2 inactivated all viruses in human femoral heads below the detection limit (at least by a factor of > or =4 log(10)). By combining a well-focussed anamnesis of the donors and serological testing for relevant infection markers (anti-HIV-1/2, HBsAg, anti-HBcore, anti-HCV, TPHA) with heat treatment of femoral heads in the Lobator sd-2 system, a high safety level is achieved. To further increase virus safety of cadaveric bone transplants, it is recommended that multi-organ donors are tested by nucleic acid testing (i.e. polymerase chain reaction) for HIV, HBV and HCV genome.


Subject(s)
Femur/virology , Hot Temperature , Tissue Banks , Virus Inactivation , Humans , Kinetics
7.
Clin Orthop Relat Res ; (324): 275-82, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8595768

ABSTRACT

The transmission of a retrovirus through transplantation of processed bone allografts was studied using the feline leukemia virus. The long bones of 4 previously infected donor cats were harvested and assigned to 1 of 3 treatment groups: single freeze/thaw cycle, double freeze/thaw cycle, or double freeze/thaw cycle with water flush to remove bone marrow. Cortical bone grafts and corticocancellous bone grafts from each treatment group were transplanted into individual specific-pathogen-free recipients. Samples of plasma were obtained weekly from all recipients and were tested with an enzyme-linked immunosorbent assay to detect viral antigen. For animals that tested consistently negative for viral antigen, plasma samples also were tested for antiviral antibody to feline leukemia virus measured by live cell immunofluorescence. The results of the antigen and antibody testing revealed that all of the cortical and corticocancellous bone allografts in each of the 3 treatment groups transmitted virus. The ability of the treated bone allografts to transmit a feline retrovirus suggests that routine processing and removal of bone marrow may not inhibit their ability to transmit other retroviruses, such as the human immunodeficiency virus.


Subject(s)
Bone Transplantation , Leukemia Virus, Feline , Retroviridae Infections/transmission , Animals , Antigens, Viral/analysis , Bone Transplantation/pathology , Cats , Culture Techniques , Enzyme-Linked Immunosorbent Assay , Extremities , Femur/pathology , Femur/transplantation , Femur/virology , Humerus/pathology , Humerus/transplantation , Humerus/virology , Leukemia Virus, Feline/immunology , Tissue Preservation , Transplantation, Homologous
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