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Frontiers in Bioscience (Landmark... Jan 2019The present study determines the cytokine gene expression in chickens following RSV-A infection, using RT-qPCR. In susceptible chickens tumors progressed to ...
The present study determines the cytokine gene expression in chickens following RSV-A infection, using RT-qPCR. In susceptible chickens tumors progressed to fulminating metastatic tumors while it regressed in regressors chickens and some resistant non-responder chickens did not respond to RSV-A infection and thus did not develop tumors at all. The expression of pro-inflammatory cytokines, Th1 cytokines and Th2 cytokines was determined at the primary site of infection, as well as in different organs of progressor, regressor and non-responder chicks at different time intervals. Our results indicated a significant upregulation of the pro-inflammatory cytokines, IL-6 and IL-8, in all the organs of progressor chicks, while they were significantly lower in regressor and non-responder chicks. The expression of the Th1 cytokines IFN-γ and TNF-α was low in all of the organs of the progressor group, except that in spleen. In contrast, regressor and non-responder groups showed high expression of IFN-γ and TNF-α. Further, there was an early upregulation of the expression of the Th2 cytokine, IL-10, TGF-β and GM-CSF, in all of the organs of progressors as compared to uninfected control.
Topics: Animals; Chickens; Cytokines; Gene Expression; Host-Pathogen Interactions; Inflammation Mediators; Rous sarcoma virus; Sarcoma, Avian; Th1 Cells; Th2 Cells; Up-Regulation
PubMed: 30468667
DOI: 10.2741/4729 -
Molecular Therapy. Nucleic Acids Mar 2022DNA-modifying technologies, such as the CRISPR-Cas9 system, are promising tools in the field of gene and cell therapies. However, high and prolonged expression of...
DNA-modifying technologies, such as the CRISPR-Cas9 system, are promising tools in the field of gene and cell therapies. However, high and prolonged expression of DNA-modifying enzymes may cause cytotoxic and genotoxic side effects and is therefore unwanted in therapeutic approaches. Consequently, development of new and potent short-term delivery methods is of utmost importance. Recently, we developed non-integrating gammaretrovirus- and MS2 bacteriophage-based Gag.MS2 (g.Gag.MS2) particles for transient transfer of non-retroviral CRISPR-Cas9 RNA into target cells. In the present study, we further improved the technique by transferring the system to the alpharetroviral vector platform (a.Gag.MS2), which significantly increased CRISPR-Cas9 delivery into target cells and allowed efficient targeted knockout of endogenous genes in primary murine fibroblasts as well as primary human fibroblasts, hepatocytes, and cord-blood-derived CD34 stem and progenitor cells. Strikingly, co-packaging of mRNA and multiple single guide RNAs (sgRNAs) into a.Gag.MS2 chimera displayed efficient targeted knockout of up to three genes. Co-transfection of single-stranded DNA donor oligonucleotides during CRISPR-Cas9 particle production generated all-in-one particles, which mediated up to 12.5% of homology-directed repair in primary cell cultures. In summary, optimized a.Gag.MS2 particles represent a versatile tool for short-term delivery of DNA-modifying enzymes into a variety of target cells, including primary murine and human cells.
PubMed: 35141043
DOI: 10.1016/j.omtn.2021.12.033 -
World Journal of Biological Chemistry Feb 2017Retroviral replication proceeds through the integration of a DNA copy of the viral RNA genome into the host cellular genome, a process that is mediated by the viral... (Review)
Review
Retroviral replication proceeds through the integration of a DNA copy of the viral RNA genome into the host cellular genome, a process that is mediated by the viral integrase (IN) protein. IN catalyzes two distinct chemical reactions: 3'-processing, whereby the viral DNA is recessed by a di- or trinucleotide at its 3'-ends, and strand transfer, in which the processed viral DNA ends are inserted into host chromosomal DNA. Although IN has been studied as a recombinant protein since the 1980s, detailed structural understanding of its catalytic functions awaited high resolution structures of functional IN-DNA complexes or intasomes, initially obtained in 2010 for the spumavirus prototype foamy virus (PFV). Since then, two additional retroviral intasome structures, from the α-retrovirus Rous sarcoma virus (RSV) and β-retrovirus mouse mammary tumor virus (MMTV), have emerged. Here, we briefly review the history of IN structural biology prior to the intasome era, and then compare the intasome structures of PFV, MMTV and RSV in detail. Whereas the PFV intasome is characterized by a tetrameric assembly of IN around the viral DNA ends, the newer structures harbor octameric IN assemblies. Although the higher order architectures of MMTV and RSV intasomes differ from that of the PFV intasome, they possess remarkably similar intasomal core structures. Thus, retroviral integration machineries have adapted evolutionarily to utilize disparate IN elements to construct convergent intasome core structures for catalytic function.
PubMed: 28289517
DOI: 10.4331/wjbc.v8.i1.32 -
Microorganisms Dec 2023The Genus contains viruses pathogenic mainly for chickens, forming the Avian Sarcoma and Leukosis Virus group (ASLV). Cells of most Galliform species, besides chickens,...
The Genus contains viruses pathogenic mainly for chickens, forming the Avian Sarcoma and Leukosis Virus group (ASLV). Cells of most Galliform species, besides chickens, contain genetic elements (endogenous retroviruses, ERVs) that could recombine with other alpharetroviruses or express proteins, complementing defective ASLV, which may successfully replicate and cause disease. However, they are quite unknown, and only ALV-F, from ring-necked pheasants, has been partially published. Upon scrutiny of 53 genomes of different avian species, we found -like sequences only in 12 different Galliformes, including six full-length (7.4-7.6 Kbp) and 27 partial sequences. Phylogenetic studies of the regions studied (LTR, , , and ) consistently resulted in five almost identical clades containing the same ERVs: Clade I (presently known ASLVs); Clade II ( spp. ERVs); Clade IIIa ( ERVs); Clade IIIb ( spp. ERVs); and Clade IV ( spp. ERVs). The low identity scores suggested that each of these Clades may be considered a different species. ORF analysis revealed that putatively encoded proteins would be very similar in length and domains to those of other alpharetroviruses and thus potentially functional. This will undoubtedly contribute to better understanding the biology of defective viruses, especially in wild Galliformes, their evolution, and the danger they may represent for other wild species and the poultry industry.
PubMed: 38257913
DOI: 10.3390/microorganisms12010086 -
Journal of Virology Sep 2022The receptor of the subgroup A avian leukosis virus (ALV-A) in chicken is Tva, which is the homologous protein of human CD320 (huCD320), contains a low-density...
The receptor of the subgroup A avian leukosis virus (ALV-A) in chicken is Tva, which is the homologous protein of human CD320 (huCD320), contains a low-density lipoprotein (LDL-A) module and is involved in the uptake of transcobalamin bound vitamin B/cobalamin (Cbl). To map the functional determinants of Tva responsible for ALV-A receptor activity, a series of chimeric receptors were created by swapping the LDL-A module fragments between huCD320 and Tva. These chimeric receptors were then used for virus entry and binding assays to map the minimal ALV-A functional domain of Tva. The results showed that Tva residues 49 to 71 constituted the minimal functional domain that directly interacted with the ALV-A gp85 protein to mediate ALV-A entry. Single-residue substitution analysis revealed that L55 and W69, which were spatially adjacent on the surface of the Tva structure, were key residues that mediate ALV-A entry. Structural alignment results indicated that L55 and W69 substitutions did not affect the Tva protein structure but abolished the interaction force between Tva and gp85. Furthermore, substituting the corresponding residues of huCD320 with L55 and W69 of Tva converted huCD320 into a functional receptor of ALV-A. Importantly, soluble huCD320 harboring Tva L55 and W69 blocked ALV-A entry. Finally, we constructed a gene-edited cell line with L55R and W69L substitutions that could fully resist ALV-A entry, while Cbl uptake was not affected. Collectively, our findings suggested that amino acids L55 and W69 of Tva were key for mediating virus entry. Retroviruses bind to cellular receptors through their envelope proteins, which is a crucial step in infection. While most retroviruses require two receptors for entry, ALV-A requires only one. Various alleles conferring resistance to ALV-A, including (C40W substitution), (frame-shifting four-nucleotide insertion), , , , and (deletion in the first intron), are known. However, the detailed entry mechanism of ALV-A in chickens remains to be explored. We demonstrated that Tva residues L55 and W69 were key for ALV-A entry and were important for correct interaction with ALV-A gp85. Soluble Tva and huCD320 harboring the Tva residues L55 and W69 effectively blocked ALV-A infection. Additionally, we constructed gene-edited cell lines targeting these two amino acids, which completely restricted ALV-A entry without affecting Cbl uptake. These findings contribute to a better understanding of the infection mechanism of ALV-A and provided novel insights into the prevention and control of ALV-A.
Topics: Amino Acids; Animals; Avian Leukosis; Avian Leukosis Virus; Avian Proteins; Chickens; Humans; Lipoproteins, LDL; Nucleotides; Receptors, Virus; Transcobalamins; Vitamin B 12
PubMed: 36069550
DOI: 10.1128/jvi.00678-22 -
Journal of Wildlife Diseases Jul 2022Growing populations of Wild Turkeys (Meleagris gallopavo) may result in increased disease transmission among wildlife and spillover to poultry. Lymphoproliferative...
Growing populations of Wild Turkeys (Meleagris gallopavo) may result in increased disease transmission among wildlife and spillover to poultry. Lymphoproliferative disease virus (LPDV) is an avian retrovirus that is widespread in Wild Turkeys of eastern North America, and infections may influence mortality and parasite co-infections. We aimed to identify individual and spatial risk factors of LPDV in Maine's Wild Turkeys. We also surveyed for co-infections between LPDV and reticuloendotheliosis virus (REV), Mycoplasma gallisepticum, and Salmonella pullorum to estimate trends in prevalence and examine covariance with LPDV. From 2017 to 2020, we sampled tissues from hunter-harvested (n=72) and live-captured (n=627) Wild Turkeys, in spring and winter, respectively, for molecular detection of LPDV and REV. In a subset of captured individuals (n=235), we estimated seroprevalence of the bacteria M. gallisepticum and S. pullorum using a plate agglutination test. Infection rates for LPDV and REV were 59% and 16% respectively, with a co-infection rate of 10%. Seroprevalence for M. gallisepticum and S. pullorum were 74% and 3.4%, with LPDV co-infection rates of 51% and 2.6%, respectively. Infection with LPDV and seroprevalence of M. gallisepticum and S. pullorum decreased, whereas REV infection increased, between 2018 and 2020. Females (64%), adults (72%), and individuals sampled in spring (76%) had higher risks of LPDV infection than males (47%), juveniles (39%), and individuals sampled in winter (57%). Furthermore, LPDV infection increased with percent forested cover (β=0.014±0.007) and decreased with percent agriculture cover for juveniles (β=-0.061±0.018) sampled in winter. These data enhance our understanding of individual and spatial predictors of LPDV infection in Wild Turkeys and aid in assessing the associated risk to Wild Turkey populations and poultry operations.
Topics: Alpharetrovirus; Animals; Animals, Wild; Bird Diseases; Coinfection; Female; Male; Poultry; Reticuloendotheliosis virus; Seroepidemiologic Studies; Turkeys; Virus Diseases
PubMed: 35704504
DOI: 10.7589/JWD-D-21-00152 -
Frontiers in Immunology 2022Avian leukosis virus (ALV) causes various diseases associated with tumor formation and decreased fertility. Moreover, ALV induces severe immunosuppression, increasing... (Review)
Review
Avian leukosis virus (ALV) causes various diseases associated with tumor formation and decreased fertility. Moreover, ALV induces severe immunosuppression, increasing susceptibility to other microbial infections and the risk of failure in subsequent vaccination against other diseases. There is growing evidence showing the interaction between ALV and the host. In this review, we will survey the present knowledge of the involvement of host factors in the important molecular events during ALV infection and discuss the futuristic perspectives from this angle.
Topics: Animals; Avian Leukosis; Avian Leukosis Virus; Chickens; Virus Replication
PubMed: 35693802
DOI: 10.3389/fimmu.2022.907287 -
Viruses Jan 2019Jan Svoboda triggered investigations on non-defective avian sarcoma viruses. These viruses were a critical factor in the genetic understanding of retroviruses. They... (Review)
Review
Jan Svoboda triggered investigations on non-defective avian sarcoma viruses. These viruses were a critical factor in the genetic understanding of retroviruses. They provided the single and unique access to the field and facilitated the discovery of the first oncogene and of the cellular origin of retroviral oncogenes. They continue to be of importance as singularly effective expression vectors that have provided insights into the molecular functions of numerous oncogenes. Combined with the contributions to the validation of the provirus hypothesis, Jan Svoboda's investigations of non-defective avian sarcoma viruses have shaped a large and important part of retrovirology.
Topics: Animals; Avian Sarcoma Viruses; Genes, Viral; Humans; Oncogenes; Proviruses
PubMed: 30669277
DOI: 10.3390/v11010080 -
Viruses Apr 2018The Czech scientist Jan Svoboda was a pioneer of Rous sarcoma virus (RSV). In the 1960s, before the discovery of reverse transcriptase, he demonstrated the long-term...
The Czech scientist Jan Svoboda was a pioneer of Rous sarcoma virus (RSV). In the 1960s, before the discovery of reverse transcriptase, he demonstrated the long-term persistence of the viral genome in non-productive mammalian cells, and he supported the DNA provirus hypothesis of Howard Temin. He showed how the virus can be rescued in the infectious form and elucidated the replication-competent nature of the Prague strain of RSV later used for the identification of the oncogene. His studies straddled molecular oncology and virology, and he remained an active contributor to the field until his death last year. Throughout the 50 years that I was privileged to know Svoboda as my mentor and friend, I admired his depth of scientific inquiry and his steadfast integrity in the face of political oppression.
Topics: Animals; History, 20th Century; History, 21st Century; Host-Pathogen Interactions; Humans; Rous sarcoma virus; Sarcoma, Avian; Virus Replication
PubMed: 29670049
DOI: 10.3390/v10040203 -
Current Protocols in Molecular Biology Jan 2018The RCAS (replication-competent avian sarcoma leukosis virus long-terminal repeat with splice acceptor)-TVA (tumor virus A) gene delivery system has been successfully...
The RCAS (replication-competent avian sarcoma leukosis virus long-terminal repeat with splice acceptor)-TVA (tumor virus A) gene delivery system has been successfully used in modeling human cancers. Based on this, we have recently developed a novel RCI-Oncogene (RCAS-Cre-IRES-Oncogene) gene delivery system that can be used to efficiently manipulate gene expression in spontaneous tumors in vivo. We used this system for tumor gene knockout (TuKO) and demonstrated a crucial role of FGFR1 in driving mammary tumor metastasis. This versatile tumor gene modification system can also be adapted into different configurations to address different questions in appropriate mutant mouse hosts. Here we describe a protocol using the TuKO approach to knock out a gene of interest in tumors in appropriate hosts. © 2018 by John Wiley & Sons, Inc.
Topics: Alpharetrovirus; Animals; Female; Gene Knockout Techniques; Gene Transfer Techniques; Humans; Mammary Neoplasms, Animal; Mice; Oncogenes; Receptor, Fibroblast Growth Factor, Type 1
PubMed: 29337371
DOI: 10.1002/cpmb.54