scholarly journals A sequence-independent in vitro transposon-based strategy for efficient cloning of genomes of large DNA viruses as bacterial artificial chromosomes

2008 ◽  
Vol 37 (1) ◽  
pp. e2-e2 ◽  
Author(s):  
Fuchun Zhou ◽  
Qiuhua Li ◽  
Shou-Jiang Gao
Keyword(s):  
Bacterial Artificial Chromosomes ◽  
Dna Viruses ◽  
Artificial Chromosomes

scholarly journals Replication-Competent Bacterial Artificial Chromosomes of Marek's Disease Virus: Novel Tools for Generation of Molecularly Defined Herpesvirus Vaccines

2003 ◽  
Vol 77 (16) ◽  
pp. 8712-8718 ◽  
Author(s):  
Lawrence Petherbridge ◽  
Ken Howes ◽  
Susan J. Baigent ◽  
Melanie A. Sacco ◽  
Simon Evans ◽  
...  
Keyword(s):  
Dna Vaccine ◽  
Marek's Disease ◽  
Published Data ◽  
Marek’S Disease ◽  
Bacterial Artificial Chromosomes ◽  
Bac Clone ◽  
Artificial Chromosomes ◽  
Bac Clones

ABSTRACT Marek's disease (MD), a highly infectious disease caused by an oncogenic herpesvirus, is one of the few herpesvirus diseases against which live attenuated vaccines are used as the main strategy for control. We have constructed bacterial artificial chromosomes (BACs) of the CVI988 (Rispens) strain of the virus, the most widely used and effective vaccine against MD. Viruses derived from the BAC clones were stable after in vitro and in vivo passages and showed characteristics and growth kinetics similar to those of the parental virus. Molecular analysis of the individual BAC clones showed differences in the structure of the meq gene, indicating that the commercial vaccine contains virus populations with distinct genomic structures. We also demonstrate that, contrary to the published data, the sequence of the L-meq of the BAC clone did not show any frameshift. Virus stocks derived from one of the BAC clones (clone 10) induced 100 percent protection against infection by the virulent strain RB1B, indicating that BAC-derived viruses could be used with efficacies similar to those of the parental CVI988 vaccines. As a DNA vaccine, this BAC clone was also able to induce protection in 6 of 20 birds. Isolation of CVI988 virus from all of these six birds suggested that immunity against challenge was probably dependent on the reconstitution of the virus in vivo and that such viruses are also as immunogenic as the in vitro-grown BAC-derived or parental vaccine viruses. Although the reasons for the induction of protection only in a proportion of birds (33.3%) that received the DNA vaccine are not clear, this is most likely to be related to the suboptimal method of DNA delivery. The construction of the CVI988 BAC is a major step towards understanding the superior immunogenic features of CVI988 and provides the opportunity to exploit the power of BAC technology for generation of novel molecularly defined vaccines.

scholarly journals Genetic Stability of Bacterial Artificial Chromosome-Derived Human Cytomegalovirus during CultureIn Vitro

2016 ◽  
Vol 90 (8) ◽  
pp. 3929-3943 ◽  
Author(s):  
Isa Murrell ◽  
Gavin S. Wilkie ◽  
Andrew J. Davison ◽  
Evelina Statkute ◽  
Ceri A. Fielding ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Human Cytomegalovirus ◽  
Artificial Chromosome ◽  
Cell Types ◽  
Clinical Strain ◽  
Minimal Risk ◽  
Bacterial Artificial Chromosomes ◽  
Wild Type ◽  
Artificial Chromosomes

ABSTRACTClinical human cytomegalovirus (HCMV) strains invariably mutate when propagatedin vitro. Mutations in gene RL13 are selected in all cell types, whereas in fibroblasts mutants in the UL128 locus (UL128L; genes UL128, UL130, and UL131A) are also selected. In addition, sporadic mutations are selected elsewhere in the genome in all cell types. We sought to investigate conditions under which HCMV can be propagated without incurring genetic defects. Bacterial artificial chromosomes (BACs) provide a stable, genetically defined source of viral genome. Viruses were generated from BACs containing the genomes of strains TR, TB40, FIX, and Merlin, as well as from Merlin-BAC recombinants containing variant nucleotides in UL128L from TB40-BAC4 or FIX-BAC. Propagation of viruses derived from TR-BAC, TB40-BAC4, and FIX-BAC in either fibroblast or epithelial cells was associated with the generation of defects around the prokaryotic vector, which is retained in the unique short (US) region of viruses. This was not observed for Merlin-BAC, from which the vector is excised in derived viruses; however, propagation in epithelial cells was consistently associated with mutations in the unique longb′ (UL/b′) region, all impacting on gene UL141. Viruses derived from Merlin-BAC in fibroblasts had mutations in UL128L, but mutations occurred less frequently with recombinants containing UL128L nucleotides from TB40-BAC4 or FIX-BAC. Viruses derived from a Merlin-BAC derivative in which RL13 and UL128L were either mutated or repressed were remarkably stable in fibroblasts. Thus, HCMV containing a wild-type gene complement can be generatedin vitroby deriving virus from a self-excising BAC in fibroblasts and repressing RL13 and UL128L.IMPORTANCEResearchers should aim to study viruses that accurately represent the causative agents of disease. This is problematic for HCMV because clinical strains mutate rapidly when propagatedin vitro, becoming less cell associated, altered in tropism, more susceptible to natural killer cells, and less pathogenic. Following isolation from clinical material, HCMV genomes can be stabilized by cloning into bacterial artificial chromosomes (BACs), and then virus is regenerated by DNA transfection. However, mutations can occur not only during isolation prior to BAC cloning but also when virus is regenerated. We have identified conditions under which BAC-derived viruses containing an intact, wild-type genome can be propagatedin vitrowith minimal risk of mutants being selected, enabling studies of viruses expressing the gene complement of a clinical strain. However, even under these optimized conditions, sporadic mutations can occur, highlighting the advisability of sequencing the HCMV stocks used in experiments.

scholarly journals Herpesvirus BACs: Past, Present, and Future

2011 ◽  
Vol 2011 ◽  
pp. 1-16 ◽  
Author(s):  
Charles Warden ◽  
Qiyi Tang ◽  
Hua Zhu
Keyword(s):  
Vaccine Development ◽  
Genetic Manipulation ◽  
Large Family ◽  
Site Directed Mutagenesis ◽  
Bacterial Artificial Chromosomes ◽  
Dna Viruses ◽  
Recombinant Viruses ◽  
Artificial Chromosomes ◽  
Viral Genes ◽  
Functional Analyses

The herpesviridae are a large family of DNA viruses with large and complicated genomes. Genetic manipulation and the generation of recombinant viruses have been extremely difficult. However, herpesvirus bacterial artificial chromosomes (BACs) that were developed approximately 10 years ago have become useful and powerful genetic tools for generating recombinant viruses to study the biology and pathogenesis of herpesviruses. For example, BAC-directed deletion mutants are commonly used to determine the function and essentiality of viral genes. In this paper, we discuss the creation of herpesvirus BACs, functional analyses of herpesvirus mutants, and future applications for studies of herpesviruses. We describe commonly used methods to create and mutate herpesvirus BACs (such as site-directed mutagenesis and transposon mutagenesis). We also evaluate the potential future uses of viral BACs, including vaccine development and gene therapy.

Preliminary Anti-Coxsackie Activity of Novel 1-[4-(5,6-dimethyl(H)- 1H(2H)-benzotriazol-1(2)-yl)phenyl]-3-alkyl(aryl)ureas

2020 ◽  
Vol 16 (5) ◽  
pp. 677-688 ◽  
Author(s):  
Sandra Piras ◽  
Paola Corona ◽  
Roberta Ibba ◽  
Federico Riu ◽  
Gabriele Murineddu ◽  
...  
Keyword(s):  
Chronic Disease ◽  
Antiviral Activity ◽  
Wide Spectrum ◽  
Immunocompromised Patients ◽  
Human Pathogen ◽  
Alkyl Aryl ◽  
Dna Viruses ◽  
Coxsackievirus B ◽  
Selective Activity

Background: Coxsackievirus infections are associated with cases of aseptic meningitis, encephalitis, myocarditis, and some chronic disease. Methods: A series of benzo[d][1,2,3]triazol-1(2)-yl derivatives (here named benzotriazol-1(2)-yl) (4a-i, 5a-h, 6a-e, g, i, j and 7a-f, h-j) were designed, synthesized and in vitro evaluated for cytotoxicity and antiviral activity against two important human enteroviruses (HEVs) members of the Picornaviridae family [Coxsackievirus B 5 (CVB-5) and Poliovirus 1 (Sb-1)]. Results: Compounds 4c (CC50 >100 μM; EC50 = 9 μM), 5g (CC50 >100 μM; EC50 = 8 μM), and 6a (CC50 >100 μM; EC50 = 10 μM) were found active against CVB-5. With the aim of evaluating the selectivity of action of this class of compounds, a wide spectrum of RNA (positive- and negativesense), double-stranded (dsRNA) or DNA viruses were also assayed. For none of them, significant antiviral activity was determined. Conclusion: These results point towards a selective activity against CVB-5, an important human pathogen that causes both acute and chronic diseases in infants, young children, and immunocompromised patients.

Exploration of the in vitro cytotoxic and antiviral activities of different medicinal plants against infectious bursal disease (IBD) virus

2014 ◽  
Vol 9 (5) ◽  
pp. 531-542 ◽  
Author(s):  
Waqas Ahmad ◽  
Sohail Ejaz ◽  
Khaleeq Anwar ◽  
Muhammad Ashraf
Keyword(s):  
Medicinal Plants ◽  
Colorimetric Assay ◽  
Rna Virus ◽  
Infectious Bursal Disease ◽  
Dna Viruses ◽  
Mtt Colorimetric Assay ◽  
Bursal Disease ◽  
Dried Fruit ◽  
Antiviral Activities

AbstractInfectious bursal disease (IBD) caused by non-enveloped double stranded RNA virus is an acute and contagious poultry disease. Outbreak of IBD could result in 10–75% mortality of the birds; hence it has gained socio-economic importance worldwide. Medicinal plants have shown broad spectrum anti-viral activities against RNA and DNA viruses. Moringa oleifera Lam (MOL), Phyllanthus emblicus Linn (PEL), Glycyrrhiza glabra Linn (GGL), and Eugenia jambolana Lam (EJL) are commonly available medicinal plants of the sub-continent and exhibited anti-viral potential against different viruses. Ethanolic extracts of the leaves of MOL and EJL, roots of GGL and dried fruit of PEL were investigated for their cytotoxic and anti-viral potential against IBD virus using MTT colorimetric assay and anti-viral assay. Significant anti-viral potential (P<0.001) was demonstrated at concentrations 12.5, 25, 50 and 100 µg ml−1 of GGL, PEL, MOL and EJL, respectively, with no cytotoxicity. Data also spotlighted that all tested plant extracts possess significant anti-viral potential and this trend was higher in GGL followed by PEL, MOL, and EJL. The data undoubtedly conclude that these medicinal plants contain several health beneficial phyto-chemicals which got significant anti-viral potential and effectively be utilized against IBD virus. Moreover, the outcomes of this study provide a platform on the way to discover novel anti-viral agents against IBD virus and other viruses from plant origin.

scholarly journals Cross-Species RNAi Rescue Platform in Drosophila melanogaster

Genetics ◽  
2009 ◽  
Vol 183 (3) ◽  
pp. 1165-1173 ◽  
Author(s):  
Shu Kondo ◽  
Matthew Booker ◽  
Norbert Perrimon
Keyword(s):  
Cell Culture ◽  
Drosophila Melanogaster ◽  
Large Scale ◽  
Transgenic Animals ◽  
Selection Marker ◽  
Bacterial Artificial Chromosomes ◽  
Loss Of Function ◽  
Artificial Chromosomes ◽  
Target Effects

RNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster, both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.

Homologous Recombination Using Bacterial Artificial Chromosomes

2015 ◽  
Vol 2015 (2) ◽  
pp. pdb.prot072397
Author(s):  
Cary Lai ◽  
Tobias Fischer ◽  
Elizabeth Munroe
Keyword(s):  
Homologous Recombination ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes

scholarly journals The Nucleoprotein Is Required for Efficient Coronavirus Genome Replication

2004 ◽  
Vol 78 (22) ◽  
pp. 12683-12688 ◽  
Author(s):  
Fernando Almazán ◽  
Carmen Galán ◽  
Luis Enjuanes
Keyword(s):  
Heterologous Protein ◽  
Cytopathic Effect ◽  
Human Cell Line ◽  
Genome Replication ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes ◽  
Restriction Sites ◽  
Unique Restriction ◽  
Transmissible Gastroenteritis ◽  
Transmissible Gastroenteritis Coronavirus

ABSTRACT The construction of a set of transmissible gastroenteritis coronavirus (TGEV)-derived replicons as bacterial artificial chromosomes is reported. These replicons were generated by sequential deletion of nonessential genes for virus replication, using a modified TGEV full-length cDNA clone containing unique restriction sites between each pair of consecutive genes. Efficient activity of TGEV replicons was associated with the presence of the nucleoprotein provided either in cis or in trans. TGEV replicons were functional in several cell lines, including the human cell line 293T, in which no or very low cytopathic effect was observed, and expressed high amounts of heterologous protein.

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