scholarly journals Rapid Construction of a Replication-Competent Infectious Clone of Human Adenovirus Type 14 by Gibson Assembly

Viruses ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 568 ◽  
Author(s):  
Haibin Pan ◽  
Yuqian Yan ◽  
Jing Zhang ◽  
Shan Zhao ◽  
Liqiang Feng ◽  
...  
Keyword(s):  
Infectious Clone ◽  
A549 Cells ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Enzyme Analysis ◽  
Gibson Assembly ◽  
Assembly Method ◽  
Rapid Construction ◽  
One Step ◽  
Genomic Variant

In 1955, Human adenovirus type 14 (HAdV-B14p) was firstly identified in a military trainee diagnosed as acute respiratory disease (ARD) in the Netherlands. Fifty years later, a genomic variant, HAdV-B14p1, re-emerged in the U.S. and caused large and fatal ARD outbreaks. Subsequently, more and more ARD outbreaks occurred in Canada, the UK, Ireland, and China, in both military and civil settings. To generate a tool for the efficient characterization of this new genomic variant, a full-length infectious genomic clone of HAdV-B14 was successfully constructed using one-step Gibson Assembly method in this study. Firstly, the full genome of HAdV-B14p1 strain GZ01, the first HAdV-B14 isolate in China, was assembled into pBR322 plasmid by Gibson Assembly. The pBRAdV14 plasmid, generated by Gibson Assembly, was analyzed and verified by PCR, restriction enzymes digestion and the sequencing. Secondly, viruses were rescued from pBRAdV14-transfected A549 cells. The integrity of the rescued viruses was identified by restriction enzyme analysis. The complete sequence of the infectious clone was further sequenced. No mutation was found in the infectious clone during the construction when compared with the parental virus and pBR322 sequences. The direct immunofluorescence assay indicated the expression of the hexon protein. Finally, typical virions were observed; the one-step growth curves further showed that the DNA replication and viral reproduction efficiency of pBRAd14 derived viruses was similar with that of wild-type HAdV-B14 strain. The successful construction of the replication-competent infectious clone of pBRAdV14 facilitates the development of vaccine and antiviral drugs against HAdV-B14, as well as provides a novel strategy for rapid construction of infectious viral clones for other large-genome DNA viruses.

scholarly journals iTRAQ-Based and Label-Free Proteomics Approaches for Studies of Human Adenovirus Infections

2013 ◽  
Vol 2013 ◽  
pp. 1-16 ◽  
Author(s):  
Hung V. Trinh ◽  
Jonas Grossmann ◽  
Peter Gehrig ◽  
Bernd Roschitzki ◽  
Ralph Schlapbach ◽  
...  
Keyword(s):  
A549 Cells ◽  
Correlation Coefficients ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Label Free ◽  
Absolute Quantitation ◽  
Software Packages ◽  
Itraq Label ◽  
Isobaric Tags ◽  
Type 3

Both isobaric tags for relative and absolute quantitation (iTRAQ) and label-free methods are widely used for quantitative proteomics. Here, we provide a detailed evaluation of these proteomics approaches based on large datasets from biological samples. iTRAQ-label-based and label-free quantitations were compared using protein lysate samples from noninfected human lung epithelial A549 cells and from cells infected for 24 h with human adenovirus type 3 or type 5. Either iTRAQ-label-based or label-free methods were used, and the resulting samples were analyzed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS). To reduce a possible bias from quantitation software, we applied several software packages for each procedure. ProteinPilot and Scaffold Q+ software were used for iTRAQ-labeled samples, while Progenesis LC-MS and ProgenesisF-T2PQ/T3PQ were employed for label-free analyses. R2 correlation coefficients correlated well between two software packages applied to the same datasets with values between 0.48 and 0.78 for iTRAQ-label-based quantitations and 0.5 and 0.86 for label-free quantitations. Analyses of label-free samples showed higher levels of protein up- or downregulation in comparison to iTRAQ-labeled samples. The concentration differences were further evaluated by Western blotting for four downregulated proteins. These data suggested that the label-free method was more accurate than the iTRAQ method.

scholarly journals Replication Properties of Human Adenovirus In Vivo and in Cultures of Primary Cells from Different Animal Species

2006 ◽  
Vol 80 (7) ◽  
pp. 3549-3558 ◽  
Author(s):  
Christian Jogler ◽  
Dennis Hoffmann ◽  
Dirk Theegarten ◽  
Thomas Grunwald ◽  
Klaus Überla ◽  
...  
Keyword(s):  
Viral Load ◽  
Animal Species ◽  
A549 Cells ◽  
Human Adenovirus ◽  
Fold Increase ◽  
Adenovirus Type ◽  
Adenoviral Vectors ◽  
Treatment Modalities ◽  
Late Gene Expression

ABSTRACT Oncolytic adenoviruses have emerged as a promising approach for the treatment of tumors resistant to other treatment modalities. However, preclinical safety studies are hampered by the lack of a permissive nonhuman host. Screening of a panel of primary cell cultures from seven different animal species revealed that porcine cells support productive replication of human adenovirus type 5 (Ad5) nearly as efficiently as human A549 cells, while release of infectious virus by cells from other animal species tested was diminished by several orders of magnitude. Restriction of productive Ad5 replication in rodent and rabbit cells seems to act primarily at a postentry step. Replication efficiency of adenoviral vectors harboring different E1 deletions or mutations in porcine cells was similar to that in A549 cells. Side-by-side comparison of the viral load kinetics in blood of swine and mice injected with Ad5 or a replication-deficient adenoviral vector failed to provide clear evidence for virus replication in mice. In contrast, evidence suggests that adenovirus replication occurs in swine, since adenoviral late gene expression produced a 13.5-fold increase in viral load in an individual swine from day 3 to day 7 and 100-fold increase in viral DNA levels in the Ad5-infected swine compared to the animal receiving a replication-deficient adenovirus. Lung histology of Ad5-infected swine revealed a severe interstitial pneumonia. Although the results in swine are based on a small number of animals and need to be confirmed, our data strongly suggest that infection of swine with human adenovirus or oncolytic adenoviral vectors is a more appropriate animal model to study adenoviral pathogenicity or pharmacodynamic and toxicity profiles of adenoviral vectors than infection of mice.

Quantitative proteomic analysis of A549 cells infected with human adenovirus type 2

2019 ◽  
Vol 91 (7) ◽  
pp. 1239-1249 ◽  
Author(s):  
Kareem R. Badr ◽  
Juliana A. Parente‐Rocha ◽  
Lilian C. Baeza ◽  
Fabiola S. Ficcadori ◽  
Menira Souza ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
A549 Cells ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Quantitative Proteomic Analysis ◽  
Adenovirus Type 2

scholarly journals Home-made enzymatic premix and Illumina sequencing allow for one-step Gibson assembly and verification of virus infectious clones

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Mingmin Zhao ◽  
Beatriz García ◽  
Araiz Gallo ◽  
Ioannis E. Tzanetakis ◽  
Carmen Simón-Mateo ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
De Novo ◽  
Rna Virus ◽  
Infectious Clone ◽  
Gibson Assembly ◽  
Infectious Clones ◽  
Universal Approach ◽  
One Step ◽  
Step Generation

AbstractAn unprecedented number of viruses have been discovered by leveraging advances in high-throughput sequencing. Infectious clone technology is a universal approach that facilitates the study of biology and role in disease of viruses. In recent years homology-based cloning methods such as Gibson assembly have been used to generate virus infectious clones. We detail herein the preparation of home-made cloning materials for Gibson assembly. The home-made materials were used in one-step generation of the infectious cDNA clone of a plant RNA virus into a T-DNA binary vector. The clone was verified by a single Illumina reaction and a de novo read assembly approach that required no primer walking, custom primers or reference sequences. Clone infectivity was finally confirmed by Agrobacterium-mediated delivery to host plants. We anticipate that the convenient home-made materials, one-step cloning and Illumina verification strategies described herein will accelerate characterization of viruses and their role in disease development.

Construction of an infectious clone of human adenovirus type 41

2012 ◽  
Vol 157 (7) ◽  
pp. 1313-1321 ◽  
Author(s):  
Duo-Ling Chen ◽  
Liu-Xin Dong ◽  
Meng Li ◽  
Xiao-Juan Guo ◽  
Min Wang ◽  
...  
Keyword(s):  
Infectious Clone ◽  
Human Adenovirus ◽  
Adenovirus Type

scholarly journals A naturally occurring human adenovirus type 7 variant with a 1743 bp deletion in the E3 cassette

2011 ◽  
Vol 92 (10) ◽  
pp. 2399-2404 ◽  
Author(s):  
Juliana C. Marinheiro ◽  
Tatiana G. dos Santos ◽  
Joselma Siqueira-Silva ◽  
Xiaoyan Lu ◽  
Daniela Carvalho ◽  
...  
Keyword(s):  
Risk Factors ◽  
Restriction Analysis ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Medical Attention ◽  
Acute Respiratory Disease ◽  
Genome Type ◽  
Genomic Variants ◽  
Naturally Occurring ◽  
Genomic Variant

Human adenovirus type 7 (HAdV-7) is an important cause of acute respiratory disease (ARD). Different genomic variants of HAdV-7 have been described, designated 7a–7l. In a previous study to investigate risk factors for ARD and wheezing, nasopharyngeal samples were collected from 90 ill children seeking medical attention in Ribeirão Preto, São Paulo, Brazil. HAdVs were identified in 31 samples and were characterized by serum neutralization and genome restriction analysis. Eleven HAdVs were identified as being HAdV-7, five of which were classified as being of genome type 7p (Gomen). Six other HAdV-7 isolates gave new restriction profiles with all enzymes used and were classified as being a new genomic variant, 7m. These isolates were further characterized by sequencing. The hexon and fiber genes of the 7m variant were nearly identical to the prototype, 7p. However, nucleotide sequences from the E3 cassette revealed a 1743 bp deletion affecting the 16.1K, 19K, 20.1K and 20.5K ORFs.

scholarly journals Fatal Neonatal Sepsis Associated with Human Adenovirus Type 56 Infection: Genomic Analysis of Three Recent Cases Detected in the United States

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1105
Author(s):  
William R. Otto ◽  
Daryl M. Lamson ◽  
Gabriel Gonzalez ◽  
Geoffrey A. Weinberg ◽  
Nicole D. Pecora ◽  
...  
Keyword(s):  
United States ◽  
Neonatal Sepsis ◽  
Fatal Case ◽  
Genomic Analysis ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
The United States ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Genome Sequences

Background: Human adenovirus (HAdV)-D56 was first described in 2011 by genomics analysis of a strain isolated in France in 2008 from a fatal case of neonatal infection. Since then, it has been reported in cases of keratoconjunctivitis and male urethritis. Three epidemiologically unrelated fatal cases of neonatal sepsis associated with infection by HAdV-D strains with a similar genetic makeup were documented in the United States between 2014 and 2020. Methods: Whole genome sequences were obtained for the isolated strains, and genomics analyses were conducted to compare them to phylogenetically related HAdV-D genomic sequences available in GenBank. Results: The three new US strains were indistinguishable by in silico restriction enzyme analysis. Their genome sequences were 99.9% identical to one another and to the prototype strain isolated in 2008 from a similar context of disease. The phylogenetic reconstruction revealed a highly supported clustering of all HAdV-D56 strains isolated in various countries since 1982. Our comparison to serologically intermediate strains 15/H9 described in the literature indicated that HAdV-D56-like viruses have circulated worldwide since the late 1950s. Conclusion: As with other HAdV-D genotypes with the ability to infect ocular and genital mucosae, the risk of severe prenatal or perinatal HAdV-D56 infection must be considered.

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