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Viruses Oct 2016Viruses must continuously evolve to hijack the host cell machinery in order to successfully replicate and orchestrate key interactions that support their persistence.... (Review)
Review
Viruses must continuously evolve to hijack the host cell machinery in order to successfully replicate and orchestrate key interactions that support their persistence. The type-1 human immunodeficiency virus (HIV-1) is a prime example of viral persistence within the host, having plagued the human population for decades. In recent years, advances in cellular imaging and molecular biology have aided the elucidation of key steps mediating the HIV-1 lifecycle and viral pathogenesis. Super-resolution imaging techniques such as stimulated emission depletion (STED) and photoactivation and localization microscopy (PALM) have been instrumental in studying viral assembly and release through both cell-cell transmission and cell-free viral transmission. Moreover, powerful methods such as Forster resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) have shed light on the protein-protein interactions HIV-1 engages within the host to hijack the cellular machinery. Specific advancements in live cell imaging in combination with the use of multicolor viral particles have become indispensable to unravelling the dynamic nature of these virus-host interactions. In the current review, we outline novel imaging methods that have been used to study the HIV-1 lifecycle and highlight advancements in the cell culture models developed to enhance our understanding of the HIV-1 lifecycle.
Topics: Cells, Cultured; HIV-1; Host-Pathogen Interactions; Humans; Optical Imaging; Staining and Labeling; Virology
PubMed: 27775563
DOI: 10.3390/v8100288 -
Viruses Aug 2016In the last 25 years, the scientific and public attention paid to bunyaviruses has increased considerably.[...].
In the last 25 years, the scientific and public attention paid to bunyaviruses has increased considerably.[...].
Topics: Bunyaviridae; History, 20th Century; History, 21st Century; Host-Pathogen Interactions; Humans; Viral Matrix Proteins; Virology; Virus Replication; Viruses
PubMed: 27517952
DOI: 10.3390/v8080224 -
Virologica Sinica Oct 2016Three-dimensional (3D) culture models are physiologically relevant, as they provide reproducible results, experimental flexibility and can be adapted for high-throughput... (Review)
Review
Three-dimensional (3D) culture models are physiologically relevant, as they provide reproducible results, experimental flexibility and can be adapted for high-throughput experiments. Moreover, these models bridge the gap between traditional two-dimensional (2D) monolayer cultures and animal models. 3D culture systems have significantly advanced basic cell science and tissue engineering, especially in the fields of cell biology and physiology, stem cell research, regenerative medicine, cancer research, drug discovery, and gene and protein expression studies. In addition, 3D models can provide unique insight into bacteriology, virology, parasitology and host-pathogen interactions. This review summarizes and analyzes recent progress in human virological research with 3D cell culture models. We discuss viral growth, replication, proliferation, infection, virus-host interactions and antiviral drugs in 3D culture models.
Topics: Animals; Cell Culture Techniques; Humans; Models, Biological; Virology; Viruses
PubMed: 27822716
DOI: 10.1007/s12250-016-3889-z -
Methods (San Diego, Calif.) Aug 2017Fluorescent tags constitute an invaluable tool in facilitating a deeper understanding of the mechanistic processes governing virus-host interactions. However, when... (Review)
Review
Fluorescent tags constitute an invaluable tool in facilitating a deeper understanding of the mechanistic processes governing virus-host interactions. However, when selecting a fluorescent tag for in vivo imaging of cells, a number of parameters and aspects must be considered. These include whether the tag may affect and interfere with protein conformation or localization, cell toxicity, spectral overlap, photo-stability and background. Cumulatively, these constitute challenges to be overcome. Bluetongue virus (BTV), a member of the Orbivirus genus in the Reoviridae family, is a non-enveloped virus that is comprised of two architecturally complex capsids. The outer capsid, composed of two proteins, VP2 and VP5, together facilitate BTV attachment, entry and the delivery of the transcriptionally active core in the cell cytoplasm. Previously, the significance of the endocytic pathway for BTV entry was reported, although a detailed analysis of the role of each protein during virus trafficking remained elusive due to the unavailability of a tagged virus. Described here is the successful modification, and validation, of a segmented genome belonging to a complex and large capsid virus to introduce tags for fluorescence visualization. The data generated from this approach highlighted the sequential dissociation of VP2 and VP5, driven by decreasing pH during the transition from early to late endosomes, and their retention therein as the virus particles progress along the endocytic pathway. Furthermore, the described tagging technology and methodology may prove transferable and allow for the labeling of other non-enveloped complex viruses.
Topics: Animals; Bluetongue virus; Host-Pathogen Interactions; Microbiological Techniques; Virology; Virus Internalization
PubMed: 28802715
DOI: 10.1016/j.ymeth.2017.08.004 -
International Journal of Molecular... Oct 2020Virus detection in natural and clinical samples is a complicated problem in research and diagnostics. There are different approaches for virus isolation and... (Review)
Review
Virus detection in natural and clinical samples is a complicated problem in research and diagnostics. There are different approaches for virus isolation and identification, including PCR, CRISPR/Cas technology, NGS, immunoassays, and cell-based assays. Following the development of genetic engineering methods, approaches that utilize cell cultures have become useful and informative. Molecular biology methods allow increases in the sensitivity and specificity of cell cultures for certain viruses and can be used to generate reporter cell lines. These cell lines express specific reporter proteins (e.g., GFP, luciferase, and CAT) in response to virus infection that can be detected in a laboratory setting. The development of genome editing and synthetic biology methods has given rise to new perspectives regarding the design of virus reporter systems in cell cultures. This review is aimed at describing both virology methods in general and examples of the development of cell-based methods that exist today.
Topics: Animals; Cell Culture Techniques; Genes, Reporter; Genetic Engineering; Humans; Synthetic Biology; Virology; Viruses
PubMed: 33121109
DOI: 10.3390/ijms21217978 -
Progress in Biophysics and Molecular... Dec 2020
Topics: Biological Evolution; COVID-19; Extracellular Vesicles; Hepacivirus; History, 20th Century; History, 21st Century; Humans; Nobel Prize; SARS-CoV-2; Virology; Virus Diseases; Virus Replication; Viruses
PubMed: 33096122
DOI: 10.1016/j.pbiomolbio.2020.10.004 -
Virology Mar 2006Some stem cell lines may contain an endogenous virus or can be contaminated with exogenous viruses (even of animal origin) and may secrete viral particles or express... (Review)
Review
Some stem cell lines may contain an endogenous virus or can be contaminated with exogenous viruses (even of animal origin) and may secrete viral particles or express viral antigens on their surface. Moreover, certain biotechnological products (e.g. bovine fetal serum, murine feeder cells) may contain prion particles. Viral and prion contamination of cell cultures and "feeder" cells, which is a common risk in all biotechnological products derived from the cell lines, is the most challenging and potentially serious outcome to address, due to the difficulty involved in virus and prion detection and the potential to cause serious disease in recipients of these cell products. Stem cell banks should introduce adequate quality assurance programs like the microbiological control program and can provide researchers with valuable support in the standardization and safety of procedures and protocols used for the viral and prion testing and in validation programs to assure the quality and safety of the cells.
Topics: Animals; Biological Specimen Banks; Cell Culture Techniques; Coculture Techniques; Humans; Prions; Safety; Stem Cells; Virology; Viruses
PubMed: 16380145
DOI: 10.1016/j.virol.2005.11.026 -
MSphere May 2021Ebola virus (EBOV) is a highly pathogenic negative-stranded RNA virus that has caused several deadly endemics in the past decades. EBOV reverse genetics systems are...
Ebola virus (EBOV) is a highly pathogenic negative-stranded RNA virus that has caused several deadly endemics in the past decades. EBOV reverse genetics systems are available for studying live viruses under biosafety level 4 (BSL-4) or subviral particles under BSL-2 conditions. However, these systems all require cotransfection of multiple plasmids expressing viral genome and viral proteins essential for EBOV replication, which is technically challenging and unable to naturally mimic virus propagation using the subviral particle. Here, we established a new EBOV reverse genetics system only requiring transfection of a single viral RNA genome into an engineered cell line that stably expresses viral nucleoprotein (NP), viral protein 35 (VP35), VP30, and large (L) proteins and has been fine-tuned for its superior permissiveness for EBOV replication. Using this system, subviral particles expressing viral VP40, glycoprotein (GP), and VP24 could be produced and continuously propagated and eventually infect the entire cell population. We demonstrated the authentic response of the subviral system to antivirals and uncovered that the VP35 amount is critical for optimal virus replication. Furthermore, we showed that fully infectious virions can be efficiently rescued by delivering the full-length EBOV genome into the same supporting cell, and the efficiency is not affected by genome polarity or virus variant specificity. In summary, our work provides a new tool for studying EBOV under different biosafety levels. Ebola virus is among the most dangerous viral pathogens, with a case fatality rate of up to 90%. Since 2013, the two largest and most complex Ebola outbreaks in Africa have revealed the lack of investigation on this notorious virus. A reverse genetics system is an important tool for studying viruses by producing mutant viruses or generating safer and convenient model systems. Here, we developed an EBOV life cycle modeling system in which subviral particles can spontaneously propagate in cell culture. In addition, this system can be employed to rescue infectious virions of homologous or heterologous EBOV isolates using either sense or antisense viral RNA genomes. In summary, we developed a new tool for EBOV research.
Topics: Cell Line; Ebolavirus; Genome, Viral; RNA, Viral; Reverse Genetics; Virology
PubMed: 33952663
DOI: 10.1128/mSphere.00235-21 -
Methods (San Diego, Calif.) Nov 2015Stress granules are induced in many different viral infections, and in turn are inhibited by the expression of viral proteins or RNAs. It is therefore evident that these... (Review)
Review
Stress granules are induced in many different viral infections, and in turn are inhibited by the expression of viral proteins or RNAs. It is therefore evident that these bodies are not compatible with efficient viral replication, but the mechanism by which they act to restrict viral gene expression or genome replication is not yet understood. This article discusses a number of methods that can be employed to gain a more complete understanding of the relationship between cellular SGs and viral RNA and protein synthesis in cells infected with diverse viruses.
Topics: Host-Pathogen Interactions; Microscopy; RNA, Viral; Ribonucleoproteins; Stress, Physiological; Viral Proteins; Virology
PubMed: 25896634
DOI: 10.1016/j.ymeth.2015.04.009 -
Viruses May 2016Commonly used tests based on wild-type viruses, such as immunostaining, cannot meet the demands for rapid detection of viral replication, high-throughput screening for... (Review)
Review
Commonly used tests based on wild-type viruses, such as immunostaining, cannot meet the demands for rapid detection of viral replication, high-throughput screening for antivirals, as well as for tracking viral proteins or virus transport in real time. Notably, the development of replicating-competent reporter-expressing viruses (RCREVs) has provided an excellent option to detect directly viral replication without the use of secondary labeling, which represents a significant advance in virology. This article reviews the applications of RCREVs in diagnostic and molecular virology, including rapid neutralization tests, high-throughput screening systems, identification of viral receptors and virus-host interactions, dynamics of viral infections in vitro and in vivo, vaccination approaches and others. However, there remain various challenges associated with RCREVs, including pathogenicity alterations due to the insertion of a reporter gene, instability or loss of the reporter gene expression, or attenuation of reporter signals in vivo. Despite all these limitations, RCREVs have become powerful tools for both basic and applied virology with the development of new technologies for generating RCREVs, the inventions of novel reporters and the better understanding of regulation of viral replication.
Topics: Animals; Gene Expression; Genes, Reporter; Humans; Molecular Biology; Staining and Labeling; Virology; Virus Replication; Viruses
PubMed: 27164126
DOI: 10.3390/v8050127