Viruses

Research

Jump to: Review

19 pages, 6582 KiB

Open AccessArticle

3D Analysis of HCMV Induced-Nuclear Membrane Structures by FIB/SEM Tomography: Insight into an Unprecedented Membrane Morphology

by Clarissa Villinger, Gregor Neusser, Christine Kranz, Paul Walther and Thomas Mertens

Viruses 2015, 7(11), 5686-5704; https://doi.org/10.3390/v7112900 - 4 Nov 2015

Cited by 36 | Viewed by 10874

Abstract

We show that focused ion beam/scanning electron microscopy (FIB/SEM) tomography is an excellent method to analyze the three-dimensional structure of a fibroblast nucleus infected with human cytomegalovirus (HCMV). We found that the previously described infoldings of the inner nuclear membrane, which are unique [...] Read more.

We show that focused ion beam/scanning electron microscopy (FIB/SEM) tomography is an excellent method to analyze the three-dimensional structure of a fibroblast nucleus infected with human cytomegalovirus (HCMV). We found that the previously described infoldings of the inner nuclear membrane, which are unique among its kind, form an extremely complex network of membrane structures not predictable by previous two-dimensional studies. In all cases they contained further invaginations (2nd and 3rd order infoldings). Quantification revealed 5498HCMV capsids within two nuclear segments, allowing an estimate of 15,000 to 30,000 capsids in the entire nucleus five days post infection. Only 0.8% proved to be enveloped capsids which were exclusively detected in 1st order infoldings (perinuclear space). Distribution of the capsids between 1st, 2nd and 3rd order infoldings is in complete agreement with the envelopment/de-envelopment model for egress of HCMV capsids from the nucleus and we confirm that capsid budding does occur at the large infoldings. Based on our results we propose the pushing membrane model: HCMV infection induces local disruption of the nuclear lamina and synthesis of new membrane material which is pushed into the nucleoplasm, forming complex membrane infoldings in a highly abundant manner, which then may be also used by nucleocapsids for budding. Full article

(This article belongs to the Special Issue Electron Microscopy in Virus Diagnostics and Research)

► Show Figures

Graphical abstract

21 pages, 6034 KiB

Open AccessArticle

The Use of Convalescent Sera in Immune-Electron Microscopy to Detect Non-Suspected/New Viral Agents

by Antonio Lavazza, Cristiana Tittarelli and Monica Cerioli

Viruses 2015, 7(5), 2683-2703; https://doi.org/10.3390/v7052683 - 22 May 2015

Cited by 15 | Viewed by 9737

Abstract

Negative staining electron microscopy methods can be employed for the diagnosis of viral particles in animal samples. In fact, negative staining electron microscopy methods are used to identify viruses, especially in minor species and wild animals, when no other methods are available and [...] Read more.

Negative staining electron microscopy methods can be employed for the diagnosis of viral particles in animal samples. In fact, negative staining electron microscopy methods are used to identify viruses, especially in minor species and wild animals, when no other methods are available and in cases of rare, emerging or re-emerging infections. In particular, immune-electron-microscopy with convalescent sera is employed to detect etiological agents when there are undiagnosed clinical outbreaks, when alternative diagnostic methods fail due to the lack of immunological reagents and primers, and when there is no indicative clinical suspect. An overview of immune-electron-microscopy with convalescent sera’s use in the diagnosis of new and unsuspected viruses in animals of domestic and wild species is provided through the descriptions of the following four diagnostic veterinary cases: (I) enteric viruses of pigs: Porcine Rotavirus, Porcine Epidemic Diarrhea Virus, Porcine Circovirus and Porcine Torovirus; (II) Rotavirus and astrovirus in young turkeys with enteritis; (III) Parvovirus-like particles in pheasants; and (IV) Lagoviruses: Rabbit Hemorrhagic Disease Virus and European Brown Hare Syndrome Virus. Full article

(This article belongs to the Special Issue Electron Microscopy in Virus Diagnostics and Research)

► Show Figures

Figure 1

14 pages, 832 KiB

Open AccessArticle

Evaluation of Virus Inactivation by Formaldehyde to Enhance Biosafety of Diagnostic Electron Microscopy

by Lars Möller, Livia Schünadel, Andreas Nitsche, Ingeborg Schwebke, Manuela Hanisch and Michael Laue

Viruses 2015, 7(2), 666-679; https://doi.org/10.3390/v7020666 - 10 Feb 2015

Cited by 53 | Viewed by 15300

Abstract

Formaldehyde (FA) fixation of infectious samples is a well-established protocol in diagnostic electron microscopy of viruses. However, published experimental data that demonstrate virus inactivation by these fixation procedures are lacking. Usually, fixation is performed immediately before the sample preparation for microscopy. The fixation [...] Read more.

Formaldehyde (FA) fixation of infectious samples is a well-established protocol in diagnostic electron microscopy of viruses. However, published experimental data that demonstrate virus inactivation by these fixation procedures are lacking. Usually, fixation is performed immediately before the sample preparation for microscopy. The fixation procedure should transform viruses in a non–infectious but nonetheless structurally intact form in order to allow a proper diagnosis based on morphology. FA provides an essential advantage in comparison to other disinfectants, because it preserves the ultrastructure of biological material without interfering significantly with the preparation (i.e., the negative staining) and the detection of viruses. To examine the efficiency of FA inactivation, we used Vaccinia virus, Human adenovirus and Murine norovirus as models and treated them with FA under various conditions. Critical parameters for the inactivation efficiency were the temperature, the duration of the FA treatment, and the resistance of the virus in question. Our results show that FA inactivation at low temperature (4 °C) bears a high risk of incomplete inactivation. Higher temperatures (25 °C) are more efficient, although they still require rather long incubation times to fully inactivate a complex and highly robust virus like Vaccinia. A protocol, which applied 2% buffered FA for 60 min and a temperature–shift from 25 to 37 °C after 30 min was efficient for the complete inactivation of all test viruses, and therefore has the potential to improve both biosafety and speed of diagnostic electron microscopy. Full article

(This article belongs to the Special Issue Electron Microscopy in Virus Diagnostics and Research)

► Show Figures

Figure 1

14 pages, 1186 KiB

Open AccessArticle

CMV-Promoter Driven Codon-Optimized Expression Alters the Assembly Type and Morphology of a Reconstituted HERV-K(HML-2)

by Oliver Hohn, Kirsten Hanke, Veronika Lausch, Anja Zimmermann, Saeed Mostafa and Norbert Bannert

Viruses 2014, 6(11), 4332-4345; https://doi.org/10.3390/v6114332 - 11 Nov 2014

Cited by 3 | Viewed by 8410

Abstract

The HERV-K(HML-2) family contains the most recently integrated and best preserved endogenized proviral sequences in the human genome. All known elements have nevertheless been subjected to mutations or deletions that render expressed particles non-infectious. Moreover, these post-insertional mutations hamper the analysis of the [...] Read more.

The HERV-K(HML-2) family contains the most recently integrated and best preserved endogenized proviral sequences in the human genome. All known elements have nevertheless been subjected to mutations or deletions that render expressed particles non-infectious. Moreover, these post-insertional mutations hamper the analysis of the general biological properties of this ancient virus family. The expression of consensus sequences and sequences of elements with reverted post-insertional mutations has therefore been very instrumental in overcoming this limitation. We investigated the particle morphology of a recently reconstituted HERV-K113 element termed oriHERV-K113 using thin-section electron microscopy (EM) and could demonstrate that strong overexpression by substitution of the 5'LTR for a CMV promoter and partial codon optimization altered the virus assembly type and morphology. This included a conversion from the regular C-type to an A-type morphology with a mass of cytoplasmic immature cores tethered to the cell membrane and the membranes of vesicles. Overexpression permitted the release and maturation of virions but reduced the envelope content. A weaker boost of virus expression by Staufen-1 was not sufficient to induce these morphological alterations. Full article

(This article belongs to the Special Issue Electron Microscopy in Virus Diagnostics and Research)

► Show Figures

Figure 1

14 pages, 2187 KiB

Open AccessArticle

A Novel Strain of Porcine Adenovirus Detected in Urinary Bladder Urothelial Cell Culture

by Urška Dragin Jerman, Marko Kolenc, Andrej Steyer, Peter Veranič, Mateja Poljšak Prijatelj and Mateja Erdani Kreft

Viruses 2014, 6(6), 2505-2518; https://doi.org/10.3390/v6062505 - 23 Jun 2014

Cited by 14 | Viewed by 11094

Abstract

Contamination of cell cultures is the most common problem encountered in cell culture laboratories. Besides the secondary cell contaminations often occurring in the cell laboratories, the contaminations originating from donor animal or human tissue are equally as common, but usually harder to recognize [...] Read more.

Contamination of cell cultures is the most common problem encountered in cell culture laboratories. Besides the secondary cell contaminations often occurring in the cell laboratories, the contaminations originating from donor animal or human tissue are equally as common, but usually harder to recognize and as such require special attention. The present study describes the detection of porcine adenovirus (PAdV), strain PAdV-SVN1 in cultures of normal porcine urothelial (NPU) cells isolated from urinary bladders of domestic pigs. NPU cell cultures were evaluated by light microscopy (LM), polymerase chain reaction (PCR), and additionally assessed by transmission electron microscopy (TEM). Characteristic ultrastructure of virions revealed the infection with adenovirus. The adenoviral contamination was further identified by the sequence analysis, which showed the highest similarity to recently described PAdV strain PAdV-WI. Additionally, the cell ultrastructural analysis confirmed the life-cycle characteristic for adenoviruses. To closely mimic the in vivo situation, the majority of research on in vitro models uses cell cultures isolated from human or animal tissue and their subsequent passages. Since the donor tissue could be a potential source of contamination, the microbiological screening of the excised tissue and harvested cell cultures is highly recommended. Full article

(This article belongs to the Special Issue Electron Microscopy in Virus Diagnostics and Research)

► Show Figures

Figure 1

Review

Jump to: Research

30 pages, 5082 KiB

Open AccessReview

Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells

by Inés Romero-Brey and Ralf Bartenschlager

Viruses 2015, 7(12), 6316-6345; https://doi.org/10.3390/v7122940 - 3 Dec 2015

Cited by 51 | Viewed by 19172

Abstract

As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and [...] Read more.

As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (EM) methods are extremely helpful. While conventional EM has given important information about virus-host cell interactions, the development of three-dimensional EM (3D-EM) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. During the last years several 3D-EM methods have been developed. Here we will provide a description of the main approaches and examples of innovative applications. Full article

(This article belongs to the Special Issue Electron Microscopy in Virus Diagnostics and Research)

► Show Figures

Figure 1

12 pages, 2064 KiB

Open AccessReview

Morphologic Differentiation of Viruses beyond the Family Level

by Cynthia S. Goldsmith

Viruses 2014, 6(12), 4902-4913; https://doi.org/10.3390/v6124902 - 9 Dec 2014

Cited by 18 | Viewed by 15214

Abstract

Electron microscopy has been instrumental in the identification of viruses by being able to characterize a virus to the family level. There are a few cases where morphologic or morphogenesis factors can be used to differentiate further, to the genus level. These include [...] Read more.

Electron microscopy has been instrumental in the identification of viruses by being able to characterize a virus to the family level. There are a few cases where morphologic or morphogenesis factors can be used to differentiate further, to the genus level. These include viruses in the families Poxviridae, Reoviridae, Retroviridae, Herpesviridae, Filoviridae, and Bunyaviridae. Full article

(This article belongs to the Special Issue Electron Microscopy in Virus Diagnostics and Research)

► Show Figures

Figure 1

14 pages, 1315 KiB

Open AccessReview

A Filtration Based Technique for Simultaneous SEM and TEM Sample Preparation for the Rapid Detection of Pathogens

by Daniel R. Beniac, Christine G. Siemens, Christine J. Wright and Tim F. Booth

Viruses 2014, 6(9), 3458-3471; https://doi.org/10.3390/v6093458 - 19 Sep 2014

Cited by 28 | Viewed by 32670

Abstract

Diagnostic electron microscopy for infectious diseases has the advantage that “everything” in the specimen can be observed, without a priori knowledge of the likely identity of the microorganisms present in the sample. The classical specimen preparation method used employs a droplet of sample, [...] Read more.

Diagnostic electron microscopy for infectious diseases has the advantage that “everything” in the specimen can be observed, without a priori knowledge of the likely identity of the microorganisms present in the sample. The classical specimen preparation method used employs a droplet of sample, which allows particles to adsorb to a support film, and is subsequently negative stained. This “grid on drop” procedure has a sensitivity range of approximately 10⁶ viruses per mL if no enrichment procedures are used. In the current investigation we present a novel use of filtration that allows us to detect viruses at concentrations as low as 10² viruses per mL. We present here methods based on filtration, in which total virus, and not virus concentration, is the limiting factor for detection. We show that filtration is more sensitive than conventional negative staining and can detect as few as 5 × 10³ particles per sample. Full article

(This article belongs to the Special Issue Electron Microscopy in Virus Diagnostics and Research)

► Show Figures

Figure 1

32 pages, 1306 KiB

Open AccessReview

Membranous Replication Factories Induced by Plus-Strand RNA Viruses

by Inés Romero-Brey and Ralf Bartenschlager

Viruses 2014, 6(7), 2826-2857; https://doi.org/10.3390/v6072826 - 22 Jul 2014

Cited by 219 | Viewed by 17367

Abstract

In this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+) RNA viruses. We discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has [...] Read more.

In this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+) RNA viruses. We discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has become more widely available. A general denominator is that two “morphotypes” of membrane alterations can be found that are exemplified by flaviviruses and hepaciviruses: membrane invaginations towards the lumen of the endoplasmatic reticulum (ER) and double membrane vesicles, representing extrusions also originating from the ER, respectively. We hypothesize that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments. Full article

(This article belongs to the Special Issue Electron Microscopy in Virus Diagnostics and Research)

► Show Figures

Graphical abstract

Journal Menu

Journal Browser

Electron Microscopy in Virus Diagnostics and Research

Share This Special Issue

Special Issue Editor

Special Issue Information

Keywords

Benefits of Publishing in a Special Issue

Published Papers (9 papers)

Research

Review

Further Information

Guidelines

MDPI Initiatives

Follow MDPI