Visualization of viral assembly in the infected cell.

The study of the virus life cycle in infected cells is a methodological challenge due to the small size and diversity of the viral components. Recent developments on preservation of fine structure and molecular localization have provided a group of powerful methods with wide applications in cell biology and virology. Among the different electron microscopy (EM) techniques available to visualize viral assembly at the intracellular level, we will focus on conventional ultrathin sections, cryosections, and freeze-substitution. For obtaining molecular information associated to ultrastructure we have now a group of methods to detect viral proteins (immunogold labeling), as well as the viral genome, through the different techniques for detection of nucleic acids (the enzyme-gold approach, in situ hybridization, and elemental mapping). We will illustrate the applications of these methods with examples of viruses that exhibit different levels of structural complexity. These new approaches help to detect and identify viruses in clinical samples and to characterize the virus life cycle and the cellular components involved, to obtain data that could help for a therapeutic intervention, and to characterize virus-like particles that can be the basis of new and safe vaccines.     

New techniques in rapid viral diagnosis

Abstract The development of new diagnostic techniques in immunology and molecular biology during the last two decades has opened up new possibilities for rapid viral diagnosis. Solid phase immunoassays for antigen and antibody detection are now widely used in diagnostic settings. Several novel techniques have been introduced and have led to commercially available tests. Diagnostic methods using nucleic acid amplification procedures are already applied in research laboratories and will be commercialized soon. Biosensor-based diagnostic techniques have the potential of generating a result nearly instantaneously and it has become possible to monitor kinetic processes. Automatization and simplified procedures are needed to allow diagnostic tests to be performed soon after the sample has been obtained from the patient. In order to evaluate the new procedures and avoid false results, rigorous quality control in diagnostic virology will have to be instituted.     

Bioinformatics: alive and kicking

Bioinformatics has become too central to biology to be left to specialist bioinformaticians. Biologists are all bioinformaticians now.     

RNA viruses: emerging vectors for vaccination and gene therapy

The application of modern molecular techniques has profoundly influenced our understanding of virus function. As a consequence, virus biology is being directly applied to medical research. It is a reflection of the current pace of virology that we are now beginning to think of our ancient foes as useful and beneficial tools.     

Bioinformatics: alive and kicking

Bioinformatics has become too central to biology to be left to specialist bioinformaticians. Biologists are all bioinformaticians now.     

AIDS-associated malignancies: research perspectives

The appearance in 1981 of a usually rare malignancy, Kaposi’s sarcoma, in homosexual men [1] was one of the first harbingers of an epidemic caused by a retrovirus, human immunodeficiency virus (HIV), which causes the acquired immunodeficiency syndrome (AIDS). Lymphoid and other malignancies were also increased, most strikingly non-Hodgkin’s lymphoma and primary central nervous system (CNS) lymphoma. Advances in molecular biology, immunology, virology, and anti-viral therapy have combined to create unique research opportunities. One developing theme is the role of viral co-infection and malignancy. Human herpes virus 8 (HHV8), Epstein-Barr virus (EBV) and papilloma virus each may have a causal role in the development of HIV-associated malignancy. New antiretroviral therapies are able to substantially reverse or delay the profound immunosuppression of HIV infection. The changes in the epidemiology of malignancies, and understanding the mechanism of action of these new therapeutics provide research opportunities to understand the pathogenesis of these malignancies. The opportunities to discover the consequences of T-cell based immunodeficiency and the interactions with specific viral pathogens will likely lead to progress in HIV treatment and new strategies for other malignancies.     

The Development of Computational Biology in South Africa: Successes Achieved and Lessons Learnt

Bioinformatics is now a critical skill in many research and commercial environments as biological data are increasing in both size and complexity. South African researchers recognized this need in the mid-1990s and responded by working with the government as well as international bodies to develop initiatives to build bioinformatics capacity in the country. Significant injections of support from these bodies provided a springboard for the establishment of computational biology units at multiple universities throughout the country, which took on teaching, basic research and support roles. Several challenges were encountered, for example with unreliability of funding, lack of skills, and lack of infrastructure. However, the bioinformatics community worked together to overcome these, and South Africa is now arguably the leading country in bioinformatics on the African continent. Here we discuss how the discipline developed in the country, highlighting the challenges, successes, and lessons learnt.     

A short history of plant virology. III. The thirties.

The Thirties testified on the outstanding development of plant virology: the new discoveries formalized the concept of virus on a physicochemical background. Plant viruses, which had received their own taxonomical position at the end of the Twenties, were no longer considered as simple "infective pathogens" as their size, shape and chemical nature were determined, particularly for one of them--tobacco mosaic virus (TMV). This paramount contribution was achieved as a consequence of a functional interaction between biology on one side, and chemistry and physics on the other side, from the development of which molecular biology was born. The chemical characterization of TMV developed from the first determination of nitrogen presence in purified virus, performed by Carl Vinson, through the identification of TMV as Wendell Stanley's infective, autoreplicative protein macromolecule, to the final discovery of its nucleoprotein nature by the British group of Frederick Bawden. Thorough analytical techniques--in particular electron microscopy--led to disclose the exact shape and size of TMV particle. These discoveries, that opened a new era of virology, were corroborated by new knowledge that, although less explosive, can be considered of great importance for the development of plant virology. The methodologies to estimate viral activity; the study of the relationships between viruses and insect vectors; the studies on virus spread within plants; the identification of non-sterile type of resistance and of correlation between single plant genes and viral pathogenesis benefited plant virology of a set of knowledge that, together with the discoveries on the physico-chemical properties of TMV, raised plant virology from a secondary branch of plant pathology to a new independent science by itself.     

Recent contributions of molecular biology to the clinical virology of myxoviruses.

Recent advances in the clinical virology of influenza are based on non-pragmatically oriented research on the genetics and biochemistry of the influenza virus. Antigenically hybrid recombinant viruses can be tailored to provide monospecific reagents for serological studies. Basic research on viral structure and the mechanism of viral replication has directly influenced the establishment of a cell culture system suitable for the isolation of most influenza viruses. Identification of viral genotype by RNA gel electrophoresis and mapping of oligonucleotides of viral RNA has already facilitated epidemiologic investigations. The clinical virologist of the future must have an understanding of the potential limitations of these techniques for specific strain identification.     

News and views from the 8th annual meeting of the Italian Society of Virology

The 8th annual meeting of the Italian Society of Virology (SIV) took place in Orvieto, Italy from the 21st to the 23rd of September 2008. The meeting covered different areas of Virology and the scientific sessions focused on: general virology and viral genetics; viral oncology, virus–host interaction and pathogenesis; emerging viruses and zoonotic, foodborne and environmental pathways of transmission; viral immunology and vaccines; viral biotechnologies and gene therapy; medical virology and antiviral therapy. The meeting had an attendance of about 160 virologists from all Italy. In this edition, a satellite workshop on “Viral biotechnologies” was organized in order to promote the role of virologists in the biotechnological research and teaching fields. A summary of the plenary lectures and oral selected presentations is reported. J. Cell. Physiol. 219: 797–799, 2009. © 2009 Wiley‐Liss, Inc.     

An evolutionary change in diagnostic virology

The earliest laboratory diagnoses of viral infections were made by microscopy just after the turn of the twentieth century. Animal and egg inoculation were the methods of choice until tissue culture and serology accelerated the field of diagnostic virology during the fifties and sixties. More rapid methods, including electron microscopy, immunoassays, and nucleic acid probes, are now available and influencing laboratory decisions and patient care. This review discusses changes in science and society which have influenced diagnostic virology and how the discipline has responded to these influences.     

Human Organoids as a Promising Platform for Fighting COVID-19

The coronavirus disease 2019 (COVID-19) global pandemic evoked by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a major public health problem with significant morbidity and mortality. Understanding the pathogenesis and molecular mechanisms underlying this novel virus is crucial for both fundamental research and clinical trials in order to devise effective therapies and vaccination regimens. Basic research on SARS-CoV-2 largely depends on ex vivo models that allow viral invasion and replication. Organoid models are now emerging as a valuable tool to investigate viral biology and disease progression, serving as an efficient platform to investigate potential therapies for COVID-19. Here, we summarize various human stem cell-derived organoid types employed in SARS-CoV-2 studies. We highlight key findings from these models, including cell tropisms and molecular mechanisms in viral infection. We also describe their use in identifying potential therapeutic agents against SARS-CoV-2. As more and more advanced organoids emerge, they will facilitate the understanding of disease pathogenesis for drug development in this dreaded pandemic.     

Newly discovered insect RNA viruses in China

Insects are a group of arthropods and the largest group of animals on Earth,with over one million species described to date.Like other life forms,insects suffer from viruses that cause disease and death.Viruses that are pathogenic to beneficial insects cause dramatic economic losses on agriculture.In contrast,viruses that are pathogenic to insect pests can be exploited as attractive biological control agents.All of these factors have led to an explosion in the amount of research into insect viruses in recent years,generating impressive quantities of information on the molecular and cellular biology of these viruses.Due to the wide variety of insect viruses,a better understanding of these viruses will expand our overall knowledge of their virology.Here,we review studies of several newly discovered RNA insect viruses in China.     

Comparative Analysis of T-Cell Spatial Proteomics and the Influence of HIV Expression

As systems biology approaches to virology have become more tractable, highly studied viruses such as HIV can now be analyzed in new unbiased ways, including spatial proteomics. We employed here a differential centrifugation protocol to fractionate Jurkat T cells for proteomic analysis by mass spectrometry; these cells contain inducible HIV-1 genomes, enabling us to look for changes in the spatial proteome induced by viral gene expression. Using these proteomics data, we evaluated the merits of several reported machine learning pipelines for classification of the spatial proteome and identification of protein translocations. From these analyses, we found that classifier performance in this system was organelle dependent, with Bayesian t-augmented Gaussian mixture modeling outperforming support vector machine learning for mitochondrial and endoplasmic reticulum proteins but underperforming on cytosolic, nuclear, and plasma membrane proteins by QSep analysis. We also observed a generally higher performance for protein translocation identification using a Bayesian model, Bayesian analysis of differential localization experiments, on row-normalized data. Comparative Bayesian analysis of differential localization experiment analysis of cells induced to express the WT viral genome versus cells induced to express a genome unable to express the accessory protein Nef identified known Nef-dependent interactors such as T-cell receptor signaling components and coatomer complex. Finally, we found that support vector machine classification showed higher consistency and was less sensitive to HIV-dependent noise. These findings illustrate important considerations for studies of the spatial proteome following viral infection or viral gene expression and provide a reference for future studies of HIV-gene-dropout viruses.     

The pollen virome: A review of pollen-associated viruses and consequences for plants and their interactions with pollinators

The movement of pollen grains from anthers to stigmas, often by insect pollinator vectors, is essential for plant reproduction. However, pollen is also a unique vehicle for viral spread. Pollen-associated plant viruses reside on the outside or inside of pollen grains, infect susceptible individuals through vertical or horizontal infection pathways, and can decrease plant fitness. These viruses are transferred with pollen between plants by pollinator vectors as they forage for floral resources; thus, pollen-associated viral spread is mediated by floral and pollen grain phenotypes and pollinator traits, much like pollination. Most of what is currently known about pollen-associated viruses was discovered through infection and transmission experiments in controlled settings, usually involving one virus and one plant species of agricultural or horticultural interest. In this review, we first provide an updated, comprehensive list of the recognized pollen-associated viruses. Then, we summarize virus, plant, pollinator vector, and landscape traits that can affect pollen-associated virus transmission, infection, and distribution. Next, we highlight the consequences of plant–pollinator–virus interactions that emerge in complex communities of co-flowering plants and pollinator vectors, such as pollen-associated virus spread between plant species and viral jumps from plant to pollinator hosts. We conclude by emphasizing the need for collaborative research that bridges pollen biology, virology, and pollination biology.     

Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses

Recent metagenomic studies have provided an unprecedented wealth of data, which are revolutionizing our understanding of virus diversity. A redrawn landscape highlights viruses as active players in the phytobiome, and surveys have uncovered their positive roles in environmental stress tolerance of plants. Viral infectious clones are key tools for functional characterization of known and newly identified viruses. Knowledge of viruses and their components has been instrumental for the development of modern plant molecular biology and biotechnology. In this review, we provide extensive guidelines built on current synthetic biology advances that streamline infectious clone assembly, thus lessening a major technical constraint of plant virology. The focus is on generation of infectious clones in binary T‐DNA vectors, which are delivered efficiently to plants by Agrobacterium. We then summarize recent applications of plant viruses and explore emerging trends in microbiology, bacterial and human virology that, once translated to plant virology, could lead to the development of virus‐based gene therapies for ad hoc engineering of plant traits. The systematic characterization of plant virus roles in the phytobiome and next‐generation virus‐based tools will be indispensable landmarks in the synthetic biology roadmap to better crops.     

The viral killer system in yeast: from molecular biology to application

Since the initial discovery of the yeast killer system almost 40 years ago, intensive studies have substantially strengthened our knowledge in many areas of biology and provided deeper insights into basic aspects of eukaryotic cell biology as well as into virus-host cell interactions and general yeast virology. Analysis of killer toxin structure, synthesis and secretion has fostered understanding of essential cellular mechanisms such as post-translational prepro-protein processing in the secretory pathway. Furthermore, investigation of the receptor-mediated mode of toxin action proved to be an effective means for dissecting the molecular structure and in vivo assembly of yeast and fungal cell walls, providing important insights relevant to combating infections by human pathogenic yeasts. Besides their general importance in understanding eukaryotic cell biology, killer yeasts, killer toxins and killer viruses are also becoming increasingly interesting with respect to possible applications in biomedicine and gene technology. This review will try to address all these aspects.     

Not Beyond Reasonable Doubt: Howard Temin’s Provirus Hypothesis Revisited

During the 1960s, Howard M. Temin (1934–1994), dared to advocate a “heretical” hypothesis that appeared to be at variance with the central dogma of molecular biology, understood by many to imply that information transfer in nature occurred only from DNA to RNA. Temin’s provirus hypothesis offered a simple explanation of both virus replication and viral-induced cancer and stated that Rous sarcoma virus, an RNA virus, is replicated via a DNA intermediate. Popular accounts of this scientific episode, written after the discovery of an RNA-directed DNA polymerase in 1970, tend to describe the reaction to his proposition as ardent opposition. Typically these accounts use a ‹molecular biology’ standpoint emphasizing the central dogma’s part in its rejection. In this article, however, this episode will be examined from a joint perspective of virology and experimental cancer research. From this perspective it is clear that Temin’s work was well within the epistemological and methodological boundaries of virology and cancer research. Still, scientists did have␣reasons to doubt the provirus hypothesis, but these do not seem to be good enough to either justify an account that portrays Temin as a renegade or his ideas as heretical.     

A microfluidic chamber for analysis of neuron-to-cell spread and axonal transport of an alpha-herpesvirus

Alpha-herpesviruses, including herpes simplex virus and pseudorabies virus (PRV), infect the peripheral nervous system (PNS) of their hosts. Here, we describe an in vitro method for studying neuron-to-cell spread of infection as well as viral transport in axons. The method centers on a novel microfluidic chamber system that directs growth of axons into a fluidically isolated environment. The system uses substantially smaller amounts of virus inoculum and media than previous chamber systems and yet offers the flexibility of applying multiple virology and cell biology assays including live-cell optical imaging. Using PRV infection of cultured PNS neurons, we demonstrate that the microfluidic chamber recapitulates all known facets of neuron-to-cell spread demonstrated in animals and other compartmented cell systems.