Basic Virology Research Articles

Recombinant Adeno-Associated Virus Gene Therapy in Light of Luxturna (and Zolgensma and Glybera): Where Are We, and How Did We Get Here?

The recent market approvals of recombinant adeno-associated virus (rAAV) gene therapies in Europe and the United States are landmark achievements in the history of modern science. These approvals are also anticipated to herald the emergence of a new class of therapies for monogenic disorders, which had hitherto been considered untreatable. These events can be viewed as stemming from the convergence of several important historical trends: the study of basic virology, the development of genomic technologies, the imperative for translational impact of National Institutes of Health-funded research, and the development of economic models for commercialization of rare disease therapies. In this review, these historical trends are described and the key developments that have enabled clinical rAAV gene therapies are discussed, along with an overview of the current state of the field and future directions.

Dynamic Perspectives on the Search for a Universal Influenza Vaccine.

A universal influenza vaccine (UIV) could considerably alleviate the public health burden of both seasonal and pandemic influenza. Although significant progress has been achieved in clarifying basic immunology and virology relating to UIV, several important questions relating to the dynamics of infection, immunity, and pathogen evolution remain unsolved. In this study, we review these gaps, which span integrative levels, from cellular to global and timescales from molecular events to decades. We argue that they can be best addressed by a tight integration of empirical (laboratory, epidemiological) research and theory and suggest fruitful areas for this synthesis. In particular, quantifying natural and vaccinal limitations on viral transmission are central to this effort.

Delivery of gene therapy to resting immune cells for an HIV cure.

The utilization of genetically modified T cells to therapeutically target to various previously incurable diseases such, as cancer, has expanded exponentially in recent years. This success now provides the motivating force in applying the same technology for incurable infectious diseases including HIV. The common bottleneck in gene therapy continues to be at the level of gene delivery. Although present approaches adapt the cell to the delivery technology, emerging techniques now focus on leaving cells in their phenotypically resting state. In doing so, engraftment and proliferation potential are retained and in turn increase the efficacy of this approach at a lowered cost. This review will outline the main efforts of gene delivery using viral vectors or nonviral vectors and challenges moving forward not only in resting T cells, but also in other resting immune cells including hematopoietic stem cells. In focusing on HIV cure efforts using gene therapy, progress on solving the challenges of gene delivery will be described for both viral and nonviral vectors. Advances in the basic virology of lentiviruses have led to the proposal of many next generation lentiviral vector platforms for resting immune cells. Moreover, we will also highlight the progress made in nonviral approaches using nanotechnology as alternatives and/or synergistic technologies to be used alongside lentiviral platforms. The innovative approaches described in these recent studies, particularly those using the natural mechanisms employed by HIV to enhance for example virus entry or virus latency, will enable future optimization of gene delivery platforms and therapeutics, which will importantly, provide a pathway toward translation into clinical practice.

The Natural Large Genomic Deletion Is Unrelated to the Increased Virulence of the Novel Genotype Fowl Adenovirus 4 Recently Emerged in China.

Since 2015, severe hydropericardium-hepatitis syndrome (HHS), caused by a highly pathogenic fowl adenovirus 4 (FAdV-4), emerged in China. In our previous study, the FAdV-4 has been identified as a novel genotype with a unique 1966-bp nucleotide deletion (1966Del) between open reading frame 42 and 43. In this study, the natural 1966Del was frequently identified among 17 clinical isolates and other reported Chinese clinical strains. To investigate the relationship between 1966Del and the increased virulence of the novel FAdV-4, a CRISPR/Cas9 operating platform for FAdV-4 was developed for the first time in this study. Based on this platform, a Re1966 strain was rescued, inserted the relative 1966Del sequence of a nonpathogenic strain KR5. In the pathogenicity study, the Re1966 strain retained high virulence for specific-pathogen-free chickens, similar to the parental wild-type HLJFAd15, although the survival time of chickens infected with Re1966 was much longer. Therefore, the natural 1966Del was identified as a non-essential site for the increased virulence of the emerged novel FAdV-4. Although further research on the virulence-determining region or point within the genome of the novel FAdV-4 is needed, the CRISPR/Cas9 operating platform for the novel FAdV-4 was developed and successfully applied to edit the genomic DNA for the first time, and it provides a novel powerful tool for both basic virology studies and vaccine vector development of FAdVs.

Characterizing replication kinetics and plaque production of type I feline infectious peritonitis virus in three feline cell lines

Investigating type I feline coronaviruses (FCoVs) in tissue culture is critical for understanding the basic virology, pathogenesis, and virus-host interactome of these important veterinary pathogens. This has been a perennial challenge as type I FCoV strains do not easily adapt to cell culture. Here we characterize replication kinetics and plaque formation of a model type I strain FIPV Black in Fcwf-4 cells established at Cornell University (Fcwf-4 CU). We determined that maximum virus titers (>107 pfu/mL) were recoverable from infected Fcwf-4 CU cell-free supernatant at 20 h post-infection. Type I FIPV Black and both biotypes of type II FCoV formed uniform and enumerable plaques on Fcwf-4 CU cells. Therefore, these cells were employable in a standardized plaque assay. Finally, we determined that the Fcwf-4 CU cells were morphologically distinct from feline bone marrow-derived macrophages and were less sensitive to exogenous type I interferon than were Fcwf-4 cells purchased from ATCC.

Potential Application of the CRISPR/Cas9 System against Herpesvirus Infections.

The CRISPR/Cas9 system has been applied in the genome editing and disruption of latent infections for herpesviruses such as the herpes simplex virus, Epstein–Barr virus, cytomegalovirus, and Kaposi’s sarcoma-associated herpesvirus. CRISPR/Cas9-directed mutagenesis can introduce similar types of mutations to the viral genome as can bacterial artificial chromosome recombination engineering, which maintains and reconstitutes the viral genome successfully. The cleavage mediated by CRISPR/Cas9 enables the manipulation of disease-associated viral strains with unprecedented efficiency and precision. Additionally, current therapies for herpesvirus productive and latent infections are limited in efficacy and cannot eradicate viruses. CRISPR/Cas9 is potentially adapted for antiviral treatment by specifically targeting viral genomes during latent infections. This review, which focuses on recently published progress, suggests that the CRISPR/Cas9 system is not only a useful tool for basic virology research, but also a promising strategy for the control and prevention of herpesvirus latent infections.

Astrovirology: Viruses at Large in the Universe.

Viruses are the most abundant biological entities on modern Earth. They are highly diverse both in structure and genomic sequence, play critical roles in evolution, strongly influence terran biogeochemistry, and are believed to have played important roles in the origin and evolution of life. However, there is yet very little focus on viruses in astrobiology. Viruses arguably have coexisted with cellular life-forms since the earliest stages of life, may have been directly involved therein, and have profoundly influenced cellular evolution. Viruses are the only entities on modern Earth to use either RNA or DNA in both single- and double-stranded forms for their genetic material and thus may provide a model for the putative RNA-protein world. With this review, we hope to inspire integration of virus research into astrobiology and also point out pressing unanswered questions in astrovirology, particularly regarding the detection of virus biosignatures and whether viruses could be spread extraterrestrially. We present basic virology principles, an inclusive definition of viruses, review current virology research pertinent to astrobiology, and propose ideas for future astrovirology research foci. Key Words: Astrobiology-Virology-Biosignatures-Origin of life-Roadmap. Astrobiology 18, 207-223.

Vaccine Development Strategies, Progresses and Challenges for Human Immunodeficiency Virus (HIV): A Review

Human immunodeficiency virus is classified under the Retroviridae family and identified as a causative agent of acquired immunodeficiency syndrome (AIDS).Since Human immunodeficiency virus (HIV) is identified as causative agent of AIDS, about 39 million people have been died and 78 million people have been infected worldwide. Despite tremendous efforts are being made to develop successful diagnosis, treatment and prevention methods, and to develop HIV vaccine the effort remains great challenge for researchers, due to extreme genetic variability of the virus. Even though vaccination of HIV is the most promising, cost effective and feasible intervention strategy to control and eradicate HIV disease route and transmission, still now we are struggling to find an elusive vaccine after thirty years and disappointing results are recorded from previous clinical trials, except the promising RV144 HIV vaccine which is phase III clinical trial with the modest vaccine efficacy. The contribution of this study is to reveal the challenges to found potent HIV vaccine during the last decades, significant lessons learnt about the basic virology, pathogenesis, immunology, HIV/AIDS treatment and HIV infection prevention and significant findings and clinical trials progresses which are currently open the way to some extent to hope that HIV vaccine development is possible.

The clinical utility of JC virus antibody index measurements in the context of progressive multifocal leukoencephalopathy.

In natalizumab-treated patients without previous immunosuppressive treatment, the JCV antibody index is used to stratify PML risk. A high index value indicates that the risk to develop PML is significantly elevated, although probably about 99% of patients with this index value will not develop PML. This minireview aimed to provide an overview of the basic virology and immunology relevant to understanding JCV infections in MS patients, with a focus on what is presently known about antibodies to JCV and how they could be of use to predict and diagnose PML.

Viral Population Changes during Murine Norovirus Propagation in RAW 264.7 Cells.

Laboratory adaptation of viruses is an essential technique for basic virology research, including the generation of attenuated vaccine strains, although the principles of cell adaptation remain largely unknown. Deep sequencing of murine norovirus (MuNoV) S7 during serial passages in RAW264.7 cells showed that the frequencies of viral variants were altered more dynamically than previously reported. Serial passages of the virus following two different multiplicity of infections gave rise to distinct haplotypes, implying that multiple cell-adaptable sequences were present in the founder population. Nucleotide variants lost during passage were assembled into a viral genome representative of that prior to cell adaptation, which was unable to generate viral particles upon infection in cultured cells. In addition, presence of the reconstructed genome interfered with production of infectious particles from viruses that were fully adapted to in vitro culture. Although the key nucleotide changes dictating cell adaptation of MuNoV S7 viral infection are yet to be elucidated, our results revealed the elaborate interplay among selected sequences of viral variants better adapted to propagation in cell culture. Such knowledge will be instrumental in understanding the processes necessary for the laboratory adaptation of viruses, especially to those without relevant cell culture systems.

An Engineered Virus Library as a Resource for the Spectrum-wide Exploration of Virus and Vector Diversity

Adenoviruses (Ads) are large human-pathogenic double-stranded DNA (dsDNA) viruses presenting an enormous natural diversity associated with a broad variety of diseases. However, only a small fraction of adenoviruses has been explored in basic virology and biomedical research, highlighting the need to develop robust and adaptable methodologies and resources. We developed a method for high-throughput direct cloning and engineering of adenoviral genomes from different sources utilizing advanced linear-linear homologous recombination (LLHR) and linear-circular homologous recombination (LCHR). We describe 34 cloned adenoviral genomes originating from clinical samples, which were characterized by next-generation sequencing (NGS). We anticipate that this recombineering strategy and the engineered adenovirus library will provide an approach to study basic and clinical virology. High-throughput screening (HTS) of the reporter-tagged Ad library in a panel of cell lines including osteosarcoma disease-specific cell lines revealed alternative virus types with enhanced transduction and oncolysis efficiencies. This highlights the usefulness of this resource.

Zika virus induced cellular remodelling.

Zika virus (ZIKV) has been associated with morbidities such as Guillain-Barré, infant microcephaly, and ocular disease. The spread of this positive-sense, single-stranded RNA virus and its growing public health threat underscore gaps in our understanding of basic ZIKV virology. To advance knowledge of the virus replication cycle within mammalian cells, we use serial section 3-dimensional electron tomography to demonstrate the widespread remodelling of intracellular membranes upon infection with ZIKV. We report extensive structural rearrangements of the endoplasmic reticulum and reveal stages of the ZIKV viral replication cycle. Structures associated with RNA genome replication and virus assembly are observed integrated within the endoplasmic reticulum, and we show viruses in transit through the Golgi apparatus for viral maturation, and subsequent cellular egress. This study characterises in detail the 3-dimensional ultrastructural organisation of the ZIKV replication cycle stages. Our results show close adherence of the ZIKV replication cycle to the existing flavivirus replication paradigm.

Ebola Through a Glass, Darkly: Ways of Knowing the State and Each Other

(ProQuest: ... denotes non-US-ASCII text omitted.)During the height of the Ebola Virus Disease (EVD) epidemic in Sierra Leone in 2014, the international community worked to vernacularize biomedical models of the disease. They did so hoping that translating biomedical models of disease into local concepts would help stop transmission (Spencer 2015:2). Health communicators encouraged people to shed the intimate behaviors of care-welcoming strangers into the home, careful personal tending to the ill, washing corpses for burial-that facilitated Ebola's spread, in addition to educating them in basic virology to reinforce behavioral changes. In spite of these mass sensitization campaigns (see Bolten 2012), the disease continued to spread. Medical education seemed helpless against pavement radio (Ellis 1989)1: people in the Bombali District of Sierra Leone, where this study took place, explained the catastrophic death toll in one village the crash of a witch plane (Spencer 2015:2-3),2 with rumors circulating in urban areas that the disease's spread was a political ploy by the opposition party to poison wells in order to undermine the President.3 Families continued to nurse their sick at home, bury their dead, and mourn with loving, personal attention. In spite of this apparently willful ignorance of public health measures, Ebola did not spread exponentially around the country, and the 500,000 deaths that had been predicted by the Center for Disease Control (CDC) did not, in fact, occur (CDC 2014). Transmission patterns were uneven, with some areas experiencing dozens of cases while others remained relatively unscathed, even if they were geographically and socially close. What accounted for this?In this article, we analyze a case study of two neighboring villages in northern Sierra Leone, which we will call Mabele and Katibana, with Mabele experiencing over 25 percent of its population falling ill, and Katibana not recording a single case. The villages are located less than 100 meters from each other on a well-traveled road, and had long enjoyed a close and cordial relationship involving mutual work and ritual activity, a situation potentially enabling the presence of Ebola in one to affect the other dramatically. That potential, however, never materialized into cases in Katibana. We argue that their divergent experiences of the epidemic occurred because the village chiefs interpreted their powers vis-a-vis outside and its purveyors in radically different ways, with one shunning the involvement of the government and the other seeing it as an opportunity to consolidate his power. Rosalind Shaw (2002:91) uses the phrase through a glass, darkly to refer to local understanding of in northern Sierra Leone as value-laden and political: information is never assessed as though it was value-free. Rather, it becomes knowledge by being interpreted one's social and political relationship to the bearer of that information, and an appraisal of what the bearer gains by disseminating it. Ebola communications from the government and NGOs were viewed this dark glass: many citizens were suspicious of the motives of health communicators and the so-called neutral information's potential to alter existing power relations. They could not see the messages as a straightforward articulation of the risks of a devastating viral hemorrhagic fever.The two chiefs handled EVD information as they would any government announcement, considering first why the government would bring Ebola to their villages, and how their own social and political positions would be influenced or changed by either embracing or rejecting these education and praxis campaigns. It was the social effects of Ebola about which people were concerned, not the itself (see Murphy 1981). Both chiefs initially acted strategically-rather than becoming compliant or resistant (as health educators would label villages)-in their handling of EVD sensitization. …

Highly Sensitive Detection of HBV RNA in Liver Tissue by In Situ Hybridization.

As soon as HBV was identified, HBV localization at the cellular level became instrumental in studying HBV infection. Multiple methodologies for detection of viral antigens and nucleic acids at the cellular level, not only in patient derived liver biopsy samples, but also in many other experimental systems, have been developed over the years. Recently, the development of highly sensitive and specific in situ hybridization systems enabled detection of cellular mRNAs at the single copy level. Adaptation of such a system (ViewRNA ISH Tissue Assay; Affymetrix, Santa Clara, CA, USA) for the detection of viral RNAs allowed us to reliably identify hepatitis C virus RNA positive cells in the liver of HCV infected patients. Similarly, this protocol enabled detection of very rare HBV positive cells in liver biopsy tissue. Here, we now describe the specifics of the protocol for detection of HBV RNA using the ViewRNA ISH system. The protocol focuses on probe set design, sample preparation and data interpretation for accurate HBV RNA detection in liver tissue samples. The methodology is straightforward and will be very useful in the study of basic HBV virology as well as in clinical applications.

Comprehensive Integration Site Analysis of Human Immunodeficiency Virus during In Vivo Infections Reveals Genomic Regions of Enrichment and Clonal Expansion

BACKGROUNDA key event in the lifecycle of Human Immunodeficiency Virus (HIV) is permanent integration into the infected cells genome. In addition to allowing long-term persistence of the virus, this results in a trackable mark carried in all infected cells. Active HIV replication represses cellular pathways, preventing further cell division. This would imply that any specific integration site (IS) which is clonally expanded either during active or repressed viral infection arises from either a dormant/inactive virus, or is perturbing local gene expression, leading to increased cell proliferation. Alternatively, a cell carrying HIV provirus could proliferate due to T-cell specific antigen stimulation. By analyzing the patterns of integration sites detected in cell cultures and tissue samples from animal models of HIV infection, we can better understand the basic virology of integration site selection and determine what may potentially drive infected cells to persist despite effective treatment regimens.METHODSJurkat reporter cell lines or primary human CD4+ cells were cultured and infected with various strains of HIV including both CCR5 and CXCR4 tropic viruses. Infected cells were cultured up to 21 days post infection, then analyzed for HIV proviral integration sites by next-generation sequencing. For in vivo studies, NSG mice were infused with human CD34+ hematopoietic stem/progenitor cells, resulting in a reconstituted human immune system including high levels of CD4+ T cells capable of sustaining HIV infection. After 16 weeks post-challenge, tissues were collected and subjected to integration site analysis for HIV proviral DNA. Identified integration sites were mapped and compared across multiple parameters to identify chromosomal regions and associated genes enriched for integration events, as well as clonally expanded cells in vivo.RESULTSGenome-wide analysis of HIV integration sites reveals a remarkably similar chromosomal landscape both in tissue culture infection of Jurkat cells and in vivo infection data (Figure 1), as well as across multiple HIV strains. As previously observed, the majority of integrations occur near or within gene coding regions thought to be actively transcribed at time of infection. However, certain areas of the genome, and even unique genes, are enriched for IS in individual samples. In addition to these genomic regions of enrichment, we also observe specific clonal outgrowth of unique integration events in genes previously unidentified in the literature. Three genes in particular exhibit a significant increase of integration events during acute infection which are 3x higher than predicted by random chance alone. We also observe integration events in genes that have been documented by other labs in HIV+ clinical patient samples, however in our active infection models, we do not see those specific genes enriched or expanded. This could indicate that these genes play a role in persistence that is only present during anti-retroviral therapy which suppresses active replication.CONCLUSIONSWe have cataloged the most extensive HIV IS library to date in both relevant tissue culture models and in vivo infection studies, including over 245,000 unique integration events and three different HIV strains commonly used in research. Genome-wide correlation studies reveal regions significantly enriched for HIV integrations and genes which repeatedly exhibit clonal outgrowth in multiple animals. These types of studies are now being applied to human patient samples to determine if latency and persistence of infection can be mapped to unique integration events or genes of interest. Such information may indicate when and how the latent HIV reservoir is seeded and what types of therapy or treatments are most effective at targeting and eliminating these populations.Circos plot comparing HIV integrations sites (IS) identified either during in vitro cell culture infections (black bars), or in vivo infection studies using humanized mice (red bars). The outer ring is composed of human chromosomes each of which are divided into 25kB fragment bins. Total number of unique integration sites identified in each bin is represented by the height of the histogram bars. The in vitro IS concentric ring scale represents increments of 25 outwards up to 250 while the in vivo IS scales inwards in increments of 2 up to 16. [Display omitted] DisclosuresAdair:Rocket Pharmaceuticals: Consultancy, Equity Ownership.

Zika Virus: The Agent and Its Biology, With Relevance to Pathology.

Once obscure, Zika virus (ZIKV) has attracted significant medical and scientific attention in the past year because of large outbreaks associated with the recent introduction of this virus into the Western hemisphere. In particular, the occurrence of severe congenital infections and cases of Guillain-Barré syndrome has placed this virus squarely in the eyes of clinical and anatomic pathologists. This review article provides a basic introduction to ZIKV, its genetics, its structural characteristics, and its biology. A multidisciplinary effort will be essential to establish clinicopathologic correlations of the basic virology of ZIKV in order to advance development of diagnostics, therapeutics, and vaccines.

Why Are Some HIV-1 Subtypes More “Wimpy” at Causing Disease?

Why Are Some HIV-1 Subtypes More “Wimpy” at Causing Disease?

Zika Fetal Neuropathogenesis: Etiology of a Viral Syndrome.

The ongoing Zika virus epidemic in the Americas and the observed association with both fetal abnormalities (primary microcephaly) and adult autoimmune pathology (Guillain–Barré syndrome) has brought attention to this neglected pathogen. While initial case studies generated significant interest in the Zika virus outbreak, larger prospective epidemiology and basic virology studies examining the mechanisms of Zika viral infection and associated pathophysiology are only now starting to be published. In this review, we analyze Zika fetal neuropathogenesis from a comparative pathology perspective, using the historic metaphor of “TORCH” viral pathogenesis to provide context. By drawing parallels to other viral infections of the fetus, we identify common themes and mechanisms that may illuminate the observed pathology. The existing data on the susceptibility of various cells to both Zika and other flavivirus infections are summarized. Finally, we highlight relevant aspects of the known molecular mechanisms of flavivirus replication.

Fungus-Derived Neoechinulin B as a Novel Antagonist of Liver X Receptor, Identified by Chemical Genetics Using a Hepatitis C Virus Cell Culture System.

Cell culture systems reproducing virus replication can serve as unique models for the discovery of novel bioactive molecules. Here, using a hepatitis C virus (HCV) cell culture system, we identified neoechinulin B (NeoB), a fungus-derived compound, as an inhibitor of the liver X receptor (LXR). NeoB was initially identified by chemical screening as a compound that impeded the production of infectious HCV. Genome-wide transcriptome analysis and reporter assays revealed that NeoB specifically inhibits LXR-mediated transcription. NeoB was also shown to interact directly with LXRs. Analysis of structural analogs suggested that the molecular interaction of NeoB with LXR correlated with the capacity to inactivate LXR-mediated transcription and to modulate lipid metabolism in hepatocytes. Our data strongly suggested that NeoB is a novel LXR antagonist. Analysis using NeoB as a bioprobe revealed that LXRs support HCV replication: LXR inactivation resulted in dispersion of double-membrane vesicles, putative viral replication sites. Indeed, cells treated with NeoB showed decreased replicative permissiveness for poliovirus, which also replicates in double-membrane vesicles, but not for dengue virus, which replicates via a distinct membrane compartment. Together, our data suggest that LXR-mediated transcription regulates the formation of virus-associated membrane compartments. Significantly, inhibition of LXRs by NeoB enhanced the activity of all known classes of anti-HCV agents, and NeoB showed especially strong synergy when combined with interferon or an HCV NS5A inhibitor. Thus, our chemical genetics analysis demonstrates the utility of the HCV cell culture system for identifying novel bioactive molecules and characterizing the virus-host interaction machinery. Hepatitis C virus (HCV) is highly dependent on host factors for efficient replication. In the present study, we used an HCV cell culture system to screen an uncharacterized chemical library. Our results identified neoechinulin B (NeoB) as a novel inhibitor of the liver X receptor (LXR). NeoB inhibited the induction of LXR-regulated genes and altered lipid metabolism. Intriguingly, our results indicated that LXRs are critical to the process of HCV replication: LXR inactivation by NeoB disrupted double-membrane vesicles, putative sites of viral replication. Moreover, NeoB augmented the antiviral activity of all known classes of currently approved anti-HCV agents without increasing cytotoxicity. Thus, our strategy directly links the identification of novel bioactive compounds to basic virology and the development of new antiviral agents.

Infection Counter: Automated Quantification of in Vitro Virus Replication by Fluorescence Microscopy.

The ability to accurately and reliably quantify viral infection is essential to basic and translational virology research. Here, we describe a simple and robust automated method for using fluorescence microscopy to estimate the proportion of virally infected cells in a monolayer. We provide details of the automated analysis workflow along with a freely available open-source ImageJ plugin, Infection Counter, for performing image quantification. Using hepatitis C virus (HCV) as an example, we have experimentally verified our method, demonstrating that it is equivalent, if not better, than the established focus-forming assay. Finally, we used Infection Counter to assess the anti-HCV activity of SMBz-CsA, a non-immunosuppressive cyclosporine analogue.