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1.  Pathogenesis and molecular biology of progressive multifocal leukoencephalopathy, the JC virus-induced demyelinating disease of the human brain. 
Clinical Microbiology Reviews  1992;5(1):49-73.
Studies of the pathogenesis and molecular biology of JC virus infection over the last two decades have significantly changed our understanding of progressive multifocal leukoencephalopathy, which can be described as a subacute viral infection of neuroglial cells that probably follows reactivation of latent infection rather than being the consequence of prolonged JC virus replication in the brain. There is now sufficient evidence to suggest that JC virus latency occurs in kidney and B cells. However, JC virus isolates from brain or kidney differ in the regulatory regions of their viral genomes which are controlled by host cell factors for viral gene expression and replication. DNA sequences of noncoding regions of the viral genome display a certain heterogeneity among isolates from brain and kidney. These data suggest that an archetypal strain of JC virus exists whose sequence is altered during replication in different cell types. The JC virus regulatory region likely plays a significant role in establishing viral latency and must be acted upon for reactivation of the virus. A developing hypothesis is that reactivation takes place from latently infected B lymphocytes that are activated as a result of immune suppression. JC virus enters the brain in the activated B cell. Evidence for this mechanism is the detection of JC virus DNA in peripheral blood lymphocytes and infected B cells in the brains of patients with progressive multifocal leukoencephalopathy. Once virus enters the brain, astrocytes as well as oligodendrocytes support JC virus multiplication. Therefore, JC virus infection of neuroglial cells may impair other neuroglial functions besides the production and maintenance of myelin. Consequently our increased understanding of the pathogenesis of progressive multifocal leukoencephalopathy suggests new ways to intervene in JC virus infection with immunomodulation therapies. Perhaps along with trials of nucleoside analogs or interferon administration, this fatal disease, for which no consensus of antiviral therapy exists, may yield to innovative treatment protocols.
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PMCID: PMC358223  PMID: 1310438
2.  Molecular Biology of Pseudorabies Virus: Impact on Neurovirology and Veterinary Medicine 
Pseudorabies virus (PRV) is a herpesvirus of swine, a member of the Alphaherpesvirinae subfamily, and the etiological agent of Aujeszky's disease. This review describes the contributions of PRV research to herpesvirus biology, neurobiology, and viral pathogenesis by focusing on (i) the molecular biology of PRV, (ii) model systems to study PRV pathogenesis and neurovirulence, (iii) PRV transsynaptic tracing of neuronal circuits, and (iv) veterinary aspects of pseudorabies disease. The structure of the enveloped infectious particle, the content of the viral DNA genome, and a step-by-step overview of the viral replication cycle are presented. PRV infection is initiated by binding to cellular receptors to allow penetration into the cell. After reaching the nucleus, the viral genome directs a regulated gene expression cascade that culminates with viral DNA replication and production of new virion constituents. Finally, progeny virions self-assemble and exit the host cells. Animal models and neuronal culture systems developed for the study of PRV pathogenesis and neurovirulence are discussed. PRV serves as a self-perpetuating transsynaptic tracer of neuronal circuitry, and we detail the original studies of PRV circuitry mapping, the biology underlying this application, and the development of the next generation of tracer viruses. The basic veterinary aspects of pseudorabies management and disease in swine are discussed. PRV infection progresses from acute infection of the respiratory epithelium to latent infection in the peripheral nervous system. Sporadic reactivation from latency can transmit PRV to new hosts. The successful management of PRV disease has relied on vaccination, prevention, and testing.
doi:10.1128/MMBR.69.3.462-500.2005
PMCID: PMC1197806  PMID: 16148307
3.  EBV Chronic Infections 
The infection from Epstein-Barr virus (EBV) or virus of infectious mononucleosis, together with other herpes viruses’ infections, represents a prototype of persistent viral infections characterized by the property of the latency. Although the reactivations of the latent infection are associated with the resumption of the viral replication and eventually with the “shedding”, it is still not clear if this virus can determine chronic infectious diseases, more or less evolutive. These diseases could include some pathological conditions actually defined as “idiopathic”and characterized by the “viral persistence” as the more credible pathogenetic factor. Among the so-called idiopathic syndromes, the “chronic fatigue syndrome” (CFS) aroused a great interest around the eighties of the last century when, just for its relationship with EBV, it was called “chronic mononucleosis” or “chronic EBV infection”.
Today CFS, as defined in 1994 by the CDC of Atlanta (USA), really represents a multifactorial syndrome characterized by a chronic course, where reactivation and remission phases alternate, and by a good prognosis. The etiopathogenetic role of EBV is demonstrated only in a well-examined subgroup of patients, while in most of the remaining cases this role should be played by other infectious agents - able to remain in a latent or persistent way in the host – or even by not infectious agents (toxic, neuroendocrine, methabolic, etc.). However, the pathogenetic substrate of the different etiologic forms seems to be the same, much probably represented by the oxidative damage due to the release of pro-inflammatory cytokines as a response to the triggering event (infectious or not infectious).
Anyway, recently the scientists turned their’s attention to the genetic predisposition of the subjects affected by the syndrome, so that in the last years the genetic studies, together with those of molecular biology, received a great impulse. Thanks to both these studies it was possibile to confirm the etiologic links between the syndrome and EBV or other herpesviruses or other persistent infectious agents.
The mechanisms of EBV latency have been carefully examined both because they represent the virus strategy to elude the response of the immune system of the host, and because they are correlated with those oncologic conditions associated to the viral persistence, particularly lymphomas and lymphoproliferative disorders. Just these malignancies, for which a pathogenetic role of EBV is clearly documented, should represent the main clinical expression of a first group of chronic EBV infections characterized by a natural history where the neoplastic event aroused from the viral persistence in the resting B cells for all the life, from the genetic predisposition of the host and from the oncogenic potentialities of the virus that chronically persists and incurs reactivations.
Really, these oncological diseases should be considered more complications than chronic forms of the illness, as well as other malignancies for which a viral – or even infectious - etiology is well recognized. The chronic diseases, in fact, should be linked in a pathogenetic and temporal way to the acute infection, from whom start the natural history of the following disease. So, as for the chronic liver diseases from HBV and HCV, it was conied the acronym of CAEBV (Chronic Active EBV infection), distinguishing within these pathologies the more severe forms (SCAEBV) mostly reported in Far East and among children or adolescents. Probably only these forms have to be considered expressions of a chronic EBV infection “sensu scrictu”, together with those forms of CFS where the etiopathogenetic and temporal link with the acute EBV infection is well documented. As for CFS, also for CAEBV the criteria for a case definition were defined, even on the basis of serological and virological findings. However, the lymphoproliferative disorders are excluded from these forms and mantain their nosographic (e.g. T or B cell or NK type lymphomas) and pathogenetic collocation, even when they occur within chronic forms of EBV infection. In the pathogenesis, near to the programs of latency of the virus, the genetic and environmental factors, independent from the real natural history of EBV infection, play a crucial role.
Finally, it was realized a review of cases - not much numerous in literature – of chronic EBV infection associated to chronic liver and neurological diseases, where the modern techniques of molecular biology should be useful to obtain a more exact etiologic definition, not always possibile to reach in the past.
The wide variety of clinical forms associated to the EBV chronic infection makes difficult the finding of a univocal pathogenetic link. There is no doubt, however, that a careful examination of the different clinical forms described in this review should be useful to open new horizons to the study of the persistent viral infections and the still not well cleared pathologies that they can induce in the human host.
doi:10.4084/MJHID.2010.022
PMCID: PMC3033110  PMID: 21415952
4.  A Bayesian Approach to Analyse Genetic Variation within RNA Viral Populations 
PLoS Computational Biology  2011;7(3):e1002027.
The development of modern and affordable sequencing technologies has allowed the study of viral populations to an unprecedented depth. This is of particular interest for the study of within-host RNA viral populations, where variation due to error-prone polymerases can lead to immune escape, antiviral resistance and adaptation to new host species. Methods to sequence RNA virus genomes include reverse transcription (RT) and polymerase chain reaction (PCR). RT-PCR is a molecular biology technique widely used to amplify DNA from an RNA template. The method itself relies on the in vitro synthesis of copy DNA from RNA followed by multiple cycles of DNA amplification. However, this method introduces artefactual errors that can act as confounding factors when the sequence data are analysed. Although there are a growing number of published studies exploring the intra- and inter-host evolutionary dynamics of RNA viruses, the complexity of the methods used to generate sequences makes it difficult to produce probabilistic statements about the likely sources of observed sequence variants. This complexity is further compounded as both the depth of sequencing and the length of the genome segment of interest increase. Here we develop a Bayesian method to characterise and differentiate between likely structures for the background viral population. This approach can then be used to identify nucleotide sites that show evidence of change in the within-host viral population structure, either over time or relative to a reference sequence (e.g. an inoculum or another source of infection), or both, without having to build complex evolutionary models. Identification of these sites can help to inform the design of more focussed experiments using molecular biology tools, such as site-directed mutagenesis, to assess the function of specific amino acids. We illustrate the method by applying to datasets from experimental transmission of equine influenza, and a pre-clinical vaccine trial for HIV-1.
Author Summary
Characterising genetic variation in viral populations can have important implications in terms of understanding how viruses evolve within infected hosts. Modern sequencing technologies allow genetic information to be obtained faster, more affordably, and in much greater quantities than before. This allows new experimental procedures to be designed to explore aspects of pathogenesis that were previously unattainable, particularly with regard to mutations that occur at particular nucleotide sites that may confer a fitness advantage to the pathogen. This information can be used to study important issues such as the development of antiviral resistance, virulence, and/or changes in host-range specificity. Nonetheless, the experimental procedures used to generate the data can incorporate artefactual errors, and in order to optimise the information obtained from these studies techniques are required to characterise which sites exhibit mutations that may alter viral fitness. As both the depth of sequencing increases and the length of the region sequenced increases (e.g. moves to whole genomes rather than smaller segments), large numbers of sites will exhibit some form of variation, and hence development of a probabilistic method to define and extract these sites-of-interest becomes more important. We tackle this problem here using a Bayesian framework.
doi:10.1371/journal.pcbi.1002027
PMCID: PMC3068928  PMID: 21483482
5.  Synergistic Interactions between the NS3hel and E Proteins Contribute to the Virulence of Dengue Virus Type 1 
Background
Dengue includes a broad range of symptoms, ranging from fever to hemorrhagic fever and may occasionally have alternative clinical presentations. Many possible viral genetic determinants of the intrinsic virulence of dengue virus (DENV) in the host have been identified, but no conclusive evidence of a correlation between viral genotype and virus transmissibility and pathogenicity has been obtained.
Methodology/Principal Findings
We used reverse genetics techniques to engineer DENV-1 viruses with subsets of mutations found in two different neuroadapted derivatives. The mutations were inserted into an infectious clone of DENV-1 not adapted to mice. The replication and viral production capacity of the recombinant viruses were assessed in vitro and in vivo. The results demonstrated that paired mutations in the envelope protein (E) and in the helicase domain of the NS3 (NS3hel) protein had a synergistic effect enhancing viral fitness in human and mosquito derived cell lines. E mutations alone generated no detectable virulence in the mouse model; however, the combination of these mutations with NS3hel mutations, which were mildly virulent on their own, resulted in a highly neurovirulent phenotype.
Conclusions/Significance
The generation of recombinant viruses carrying specific E and NS3hel proteins mutations increased viral fitness both in vitro and in vivo by increasing RNA synthesis and viral load (these changes being positively correlated with central nervous system damage), the strength of the immune response and animal mortality. The introduction of only pairs of amino acid substitutions into the genome of a non-mouse adapted DENV-1 strain was sufficient to alter viral fitness substantially. Given current limitations to our understanding of the molecular basis of dengue neuropathogenesis, these results could contribute to the development of attenuated strains for use in vaccinations and provide insights into virus/host interactions and new information about the mechanisms of basic dengue biology.
Author Summary
Dengue virus constitutes a significant public health problem in tropical regions of the world. Despite the high morbidity and mortality of this infection, no effective antiviral drugs or vaccines are available for the treatment or prevention of dengue infections. The profile of clinical signs associated with dengue infection has changed in recent years with an increase in the number of episodes displaying unusual signs. We use reverse genetics technology to engineer DENV-1 viruses with subsets of mutations previously identified in highly neurovirulent strains to provide insights into the molecular mechanisms underlying dengue neuropathogenesis. We found that single mutations affecting the E and NS3hel proteins, introduced in a different genetic context, had a synergistic effect increasing DENV replication capacity in human and mosquito derived cells in vitro. We also demonstrated correlations between the presence of these mutations and viral replication efficiency, viral loads, the induction of innate immune response genes and pathogenesis in a mouse model. These results should improve our understanding of the DENV-host cell interaction and contribute to the development of effective antiviral strategies.
doi:10.1371/journal.pntd.0001624
PMCID: PMC3328427  PMID: 22530074
6.  Dengue Virus Capsid Protein Usurps Lipid Droplets for Viral Particle Formation 
PLoS Pathogens  2009;5(10):e1000632.
Dengue virus is responsible for the highest rates of disease and mortality among the members of the Flavivirus genus. Dengue epidemics are still occurring around the world, indicating an urgent need of prophylactic vaccines and antivirals. In recent years, a great deal has been learned about the mechanisms of dengue virus genome amplification. However, little is known about the process by which the capsid protein recruits the viral genome during encapsidation. Here, we found that the mature capsid protein in the cytoplasm of dengue virus infected cells accumulates on the surface of ER-derived organelles named lipid droplets. Mutagenesis analysis using infectious dengue virus clones has identified specific hydrophobic amino acids, located in the center of the capsid protein, as key elements for lipid droplet association. Substitutions of amino acid L50 or L54 in the capsid protein disrupted lipid droplet targeting and impaired viral particle formation. We also report that dengue virus infection increases the number of lipid droplets per cell, suggesting a link between lipid droplet metabolism and viral replication. In this regard, we found that pharmacological manipulation of the amount of lipid droplets in the cell can be a means to control dengue virus replication. In addition, we developed a novel genetic system to dissociate cis-acting RNA replication elements from the capsid coding sequence. Using this system, we found that mislocalization of a mutated capsid protein decreased viral RNA amplification. We propose that lipid droplets play multiple roles during the viral life cycle; they could sequester the viral capsid protein early during infection and provide a scaffold for genome encapsidation.
Author Summary
Dengue virus is the single most significant arthropod-borne virus pathogen in humans. In spite of the urgent medical need to control dengue infections, vaccines are still unavailable, and many aspects of dengue virus biology and pathogenesis remain elusive. We discovered a link between dengue virus replication and ER-derived organelles known as lipid droplets (LDs). Dengue infection increases the amount of LDs per cell and pharmacological inhibition of LD formation greatly reduces dengue virus replication. In addition, we have found that the viral capsid protein in infected cells accumulates on the surface of LDs. Manipulation of infectious clones and generation of new reporter dengue viruses allowed us to define the molecular basis of capsid protein association to LDs. Specific amino acids on the α2 helix, located in the center of the capsid protein, were found to be crucial for both accumulation of capsid protein on LDs and dengue virus infectious particle formation. We propose that LDs facilitate viral replication providing a platform for nucleocapsid formation during encapsidation. Our findings begin to unravel the complex mechanism by which dengue virus usurps cellular organelles to coordinate different steps of the viral life cycle.
doi:10.1371/journal.ppat.1000632
PMCID: PMC2760139  PMID: 19851456
7.  Stabilization of Myc through Heterotypic Poly-Ubiquitination by mLANA Is Critical for γ-Herpesvirus Lymphoproliferation 
PLoS Pathogens  2013;9(8):e1003554.
Host colonization by lymphotropic γ-herpesviruses depends critically on expansion of viral genomes in germinal center (GC) B-cells. Myc is essential for the formation and maintenance of GCs. Yet, the role of Myc in the pathogenesis of γ-herpesviruses is still largely unknown. In this study, Myc was shown to be essential for the lymphotropic γ-herpesvirus MuHV-4 biology as infected cells exhibited increased expression of Myc signature genes and the virus was unable to expand in Myc defficient GC B-cells. We describe a novel strategy of a viral protein activating Myc through increased protein stability resulting in increased progression through the cell cycle. This is acomplished by modulating a physiological post-translational regulatory pathway of Myc. The molecular mechanism involves Myc heterotypic poly-ubiquitination mediated via the viral E3 ubiquitin-ligase mLANA protein. EC5SmLANA modulates cellular control of Myc turnover by antagonizing SCFFbw7 mediated proteasomal degradation of Myc, mimicking SCFβ-TrCP. The findings here reported reveal that modulation of Myc is essential for γ-herpesvirus persistent infection, establishing a link between virus induced lymphoproliferation and disease.
Author Summary
Being obligatory intracellular parasites, it is not surprising that viruses have evolved mechanisms to induce cellular proliferation to promote their own life cycle. This is notorious in the case of γ-herpesviruses, such as Epstein-Barr virus (EBV) and Kaposi's sarcoma virus (KSHV), which are human pathogens associated with lymphoproliferative disease and several tumors. Host colonization by γ-herpesviruses is critically dependent on the ability to expand latent infection in proliferating B-cells. Virus-induced cellular proliferation is a process mediated by the expression of specific viral proteins. One of such proteins is the latency-associated protein (LANA) of KSHV. In this study, we use murid herpesvirus-4 (MuHV-4) as a mouse model of γ-herpesvirus pathogenesis. We show that the MuHV-4 LANA (mLANA) stabilizes the cellular oncogene Myc, increasing its half-life, thus promoting its activity as a potent inducer of cellular proliferation. The molecular mechanism involves heterotypic poly-ubiquitination of Myc mediated via mLANA. The findings here reported demonstrate that modulation of Myc is essential for γ-herpesvirus persistent infection, establishing a link between virus induced lymphoproliferation and disease. The implication is that revealing a critical function of a viral protein possibly allows the development of small molecule probes to disrupt mLANA-Myc interaction, therefore inhibit virus induced lyhophoproliferative disease.
doi:10.1371/journal.ppat.1003554
PMCID: PMC3738482  PMID: 23950719
8.  Dissecting the HIV-specific immune response: a systems biology approach 
Purpose of review
Several unique HIV-infected or HIV-resistant cohorts have been studied over the years to try and delineate the correlates of protection. Although several mechanisms have been put forward, studies aiming to integrate the different mechanisms into a comprehensive model are still lacking. Current systems biology approaches emphasize the importance of unifying independent datasets, provide tools that facilitate hypothesis formulation and testing, and direct us toward uncovering novel therapeutic targets by defining molecular networks perturbed during disease. This review will focus on the current findings that utilized systems biology techniques in order to identify correlates of protection from HIV disease progression and resistance to infection in unique cohorts of individuals as well as in nonhuman primate models of SIV infection.
Recent findings
Using systems biology technologies and data analysis tools, the studies described herein have found that pathways implicated in survival, cell cycling, inflammation, and oxidative stress work in unison to limit pathology caused by chronic immune activation. This situation favors the survival of effector lymphocytes and limits the dissemination of viral particles in HIV elite controllers, exposed-uninfected individuals, and natural hosts of SIV infection.
Summary
Systems and computational biology tools have clearly expanded our understanding of HIV pathogenesis by unifying independent observations and by giving us novel molecular targets to pursue. These molecular signatures have the potential to uncover correlates of protection in HIV disease and, in the era of personalized medicine, to determine predictive signatures of treatment efficacy and/or failure.
doi:10.1097/COH.0b013e32834ddb0e
PMCID: PMC3627479  PMID: 22134339
elite controllers; exposed-uninfected; HIV; networks; pathways; resistance; systems biology
9.  Regulation of bovine papillomavirus type 1 gene expression by RNA processing 
Bovine papillomavirus type 1 (BPV-1) has served as a prototype for studying the molecular biology and pathogenesis of papillomaviruses. The expression of BPV-1 early and late genes is highly regulated at both transcription and post-transcriptional levels and strictly tied to the differentiation of keratinocytes. BPV-1 infects keratinocytes in the basal layer of the skin and replicates in the nucleus of infected cells in a differentiation-dependent manner. Although viral early genes begin to be expressed from the infected, undifferentiated basal cells, viral late genes are not expressed until the infected cells enter the terminal differentiation stage. Both BPV-1 early and late transcripts are intron-containing bicistronic or polycistronic RNAs, bearing more than one open reading frame and are polyadenylated at either an early or late poly (A) site. Nuclear RNA processing of these transcripts by RNA splicing and poly (A) site selection has been extensively analyzed in the past decade and various viral cis-elements and cellular factors involved in regulation of viral RNA processing were discovered, leading to our better understanding of the gene expression and biology of human papillomaviruses.
PMCID: PMC2654602  PMID: 19273129
Papillomaviruses; Gene expression; RNA splicing; RNA polyadenylation; Post-transcriptional regulation; Review
10.  Rotaviruses: from pathogenesis to vaccination 
Gastroenterology  2009;136(6):1939-1951.
Rotaviruses cause life-threatening gastroenteritis in children worldwide; the enormous disease burden has focused efforts to develop vaccines and led to the discovery of novel mechanisms of gastrointestinal virus pathogenesis and host responses to infection. Two live-attenuated vaccines for gastroenteritis (Rotateq and Rotarix) have been licensed in many countries. This review summarizes the latest data on these vaccines, their effectiveness and challenges to global vaccination. Recent insights into rotavirus pathogenesis are also discussed, including information on extra-intestinal infection, viral antagonists of the interferon response and the first described viral enterotoxin. Rotavirus-induced diarrhea is now considered to be a disease that can be prevented through vaccination, although there are many challenges to achieving global effectiveness. Molecular biology studies of rotavirus replication and pathogenesis have identified unique viral targets that might be useful in developing therapies for immunocompromised children with chronic infections.
doi:10.1053/j.gastro.2009.02.076
PMCID: PMC3690811  PMID: 19457420
11.  Memories of John N. Brady: scientist, mentor and friend 
Retrovirology  2009;6:48.
Friends and colleagues remember John N. Brady, Ph.D., Chief of the Virus Tumor Biology Section of the Laboratory of Cellular Oncology, who died much too young at the age of 57 on April 27, 2009 of colon cancer. John grew up in Illinois and received his Ph.D. with Dr. Richard Consigli at Kansas State University studying the molecular structure of polyomavirus. In 1984 John came to the National Institutes of Health as a Staff Fellow in the laboratory of Dr. Norman Salzman, Laboratory of Biology of Viruses NIAID, where he was among the first to analyze SV40 transcription using in vitro transcription systems and to analyze regulatory sequences for SV40 late transcription. He then trained with Dr. George Khoury in the Laboratory of Molecular Virology NCI, where he identified SV40 T-antigen as a transcriptional activator protein. His research interests grew to focus on the human retroviruses: human T-cell lymphotropic virus type I (HTLV-I) and human immunodeficiency virus (HIV), analyzing how interactions between these viruses and the host cell influence viral gene regulation, viral pathogenesis and viral transformation. His research also impacted the fields of eukaryotic gene regulation and tumor suppressor proteins. John is survived by his wife, Laraine, and two sons, Matt and Kevin.
doi:10.1186/1742-4690-6-48
PMCID: PMC2693500  PMID: 19454030
12.  The First Endogenous Herpesvirus, Identified in the Tarsier Genome, and Novel Sequences from Primate Rhadinoviruses and Lymphocryptoviruses 
PLoS Genetics  2014;10(6):e1004332.
Herpesviridae is a diverse family of large and complex pathogens whose genomes are extremely difficult to sequence. This is particularly true for clinical samples, and if the virus, host, or both genomes are being sequenced for the first time. Although herpesviruses are known to occasionally integrate in host genomes, and can also be inherited in a Mendelian fashion, they are notably absent from the genomic fossil record comprised of endogenous viral elements (EVEs). Here, we combine paleovirological and metagenomic approaches to both explore the constituent viral diversity of mammalian genomes and search for endogenous herpesviruses. We describe the first endogenous herpesvirus from the genome of the Philippine tarsier, belonging to the Roseolovirus genus, and characterize its highly defective genome that is integrated and flanked by unambiguous host DNA. From a draft assembly of the aye-aye genome, we use bioinformatic tools to reveal over 100,000 bp of a novel rhadinovirus that is the first lemur gammaherpesvirus, closely related to Kaposi's sarcoma-associated virus. We also identify 58 genes of Pan paniscus lymphocryptovirus 1, the bonobo equivalent of human Epstein-Barr virus. For each of the viruses, we postulate gene function via comparative analysis to known viral relatives. Most notably, the evidence from gene content and phylogenetics suggests that the aye-aye sequences represent the most basal known rhadinovirus, and indicates that tumorigenic herpesviruses have been infecting primates since their emergence in the late Cretaceous. Overall, these data show that a genomic fossil record of herpesviruses exists despite their extremely large genomes, and expands the known diversity of Herpesviridae, which will aid the characterization of pathogenesis. Our analytical approach illustrates the benefit of intersecting evolutionary approaches with metagenomics, genetics and paleovirology.
Author Summary
Herpesviridae is a family of DNA viruses that have characteristically large and complex genomes. This defining feature is also responsible for bioinformatic challenges that complicate herpesvirus genomics, and why an endogenous herpesvirus remains elusive. Given that several species of herpesvirus are clinically relevant to humans, there is a pressing demand for techniques capable of generating and managing large quantities of herpesvirus genome data. This is coupled with a need to explore herpesvirus diversity in order to understand pathogenesis within an evolutionary context. Lessons from the study of ancient viral integrations have also highlighted the need to include information offered by paleoviruses. Using perspectives from paleovirology and metagenomics, we identify three herpesviruses within the genome data of their primate hosts, including the first endogenous herpesvirus. All three viruses are closely related to important human pathogens and two of them are entirely new species. Both comparative molecular biology and evolutionary analysis were applied to examine our results for their clinical relevance. Furthermore, we demonstrate how this analytical approach was also used for the data collection itself, by treating nucleotide databases in their entirety as a single metagenomic resource.
doi:10.1371/journal.pgen.1004332
PMCID: PMC4063692  PMID: 24945689
13.  Schmallenberg Virus Pathogenesis, Tropism and Interaction with the Innate Immune System of the Host 
PLoS Pathogens  2013;9(1):e1003133.
Schmallenberg virus (SBV) is an emerging orthobunyavirus of ruminants associated with outbreaks of congenital malformations in aborted and stillborn animals. Since its discovery in November 2011, SBV has spread very rapidly to many European countries. Here, we developed molecular and serological tools, and an experimental in vivo model as a platform to study SBV pathogenesis, tropism and virus-host cell interactions. Using a synthetic biology approach, we developed a reverse genetics system for the rapid rescue and genetic manipulation of SBV. We showed that SBV has a wide tropism in cell culture and “synthetic” SBV replicates in vitro as efficiently as wild type virus. We developed an experimental mouse model to study SBV infection and showed that this virus replicates abundantly in neurons where it causes cerebral malacia and vacuolation of the cerebral cortex. These virus-induced acute lesions are useful in understanding the progression from vacuolation to porencephaly and extensive tissue destruction, often observed in aborted lambs and calves in naturally occurring Schmallenberg cases. Indeed, we detected high levels of SBV antigens in the neurons of the gray matter of brain and spinal cord of naturally affected lambs and calves, suggesting that muscular hypoplasia observed in SBV-infected lambs is mostly secondary to central nervous system damage. Finally, we investigated the molecular determinants of SBV virulence. Interestingly, we found a biological SBV clone that after passage in cell culture displays increased virulence in mice. We also found that a SBV deletion mutant of the non-structural NSs protein (SBVΔNSs) is less virulent in mice than wild type SBV. Attenuation of SBV virulence depends on the inability of SBVΔNSs to block IFN synthesis in virus infected cells. In conclusion, this work provides a useful experimental framework to study the biology and pathogenesis of SBV.
Author Summary
Schmallenberg virus (SBV) was discovered in Germany (near the town of Schmallenberg) in November 2011 and since then has been found to be the cause of malformations and stillbirths in ruminants. SBV has spread very rapidly to many European countries including the Netherlands, Belgium, France and the United Kingdom. Very little is known about the biological properties of this virus and there is no vaccine available. In this study (i) we developed an approach (called reverse genetics) that allows the recovery of “synthetic” SBV under laboratory conditions; (ii) we developed a mouse model of infection for SBV; (iii) we showed that SBV replicates in neurons of experimentally infected mice similar to naturally infected lambs and calves; (iv) we developed viral mutants that are not as pathogenic as the original virus due to the inability to counteract the host cell defenses; and v) we identified mutations that are associated with increased virulence. This work provides the experimental tools to understand how this newly emerged virus causes disease in ruminants. In addition, it will now be possible to manipulate the SBV genome in order to develop highly effective vaccines.
doi:10.1371/journal.ppat.1003133
PMCID: PMC3542112  PMID: 23326235
14.  Differential Regulation of Type I Interferon and Epidermal Growth Factor Pathways by a Human Respirovirus Virulence Factor 
PLoS Pathogens  2009;5(9):e1000587.
A number of paramyxoviruses are responsible for acute respiratory infections in children, elderly and immuno-compromised individuals, resulting in airway inflammation and exacerbation of chronic diseases like asthma. To understand the molecular pathogenesis of these infections, we searched for cellular targets of the virulence protein C of human parainfluenza virus type 3 (hPIV3-C). We found that hPIV3-C interacts directly through its C-terminal domain with STAT1 and GRB2, whereas C proteins from measles or Nipah viruses failed to do so. Binding to STAT1 explains the previously reported capacity of hPIV3-C to block type I interferon signaling, but the interaction with GRB2 was unexpected. This adaptor protein bridges Epidermal Growth Factor (EGF) receptor to MAPK/ERK pathway, a signaling cascade recently found to be involved in airway inflammatory response. We report that either hPIV3 infection or transient expression of hPIV3-C both increase cellular response to EGF, as assessed by Elk1 transactivation and phosphorylation levels of ERK1/2, 40S ribosomal subunit protein S6 and translation initiation factor 4E (eIF4E). Furthermore, inhibition of MAPK/ERK pathway with U0126 prevented viral protein expression in infected cells. Altogether, our data provide molecular basis to explain the role of hPIV3-C as a virulence factor and determinant of pathogenesis and demonstrate that Paramyxoviridae have evolved a single virulence factor to block type I interferon signaling and to boost simultaneous cellular response to growth factors.
Author Summary
Respiroviruses are important pathogens responsible for acute respiratory tract infections associated with severe airway inflammation in children, elderly and immuno-compromised individuals. Their RNA genome encodes for structural proteins that compose viral particles, but also for virulence factors that alter cell biology to enhance virus replication and spreading. Among them, the C protein plays a critical role by blocking cellular response to type I interferons, the main antiviral cytokines secreted during virus infections. To provide molecular basis to this activity, we found that the C protein of human parainfluenza virus type 3 (hPIV3-C), the most frequent human Respirovirus, interacts with STAT1, a key component of type I interferon receptor complex. But hPIV3-C was also found to interact with GRB2, an adaptor molecule involved in cellular response to Epidermal Growth Factor (EGF), and to enhance cell response to this cytokine. This pathway increases protein translation, promotes cell survival and contributes to airway inflammation and mucus secretion. Thus, our findings show that hPIV3-C not only inhibits the antiviral response but also stimulates cellular response to EGF, which benefits virus replication and induces an excessive inflammation of airways during infection.
doi:10.1371/journal.ppat.1000587
PMCID: PMC2736567  PMID: 19806178
15.  Ago HITS-CLIP Expands Understanding of Kaposi's Sarcoma-associated Herpesvirus miRNA Function in Primary Effusion Lymphomas 
PLoS Pathogens  2012;8(8):e1002884.
KSHV is the etiological agent of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and a subset of multicentricCastleman's disease (MCD). The fact that KSHV-encoded miRNAs are readily detectable in all KSHV-associated tumors suggests a potential role in viral pathogenesis and tumorigenesis. MiRNA-mediated regulation of gene expression is a complex network with each miRNA having many potential targets, and to date only few KSHV miRNA targets have been experimentally determined. A detailed understanding of KSHV miRNA functions requires high-through putribonomics to globally analyze putative miRNA targets in a cell type-specific manner. We performed Ago HITS-CLIP to identify viral and cellular miRNAs and their cognate targets in two latently KSHV-infected PEL cell lines. Ago HITS-CLIP recovered 1170 and 950 cellular KSHVmiRNA targets from BCBL-1 and BC-3, respectively. Importantly, enriched clusters contained KSHV miRNA seed matches in the 3′UTRs of numerous well characterized targets, among them THBS1, BACH1, and C/EBPβ. KSHV miRNA targets were strongly enriched for genes involved in multiple pathways central for KSHV biology, such as apoptosis, cell cycle regulation, lymphocyte proliferation, and immune evasion, thus further supporting a role in KSHV pathogenesis and potentially tumorigenesis. A limited number of viral transcripts were also enriched by HITS-CLIP including vIL-6 expressed only in a subset of PEL cells during latency. Interestingly, Ago HITS-CLIP revealed extremely high levels of Ago-associated KSHV miRNAs especially in BC-3 cells where more than 70% of all miRNAs are of viral origin. This suggests that in addition to seed match-specific targeting of cellular genes, KSHV miRNAs may also function by hijacking RISCs, thereby contributing to a global de-repression of cellular gene expression due to the loss of regulation by human miRNAs. In summary, we provide an extensive list of cellular and viral miRNA targets representing an important resource to decipher KSHV miRNA function.
Author Summary
Kaposi's sarcoma-associated herpesvirus is the etiological agent of KS and two lymphoproliferative diseases: multicentricCastleman's disease and primary effusion lymphomas (PEL). KSHV tumors are the most prevalent AIDS malignancies and within Sub-Saharan Africa KS is the most common cancer in males, both in the presence and absence of HIV infection. KSHV encodes 12 miRNA genes whose function is largely unknown. Viral miRNAs are incorporated into RISCs, which regulate gene expression mostly by binding to 3′UTRs of mRNAs to inhibit their translation and/or induce degradation. The small subset of viral miRNA targets identified to date suggests that these small posttranscriptional regulators target important cellular pathways involved in pathogenesis and tumorgenesis. Using Ago HITS-CLIP, a technique which combines UV cross-linking, immunoprecipitation of Ago-miRNA-mRNA complexes, and high throughput sequencing, we performed a detailed analysis of the KSHV miRNA targetome in two commonly studied PEL cell lines, BCBL-1 and BC-3 and identified 1170 and 950 putative miRNA targets, respectively. This data set provides a valuable resource to decipher how KSHV miRNAs contribute to viral biology and pathogenesis.
doi:10.1371/journal.ppat.1002884
PMCID: PMC3426530  PMID: 22927820
16.  Live-Cell Characterization and Analysis of a Clinical Isolate of Bovine Respiratory Syncytial Virus, Using Molecular Beacons 
Journal of Virology  2006;80(2):682-688.
Understanding viral pathogenesis is critical for prevention of outbreaks, development of antiviral drugs, and biodefense. Here, we utilize molecular beacons to directly detect the viral genome and characterize a clinical isolate of bovine respiratory syncytial virus (bRSV) in living cells. Molecular beacons are dual-labeled, hairpin oligonucleotide probes with a reporter fluorophore at one end and a quencher at the other; they are designed to fluoresce only when hybridizing to a complementary target. By imaging the fluorescence signal of molecular beacons, the spread of bRSV was monitored for 7 days with a signal-to-noise ratio of 50 to 200, and the measured time course of infection was quantified with a mathematical model for viral growth. We found that molecular beacon signal could be detected in single living cells infected with a viral titer of 2 × 103.6 50% tissue culture infective doses/ml diluted 1,000 fold, demonstrating high detection sensitivity. Low background in uninfected cells and simultaneous staining of fixed cells with molecular beacons and antibodies showed high detection specificity. Furthermore, using confocal microscopy to image the viral genome in live, infected cells, we observed a connected, highly three-dimensional, amorphous inclusion body structure not seen in fixed cells. Taken together, the use of molecular beacons for active virus imaging provides a powerful tool for rapid viral infection detection, the characterization of RNA viruses, and the design of new antiviral drugs.
doi:10.1128/JVI.80.2.682-688.2006
PMCID: PMC1346841  PMID: 16378971
17.  Comparative Pathogenesis and Systems Biology for Biodefense Virus Vaccine Development 
Developing vaccines to biothreat agents presents a number of challenges for discovery, preclinical development, and licensure. The need for high containment to work with live agents limits the amount and types of research that can be done using complete pathogens, and small markets reduce potential returns for industry. However, a number of tools, from comparative pathogenesis of viral strains at the molecular level to novel computational approaches, are being used to understand the basis of viral attenuation and characterize protective immune responses. As the amount of basic molecular knowledge grows, we will be able to take advantage of these tools not only to rationally attenuate virus strains for candidate vaccines, but also to assess immunogenicity and safety in silico. This review discusses how a basic understanding of pathogenesis, allied with systems biology and machine learning methods, can impact biodefense vaccinology.
doi:10.1155/2010/236528
PMCID: PMC2896660  PMID: 20617142
18.  The Cell Adhesion Molecule “CAR” and Sialic Acid on Human Erythrocytes Influence Adenovirus In Vivo Biodistribution 
PLoS Pathogens  2009;5(1):e1000277.
Although it has been known for 50 years that adenoviruses (Ads) interact with erythrocytes ex vivo, the molecular and structural basis for this interaction, which has been serendipitously exploited for diagnostic tests, is unknown. In this study, we characterized the interaction between erythrocytes and unrelated Ad serotypes, human 5 (HAd5) and 37 (HAd37), and canine 2 (CAV-2). While these serotypes agglutinate human erythrocytes, they use different receptors, have different tropisms and/or infect different species. Using molecular, biochemical, structural and transgenic animal-based analyses, we found that the primary erythrocyte interaction domain for HAd37 is its sialic acid binding site, while CAV-2 binding depends on at least three factors: electrostatic interactions, sialic acid binding and, unexpectedly, binding to the coxsackievirus and adenovirus receptor (CAR) on human erythrocytes. We show that the presence of CAR on erythrocytes leads to prolonged in vivo blood half-life and significantly reduced liver infection when a CAR-tropic Ad is injected intravenously. This study provides i) a molecular and structural rationale for Ad–erythrocyte interactions, ii) a basis to improve vector-mediated gene transfer and iii) a mechanism that may explain the biodistribution and pathogenic inconsistencies found between human and animal models.
Author Summary
In most cases, adenoviruses are thought to initially enter the host via contact with epithelial cells and spread within the host via an unknown mechanism. Most adenovirus serotypes use a cell adhesion molecule dubbed “CAR” to attach to cells. To assess, predict and understand adenovirus biology and vectorology, many in vivo studies use mice and monkeys. These animal models have been considered reliable models in the realm of viral pathogenesis and gene transfer. One of the implications of our study suggests that the rat may be a more appropriate model during intravenous adenovirus delivery because like humans, and unlike mice and monkeys, they also express CAR on their erythrocytes. The identification of CAR on human erythrocytes explains a 50-year-old enigma of adenovirus hemagglutination, helps us better understand adenovirus in vivo biology and may open new avenues to understand the role of cell adhesion molecules during erythropoiesis.
doi:10.1371/journal.ppat.1000277
PMCID: PMC2607015  PMID: 19119424
19.  Chronic viral diseases. 
Until 20 years ago the only chronic viral diseases known were those considered to be confined to the nervous system. As a result of recent advances in epidemiology, molecular biology and immunology, new viral diseases have been recognized and their clinical features and pathogenesis elucidated. Chronic disease may result from infection with the hepatitis B and D viruses and whatever agent or agents cause hepatitis non-A, non-B, the herpesviruses, Epstein-Barr virus, cytomegalovirus and human T-lymphotropic virus type III. These diseases have common features, including long-term or even lifetime asymptomatic carriage, viremia, with virus free in the plasma or attached to circulating mononuclear cells, presence of virus in body secretions, irreversible tissue injury in target organs and oncogenic potential. New information on these diseases is reviewed. Other chronic diseases for which the cause is currently unknown may eventually prove to be due to viral infection. In addition, vaccines may be developed for prophylaxis of some chronic viral diseases and associated malignant diseases.
PMCID: PMC1491381  PMID: 3022903
20.  Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome 
PLoS ONE  2011;6(12):e29112.
Merkel Cell Polyomavirus (MCPyV) genomes are clonally integrated in tumor tissues of approximately 85% of all Merkel cell carcinoma (MCC) cases, a highly aggressive tumor of the skin which predominantly afflicts elderly and immunosuppressed patients. All integrated viral genomes recovered from MCC tissue or MCC cell lines harbor signature mutations in the early gene transcript encoding for the large T-Antigen (LT-Ag). These mutations selectively abrogate the ability of LT-Ag to support viral replication while still maintaining its Rb-binding activity, suggesting a continuous requirement for LT-Ag mediated cell cycle deregulation during MCC pathogenesis. To gain a better understanding of MCPyV biology, in vitro MCPyV replication systems are required. We have generated a synthetic MCPyV genomic clone (MCVSyn) based on the consensus sequence of MCC-derived sequences deposited in the NCBI database. Here, we demonstrate that transfection of recircularized MCVSyn DNA into some human cell lines recapitulates efficient replication of the viral genome, early and late gene expression together with virus particle formation. However, serial transmission of infectious virus was not observed. This in vitro culturing system allows the study of viral replication and will facilitate the molecular dissection of important aspects of the MCPyV lifecycle.
doi:10.1371/journal.pone.0029112
PMCID: PMC3246459  PMID: 22216177
21.  KSHV 2.0: A Comprehensive Annotation of the Kaposi's Sarcoma-Associated Herpesvirus Genome Using Next-Generation Sequencing Reveals Novel Genomic and Functional Features 
PLoS Pathogens  2014;10(1):e1003847.
Productive herpesvirus infection requires a profound, time-controlled remodeling of the viral transcriptome and proteome. To gain insights into the genomic architecture and gene expression control in Kaposi's sarcoma-associated herpesvirus (KSHV), we performed a systematic genome-wide survey of viral transcriptional and translational activity throughout the lytic cycle. Using mRNA-sequencing and ribosome profiling, we found that transcripts encoding lytic genes are promptly bound by ribosomes upon lytic reactivation, suggesting their regulation is mainly transcriptional. Our approach also uncovered new genomic features such as ribosome occupancy of viral non-coding RNAs, numerous upstream and small open reading frames (ORFs), and unusual strategies to expand the virus coding repertoire that include alternative splicing, dynamic viral mRNA editing, and the use of alternative translation initiation codons. Furthermore, we provide a refined and expanded annotation of transcription start sites, polyadenylation sites, splice junctions, and initiation/termination codons of known and new viral features in the KSHV genomic space which we have termed KSHV 2.0. Our results represent a comprehensive genome-scale image of gene regulation during lytic KSHV infection that substantially expands our understanding of the genomic architecture and coding capacity of the virus.
Author Summary
Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-causing agent in immunocompromised patients that establishes long-lasting infections in its hosts. Initially described in 1994 and extensively studied ever since, KSHV molecular biology is understood in broad outline, but many detailed questions are still to be resolved. After almost two decades, specific aspects pertaining to the organization of the KSHV genome as well as the fate of the viral transcripts during the productive stages of infection remain unexplored. Here we use a systematic genome-wide approach to investigate changes in gene and protein expression during the productive stage of infection known as the lytic cycle. We found that the viral genome has a large coding capacity, capable of generating at least 45% more products than initially anticipated by bioinformatic analyses alone, and that it uses multiple strategies to expand its coding capacity well beyond what is determined solely by the DNA sequence of its genome. We also provide an expanded and highly detailed annotation of known and new genomic features in KSHV. We have termed this new architectural and functional annotation KSHV 2.0. Our results indicate that viral genomes are more complex than anticipated, and that they are subject to tight mechanisms of regulation to ensure correct gene expression.
doi:10.1371/journal.ppat.1003847
PMCID: PMC3894221  PMID: 24453964
22.  SARS CORONAVIRUS AND INNATE IMMUNITY 
Virus research  2007;133(1):101-112.
The emergence of the highly pathogenic SARS coronavirus (SARS-CoV) has reignited interest in coronavirus biology and pathogenesis. An emerging theme in coronavirus pathogenesis is that the interaction between specific viral genes and the host immune system, specifically the innate immune system, functions as a key determinant in regulating virulence and disease outcomes. Using SARS-CoV as a model, we will review the current knowledge of the interplay between coronavirus infection and the host innate immune system in vivo, and then discuss the mechanisms by which specific gene products antagonize the host innate immune response in cell culture models. Our data suggests that the SARS-CoV uses specific strategies to evade and antagonize the sensing and signaling arms of the interferon pathway. We summarize by identifying future points of consideration that will contribute greatly to our understanding of the molecular mechanisms governing coronavirus pathogenesis and virulence, and the development of severe disease in humans and animals.
doi:10.1016/j.virusres.2007.03.015
PMCID: PMC2292640  PMID: 17451827
23.  Junín Virus Pathogenesis and Virus Replication 
Viruses  2012;4(10):2317-2339.
Junín virus, the etiological agent of Argentine hemorrhagic fever, causes significant morbidity and mortality. The virus is spread through the aerosolization of host rodent excreta and endemic to the humid pampas of Argentina. Recently, significant progress has been achieved with the development of new technologies (e.g. reverse genetics) that have expanded knowledge about the pathogenesis and viral replication of Junín virus. We will review the pathogenesis of Junín virus in various animal models and the role of innate and adaptive immunity during infection. We will highlight current research regarding the role of molecular biology of Junín virus in elucidating virus attenuation. We will also summarize current knowledge on Junín virus pathogenesis focusing on the recent development of vaccines and potential therapeutics.
doi:10.3390/v4102317
PMCID: PMC3497054  PMID: 23202466
arenavirus; Junín virus; pathogenesis
24.  Rates of Viral Evolution Are Linked to Host Geography in Bat Rabies 
PLoS Pathogens  2012;8(5):e1002720.
Rates of evolution span orders of magnitude among RNA viruses with important implications for viral transmission and emergence. Although the tempo of viral evolution is often ascribed to viral features such as mutation rates and transmission mode, these factors alone cannot explain variation among closely related viruses, where host biology might operate more strongly on viral evolution. Here, we analyzed sequence data from hundreds of rabies viruses collected from bats throughout the Americas to describe dramatic variation in the speed of rabies virus evolution when circulating in ecologically distinct reservoir species. Integration of ecological and genetic data through a comparative Bayesian analysis revealed that viral evolutionary rates were labile following historical jumps between bat species and nearly four times faster in tropical and subtropical bats compared to temperate species. The association between geography and viral evolution could not be explained by host metabolism, phylogeny or variable selection pressures, and instead appeared to be a consequence of reduced seasonality in bat activity and virus transmission associated with climate. Our results demonstrate a key role for host ecology in shaping the tempo of evolution in multi-host viruses and highlight the power of comparative phylogenetic methods to identify the host and environmental features that influence transmission dynamics.
Author Summary
Rapid evolution of RNA viruses is intimately linked to their success in overcoming the defenses of their hosts. Several studies have shown that rates of viral evolution can vary dramatically among distantly related viral families. Variability in the speed of evolution among closely related viruses has received less attention, but could be an important determinant of the geographic or host species origins of viral emergence if certain species or regions promote especially rapid evolution. Here, using a dataset of rabies virus sequences collected from bat species throughout the Americas, we test the role of inter-specific differences in reservoir host biology on the tempo of viral evolution. We show the annual rate of molecular evolution to be a malleable trait of viruses that is accelerated in subtropical and tropical bats compared to temperate species. The association between geography and the speed of evolution appears to reflect differences in the seasonality of rabies virus transmission in different climatic zones. Our results illustrate that the viral mechanisms that are commonly invoked to explain heterogeneous rates of evolution among viral families may be insufficient to explain evolution in multi-host viruses and indicate a role for host biology in shaping the speed of viral evolution.
doi:10.1371/journal.ppat.1002720
PMCID: PMC3355098  PMID: 22615575
25.  The Biology of Tobacco and Nicotine: Bench to Bedside 
Strong epidemiologic evidence links smoking and cancer. An increased understanding of the molecular biology of tobacco-related cancers could advance progress toward improving smoking cessation and patient management. Knowledge gaps between tobacco addiction, tumorigenesis, and cancer brought an interdisciplinary group of investigators together to discuss “The Biology of Nicotine and Tobacco: Bench to Bedside.” Presentations on the signaling pathways and pathogenesis in tobacco-related cancers, mouse models of addiction, imaging and regulation of nicotinic receptors, the genetic basis for tobacco carcinogenesis and development of lung cancer, and molecular mechanisms of carcinogenesis were heard. Importantly, new opportunities to use molecular biology to identify and abrogate tobacco-mediated carcinogenesis and to identify high-risk individuals were recognized.
doi:10.1158/1055-9965.EPI-04-0652
PMCID: PMC3459058  PMID: 15824140

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