Emerging RNA Viruses Research Articles

Characterization and phylogenetic analysis of new bat astroviruses detected in Gabon, Central Africa.

Astroviruses are emerging RNA viruses that cause enteropathogenic infections in humans and in other mammals. The identification of astroviruses in a wide range of animals highlights the zoonotic importance of these viruses. Bats can harbor many different viruses, among which some are highly pathogenic for humans (for instance, Nipah, Ebola and SARS coronavirus), and also several astroviruses. As some RNA viruses can be directly transmitted from bats to humans, it is crucial to collect data about their frequency, genetic diversity and phylogenetic characterization. In this study, we report the molecular identification of 44 new astroviruses (with a detection rate of 4.5%) in 962 apparently healthy bats that belong to five different species and that were captured in different caves in North-East Gabon, Central Africa. Our results show that bat astroviruses form a group that is genetically distinct from astroviruses infecting other mammals. Moreover, these astroviruses showed an important genetic diversity and low host restriction in bat species.

Fidelity of Nucleotide Incorporation by the RNA-Dependent RNA Polymerase from Poliovirus.

Using poliovirus (PV) and its RNA-dependent RNA polymerase (RdRp) as our primary model system, we have advanced knowledge fundamental to the chemistry and fidelity of nucleotide addition by nucleic acid polymerase. Two fidelity checkpoints exist prior to nucleotide addition. The first toggles the enzyme between a nucleotide binding-occluded state and a nucleotide binding-competent state. The second represents an ensemble of conformational states of conserved structural motifs that permits retention of the incoming nucleotide in a state competent for phosphoryl transfer long enough for chemistry to occur. Nucleophilic attack of the alpha-phosphorous atom of the incoming nucleotide produces a pentavalent transition state, collapse of which is facilitated by protonation of the pyrophosphate leaving group by a general acid. All of the relevant conformational states of the enzyme are controlled by a network of interacting residues that permits remote-site residues to control active-site function. The current state of the art for PV RdRp enzymology is such that mechanisms governing fidelity of this enzyme can now be targeted genetically and chemically for development of attenuated viruses and antiviral agents, respectively. Application of the knowledge obtained with the PV RdRp to the development of vaccines and antivirals for emerging RNA viruses represents an important goal for the future.

The future of antivirals: broad-spectrum inhibitors.

Potent antivirals are successfully used for the treatment of infections with herpesviruses, hepatitis B and C viruses, HIV, and with some success for influenza viruses. However, no selective inhibitors are available for a multitude of medically important viruses, most of which are (re-)emerging RNA viruses. As it is impossible to develop drugs against each of these viruses, broad-spectrum antiviral agents (BSAA) are a prime strategy to cope with this challenge. We propose four categories of antiviral molecules that hold promise as BSAA. Several nucleoside analogues with broad antiviral activity have been described and given the relatively conserved nature of viral polymerases, it may be possible to develop more broad-spectrum nucleoside analogues. A number of viral proteins are relatively conserved between families and may also be interesting targets. Host-targeting antiviral drugs such as modulators of lipid metabolism and cyclophilin inhibitors can be explored as well. Finally, the potent and broad antiviral function of the immune system can be exploited by the development of immune-modulating BSAA. Despite the recent advances, the BSAA field is still in its infancy. Nevertheless, the discovery and development of such molecules will be a key aim of antiviral research in the coming decades.

Diversity in a honey bee pathogen: first report of a third master variant of the Deformed Wing Virus quasispecies.

Treatment of emerging RNA viruses is hampered by the high mutation and replication rates that enable these viruses to operate as a quasispecies. Declining honey bee populations have been attributed to the ectoparasitic mite Varroa destructor and its affiliation with Deformed Wing Virus (DWV). In the current study we use next-generation sequencing to investigate the DWV quasispecies in an apiary known to suffer from overwintering colony losses. We show that the DWV species complex is made up of three master variants. Our results indicate that a new DWV Type C variant is distinct from the previously described types A and B, but together they form a distinct clade compared with other members of the Iflaviridae. The molecular clock estimation predicts that Type C diverged from the other variants ∼319 years ago. The discovery of a new master variant of DWV has important implications for the positive identification of the true pathogen within global honey bee populations.

Isolation and characterization of a novel alphaherpesvirus in fruit bats.

Bats are known to harbor emerging RNA viruses. Recent studies have used high-throughput sequencing technology to identify various virus species, including DNA viruses that are harbored by bats; however, little is known about the nature of these potentially novel viruses. Here, we report the characterization of a novel herpesvirus isolated from an Indonesian pteropodid bat. The virus, tentatively named fruit bat alphaherpesvirus 1 (FBAHV1), has a double-stranded DNA genome of 149,459 bp. The phylogenetic analyses suggested that FBAHV1 is phylogenetically grouped with simplexviruses within the subfamily Alphaherpesvirinae. Inoculation of FBAHV1 into laboratory mice caused a lethal infection. Virus infection was observed in lung, liver, and brain tissue. Serological and PCR screening revealed that fruit bats infected with FBAHV1 or its related virus are widely distributed in Indonesia. The identification of FBAHV1 makes a considerable contribution to our understanding of simplexviruses associated with bats. Bats are known to harbor emerging viruses, such as lyssaviruses, henipaviruses, severe acute respiratory syndrome-like coronaviruses, and filoviruses. Although alphaherpesviruses are disseminated in humans and other animals, there is little information about their distribution in bats. Here, we isolated a previously unknown alphaherpesvirus from an Indonesian fruit bat. Genome sequence analysis suggested that the virus is a member of the genus Simplexvirus within the subfamily Alphaherpesvirinae, which also includes common human viruses, such as herpes simplex virus 1 and herpes simplex virus 2. FBAHV1 is the first bat-derived alphaherpesvirus whose complete genome has been sequenced.

Small-molecule probes targeting the viral PPxY-host Nedd4 interface block egress of a broad range of RNA viruses.

Budding of filoviruses, arenaviruses, and rhabdoviruses is facilitated by subversion of host proteins, such as Nedd4 E3 ubiquitin ligase, by viral PPxY late (L) budding domains expressed within the matrix proteins of these RNA viruses. As L domains are important for budding and are highly conserved in a wide array of RNA viruses, they represent potential broad-spectrum targets for the development of antiviral drugs. To identify potential competitive blockers, we used the known Nedd4 WW domain-PPxY interaction interface as the basis of an in silico screen. Using PPxY-dependent budding of Marburg (MARV) VP40 virus-like particles (VLPs) as our model system, we identified small-molecule hit 1 that inhibited Nedd4-PPxY interaction and PPxY-dependent budding. This lead candidate was subsequently improved with additional structure-activity relationship (SAR) analog testing which enhanced antibudding activity into the nanomolar range. Current lead compounds 4 and 5 exhibit on-target effects by specifically blocking the MARV VP40 PPxY-host Nedd4 interaction and subsequent PPxY-dependent egress of MARV VP40 VLPs. In addition, lead compounds 4 and 5 exhibited antibudding activity against Ebola and Lassa fever VLPs, as well as vesicular stomatitis and rabies viruses (VSV and RABV, respectively). These data provide target validation and suggest that inhibition of the PPxY-Nedd4 interaction can serve as the basis for the development of a novel class of broad-spectrum, host-oriented antivirals targeting viruses that depend on a functional PPxY L domain for efficient egress. There is an urgent and unmet need for the development of safe and effective therapeutics against biodefense and high-priority pathogens, including filoviruses (Ebola and Marburg) and arenaviruses (e.g., Lassa and Junin) which cause severe hemorrhagic fever syndromes with high mortality rates. We along with others have established that efficient budding of filoviruses, arenaviruses, and other viruses is critically dependent on the subversion of host proteins. As disruption of virus budding would prevent virus dissemination, identification of small-molecule compounds that block these critical viral-host interactions should effectively block disease progression and transmission. Our findings provide validation for targeting these virus-host interactions as we have identified lead inhibitors with broad-spectrum antiviral activity. In addition, such inhibitors might prove useful for newly emerging RNA viruses for which no therapeutics would be available.

Molecular evolutionary signatures reveal the role of host ecological dynamics in viral disease emergence and spread

RNA viruses account for numerous emerging and perennial infectious diseases, and are characterized by rapid rates of molecular evolution. The ecological dynamics of most emerging RNA viruses are still poorly understood and difficult to ascertain. The availability of genome sequence data for many RNA viruses, in principle, could be used to infer ecological dynamics if changes in population numbers produced a lasting signature within the pattern of genome evolution. As a result, the rapidly emerging phylogeographic structure of a pathogen, shaped by the rise and fall in the number of infections and their spatial distribution, could be used as a surrogate for direct ecological assessments. Based on rabies virus as our example, we use a model combining ecological and evolutionary processes to test whether variation in the rate of host movement results in predictive diagnostic patterns of pathogen genetic structure. We identify several linearizable relationships between host dispersal rate and measures of phylogenetic structure suggesting genetic information can be used to directly infer ecological process. We also find phylogenetic structure may be more revealing than demography for certain ecological processes. Our approach extends the reach of current analytic frameworks for infectious disease dynamics by linking phylogeography back to underlying ecological processes.

Characterization of the Interaction between Hantavirus Nucleocapsid Protein (N) and Ribosomal Protein S19 (RPS19)

Hantaviruses, members of the Bunyaviridae family, are negative-stranded emerging RNA viruses and category A pathogens that cause serious illness when transmitted to humans through aerosolized excreta of infected rodent hosts. Hantaviruses have evolved a novel translation initiation mechanism, operated by nucleocapsid protein (N), which preferentially facilitates the translation of viral mRNAs. N binds to the ribosomal protein S19 (RPS19), a structural component of the 40 S ribosomal subunit. In addition, N also binds to both the viral mRNA 5' cap and a highly conserved triplet repeat sequence of the viral mRNA 5' UTR. The simultaneous binding of N at both the terminal cap and the 5' UTR favors ribosome loading on viral transcripts during translation initiation. We characterized the binding between N and RPS19 and demonstrate the role of the N-RPS19 interaction in N-mediated translation initiation mechanism. We show that N specifically binds to RPS19 with high affinity and a binding stoichiometry of 1:1. The N-RPS19 interaction is an enthalpy-driven process. RPS19 undergoes a conformational change after binding to N. Using T7 RNA polymerase, we synthesized the hantavirus S segment mRNA, which matches the transcript generated by the viral RNA-dependent RNA polymerase in cells. We show that the N-RPS19 interaction plays a critical role in the translation of this mRNA both in cells and rabbit reticulocyte lysates. Our results demonstrate that the N-mediated translation initiation mechanism, which lures the host translation machinery for the preferential translation of viral transcripts, primarily depends on the N-RPS19 interaction. We suggest that the N-RPS19 interaction is a novel target to shut down the N-mediated translation strategy and hence virus replication in cells.

Virus Budding/Host Interactions

Viruses have developed unique and oftentimes complex molecular mechanisms to ensure efficient egress of mature virions from infected cells. Over the years, virologists have begun to unravel and appreciate the intricate roles of both viral and host proteins in this process, and particularly the specific recruitment of host factors to promote efficient budding of infectious virus. A better understanding of these virus-host interactions and the mechanisms of virus budding not only will provide fundamental insights into the functions of both viral and host proteins, but also will lead to the emergence of novel strategies to inhibit virion egress and spread. In this special issue on virus budding/host interactions, we have invited both review articles to summarize recent findings in the field as well as original research articles to provide new insights into this late stage of virus replication. The first paper of this special issue is a review article that focuses on the current knowledge of arenavirus budding and the critical role that the small RING finger Z protein plays in this process. S. Urata and J. C. de la Torre present a comprehensive discussion on the structure and function of Z and highlight the various L-domain motifs conserved in arenavirus Z proteins and in the matrix proteins of other emerging RNA viruses. Viral L-domains are of particular interest since they mediate recruitment of host proteins to promote virus budding and, as such, constitute attractive and potentially broad-spectrum targets for antiviral drugs designed to inhibit virus egress. Indeed, the authors discuss several strategies and ongoing efforts to identify and screen candidate budding inhibitors. The viral L-domain/host interaction theme continues in the second paper, where Y. Liu et al. have utilized a bimolecular complementation (BiMC) approach to detect, localize, and follow filovirus VP40-host complexes in live mammalian cells in real time. The authors postulate that adaptation of the BiMC approach to the study of virus-host interactions and budding may help address gaps in our understanding of the dynamics, kinetics, and trafficking patterns of virus-host complexes involved in the budding process. In addition, the BiMC approach was used in conjunction with the more established filovirus VP40 virus-like particle (VLP) budding assay to test candidate small molecule inhibitors (identified by in silico screening) of L-domain/host interactions for their ability to inhibit particle release. The mechanisms by which viral and host proteins are trafficked and/or targeted to the site of budding are of great interest. The third paper summarizes experimental data in support of a model for assembly and budding of influenza virus from viral bud zones and raft-enriched domains at the plasma membrane. Indeed, assembly and budding at raft domains appears to be a characteristic shared by several RNA virus families. M. Veit and B. Thaa discuss the raft-targeting features of the hemagglutinin (HA) and neuraminidase (NA) glycoproteins of influenza virus, along with the critical roles of M1 and M2 proteins in orchestrating virus assembly and in membrane bending/particle scission, respectively. Little is known about the role of human respiratory syncytial virus (HRSV) glycoproteins during assembly and budding of mature virions. In the fourth paper, M. Batonick and G. W. Wertz address this gap by engineering recombinant viruses and using microscopic and biochemical analyses to demonstrate that deletion of the G and F proteins affected the incorporation of other viral proteins into budding virions; however, their absence did not directly affect the efficiency of virion egress. Thus, this study ascribes a novel role for the G and F glycoproteins during the late stage of HRSV replication. In the fifth paper, Snyder et al. describe a novel and intriguing cell lysis system utilized by two archaeal viruses, Sulfolobus turreted icosahedral virus (STIV) and Sulfolobus islandicus rod-shaped virus 2 (SIRV2). The authors demonstrate that the STIV c92 protein forms unique pyramid-like structures on the cell surface through which newly assembled virions are released during cell lysis. The authors speculate that this new lysis system may be common within other archaeal viral populations present in acidic hot springs. Ronald N. Harty Anthony P. Schmitt Fadila Bouamr Carolina B. Lopez Claude Krummenacher

Pathogenesis of noroviruses, emerging RNA viruses.

Human noroviruses in the family Caliciviridae are a major cause of epidemic gastroenteritis. They are responsible for at least 95% of viral outbreaks and over 50% of all outbreaks worldwide. Transmission of these highly infectious plus-stranded RNA viruses occurs primarily through contaminated food or water, but also through person-to-person contact and exposure to fomites. Norovirus infections are typically acute and self-limited. However, disease can be much more severe and prolonged in infants, elderly, and immunocompromised individuals. Norovirus outbreaks frequently occur in semi-closed communities such as nursing homes, military settings, schools, hospitals, cruise ships, and disaster relief situations. Noroviruses are classified as Category B biodefense agents because they are highly contagious, extremely stable in the environment, resistant to common disinfectants, and associated with debilitating illness. The number of reported norovirus outbreaks has risen sharply since 2002 suggesting the emergence of more infectious strains. There has also been increased recognition that noroviruses are important causes of childhood hospitalization. Moreover, noroviruses have recently been associated with multiple clinical outcomes other than gastroenteritis. It is unclear whether these new observations are due to improved norovirus diagnostics or to the emergence of more virulent norovirus strains. Regardless, it is clear that human noroviruses cause considerable morbidity worldwide, have significant economic impact, and are clinically important emerging pathogens. Despite the impact of human norovirus-induced disease and the potential for emergence of highly virulent strains, the pathogenic features of infection are not well understood due to the lack of a cell culture system and previous lack of animal models. This review summarizes the current understanding of norovirus pathogenesis from the histological to the molecular level, including contributions from new model systems.

Evidence of Henipavirus Infection in West African Fruit Bats

Henipaviruses are emerging RNA viruses of fruit bat origin that can cause fatal encephalitis in man. Ghanaian fruit bats (megachiroptera) were tested for antibodies to henipaviruses. Using a Luminex multiplexed microsphere assay, antibodies were detected in sera of Eidolon helvum to both Nipah (39%, 95% confidence interval: 27–51%) and Hendra (22%, 95% CI: 11–33%) viruses. Virus neutralization tests further confirmed seropositivity for 30% (7/23) of Luminex positive serum samples. Our results indicate that henipavirus is present within West Africa.

Temporal dynamics of European bat Lyssavirus type 1 and survival of Myotis myotis bats in natural colonies.

Many emerging RNA viruses of public health concern have recently been detected in bats. However, the dynamics of these viruses in natural bat colonies is presently unknown. Consequently, prediction of the spread of these viruses and the establishment of appropriate control measures are hindered by a lack of information. To this aim, we collected epidemiological, virological and ecological data during a twelve-year longitudinal study in two colonies of insectivorous bats (Myotis myotis) located in Spain and infected by the most common bat lyssavirus found in Europe, the European bat lyssavirus subtype 1 (EBLV-1). This active survey demonstrates that cyclic lyssavirus infections occurred with periodic oscillations in the number of susceptible, immune and infected bats. Persistence of immunity for more than one year was detected in some individuals. These data were further used to feed models to analyze the temporal dynamics of EBLV-1 and the survival rate of bats. According to these models, the infection is characterized by a predicted low basic reproductive rate (R0 = 1.706) and a short infectious period (D = 5.1 days). In contrast to observations in most non-flying animals infected with rabies, the survival model shows no variation in mortality after EBLV-1 infection of M. myotis. These findings have considerable public health implications in terms of management of colonies where lyssavirus-positive bats have been recorded and confirm the potential risk of rabies transmission to humans. A greater understanding of the dynamics of lyssavirus in bat colonies also provides a model to study how bats contribute to the maintenance and transmission of other viruses of public health concern.

DNA Bacteriophage as Controls for Clinical Viral Testing

In the mid- to late-1980s, a revolution in molecular diagnostics began with the introduction of innovative methods for the detection of nucleic acids. In retrospect, the appearance of these technologies roughly coincided with the debuts of new pathogenic viruses, such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV). Techniques such as PCR, transcription-mediated amplification (TMA), and branched DNA (bDNA) were applied to the detection and quantification of these viruses. Eventually, these tests were integrated into routine clinical laboratories for diagnosing and monitoring the treatment of patients infected with these viruses. Quantification data (“viral load”) indicated whether the current drug cocktail was having the desired effect on the virus (i.e., lowering the viral load). If not, then another drug course could be prescribed. Today, the main advantages of these assays are great sensitivity (measuring as few as 50 copies/mL of plasma), ease of use for quantification, and early detection of viral nucleic acid in the peripheral blood before an antibody response develops, an application that has proven to be especially important in screening of human blood products. These technologies were focused not only on the newly emerging RNA viruses but also on the better-known hepatitis B virus (HBV), cytomegalovirus (CMV), herpes simplex virus (HSV), varicella zoster virus (VZV), human papillomavirus (HPV), and Epstein–Barr virus (EBV). In developing DNA-based tests for these viruses, laboratories incorporated positive controls from one of three potential sources: plasmid DNA, positive patient specimens, or commercially available viral preparations. None of these formats is ideal. Plasmid DNA cannot be used until after the specimen analyte has been extracted. Patient specimens have become more difficult to use in the US after the introduction of new “HIPAA” regulations for protection of patient privacy. Moreover, viral nucleic acids in patient specimens are degraded during multiple freeze–thaw cycles. Lastly, the …