scholarly journals Inhibition of Nipah Virus by Defective Interfering Particles

2020 ◽  
Vol 221 (Supplement_4) ◽  
pp. S460-S470 ◽  
Author(s):  
Stephen R Welch ◽  
Natasha L Tilston ◽  
Michael K Lo ◽  
Shannon L M Whitmer ◽  
Jessica R Harmon ◽  
...  
Keyword(s):  
Rna Virus ◽  
Nipah Virus ◽  
Vero Cells ◽  
Viral Population ◽  
Alternative Source ◽  
Highly Pathogenic ◽  
Defective Interfering Particles ◽  
Viral Genomes ◽  
Antiviral Control

Abstract The error-prone nature of RNA-dependent RNA polymerases drives the diversity of RNA virus populations. Arising within this diversity is a subset of defective viral genomes that retain replication competency, termed defective interfering (DI) genomes. These defects are caused by aberrant viral polymerase reinitiation on the same viral RNA template (deletion DI species) or the nascent RNA strand (copyback DI species). DI genomes have previously been shown to alter the dynamics of a viral population by interfering with normal virus replication and/or by stimulating the innate immune response. In this study, we investigated the ability of artificially produced DI genomes to inhibit Nipah virus (NiV), a highly pathogenic biosafety level 4 paramyxovirus. High multiplicity of infection passaging of both NiV clinical isolates and recombinant NiV in Vero cells generated an extensive DI population from which individual DIs were identified using next-generation sequencing techniques. Assays were established to generate and purify both naturally occurring and in silico-designed DIs as fully encapsidated, infectious virus-like particles termed defective interfering particles (DIPs). We demonstrate that several of these NiV DIP candidates reduced NiV titers by up to 4 logs in vitro. These data represent a proof-of-principle that a therapeutic application of DIPs to combat NiV infections may be an alternative source of antiviral control for this disease.

scholarly journals Defective viral genomes from chikungunya virus are broad-spectrum antivirals and prevent virus dissemination in mosquitoes

2021 ◽  
Vol 17 (2) ◽  
pp. e1009110
Author(s):  
Laura I. Levi ◽  
Veronica V. Rezelj ◽  
Annabelle Henrion-Lacritick ◽  
Diana Erazo ◽  
J Boussier ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Broad Spectrum ◽  
Chikungunya Virus ◽  
Rna Virus ◽  
Viral Genomes ◽  
Mosquito Cells ◽  
Spectrum Activity ◽  
Broad Spectrum Activity

Defective viral genomes (DVGs) are truncated and/or rearranged viral genomes produced during virus replication. Described in many RNA virus families, some of them have interfering activity on their parental virus and/or strong immunostimulatory potential, and are being considered in antiviral approaches. Chikungunya virus (CHIKV) is an alphavirus transmitted by Aedes spp. that infected millions of humans in the last 15 years. Here, we describe the DVGs arising during CHIKV infection in vitro in mammalian and mosquito cells, and in vivo in experimentally infected Aedes aegypti mosquitoes. We combined experimental and computational approaches to select DVG candidates most likely to have inhibitory activity and showed that, indeed, they strongly interfere with CHIKV replication both in mammalian and mosquito cells. We further demonstrated that some DVGs present broad-spectrum activity, inhibiting several CHIKV strains and other alphaviruses. Finally, we showed that pre-treating Aedes aegypti with DVGs prevented viral dissemination in vivo.

scholarly journals Coxsackieviruses Undergo Intercellular Transmission as Pools of Sibling Viral Genomes Associated to Membranes

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 120
Author(s):  
Juan-Vicente Bou ◽  
Ron Geller ◽  
Rafael Sanjuán
Keyword(s):  
Genetic Variants ◽  
Social Evolution ◽  
Viral Population ◽  
Multiplicity Of Infection ◽  
Defective Interfering Particles ◽  
Viral Genomes ◽  
Viral Particles ◽  
A Cell ◽  
Infected Cells ◽  
Population Sequence

Some viruses are released from cells as pools of membrane-associated virions. By increasing the multiplicity of infection, this type of collective dispersal could favor viral cooperation, but also the emergence of cheater-like viruses, such as defective interfering particles. To better understand this process, we examined the genetic diversity of membrane-associated coxsackievirus infectious units. We found that infected cells released large membranous structures containing 8–21 infectious particles on average, including vesicles. However, in most cases (62–93%), these structures did not promote the co-transmission of different viral genetic variants present in a cell. Furthermore, collective dispersal had no effect on viral population sequence diversity. Our results indicate that membrane-associated collective infectious units typically contain viral particles derived from the same parental genome. Hence, if cooperation occurred, it should probably involve sibling viral particles rather than different variants. As shown by social evolution theory, cooperation among siblings should be robust against cheater invasion.

scholarly journals Heparan Sulfate-Dependent Enhancement of Henipavirus Infection

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Cyrille Mathieu ◽  
Kévin P. Dhondt ◽  
Marie Châlons ◽  
Stéphane Mély ◽  
Hervé Raoul ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Anticoagulant Activity ◽  
Antiviral Treatment ◽  
Mammalian Species ◽  
Nipah Virus ◽  
Hendra Virus ◽  
Host Cells ◽  
Emerging Infections ◽  
Highly Pathogenic

ABSTRACTNipah virus and Hendra virus are emerging, highly pathogenic, zoonotic paramyxoviruses that belong to the genusHenipavirus. They infect humans as well as numerous mammalian species. Both viruses use ephrin-B2 and -B3 as cell entry receptors, and following initial entry into an organism, they are capable of rapid spread throughout the host. We have previously reported that Nipah virus can use another attachment receptor, different from its entry receptors, to bind to nonpermissive circulating leukocytes, thereby promoting viral dissemination within the host. Here, this attachment molecule was identified as heparan sulfate for both Nipah virus and Hendra virus. Cells devoid of heparan sulfate were not able to mediate henipavirustrans-infection and showed reduced permissivity to infection. Virus pseudotyped with Nipah virus glycoproteins bound heparan sulfate and heparin but no other glycosaminoglycans in a surface plasmon resonance assay. Furthermore, heparin was able to inhibit the interaction of the viruses with the heparan sulfate and to block cell-mediatedtrans-infection of henipaviruses. Moreover, heparin was shown to bind to ephrin-B3 and to restrain infection of permissive cellsin vitro. Consequently, treatment with heparin devoid of anticoagulant activity improved the survival of Nipah virus-infected hamsters. Altogether, these results reveal heparan sulfate as a new attachment receptor for henipaviruses and as a potential therapeutic target for the development of novel approaches against these highly lethal infections.IMPORTANCETheHenipavirusgenus includes two closely related, highly pathogenic paramyxoviruses, Nipah virus and Hendra virus, which cause elevated morbidity and mortality in animals and humans. Pathogenesis of both Nipah virus and Hendra virus infection is poorly understood, and efficient antiviral treatment is still missing. Here, we identified heparan sulfate as a novel attachment receptor used by both viruses to bind host cells. We demonstrate that heparin was able to inhibit the interaction of the viruses with heparan sulfate and to block cell-mediatedtrans-infection of henipaviruses. Moreover, heparin also bound to the viral entry receptor and thereby restricted infection of permissive cellsin vitro. Consequently, heparin treatment improved survival of Nipah virus-infected hamsters. These results uncover an important role of heparan sulfate in henipavirus infection and open novel perspectives for the development of heparan sulfate-targeting therapeutic approaches for these emerging infections.

scholarly journals Identification and characterization of defective viral genomes in Ebola virus infected rhesus macaques

2021 ◽  
Author(s):  
Rebecca I. Johnson ◽  
Beata Boczkowska ◽  
Kendra Alfson ◽  
Taylor Weary ◽  
Heather Menzie ◽  
...  
Keyword(s):  
Nonhuman Primates ◽  
Ebola Virus ◽  
Sequence Data ◽  
Rna Virus ◽  
Hemorrhagic Fever ◽  
Marburg Virus ◽  
Immunostimulatory Activity ◽  
Viral Genomes

Ebola virus (EBOV), of the family Filoviridae, is an RNA virus that can cause hemorrhagic fever with a high mortality rate. Defective viral genomes (DVGs) are truncated genomes that have been observed during multiple RNA virus infections, including  in vitro EBOV infection, and have previously been associated with viral persistence and immunostimulatory activity. As DVGs have been detected in cells persistently infected with EBOV, we hypothesized that DVGs may also accumulate during viral replication in filovirus-infected hosts. Therefore, we interrogated sequence data from serum and tissues using a bioinformatics tool in order to identify the presence of DVGs in nonhuman primates (NHPs) infected with EBOV, Sudan virus (SUDV) or Marburg virus (MARV). Multiple 5’ copy-back DVGs (cbDVGs) were detected in NHP serum during the acute phase of filovirus infection. While the relative abundance of total DVGs in most animals was low, serum collected during acute EBOV and SUDV infections, but not MARV infection, contained a higher proportion of short trailer sequence cbDVGs than the challenge stock. This indicated an accumulation of these DVGs throughout infection, potentially due to the preferential replication of short DVGs over the longer viral genome. Using RT-PCR and deep sequencing, we also confirmed the presence of 5’ cbDVGs in EBOV-infected NHP testes, which is of interest due to EBOV persistence in semen of male survivors of infection. This work suggests that DVGs play a role in EBOV infection in vivo and further study will lead to a better understanding of EBOV pathogenesis. Importance The study of filovirus pathogenesis is critical for understanding the consequences of infection and the development of strategies to ameliorate future outbreaks. Defective viral genomes (DVGs) have been detected during EBOV infections in vitro , however their presence in in vivo infections remains unknown. In this study, DVGs were detected in samples collected from EBOV- and SUDV-infected nonhuman primates (NHPs). The accumulation of these DVGs in the trailer region of the genome during infection indicates a potential role in EBOV and SUDV pathogenesis. In particular, the presence of DVGs in the testes of infected NHPs requires further investigation as it may be linked to the establishment of persistence.

scholarly journals Reovirus RNA recombination is sequence directed and generates internally deleted defective genome segments during passage

2020 ◽  
Author(s):  
Sydni Caet Smith ◽  
Jennifer Gribble ◽  
Julia R. Diller ◽  
Michelle A. Wiebe ◽  
Timothy W. Thoner ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Rna Virus ◽  
Serial Passage ◽  
Viral Population ◽  
Viral Gene ◽  
Rna Recombination ◽  
Chronic Infections ◽  
Rna Sequences ◽  
Mammalian Orthoreovirus

ABSTRACTFor viruses with segmented genomes, genetic diversity is generated by genetic drift, reassortment, and recombination. Recombination produces RNA populations distinct from full-length gene segments and can influence viral population dynamics, persistence, and host immune responses. Viruses in the Reoviridae family, including rotavirus and mammalian orthoreovirus (reovirus), have been reported to package segments containing rearrangements or internal deletions. Rotaviruses with RNA segments containing rearrangements have been isolated from immunocompromised and immunocompetent children and in vitro following serial passage at high multiplicity. Reoviruses that package small, defective RNA segments have established chronic infections in cells and in mice. However, the mechanism and extent of Reoviridae RNA recombination are undefined. Towards filling this gap in knowledge, we determined the titers and RNA segment profiles for reovirus and rotavirus following serial passage in cultured cells. The viruses exhibited occasional titer reductions characteristic of interference. Reovirus strains frequently accumulated segments that retained 5′ and 3′ terminal sequences and featured large internal deletions, while similar segments were rarely detected in rotavirus populations. Using next-generation RNA-sequencing to analyze RNA molecules packaged in purified reovirus particles, we identified distinct recombination sites within individual viral gene segments. Recombination junction sites were frequently associated with short regions of identical sequence. Taken together, these findings suggest that reovirus accumulates defective gene segments featuring internal deletions during passage and undergoes sequence-directed recombination at distinct sites.IMPORTANCEViruses in the Reoviridae family include important pathogens of humans and other animals and have segmented RNA genomes. Recombination in RNA virus populations can facilitate novel host exploration and increased disease severity. The extent, patterns, and mechanisms of Reoviridae recombination and the functions and effects of recombined RNA products are poorly understood. Here, we provide evidence that mammalian orthoreovirus regularly synthesizes RNA recombination products that retain terminal sequences but contain internal deletions, while rotavirus rarely synthesizes such products. Recombination occurs more frequently at specific sites in the mammalian orthoreovirus genome, and short regions of identical sequence are often detected at junction sites. These findings suggest that mammalian orthoreovirus recombination events are directed in part by RNA sequences. An improved understanding of recombined viral RNA synthesis may enhance our capacity to engineer improved vaccines and virotherapies in the future.

scholarly journals Rescue of codon-pair deoptimized respiratory syncytial virus by the emergence of genomes with very large internal deletions that complemented replication

2021 ◽  
Vol 118 (13) ◽  
pp. e2020969118
Author(s):  
Cyril Le Nouën ◽  
Thomas McCarty ◽  
Lijuan Yang ◽  
Michael Brown ◽  
Eckard Wimmer ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
De Novo ◽  
Point Mutations ◽  
Surface Glycoprotein ◽  
Temperature Sensitive ◽  
Defective Interfering Particles ◽  
Viral Genomes ◽  
Codon Pair ◽  
Syncytial Virus

Recoding viral genomes by introducing numerous synonymous but suboptimal codon pairs—called codon-pair deoptimization (CPD)—provides new types of live-attenuated vaccine candidates. The large number of nucleotide changes resulting from CPD should provide genetic stability to the attenuating phenotype, but this has not been rigorously tested. Human respiratory syncytial virus in which the G and F surface glycoprotein ORFs were CPD (called Min B) was temperature-sensitive and highly restricted in vitro. When subjected to selective pressure by serial passage at increasing temperatures, Min B substantially regained expression of F and replication fitness. Whole-genome deep sequencing showed many point mutations scattered across the genome, including one combination of six linked point mutations. However, their reintroduction into Min B provided minimal rescue. Further analysis revealed viral genomes bearing very large internal deletions (LD genomes) that accumulated after only a few passages. The deletions relocated the CPD F gene to the first or second promoter-proximal gene position. LD genomes amplified de novo in Min B–infected cells were encapsidated, expressed high levels of F, and complemented Min B replicationin trans. This study provides insight on a variation of the adaptability of a debilitated negative-strand RNA virus, namely the generation of defective minihelper viruses to overcome its restriction. This is in contrast to the common “defective interfering particles” that interfere with the replication of the virus from which they originated. To our knowledge, defective genomes that promote rather than inhibit replication have not been reported before in RNA viruses.

scholarly journals Reovirus RNA recombination is sequence directed and generates internally deleted defective genome segments during passage

2021 ◽  
Author(s):  
Sydni Caet Smith ◽  
Jennifer Gribble ◽  
Julia R. Diller ◽  
Michelle A. Wiebe ◽  
Timothy W. Thoner ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Rna Virus ◽  
Serial Passage ◽  
Viral Population ◽  
Rna Recombination ◽  
Chronic Infections ◽  
Rna Sequences ◽  
Rna Molecules ◽  
Mammalian Orthoreovirus

For viruses with segmented genomes, genetic diversity is generated by genetic drift, reassortment, and recombination. Recombination produces RNA populations distinct from full-length gene segments and can influence viral population dynamics, persistence, and host immune responses. Viruses in the Reoviridae family, including rotavirus and mammalian orthoreovirus (reovirus), have been reported to package segments containing rearrangements or internal deletions. Rotaviruses with RNA segments containing rearrangements have been isolated from immunocompromised and immunocompetent children and in vitro following serial passage at relatively high multiplicity. Reoviruses that package small, defective RNA segments have established chronic infections in cells and in mice. However, the mechanism and extent of Reoviridae RNA recombination are undefined. Towards filling this gap in knowledge, we determined the titers and RNA segment profiles for reovirus and rotavirus following serial passage in cultured cells. The viruses exhibited occasional titer reductions characteristic of interference. Reovirus strains frequently accumulated segments that retained 5′ and 3′ terminal sequences and featured large internal deletions, while similarly fragmented segments were rarely detected in rotavirus populations. Using next-generation RNA-sequencing to analyze RNA molecules packaged in purified reovirus particles, we identified distinct recombination sites within individual viral genome segments. Recombination junctions were frequently but not always characterized by short direct sequence repeats upstream and downstream that spanned junction sites. Taken together, these findings suggest that reovirus accumulates defective gene segments featuring internal deletions during passage and undergoes sequence-directed recombination at distinct sites. IMPORTANCE Viruses in the Reoviridae family include important pathogens of humans and other animals and have segmented RNA genomes. Recombination in RNA virus populations can facilitate novel host exploration and increased disease severity. The extent, patterns, and mechanisms of Reoviridae recombination and the functions and effects of recombined RNA products are poorly understood. Here, we provide evidence that mammalian orthoreovirus regularly synthesizes RNA recombination products that retain terminal sequences but contain internal deletions, while rotavirus rarely synthesizes such products. Recombination occurs more frequently at specific sites in the mammalian orthoreovirus genome, and short regions of identical sequence are often detected at junction sites. These findings suggest that mammalian orthoreovirus recombination events are directed in part by RNA sequences. An improved understanding of recombined viral RNA synthesis may enhance our capacity to engineer improved vaccines and virotherapies in the future.

scholarly journals A Novel Factor I Activity in Nipah Virus Inhibits Human Complement Pathways through Cleavage of C3b

2014 ◽  
Vol 89 (2) ◽  
pp. 989-998 ◽  
Author(s):  
John B. Johnson ◽  
Viktoriya Borisevich ◽  
Barry Rockx ◽  
Griffith D. Parks
Keyword(s):  
Protease Activity ◽  
Nipah Virus ◽  
Factor H ◽  
Human Complement ◽  
Emerging Viruses ◽  
Highly Pathogenic ◽  
Enveloped Virus ◽  
Factor I ◽  
Complement Pathways

ABSTRACTComplement is an innate immune system that most animal viruses must face during natural infections. Given that replication and dissemination of the highly pathogenic Nipah virus (NiV) include exposure to environments rich in complement factors, we tested thein vitrosensitivity of NiV to complement-mediated neutralization. Here we show that NiV was completely resistant toin vitroneutralization by normal human serum (NHS). Treatment of purified NiV with NHS activated complement pathways, but there was very little C3 deposition on virus particles. Inin vitroreconstitution experiments, NiV particles provided time- and dose-dependent factor I-like protease activity capable of cleaving C3b into inactive C3b (iC3b). NiV-dependent inactivation of C3b only occurred with the cofactors factor H and soluble CR1 but not with CD46. Purified NiV particles did not support C4b cleavage. Electron microscopy of purified NiV particles showed immunogold labeling with anti-factor I antibodies. Our results suggest a novel mechanism by which NiV evades the human complement system through a unique factor I-like activity.IMPORTANCEViruses have evolved mechanisms to limit complement-mediated neutralization, some of which involve hijacking cellular proteins involved in control of inappropriate complement activation. Here we report a previously unknown mechanism whereby NiV provides a novel protease activity capable ofin vitrocleavage and inactivation of C3b, a key component of the complement cascade. These data help to explain how an enveloped virus such as NiV can infect and disseminate through body fluids that are rich in complement activity. Disruption of the ability of NiV to recruit complement inhibitors could form the basis for the development of effective therapies and safer vaccines to combat these highly pathogenic emerging viruses.

scholarly journals Virus Population Bottlenecks: What Are They Telling Us?

2020 ◽  
Author(s):  
Feng Qu ◽  
Limin Zheng ◽  
Shaoyan Zhang ◽  
Rong Sun ◽  
Jason Slot
Keyword(s):  
Rna Virus ◽  
Viral Population ◽  
Dependent Manner ◽  
Population Bottlenecks ◽  
Replication Proteins ◽  
Concentration Dependent Manner ◽  
Beneficial Mutations ◽  
Viral Genomes ◽  
Infected Cells ◽  
New Hypothesis

Stringent, stochastic viral population bottlenecks have been observed in the infections of many 5 viruses, but exactly how and why they occur is unclear. A critical review of recent literature prompts us 6 to propose a new hypothesis, designated Isolate, Amplify, and Select (IAS), that satisfactorily explains 7 the bottlenecks in single-stranded, positive sense (+) RNA virus infections. This new hypothesis 8 postulates that, unlike those in free-living organisms, the viral population bottlenecks are imposed by 9 viruses themselves, inside the infected cells, through virus-encoded bottleneck-enforcing proteins 10 (BNEPs) that function in a concentration-dependent manner. Most BNEPs are directly translated from 11 the (+) RNA genomes of invading viruses, so that if numerous virions of the same virus invade a cell 12 simultaneously, the bottleneck-ready concentration of BNEPs would be reached sufficiently early to 13 arrest nearly all internalized viral genomes. As a result, in these cells very few (as few as one) viral 14 genomes escape from the bottlenecks stochastically to initiate viral reproduction. Repetition of this 15 process in successively infected cells ensures the progeny genomes in each cell descend from the same 16 parental genome(s), hence isolating different mutant genomes in separate cells. This isolation precludes 17 mutant viral genomes encoding defective replication proteins from exploiting the complementing 18 proteins synthesized by sister genomes, leading to the prompt elimination of such mutants. Conversely, 19 the IAS model ensures replication proteins with beneficial mutations exclusively amplify the viral 20 genomes that harbor the very mutations. Reiteration of this process in consecutively infected cells 21 enriches such beneficial mutations in the virus pool. In conclusion, the IAS hypothesis provides a 22 compelling evolutionary model for population bottlenecks of (+) RNA viruses.

scholarly journals CryoEM structure of the Nipah virus nucleocapsid assembly

2020 ◽  
Author(s):  
De-Sheng Ker ◽  
Huw T. Jenkins ◽  
Sandra J. Greive ◽  
Alfred A. Antson
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Rna Virus ◽  
Nipah Virus ◽  
Outer Edge ◽  
Highly Pathogenic ◽  
N Protein ◽  
Loop Regions ◽  
Intersubunit Interactions

AbstractNipah virus is a highly pathogenic zoonotic RNA virus, causing fatal encephalitis in humans. Like other negative-strand RNA viruses including Ebola and measles, its genome is wrapped by the nucleocapsid (N) protein forming a helical assembly. Here we report the CryoEM structure of the Nipah nucleocapsid protein-RNA assembly, at near atomic resolution. The N protein wraps the RNA genome with a periodicity of six nucleotides per protomer, around the outer edge of the helical assembly, in common with other paramyxoviruses. This structure uncovers details of the nucleocapsid assembly, demonstrating the role of the N-terminal arm of the N protein in the formation of the helical assembly and revealing details of the sequence-independent coordination of RNA binding in the “3-bases-in, 3-bases-out” conformation. CryoEM analysis also reveals formation of clam-shaped assemblies of the N-protein, mediated by intersubunit interactions involving several N protein loop regions.

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