Interspecies protein substitution to investigate the role of the lyssavirus glycoprotein

Marston, Denise A.; McElhinney, Lorraine M.; Banyard, Ashley C.; Horton, Daniel L.; Núñez, Alejandro; Koser, Martin L.; Schnell, Matthias J.

doi:10.1099/vir.0.048827-0

Cited by 11 publications

(31 citation statements)

References 41 publications

(60 reference statements)

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“…Indeed, for RV20 the total amount of RNA was too low to obtain 200 ng RNA for fragmentation. The virus titer of RV1787 and RV20 has been calculated previously [23,24] with RV1787 (EBLV-2) approximately 1 log lower than RV20, therefore the difference in the percentage of viral reads is likely to be a reflection of this. Despite the marked difference between the percentage of viral reads of RV20 and RV1787, the difference within samples regarding whether the RNA was depleted or not, is not so obvious.…”

Section: Resultsmentioning

confidence: 99%

Next generation sequencing of viral RNA genomes

et al. 2013

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BackgroundWith the advent of Next Generation Sequencing (NGS) technologies, the ability to generate large amounts of sequence data has revolutionized the genomics field. Most RNA viruses have relatively small genomes in comparison to other organisms and as such, would appear to be an obvious success story for the use of NGS technologies. However, due to the relatively low abundance of viral RNA in relation to host RNA, RNA viruses have proved relatively difficult to sequence using NGS technologies. Here we detail a simple, robust methodology, without the use of ultra-centrifugation, filtration or viral enrichment protocols, to prepare RNA from diagnostic clinical tissue samples, cell monolayers and tissue culture supernatant, for subsequent sequencing on the Roche 454 platform.ResultsAs representative RNA viruses, full genome sequence was successfully obtained from known lyssaviruses belonging to recognized species and a novel lyssavirus species using these protocols and assembling the reads using de novo algorithms. Furthermore, genome sequences were generated from considerably less than 200 ng RNA, indicating that manufacturers’ minimum template guidance is conservative. In addition to obtaining genome consensus sequence, a high proportion of SNPs (Single Nucleotide Polymorphisms) were identified in the majority of samples analyzed.ConclusionsThe approaches reported clearly facilitate successful full genome lyssavirus sequencing and can be universally applied to discovering and obtaining consensus genome sequences of RNA viruses from a variety of sources.

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Section: Resultsmentioning

confidence: 99%

Next generation sequencing of viral RNA genomes

et al. 2013

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“…The neurotropism of RABV is conferred by its single attachment and fusion glycoprotein (G) [ 1 ]. Virulence of specific RABV strains correlates with the neuroinvasiveness of their G proteins [ 2 ], such that exchange of G of an attenuated strain with that of a pathogenic strain and vice versa confers the corresponding level of pathogenicity [ 1 , 3 – 5 ]. Although differential glycosylation [ 6 , 7 ], dysregulation of G expression levels [ 8 , 9 ], and increased induction of apoptosis [ 8 ] all contribute to G-dependent attenuation of RABV strains, it is apparent that a predominant mechanism by which G modulates rabies virulence is by dictating affinity for and spread between neurons.…”

Section: Introductionmentioning

confidence: 99%

Rabies Internalizes into Primary Peripheral Neurons via Clathrin Coated Pits and Requires Fusion at the Cell Body

Piccinotti

Whelan

2016

PLoS Pathog

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The single glycoprotein (G) of rabies virus (RABV) dictates all viral entry steps from receptor engagement to membrane fusion. To study the uptake of RABV into primary neuronal cells in culture, we generated a recombinant vesicular stomatitis virus in which the G protein was replaced with that of the neurotropic RABV CVS-11 strain (rVSV CVS G). Using microfluidic compartmentalized culture, we examined the uptake of single virions into the termini of primary neurons of the dorsal root ganglion and ventral spinal cord. By pharmacologically disrupting endocytosis at the distal neurites, we demonstrate that rVSV CVS G uptake and infection are dependent on dynamin. Imaging of single virion uptake with fluorescent endocytic markers further identifies endocytosis via clathrin-coated pits as the predominant internalization mechanism. Transmission electron micrographs also reveal the presence of viral particles in vesicular structures consistent with incompletely coated clathrin pits. This work extends our previous findings of clathrin-mediated uptake of RABV into epithelial cells to two neuronal subtypes involved in rabies infection in vivo. Chemical perturbation of endosomal acidification in the neurite or somal compartment further shows that establishment of infection requires pH-dependent fusion of virions at the cell body. These findings correlate infectivity to existing single particle evidence of long-range endosomal transport of RABV and clathrin dependent uptake at the plasma membrane.

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“…The rabies vaccine strain (SN) reverse genetics DNA backbone was utilised for all recombinant virus genome clone assembly. The SN strain is based on the street Alabama Dufferin (SAD) B19 vaccine strain of rabies as described previously [ 20 , 21 , 22 , 23 , 24 ]. Constructs were generated by exchanging the vaccine strain G with that of the highly divergent West Caucasian Bat virus G and the Ikoma virus G ( Figure S1 ).…”

Section: Methodsmentioning

confidence: 99%

Utilisation of Chimeric Lyssaviruses to Assess Vaccine Protection against Highly Divergent Lyssaviruses

Evans

Selden

et al. 2018

Viruses

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Lyssaviruses constitute a diverse range of viruses with the ability to cause fatal encephalitis known as rabies. Existing human rabies vaccines and post exposure prophylaxes (PEP) are based on inactivated preparations of, and neutralising antibody preparations directed against, classical rabies viruses, respectively. Whilst these prophylaxes are highly efficient at neutralising and preventing a productive infection with rabies virus, their ability to neutralise other lyssaviruses is thought to be limited. The remaining 15 virus species within the lyssavirus genus have been divided into at least three phylogroups that generally predict vaccine protection. Existing rabies vaccines afford protection against phylogroup I viruses but offer little to no protection against phylogroup II and III viruses. As such, work involving sharps with phylogroup II and III must be considered of high risk as no PEP is thought to have any effect on the prevention of a productive infection with these lyssaviruses. Whilst rabies virus itself has been characterised in a number of different animal models, data on the remaining lyssaviruses are scarce. As the lyssavirus glycoprotein is considered to be the sole target of neutralising antibodies we generated a vaccine strain of rabies using reverse genetics expressing highly divergent glycoproteins of West Caucasian Bat lyssavirus and Ikoma lyssavirus. Using these recombinants, we propose that recombinant vaccine strain derived lyssaviruses containing heterologous glycoproteins may be a suitable surrogate for wildtype viruses when assessing vaccine protection for the lyssaviruses.

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Interspecies protein substitution to investigate the role of the lyssavirus glycoprotein

Cited by 11 publications

References 41 publications

Next generation sequencing of viral RNA genomes

Next generation sequencing of viral RNA genomes

Rabies Internalizes into Primary Peripheral Neurons via Clathrin Coated Pits and Requires Fusion at the Cell Body

Utilisation of Chimeric Lyssaviruses to Assess Vaccine Protection against Highly Divergent Lyssaviruses

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