Rabies Virus Populations in Humans and Mice Show Minor Inter-Host Variability within Various Central Nervous System Regions and Peripheral Tissues (original) (raw)
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Ultra-Deep Sequencing of Intra-host Rabies Virus Populations during Cross-species Transmission
PLoS Neglected Tropical Diseases, 2013
One of the hurdles to understanding the role of viral quasispecies in RNA virus cross-species transmission (CST) events is the need to analyze a densely sampled outbreak using deep sequencing in order to measure the amount of mutation occurring on a small time scale. In 2009, the California Department of Public Health reported a dramatic increase (.350%) in the number of gray foxes infected with a rabies virus variant for which striped skunks serve as a reservoir host in Humboldt County. To better understand the evolution of rabies, deep-sequencing was applied to 40 unpassaged rabies virus samples from the Humboldt outbreak. For each sample, approximately 11 kb of the 12 kb genome was amplified and sequenced using the Illumina platform. Average coverage was 17,4486and this allowed characterization of the rabies virus population present in each sample at unprecedented depths. Phylogenetic analysis of the consensus sequence data demonstrated that samples clustered according to date (1995 vs. 2009) and geographic location (northern vs. southern). A single amino acid change in the G protein distinguished a subset of northern foxes from a haplotype present in both foxes and skunks, suggesting this mutation may have played a role in the observed increased transmission among foxes in this region. Deepsequencing data indicated that many genetic changes associated with the CST event occurred prior to 2009 since several nonsynonymous mutations that were present in the consensus sequences of skunk and fox rabies samples obtained from 2003-2010 were present at the sub-consensus level (as rare variants in the viral population) in skunk and fox samples from 1995. These results suggest that analysis of rare variants within a viral population may yield clues to ancestral genomes and identify rare variants that have the potential to be selected for if environment conditions change. Citation: Borucki MK, Chen-Harris H, Lao V, Vanier G, Wadford DA, et al. (2013) Ultra-Deep Sequencing of Intra-host Rabies Virus Populations during Cross-species Transmission. PLoS Negl Trop Dis 7(11): e2555.
Retrospective diagnosis of two rabies cases in humans by high throughput sequencing
Journal of Clinical Virology, 2016
Highlights Two cases of rabies among humans were detected using a broad-range PCR analysis followed by high throughput sequencing. Diagnoses were subsequently confirmed using a fluorescent antibody test, an enzyme-linked immunosorbent assay (ELISA) and a mouse inoculation test. Two strains of rabies virus were isolated and characterized using virological methods. The entire genome of each strain was sequenced.
Rabies virus RNA was detected in mouse and human brains by in situ hybridization. 3H-labeled single-stranded RNA probes were prepared which were specific for genomic RNA and mRNAs coding for the five rabies virus proteins (N, NS, M, G, and L). Paraffin-embedded brain tissues from human cases of rabies and mice experimentally infected with the challenge virus standard (CVS)-11 strain of rabies virus and street rabies virus were examined. In CVS-infected mice, genomic RNA had a multifocal distribution in the perikarya of infected neurons, perhaps reflecting concentration of genomic RNA in viral factories. The mRNAs were more abundant than genomic RNAs in CVSand street virus-infected mouse brains and had a diffuse distribution in the perikarya. Similar amounts of signal were present in infected neurons for mRNAs coding for different rabies virus proteins. In brain tissues from human cases of rabies, genomic RNA was much more abundant than the mRNAs in infected neurons. This finding suggests either a relative block at the level of transcription or greater loss of mRNAs than of genomic RNA during the agonal period, postmortem interval, or prior to penetration of fixative during immersion fixation.
One-step protocol for amplification of near full-length cDNA of the rabies virus genome
Journal of Virological Methods, 2011
Full-length genome sequencing of the rabies virus is not a routine laboratory procedure. To understand fully the epidemiology, genetic variation and evolution of the rabies virus, full-length viral genomes need to be obtained. For rabies virus studies, cDNA synthesis is usually performed using nonspecific oligonucleotides followed by cloning. When specific primers are used, the cDNA obtained is only partial and is limited to the coding regions. Therefore, the development of methods for synthesizing long cDNA using rabies virus-specific primers is of fundamental importance. A new protocol for the synthesis of long cDNA and the development of 19 new primers are described in this study. This procedure allowed the efficient amplification of the full-length genome of the rabies virus variant maintained by hematophagous bat (Desmodus rotundus) populations following the synthesis of a complete long cDNA. Partial sequencing of the rabies virus genome was performed to confirm rabies-specific PCR amplification. Because degenerate primers were employed, this technique can be adapted easily to other variants. Importantly, this new method is faster and less expensive than cloning methods.► Full-length genome of the rabies virus is not a routine laboratory procedure. ► A new protocol for the synthesis of long cDNA and the development of 19 new primers. ► The efficient synthesis of a complete long cDNA was performed. ► This technique can be adapted easily to other variants. ► This new method is faster and less expensive than cloning methods.
The rabies virus genome: an overview
The Onderstepoort journal of veterinary research, 1993
The replication strategy, genome organization and extent of variation within the genome of the genus Lyssavirus is briefly reviewed. Strategies used in the approach to genome studies are discussed.
Sequence Analysis of Rabies Virus in Humans Exhibiting Encephalitic or Paralytic Rabies
Journal of Infectious Diseases, 2003
Two distinct clinical patterns, encephalitic (furious) and paralytic (dumb), have been recognized in human rabies. It has been postulated that different rabies virus variants associated with particular vectors may be responsible for these different clinical manifestations. Analysis of the glycoprotein (G), nucleoprotein (N), and phosphoprotein (P) genes of rabies viruses from 2 human cases of encephalitic rabies and from 2 human cases of paralytic rabies demonstrated only minor nucleotide differences. Deduced amino-acid patterns of the N protein were identical in both human and canine samples that came from the same geographic location, regardless of the clinical form. All differences in amino-acid patterns of the G protein were found outside the ectodomain, in either the signal peptide or the transmembrane and endodomains. None of the amino-acid differences of the P protein was within the interactive site with dynein. These findings support the concept that clinical manifestations of rabies are not explained solely by the associated rabies virus variant.