New astrovirus in human feces from Burkina Faso

Phan, Tung Gia; Nordgren, Johan; Ouermi, Djénèba; Simporé, Jacques; Nitiema, Léon W.; Deng, Xutao; Delwart, Eric

doi:10.1016/j.jcv.2014.03.024

Cited by 36 publications

(23 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As viral metagenomics is virus-sequence independent, potentially any virus, cultivable or uncultivable, known or novel, can be readily detected and the method can be applied to all types of virus genomes, including single-stranded DNA and RNA. Several research studies have used this technology in recent years to explore the breadth of the virome in diverse biological and environmental samples including human and animal feces [4][5][6][7][8][9][10][11][12], blood [13,14], animal and human tissues [15][16][17], and human respiratory tract secretions [18][19][20][21][22] and highlighted the validity of the approach to detect rare and novel viruses.…”

Section: Introductionmentioning

confidence: 99%

Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples

et al. 2017

View full text Add to dashboard Cite

BackgroundSequence-specific PCR is the most common approach for virus identification in diagnostic laboratories. However, as specific PCR only detects pre-defined targets, novel virus strains or viruses not included in routine test panels will be missed. Recently, advances in high-throughput sequencing allow for virus-sequence-independent identification of entire virus populations in clinical samples, yet standardized protocols are needed to allow broad application in clinical diagnostics. Here, we describe a comprehensive sample preparation protocol for high-throughput metagenomic virus sequencing using random amplification of total nucleic acids from clinical samples.ResultsIn order to optimize metagenomic sequencing for application in virus diagnostics, we tested different enrichment and amplification procedures on plasma samples spiked with RNA and DNA viruses. A protocol including filtration, nuclease digestion, and random amplification of RNA and DNA in separate reactions provided the best results, allowing reliable recovery of viral genomes and a good correlation of the relative number of sequencing reads with the virus input. We further validated our method by sequencing a multiplexed viral pathogen reagent containing a range of human viruses from different virus families. Our method proved successful in detecting the majority of the included viruses with high read numbers and compared well to other protocols in the field validated against the same reference reagent. Our sequencing protocol does work not only with plasma but also with other clinical samples such as urine and throat swabs.ConclusionsThe workflow for virus metagenomic sequencing that we established proved successful in detecting a variety of viruses in different clinical samples. Our protocol supplements existing virus-specific detection strategies providing opportunities to identify atypical and novel viruses commonly not accounted for in routine diagnostic panels.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-017-0317-z) contains supplementary material, which is available to authorized users.

show abstract

Section: Introductionmentioning

confidence: 99%

Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples

et al. 2017

View full text Add to dashboard Cite

show abstract

“…Application of molecular methods, including high-throughput sequencing and consensus PCR assays, has identified several novel astroviruses in human stool specimens, including the prototype species of mamastrovirus 6 (representative astrovirus strain MLB1), mamastrovirus 8 (representative astrovirus strain VA2/HMO-A), and mamastrovirus 9 (representative astrovirus strain VA1/HMO-C [VA1]) (21)(22)(23)(24)(25)(26)(27)(28). Humans are frequently exposed to these novel astroviruses, as seroprevalence in adults ranges from 65 to 100% (29,30).…”

mentioning

confidence: 99%

Propagation of Astrovirus VA1, a Neurotropic Human Astrovirus, in Cell Culture

Janowski

Bauer

Holtz

et al. 2017

J Virol

View full text Add to dashboard Cite

Astrovirus VA1/HMO-C (VA1; mamastrovirus 9) is a recently discovered astrovirus genotype that is divergent from the classic human astroviruses (mamastrovirus 1). The gastrointestinal tract is presumed to be the primary site of infection and pathogenicity for astroviruses. However, VA1 has been independently detected in brain tissue of five cases of human encephalitis. Studies of the pathogenicity of VA1 are currently impossible because there are no reported cell culture systems or in vivo models that support VA1 infection. Here, we describe successful propagation of VA1 in multiple human cell lines. The initial inoculum, a filtered clinical stool sample from the index gastroenteritis case cluster that led to the discovery of VA1, was first passaged in Vero cells. Serial blind passage in Caco-2 cells yielded increasing copies of VA1 RNA, and multistep growth curves demonstrated a Ͼ100-fold increase in VA1 RNA 72 h after inoculation. The full-length genomic and subgenomic RNA strands were detected by Northern blotting, and crystalline lattices of viral particles of ϳ26-nm diameter were observed by electron microscopy in infected Caco-2 cells. Unlike other human astrovirus cell culture systems, which require addition of exogenous trypsin for continued propagation, VA1 could be propagated equally well with or without the addition of trypsin. Furthermore, VA1 was sensitive to the type I interferon (IFN-I) response, as VA1 RNA levels were reduced by pretreatment of Caco-2 cells with IFN-␤1a. The ability to propagate VA1 in cell culture will facilitate studies of the neurotropism and neuropathogenesis of VA1.IMPORTANCE Astroviruses are an emerging cause of central nervous system infections in mammals, and astrovirus VA1/HMO-C is the most prevalent astrovirus in cases of human encephalitis. This virus has not been previously propagated, preventing elucidation of the biology of this virus. We describe the first cell culture system for VA1, a key step necessary for the study of its ability to cause disease.KEYWORDS astrovirus, astrovirus VA1, cell culture, electron microscopy, encephalitis, pathogenesis, subgenomic RNA, viral propagation I nitially identified in 1975, members of the single-stranded, positive-sense RNA viral family Astroviridae have been frequently detected in vertebrate stool samples (1, 2). In humans, the first identified astrovirus species was mamastrovirus 1 (classic human astrovirus), and eight serotypes of this virus have been described (2-4). Most humans are exposed to the classic human astroviruses with seroprevalence as high as 94% to specific serotypes (5-9). The classic human astroviruses predominantly cause a selflimited gastrointestinal illness and have been characterized as the third-to fifth-mostcommon viral etiology of diarrhea and gastroenteritis in humans (10)(11)(12)(13)(14).Astroviruses contain three open reading frames (ORFs) and based on conserved genomic elements are hypothesized to share common mechanisms of replication (2). Astroviruses encode a slippery sequence and stem-...

show abstract

“…In the present case-control study, viruses, bacteria and/or parasites were found in stool specimens obtained from children with AGE in 91.2% using broad range molecular testing, and in an additional 5.8% cases using NGS, resulting in diagnosis of 97% cases. NGS is an important tool in pathogen discovery, since many new or emerging viruses have already been successfully detected with this method (37)(38)(39)(40)(41)(42). In our case, NGS provided important information on viral pathogens, which were either not detected with our existing method or represented a potentially new enteric pathogen.…”

Section: Discussionmentioning

confidence: 84%

Narrowing of the Diagnostic Gap of Acute Gastroenteritis in Children 0–6 Years of Age Using a Combination of Classical and Molecular Techniques, Delivers Challenges in Syndromic Approach Diagnostics

Steyer

Jevšnik

Petrovec

et al. 2016

Pediatric Infectious Disease Journal

View full text Add to dashboard Cite

Background 25%-50% of acute gastroenteritis (AGE) cases remain etiologically undiagnosed. Our main aim was to determine the most appropriate list of enteric pathogens to be included in the daily diagnostics scheme of AGE, ensuring the lowest possible diagnostic gap. Methods 297 children ≤6 years of age, admitted to hospital in Slovenia, October 2011 – October 2012, with AGE, and 88 ≤6 year old healthy children, were included in the study. A broad spectrum of enteric pathogens was targeted with molecular methods, including 8 viruses, 6 bacteria and 2 parasites. Results At least one enteric pathogen was detected in 91.2% of cases with AGE and 27.3% of controls. Viruses were the most prevalent (82.5% and 15.9%), followed by bacteria (27.3% and 10.2%) and parasites (3.0% and 1.1%) in cases and controls, respectively. A high proportion (41.8%) of mixed infections was observed in the cases. For cases with undetermined etiology (8.8%), stool samples were analyzed with next generation sequencing and a potential viral pathogen was detected in 17 additional samples (5.8%). Conclusions Our study suggests that tests for rotaviruses, noroviruses genogroup II, adenoviruses 40/41, astroviruses, Campylobacter spp. and Salmonella sp. should be included in the initial diagnostic algorithm, which revealed the etiology in 83.5% of children tested. The use of molecular methods in diagnostics of gastroenteritis is preferable because of their high sensitivity, specificity, fast performance and the possibility of establishing the concentration of the target. The latter may be valuable for assessing the clinical significance of the detected enteric, particularly viral pathogens.

show abstract

New astrovirus in human feces from Burkina Faso

Cited by 36 publications

References 24 publications

Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples

Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples

Propagation of Astrovirus VA1, a Neurotropic Human Astrovirus, in Cell Culture

Narrowing of the Diagnostic Gap of Acute Gastroenteritis in Children 0–6 Years of Age Using a Combination of Classical and Molecular Techniques, Delivers Challenges in Syndromic Approach Diagnostics

Contact Info

Product

Resources

About