Next generation sequencing study on RNA viruses of Vespa velutina and Apis mellifera sharing the same foraging area

2020 ◽  
Author(s):  
Filippo Marzoli ◽  
Mario Forzan ◽  
Laura Bortolotti ◽  
Maria Irene Pacini ◽  
María Shantal Rodríguez‐Flores ◽  
...  
Keyword(s):  
Apis Mellifera ◽  
Next Generation Sequencing ◽  
Rna Viruses ◽  
Next Generation ◽  
Vespa Velutina ◽  
Foraging Area ◽  
Generation Sequencing

scholarly journals A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses

mSystems ◽  
2016 ◽  
Vol 1 (3) ◽  
Author(s):  
Lindsey A. Moser ◽  
Lisbeth Ramirez-Carvajal ◽  
Vinita Puri ◽  
Steven J. Pauszek ◽  
Krystal Matthews ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Operating Procedure ◽  
Rna Viruses ◽  
Standard Operating Procedure ◽  
Next Generation ◽  
Standard Operating ◽  
Level 3 ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Biosafety Level

ABSTRACT This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories. Several biosafety level 3 and/or 4 (BSL-3/4) pathogens are high-consequence, single-stranded RNA viruses, and their genomes, when introduced into permissive cells, are infectious. Moreover, many of these viruses are select agents (SAs), and their genomes are also considered SAs. For this reason, cDNAs and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral RNA before transfer out of the BSL-3/4 and/or SA laboratory. This tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (NGS). Here, we present a sequence-independent method to rapidly amplify viral genomic RNA while simultaneously abolishing both viral and genomic RNA infectivity across multiple single-stranded positive-sense RNA (ssRNA+) virus families. The process generates barcoded DNA amplicons that range in length from 300 to 1,000 bp, which cannot be used to rescue a virus and are stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded samples to be pooled into a single library and run across various NGS platforms without potential reconstitution of the viral genome. Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. In summary, we describe a rapid, universal standard operating procedure that generates high-quality NGS libraries free of infectious virus and infectious viral RNA. IMPORTANCE This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories.

scholarly journals Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses

Viruses ◽  
2016 ◽  
Vol 8 (3) ◽  
pp. 70 ◽  
Author(s):  
Hironobu Yanagisawa ◽  
Reiko Tomita ◽  
Koji Katsu ◽  
Takuya Uehara ◽  
Go Atsumi ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Rna Viruses ◽  
Next Generation ◽  
Efficient Detection ◽  
Generation Sequencing

scholarly journals Inter- and Intra-Host Nucleotide Variations in Hepatitis A Virus in Culture and Clinical Samples Detected by Next-Generation Sequencing

Viruses ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 619 ◽  
Author(s):  
Zhihui Yang ◽  
Mark Mammel ◽  
Chris Whitehouse ◽  
Diana Ngo ◽  
Michael Kulka
Keyword(s):  
Next Generation Sequencing ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Rna Viruses ◽  
Sequence Variant ◽  
Single Individual ◽  
Next Generation ◽  
Source Attribution ◽  
Clinical Specimens ◽  
Generation Sequencing

The accurate virus detection, strain discrimination, and source attribution of contaminated food items remains a persistent challenge because of the high mutation rates anticipated to occur in foodborne RNA viruses, such as hepatitis A virus (HAV). This has led to predictions of the existence of more than one sequence variant between the hosts (inter-host) or within an individual host (intra-host). However, there have been no reports of intra-host variants from an infected single individual, and little is known about the accuracy of the single nucleotide variations (SNVs) calling with various methods. In this study, the presence and identity of viral SNVs, either between HAV clinical specimens or among a series of samples derived from HAV clone1-infected FRhK4 cells, were determined following analyses of nucleotide sequences generated using next-generation sequencing (NGS) and pyrosequencing methods. The results demonstrate the co-existence of inter- and intra-host variants both in the clinical specimens and the cultured samples. The discovery and confirmation of multi-viral RNAs in an infected individual is dependent on the strain discrimination at the SNV level, and critical for successful outbreak traceback and source attribution investigations. The detection of SNVs in a time series of HAV infected FRhK4 cells improved our understanding on the mutation dynamics determined probably by different selective pressures. Additionally, it demonstrated that NGS could potentially provide a valuable investigative approach toward SNV detection and identification for other RNA viruses.

scholarly journals Metagenomic Analysis of the Virome of Mosquito Excreta

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Ana L. Ramírez ◽  
Agathe M. G. Colmant ◽  
David Warrilow ◽  
Bixing Huang ◽  
Alyssa T. Pyke ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Rna Viruses ◽  
Full Length ◽  
Metagenomic Analysis ◽  
Virus Discovery ◽  
Next Generation ◽  
West Nile ◽  
Arbovirus Surveillance ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

ABSTRACT Traditional screening for arboviruses in mosquitoes requires a priori knowledge and the utilization of appropriate assays for their detection. Mosquitoes can also provide other valuable information, including unexpected or novel arboviruses, nonarboviral pathogens ingested from hosts they feed on, and their own genetic material. Metagenomic analysis using next-generation sequencing (NGS) is a rapidly advancing technology that allows us to potentially obtain all this information from a mosquito sample without any prior knowledge of virus, host, or vector. Moreover, it has been recently demonstrated that pathogens, including arboviruses and parasites, can be detected in mosquito excreta by molecular methods. In this study, we investigated whether RNA viruses could be detected in mosquito excreta by NGS. Excreta samples were collected from Aedes vigilax and Culex annulirostris experimentally exposed to either Ross River or West Nile viruses and from field mosquitoes collected across Queensland, Australia. Total RNA was extracted from the excreta samples, reverse transcribed to cDNA, and sequenced using the Illumina NextSeq 500 platform. Bioinformatic analyses from the generated reads demonstrate that mosquito excreta provide sufficient RNA for NGS, allowing the assembly of near-full-length viral genomes. We detected Australian Anopheles totivirus, Wuhan insect virus 33, and Hubei odonate virus 5 and identified seven potentially novel viruses closely related to members of the order Picornavirales (2/7) and to previously described, but unclassified, RNA viruses (5/7). Our results suggest that metagenomic analysis of mosquito excreta has great potential for virus discovery and for unbiased arbovirus surveillance in the near future. IMPORTANCE When a mosquito feeds on a host, it ingests not only its blood meal but also an assortment of microorganisms that are present in the blood, thus acting as an environmental sampler. By using specific tests, it is possible to detect arthropod-borne viruses (arboviruses) like dengue and West Nile viruses in mosquito excreta. Here, we explored the use of next-generation sequencing (NGS) for unbiased detection of RNA viruses present in excreta from experimentally infected and field-collected mosquitoes. We have demonstrated that mosquito excreta provide a suitable template for NGS and that it is possible to recover and assemble near-full-length genomes of both arboviruses and insect-borne viruses, including potentially novel ones. These results importantly show the direct practicality of the use of mosquito excreta for NGS, which in the future could be used for virus discovery, environmental virome sampling, and arbovirus surveillance.

Microsatellite markers developed by next-generation sequencing differentiate inbred lines of Apis mellifera

2015 ◽  
Vol 18 (4) ◽  
pp. 801-805 ◽  
Author(s):  
Hye-Kyung Kim ◽  
Iksoo Kim ◽  
Myeong-Lyeol Lee ◽  
Yong-Soo Choi ◽  
Byung Rae Jin
Keyword(s):  
Apis Mellifera ◽  
Next Generation Sequencing ◽  
Microsatellite Markers ◽  
Inbred Lines ◽  
Next Generation ◽  
Generation Sequencing

scholarly journals Inter- and Intra-Host Nucleotide Variations in Hepatitis A Virus in Culture and Clinical Samples Detected by Next-Generation Sequencing

2018 ◽  
Author(s):  
Zhihui Yang ◽  
Mark Mammel ◽  
Chris A. Whitehouse ◽  
Diana Ngo ◽  
Michael Kulka
Keyword(s):  
Next Generation Sequencing ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Rna Viruses ◽  
Sequence Variant ◽  
Single Individual ◽  
Next Generation ◽  
Source Attribution ◽  
Clinical Specimens ◽  
Generation Sequencing

The accurate virus detection, strain discrimination, and source attribution of contaminated food items remains a persistent challenge because of the high mutation rates anticipated to occur in foodborne RNA viruses, such as Hepatitis A virus (HAV). This has led to predictions of the existence of more than one sequence variant between the hosts (inter-host) or within an individual host (intra-host). However, there have been no reports of intra-host variants from an infected single individual, and little is known about the accuracy of the single nucleotide variations (SNVs) calling with various methods. In this study, the presence and identity of viral SNVs, either between HAV clinical specimens or among a series of samples derived from HAV clone1-infected FRhK4 cells, were determined following analyses of nucleotide sequences generated using next-generation sequencing (NGS) and pyrosequencing methods. The results demonstrate the co-existence of inter- and intra-host variants both in the clinical specimens and the cultured samples. The discovery and confirmation of multi-viral RNAs in an infected individual is dependent on the strain discrimination at the SNV level, and critical for successful outbreak traceback and source attribution investigations. The detection of SNVs in a time series of HAV infected FRhK4 cells improved our understanding on the mutation dynamics determined probably by different selective pressures. Additionally, it demonstrated that NGS could potentially provide a valuable investigative approach toward SNV detection and identification for other RNA viruses.

scholarly journals Quality control implementation for universal characterization of DNA and RNA viruses in clinical respiratory samples using single metagenomic next-generation sequencing workflow

2018 ◽  
Author(s):  
A. Bal ◽  
M. Pichon ◽  
C. Picard ◽  
JS. Casalegno ◽  
M. Valette ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Real Time ◽  
Real Time Pcr ◽  
Viral Genome ◽  
Viral Infections ◽  
Rna Viruses ◽  
Next Generation ◽  
Dna And Rna ◽  
Generation Sequencing

AbstractBackgroundIn recent years, metagenomic Next-Generation Sequencing (mNGS) has increasingly been used for an accurate assumption-free virological diagnosis. However, the systematic workflow evaluation on clinical respiratory samples and implementation of quality controls (QCs) is still lacking.MethodsA total of 3 QCs were implemented and processed through the whole mNGS workflow: a notemplate-control to evaluate contamination issues during the process; an internal and an external QC to check the integrity of the reagents, equipment, the presence of inhibitors, and to allow the validation of results for each sample. The workflow was then evaluated on 37 clinical respiratory samples from patients with acute respiratory infections previously tested for a broad panel of viruses using semi-quantitative real-time PCR assays (28 positive samples including 6 multiple viral infections; 9 negative samples). Selected specimens included nasopharyngeal swabs (n = 20), aspirates (n = 10), or sputums (n = 7).ResultsThe optimal spiking level of the internal QC was first determined in order to be sufficiently detected without overconsumption of sequencing reads. According to QC validation criteria, mNGS results were validated for 34/37 selected samples. For valid samples, viral genotypes were accurately determined for 36/36 viruses detected with PCR (viral genome coverage ranged from 0.6% to 100%, median = 67.7%). This mNGS workflow allowed the detection of DNA and RNA viruses up to a semi-quantitative PCR Ct value of 36. The six multiple viral infections involving 2 to 4 viruses were also fully characterized. A strong correlation between results of mNGS and real-time PCR was obtained for each type of viral genome (R2 ranged from 0.72 for linear single-stranded (ss) RNA viruses to 0.98 for linear ssDNA viruses).ConclusionsAlthough the potential of mNGS technology is very promising, further evaluation studies are urgently needed for its routine clinical use within a reasonable timeframe. The approach described herein is crucial to bring standardization and to ensure the quality of the generated sequences in clinical setting. We provide an easy-to-use single protocol successfully evaluated for the characterization of a broad and representative panel of DNA and RNA respiratory viruses in various types of clinical samples.

Towards next-generation sequencing analytics for foodborne RNA viruses: Examining the effect of RNA input quantity and viral RNA purity

2016 ◽  
Vol 236 ◽  
pp. 221-230 ◽  
Author(s):  
Zhihui Yang ◽  
Susan R. Leonard ◽  
Mark K. Mammel ◽  
Christopher A. Elkins ◽  
Michael Kulka
Keyword(s):  
Next Generation Sequencing ◽  
Rna Viruses ◽  
Viral Rna ◽  
Next Generation ◽  
Input Quantity ◽  
Generation Sequencing

scholarly journals European foulbrood in Czechia after 40 years: application of next-generation sequencing to analyze Melissococcus plutonius transmission and influence on the bacteriome of Apis mellifera

2016 ◽  
Author(s):  
Tomas Erban ◽  
Ondrej Ledvinka ◽  
Martin Kamler ◽  
Bronislava Hortova ◽  
Marta Nesvorna ◽  
...  
Keyword(s):  
Apis Mellifera ◽  
Next Generation Sequencing ◽  
Pathogenic Bacteria ◽  
Clinical Symptoms ◽  
Rrna Gene ◽  
Next Generation ◽  
Colony Losses ◽  
Melissococcus Plutonius ◽  
European Foulbrood ◽  
Generation Sequencing

Worker honeybees (Apis mellifera) transmit Melissococcus plutonius between colonies. However, the transmission of M. plutonius, which causes European foulbrood (EFB), is poorly understood. To analyze the first EFB outbreak in 40 years in Czechia, we collected 49 hive worker samples from 18 beehives in two diseased apiaries for bacteriome analysis of the V1-V3 portion of the 16S rRNA gene. When we compared control samples obtained outside of the EFB zone, bees from an EFB apiaries containing colonies without clinical symptoms and bees from colonies with EFB clinical symptoms, there was a 100-fold higher occurrence of M. plutonius in colonies with EFB symptoms. The presence of M. plutonius in controls indicated that this pathogen exists in an enzootic state. EFB influenced the core bacteria in the worker bacteriome because the number of Snodgrassella alvi, Lactobacillus mellis, Lactobacillus melliventris, and Fructobacillus fructosus sequences increased, while Bartonella apis, Frischella perrara, and Commensalibacter intestine sequences decreased. Together, the results of this study suggest worker bees from EFB-diseased apiaries serve as vectors of M. plutonius, and eliminating such colonies is an appropriate method to overcome disease outbreaks. Because M. plutonius exists in honeybee colonies in an enzootic state, there may be similar abundances in control colonies outside the EFB zone to those in asymptomatic colonies from EFB apiaries. High-throughput Illumina next-generation sequencing permitted the quantitative interpretation of M. plutonius within the honeybee worker bacteriome. Future studies focusing on honeybee diseases, colony losses, detection of bacterial pathogens and interactions of bacteriome with pathogenic bacteria will benefit of this study.

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