scholarly journals High levels of unreported intraspecific diversity among RNA viruses in faeces of neonatal piglets with diarrhoea

2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Martí Cortey ◽  
Ivan Díaz ◽  
Anna Vidal ◽  
Gerard Martín-Valls ◽  
Giovanni Franzo ◽  
...  
Keyword(s):  
Early Life ◽  
Rna Viruses ◽  
Intraspecific Diversity ◽  
Virus Species ◽  
Neonatal Piglets ◽  
Porcine Epidemic Diarrhoea Virus ◽  
Diarrhoea Virus ◽  
High Level ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Background Diarrhoea is a major cause of death in neonate pigs and most of the viruses that cause it are RNA viruses. Next Generation Sequencing (NGS) deeply characterize the genetic diversity among rapidly mutating virus populations at the interspecific as well as the intraspecific level. The diversity of RNA viruses present in faeces of neonatal piglets suffering from diarrhoea in 47 farms, plus 4 samples from non-diarrhoeic piglets has been evaluated by NGS. Samples were selected among the cases submitted to the Veterinary Diagnostic Laboratories of Infectious Diseases of the Universitat Autònoma de Barcelona (Barcelona, Spain) and Universidad de León (León, Spain). Results The analyses identified the presence of 12 virus species corresponding to 8 genera of RNA viruses. Most samples were co-infected by several viruses. Kobuvirus and Rotavirus were more commonly reported, with Sapovirus, Astrovirus 3, 4 and 5, Enterovirus G, Porcine epidemic diarrhoea virus, Pasivirus and Posavirus being less frequently detected. Most sequences showed a low identity with the sequences deposited in GenBank, allowing us to propose several new VP4 and VP7 genotypes for Rotavirus B and Rotavirus C. Conclusions Among the cases analysed, Rotaviruses were the main aetiological agents of diarrhoea in neonate pigs. Besides, in a small number of cases Kobuvirus and Sapovirus may also have an aetiological role. Even most animals were co-infected in early life, the association with enteric disease among the other examined viruses was unclear. The NGS method applied successfully characterized the RNA virome present in faeces and detected a high level of unreported intraspecific diversity.

scholarly journals MET Amplification in Non-Small Cell Lung Cancer (NSCLC)—A Consecutive Evaluation Using Next-Generation Sequencing (NGS) in a Real-World Setting

Cancers ◽  
2021 ◽  
Vol 13 (19) ◽  
pp. 5023
Author(s):  
Christoph Schubart ◽  
Robert Stöhr ◽  
Lars Tögel ◽  
Florian Fuchs ◽  
Horia Sirbu ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Next Generation Sequencing ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
Resistance Mechanism ◽  
Small Cell ◽  
Small Cell Lung ◽  
High Level ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

In non-small cell lung cancer (NSCLC), approximately 1–3% of cases harbor an increased gene copy number (GCN) of the MET gene. This alteration can be due to de novo amplification of the MET gene or can represent a secondary resistance mechanism in response to targeted therapies. To date, the gold standard method to evaluate the GCN of MET is fluorescence in situ hybridization (FISH). However, next-generation sequencing (NGS) is becoming more relevant to optimize therapy by revealing the mutational profile of each NSCLC. Using evaluable n = 205 NSCLC cases of a consecutive cohort, this study addressed the question of whether an amplicon based NGS assay can completely replace the FISH method regarding the classification of MET GCN status. Out of the 205 evaluable cases, only n = 9 cases (43.7%) of n = 16 high-level MET amplified cases assessed by FISH were classified as amplified by NGS. Cases harboring a MET GCN > 10 showed the best concordance when comparing FISH versus NGS (80%). This study confirms that an amplicon-based NGS assessment of the MET GCN detects high-level MET amplified cases harboring a MET GCN > 10 but fails to detect the various facets of MET gene amplification in the context of a therapy-induced resistance mechanism.

Dual infection with PCV-2 and porcine epidemic diarrhoea virus in neonatal piglets

2001 ◽  
Vol 148 (15) ◽  
pp. 482-484 ◽  
Author(s):  
T. Hirai ◽  
T. Nunoya ◽  
T. Ihara ◽  
K. Kusanagi ◽  
K. Shibuya
Keyword(s):  
Dual Infection ◽  
Neonatal Piglets ◽  
Porcine Epidemic Diarrhoea Virus ◽  
Diarrhoea Virus

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 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.

scholarly journals The study of hybridization processes within genus Sparganium L. Subgenus Xanthosparganium holmb. Based on data of next generation sequencing (NGS)

2019 ◽  
Vol 17 (4) ◽  
pp. 27-35
Author(s):  
Evgeniy A. Belyakov ◽  
Eduard M. Machs ◽  
Yulia V. Mikhailova ◽  
Aleksandr V. Rodionov
Keyword(s):  
Next Generation Sequencing ◽  
North American ◽  
Illumina Miseq ◽  
Rrna Genes ◽  
Next Generation ◽  
Close Relationship ◽  
Species Characteristics ◽  
High Level ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

The study represents the results of research of intragenic polymorphism in transcribed spacer ITS1 of the 35S rRNA genes in representatives of subgenus Xanthosparganium genus Sparganium which were obtained by means of locus-specific next generation sequencing on the platform Illumina MiSeq. It was shown that ribotype variations in studied samples generally correspond to the division of this genus into three sections Erecta (subgenus Sparganium), Natantia and Minima (subgenus Xanthosparganium). High level of intragenic polymorphism was revealed in S. hyperboreum, with ribotypes distributed among several groups. Genome of this species includes ribotypes which are typical for other species in subgenus Xanthosparganium. For two investigated S. glomeratum samples, there were no ribotypes similar to such ribotypes in other species of Natantia section. S. glomeratum has got ribotypes identical with S. hyperboreum of Minima section. This feature may be the evidence of ancient intersectional hybridization of these two species. Characteristics of rDNA in S. glomeratum are in favor of putting this species into Minima section. It was suggested that speciation processes within the genus could be based not only on hybridization but also went on in allopatric way. The fist statement is supported by the presence of similar and identical ribotypes in S. emersum, S. longifolium, S. gramineum and S. hyperboreum, the second as it was mentioned by other researchers, is due to close relationship between North American and Eurasian taxa.

scholarly journals Pathogenesis comparison between the United States porcine epidemic diarrhoea virus prototype and S-INDEL-variant strains in conventional neonatal piglets

2016 ◽  
Vol 97 (5) ◽  
pp. 1107-1121 ◽  
Author(s):  
Qi Chen ◽  
Phillip C. Gauger ◽  
Molly R. Stafne ◽  
Joseph T. Thomas ◽  
Darin M. Madson ◽  
...  
Keyword(s):  
United States ◽  
The United States ◽  
Neonatal Piglets ◽  
Porcine Epidemic Diarrhoea Virus ◽  
Diarrhoea Virus

Phylogeny and expression of the nucleocapsid gene of porcine epidemic diarrhoea virus

2013 ◽  
Vol 61 (2) ◽  
pp. 257-269 ◽  
Author(s):  
Wei Yang ◽  
Guangxing Li ◽  
Yudong Ren ◽  
Siqingaowa Suo ◽  
Xiaofeng Ren
Keyword(s):  
Escherichia Coli ◽  
Protein Sequences ◽  
N Gene ◽  
Severe Diarrhoea ◽  
High Level Expression ◽  
Nucleocapsid Gene ◽  
N Protein ◽  
Porcine Epidemic Diarrhoea Virus ◽  
Diarrhoea Virus ◽  
High Level

Porcine epidemic diarrhoea virus (PEDV) is one of the important pathogens that may cause severe diarrhoea in piglets. In this study, the nucleocapsid (N) gene of a Chinese PEDV isolate designated HLJBY was cloned. The phylogeny of PEDV strains was investigated by constructing a phylogenetic tree based on the N protein sequences. The results indicate that there are two major groups of Chinese PEDVs, a Japanese PEDV group and a Korean PEDV group. High-level expression of the N protein was achieved inEscherichia coli. The immunoreactivity between PEDV particles or the bacterially expressed N protein and rabbit anti-PEDV serum was confirmed by immunofluorescence assays and Western blot. Both PEDV N protein and the polyclonal antibody generated in this study are valuable diagnostic reagents for PEDV surveillance.

scholarly journals Genomic evidence for divergent co-infections of SARS-CoV-2 lineages

2021 ◽  
Author(s):  
Hang-Yu Zhou ◽  
Ye-Xiao Cheng ◽  
Lin Xu ◽  
Jia-Ying Li ◽  
Chen-Yue Tao ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Large Scale ◽  
Rna Viruses ◽  
Average Rate ◽  
Genomic Analysis ◽  
Distribution Method ◽  
Reference Framework ◽  
Next Generation Sequencing Ngs ◽  
Ngs Data ◽  
Generation Sequencing

Recently, patients co-infected by two SARS-CoV-2 lineages have been sporadically reported. Concerns are raised because previous studies have demonstrated co-infection may contribute to the recombination of RNA viruses and cause severe clinic symptoms. In this study, we have estimated the compositional lineage(s), tendentiousness, and frequency of co-infection events in population from a large-scale genomic analysis for SARS-CoV-2 patients. SARS-CoV-2 lineage(s) infected in each sample have been recognized from the assignment of within-host site variations into lineage-defined feature variations by introducing a hypergeometric distribution method. Of all the 29,993 samples, 53 (~0.18%) co-infection events have been identified. Apart from 52 co-infections with two SARS-CoV-2 lineages, one sample with co-infections of three SARS-CoV-2 lineages was firstly identified. As expected, the co-infection events mainly happened in the regions where have co-existed more than two dominant SARS-CoV-2 lineages. However, co-infection of two sub-lineages in Delta lineage were detected as well. Our results provide a useful reference framework for the high throughput detecting of SARS-CoV-2 co-infection events in the Next Generation Sequencing (NGS) data. Although low in average rate, the co-infection events showed an increasing tendency with the increased diversity of SARS-CoV-2. And considering the large base of SARS-CoV-2 infections globally, co-infected patients would be a nonnegligible population. Thus, more clinical research is urgently needed on these patients.

scholarly journals Pro197Thr Substitution in Ahas Gene Causing Resistance to Pyroxsulam Herbicide in Rigid Ryegrass (Lolium Rigidum Gaud.)

2021 ◽  
Vol 13 (12) ◽  
pp. 6648
Author(s):  
Barbara Kutasy ◽  
Zsolt Takács ◽  
Judit Kovács ◽  
Verëlindë Bogaj ◽  
Syafiq A. Razak ◽  
...  
Keyword(s):  
Amino Acid ◽  
De Novo ◽  
Acetohydroxyacid Synthase ◽  
Lolium Rigidum ◽  
High Genetic Diversity ◽  
Reading Frame ◽  
Rigid Ryegrass ◽  
High Level ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Lolium rigidum Gaud. is a cross-pollinated species characterized by high genetic diversity and it was detected as one of the most herbicide resistance-prone weeds, globally. Acetohydroxyacid synthase (AHAS) resistant populations cause significant problems in cereal production; therefore, monitoring the development of AHAS resistance is widely recommended. Using next-generation sequencing (NGS), a de novo transcriptome sequencing dataset was presented to identify the complete open reading frame (ORF) of AHAS enzyme in L. rigidum and design markers to amplify fragments consisting of all of the eight resistance-conferring amino acid mutation sites. Pro197Thr, Pro197Ala, Pro197Ser, Pro197Gln, and Trp574Leu amino acid substitutions have been observed in samples. Although the Pro197Thr amino acid substitution was already described in SU and IMI resistant populations, this is the first report to reveal that the Pro197Thr in AHAS enzyme confers a high level of resistance (ED50 3.569) to pyroxsulam herbicide (Triazolopyrimidine).

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