Pod pepper vein yellows virus, a new recombinant polerovirus infecting Capsicum frutescens in Yunnan province, China

AbstractPepper vein yellows viruses (PeVYV) are phloem-restricted viruses in the genus Polerovirus, family Luteoviridae. Typical viral symptoms of PeVYV including interveinal yellowing of leaves and upward leaf curling were observed in pod pepper plants (Capsicum frutescens) growing in Wenshan city, Yunnan province, China. The complete genome sequence of a virus from a sample of these plants was determined by next-generation sequencing and RT-PCR. Pod pepper vein yellows virus (PoPeVYV) (MT188667) has a genome of 6015 nucleotides, and the characteristic genome organization of a member of the genus Polerovirus. In the 5′ half of its genome (encoding P0 to P4), PoPeVYV is most similar (93.1% nt identity) to PeVYV-3 (Pepper vein yellows virus 3) (KP326573) but diverges greatly in the 3′-part encoding P5, where it is most similar (91.7% nt identity) to tobacco vein distorting virus (TVDV, EF529624) suggesting a recombinant origin. Recombination analysis predicted a single recombination event affecting nucleotide positions 4126 to 5192 nt, with PeVYV-3 as the major parent but with the region 4126–5192 nt derived from TVDV as the minor parent. A full-length clone of PoPeVYV was constructed and shown to be infectious in C. frutescens by RT-PCR and the presence of icosahedral viral particles.

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Pod pepper vein yellows virus, a new recombinant polerovirus infecting Capsicum frutescens in Yunnan province, China

10.21203/rs.3.rs-103456/v2 ◽

2021 ◽

Author(s):

Kuangjie Zhao ◽

Yueyan Yin ◽

Mengying Hua ◽

Shaoxiang Wang ◽

Xiaohan Mo ◽

...

Keyword(s):

Genome Organization ◽

Yunnan Province ◽

Capsicum Frutescens ◽

Rt Pcr ◽

Minor Parent ◽

Viral Particles ◽

A Genome ◽

Pepper Vein Yellows Virus ◽

Generation Sequencing ◽

Full Length Clone

Abstract Pepper vein yellows viruses (PeVYV) are phloem-restricted viruses in the genus Polerovirus, family Luteoviridae. Typical viral symptoms of PeVYV including interveinal yellowing of leaves and upward leaf curling were observed in pod pepper plants (Capsicum frutescens) growing in Wenshan city, Yunnan province, China. The complete genome sequence of a virus from a sample of these plants was determined by next-generation sequencing and RT-PCR. Pod pepper vein yellows virus (PoPeVYV) (MT188667) has a genome of 6015 nucleotides, and the characteristic genome organization of a member of the genus Polerovirus. In the 5’ half of its genome (encoding P0 to P4), PoPeVYV is most similar (93.1% nt identity) to PeVYV-3 (Pepper vein yellows virus 3) (KP326573) but diverges greatly in the 3’-part encoding P5, where it is most similar (91.7% nt identity) to tobacco vein distorting virus (TVDV, EF529624) suggesting a recombinant origin. Recombination analysis predicted a single recombination event affecting nucleotide positions 4126 to 5192 nt, with PeVYV-3 as the major parent but with the region 4126-5192 nt derived from TVDV as the minor parent. A full-length clone of PoPeVYV was constructed and shown to be infectious in C. frutescens by RT-PCR and the presence of icosahedral viral particles.

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Pod Pepper Vein Yellows Virus, a New Recombinant Polerovirus Infecting Pod Pepper in Yunnan Province, China

10.21203/rs.3.rs-103456/v1 ◽

2020 ◽

Author(s):

Kuangjie Zhao ◽

Yueyan Yin ◽

Mengying Hua ◽

Shaoxiang Wang ◽

Xiaohan Mo ◽

...

Keyword(s):

Genome Organization ◽

Recombination Event ◽

Yunnan Province ◽

Capsicum Frutescens ◽

Rt Pcr ◽

Viral Particles ◽

A Genome ◽

Pepper Vein Yellows Virus ◽

Generation Sequencing ◽

Full Length Clone

Abstract Pepper vein yellows viruses (PeVYV) are phloem-restricted viruses in the genus Polerovirus, family Luteoviridae. Typical viral symptoms of PeVYV including interveinal yellowing of leaves and upward leaf curling were observed in pod pepper plants (Capsicum frutescens) growing in Wenshan city, Yunnan province, China. The complete genome sequence of a virus from a sample of these plants was determined by next-generation sequencing and RT-PCR. Pod pepper vein yellows virus (PoPeVYV) (MT188667) has a genome of 6015 nucleotides, and the characteristic genome organization of a member of the genus Polerovirus. In the 5’ half of its genome (encoding P0 to P4), PoPeVYV is most similar (94% nt identity) to PeVYV-3 (Pepper vein yellows virus 3) (KP326573) but diverges greatly in the 3’-part encoding P5, where it is most similar (88.8% nt identity) to tobacco vein distorting virus (TVDV, EF529624) suggesting a recombinant origin. Recombination analysis predicted a single recombination event at nucleotide positions 4126 to 5192 nt, with TVDV and PeVYV-3 as the minor and major parents, respectively. A full-length clone of PoPeVYV was constructed and shown to be infectious in C. frutescens by RT-PCR and the presence of icosahedral viral particles.

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Biological and Genetic Characterization of Pod Pepper Vein Yellows Virus-Associated RNA From Capsicum frutescens in Wenshan, China

Frontiers in Microbiology ◽

10.3389/fmicb.2021.662352 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jiejun Peng ◽

Shan Bu ◽

Yueyan Yin ◽

Mengying Hua ◽

Kuangjie Zhao ◽

...

Keyword(s):

Yunnan Province ◽

Genetic Characterization ◽

Biological Properties ◽

Capsicum Frutescens ◽

Chain Reaction ◽

Positive Sense ◽

Polymerase Chain ◽

Pepper Vein Yellows Virus ◽

Generation Sequencing

Tombusvirus-like associated RNAs (tlaRNAs) are positive-sense single-stranded RNAs found in plants co-infected with some viruses of the genus Polerovirus. Pod pepper vein yellows virus (PoPeVYV) was recently reported as a new recombinant polerovirus causing interveinal yellowing, stunting, and leaf rolling in Capsicum frutescens plants at Wenshan city, Yunnan province, China. The complete genome sequence of its associated RNA has now been determined by next-generation sequencing and reverse transcription (RT) polymerase chain reaction (PCR). PoPeVYV-associated RNA (PoPeVYVaRNA) (GenBank Accession No. MW323470) has 2970 nucleotides and is closely related to other group II tlaRNAs, particularly tobacco bushy top disease-associated RNA (TBTDaRNA, GenBank Accession No. EF529625). In infection experiments on Nicotiana benthamiana and C. frutescens plants, synergism between PoPeVYVaRNA and PoPeVYV was demonstrated, leading to severe interveinal yellowing of leaves and stunting of plants. The results provide further information on the genetic and biological properties of the various agents associated with pepper vein yellows disease (PeVYD).

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Detection of SARS-CoV-2 from patient fecal samples by whole genome sequencing

Gut Pathogens ◽

10.1186/s13099-021-00398-5 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Andreas Papoutsis ◽

Thomas Borody ◽

Siba Dolai ◽

Jordan Daniels ◽

Skylar Steinberg ◽

...

Keyword(s):

Next Generation Sequencing ◽

Nasopharyngeal Swab ◽

Whole Genome ◽

Rt Pcr ◽

Whole Genome Analysis ◽

Fecal Samples ◽

Oral Transmission ◽

Study Participants ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Abstract Background SARS-CoV-2 has been detected not only in respiratory secretions, but also in stool collections. Here were sought to identify SARS-CoV-2 by enrichment next-generation sequencing (NGS) from fecal samples, and to utilize whole genome analysis to characterize SARS-CoV-2 mutational variations in COVID-19 patients. Results Study participants underwent testing for SARS-CoV-2 from fecal samples by whole genome enrichment NGS (n = 14), and RT-PCR nasopharyngeal swab analysis (n = 12). The concordance of SARS-CoV-2 detection by enrichment NGS from stools with RT-PCR nasopharyngeal analysis was 100%. Unique variants were identified in four patients, with a total of 33 different mutations among those in which SARS-CoV-2 was detected by whole genome enrichment NGS. Conclusion These results highlight the potential viability of SARS-CoV-2 in feces, its ongoing mutational accumulation, and its possible role in fecal–oral transmission. This study also elucidates the advantages of SARS-CoV-2 enrichment NGS, which may be a key methodology to document complete viral eradication. Trial registration ClinicalTrials.gov, NCT04359836, Registered 24 April 2020, https://clinicaltrials.gov/ct2/show/NCT04359836?term=NCT04359836&draw=2&rank=1).

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Development of an RNA Extraction Protocol for a Norovirus from Raw Oysters and Detection by qRT-PCR and Droplet-Digital RT-PCR

Foods ◽

10.3390/foods10081804 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1804

Author(s):

Daniel Plante ◽

Julio Alexander Bran Barrera ◽

Maude Lord ◽

Irène Iugovaz ◽

Neda Nasheri

Keyword(s):

Hepatitis A Virus ◽

Rna Extraction ◽

Complex Nature ◽

Murine Norovirus ◽

Rt Pcr ◽

Pcr Inhibitors ◽

Qrt Pcr ◽

Extraction Protocol ◽

Viral Particles ◽

Hands On

Foodborne viruses such as norovirus and hepatitis A virus cause frequent outbreaks associated with the consumption of raw or undercooked oysters. Viral particles are bioaccumulated in the oyster’s digestive glands, making RNA extraction and RT-PCR detection difficult due to the complex nature of the food matrix and the presence of RT-PCR inhibitors. Herein, we have developed a viral RNA extraction protocol from raw oysters using murine norovirus (MNV) as a surrogate for human noroviruses. The method combines lysis in Tri-Reagent reagent, followed by RNA extraction using Direct-Zol purification columns and lithium chloride precipitation. Viral load quantification was performed by both qRT-PCR and droplet-digital RT-PCR. We have demonstrated that this method can efficiently remove RT-PCR inhibitors, and is sensitive enough to reliably detect viral contamination at 25 PFU/0.2 g. We have also compared the efficiency of this method with the ISO 15216-1:2017 method and Method E developed by Quang and colleagues, and observed significantly higher efficiency compared with the ISO 15216-1 method and comparable efficiency with Method E, with less steps, and shorter hands-on time.

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Multiplexed detection of SARS-CoV-2 and other respiratory infections in high throughput by SARSeq

Nature Communications ◽

10.1038/s41467-021-22664-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ramesh Yelagandula ◽

◽

Aleksandr Bykov ◽

Alexander Vogt ◽

Robert Heinen ◽

...

Keyword(s):

Influenza A ◽

Respiratory Infections ◽

High Sensitivity ◽

Cost Effective ◽

Massively Parallel ◽

Rt Pcr ◽

Viral Spread ◽

Saliva Analysis ◽

Double Blinded ◽

Generation Sequencing

AbstractThe COVID-19 pandemic has demonstrated the need for massively-parallel, cost-effective tests monitoring viral spread. Here we present SARSeq, saliva analysis by RNA sequencing, a method to detect SARS-CoV-2 and other respiratory viruses on tens of thousands of samples in parallel. SARSeq relies on next generation sequencing of multiple amplicons generated in a multiplexed RT-PCR reaction. Two-dimensional, unique dual indexing, using four indices per sample, enables unambiguous and scalable assignment of reads to individual samples. We calibrate SARSeq on SARS-CoV-2 synthetic RNA, virions, and hundreds of human samples of various types. Robustness and sensitivity were virtually identical to quantitative RT-PCR. Double-blinded benchmarking to gold standard quantitative-RT-PCR performed by human diagnostics laboratories confirms this high sensitivity. SARSeq can be used to detect Influenza A and B viruses and human rhinovirus in parallel, and can be expanded for detection of other pathogens. Thus, SARSeq is ideally suited for differential diagnostic of infections during a pandemic.

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LncRNA:DNA triplex-forming sites are positioned at specific areas of genome organization and are predictors for Topologically Associated Domains

BMC Genomics ◽

10.1186/s12864-021-07727-7 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Benjamin Soibam ◽

Ayzhamal Zhamangaraeva

Keyword(s):

Genome Organization ◽

Chromatin Organization ◽

Ctcf Binding ◽

General Mechanism ◽

Promoter Regions ◽

Cellular Processes ◽

A Genome ◽

Topologically Associated Domains ◽

Multiple Cell ◽

Genomic Regions

Abstract Background Chromosomes are organized into units called topologically associated domains (TADs). TADs dictate regulatory landscapes and other DNA-dependent processes. Even though various factors that contribute to the specification of TADs have been proposed, the mechanism is not fully understood. Understanding the process for specification and maintenance of these units is essential in dissecting cellular processes and disease mechanisms. Results In this study, we report a genome-wide study that considers the idea of long noncoding RNAs (lncRNAs) mediating chromatin organization using lncRNA:DNA triplex-forming sites (TFSs). By analyzing the TFSs of expressed lncRNAs in multiple cell lines, we find that they are enriched in TADs, their boundaries, and loop anchors. However, they are evenly distributed across different regions of a TAD showing no preference for any specific portions within TADs. No relationship is observed between the locations of these TFSs and CTCF binding sites. However, TFSs are located not just in promoter regions but also in intronic, intergenic, and 3’UTR regions. We also show these triplex-forming sites can be used as predictors in machine learning models to discriminate TADs from other genomic regions. Finally, we compile a list of important “TAD-lncRNAs” which are top predictors for TADs identification. Conclusions Our observations advocate the idea that lncRNA:DNA TFSs are positioned at specific areas of the genome organization and are important predictors for TADs. LncRNA:DNA triplex formation most likely is a general mechanism of action exhibited by some lncRNAs, not just for direct gene regulation but also to mediate 3D chromatin organization.

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Screening of Mosquitoes for West Nile Virus and Usutu Virus in Croatia, 2015–2020

Tropical Medicine and Infectious Disease ◽

10.3390/tropicalmed6020045 ◽

2021 ◽

Vol 6 (2) ◽

pp. 45

Author(s):

Ana Klobucar ◽

Vladimir Savic ◽

Marcela Curman Posavec ◽

Suncica Petrinic ◽

Urska Kuhar ◽

...

Keyword(s):

West Nile Virus ◽

Culex Pipiens ◽

Rt Pcr ◽

West Nile ◽

Usutu Virus ◽

Culex Pipiens Complex ◽

Mammalian Origin ◽

Mosquito Pool ◽

The City ◽

Generation Sequencing

In the period from 2015 to 2020, an entomological survey for the presence of West Nile virus (WNV) and Usutu virus (USUV) in mosquitoes was performed in northwestern Croatia. A total of 20,363 mosquitoes were sampled in the City of Zagreb and Međimurje county, grouped in 899 pools and tested by real-time RT-PCR for WNV and USUV RNA. All pools were negative for WNV while one pool each from 2016 (Aedes albopictus), 2017 (Culex pipiens complex), 2018 (Cx. pipiens complex), and 2019 (Cx. pipiens complex), respectively, was positive for USUV. The 2018 and 2019 positive pools shared 99.31% nucleotide homology within the USUV NS5 gene and both clustered within USUV Europe 2 lineage. The next-generation sequencing of one mosquito pool (Cx. pipiens complex) collected in 2018 in Zagreb confirmed the presence of USUV and revealed several dsDNA and ssRNA viruses of insect, bacterial and mammalian origin.

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