Functional annotation of 19,841 Populus nigra full-length enriched cDNA clones

ABSTRACT The 5′-terminal end of the genomic RNA of the Lelystad virus isolate (LV) of porcine reproductive and respiratory syndrome virus was determined. To construct full-length cDNA clones, the 5′-terminal sequence was ligated to cDNA clones covering the complete genome of LV. When RNA that was transcribed in vitro from these full-length cDNA clones was transfected into BHK-21 cells, infectious LV was produced and secreted. The virus was rescued by passage to porcine alveolar lung macrophages or CL2621 cells. When infectious transcripts were transfected to porcine alveolar lung macrophages or CL2621 cells, no infectious virus was produced due to the poor transfection efficiency of these cells. The growth properties of the viruses produced by BHK-21 cells transfected with infectious transcripts of LV cDNA resembled the growth properties of the parental virus from which the cDNA was derived. Two nucleotide changes leading to a unique PacI restriction site directly downstream of the ORF7 gene were introduced in the genome-length cDNA clone. The virus recovered from this mutated cDNA clone retained the PacI site, which confirmed the de novo generation of infectious LV from cloned cDNA. These results indicate that the infectious clone of LV enables us to mutagenize the viral genome at specific sites and that it will therefore be useful for detailed molecular characterization of the virus, as well as for the development of a safe and effective live vaccine for use in pigs.

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Large-scale RT-PCR recovery of full-length cDNA clones

BioTechniques ◽

10.2144/04364dd03 ◽

2004 ◽

Vol 36 (4) ◽

pp. 690-700 ◽

Cited By ~ 4

Author(s):

Jia Qian Wu ◽

Angela M. Garcia ◽

Steven Hulyk ◽

Anna Sneed ◽

Carla Kowis ◽

...

Keyword(s):

Large Scale ◽

Full Length ◽

Cdna Clones ◽

Rt Pcr ◽

Full Length Cdna

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Construction of full-length infectious cDNA clones of Apple stem grooving virus using Gibson Assembly method

Virus Research ◽

10.1016/j.virusres.2019.197790 ◽

2020 ◽

Vol 276 ◽

pp. 197790

Author(s):

Zheng-Nan Li ◽

Wilhelm Jelkmann ◽

Ping-ping Sun ◽

Lei Zhang

Keyword(s):

Full Length ◽

Cdna Clones ◽

Gibson Assembly ◽

Assembly Method ◽

Apple Stem Grooving Virus ◽

Apple Stem

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Complete Nucleotide Sequence and Genome Organization of Hibiscus Chlorotic Ringspot Virus, a New Member of the Genus Carmovirus: Evidence for the Presence and Expression of Two Novel Open Reading Frames

Journal of Virology ◽

10.1128/jvi.74.7.3149-3155.2000 ◽

2000 ◽

Vol 74 (7) ◽

pp. 3149-3155 ◽

Cited By ~ 38

Author(s):

Mei Huang ◽

Dora Chin-Yen Koh ◽

Li-Juan Weng ◽

Min-Li Chang ◽

Yun-Kiam Yap ◽

...

Keyword(s):

Nucleotide Sequence ◽

Genome Organization ◽

Complete Nucleotide Sequence ◽

Full Length ◽

Open Reading Frames ◽

Ringspot Virus ◽

In Vitro Translation ◽

Cdna Clones ◽

High Amino Acid ◽

Reading Frames

ABSTRACT The complete nucleotide sequence of hibiscus chlorotic ringspot virus (HCRSV) was determined. The genomic RNA (gRNA) is 3,911 nucleotides long and has the potential to encode seven viral proteins in the order of 28 (p28), 23 (p23), 81 (p81), 8 (p8), 9 (p9), 38 (p38), and 25 (p25) kDa. Excluding two unique open reading frames (ORFs) encoding p23 and p25, the ORFs encode proteins with high amino acid similarity to those of carmoviruses. In addition to gRNA, two 3′-coterminated subgenomic RNA (sgRNA) species were identified. Full-length cDNA clones derived from gRNA and sgRNA were constructed under the control of a T7 promoter. Both capped and uncapped transcripts derived from the full-length genomic cDNA clone were infectious. In vitro translation and mutagenesis assays confirmed that all the predicted ORFs except the ORF encoding p8 are translatable, and the two novel ORFs (those encoding p23 and p25) may be functionally indispensable for the viral infection cycle. Based on virion morphology and genome organization, we propose that HCRSV be classified as a new member of the genus Carmovirus in familyTombusviridae.

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Construction of Full-Length cDNA Clones to Soil-Borne Wheat Mosaic Virus RNA1 and RNA2, from Which Infectious RNAs Are Transcribed In Vitro: Virion Formation and Systemic Infection without Expression of the N-Terminal and C-Terminal Extensions to the Capsid Protein

Virology ◽

10.1006/viro.2000.0587 ◽

2000 ◽

Vol 277 (1) ◽

pp. 66-75 ◽

Cited By ~ 17

Author(s):

Aya Yamamiya ◽

Yukio Shirako

Keyword(s):

Mosaic Virus ◽

Capsid Protein ◽

Systemic Infection ◽

Full Length ◽

Cdna Clones ◽

Full Length Cdna ◽

Virion Formation

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Inferring Higher Functional Information for RIKEN Mouse Full-Length cDNA Clones With FACTS

Genome Research ◽

10.1101/gr.1019903 ◽

2003 ◽

Vol 13 (6) ◽

pp. 1520-1533 ◽

Cited By ~ 4

Author(s):

T. Nagashima

Keyword(s):

Full Length ◽

Cdna Clones ◽

Functional Information ◽

Full Length Cdna

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A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy

Viruses ◽

10.3390/v10070368 ◽

2018 ◽

Vol 10 (7) ◽

pp. 368 ◽

Cited By ~ 16

Author(s):

Maximilian Münster ◽

Anna Płaszczyca ◽

Mirko Cortese ◽

Christopher Neufeldt ◽

Sarah Goellner ◽

...

Keyword(s):

Zika Virus ◽

In Silico ◽

Reverse Genetics ◽

Molecular Mechanisms ◽

Full Length ◽

Cdna Clones ◽

In Silico Prediction ◽

Fluorescent Reporter ◽

Full Length Cdna ◽

Spurious Transcription

The Zika virus (ZIKV) has recently attracted major research interest as infection was unexpectedly associated with neurological manifestations in developing foetuses and with Guillain-Barré syndrome in infected adults. Understanding the underlying molecular mechanisms requires reverse genetic systems, which allow manipulation of infectious cDNA clones at will. In the case of flaviviruses, to which ZIKV belongs, several reports have indicated that the construction of full-length cDNA clones is difficult due to toxicity during plasmid amplification in Escherichia coli. Toxicity of flaviviral cDNAs has been linked to the activity of cryptic prokaryotic promoters within the region encoding the structural proteins leading to spurious transcription and expression of toxic viral proteins. Here, we employ an approach based on in silico prediction and mutational silencing of putative promoters to generate full-length cDNA clones of the historical MR766 strain and the contemporary French Polynesian strain H/PF/2013 of ZIKV. While for both strains construction of full-length cDNA clones has failed in the past, we show that our approach generates cDNA clones that are stable on single bacterial plasmids and give rise to infectious viruses with properties similar to those generated by other more complex assembly strategies. Further, we generate luciferase and fluorescent reporter viruses as well as sub-genomic replicons that are fully functional and suitable for various research and drug screening applications. Taken together, this study confirms that in silico prediction and silencing of cryptic prokaryotic promoters is an efficient strategy to generate full-length cDNA clones of flaviviruses and reports novel tools that will facilitate research on ZIKV biology and development of antiviral strategies.

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