scholarly journals Molecular Identification of Prune Dwarf Virus (PDV) Infecting Sweet Cherry in Canada and Development of a PDV Full-Length Infectious cDNA Clone

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2025
Author(s):  
Aaron J. Simkovich ◽  
Yinzi Li ◽  
Susanne E. Kohalmi ◽  
Jonathan S. Griffiths ◽  
Aiming Wang

Prune dwarf virus (PDV) is a member of ilarviruses that infects stone fruit species such as cherry, plum and peach, and ornamentally grown trees worldwide. The virus lacks an RNA silencing suppressor. Infection by PDV either alone, or its mixed infection with other viruses causes deteriorated fruit marketability and reduced fruit yields. Here, we report the molecular identification of PDV from sweet cherry in the prominent fruit growing region of Ontario, Canada known as the Niagara fruit belt using next generation sequencing of small interfering RNAs (siRNAs). We assessed its incidence in an experimental farm and determined the full genome sequence of this PDV isolate. We further constructed an infectious cDNA clone. Inoculation of the natural host cherry with this clone induced a dwarfing phenotype. We also examined its infectivity on several common experimental hosts. We found that it was infectious on cucurbits (cucumber and squash) with clear symptoms and Nicotiana benthamiana without causing noticeable symptoms, and it was unable to infect Arabidopsis thaliana. As generating infectious clones for woody plants is very challenging with limited success, the PDV infectious clone developed from this study will be a useful tool to facilitate molecular studies on PDV and related Prunus-infecting viruses.

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1050
Author(s):  
Liqin Tu ◽  
Shuhua Wu ◽  
Danna Gao ◽  
Yong Liu ◽  
Yuelin Zhu ◽  
...  

Tomato mottle mosaic virus (ToMMV) is a noteworthy virus which belongs to the Virgaviridae family and causes serious economic losses in tomato. Here, we isolated and cloned the full-length genome of a ToMMV Chinese isolate (ToMMV-LN) from a naturally infected tomato (Solanum lycopersicum L.). Sequence analysis showed that ToMMV-LN contains 6399 nucleotides (nts) and is most closely related to a ToMMV Mexican isolate with a sequence identity of 99.48%. Next, an infectious cDNA clone of ToMMV was constructed by a homologous recombination approach. Both the model host N. benthamiana and the natural hosts tomato and pepper developed severe symptoms upon agroinfiltration with pToMMV, which had a strong infectivity. Electron micrographs indicated that a large number of rigid rod-shaped ToMMV virions were observed from the agroinfiltrated N. benthamiana leaves. Finally, our results also confirmed that tomato plants inoculated with pToMMV led to a high infection rate of 100% in 4–5 weeks post-infiltration (wpi), while pepper plants inoculated with pToMMV led to an infection rate of 40–47% in 4–5 wpi. This is the first report of the development of a full-length infectious cDNA clone of ToMMV. We believe that this infectious clone will enable further studies of ToMMV genes function, pathogenicity and virus–host interaction.


2004 ◽  
Vol 85 (4) ◽  
pp. 1007-1018 ◽  
Author(s):  
Daisuke Hayasaka ◽  
Tamara S. Gritsun ◽  
Kentarou Yoshii ◽  
Tomotaka Ueki ◽  
Akiko Goto ◽  
...  

A stable full-length infectious cDNA clone of the Oshima strain of Tick-borne encephalitis virus (Far-Eastern subtype) was developed by a long high-fidelity RT-PCR and one-step cloning procedure. The infectious clone (O-IC) had four amino acid substitutions and produced smaller plaques when compared with the parent Oshima 5-10 strain. Using site-directed mutagenesis, the substitutions were reverted to restore the parent virus sequence (O-IC-pt). Although genetically identical, parent virus Oshima 5-10 and virus recovered from O-IC-pt demonstrated some biological differences that are possibly explained by the presence of quasispecies with differing virulence characteristics within the original virus population. These observations may have implications for vaccines based on modified infectious clones. It was also demonstrated that the amino acid substitution E-S40→P at position 40 in the envelope (E) glycoprotein was responsible for plaque size reduction, reduced infectious virus yields in cell culture and reduced mouse neurovirulence. Additionally, two amino acid substitutions in the non-structural (NS)5 protein (virus RNA-dependent RNA polymerase) NS5-V378→A and NS5-R674→K also contributed to attenuation of virulence in mice, but did not demonstrate a noticeable biological effect in baby hamster kidney cell culture. Comparative neurovirulence tests revealed how the accumulation of individual mutations (E-S40→P, NS5-V378→A and NS5-R674→K) can result in the attenuation of a virus.


2006 ◽  
Vol 80 (21) ◽  
pp. 10900-10906 ◽  
Author(s):  
Fernando Almazán ◽  
Marta L. DeDiego ◽  
Carmen Galán ◽  
David Escors ◽  
Enrique Álvarez ◽  
...  

ABSTRACT The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli. Using the SARS-CoV replicon, we have shown that the recently described RNA-processing enzymes exoribonuclease, endoribonuclease, and 2′-O-ribose methyltransferase were essential for efficient coronavirus RNA synthesis. The SARS reverse genetic system developed as a BAC constitutes a useful tool for the study of fundamental viral processes and also for developing genetically defined vaccines.


1998 ◽  
pp. 249-256 ◽  
Author(s):  
S. Spiegel ◽  
A. Rosner ◽  
Y. Tam ◽  
S. Zilkah ◽  
E. Faingersh ◽  
...  

2000 ◽  
Vol 31 (1) ◽  
pp. 59-60
Author(s):  
P. J.G.M. Steverink ◽  
J. M.A. Pol ◽  
J.N.A. Bos-de Ruijter ◽  
J. J.M. Meulenberg

Author(s):  
Hongqing Zhu ◽  
Yin Liu ◽  
Yingyun Cai ◽  
Dongdong Yu ◽  
Yinghui Pu ◽  
...  

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