scholarly journals Generation of an Infectious Clone of VR-2332, a Highly Virulent North American-Type Isolate of Porcine Reproductive and Respiratory Syndrome Virus

2003 ◽  
Vol 77 (6) ◽  
pp. 3702-3711 ◽  
Author(s):  
H. S. Nielsen ◽  
G. Liu ◽  
J. Nielsen ◽  
M. B. Oleksiewicz ◽  
A. Bøtner ◽  
...  
Keyword(s):  
Growth Kinetics ◽  
Cdna Clone ◽  
North American ◽  
Molecular Mechanisms ◽  
Full Length ◽  
Respiratory Syndrome Virus ◽  
Full Length Cdna ◽  
Kinetics Of ◽  
Highly Virulent

ABSTRACT A full-length cDNA clone of the prototypical North American porcine reproductive and respiratory syndrome virus (PRRSV) isolate VR-2332 was assembled in the plasmid vector pOK12. To rescue infectious virus, capped RNA was transcribed in vitro from the pOK12 clone and transfected into BHK-21C cells. The supernatant from transfected monolayers were serially passaged on Marc-145 cells and porcine pulmonary alveolar macrophages. Infectious PRRSV was recovered on Marc-145 cells as well as porcine pulmonary macrophages; thus, the cloned virus exhibited the same cell tropism as the parental VR-2332 strain. However, the cloned virus was clearly distinguishable from the parental VR-2332 strain by an engineered marker, a BstZ17I restriction site. The full-length cDNA clone had 11 nucleotide changes, 2 of which affected coding, compared to the parental VR-2332 strain. Additionally, the transcribed RNA had an extra G at the 5′ end. To examine whether these changes influenced viral replication, we examined the growth kinetics of the cloned virus in vitro. In Marc-145 cells, the growth kinetics of the cloned virus reflected those of the parental isolate, even though the titers of the cloned virus were consistently slightly lower. In experimentally infected 5.5-week-old pigs, the cloned virus produced blue discoloration of the ears, a classical clinical symptom of PRRSV. Also, the seroconversion kinetics of pigs infected with the cloned virus and VR-2332 were very similar. Hence, virus derived from the full-length cDNA clone appeared to recapitulate the biological properties of the highly virulent parental VR-2332 strain. This is the first report of an infectious cDNA clone based on American-type PRRSV. The availability of this cDNA clone will allow examination of the molecular mechanisms behind PRRSV virulence and attenuation, which might in turn allow the production of second-generation, genetically engineered PRRSV vaccines.

scholarly journals Infectious Transcripts from Cloned Genome-Length cDNA of Porcine Reproductive and Respiratory Syndrome Virus

1998 ◽  
Vol 72 (1) ◽  
pp. 380-387 ◽  
Author(s):  
J. J. M. Meulenberg ◽  
J. N. A. Bos-de Ruijter ◽  
R. van de Graaf ◽  
G. Wensvoort ◽  
R. J. M. Moormann
Keyword(s):  
Cdna Clone ◽  
De Novo ◽  
Infectious Clone ◽  
Full Length ◽  
Respiratory Syndrome Virus ◽  
Cdna Clones ◽  
Genome Length ◽  
Full Length Cdna ◽  
Lung Macrophages ◽  
Growth Properties

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.

Rescue of an infection clone of barley yellow dwarf virus -GAV

2021 ◽  
Author(s):  
Xingan Hao ◽  
Shuang Song ◽  
Qinrong Zhong ◽  
Jamal-U-Ddin Hajano ◽  
Jie Guo ◽  
...  
Keyword(s):  
Cdna Clone ◽  
Molecular Mechanisms ◽  
Barley Yellow Dwarf Virus ◽  
Full Length ◽  
Yellow Dwarf ◽  
Biologically Active ◽  
Dwarf Virus ◽  
Full Length Cdna ◽  
Barley Yellow Dwarf ◽  
Yellow Dwarf Virus

Barley yellow dwarf virus-GAV (BYDV-GAV) is one of the most prevalent viruses causing yellow dwarf disease in wheat in China. The biology and pathology of BYDV-GAV are well studied; however, gene functions and molecular mechanisms of BYDV-GAV disease development are unclear due to the lack of a reverse genetics system. In this study, a full-length cDNA clone of BYDV-GAV was constructed and expressed using Agrobacterium-mediated inoculation of Nicotiana benthamiana. Virions produced by BYDV-GAV in N. benthamiana were transmitted to wheat by an aphid vector after acquisition via a sandwich feeding method. Infectivity of the cDNA clone in wheat was verified through RT-PCR and Western blot assays, and the recombinant virus elicited typical reddening symptoms in oats and was transmitted between wheat plants. These results confirm the production of biologically active transmissible virions. Using the BYDV-GAV infectious clone, we demonstrate that the viral protein P4 was involved in cell-to-cell movement and stunting symptoms in wheat. This is the first report describing the development of an infectious full-length cDNA clone of BYDV-GAV and provides a useful tool for virus-host-vector interaction studies.

scholarly journals Infectious genomic RNA of Rhopalosiphum padi virus transcribed in vitro from a full-length cDNA clone

Virology ◽  
2008 ◽  
Vol 375 (2) ◽  
pp. 401-411 ◽  
Author(s):  
Sandhya Boyapalle ◽  
Randy J. Beckett ◽  
Narinder Pal ◽  
W. Allen Miller ◽  
Bryony C. Bonning
Keyword(s):  
Cdna Clone ◽  
Rhopalosiphum Padi ◽  
Full Length ◽  
Genomic Rna ◽  
Full Length Cdna

scholarly journals A Point Mutation within the Replicase Gene Differentially Affects Coronavirus Genome versus Minigenome Replication

2005 ◽  
Vol 79 (24) ◽  
pp. 15016-15026 ◽  
Author(s):  
Carmen Galán ◽  
Luis Enjuanes ◽  
Fernando Almazán
Keyword(s):  
Amino Acid ◽  
Point Mutation ◽  
Cdna Clone ◽  
Reverse Genetics ◽  
Point Mutations ◽  
Full Length ◽  
Transmissible Gastroenteritis Virus ◽  
Replicase Gene ◽  
Full Length Cdna

ABSTRACT During the construction of the transmissible gastroenteritis virus (TGEV) full-length cDNA clone, a point mutation at position 637 that was present in the defective minigenome DI-C was maintained as a genetic marker. Sequence analysis of the recovered viruses showed a reversion at this position to the original virus sequence. The effect of point mutations at nucleotide 637 was analyzed by reverse genetics using a TGEV full-length cDNA clone and cDNAs from TGEV-derived minigenomes. The replacement of nucleotide 637 of TGEV genome by a T, as in the DI-C sequence, or an A severely affected virus recovery from the cDNA, yielding mutant viruses with low titers and small plaques compared to those of the wild type. In contrast, T or A at position 637 was required for minigenome rescue in trans by the helper virus. No relationship between these observations and RNA secondary-structure predictions was found, indicating that mutations at nucleotide 637 most likely had an effect at the protein level. Nucleotide 637 occupies the second codon position at amino acid 108 of the pp1a polyprotein. This position is predicted to map in the N-terminal polyprotein papain-like proteinase (PLP-1) cleavage site at the p9/p87 junction. Replacement of G-637 by A, which causes a drastic amino acid change (Gly to Asp) at position 108, affected PLP-1-mediated cleavage in vitro. A correlation was found between predicted cleaving and noncleaving mutations and efficient virus rescue from cDNA and minigenome amplification, respectively.

Recombinant expression and characterization of lemon (Citrus limon) peroxidase

2020 ◽  
Vol 27 ◽  
Author(s):  
Veda P. Pandey ◽  
Apoorvi Tyagi ◽  
Shagoofa Ali ◽  
Kusum Yadav ◽  
Anurag Yadav ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plant Defense ◽  
Cdna Clone ◽  
Protein Function ◽  
In Silico ◽  
Full Length ◽  
Function Annotation ◽  
Full Length Cdna ◽  
Protein Function Annotation

Background: Class III plant peroxidases play important role in a number of physiological processes in plant such as lignin biosynthesis, suberization, cell wall biosynthesis, reactive oxygen species metabolism and plant defense against pathogens. Peroxidases are also of significance in several industrial applications. In view of this, the production and identification of novel peroxidases having resistance towards temperature, pH, salts is desirable. Objective: The objective of the present work was to clone and characterize a novel plant peroxidase suitable for industrial application. Methods: A full length cDNA clone of lemon peroxidase was isolated using PCR and RACE approaches, characterized and heterologously expressed in Escherichia coli using standard protocols. The expressed peroxidase was purified using Ni-NTA agarose column and biochemically characterized using standard protocols. The peroxidase was also in-silico characterized at nucleotide as well as protein levels using standard protocols. Results: A full length cDNA clone of lemon peroxidase was isolated and expressed heterologously expressed in Escherichia coli. The expressed recombinant lemon peroxidase (LPRX) was activated by in-vitro refolding and purified. The purified LPRX exhibited pH and temperature optima of pH 7.0 and 50°C, respectively. The LPRX was found to be activated by metal ions (Na+ , Ca2+, Mg2+ and Mn2+) at lower concentration. The expressional analysis of the transcripts suggested involvement of lemon peroxidase in plant defense. The lemon peroxidase was in silico modelled and docked with the substrates guaiacol, and pyrogallol and results show the favourability of pyrogallol over guaiacol, which is in agreement with the in-vitro findings. The protein function annotation analyses suggested the involvement of lemon peroxidase in the phenylpropanoid biosynthesis pathway and plant defense mechanisms. Conclusion: Based on the biochemical characterization, the purified peroxidase was found to be resistant towards the salts and thus, might be a good candidate for industrial exploitation. The in-silico protein function annotation and transcript analyses highlighted the possible involvement of the lemon peroxidase in plant defense response.

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