Virus infectious clone

PVY is transmitted by more than 40 aphid family Aphididae species in a non-persistent manner. Transmission by worldwide distributed aphid Myzus persicae Sulz. Phylogenetic and epidemiologic analyses indicate that PVY originated in the Americas, but the virus is currently distributed worldwide Quenouille et al. This genomic RNA is encapsidated by approximately copies of a single coat protein CP in elongated and flexuous virions. An additional product P3N-PIPO results from a frameshift at the P3 cistron, recently shown to be generated through a polymerase slippage mechanism Olspert et al.

Rosea1 Ros1 is indicated by a red box. Arrowheads indicate the cleavage site. The nucleotides in red correspond to silent mutations to avoid homologous repetitions in the PVY-Ros1sequence.

We wondered whether the Rosea1 visual marker could be applied to such a relevant virus as PVY, and, particularly whether the marker confers any advantage to either the aphid transmission analysis or the effect of antiviral treatments, among others. We observed that this recombinant clone induced red spots in mechanically inoculated tissue, which is indicative of infection foci, and solid dark red coloration on upper non-inoculated leaves when systemic infection was established.

Using silver nanoparticles, which are an easy-to-produce novel nanomaterial with exciting antimicrobial activities, we proved that PVY-Ros1 allows the very precise quantification of the effect of antiviral treatments in plants. Interestingly, aphids that fed on red-colored tissue efficiently transmitted the disease during a process that can be easily tracked thanks induction of discrete visible infection foci, which presumably surround the aphid puncture site during inoculation. Finally, we observed that PVY-Ros1 can be used in molecular farming to very quickly induce the production of large amounts of anthocyanins, antioxidant compounds with nutritional, pharmaceutical and industrial applications, in biofactory crops.

Transformed A. Liquid cultures of the transformed A. Cells were recovered by centrifugation and were resuspended at an OD of 0. For the mechanical inoculation of plants, crude extracts of infected tissues, obtained in 20 volumes of 50 mM of potassium phosphate, pH 8. Five-week-old tobacco N.

Xanthi nc , 4-week-old tomato S. Marglobe and 6-week-old potato traditional cultivar from Soria, Spain plants were used for mechanical inoculation. For aphid transmission, standard plant-to-plant transmission experiments were performed, as previously described Atreya et al.

Apterae individuals from a colony of M. Aphids were allowed a min acquisition access period on the PVY-Ros1-infected leaves, and were then transferred to the test plants, either individually or in groups of 5 or After an overnight inoculation access period 14—18 h , aphids were killed by spraying with insecticide, and plants were placed in a growth chamber for symptoms to develop. The culture exudate was recovered by filtration with Whatman number 1 paper and was mixed with ml of 1 mM AgNO 3.

The mixture was then incubated in the dark at room temperature until color changed. The silver reduction reaction and the formation of silver nanoparticles were monitored for approximately 1 week by a spectrophotometric analysis. The resulting silver nanoparticles were purified, dried and characterized by spectroscopic and microscopic analyses, as previously described Abdel-Hafez et al. A fine dispersion of silver nanoparticles in water at parts per million ppm was obtained by three cycles of 5-min stirring and 5-min sonication.

From this dispersion, a dilution in water ppm was prepared. These silver nanoparticle preparations were sprayed on 5-week-old tobacco cv. Xanthi nc plants.

Two days later, the true leaves 5 and 6 of these plants were mechanically inoculated with PVY-Ros1 in the presence of carborundum, as described above. While frozen, these tissue samples were mechanically ground and mixed. Extracts were vigorously vortexed, incubated on ice for 1 h with sporadic vortexing, and finally clarified by centrifugation for 10 min at g. Supernatants were diluted to in extraction solution final ratio tissue:extraction solution and analyzed spectrophotometrically UVPC, VWR.

The flow rate was 0. UV-visible spectra were acquired within the wavelength range of to nm with 1. The MS analysis was performed by electrospray ionization in the negative mode. MS was calibrated using sodium formate, and leu-enkephalin was used as the lock mass with a LockSpray exact mass ionization source.

MassLynx version 4. To analyze whether the Rosea1 marker is operational to visually reveal PVY infected tissues, starting with a wild isolate of this virus, we constructed a full-length clone in which a cDNA coding for A. The sequences were Note that the GA silent mutation was introduced into the recombinant clone in order to eliminate an endogenous Eco 31I recognition site and to facilitate manipulation.

Three N. One of the three agroinoculated plants showed infection symptoms at 26 dpi in upper non-inoculated leaves. Interestingly, symptomatic tissues turned red Figure 2A. Considering the relatively low infection rate observed in this agroinoculation experiment, a similar set of three N. One of the three mechanically inoculated plants also displayed infection symptoms at 31 dpi, which also turned red.

We used infected tissue from these plants to prepare crude extracts to mechanically inoculate tobacco N. All the mechanically inoculated leaves started to show red infectious foci at 5 dpi, which were clearly visible at 6 dpi Figure 2B.

All the mechanically inoculated plants started to show infection symptoms on their upper non-inoculated leaves at 6 dpi, which were easily recognized thanks to the simultaneous red pigmentation. In all the inoculated plants, symptomatic tissue showed intense solid dark red coloration at 12 dpi Figure 2C. When these same infectious extracts were used to mechanically inoculate potato and tomato plants, all the plants also became infected and exhibited red pigmentation in infected tissue Figures 2D,E.

Plants infected with PVY-Ros1 that show red pigmentation. The picture was taken at 6 dpi. C Detail of an N. Pictures were taken at 6 and 19 dpi, respectively. The electrophoretic analysis of the amplification product revealed a specific band whose size matched the bp size expected for the PVY cistron CP in the samples that corresponded to the PVY-Ros1-infected plants Figure 3.

Amplification products from the RNA preparations of three plants mock-inoculated lanes 1 to 3 , and inoculated with PVY-Ros1 lanes 4 to 6 , were separated by electrophoresis in agarose gel and revealed by ethidium bromide staining. Lane 7 contains a DNA marker with sizes in bp on the right.

However, the results also suggested that pGPVY-Ros1 was rather an unstable plasmid because, when starting with the plasmid, the infection rate obtained by either agroinoculation or direct mechanical inoculation was low. In addition to low infectivity, infection from plasmid either by agroinoculation or mechanical inoculation also showed an extended delay compared to the mechanical inoculation using an infectious extract.

This suggests poor expression efficiency of viral full-length cDNA. As the expression in this construct was driven by the very strong Cauliflower mosaic virus CaMV 35S promoter and terminator, one possible explanation for this low infectivity could be presence of cryptic introns in the PVY sequence, which could result in undesired splicing in most transcripts.

The undesired presence of cryptic introns has been previously shown in Tobacco mosaic virus Marillonnet et al.

Most importantly, after obtaining some initial infected tissue, it could be used as a fast and efficient source of PVY-Ros1 inoculum for successive experiments.

Nonetheless, infection efficiency of agroinoculated PVY-Ros1 may be improved by the co-expression of tombusvirus p19 silencing suppressor Saxena et al. Recently, a PVY clone tagged with GFP has been proposed as a versatile tool for the functional analysis of plant-virus interaction Rupar et al. Based on direct visual detection, PVY-Ros1 must be able to complement this tool with new experimental possibilities.

The speed and easiness with which PVY-Ros1-derived red spots and patterns are monitored or recorded in real time without having to resort to complex instrumentation make this recombinant virus an excellent tool to analyze the effect of environmental growth conditions, the host genetic background or phytosanitary treatments in virus infection.

To acquire proof of this concept, we thought about treating tobacco plants with silver nanoparticles, a novel kind of nanomaterial for which exciting antimicrobial activities have been reported Mishra and Singh, , and then inoculating the treated tissues with PVY-Ros1.

Sets of six tobacco plants 6 weeks old were sprayed with a preparation of C. In this experiment, we included a set of mock-sprayed plants. Two days later, two leaves per plant were mechanically inoculated with aliquots of the same PVY-Ros1 inoculum. Red infection foci were clearly visible at 6 dpi Figure 4A.

The inoculated leaves were harvested and foci were counted Supplementary Table S2. Whereas an average of individual infection foci per leaf was observed on the mock inoculated leaves, only an average of 87 and 22 foci was observed on the leaves treated with and ppm of silver nanoparticles, respectively Figure 4B.

Antiviral activity of silver nanoparticles has been repeatedly shown in human viruses Lu et al. It is interesting to note that while in this report authors did not detect any beneficial effect of silver nanoparticles in pre-infection treatment and only remarkable positive results were observed in post-infection treatment Elbeshehy et al.

Effect of silver nanoparticles on PVY-Ros1 infectivity. A Pictures of representative leaves of tobacco plants mock-treated or sprayed with a preparation of silver nanoparticles in water at and ppm, as indicated.

Two days after treatment, leaves were mechanically inoculated with PVY-Ros1. Pictures were taken at 6 dpi with PVY-Ros1.

B Histogram of the average number of infection foci per leaf of the six plants treated with silver nanoparticles, as indicated, and mechanically inoculated with PVY-Ros1 2 days later. Foci were quantified at 6 dpi with PVY-Ros1. Error bars represent the standard error median. Most plant viruses are transmitted by vectors. In the particular case of potyviruses, transmission by aphids is a distinctive trait of the genus. We wondered whether the Rosea1 marker would allow the visual monitoring of the aphid transmission process.

To this end, and after a standard starvation period, aphid populations were allowed to feed on red-colored PVY-Ros1-infected tobacco tissue for 10 min to acquire the virus. Next they were individually transferred at different densities to healthy tobacco test plantlets and confined there for overnight inoculation.

Unfortunately, at present there is no specific treatment or vaccine available for ZIKV infection and its pathogenic mechanisms are unclear Ferraris et al. It is thus important that we continue to research and improve our understanding of the ZIKV genome. In our study, one of the earliest ZIKV strains, which was imported into China in , was used to construct an infectious clone. It is one of the standard strains used to perform sequence analyses of the ZIKV. To date, three methods to construct infectious clones have been utilized and were described in the Introduction.

Each method has its advantages and disadvantages. For strategy i , the viral RNA is directly transfected into cells, and usually has a high recovery rate due to the production efficiency of the virus Aubry et al.

However, it requires additional in vitro RNA transcription steps, and genome instability of the cDNA clones in bacteria has been reported Widman et al.

To address these problems, we used an RNase cleaner to inactivate the RNase on the surface of the pipettor and the other furniture prior to conducting the RNA experiments. Furthermore, RNase-free consumables were also used. After T7 transcription, the RNase inhibitor was used in the transcript. For strategy ii , the plasmid containing the viral genome is directly transfected into the cells so that in vitro transcription is not required Liu et al.

However, the problem of genome instability remains. In strategy iii plasmids are rapidly and easily constructed but nonhomogeneous populations can be produced due to errors in the recombination events Tsetsarkin et al. In our study, we used strategy i and inserted three adenines in between the T7 promoter and the 5' end of the ZIKV to achieve high efficiency levels during in vitro transcription.

We also tried to use different methods during the construction of the infectious clones to achieve a faster virus recovery. The basic methodology of this investigation was based on classical methods, which were then combined with more modern methods.

Ultimately, we used three methods to improve the efficiency of infectious clone construction. During the plasmid construction, we used homologous recombination clones instead of a classical restriction enzyme ligation method, as they did not require restriction site cutting and could increase the chance of successful plasmid construction.

We selected 10 clones after the transformation of the XL10 competent cells and used the 4 PCR fragments to carry out primary selection prior to sequencing. The results showed that 2 of the 10 clones were positive for all 4 fragments, which contained the complete ZIKV genome. Based on the results of our literature search, the use of a PCR product as a T7 transcription template has not been reported earlier since the length of the amplified product may hinder its accurate amplification.

We examined the accuracy of our method over five rounds and the results showed that no additional mutations were generated. In contrast to the methodology of restriction enzyme cutting and purifying, the use of purified PCR products could obtain high concentrations of the transcription template in a shorter time without using a large centrifuge to conduct large scale plasmid extraction. Electro-transfection is the most classical method used to conduct large scale RNA transfection.

The use of the chemical transfection method for transfecting viral genomic RNA during the acquisition of the recovery virus has not been reported in the literature. In our study, however, we used a chemical transfection method for long RNA transfection, which was cheaper according to our accounts, but were still able to recover the virus.

Based on the results of this study, we recommend the following procedure for developing infectious clones: i Homologous recombination should be performed during plasmid construction ii Purified PCR products synthesized using a high fidelity polymerase should be employed as a transcription template to obtain viral genomic RNA iii Chemical transfection for long-length RNA should be performed for transfecting RNA into the susceptible cells.

Significantly, time-consuming traditional restriction enzyme digestion, further processing using ligation, high-speed centrifugation for large-scale plasmid extraction, and relatively expensive electroporation methods can be avoided using the methodology described in this study.

When compared with strategy iii , our method showed a relatively higher mutation rate, which may lead to failures when trying to recover the virus. The topological model of the NS2A protein constructed using bioinformatics indicates that the NS2A protein contains a single trans-membrane domain across the endoplasmic reticulum membrane and that six membrane-related regions and the sites that affect RNA replication or virus assembly may be concentrated in the outer membrane region Zhang et al.

Thus we first analyzed mutation 3,, and found that it may lead to an shorter trans-membrane domain, which makes the structure less stable and may lead to failures while trying to obtain rZKC2. After back mutations of the 3, mutation, rZKC2 was successfully obtained.

It could also possibly be a target site for the development of antiviral medications. Because of the greater ISGs signal, lower viral replication efficiencies were observed.

Based on the analysis for protein structure, the mutation AV in NS2A resulted in a different tertiary structure compared to the parental virus, which may impair the function of NS2A. Therefore, we hypothesize that the AV mutation impairs the function of NS2A, thereby inducing a greater innate immune response and inhibiting rZKC2 replication. The NS2A D62G mutation has huge impacts for obtaining recovery virus and may become a target for antiviral medications in the future.

Improved methods for the process of infectious clone construction were developed. The results show that the use of homologous recombination clones during plasmid construction, PCR products as templates for T7 transcription, and lipotransfection for large RNA transfections can be utilized for cheaper and faster infectious clone construction, while using the RNA transcripts from a cDNA clone strategy.

All authors contributed to the study design and data analysis. ZQ and WZ contributed to the manuscript preparation.

XL contributed to the additional study during interactive review. Also, we would like to thank Editage www. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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National Center for Biotechnology Information , U. Front Microbiol. Published online Oct Author information Article notes Copyright and License information Disclaimer. Wei Zhao, nc. This article was submitted to Virology, a section of the journal Frontiers in Microbiology. Received Aug 3; Accepted Sep The use, distribution or reproduction in other forums is permitted, provided the original author s and the copyright owner s are credited and that the original publication in this journal is cited, in accordance with accepted academic practice.

No use, distribution or reproduction is permitted which does not comply with these terms. Keywords: Zika virus, homologous recombination, infectious clone, recovery virus, mutation. Introduction Zika virus ZIKV is an important new mosquito-borne virus that is a member of the Flavivirus genus within the Flaviviridae family. Primer Design and Virus Genome Amplification An online software supported by Takara 1 was used to design the primers for homologous recombination Table 1.

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HL and QL conceived and designed the study and the experiments. QL contributed to data analysis and manuscript preparation. HL and ZM provided critique to the manuscript. All authors have read and approved the manuscript. Correspondence to Zhijing Mo or Hongbo Liu. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Reprints and Permissions.