Reverse genetics approach for arteriviruses using circular polymerase extension reaction (CPER)
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The circular polymerase extension reaction (CPER) has emerged as a high-fidelity, versatile, and rapid tool for generating infectious complementary DNA (cDNA) clones of positive-sense, single-stranded RNA viruses. Here, we implemented this strategy to generate infectious cDNA clones of two closely related members of the family Arteriviridae: equine arteritis virus KY84 strain (EAV KY84) and porcine reproductive and respiratory syndrome virus VR2332 strain (PRRSV VR2332). Overlapping cDNA fragments spanning the entire viral genomes were generated and assembled with a linker sequence incorporating critical expression elements, yielding a circularized, full-length viral cDNA genome ready for direct transfection into permissive cells. Following transfection, infectious rEAV KY84 and rPRRSV VR2332 strains were recovered and characterized. Similarly, we generated reporter viruses by inserting the mCherry gene downstream of the nsp1/nsp2 cleavage junction in EAV KY84 ORF1a and the GFP gene downstream of ORF1b of PRRSV VR2332. All CPER-generated viruses showed high identity to their parental strains by next-generation sequencing. Growth kinetics of CPER-generated non-reporter and rEAV KY84-RFP mCherry viruses were comparable to their parental strains; however, the reporter rPRRSV VR2332-GFP showed a significant reduction in viral titers despite its stability for at least 15 passages. In contrast, mCherry expression from rEAV KY84-mCherry was negligible at passage 15 and associated with the deletion of the reporter gene. Taken together, the CPER-based method can be used as a rapid tool to generate infectious cDNA clones of arteriviruses, allowing the easy generation of mutants or reporter viruses that can facilitate the investigation of viral gene functions and their interactions with the host.
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Thank for submitting your revised manuscript to Access Microbiology addressing the reviewers' suggestions. I am pleased to let you know that your manuscript is now accepted for publication. Congratulations!
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Thank you for submitting your manuscript to Access Microbiology. It has now been reviewed by two experts in the field whose comments are attached bellow. They have provided a number of suggestions to improve the manuscript, including further justification of methodologies and discussion. Please address then carefully in a revised manuscript. Please provide your revised manuscript (including a tracked-changes document) along with a point-by-point response to the reviewer comments within one month.
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Comments to Author
This manuscript describes the implementation of a circular polymerase extension reaction (CPER)-based reverse genetics system to generate infectious clones of two arteriviruses, equine arteritis virus (EAV KY84) and porcine reproductive and respiratory syndrome virus (PRRSV VR2332), including the construction of reporter viruses expressing mCherry and GFP. The study demonstrates successful virus rescue from overlapping cDNA fragments assembled via CPER and provides characterization of rescued viruses through immunofluorescence, growth kinetics, and next-generation sequencing. The work is technically sound and demonstrates that CPER can be adapted for members of the family Arteriviridae, which is valuable for reverse genetics and reporter virus generation. However, several aspects of the manuscript require …
Comments to Author
This manuscript describes the implementation of a circular polymerase extension reaction (CPER)-based reverse genetics system to generate infectious clones of two arteriviruses, equine arteritis virus (EAV KY84) and porcine reproductive and respiratory syndrome virus (PRRSV VR2332), including the construction of reporter viruses expressing mCherry and GFP. The study demonstrates successful virus rescue from overlapping cDNA fragments assembled via CPER and provides characterization of rescued viruses through immunofluorescence, growth kinetics, and next-generation sequencing. The work is technically sound and demonstrates that CPER can be adapted for members of the family Arteriviridae, which is valuable for reverse genetics and reporter virus generation. However, several aspects of the manuscript require clarification and improvement, particularly regarding experimental design justification and interpretation of reporter stability. I will list the issues below. Major Comments: 1. Novelty relative to previous CPER applications should be clarified CPER has already been applied to several positive-sense RNA viruses and more recently to some larger viral genomes, including SARS-CoV-2 and mononegaviruses. While the present work successfully adapts the method to arteriviruses, the manuscript would benefit from a clearer statement of what specifically distinguishes this implementation from previously published CPER systems. For example, it would be useful to emphasize whether particular challenges related to arterivirus genome organization, size, or instability were encountered and how the CPER strategy addressed them. Clarifying the methodological novelty and the advantages of CPER over existing reverse genetics. In addition, the authors may consider expanding the discussion of previous CPER-based reverse genetics systems. CPER has been widely applied to several RNA viruses, including flaviviruses and coronaviruses, and a brief overview of these studies would help contextualize the current work. Including additional representative references would improve the completeness of the background and highlight how the present implementation for arteriviruses relates to previously reported CPER systems. 2. In previously reported CPER-based reverse genetics systems for other positive-sense RNA viruses, such as coronaviruses or flaviviruses, viral genomes are typically assembled from a larger number of overlapping fragments (e.g., 7-9 fragments). In the present study, the authors assembled the arterivirus genomes using only five fragments. It would be helpful if the authors could clarify the rationale behind this fragment design. In CPER systems, fragment number and fragment length can influence amplification efficiency, assembly fidelity, and overall rescue efficiency. A brief explanation of how the number of fragments was determined (e.g., based on genome size, PCR efficiency, or empirical optimization) would improve reproducibility and provide useful guidance for readers interested in applying CPER to other arteriviruses or related RNA viruses. 3. The stability of reporter constructs differs substantially between the two viruses tested. The rEAV-KY84-mCherry virus lost reporter expression after approximately 15 passages, whereas rPRRSV-VR2332-GFP remained stable over the same period. The manuscript reports these observations but does not sufficiently explore possible explanations. The Discussion would benefit from addressing potential reasons for this difference, such as genome architecture, insertion site constraints, selective pressure against reporter expression, or differences in replication fitness between the two viruses. Relating to this aspect, in Figure 8D, the merged DIC and fluorescence images make it somewhat difficult to clearly evaluate the fluorescent protein (FP) signal and the extent of infection. Presenting the DIC and fluorescence channels as separate panels, in addition to the merged image, may improve the clarity and interpretability of the data. In addition, if feasible, a quantitative assessment of FP-positive cells using flow cytometry (e.g., FACS) could provide a more objective measure of reporter stability across passages. Such an analysis would complement the microscopy-based observations and help to better characterize changes in the proportion of FP-expressing cells over time. 4. The authors conclude that CPER represents a "high-fidelity" reverse genetics approach based on the high sequence identity of rescued viruses relative to parental strains. While the sequencing results indeed show high identity, the presence of several nonsynonymous substitutions in some recombinant viruses suggests that PCR-derived mutations can still occur. The manuscript should therefore moderate the language slightly and clarify that CPER reduces—but does not completely eliminate—the possibility of polymerase-induced mutations. 5. Both viruses were initially transfected into BHK-21 cells, after which PRRSV rescue required transfer of supernatant into MARC-145 cells before clear cytopathic effect was observed. The rationale for using BHK-21 cells as the primary transfection host for PRRSV should be explained more explicitly, particularly since MARC-145 cells are known to support PRRSV replication more efficiently. Providing a short justification would improve clarity for readers who may attempt to reproduce the protocol. Minor comments: Line 252: 5% CO2 for 96 hours Lines 276: 1 hour incubation at RT Line 334: every 24 hours Lines 387-388: Titers were calculated using the Reed and Muench method. Line 541: PRRSV VR2332 Line 549: insertions, such as GFP, into the SARS-CoV-2 genome
Please confirm that no generative AI tools or large language models have been used to generate this peer review report or to assist with any part of the peer review process.
I confirm no generative AI tools were used in preparation of this review.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
Clearly written paper. This is a good description of a novel approach for constructing infectious clones. A thorough analysis of the viral products
Please confirm that no generative AI tools or large language models have been used to generate this peer review report or to assist with any part of the peer review process.
I confirm no generative AI tools were used in preparation of this review.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If …
Comments to Author
Clearly written paper. This is a good description of a novel approach for constructing infectious clones. A thorough analysis of the viral products
Please confirm that no generative AI tools or large language models have been used to generate this peer review report or to assist with any part of the peer review process.
I confirm no generative AI tools were used in preparation of this review.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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