Non-permissive CHO cells; A rapid approach for purification of recombinant Herpes Simplex Virus-1

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It has been suggested as a new platform for cancer therapy. Gene-modification techniques such as conventional homologous recombination or CRISPR/Cas9 system are utilized to introduce site-specific mutations in targeted viral genes. Although, the CRISPR-Cas9 system could significantly increase the efficiency of homologous recombination; nevertheless the process of purifying recombinant variants can be tedious. Here we present a rapid, innovative method using non-permissive hamster ovary (CHO) cells which is a remarkable improvement on the previously mentioned tedious process. Using this strategy, only 1–2 rounds of plaque purification would suffice. Our proposed protocol demonstrated high potential as a worthy alternative way for the current approaches of the isolation and purification of the fluorescent reporter genes-expressing recombinant HSV-1 by plaque assay using CHO cells. Fluorescent reporter CHO cells Oncolytic HSV-1 Purification plaque BHK-21 Non-permissive Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Human alphaherpesvirus 1 (HSV-1) is a ubiquitous eukaryotic pathogen, from the Herpesviridae family that has a relatively large 152-kb linear double-stranded genome which codes about 80 proteins, half of which are not essential for virus replication (Conner, Rixon et al. 2005 , Roehm, Shekarabi et al. 2016 , Oh, Neuhausser et al. 2019 ). Recombinant HSV-1 has emerged as a new platform in the development of tumor-targeted replicating oncolytic. Considering that the viral genome is not incorporated into the human genome, it has abilities such as foreign gene cloning, efficient replication, broad cell host range, and safety (Lin, Li et al. 2016 ). These viruses are especially helpful in combating cancers related to the nervous system due to the original virus’s natural infection site. Currently, different versions of oncolytic HSV-1 have shown promising results in clinical trials (Fu and Zhang 2001 , Varghese and Rabkin 2002 , Gianni, Campadelli-Fiume et al. 2004 , Kagabu, Yoshino et al. 2021 ). However, shuttle vector homologous recombination is one of the standard methods of HSV-1 genome engineering. The shuttle vector contains the sequence homolog which recombines with HSV-1 in pre-infected cells (Russell, Stefanovic et al. 2015 , Lin, Li et al. 2016 , Lee 2019 ). Homologous integration efficiency and editing precision of viral genes can be drastically enhanced with the use of the recently developed CRISPR-Cas9 system. The system was originally discovered as one of the anti-viral defense mechanisms in bacteria. The use of this powerful and reliable system has resulted in the development of antiviral agents, oncolytic viruses, and vaccines (Suenaga, Kohyama et al. 2014 , Yuan, Wang et al. 2016 ). Currently, further in-progress studies demonstrate that even with the massive improvement in genome editing and gene replacement by CRISPR/Cas9, challenges remain in the isolation and purification of recombinant HSV-1 (Walker 1977 , Russell, Stefanovic et al. 2015 , Yuan, Wang et al. 2016 , Li, Bi et al. 2018 ). The use of fluorescent reporter genes-expressing shuttle vectors such as GFP (Liu, Robinson et al. 2003 )(green fluorescent protein), JRed (Ramachandran, Knickelbein et al. 2008 ) and mCherry (Ramachandran, Knickelbein et al. 2008 ) (red fluorescent proteins) and YFP (Miyagawa, Marino et al. 2015 ) (yellow fluorescent protein) has become a common strategy for screening successfully the recombinant HSV-1 which expresses these fluorescent proteins. These viruses could be easily detected by fluorescent microscopy and separated from other viruses using plaque purification method and are used as a basic platform for construction of higher oHSV generations, vaccine design and cancer therapy (Abdoli, Roohvand et al. 2017 , Scanlan, Coffman et al. 2022 ). However, plaque purification is an issue when the gene entry in the insertion site of the desired gene results in a reduction of progeny virus replication and a low titer of cell-released virus compared to the parental virus. At least 15 − 10 tissue culture dishes (10 cm) are usually required, which results in a tedious first-round screening. So, the plaque purification process can be time-consuming and labor-intensive. Pointing to the need for improvement in the screening procedure (Walker 1977 , Russell, Stefanovic et al. 2015 , Yuan, Wang et al. 2016 ), we offer an practical screening option for overcoming these obstacles and facilitating the process using non-permissive CHO cells, which will significantly improve the isolation and purification efficiency of desired recombinant viruses. Using this method, 1–2 rounds of plaque purification might be sufficient. In this paper, the US12 gene of the ICP34.5 deleted HSV-1, as an example for describing this method, was manipulated with the insertion of the EGFP expression cassette using homologous recombination technique. ICP34.5 protein, is important for viral replication, viral, exit from infected cells, prevention of the premature shut-off of protein synthesis in the infected host, and neurovirulence. ICP47 usually functions to block antigen presentation in HSV-infected cells so its disruption leads to a virus that does not confer on infected tumour cells properties that might protect them from the host's immune system when infected with HSV. This product can be used in oncolytic virotherapy research and further recombinant HSV construction (Liu, Robinson et al. 2003 ). Materials and methods Cell Lines In this study, Vero (African green monkey kidney, NCBI-C101 ) , BHK-21 (Baby hamster kidney, NCBI-C107 ) , and CHO-K1 (Chinese hamster ovary; NCBI-C644), cell lines were obtained from the National Cell Bank of Pasteur Institute of Iran and were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco), 2 mM L-glutamine and 1% penicillin/streptomycin (Gibco, Germany) at 37° C with a humidified atmosphere containing 5% CO2. All cell lines were free from the contaminations. Viruses The principal virus used in this study was Δγ34.5/HSV-1 (Red). Briefly, an ICP34.5-null mutant that both copies of ICP34.5 carrying a BleCherry reporter gene driven by the cytomegalovirus promoter was used as a reporter virus for the identification and confirmation of homologous recombination (Abdoli, Roohvand et al. 2017 , Haghighi-Najafabadi, Roohvand et al. 2021 ). Δγ34.5/HSV-1 had been constructed by a standard homologous recombination-based method from HSV-1 (a generous gift from Dr. Houriyeh Soleimanjahi, Tarbiat Modarres University, Tehran, Iran) and it was available at the Virology Department of Pasteur Institute of Iran (Abdoli, Roohvand et al. 2017 , Haghighi-Najafabadi, Roohvand et al. 2021 ). For propagation, the viruses were cultured in Vero cells containing DMEM media, supplemented with 10% FBS and 1% penicillin/streptomycin at 37° C with a humidified atmosphere containing 5% CO2. After 24–72 h and with the observation of cytopathic effects, cells were harvested and freeze-thawed three times. The viral supernatant was then filtered through 0.45 µm pores, titered using plaque assay, aliquoted, and stored at -80°C. Infection of BHK cells with Δγ34.5/HSV-1 At first, BHK cells were infected with Δγ34.5/HSV-1 at a multiplicity of infection (MOI) of 1. The cells were harvested after 48 h when cytopathic effects (CPE) were observed, then viral supernatant was filtered, aliquoted, titered, and stored at -80°C. DNA extraction DNA was purified from virus-infected BHK cells stock by High Pure Extraction Kit) Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. ICP47 shuttle vector construction for homologous recombination (HR) The ICP47 shuttle vector was constructed by Haghighi-Najafabadi et al. and was available at the Virology Department of the Pasteur Institute of Iran. Briefly, two upstream and downstream homologous fragments (encoding 1638 and 1502 nucleotides, respectively) of the genome flanking specific regions (within the US12 CDS of HSV-1 and the EGFP expression cassette under the control of the CMV promoter and BGH terminator) were amplified and subcloned into pSL1180 to generate pSL1180-ARM1&2-ICP47-EGFP (Haghighi-Najafabadi, Roohvand et al. 2021 ). Generation of Δ47/Δγ34.5 HSV-1 through homologous recombination Transient transfections were performed on BHK cells using ScreenFect™ A plus - Fujifilm WAKO according to the manufacturer protocol. The reagent: DNA ratio of 2:1 was used using 1µg of DNA per well in a 24-well plate as the optimal ratio for all the experiments. Briefly, the cells were seeded into 24-well plates (SPL Life Sciences, Korea) at a density of 0.05 x 10 6 cells/well to achieve 80% confluency for transfection. Cells were then transfected with shuttle vector pSL1180-ARM1&2-ICP47-EGFP plasmid, containing the homologous sequence) and placed in a 37°C incubator with 5% CO2 to be expressed for 24h. The next day, the same transfection procedure was repeated with the exception that after lipoplex formation, Δγ34.5/HSV-1 (MOI of 1) was added to the complex and then the complexes (shuttle vector + virus) were added to the same BHK monolayers that were cultured in 24-well plates in 0.35 mL complete medium. The infection/transfection mixture from BHK cells was harvested at 24–48 h, upon reaching 80% cytopathic effect of the cell monolayer. After three freeze-thaw cycles, the cells were stored in aliquots at − 80°C. Isolation and expansion of recombinant virus using non-permissive (CHO) cells (two Rounds) The aliquot vials expected to contain mutated viruses were serially diluted two-fold and mixed with CHO cells (10 4 cells/well), then seeded in a 96-well tissue culture. The plate was monitored daily using an inverted fluorescent microscope for EGFP expression at 1– 3 days post-infection. The wells containing EGFP-expressing cells were covered with trypsin to detach the cells. The cells were then transferred into a 100-mm cell culture dish preseeded with CHO cells (5x10 5 cells per 100-mm dish) to increase the distance between fluorescent positive cells. The single cells positive for both BleCherry and GFP signals (cells containing the recombinant green/red virus) were selected and detached using a cell scraper and were then propagated in BHK cells (or Vero) following three freeze-thaw cycles. Henceforth, the new virus which was designated as Δ47 (GFP) /Δγ34.5 (BleCherry) HSV-1. The mutant virus stock was aliquoted and after three freeze-thaw cycles, stored in aliquots at − 80°C. PCR analysis and sequencing for verification of homologous recombination To verify the recombination process, the mutant virus DNA was extracted with High Pure Extraction Kit )Roche Diagnostics GmbH, Mannheim, Germany) and PCR was performed using a PCR master mix kit (Taq DNA Polymerase Master Mix RED 2x, Ampliqon, Denmark) in a total volume of 25 containing 12.5 µl Taq DNA Polymerase, 1x Master Mix RED, ( ~ 100–150 ng) of HSV-1 DNA and 0.2 µM of each forward and reverse test primers given in Table 1 , then programmed as follows: 95°C for 5 min; 30 cycles of 95°C for 60 s, 55°C for 30 s, 72°C for 2 min; followed by a final extension step of 72°C for 5 min. The PCR products were loaded on a 1% agarose gel and were visualized by exposing them to ultraviolet (UV) light (1- kb DNA ladder, CinnaGen) (Haghighi-Najafabadi, Roohvand et al. 2021 ). Result Construction of Δ47/Δγ34.5 HSV-1 and verification of homologous recombination For the construction of the recombinant virus, the transfection were performed on BHK cells with the vector containing the pSL1180-ARM1&2-ICP47-EGFP fragment for homologous recombination. The next day, the same transfection procedure was repeated (Fig. 1 ). The infection/transfection mixture from BHK cells mixed with CHO cells and seeded in a 96-well tissue culture. The plate was monitored daily using an inverted fluorescent microscope for EGFP expression at 1– 3 days post-infection (Fig. 2 ). The wells containing EGFP-expressing cells were transferred into a 100-mm cell culture dish preseeded with CHO cells (Fig. 3 ). The single cells positive for both BleCherry and GFP signals were propagated in BHK cells (Fig. 3 ) and confirmed by PCR. As expected, the PCR product of the test primers in Δγ34.5/ HSV-1 parental virus validated the EGFP expression cassette in the eliminated region which increase the size of PCR products at ICP47 loci in comparison to the Δγ34.5/ HSV-1 parental virus (Haghighi-Najafabadi, Roohvand et al. 2021 ) Discussion In the present study, the HSV-1 was manipulated by replacing US12 (ICP47) with green fluorescence protein in the Δγ34.5/HSV-1 virus (lacking both copies of the Δγ34.5 gene which functions as a virulence factor during HSV1 infection (Haghighi-Najafabadi, Roohvand et al. 2021 ). The operation was done in order to generate a replication-conditional mutant of HSV-1. The current study is providing novel insights into the use of CHO cells for the first time. For instance, to construct and purify Δ47/Δγ34.5 HSV-1. It additionally proposes CHO cells as an appealing candidate to purge HSV1, utilizing an efficient method which circumvents these problems. We explain comprehensively how this process will advance the production and purification efficiency of mutant HSV-1 concerning the purpose of this study. CHO-K1 cells are typically regarded as nonpermissive cells for HSV-1 infection since they naturally lack gD receptors, hence resistant to HSV infections (Brown, Harland et al. 1994 , Roller and Herold 1997 , Conner, Rixon et al. 2005 ). As submitted in previous studies, the virus’s ability to infect CHO cells is influenced by the cell type used for virus propagation. The cause of this matter is currently unknown, nevertheless, based on the documents, the cell and the structural features of receptors play an undeniably major role in the process of virus infection. Incipiently, we investigated the virus infectability of CHO cells with Δγ34.5/HSV-1that expresses the BleCherry reporter gene, afterwards the US12 gene of Δγ34.5/HSV-1 was modified as the target region using the conventional homologous recombination method. The results of BleCherry expression and titration demonstrated that Δγ34.5/HSV-1 propagated on BHK (Δγ34.5/HSV-1/BHK) but not on Vero (Δγ34.5/HSV-1/Vero) cells. Therefore the subject was able to infect CHO cells and progeny virion was productive (data not shown). The results of our operation were in accordance with other discoveries which are based on studies using electron microscopy reporting nucleocapsids and enveloped virions that were visible in the nucleus and cytoplasm of HSV-1/BHK-infected CHO cells (Nicola and Straus 2004 , Conner, Rixon et al. 2005 ). On that account, the BHK-21 cell line was selected for the transfection/infection process in our study. Consequently, the parental/mutant virus mixture will be able to infect CHO cells(Conner, Rixon et al. 2005 ). The data suggest that transfection efficiency is notably increased by double transfection of the cells. Adding Δγ34.5/HSV-1 to the complex after lipoplex formation during the second transfection can remarkably enhance it. Of note, the mixtures should be tested at various doses of the virus to measure the transfection process. (Fig. 1 ) (Ishikawa and Homcy 1992 , Fu and Zhang 2001 , Burnham, Jaishankar et al. 2016 ). The aliquot vials that were expected to contain mutated viruses were serially diluted two-fold and mixed with CHO cells (10 4 cells/well ), then seeded in a 96-well tissue culture. Our results indicate that Δγ34.5/HSV-1 produced from BHK cells with a titer of 10 7 PFU/ml can infect approximately 30 to 40 percent of exponentially growing CHO cells, and simultaneous introduction of serum and virus after 24 h serum starvation of the CHO cells, infects up to 70% of cells (data not shown), suggesting that the vulnerability of CHO cells to HSV-1 infection depend on virus titers and cell cycle status. So, the supernatants contain the viruses with a titer of at least 3 PFU/cell. They are mixed with CHO cells during cell seeding for optimal infection of the cells. Similar findings have been reported previously by Conner et al (Conner, Rixon et al. 2005 ). This protocol offers several advantages for the following reasons: The amount of the infectious virions released into supernatants of Δγ34.5/HSV-1/BHK CHO infected cells is too low to be detected (Conner, Rixon et al. 2005 , Berting, Farcet et al. 2010 ), however, they are readily detectable when cells and supernatants are harvested together; suggesting the fact that progeny viruses are not released efficiently by the CHO cells in comparison to BHK or Vero cells. In addition, the released virus from CHO cells is unable to infect CHO or spread to adjacent cells, and the infection is limited to a single round of replication in the cells, whereas this ability is present in BHK or Vero cells (Fig. 5) (Roller and Herold 1997 , Conner, Rixon et al. 2005 , Berting, Farcet et al. 2010 ). Figure 5 Schematic illustration of the Characteristics of CHO cell; the virus acquired from CHO cells is unable to infect CHO or spread to adjacent cells and infection is limited to a single round of replication in CHO cells whereas this ability is obtained in Vero cells. These findings are noteworthy since site - specific genome editing may lead to reduced replication of progeny virus and lower titers of cell-released virus due to the critical roles of the gene in virus replication phenotype (Oyama, Ohigashi et al. 2000 , Fu and Zhang 2002 , Mullen, Kasuya et al. 2002 , Russell, Stefanovic et al. 2015 ), this presents an issue, as the amplification of Δγ34.5/HSV-1 (parental virus) during plaque growth and direct cell-to-cell spread (Yuan, Wang et al. 2016 , Li, Bi et al. 2018 , Lee 2019 ), amounts to a decreased distinct growth of the progressive plaques and usually about 15 − 10 tissue culture dishes (10 cm) are needed to isolate plaques that contain desired recombinant virus variants, which makes first-round screening tedious (Fig. 6). Figure 6 Challenges in screening procedure of recombinant HSV-1 in permissive cell lines (Exemplified in Figs. A, B, C and D); the plaque purification process can be problematic when the percentage of HSV-1 recombinants of interest be too low in compared with parental virus (10 − 2 –2%, depend on the conventional homologous recombination method or crispr-cas9 genome editing) 2) The CHO system can be more financially reasonable than the plaque purification technique, as no semisolid overlay substrates such as agarose or carboxymethyl cellulose are required to prevent indiscriminate infection which may occur through the liquid growth medium. As a result, challenges of single plaque pick-up through the agarose layer will be overcome (Fig. 7) (Fu and Zhang 2002 , Liang, Xu et al. 2015 , Ebrahimi, Makvandi et al. 2020 ). This protocol demonstrated the successful use of CHO cells as a worthy replacement for the basic protocol for the isolation and purification of recombinant HSV-1 by plaque assay (Fig. 7). Figure 7a Graphical protocol overview of two protocols for isolation and purification of recombinant HSV-1. b Characteristics and comparison of the two protocols. Declarations Acknowledgements The authors thank all the personnel of Virology Department and Laboratory of Regenerative Medicine and Biomedical Innovations, Pasteur Institute of Iran for their assistance in this project. Authors' contributions All authors discussed the results and implications and commented on the manuscript at all stages. Funding None Availability of data and material The data used to support the fndings of this study are included within the article and The nucleotide sequence data are available in the GenBank databases. Ethics approval and consent to participate Not applicable Consent for publication Not Applicable Competing interests No competing interests declared. References Abdoli, S., F. Roohvand, L. Teimoori-Toolabi, M. A. Shokrgozar, M. Bahrololoumi and K. Azadmanesh (2017). 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ACM Transactions on Mathematical Software (TOMS) 3(3): 253-256. https://doi.org/10.1145/355744.355749 Yuan, M., P. Wang, L. S. Chard, N. R. Lemoine and Y. Wang (2016). "A simple and efficient approach to construct mutant vaccinia virus vectors." JoVE (Journal of Visualized Experiments)(116): e54171. https://doi: 10.3791/54171. Cite Share Download PDF Status: Published Journal Publication published 09 May, 2024 Read the published version in AMB Express → Version 1 posted Reviewers agreed at journal 21 Oct, 2023 Reviewers invited by journal 02 Jul, 2023 Editor assigned by journal 31 May, 2023 First submitted to journal 30 May, 2023 Editorial decision: Major Revision 27 May, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2966832","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":215072354,"identity":"7c9def81-2681-4f62-b6ed-e905f6e07674","order_by":0,"name":"Mishar Kelishadi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYBACCQbmhgOMDQyMDewNDMxEamGEauE5QIIWCJJIIFKLZHtj48GfO+xkN9x8Y/i5oMKGgb+9OwGvFmmegw2Hec8kG2+4nWMsPeNMGoPEmbMb8GqRk0hsOMzYxpwI1GIgzdt2mMFAIpeAFvmHDQd/ttUnbrh5xvg3UVqkJYAhBlSZuOEGjxlxtkj2AB3G23bceOaZtDJrnjNpPAT9InH88OGPP9uqZfuOH958m6fCRo6/vRe/FjhQOMBhAKJ5iFMOAvIN7A+IVz0KRsEoGAUjCgAAl5FP00NwWHwAAAAASUVORK5CYII=","orcid":"","institution":"Pasteur Institute of Iran","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Mishar","middleName":"","lastName":"Kelishadi","suffix":""},{"id":215072355,"identity":"db9a6cf6-849b-414c-ad0e-aded3a2682f2","order_by":1,"name":"Hosein Shahsavarani","email":"","orcid":"","institution":"Shahid Beheshti University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hosein","middleName":"","lastName":"Shahsavarani","suffix":""},{"id":215072356,"identity":"e06aa037-ba45-463d-a0bb-6142d07cace3","order_by":2,"name":"Alijan Tabarraei","email":"","orcid":"","institution":"Golestan University of Medical Sciences and Health Services School of Health and Paramedicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Alijan","middleName":"","lastName":"Tabarraei","suffix":""},{"id":215072357,"identity":"06eaba27-fd02-4aa1-b27b-8afd3d813837","order_by":3,"name":"Mohammad Ali Shokrgozar","email":"","orcid":"","institution":"Pasteur Institute of Iran","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Ali","lastName":"Shokrgozar","suffix":""},{"id":215072358,"identity":"34423e2b-76ac-4c34-9e9e-9678ecc5a63c","order_by":4,"name":"Amirabbas Rahimi Rahimi","email":"","orcid":"","institution":"Pasteur Institute of Iran","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Amirabbas","middleName":"Rahimi","lastName":"Rahimi","suffix":""},{"id":215072359,"identity":"af9723c1-c0b3-4ca9-be7e-ac373a2510f1","order_by":5,"name":"Ladan Teimoori-Toolabi","email":"","orcid":"","institution":"Pasteur Institute of Iran","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ladan","middleName":"","lastName":"Teimoori-Toolabi","suffix":""},{"id":215072360,"identity":"8eecbaae-e3eb-4766-8620-1d51cb21af57","order_by":6,"name":"Kayhan Azadmanesh","email":"","orcid":"","institution":"Pasteur Institute of Iran","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kayhan","middleName":"","lastName":"Azadmanesh","suffix":""}],"badges":[],"createdAt":"2023-05-22 13:34:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2966832/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2966832/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13568-024-01709-0","type":"published","date":"2024-05-09T21:18:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":39696223,"identity":"15f13362-e6eb-4601-b550-41e4c62df537","added_by":"auto","created_at":"2023-07-07 13:38:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":644584,"visible":true,"origin":"","legend":"\u003cp\u003eExpansion of the same single cell in BHK cell line. Fluorescent analysis of green/ red Δ47/Δ134.5 HSV-1 by: \u003cstrong\u003ea\u003c/strong\u003e Green excitation filter for Blecherry detection \u003cstrong\u003eb\u003c/strong\u003e Blue excitation filter for Blecherry detection.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-2966832/v1/a5f08957a8958ce5aa6885bc.png"},{"id":39698120,"identity":"1bccca8c-554b-434c-ad86-c815bcfa3414","added_by":"auto","created_at":"2023-07-07 13:46:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":453716,"visible":true,"origin":"","legend":"\u003cp\u003eA blend of single cells containing the parent green Δ34.5 HSV-1 or recombinant Δ47 (green) /Δ34.5 (red) HSV-1;\u003cstrong\u003e \u003c/strong\u003eThe transfection/infection supernatant harvest was mixed with CHO cells after three freeze-thaw cycles and seeded in 96-well tissue culture plates and monitored daily for EGFP expression. Fluorescent imaging was done 24 h post infection; \u003cstrong\u003ea \u003c/strong\u003eGreen excitation filter for BleCherry detection vision\u003cstrong\u003e b \u003c/strong\u003eLight + Green excitation filter for BleCherry detection vision\u003cstrong\u003e c \u003c/strong\u003eBlue excitation filter for EGFP detection vision \u003cstrong\u003ed\u003c/strong\u003e Light + Blue excitation filter for EGFP detection vision (Magnification × 200).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-2966832/v1/a9b0154de75c681cd5f9886b.png"},{"id":39698121,"identity":"b2612f7a-e206-48ba-aaf3-5a91e1e40de3","added_by":"auto","created_at":"2023-07-07 13:46:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":387905,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescent microscopy analysis of the single cell containing Δ47 (green) /Δ134.5 (red) HSV-1 after transferring into 100-mm cell culture dish pre-seeded with CHO cells;\u003cstrong\u003e \u0026nbsp;a\u003c/strong\u003e UV vision ( Green excitation filter for Blecherry) \u003cstrong\u003eb\u003c/strong\u003e Light vision\u003cstrong\u003e + \u003c/strong\u003eUV vision\u003cstrong\u003e \u003c/strong\u003e(Green excitation filter for Blecherry)\u003cstrong\u003e c\u003c/strong\u003e UV vision\u003cstrong\u003e d\u003c/strong\u003eLight vision\u003cstrong\u003e +\u003c/strong\u003e UV vision (Green excitation filter for Blecherry) (magnification × 200).\u003cstrong\u003e \u003c/strong\u003eThe virus acquired from CHO cells is unable to infect CHO or spread to adjacent cells and infection is limited to a single cell of replication in CHO. So, EGFP-expressing cells are easily isolated.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-2966832/v1/8083b23f395f9b9546693bee.png"},{"id":39694926,"identity":"acdfc3fc-3cb8-437c-b225-32017f41097c","added_by":"auto","created_at":"2023-07-07 13:30:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":526836,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eLegend not included with this version\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-2966832/v1/6cf1f0f343b57f02bfa8d93f.png"},{"id":39694928,"identity":"05b0b822-f9d9-4756-bb82-52bf8b4cfaa8","added_by":"auto","created_at":"2023-07-07 13:30:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6369276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSchematic illustration\u003c/em\u003e of the Characteristicsof CHO cell;\u003cstrong\u003e \u003c/strong\u003ethe virus acquired from CHO cells is unable to infect CHO or spread to adjacent cells and infection is limited to a single round of replication in CHO cells whereas this ability is obtained in Vero cells.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-2966832/v1/dce2baad46c3064441975afe.png"},{"id":39694929,"identity":"63bcccd6-de6f-4cb6-b93e-39bd72eddb3e","added_by":"auto","created_at":"2023-07-07 13:30:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":261778,"visible":true,"origin":"","legend":"\u003cp\u003eChallenges in screening procedure of recombinant HSV-1 in permissive cell lines (Exemplified in Figs. A, B, C and D); the plaque purification process can be problematic when the percentage of HSV-1 recombinants of interest be too low in compared with parental virus (10\u003csup\u003e-2\u003c/sup\u003e–2%, depend on the conventional homologous recombination method or crispr-cas9 genome editing)\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-2966832/v1/6828ab3dac53c5525aa33d1f.png"},{"id":39694931,"identity":"9df0f8f5-f5c7-414e-9745-ceb3e9fd8d20","added_by":"auto","created_at":"2023-07-07 13:30:47","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4570058,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eGraphical protocol overview of two protocols for isolation and purification of recombinant HSV-1.\u003cstrong\u003e b\u003c/strong\u003e Characteristics and comparison of the two protocols.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-2966832/v1/23d3a58d9501aaa88ab50d04.png"},{"id":56488276,"identity":"e1832c60-71ad-4583-91c2-c44da61b7f15","added_by":"auto","created_at":"2024-05-14 21:31:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4718005,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2966832/v1/6616c802-66b9-4d11-9257-27652e466b5a.pdf"}],"financialInterests":"","formattedTitle":"Non-permissive CHO cells; A rapid approach for purification of recombinant Herpes Simplex Virus-1","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHuman alphaherpesvirus 1 (HSV-1) is a ubiquitous eukaryotic pathogen, from the Herpesviridae family that has a relatively large 152-kb linear double-stranded genome which codes about 80 proteins, half of which are not essential for virus replication (Conner, Rixon et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Roehm, Shekarabi et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Oh, Neuhausser et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Recombinant HSV-1 has emerged as a new platform in the development of tumor-targeted replicating oncolytic. Considering that the viral genome is not incorporated into the human genome, it has abilities such as foreign gene cloning, efficient replication, broad cell host range, and safety (Lin, Li et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These viruses are especially helpful in combating cancers related to the nervous system due to the original virus\u0026rsquo;s natural infection site. Currently, different versions of oncolytic HSV-1 have shown promising results in clinical trials (Fu and Zhang \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, Varghese and Rabkin \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Gianni, Campadelli-Fiume et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Kagabu, Yoshino et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, shuttle vector homologous recombination is one of the standard methods of HSV-1 genome engineering. The shuttle vector contains the sequence homolog which recombines with HSV-1 in pre-infected cells (Russell, Stefanovic et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Lin, Li et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Lee \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHomologous integration efficiency and editing precision of viral genes can be drastically enhanced with the use of the recently developed CRISPR-Cas9 system. The system was originally discovered as one of the anti-viral defense mechanisms in bacteria. The use of this powerful and reliable system has resulted in the development of antiviral agents, oncolytic viruses, and vaccines (Suenaga, Kohyama et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Yuan, Wang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCurrently, further in-progress studies demonstrate that even with the massive improvement in genome editing and gene replacement by CRISPR/Cas9, challenges remain in the isolation and purification of recombinant HSV-1 (Walker \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1977\u003c/span\u003e, Russell, Stefanovic et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Yuan, Wang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Li, Bi et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe use of fluorescent reporter genes-expressing shuttle vectors such as GFP (Liu, Robinson et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e)(green fluorescent protein), JRed (Ramachandran, Knickelbein et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) and mCherry (Ramachandran, Knickelbein et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) (red fluorescent proteins) and YFP (Miyagawa, Marino et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) (yellow fluorescent protein) has become a common strategy for screening successfully the recombinant HSV-1 which expresses these fluorescent proteins. These viruses could be easily detected by fluorescent microscopy and separated from other viruses using plaque purification method and are used as a basic platform for construction of higher oHSV generations, vaccine design and cancer therapy (Abdoli, Roohvand et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Scanlan, Coffman et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, plaque purification is an issue when the gene entry in the insertion site of the desired gene results in a reduction of progeny virus replication and a low titer of cell-released virus compared to the parental virus. At least 15\u0026thinsp;\u0026minus;\u0026thinsp;10 tissue culture dishes (10 cm) are usually required, which results in a tedious first-round screening. So, the plaque purification process can be time-consuming and labor-intensive. Pointing to the need for improvement in the screening procedure (Walker \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1977\u003c/span\u003e, Russell, Stefanovic et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Yuan, Wang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), we offer an practical screening option for overcoming these obstacles and facilitating the process using non-permissive CHO cells, which will significantly improve the isolation and purification efficiency of desired recombinant viruses. Using this method, 1\u0026ndash;2 rounds of plaque purification might be sufficient.\u003c/p\u003e \u003cp\u003eIn this paper, the US12 gene of the ICP34.5 deleted HSV-1, as an example for describing this method, was manipulated with the insertion of the EGFP expression cassette using homologous recombination technique. ICP34.5 protein, is important for viral replication, viral, exit from infected cells, prevention of the premature shut-off of protein synthesis in the infected host, and neurovirulence. ICP47 usually functions to block antigen presentation in HSV-infected cells so its disruption leads to a virus that does not confer on infected tumour cells properties that might protect them from the host's immune system when infected with HSV. This product can be used in oncolytic virotherapy research and further recombinant HSV construction (Liu, Robinson et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eCell Lines\u003c/p\u003e\n\u003cp\u003eIn this study, Vero (African green monkey kidney, NCBI-C101\u003cem\u003e)\u003c/em\u003e, BHK-21 (Baby hamster kidney, NCBI-C107\u003cem\u003e)\u003c/em\u003e, and CHO-K1 (Chinese hamster ovary; NCBI-C644), cell lines were obtained from the National Cell Bank of Pasteur Institute of Iran and were cultured in high glucose Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) (Gibco, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco), 2 mM L-glutamine and 1% penicillin/streptomycin (Gibco, Germany) at 37\u0026deg;\u003csup\u003eC\u003c/sup\u003e with a humidified atmosphere containing 5% CO2. All cell lines were free from the contaminations.\u003c/p\u003e\n\u003cp\u003eViruses\u003c/p\u003e\n\u003cp\u003eThe principal virus used in this study was \u0026Delta;\u0026gamma;34.5/HSV-1 (Red). Briefly, an ICP34.5-null mutant that both copies of ICP34.5 carrying a BleCherry reporter gene driven by the cytomegalovirus promoter was used as a reporter virus for the identification and confirmation of homologous recombination (Abdoli, Roohvand et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e, Haghighi-Najafabadi, Roohvand et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026Delta;\u0026gamma;34.5/HSV-1 had been constructed by a standard homologous recombination-based method from HSV-1 (a generous gift from Dr. Houriyeh Soleimanjahi, Tarbiat Modarres University, Tehran, Iran) and it was available at the Virology Department of Pasteur Institute of Iran (Abdoli, Roohvand et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e, Haghighi-Najafabadi, Roohvand et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eFor propagation, the viruses were cultured in Vero cells containing DMEM media, supplemented with 10% FBS and 1% penicillin/streptomycin at 37\u0026deg;\u003csup\u003eC\u003c/sup\u003e with a humidified atmosphere containing 5% CO2. After 24\u0026ndash;72 h and with the observation of cytopathic effects, cells were harvested and freeze-thawed three times. The viral supernatant was then filtered through 0.45 \u0026micro;m pores, titered using plaque assay, aliquoted, and stored at -80\u0026deg;C.\u003c/p\u003e\n\u003cp\u003eInfection of BHK cells with \u0026Delta;\u0026gamma;34.5/HSV-1\u003c/p\u003e\n\u003cp\u003eAt first, BHK cells were infected with \u0026Delta;\u0026gamma;34.5/HSV-1 at a multiplicity of infection (MOI) of 1. The cells were harvested after 48 h when cytopathic effects (CPE) were observed, then viral supernatant was filtered, aliquoted, titered, and stored at -80\u0026deg;C.\u003c/p\u003e\n\u003cp\u003eDNA extraction\u003c/p\u003e\n\u003cp\u003eDNA was purified from virus-infected BHK cells stock by High Pure Extraction Kit) Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003eICP47 shuttle vector construction for homologous recombination (HR)\u003c/p\u003e\n\u003cp\u003eThe ICP47 shuttle vector was constructed by Haghighi-Najafabadi et al. and was available at the Virology Department of the Pasteur Institute of Iran. Briefly, two upstream and downstream homologous fragments (encoding 1638 and 1502 nucleotides, respectively) of the genome flanking specific regions (within the US12 CDS of HSV-1 and the EGFP expression cassette under the control of the CMV promoter and BGH terminator) were amplified and subcloned into pSL1180 to generate pSL1180-ARM1\u0026amp;2-ICP47-EGFP (Haghighi-Najafabadi, Roohvand et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eGeneration of \u0026Delta;47/\u0026Delta;\u0026gamma;34.5 HSV-1 through homologous recombination\u003c/p\u003e\n\u003cp\u003eTransient transfections were performed on BHK cells using ScreenFect\u0026trade; A plus - Fujifilm WAKO according to the manufacturer protocol. The reagent: DNA ratio of 2:1 was used using 1\u0026micro;g of DNA per well in a 24-well plate as the optimal ratio for all the experiments.\u003c/p\u003e\n\u003cp\u003eBriefly, the cells were seeded into 24-well plates (SPL Life Sciences, Korea) at a density of 0.05 x 10\u003csup\u003e6\u003c/sup\u003e cells/well to achieve 80% confluency for transfection. Cells were then transfected with shuttle vector pSL1180-ARM1\u0026amp;2-ICP47-EGFP plasmid, containing the homologous sequence) and placed in a 37\u0026deg;C incubator with 5% CO2 to be expressed for 24h.\u003c/p\u003e\n\u003cp\u003eThe next day, the same transfection procedure was repeated with the exception that after lipoplex formation, \u0026Delta;\u0026gamma;34.5/HSV-1 (MOI of 1) was added to the complex and then the complexes (shuttle vector\u0026thinsp;+\u0026thinsp;virus) were added to the same BHK monolayers that were cultured in 24-well plates in 0.35 mL complete medium.\u003c/p\u003e\n\u003cp\u003eThe infection/transfection mixture from BHK cells was harvested at 24\u0026ndash;48 h, upon reaching 80% cytopathic effect of the cell monolayer. After three freeze-thaw cycles, the cells were stored in aliquots at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e\n\u003cp\u003eIsolation and expansion of recombinant virus using non-permissive (CHO) cells (two Rounds)\u003c/p\u003e\n\u003cp\u003eThe aliquot vials expected to contain mutated viruses were serially diluted two-fold and mixed with CHO cells (10\u003csup\u003e4\u003c/sup\u003e cells/well), then seeded in a 96-well tissue culture. The plate was monitored daily using an inverted fluorescent microscope for EGFP expression at 1\u0026ndash; 3 days post-infection.\u003c/p\u003e\n\u003cp\u003eThe wells containing EGFP-expressing cells were covered with trypsin to detach the cells. The cells were then transferred into a 100-mm cell culture dish preseeded with CHO cells (5x10\u003csup\u003e5\u003c/sup\u003e cells per 100-mm dish) to increase the distance between fluorescent positive cells.\u003c/p\u003e\n\u003cp\u003eThe single cells positive for both BleCherry and GFP signals (cells containing the recombinant green/red virus) were selected and detached using a cell scraper and were then propagated in BHK cells (or Vero) following three freeze-thaw cycles. Henceforth, the new virus which was designated as \u0026Delta;47 (GFP) /\u0026Delta;\u0026gamma;34.5 (BleCherry) HSV-1. The mutant virus stock was aliquoted and after three freeze-thaw cycles, stored in aliquots at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e\n\u003cp\u003ePCR analysis and sequencing for verification of homologous recombination\u003c/p\u003e\n\u003cp\u003eTo verify the recombination process, the mutant virus DNA was extracted with High Pure Extraction Kit )Roche Diagnostics GmbH, Mannheim, Germany) and PCR was performed using a PCR master mix kit (Taq DNA Polymerase Master Mix RED 2x, Ampliqon, Denmark) in a total volume of 25 containing 12.5 \u0026micro;l Taq DNA Polymerase, 1x Master Mix RED, (\u003csub\u003e~\u003c/sub\u003e100\u0026ndash;150 ng) of HSV-1 DNA and 0.2 \u0026micro;M of each forward and reverse test primers given in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, then programmed as follows: 95\u0026deg;C for 5 min; 30 cycles of 95\u0026deg;C for 60 s, 55\u0026deg;C for 30 s, 72\u0026deg;C for 2 min; followed by a final extension step of 72\u0026deg;C for 5 min. The PCR products were loaded on a 1% agarose gel and were visualized by exposing them to ultraviolet (UV) light (1- kb DNA ladder, CinnaGen) (Haghighi-Najafabadi, Roohvand et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1688682526.png\"\u003e\u003cbr\u003e\u003c/p\u003e\n\n"},{"header":"Result","content":"\u003cp\u003eConstruction of Δ47/Δγ34.5 HSV-1 and verification of homologous recombination\u003c/p\u003e \u003cp\u003eFor the construction of the recombinant virus, the transfection were performed on BHK cells with the vector containing the pSL1180-ARM1\u0026amp;2-ICP47-EGFP fragment for homologous recombination. The next day, the same transfection procedure was repeated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe infection/transfection mixture from BHK cells mixed with CHO cells and seeded in a 96-well tissue culture. The plate was monitored daily using an inverted fluorescent microscope for EGFP expression at 1\u0026ndash; 3 days post-infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe wells containing EGFP-expressing cells were transferred into a 100-mm cell culture dish preseeded with CHO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe single cells positive for both BleCherry and GFP signals were propagated in BHK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and confirmed by PCR. As expected, the PCR product of the test primers in Δγ34.5/ HSV-1 parental virus validated the EGFP expression cassette in the eliminated region which increase the size of PCR products at ICP47 loci in comparison to the Δγ34.5/ HSV-1 parental virus (Haghighi-Najafabadi, Roohvand et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, the HSV-1 was manipulated by replacing US12 (ICP47) with green fluorescence protein in the Δγ34.5/HSV-1 virus (lacking both copies of the Δγ34.5 gene which functions as a virulence factor during HSV1 infection (Haghighi-Najafabadi, Roohvand et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The operation was done in order to generate a replication-conditional mutant of HSV-1.\u003c/p\u003e \u003cp\u003eThe current study is providing novel insights into the use of CHO cells for the first time. For instance, to construct and purify Δ47/Δγ34.5 HSV-1. It additionally proposes CHO cells as an appealing candidate to purge HSV1, utilizing an efficient method which circumvents these problems.\u003c/p\u003e \u003cp\u003eWe explain comprehensively how this process will advance the production and purification efficiency of mutant HSV-1 concerning the purpose of this study.\u003c/p\u003e \u003cp\u003eCHO-K1 cells are typically regarded as nonpermissive cells for HSV-1 infection since they naturally lack gD receptors, hence resistant to HSV infections (Brown, Harland et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1994\u003c/span\u003e, Roller and Herold \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1997\u003c/span\u003e, Conner, Rixon et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs submitted in previous studies, the virus\u0026rsquo;s ability to infect CHO cells is influenced by the cell type used for virus propagation. The cause of this matter is currently unknown, nevertheless, based on the documents, the cell and the structural features of receptors play an undeniably major role in the process of virus infection.\u003c/p\u003e \u003cp\u003eIncipiently, we investigated the virus infectability of CHO cells with Δγ34.5/HSV-1that expresses the BleCherry reporter gene, afterwards the US12 gene of Δγ34.5/HSV-1 was modified as the target region using the conventional homologous recombination method.\u003c/p\u003e \u003cp\u003eThe results of BleCherry expression and titration demonstrated that Δγ34.5/HSV-1 propagated on BHK (Δγ34.5/HSV-1/BHK) but not on Vero (Δγ34.5/HSV-1/Vero) cells. Therefore the subject was able to infect CHO cells and progeny virion was productive (data not shown).\u003c/p\u003e \u003cp\u003eThe results of our operation were in accordance with other discoveries which are based on studies using electron microscopy reporting nucleocapsids and enveloped virions that were visible in the nucleus and cytoplasm of HSV-1/BHK-infected CHO cells (Nicola and Straus \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Conner, Rixon et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOn that account, the BHK-21 cell line was selected for the transfection/infection process in our study. Consequently, the parental/mutant virus mixture will be able to infect CHO cells(Conner, Rixon et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe data suggest that transfection efficiency is notably increased by double transfection of the cells. Adding Δγ34.5/HSV-1 to the complex after lipoplex formation during the second transfection can remarkably enhance it. Of note, the mixtures should be tested at various doses of the virus to measure the transfection process. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Ishikawa and Homcy \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1992\u003c/span\u003e, Fu and Zhang \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, Burnham, Jaishankar et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe aliquot vials that were expected to contain mutated viruses were serially diluted two-fold and mixed with CHO cells (10\u003csup\u003e4\u003c/sup\u003e cells/well ), then seeded in a 96-well tissue culture.\u003c/p\u003e \u003cp\u003eOur results indicate that Δγ34.5/HSV-1 produced from BHK cells with a titer of 10\u003csup\u003e7\u003c/sup\u003e PFU/ml can infect approximately 30 to 40 percent of exponentially growing CHO cells, and simultaneous introduction of serum and virus after 24 h serum starvation of the CHO cells, infects up to 70% of cells (data not shown), suggesting that the vulnerability of CHO cells to HSV-1 infection depend on virus titers and cell cycle status. So, the supernatants contain the viruses with a titer of at least 3 PFU/cell. They are mixed with CHO cells during cell seeding for optimal infection of the cells. Similar findings have been reported previously by Conner et al (Conner, Rixon et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis protocol offers several advantages for the following reasons:\u003c/p\u003e \u003cp\u003eThe amount of the infectious virions released into supernatants of Δγ34.5/HSV-1/BHK CHO infected cells is too low to be detected (Conner, Rixon et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Berting, Farcet et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), however, they are readily detectable when cells and supernatants are harvested together; suggesting the fact that progeny viruses are not released efficiently by the CHO cells in comparison to BHK or Vero cells. In addition, the released virus from CHO cells is unable to infect CHO or spread to adjacent cells, and the infection is limited to a single round of replication in the cells, whereas this ability is present in BHK or Vero cells (Fig.\u0026nbsp;5) (Roller and Herold \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1997\u003c/span\u003e, Conner, Rixon et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Berting, Farcet et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;5\u003c/b\u003e Schematic illustration of the Characteristics of CHO cell; the virus acquired from CHO cells is unable to infect CHO or spread to adjacent cells and infection is limited to a single round of replication in CHO cells whereas this ability is obtained in Vero cells.\u003c/p\u003e \u003cp\u003eThese findings are noteworthy since site\u003cem\u003e-\u003c/em\u003especific genome editing may lead to reduced replication of progeny virus and lower titers of cell-released virus due to the critical roles of the gene in virus replication phenotype (Oyama, Ohigashi et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Fu and Zhang \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Mullen, Kasuya et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Russell, Stefanovic et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), this presents an issue, as the amplification of Δγ34.5/HSV-1 (parental virus) during plaque growth and direct cell-to-cell spread (Yuan, Wang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Li, Bi et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Lee \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), amounts to a decreased distinct growth of the progressive plaques and usually about 15\u0026thinsp;\u0026minus;\u0026thinsp;10 tissue culture dishes (10 cm) are needed to isolate plaques that contain desired recombinant virus variants, which makes first-round screening tedious (Fig.\u0026nbsp;6).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;6\u003c/b\u003e Challenges in screening procedure of recombinant HSV-1 in permissive cell lines (Exemplified in Figs. A, B, C and D); the plaque purification process can be problematic when the percentage of HSV-1 recombinants of interest be too low in compared with parental virus (10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026ndash;2%, depend on the conventional homologous recombination method or crispr-cas9 genome editing)\u003c/p\u003e \u003cp\u003e2) The CHO system can be more financially reasonable than the plaque purification technique, as no semisolid overlay substrates such as agarose or carboxymethyl cellulose are required to prevent indiscriminate infection which may occur through the liquid growth medium. As a result, challenges of single plaque pick-up through the agarose layer will be overcome (Fig.\u0026nbsp;7) (Fu and Zhang \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Liang, Xu et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Ebrahimi, Makvandi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis protocol demonstrated the successful use of CHO cells as a worthy replacement for the basic protocol for the isolation and purification of recombinant HSV-1 by plaque assay (Fig.\u0026nbsp;7).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;7a\u003c/b\u003e Graphical protocol overview of two protocols for isolation and purification of recombinant HSV-1. \u003cb\u003eb\u003c/b\u003e Characteristics and comparison of the two protocols.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank all the personnel\u0026nbsp;of Virology Department and\u0026nbsp;Laboratory of Regenerative Medicine and Biomedical Innovations, Pasteur Institute of Iran for their assistance in this project.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors discussed the results and implications and commented on the manuscript at all stages.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used to support the fndings of this study are included within the\u0026nbsp;article and The nucleotide sequence data are available in the GenBank databases.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo competing interests declared. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdoli, S., F. 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Arase (2014). \u0026quot;Engineering large viral DNA genomes using the CRISPR‐Cas9 system.\u0026quot; Microbiology and immunology 58(9): 513-522. https://doi: 10.1111/1348-0421.12180.\u003c/li\u003e\n\u003cli\u003eVarghese, S. and S. D. Rabkin (2002). \u0026quot;Oncolytic herpes simplex virus vectors for cancer virotherapy.\u0026quot; Cancer gene therapy 9(12): 967-978. https://doi: 10.3390/cells10061541\u003c/li\u003e\n\u003cli\u003eWalker, A. J. (1977). \u0026quot;An efficient method for generating discrete random variables with general distributions.\u0026quot; ACM Transactions on Mathematical Software (TOMS) 3(3): 253-256. https://doi.org/10.1145/355744.355749\u003c/li\u003e\n\u003cli\u003eYuan, M., P. Wang, L. S. Chard, N. R. Lemoine and Y. Wang (2016). \u0026quot;A simple and efficient approach to construct mutant vaccinia virus vectors.\u0026quot; JoVE (Journal of Visualized Experiments)(116): e54171. https://doi: 10.3791/54171.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"amb-express","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ambe","sideBox":"Learn more about [AMB Express](http://amb-express.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/AMBE/default.aspx","title":"AMB Express","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Fluorescent reporter, CHO cells, Oncolytic HSV-1, Purification plaque, BHK-21, Non-permissive","lastPublishedDoi":"10.21203/rs.3.rs-2966832/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2966832/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExploiting herpes simplex virus type 1 (HSV-1) has recently emerged as a new strategy to improve the treatment of patients with various cancers resistant to chemotherapy and associated with a poor prognosis due to its ability to infect tumor cells without causing harm to healthy cells. It has been suggested as a new platform for cancer therapy. Gene-modification techniques such as conventional homologous recombination or CRISPR/Cas9 system are utilized to introduce site-specific mutations in targeted viral genes.\u003c/p\u003e \u003cp\u003eAlthough, the CRISPR-Cas9 system could significantly increase the efficiency of homologous recombination; nevertheless the process of purifying recombinant variants can be tedious.\u003c/p\u003e \u003cp\u003eHere we present a rapid, innovative method using non-permissive hamster ovary (CHO) cells which is a remarkable improvement on the previously mentioned tedious process. Using this strategy, only 1\u0026ndash;2 rounds of plaque purification would suffice. Our proposed protocol demonstrated high potential as a worthy alternative way for the current approaches of the isolation and purification of the fluorescent reporter genes-expressing recombinant HSV-1 by plaque assay using CHO cells.\u003c/p\u003e","manuscriptTitle":"Non-permissive CHO cells; A rapid approach for purification of recombinant Herpes Simplex Virus-1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-07-07 13:30:42","doi":"10.21203/rs.3.rs-2966832/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-10-21T06:54:47+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-07-02T20:26:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-05-31T17:00:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"AMB Express","date":"2023-05-30T05:47:13+00:00","index":"","fulltext":""},{"type":"decision","content":"Major Revision","date":"2023-05-27T19:49:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"amb-express","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ambe","sideBox":"Learn more about [AMB Express](http://amb-express.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/AMBE/default.aspx","title":"AMB Express","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"355a835d-54a5-4f47-9ac9-563b7a23de72","owner":[],"postedDate":"July 7th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-14T21:26:19+00:00","versionOfRecord":{"articleIdentity":"rs-2966832","link":"https://doi.org/10.1186/s13568-024-01709-0","journal":{"identity":"amb-express","isVorOnly":false,"title":"AMB Express"},"publishedOn":"2024-05-09 21:18:37","publishedOnDateReadable":"May 9th, 2024"},"versionCreatedAt":"2023-07-07 13:30:42","video":"","vorDoi":"10.1186/s13568-024-01709-0","vorDoiUrl":"https://doi.org/10.1186/s13568-024-01709-0","workflowStages":[]},"version":"v1","identity":"rs-2966832","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2966832","identity":"rs-2966832","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}