An immortalized porcine mesangial cell line competent for the cultivation of Japanese encephalitis virus

An immortalized porcine mesangial cell line competent for the cultivation of Japanese encephalitis virus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article An immortalized porcine mesangial cell line competent for the cultivation of Japanese encephalitis virus Shengkui Xu, Shucheng Zong, Xinze Liu, Wen Pan, Fucheng Guo, Jinyu FU, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4276480/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Japanese encephalitis virus causes a huge threat to the pig-raising industry and attenuated-live vaccines are widely used to prevent JEV infection. Nowadays, primary hamster kidney cells and Vero cells are mostly used for the propagation of JEV attenuated vaccines. In this research, we constructed porcine-derived-immortalized mesangial cells (PMCs) for JEV cultivation, and their characteristics were also studied. The prepared PMCs were uniform in shape and without variation in chromosome numbers. Furthermore, PMCs showed strong division and proliferation ability, and JEV could replicate efficiently as well causing a cytopathic effect similar to that in Vero cells. The viral tiler results showed that JEV grows faster in PMCs compared with that in Vero cells. Taken together, we generated an immortalized PMC cell line competent for the cultivation of JEV, which has significance for the production of pig JEV attenuated vaccines. Figures Figure 1 Figure 2 Figure 3 Introduction Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic virus, which invades the central nervous system and causes encephalitis in humans and causes reproductive disorders in pigs[1]. Taxonomically, JEV, with a single-stranded positive-sense RNA genome, belongs to a member of the genus Flavivirus in the family Flaviviridae and is closely related to other animal flaviviruses, including classical swine fever virus (CSFV), yellow fever virus (YFV), dengue virus[2, 3]. The infection of vertebrate animals by JEV is largely attributed to the virus-amplifying host pigs, which not only develop high titers and long-lasting viremia but also are in immediate contact with human habitats[4, 5]. Thus, JEV prevention in pigs not only helps to reduce the economic losses but also cuts off the JEV transmission to humans. Vaccines are the major countermeasures to prevent JEV infection due to the lack of specific drugs [6]. Currently, mouse brain-derived inactivated vaccine, cell culture-derived live-attenuated vaccine, and cell culture-derived inactivated vaccine are three different types of JE vaccines [7-9]. The vaccines prepared by rat brain tissue and gopher kidney primary cells have shortcomings, such as the tight source of experimental animals, high production cost, low yield, exogenous factors, and batch differences [10]. Although the attenuated vaccine prepared by Vero cells solves the problems partially, Vero cells are non-porcine cells and have the problem of exogenous factors contamination. Thus, the construction of porcine-derived immortalized cells will be more suitable for the JEV vaccine for pigs. In the present study, we prepared the PMCs cell lines and examined their suitability for JEV vaccine production. Materials And Methods 2.1 Cell lines and viruses Vero and PMCs were all cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) containing 10% (V/V) fetal bovine serum (FBS) in a humidified 37℃, 5% CO 2 incubator. The JEV used in this study was the seventh generation of SA14-14-2 ( GenBank accession number: MK585066.1 ). The virus was propagated in Vero cells and the virus titers were determined by a 50% tissue culture infectious dose (TCID 50 ) assay on Vero cells. 2.2 Antibodies and chemicals The vimentin primary antibody used in this study was purchased from ProteinTech (Beijing, China). Alexa Fluor 488-conjugated goat anti-rabbit IgG (H L) F(ab=)2 fragment was purchased from Molecular Probes (Thermo Fisher Scientific, USA). 2.3 The preparation and purification of PMCs The preparation of the porcine mesangial cell line (PMC) was according to the following tips: Take the intact kidneys of fetal pig out and disinfect it in 75% ethanol for 2 min; Remove the coated blood vessels carefully and then cut the kidney into small pieces; Transfer these pieces into 50 mL tubes and remove the blood cells by repeated washes with PBS; Transfer the tissues to an 80-mesh grind carefully and wash them with PBS at the same time; Collect the filtered liquid and then filter the collected liquid with a 100-mesh screen; Transfer the filtered liquid to a new 50 mL centrifuge tube and centrifuge at 1500 rpm for 10 min; Discard the supernatant，suspend and digest the glomeruli with 0.05%-0.1% type I and type IV collagenase for 40 min; Filter the digestive solution with a 100 μm cell screen, and centrifuge at 1500 rpm for 5min; Discard the supernatant and culture the cells with DMEM with 10%FBS and in T25 cell culture bottles. After several passages, most of the cells were adhered to the cell bottle surface under an inverted microscope. To acquire homogeneous PMC, the cell populations were purified by different digestion according to the different sensitivity between PMC and endothelial cells to trypsin. PMCs are sensitive to trypsin and detached earlier than glomerular endothelial cells. Then the cell suspension was diluted to 10 cells /mL by 10 times gradient and the diluted cells were seeded to the 96-well plate with 0.1mL/ well. The cell wells containing only single cells were selected for further selection. 2.4 Immunofluorescence assay (IFA) The IFA was carried out as described previously [11]. Briefly, Vero or PMC cells grown in 6-well plates to 80-90% confluence were infected with JEV. At indicated time points, the cells were fixed in cold absolute alcohol for 20 min. Then the cells were incubated with primary antibodies at room temperature (RT) for 1 h and then washed with PBS three times. Finally, the cells were stained with Alexa Fluor 488-conjugated goat anti-rabbit IgG (H L) F(ab=)2 fragment (Thermo Fisher Scientific, USA) secondary antibody at RT for 1 h in a humid chamber. After one wash, nuclear was stained with 4’, 6’-diamidino-2-phenylindole (DAPI) (Molecular Probes) for 10 min and washed with PBS 5 times for 5 min each. The cells were observed with a Nikon A1 microscope or laser confocal microscope. 2.5 Virus titration Viruses were 10-fold serially diluted with DMEM containing 2% FBS and inoculated into Vero cells at 80-90% confluence in 96-well culture plates. 72 h later, the virus titers were calculated based on the cytopathogenic effects (CPE) according to the Reed-Muench method. Virus titers were determined from three independent experiments. 2.6 Statistical analysis All data was processed with GraphPad Prism 6 (GraphPad Software Inc.). A student T -test or nonparametric test was used to analyze the difference between the values of the two groups. A value of p< 0.05 was considered statistically significant. 2.7 Acknowledgements This research was supported by the National Natural Science Foundation of China (32202825), the R&D Program of the Beijing Municipal Education Commission (KM202310020004), and the Young Teachers’ Research and Innovation Ability Improvement Program of Beijing University of Agriculture (QJKC2022030). Results 3.1 The isolation and purification of immortalized fetal pig mesangial cell line To produce the pig mesangial cell line (PMCs), the intact kidneys of fetal pigs were taken out aseptically and disinfected in 75% ethanol. Remove the coated blood vessels carefully and then cut the kidney into small pieces as described above (Fig 1A). The cells were collected and then cultured in DMEM with 10% FBS and in T25 cell culture bottles. After 4 days cultivation, most of the cells adhered to the cell bottle surface (Fig 1B) under an inverted microscope. There are some glomerular endothelial cells scattered among glomerular endothelial cells. PMCs are sensitive to trypsin and detached earlier than glomerular endothelial cells. To acquire homogeneous PMCs, the cell populations were purified according to their different sensitivity to trypsin. The PMCs were earlier detached than glomerular endothelial cells (Fig 1C) and this process was conducted three times to acquire pure PMCs. Furthermore, a limited dilution assay was used to screen for monoclonal cells in the 96-well plate. Finally, a self-immortalized fetal porcine mesangial cell line with high purity and stable passage was obtained and designated as PMCs. Meanwhile, PMCs still retained the morphological characteristics of the original cells after 30 and 60 passages (Fig. 1D, E). Figure 1 Primary fetal pig mesangial cells (PMCs) isolation and culture (A) The schematic diagram Take out the fetal pig kidney and cut it into small pieces; (B) The heterogeneous PMCs after 4 days of cultivation in DMEM; (C) The purified PMCs by different sensitivity to trypsin; (D, E) The PMCs show similar morphological characteristics after 30 and 60 passages. 3.2 The Characterization of PMCs Vimentin is highly expressed in the cytoplasm of mesangial cells, which can serve as the marker of PMCs. To analyze the homogeneousness of cells, IFA was applied with a monoclonal antibody against vimentin 9 (V9) to detect vimentin and the cell nucleus was stained with Hoechst 33342. The IFA results showed that the vimentin was highly expressed and the cells showed a uniform shape, indicating the PMCs are homogeneous (Fig 2A). There are 19 pairs of chromosomes in normal pig cells, then chromosomal karyotype analysis was performed using the Video Test to count the chromosomes. The results showed that the spontaneously immortalized fetal PMCs contain 19 pairs of 38 chromosomes and there is no variation in chromosome number (Fig 2B). Finally, the cell cycle was determined by flow cytometry to study the proliferation capacity of PMCs. As shown in Figure 2C, the proportion of cells in the G1 phase, S phase, and G2 phase were 65.68%, 28.19%, and 6.13%, respectively, indicating the PMCs division and proliferation ability is strong. 3.3 Susceptibility of PMCs to JEV To examine the susceptibility of the PMCs to JEV, PMCs were seeded into 6-well plates and inoculated with vaccine-candidate strains JEV at 0.1 MOI. Firstly, the cytopathic effect was monitored during the whole infection course to 96 h. At 24 hours post-inoculation (hpi), cytopathic effects (CPE) appeared and were clearly observed under a microscope at 48 hpi (Figure 3A). The results showed that most cells were infected, indicating JEV replicates efficiently in PMCs. Secondly, plaque forming assay was performed in JEV-infected PMCs at 36 hpi and the results showed that JEV could cause plaque in PMCs similar to Vero cells (Fig 3B, 3C). These results indicated that PMCs are susceptible to the JEV vaccine strain. To further evaluate the viral replication efficiency on PMCs, we compared the viral titers produced in the PMCs and Vero cell cultures over time (0–96 hpi). The JEV isolates were propagated in PMCs cell cultures and reached maximal titers of 10 7.5 TCID50/mL at 72 hpi. In contrast, JEV propagated slowly in Vero cells and peaked at 96 h with a titer of 10 7 TCID50/mL(Fig 3D). Discussion Vaccination is the major strategy for JEV prevention and inactivated-vaccine, widely used in pigs nowadays, requires a steady and safe supply of JEV production. A major weakness of JEV propagation is the viruses are usually cultivated in hamster-derived Vero cells, which may possess external factor risk. Thus, the development of a porcine-derived cell line with high viral titers will not only avoid contamination by external factors but also reduce the vaccine production cost. In this study, we developed a porcine-derived-immortalized mesangial cell line (PMCs) for JEV cultivation with higher viral titers compared with that in Vero cells and high stability, indicating the prepared immortalized PMCs have significance for the production of pig JEV attenuated vaccines. An infinite cell line is one of the most favored apparatuses for virus production and this can be achieved by various ways, including physical and chemical stimulations, heterologous expression of viral oncogenes, increased telomerase activity, and spontaneous formation[12]. Spontaneous immortalization is driven by chromosomal rearrangements and epigenetic changes spontaneously during cell divisions without exogenous gene or chemical involvement[12, 13]. The purification, morphology, and characteristics of immortalized PMCs are critical to evaluating the quality of the prepared cell line. Firstly, Differential digestion and limited dilution assay were performed to ensure the purification of PMCs in our research, meanwhile, the morphology and homogeneity of PMCs were observed by microscope and IFA. Then the chromosome morphology and number were confirmed by using a Video Test. Finally, flow cytometry was used to study the cell viability, which was critical for virus proliferation. The main purpose of this study was to use the immortalized PMCs for the production of porcine-derived virus vaccines and various cell lines had been developed successfully [14-16]. JEV infection in Vero cells usually causes cell cytopathogenic effects and forms plaques[17]. When tested in PMCs, JEV also causes cytopathogenic effects and forms similar plaques in size and morphology. IFA was also conducted to observe the replication efficiency of JEV in PMCs. Another critical factor in determining the potential of vaccine apparatus is the high-yield virus production. Then the viral titers were determined at various time points. The results showed that viral titers peak at 72 hpi in PMCs with 10 7.5 TCID 50 /mL while 96 hpi in Vero cells with 10 7 TCID 50 /mL. Notably, the viral genome stays stable during propagation in PMCs. Taken together, the immortalized PMCs prepared in this study have normal characteristics in morphology and chromosomes and they are favorable for JEV cultivation with higher viral titers, which has significance for the production of pig JEV attenuated or inactivated vaccines. References Le Flohic, G., et al., Review of climate, landscape, and viral genetics as drivers of the Japanese encephalitis virus ecology. PLoS Negl Trop Dis, 2013. 7 (9): p. e2208. Zhang, J.S., et al., Genomic sequence of a Japanese encephalitis virus isolate from southern China. Arch Virol, 2009. 154 (7): p. 1177-80. Sharma, K.B., S. Vrati, and M. Kalia, Pathobiology of Japanese encephalitis virus infection. Mol Aspects Med, 2021. 81 : p. 100994. Su, C.L., et al., Molecular epidemiology of Japanese encephalitis virus in mosquitoes in Taiwan during 2005-2012. PLoS Negl Trop Dis, 2014. 8 (10): p. e3122. Teng, M., et al., Molecular characterization of Japanese encephalitis viruses circulating in pigs and mosquitoes on pig farms in the Chinese province of Henan. Virus Genes, 2013. 46 (1): p. 170-4. Hegde, N.R. and M.M. Gore, Japanese encephalitis vaccines: Immunogenicity, protective efficacy, effectiveness, and impact on the burden of disease. Hum Vaccin Immunother, 2017. 13 (6): p. 1-18. Yun, S.I. and Y.M. Lee, Japanese encephalitis: the virus and vaccines. Hum Vaccin Immunother, 2014. 10 (2): p. 263-79. Lee, P.I., et al., Recommendations for the use of Japanese encephalitis vaccines. Pediatr Neonatol, 2020. 61 (1): p. 3-8. Chen, T., et al., Protective Immune Responses Induced by an mRNA-LNP Vaccine Encoding prM-E Proteins against Japanese Encephalitis Virus Infection. Viruses, 2022. 14 (6). Momose, H., et al., Induction of indistinguishable gene expression patterns in rats by Vero cell-derived and mouse brain-derived Japanese encephalitis vaccines. Jpn J Infect Dis, 2010. 63 (1): p. 25-30. Song, J., et al., Mapping the Nonstructural Protein Interaction Network of Porcine Reproductive and Respiratory Syndrome Virus. J Virol, 2018. 92 (24). Guo, D., et al., Establishment methods and research progress of livestock and poultry immortalized cell lines: A review. Front Vet Sci, 2022. 9 : p. 956357. Fu, Y., et al., High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol, 2013. 31 (9): p. 822-6. Wang, J., et al., Construction of a peacock immortalized fibroblast cell line for avian virus production. Poult Sci, 2022. 101 (12): p. 102147. Dollery, S.J., et al., iTIME.219: An Immortalized KSHV Infected Endothelial Cell Line Inducible by a KSHV-Specific Stimulus to Transition From Latency to Lytic Replication and Infectious Virus Release. Front Cell Infect Microbiol, 2021. 11 : p. 654396. Masujin, K., et al., An immortalized porcine macrophage cell line competent for the isolation of African swine fever virus. Sci Rep, 2021. 11 (1): p. 4759. Ma, X., et al., Construction of a Recombinant Japanese Encephalitis Virus with a Hemagglutinin-Tagged NS2A: A Model for an Analysis of Biological Characteristics and Functions of NS2A during Viral Infection. Viruses, 2022. 14 (4). Cite Share Download PDF Status: Posted Version 1 posted 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-4276480","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":295907570,"identity":"0865b3b9-81a8-425e-8a65-8fe71b052400","order_by":0,"name":"Shengkui Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIie3QMQrCMBSA4ZRApqddUyLUIzwpFIeAB3FJETLpDToUCnUpdO1xCoFMLa6OPUK9gNrVQRo3h3zwtvzwXgjxvH8E82RPCSGlZnRPJqY30ZVpdE6ClhmJN9hypyKu+1QAUEgMECS5PC4mOFRaAGeQmlU3EqsvxWISUisAYU7WCoPCLCdxE1QCFIekBOROCRlKG7UdAlLXBHt72j8KBdzMn6xcbonr8+6eFa9D2BgzTrl0WOyD+u2553me980bF5Q3fZB6+k8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0003-4698-8392","institution":"Beijing University of Agriculture","correspondingAuthor":true,"prefix":"","firstName":"Shengkui","middleName":"","lastName":"Xu","suffix":""},{"id":295907571,"identity":"8ae009bd-8986-40fb-84a1-ff83963d2e9c","order_by":1,"name":"Shucheng Zong","email":"","orcid":"","institution":"Zhongmu Institutes of China Animal Husbandary Industry","correspondingAuthor":false,"prefix":"","firstName":"Shucheng","middleName":"","lastName":"Zong","suffix":""},{"id":295907572,"identity":"6c0c9c68-1d51-4e02-8af4-1f8c87a231de","order_by":2,"name":"Xinze Liu","email":"","orcid":"","institution":"Zhongmu Institutes of China Animal Husbandry Industry","correspondingAuthor":false,"prefix":"","firstName":"Xinze","middleName":"","lastName":"Liu","suffix":""},{"id":295907573,"identity":"229ac26a-7110-4663-ad5c-c416863b1479","order_by":3,"name":"Wen Pan","email":"","orcid":"","institution":"Zhongmu Institutes of China Animal Husbandary Induatry","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Pan","suffix":""},{"id":295907574,"identity":"784604f8-2fe3-484c-bf87-8cc79ef67099","order_by":4,"name":"Fucheng Guo","email":"","orcid":"","institution":"Zhongmu Institutes of China Animal Husbandary Industry","correspondingAuthor":false,"prefix":"","firstName":"Fucheng","middleName":"","lastName":"Guo","suffix":""},{"id":295907575,"identity":"76b1d2c7-08d8-4b9c-bd10-6357c7d24482","order_by":5,"name":"Jinyu FU","email":"","orcid":"","institution":"Zhongmu Institutes of China Animal Husbandry Industry","correspondingAuthor":false,"prefix":"","firstName":"Jinyu","middleName":"","lastName":"FU","suffix":""},{"id":295907576,"identity":"e92d6c02-12b8-49c2-92a7-12024383a9c2","order_by":6,"name":"Zhongyin Liu","email":"","orcid":"","institution":"Beijing University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Zhongyin","middleName":"","lastName":"Liu","suffix":""},{"id":295907577,"identity":"8b25c8d1-26b6-41dd-8559-89bda7586963","order_by":7,"name":"Jin Xiao","email":"","orcid":"","institution":"China Animal Husbandry Industry Co.Ltd","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Xiao","suffix":""}],"badges":[],"createdAt":"2024-04-16 13:50:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4276480/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4276480/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55726318,"identity":"b3a3b28c-22aa-41fe-b7ab-6460abca9e71","added_by":"auto","created_at":"2024-05-02 10:08:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1479680,"visible":true,"origin":"","legend":"\u003cp\u003ePrimary fetal pig mesangial cells (PMCs) isolation and culture\u003c/p\u003e\n\u003cp\u003e(A) The schematic diagram Take out the fetal pig kidney and cut it into small pieces; (B) The heterogeneous PMCs after 4 days of cultivation in DMEM; (C) The purified PMCs by different sensitivity to trypsin; (D, E) The PMCs show similar morphological characteristics after 30 and 60 passages.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4276480/v1/6636e9daaa7475fc197c749c.png"},{"id":55726317,"identity":"7d874d5d-c12a-4753-84e8-122295a1a8ad","added_by":"auto","created_at":"2024-05-02 10:08:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":612018,"visible":true,"origin":"","legend":"\u003cp\u003eThe Characterization of PMCs\u003c/p\u003e\n\u003cp\u003e(A) The expression of vimentin in PMCs; (B) The morphology and number of PMCs genome; (C) The proliferation capacity of PMCs.\u003c/p\u003e","description":"","filename":"Figure2TheCharacterizationofPMCs.png","url":"https://assets-eu.researchsquare.com/files/rs-4276480/v1/a405f83d06ec76b9b60828f4.png"},{"id":55726673,"identity":"8989ea33-bdb1-4576-b2bd-d6489d3bb890","added_by":"auto","created_at":"2024-05-02 10:16:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1323215,"visible":true,"origin":"","legend":"\u003cp\u003eJEV replicates efficiently in PMCs\u003c/p\u003e\n\u003cp\u003e(A) The replication of JEV on Vero and PMCs; (B) The plaque forming assay of JEV on Vero and PMCs; (C) The statistical analysis of the plaques; (D) JEV growth curve on Vero and PMCs.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4276480/v1/b18219b090f98cc419068463.png"},{"id":58902042,"identity":"beb64301-4b9c-4cfc-92f1-32f5d7697411","added_by":"auto","created_at":"2024-06-24 01:23:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3665338,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4276480/v1/cd492e74-2c66-4e61-ac3b-d7cafa37c5ff.pdf"}],"financialInterests":"","formattedTitle":"An immortalized porcine mesangial cell line competent for the cultivation of Japanese encephalitis virus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eJapanese encephalitis virus (JEV) is a mosquito-borne zoonotic virus, which invades the central nervous system and causes encephalitis in humans and causes reproductive disorders in pigs[1]. Taxonomically, JEV, with a single-stranded positive-sense RNA genome, belongs to a member of the genus \u003cem\u003eFlavivirus\u003c/em\u003e in the family\u003cem\u003e\u0026nbsp;Flaviviridae\u0026nbsp;\u003c/em\u003eand is closely related to other animal flaviviruses, including classical swine fever virus (CSFV), yellow fever virus (YFV), dengue virus[2, 3]. The infection of vertebrate animals by JEV is largely attributed to the virus-amplifying host pigs, which not only develop high titers and long-lasting viremia but also are in immediate contact with human habitats[4, 5]. Thus, JEV prevention in pigs not only helps to reduce the economic losses but also cuts off the JEV transmission to humans.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVaccines are the major countermeasures to prevent JEV infection due to the lack of specific drugs [6]. Currently, mouse brain-derived inactivated vaccine, cell culture-derived live-attenuated vaccine, and cell culture-derived inactivated vaccine are three different types of JE vaccines [7-9]. The vaccines prepared by rat brain tissue and gopher kidney primary cells have shortcomings, such as the tight source of experimental animals, high production cost, low yield, exogenous factors, and batch differences [10]. Although the attenuated vaccine prepared by Vero cells solves the problems partially, Vero cells are non-porcine cells and have the problem of exogenous factors contamination. Thus, the construction of porcine-derived immortalized cells will be more suitable for the JEV vaccine for pigs. In the present study, we prepared the PMCs cell lines and examined their suitability for JEV vaccine production.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e2.1 Cell lines and viruses\u003c/p\u003e\n\u003cp\u003eVero and PMCs were all cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM; Invitrogen, Carlsbad, CA, USA) containing 10% (V/V) fetal bovine serum (FBS) in a humidified 37℃, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. The JEV used in this study was the seventh generation of SA14-14-2 ( GenBank accession number: MK585066.1 ). The virus was propagated in Vero cells and the virus titers were determined by a 50% tissue culture infectious dose (TCID\u003csub\u003e50\u003c/sub\u003e) assay on Vero cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Antibodies and chemicals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe vimentin primary antibody used in this study was purchased from ProteinTech (Beijing, China). Alexa Fluor 488-conjugated goat anti-rabbit IgG (H L) F(ab=)2 fragment was purchased from Molecular Probes (Thermo Fisher Scientific, USA).\u003c/p\u003e\n\u003cp\u003e2.3 The preparation and purification of PMCs\u003c/p\u003e\n\u003cp\u003eThe preparation of the porcine mesangial cell line (PMC) was according to the following tips: Take the intact kidneys of fetal pig out and disinfect it in 75% ethanol for 2 min; Remove the coated blood vessels carefully and then cut the kidney into small pieces; Transfer these pieces into 50 mL tubes and remove the blood cells by repeated washes with PBS; \u0026nbsp;Transfer the tissues to an 80-mesh grind carefully and wash them with PBS at the same time; Collect the filtered liquid and then filter the collected liquid with a 100-mesh screen; Transfer the filtered liquid to a new 50 mL centrifuge tube and centrifuge at 1500 rpm for 10 min; Discard the supernatant，suspend and digest the glomeruli with 0.05%-0.1% type I and type IV collagenase for 40 min; Filter the digestive solution with a 100 \u0026mu;m cell screen, and centrifuge at 1500 rpm for 5min; Discard the supernatant and culture the cells with DMEM with 10%FBS and in T25 cell culture bottles. After several passages, most of the cells were adhered to the cell bottle surface under an inverted microscope.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo acquire homogeneous PMC, the cell populations were purified by different digestion according to the different sensitivity between PMC and endothelial cells to trypsin. PMCs are sensitive to trypsin and detached earlier than glomerular endothelial cells. Then the cell suspension was diluted to 10 cells /mL by 10 times gradient and the diluted cells were seeded to the 96-well plate with 0.1mL/ well. The cell wells containing only single cells were selected for further selection.\u003c/p\u003e\n\u003cp\u003e2.4 Immunofluorescence assay (IFA)\u003c/p\u003e\n\u003cp\u003eThe IFA was carried out as described previously\u0026nbsp;[11]. Briefly, Vero or PMC cells grown in 6-well plates to 80-90% confluence were infected with JEV. At indicated time points, the cells were fixed in cold absolute alcohol for 20 min. Then the cells were incubated with primary antibodies at room temperature (RT) for 1 h and then washed with PBS three times. Finally, the cells were stained with Alexa Fluor 488-conjugated goat anti-rabbit IgG (H L) F(ab=)2 fragment (Thermo Fisher Scientific, USA) secondary antibody at RT for 1 h in a humid chamber. \u0026nbsp;After one wash, nuclear was stained with 4\u0026rsquo;, 6\u0026rsquo;-diamidino-2-phenylindole (DAPI) (Molecular Probes) for 10 min and washed with PBS 5 times for 5 min each. The cells were observed with a Nikon A1 microscope or laser confocal microscope.\u003c/p\u003e\n\u003cp\u003e2.5 Virus titration\u003c/p\u003e\n\u003cp\u003eViruses were 10-fold serially diluted with DMEM containing 2% FBS and inoculated into Vero cells at 80-90% confluence in 96-well culture plates. 72 h later, the virus titers were calculated based on the cytopathogenic effects (CPE) according to the Reed-Muench method. Virus titers were determined from three independent experiments.\u003c/p\u003e\n\u003cp\u003e2.6 Statistical analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll data was processed with GraphPad Prism 6 (GraphPad Software Inc.). A student \u003cem\u003eT\u003c/em\u003e-test or nonparametric test was used to analyze the difference between the values of the two groups. A value of p\u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e\n\u003cp\u003e2.7 Acknowledgements\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Natural Science Foundation of China (32202825), the R\u0026amp;D Program of the Beijing Municipal Education Commission (KM202310020004), and the Young Teachers\u0026rsquo; Research and Innovation Ability Improvement Program of Beijing University of Agriculture (QJKC2022030).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 The isolation and purification of immortalized fetal pig mesangial cell line\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo produce the pig mesangial cell line (PMCs), the intact kidneys of fetal pigs were taken out aseptically and disinfected in 75% ethanol. Remove the coated blood vessels carefully and then cut the kidney into small pieces as described above (Fig 1A).\u0026nbsp;The cells were collected and then cultured in DMEM with 10% FBS and in T25 cell culture bottles. After 4 days cultivation, most of the cells adhered to the cell bottle surface (Fig 1B) under an inverted microscope.\u003c/p\u003e\n\u003cp\u003eThere are some glomerular endothelial cells scattered among glomerular endothelial cells. PMCs are sensitive to trypsin and detached earlier than glomerular endothelial cells. To acquire homogeneous PMCs, the cell populations were purified according to their different sensitivity to trypsin. The PMCs were earlier detached than glomerular endothelial cells (Fig 1C) and this process was conducted three times to acquire pure PMCs. Furthermore, a limited dilution assay was used to screen for monoclonal cells in the 96-well plate. Finally, a self-immortalized fetal porcine mesangial cell line with high purity and stable passage was obtained and designated as PMCs. Meanwhile, PMCs still retained the morphological characteristics of the original cells after 30 and 60 passages (Fig. 1D, E).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Figure 1 Primary fetal pig mesangial cells (PMCs) isolation and culture\u003c/p\u003e\n\u003cp\u003e(A) The schematic diagram Take out the fetal pig kidney and cut it into small pieces; (B) The heterogeneous PMCs after 4 days of cultivation in DMEM; (C) The purified PMCs by different sensitivity to trypsin; (D, E) The PMCs show similar morphological characteristics after 30 and 60 passages.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e3.2 The\u003c/strong\u003e \u003cstrong\u003eCharacterization of PMCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVimentin is highly expressed in the cytoplasm of mesangial cells, which can serve as the marker of PMCs. To analyze the homogeneousness of cells, IFA was applied with a monoclonal antibody against vimentin 9 (V9) to detect vimentin and the cell nucleus was stained with Hoechst 33342. The IFA results showed that the vimentin was highly expressed and the cells showed a uniform shape, indicating the PMCs are homogeneous (Fig 2A).\u003c/p\u003e\n\u003cp\u003eThere are 19 pairs of chromosomes in normal pig cells, then chromosomal karyotype analysis was performed using the Video Test to count the chromosomes. The results showed that the spontaneously immortalized fetal PMCs contain 19 pairs of 38 chromosomes and there is no variation in chromosome number (Fig 2B).\u003c/p\u003e\n\u003cp\u003eFinally, the cell cycle was determined by flow cytometry to study the proliferation capacity of PMCs. As shown in Figure 2C, the proportion of cells in the G1 phase, S phase, and G2 phase were 65.68%, 28.19%, and 6.13%, respectively, indicating the PMCs division and proliferation ability is strong.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Susceptibility of PMCs to JEV\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo examine the susceptibility of the PMCs to JEV, PMCs were seeded into 6-well plates and inoculated with vaccine-candidate strains JEV at 0.1 MOI. Firstly, the cytopathic effect was monitored during the whole infection course to 96 h. At 24 hours post-inoculation (hpi), cytopathic effects (CPE) appeared and were clearly observed under a microscope at 48 hpi (Figure 3A). The results showed that most cells were infected, indicating JEV replicates efficiently in PMCs.\u003c/p\u003e\n\u003cp\u003eSecondly, plaque forming assay was performed in JEV-infected PMCs at 36 hpi and the results showed that JEV could cause plaque in PMCs similar to Vero cells (Fig 3B, 3C). These results indicated that PMCs are susceptible to the JEV vaccine strain.\u003c/p\u003e\n\u003cp\u003eTo further evaluate the viral replication efficiency on PMCs, we compared the viral titers produced in the PMCs and Vero cell cultures over time (0\u0026ndash;96 hpi). The JEV isolates were propagated in PMCs cell cultures and reached maximal titers of 10\u003csup\u003e7.5\u003c/sup\u003e TCID50/mL at 72 hpi. In contrast, JEV propagated slowly in Vero cells and peaked at 96 h with a titer of 10\u003csup\u003e7\u003c/sup\u003e TCID50/mL(Fig 3D).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eVaccination is the major strategy for JEV prevention and inactivated-vaccine, widely used in pigs nowadays, requires a steady and safe supply of JEV production. A major weakness of JEV propagation is the viruses are usually cultivated in hamster-derived Vero cells, which may possess external factor risk. Thus, the development of a porcine-derived cell line with high viral titers will not only avoid contamination by external factors but also reduce the vaccine production cost. In this study, we developed a porcine-derived-immortalized mesangial cell line (PMCs) for JEV cultivation with higher viral titers compared with that in Vero cells and high stability, indicating\u0026nbsp;the prepared immortalized PMCs have significance for the production of pig JEV attenuated vaccines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAn infinite cell line is one of the most favored apparatuses for virus production and this can be achieved by various ways, including physical and chemical stimulations, heterologous expression of viral oncogenes, increased telomerase activity, and spontaneous formation[12]. Spontaneous immortalization is driven by chromosomal rearrangements and epigenetic changes spontaneously during cell divisions without exogenous gene or chemical involvement[12, 13]. The purification, morphology, and characteristics of immortalized PMCs are critical to evaluating the quality of the prepared cell line. Firstly, Differential digestion and limited dilution assay were performed to ensure the purification of PMCs in our research, meanwhile, the morphology and homogeneity of PMCs were observed by microscope and IFA. Then the chromosome morphology and number were confirmed by using a Video Test. Finally, flow cytometry was used to study the cell viability, which was critical for virus proliferation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe main purpose of this study was to use the immortalized PMCs for the production of porcine-derived virus vaccines and various cell lines had been developed successfully\u0026nbsp;[14-16]. JEV infection in Vero cells usually causes cell cytopathogenic effects and forms plaques[17]. When tested in PMCs, JEV also causes cytopathogenic effects and forms similar plaques in size and morphology. IFA was also conducted to observe the replication efficiency of JEV in PMCs. Another critical factor in determining the potential of vaccine apparatus is the high-yield virus production. Then the viral titers were determined at various time points. The results showed that viral titers peak at 72 hpi in PMCs with 10\u003csup\u003e7.5\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/mL while 96 hpi in Vero cells with 10\u003csup\u003e7\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/mL. Notably, the viral genome stays stable during propagation in PMCs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTaken together, the immortalized PMCs prepared in this study have normal characteristics in morphology and chromosomes and they are favorable for JEV cultivation with higher viral titers, which has significance for the production of pig JEV attenuated or inactivated vaccines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLe Flohic, G., et al., \u003cem\u003eReview of climate, landscape, and viral genetics as drivers of the Japanese encephalitis virus ecology.\u003c/em\u003e PLoS Negl Trop Dis, 2013. \u003cstrong\u003e7\u003c/strong\u003e(9): p. e2208.\u003c/li\u003e\n\u003cli\u003eZhang, J.S., et al., \u003cem\u003eGenomic sequence of a Japanese encephalitis virus isolate from southern China.\u003c/em\u003e Arch Virol, 2009. \u003cstrong\u003e154\u003c/strong\u003e(7): p. 1177-80.\u003c/li\u003e\n\u003cli\u003eSharma, K.B., S. Vrati, and M. Kalia, \u003cem\u003ePathobiology of Japanese encephalitis virus infection.\u003c/em\u003e Mol Aspects Med, 2021. \u003cstrong\u003e81\u003c/strong\u003e: p. 100994.\u003c/li\u003e\n\u003cli\u003eSu, C.L., et al., \u003cem\u003eMolecular epidemiology of Japanese encephalitis virus in mosquitoes in Taiwan during 2005-2012.\u003c/em\u003e PLoS Negl Trop Dis, 2014. \u003cstrong\u003e8\u003c/strong\u003e(10): p. e3122.\u003c/li\u003e\n\u003cli\u003eTeng, M., et al., \u003cem\u003eMolecular characterization of Japanese encephalitis viruses circulating in pigs and mosquitoes on pig farms in the Chinese province of Henan.\u003c/em\u003e Virus Genes, 2013. \u003cstrong\u003e46\u003c/strong\u003e(1): p. 170-4.\u003c/li\u003e\n\u003cli\u003eHegde, N.R. and M.M. Gore, \u003cem\u003eJapanese encephalitis vaccines: Immunogenicity, protective efficacy, effectiveness, and impact on the burden of disease.\u003c/em\u003e Hum Vaccin Immunother, 2017. \u003cstrong\u003e13\u003c/strong\u003e(6): p. 1-18.\u003c/li\u003e\n\u003cli\u003eYun, S.I. and Y.M. Lee, \u003cem\u003eJapanese encephalitis: the virus and vaccines.\u003c/em\u003e Hum Vaccin Immunother, 2014. \u003cstrong\u003e10\u003c/strong\u003e(2): p. 263-79.\u003c/li\u003e\n\u003cli\u003eLee, P.I., et al., \u003cem\u003eRecommendations for the use of Japanese encephalitis vaccines.\u003c/em\u003e Pediatr Neonatol, 2020. \u003cstrong\u003e61\u003c/strong\u003e(1): p. 3-8.\u003c/li\u003e\n\u003cli\u003eChen, T., et al., \u003cem\u003eProtective Immune Responses Induced by an mRNA-LNP Vaccine Encoding prM-E Proteins against Japanese Encephalitis Virus Infection.\u003c/em\u003e Viruses, 2022. \u003cstrong\u003e14\u003c/strong\u003e(6).\u003c/li\u003e\n\u003cli\u003eMomose, H., et al., \u003cem\u003eInduction of indistinguishable gene expression patterns in rats by Vero cell-derived and mouse brain-derived Japanese encephalitis vaccines.\u003c/em\u003e Jpn J Infect Dis, 2010. \u003cstrong\u003e63\u003c/strong\u003e(1): p. 25-30.\u003c/li\u003e\n\u003cli\u003eSong, J., et al., \u003cem\u003eMapping the Nonstructural Protein Interaction Network of Porcine Reproductive and Respiratory Syndrome Virus.\u003c/em\u003e J Virol, 2018. \u003cstrong\u003e92\u003c/strong\u003e(24).\u003c/li\u003e\n\u003cli\u003eGuo, D., et al., \u003cem\u003eEstablishment methods and research progress of livestock and poultry immortalized cell lines: A review.\u003c/em\u003e Front Vet Sci, 2022. \u003cstrong\u003e9\u003c/strong\u003e: p. 956357.\u003c/li\u003e\n\u003cli\u003eFu, Y., et al., \u003cem\u003eHigh-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.\u003c/em\u003e Nat Biotechnol, 2013. \u003cstrong\u003e31\u003c/strong\u003e(9): p. 822-6.\u003c/li\u003e\n\u003cli\u003eWang, J., et al., \u003cem\u003eConstruction of a peacock immortalized fibroblast cell line for avian virus production.\u003c/em\u003e Poult Sci, 2022. \u003cstrong\u003e101\u003c/strong\u003e(12): p. 102147.\u003c/li\u003e\n\u003cli\u003eDollery, S.J., et al., \u003cem\u003eiTIME.219: An Immortalized KSHV Infected Endothelial Cell Line Inducible by a KSHV-Specific Stimulus to Transition From Latency to Lytic Replication and Infectious Virus Release.\u003c/em\u003e Front Cell Infect Microbiol, 2021. \u003cstrong\u003e11\u003c/strong\u003e: p. 654396.\u003c/li\u003e\n\u003cli\u003eMasujin, K., et al., \u003cem\u003eAn immortalized porcine macrophage cell line competent for the isolation of African swine fever virus.\u003c/em\u003e Sci Rep, 2021. \u003cstrong\u003e11\u003c/strong\u003e(1): p. 4759.\u003c/li\u003e\n\u003cli\u003eMa, X., et al., \u003cem\u003eConstruction of a Recombinant Japanese Encephalitis Virus with a Hemagglutinin-Tagged NS2A: A Model for an Analysis of Biological Characteristics and Functions of NS2A during Viral Infection.\u003c/em\u003e Viruses, 2022. \u003cstrong\u003e14\u003c/strong\u003e(4).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4276480/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4276480/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Japanese encephalitis virus causes a huge threat to the pig-raising industry and attenuated-live vaccines are widely used to prevent JEV infection. Nowadays, primary hamster kidney cells and Vero cells are mostly used for the propagation of JEV attenuated vaccines. In this research, we constructed porcine-derived-immortalized mesangial cells (PMCs) for JEV cultivation, and their characteristics were also studied. The prepared PMCs were uniform in shape and without variation in chromosome numbers. Furthermore, PMCs showed strong division and proliferation ability, and JEV could replicate efficiently as well causing a cytopathic effect similar to that in Vero cells. The viral tiler results showed that JEV grows faster in PMCs compared with that in Vero cells. Taken together, we generated an immortalized PMC cell line competent for the cultivation of JEV, which has significance for the production of pig JEV attenuated vaccines.","manuscriptTitle":"An immortalized porcine mesangial cell line competent for the cultivation of Japanese encephalitis virus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-02 10:08:54","doi":"10.21203/rs.3.rs-4276480/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bc92cbe0-d17b-4361-8591-f502fd197be8","owner":[],"postedDate":"May 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-24T01:15:12+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-02 10:08:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4276480","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4276480","identity":"rs-4276480","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}