Porvac® subunit vaccine induces neutralizing antibodies against all three main classical swine fever virus genotypes

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The current efforts to control the disease include vaccination with Porvac®, a subunit marker vaccine. Although the efficacy of Porvac ® against subgenotype 1.4 has been extensively documented, little is known about the abilty of the antibodies induced by this vaccine to neutralize other genotypes. Sera collected frm three pigs vaccinated with Porvac ® were able to efficiently neutralize CSFV strains belonging to genotypes 1, 2 and 3. Porvac ® -induced antibodies also neutralized bovine viral diarrhea virus and border disease virus. The results suggest that Porvac ® marker vaccine could be used for controlling CSF globally. Full Text Classical swine fever (CSF) is one of the main constraints to the international trade of live pigs and pork products and therefore poses one of the greatest economic impacts on the global swine industry. The etiological agent, classical swine fever virus (CSFV), is a positive-sense single-stranded RNA virus belonging to the genus Pestivirus of the family Flaviviridae [2]. Other members of the genus Pestivirus include border disease virus (BDV) and bovine viral diarrhea virus (BVDV). Historically, CSFV has been classified into three main genotypes (1-3), and each genotype into several sub-genotypes. The different sub-genotypes of CSFV follow a specific geographical distribution [12]. Six sub-genotypes have been described for genotype 1 (1.1 to 1.6), three for genotype 2 (2.1 to 2.3), and four for genotype 3 (3.1 to 3.4) [4, 15]. Recently, a new classification system, dividing CSFV strains into five genotypes (one to five) has been proposed to accommodate the strain known as "congenital myoclonus" (Great Britain/1964) and two strains from Korea (KR/1998, KR/1999). A tendency towards a shift in the circulating virus populations from genotypes 1 and 3 to genotype 2 has been reported in Europe [24] and Asia [4, 10]. While most of the genotype 1 CSFV strains are highly virulent viruses that cause acute classical swine fever, most genotype 2 viruses cause subacute or chronic CSF [6, 12]. While vaccines play an important role in the control of CSF, there has been an increased incidence of subacute and chronic manifestations of the disease in the past few decades within the geographic areas where modified live vaccines (MLV) have been applied [22]. These subacute and chronic infections are associated with atypical clinical signs, relatively low morbidity, and long duration of the disease. In Cuba, CSFV is endemic and is the main health problem of swine farming in the country [5] Since 1965, massive vaccination campaigns have been carried out using a lapinized C-strain (Labiofam strain) in Cuba. However, since 1993, swine industry in Cuba has been stricken by increasing number of CSFV outbreaks. The Cuban CSFV isolates responsible for these outbreaks were phylogenetically related to the Cuban highly-virulent ancestral strain “Margarita/1958” that was routinely used in challenge experiments for the lapinized C-strain vaccine batch release [14]. Recently, a subunit vaccine, Porvac ® , which contains CSFV E2 antigen fused with the pig CD154 as molecular adjuvant was developed. The subunit vaccine is highly immunogenic and well tolerated in pigs of all ages. It induced rapid protection, similar to that induced by the lapinized C-strain, and protected piglets against vertical transmission [9, 19, 20]. Porvac ® was licensed and is currently being used for the control of CSF in Cuba. Challenge studies and in vitro neutralization tests with sera from animals vaccinated with Porvac ® , have been carried out to date with the CSFV genotype 1 viruses, and therefore the degree of efficacy of this vaccine against viruses of other genotypes and sub-genotypes is unknown. Therefore, the objective of this study was to assess the ability of sera from pigs immunized with Porvac ® to neutralize CSFV strains belonging to other CSFV genotypes. Table 1 summarizes the information about the CSFV isolates used in the in vitro neutralization experiments. Table 1. CSFV isolates used in the neutralization experiments CSFV isolate Sub-genotype Geographic location Gene Bank Accession number Alfort/187 1.1 China, Argentina, Brazil, Colombia, México YP_009508222.1 Brescia 1.2 Europa AAA43843.1 Margarita 1.4 Cuba AFP86048.1 Paderborn 2.1 Europa,India,China,Korea AAL68894.1 2016/Pinillos 2.6 Colombia,Vietnam, China, Thailand OL963696.1 Diepholz 2.3 Europa,China AFJ79215.1 Congenital Tremor 3.1 Asia AFJ79226.1 Kanagawa 3.4 Asia AFJ79222.1 All viruses were titrated by end point dilution in the pig kidney (PK-15) cell line (ATCC CCL-33) using a Peroxidase Immunostaining Assay. The PK-15 cell was also used in the Neutralizing Peroxidase Linked Assay. The cells were grown in Dulbecco's Modified Eagle Medium (DMEM. ThemoFisher Scientific, Burlington, ON) supplemented with 10% Fetal Calf Serum (Millipore-Sigma, Oakville, ON) and antibiotics (10,000 U/ mL of penicillin/10 mg/mL streptomycin (ThemoFisher). Porvac ® subunit vaccine was kindly supplied by the Center for Genetic Engineering and Biotechnology of Camagüey (Camagüey, Cuba). The vaccine contains the chimeric E2-CD154 fusion protein, formed by the fusion of the extracellular region of the E2 glycoprotein of the CSFV “Margarita” strain and the extracellular segment of the swine CD154 molecule. The fusion protein was expressed in HEK 293 cell line (ATCC CRL1573) using lentivirus-based gene delivery system as described elsewhere [20]. E2-CD154 fusion protein was formulated in Montanide TM ISA50 V2 (SEPPIC, La Garenne-Colombes, France) using a 60/40 proportion of aqueous/oil phase and a SD-41 homogenizer (IKA, Königswinter, Germany). The concentration of E2-CD154 in the final emulsion was 25 μg/mL. Three healthy nine-week-old crossbred Duroc/Yorkshire pigs (25–30 kg) were purchased from from a non-immunized herd in Cordovanal farm (Villa Clara, Cuba). All animals were seronegative for CSFV antibodies. The animals received feed (2 kg/per animal/per day) and water ad libitum . They were ear tagged and housed in separate experimental paddocks at the National Parasitology Laboratory (Artemisa, Cuba), following the animal welfare regulations and standards established by the Animal Health Department, Ministry of Agriculture, Cuba. Vaccination experiments were conducted according to the animal welfare regulations and standards following the EU Directive 2010/63/EU and Good Clinical Practices [23]. The protocols were approved and supervised by the Ethical and Animal Welfare Committee of CIGB. The Porvac ® vaccine (50 μg of E2-CD154 in 2 ml of Montanide TM 50V2 emulsion) was injected to the three pigs intramuscularly in the neck posterior to the ear using 18 gauge 1-inch needles. A two-dose immunization schedule was followed on days 0 and 21, and serum was collected 15 days after the second immunization from the ocular plexus. The serum samples were aliquoted and stored at -20°C in 1.5 mL aliquots until use. The commercial IDEXX CSFV Ab Test (IDEXX Laboratories, Inc, Maine, USA) was used to detect the presence of antibodies against E2 protein of CSFV in the sera after vaccination. The assay was conducted according to the manufacturer´s instructions. The test sample is positive if the blocking percentage of the sample is higher than 40. The serum samples were tested for the ability to neutralize the CSFV isolates in a Neutralizing Peroxidase-Linked Assay (NPLA). The NPLA assay for the Margarita strain was conducted at CIGB as previously described [16]. The neutralization assay for CSFV Brescia, Paderbom, 2016/Pinillos, Diepholz, Congenital Tremor, Kanagawa and the pestiviruses (BDV and BVDV) was performed at the National Centre for Foreign Animal Disease (NCFAD), a WOAH reference laboratory for CSF, according to the NCFAD NPLA protocol. Neutralization against CSFV Alfort/187 strain was evaluated in both laboratories. The three pigs immunized with Porvac ® exhibited strong antibody responses against CSFV E2 protein by 15 days booster vaccination (31 days post-primary vaccination) as assessed with the competitive IDEXX ELISA (Table 2). Table 2. Anti-E2 Antibody titers in Porvac ® -immunized pigs Animals IDEXX diagnostic kit % Blocking Result 2 92.42 Positive 3 94.35 Positive 4 95.16 Positive Anti-E2 antibodies were measured with IDEXX CSFV Ab Test kit. Blocking % higher than 40 is regarded as positive. As reported before, high levels of neutralizing antibody (nAb) titers against the homologous CSFV Margarita strain were elicited days after the booster vaccination with Porvac ® (Table 3). In the NPLA experiments performed at the NCFAD, the sera from animals immunized with Porvac ® were also able to neutralize CSFV isolates belong to the six sub-genotypes representing the three main genotypes of CSFV (Table 3). The nAb titers against these viruses were above the protection threshold of 1:50 [21]. Even for the CSFV Paderbom strain, for which the lowest neutralizing titers observed, nAb titers were equal or higher than 1:320. The absence of serotypes in CSFV due to the high degree of cross-neutralization or even cross protection among isolates from different genotypes has been documented [7, 8, 12, 17, 32]. The degree of cross-neutralization between CSFV isolates can be more related to the immune status of the individual animals and the immunogenic properties of the vaccine administered than to the antigenic variation among the E2 protein of the different viruses [17]. However, the antigenic differences among CSFV isolates seems to significantly impair the efficacy of vaccines and differences in the nAb titers between the homologous virus and other more genetically distant isolates can be observed [17]. Moreover, escape mutant variants has been described in virus under strong selective antibody pressure in cell culture [10] or in the field [9, 20]. Therefore, the capacity of CSF subunit vaccines to protect against genetically distant isolates must be always assessed. Table 3. Neutralizing antibody titers against diverse CSFV strains and other pestiviruses Sera # Strains Sub-genotype 2 3 4 Back titer CSFV isolates Alfort 187 (NCFAD) (1.1) >1280 >1280 >1280 179 Alfort 187 (CIGB) (1.1) 1600 3200 3200 213 Brescia (1.2) 640 >1280 >1280 381 Margarita (1.4) 9600 12800 12800 263 Paderbom (2.1) 320 640 640 256 2016/Pinillos* (2.6) >1280 >1280 >1280 40 Diepholz (2.3) >1280 >1280 >1280 32 Congenital Tremor (3.2) >1280 >1280 >1280 32 Kanagawa (3.4) 640 >1280 >1280 55 Other pestiviruses BDV - 10 80 80 56 BVDV - 80 320 640 230 The back titer is an internal control run in parallel for each virus.* CSFV 2016/Pinillos is field isolate from 2016 CSF outbreak in Colombia [15] The antibodies elicited by Porvac ® also cross neutralized other pestiviruses, such as BVDV and BDV (Table 3). This was expected, because the presence of common epitopes among different pestivirus species is well-documented [3, 7, 11, 13]. Previous infection with BVDV primed the immune system of pigs to a challenge with CSFV, resulting in an anamnestic response [18]. Cross-protection among pestiviruses has also been documented [1, 17]. These results suggest that Porvac ® not only provides protection against CSFV but could also protect against related pestiviruses that can occasionally infect pigs. The nAb titers induced by Porvac ® were higher against BVDV as compared to BDV. BDV and BVDV display more genetic and antigenic variability than CSFV. Therefore the extent of Porvac ® -induced cross-reactivity against the viruses cannot be accurately estimated with a single isolate [8, 11]. In summary, the experimental corroboration of the ability of the sera from animals vaccinated with Porvac ® to in vitro neutralize seven additional CSFV isolates belong to genotypes 1, 2 and 3 of CSFV supports its use, not only in the Caribbean, but also in other geographic regions, where circulating CSFV isolates are more genetically distant from the vaccine strain. Declarations Acknowledgments We would like to thank all researchers and technicians from the National Parasitology Laboratory (Artemisa, Cuba) for their unconditional support in handling and caring for the animals. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript Competing Interests: The authors have no relevant financial or non-financial interests to disclose. Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Yusmel Sordo-Puga , Elaine Santana-Rodríguez, Danny Pérez-Pérez, Mary Karla Méndez-Orta, Talía Sardina-González, Milagros Vargas-Hernández and Aruna Ambagala. The first draft of the manuscript was written by Carlos A. Duarte and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. References Beckenhauer WH, Brown A, Lidolph A, Norden C (1961) Immunization of swine against hog cholera with a bovine enterovirus. Veterinary Med 56:108–112 Blome S, Staubach C, Henke J, Carlson J, Beer M (2017) Classical swine fever—an updated review. Viruses 9:86 Darbyshire J (1960) A serological relationship between swine fever and mucosal disease of cattle. Veterinary record 72 Fatima M, Luo Y, Zhang L, Wang P-Y, Song H, Fu Y, Li Y, Sun Y, Li S, Bao Y-J (2021) Genotyping and molecular characterization of classical swine fever virus isolated in China during 2016–2018. 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Proceedings of the Society for Experimental Biology and Medicine 109:349–352 Sordo-Puga Y, Suárez-Pedroso M, Naranjo-Valdéz P, Pérez-Pérez D, Santana-Rodríguez E, Sardinas-Gonzalez T, Mendez-Orta MK, Duarte-Cano CA, Estrada-Garcia MP, Rodríguez-Moltó MP (2021) Porvac(®) Subunit Vaccine E2-CD154 Induces Remarkable Rapid Protection against Classical Swine Fever Virus. Vaccines (Basel) 9 Suárez M, Sordo Y, Prieto Y, Rodríguez MP, Méndez L, Rodríguez EM, Rodríguez-Mallon A, Lorenzo E, Santana E, González N (2017) A single dose of the novel chimeric subunit vaccine E2-CD154 confers early full protection against classical swine fever virus. Vaccine Terpstra C, Wensvoort G (1988) The protective value of vaccine-induced neutralising antibody titres in swine fever. Vet Microbiol 16:123–128 Tu C, Lu Z, Li H, Yu X, Liu X, Li Y, Zhang H, Yin Z (2001) Phylogenetic comparison of classical swine fever virus in China. Virus Res 81:29–37 VICH (2000) Good clinical practices, GL09 Wonnemann H, Floegel-Niesmann G, Moennig V, Greiser-Wilke I (2001) Genetic typing of German isolates of classical swine fever virus. Dtsch Tierarztl Wochenschr 108:252–256 Cite Share Download PDF Status: Published Journal Publication published 17 Dec, 2024 Read the published version in Archives of Virology → Version 1 posted Editorial decision: Major Revision 31 Aug, 2024 Reviewers agreed at journal 01 Jul, 2024 Reviewers invited by journal 07 Jun, 2024 Editor assigned by journal 07 Jun, 2024 First submitted to journal 05 Jun, 2024 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-4535562","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":311764470,"identity":"13c071e0-810b-480b-998f-1aaf1f32ba76","order_by":0,"name":"Yusmel Sordo-Puga","email":"","orcid":"","institution":"Centro de Ingenieria Genetica y Biotecnologia","correspondingAuthor":false,"prefix":"","firstName":"Yusmel","middleName":"","lastName":"Sordo-Puga","suffix":""},{"id":311764471,"identity":"0bc45ec7-243c-43ab-ba92-891bb247b563","order_by":1,"name":"Elaine Santana-Rodríguez","email":"","orcid":"","institution":"Centro de Ingenieria Genetica y Biotecnologia","correspondingAuthor":false,"prefix":"","firstName":"Elaine","middleName":"","lastName":"Santana-Rodríguez","suffix":""},{"id":311764472,"identity":"242ba7db-d3a0-4503-97e0-85de5f9cd2c3","order_by":2,"name":"Danny Pérez-Pérez","email":"","orcid":"","institution":"Centro de Ingenieria Genetica y Biotecnologia","correspondingAuthor":false,"prefix":"","firstName":"Danny","middleName":"","lastName":"Pérez-Pérez","suffix":""},{"id":311764473,"identity":"41d02575-259d-43c1-9f8a-ccc0700b9619","order_by":3,"name":"Mary Karla Méndez-Orta","email":"","orcid":"","institution":"Centro de Ingenieria Genetica y Biotecnologia","correspondingAuthor":false,"prefix":"","firstName":"Mary","middleName":"Karla","lastName":"Méndez-Orta","suffix":""},{"id":311764474,"identity":"ada4aa44-446d-456e-a85a-3c5ca516a4f3","order_by":4,"name":"Talía Sardina-González","email":"","orcid":"","institution":"Centro de Ingenieria Genetica y Biotecnologia","correspondingAuthor":false,"prefix":"","firstName":"Talía","middleName":"","lastName":"Sardina-González","suffix":""},{"id":311764475,"identity":"6c234598-3cf0-47f8-a477-6c1ee5bd07d6","order_by":5,"name":"Milagros Vargas-Hernández","email":"","orcid":"","institution":"Centro de Ingenieria Genetica y Biotecnologia","correspondingAuthor":false,"prefix":"","firstName":"Milagros","middleName":"","lastName":"Vargas-Hernández","suffix":""},{"id":311764476,"identity":"6bcaecb8-fbcd-4492-881a-e6ab1523f706","order_by":6,"name":"Carlos A. 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The etiological agent, classical swine fever virus (CSFV), is a positive-sense single-stranded RNA virus belonging to the genus \u003cem\u003ePestivirus\u003c/em\u003e of the family \u003cem\u003eFlaviviridae\u0026nbsp;\u003c/em\u003e[2]. Other members of the genus \u003cem\u003ePestivirus\u0026nbsp;\u003c/em\u003einclude border disease virus (BDV) and bovine viral diarrhea virus (BVDV).\u003c/p\u003e\n\u003cp\u003eHistorically, CSFV has been classified into three main genotypes (1-3), and each genotype into several sub-genotypes. The different sub-genotypes of CSFV follow a specific geographical distribution [12]. Six sub-genotypes have been described for genotype 1 (1.1 to 1.6), three for genotype 2 (2.1 to 2.3), and four for genotype 3 (3.1 to 3.4) [4, 15]. Recently, a new classification system, dividing CSFV strains into five genotypes (one to five) has been proposed to accommodate the strain known as \u0026quot;congenital myoclonus\u0026quot; (Great Britain/1964) and two strains from Korea (KR/1998, KR/1999). A tendency towards a shift in the circulating virus populations from genotypes 1 and 3 to genotype 2 has been reported in Europe [24] and Asia [4, 10]. While most of the genotype 1 CSFV strains are highly virulent viruses that cause acute classical swine fever, most genotype 2 viruses cause subacute or chronic CSF [6, 12].\u003c/p\u003e\n\u003cp\u003eWhile vaccines play an important role in the control of CSF, there has been an increased incidence of subacute and chronic manifestations of the disease in the past few decades within the geographic areas where modified live vaccines (MLV) have been applied [22]. These subacute and chronic infections are associated with atypical clinical signs, relatively low morbidity, and long duration of the disease.\u003c/p\u003e\n\u003cp\u003eIn Cuba, CSFV is endemic and is the main health problem of swine farming in the country [5] Since 1965, massive vaccination campaigns have been carried out using a lapinized C-strain (Labiofam strain) in Cuba. However, since 1993, swine industry in Cuba has been stricken by increasing number of CSFV outbreaks. The Cuban CSFV isolates responsible for these outbreaks were phylogenetically related to the Cuban highly-virulent ancestral strain \u0026ldquo;Margarita/1958\u0026rdquo; that was routinely used in challenge experiments for the lapinized C-strain vaccine batch release [14].\u003c/p\u003e\n\u003cp\u003eRecently, a subunit vaccine, Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e, which contains CSFV E2 antigen fused with the pig CD154 as molecular adjuvant was developed. The subunit vaccine is highly immunogenic and well tolerated in pigs of all ages. It induced rapid protection, similar to that induced by the lapinized C-strain, and protected piglets against vertical transmission [9, 19, 20]. Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e was licensed and is currently being used for the control of CSF in Cuba.\u003c/p\u003e\n\u003cp\u003eChallenge studies and \u003cem\u003ein vitro\u003c/em\u003e neutralization tests with sera from animals vaccinated with Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e, have been carried out to date with the CSFV genotype 1 viruses, and therefore the degree of efficacy of this vaccine against viruses of other genotypes and sub-genotypes is unknown.\u003c/p\u003e\n\u003cp\u003eTherefore, the objective of this study was to assess the ability of sera from pigs immunized with Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e to neutralize CSFV strains belonging to other CSFV genotypes.\u003c/p\u003e\n\u003cp\u003eTable 1 summarizes the information about the CSFV isolates used in the \u003cem\u003ein vitro\u003c/em\u003e neutralization experiments.\u003c/p\u003e\n\u003cp\u003eTable 1. CSFV isolates used in the neutralization experiments\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.884615384615383%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCSFV isolate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91025641025641%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSub-genotype\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.37179487179487%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGeographic location\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.833333333333332%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene Bank Accession number\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.884615384615383%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAlfort/187\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91025641025641%\" valign=\"top\"\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.37179487179487%\" valign=\"top\"\u003e\n \u003cp\u003eChina, Argentina, Brazil, Colombia, M\u0026eacute;xico\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.833333333333332%\" valign=\"top\"\u003e\n \u003cp\u003eYP_009508222.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.884615384615383%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBrescia\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91025641025641%\" valign=\"top\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.37179487179487%\" valign=\"top\"\u003e\n \u003cp\u003eEuropa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.833333333333332%\" valign=\"top\"\u003e\n \u003cp\u003eAAA43843.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.884615384615383%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMargarita\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91025641025641%\" valign=\"top\"\u003e\n \u003cp\u003e1.4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.37179487179487%\" valign=\"top\"\u003e\n \u003cp\u003eCuba\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.833333333333332%\" valign=\"top\"\u003e\n \u003cp\u003eAFP86048.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.884615384615383%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePaderborn\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91025641025641%\" valign=\"top\"\u003e\n \u003cp\u003e2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.37179487179487%\" valign=\"top\"\u003e\n \u003cp\u003eEuropa,India,China,Korea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.833333333333332%\" valign=\"top\"\u003e\n \u003cp\u003eAAL68894.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.884615384615383%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e2016/Pinillos\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91025641025641%\" valign=\"top\"\u003e\n \u003cp\u003e2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.37179487179487%\" valign=\"top\"\u003e\n \u003cp\u003eColombia,Vietnam, China, Thailand\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.833333333333332%\" valign=\"top\"\u003e\n \u003cp\u003eOL963696.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.884615384615383%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDiepholz\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91025641025641%\" valign=\"top\"\u003e\n \u003cp\u003e2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.37179487179487%\" valign=\"top\"\u003e\n \u003cp\u003eEuropa,China\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.833333333333332%\" valign=\"top\"\u003e\n \u003cp\u003eAFJ79215.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.884615384615383%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCongenital Tremor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91025641025641%\" valign=\"top\"\u003e\n \u003cp\u003e3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.37179487179487%\" valign=\"top\"\u003e\n \u003cp\u003eAsia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.833333333333332%\" valign=\"top\"\u003e\n \u003cp\u003eAFJ79226.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.884615384615383%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eKanagawa\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.91025641025641%\" valign=\"top\"\u003e\n \u003cp\u003e3.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.37179487179487%\" valign=\"top\"\u003e\n \u003cp\u003eAsia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.833333333333332%\" valign=\"top\"\u003e\n \u003cp\u003eAFJ79222.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAll viruses were titrated by end point dilution in the pig kidney (PK-15) cell line (ATCC CCL-33) using a Peroxidase Immunostaining Assay. The PK-15 cell was also used in the Neutralizing Peroxidase Linked Assay. The cells were grown in Dulbecco\u0026apos;s Modified Eagle Medium (DMEM. ThemoFisher Scientific, Burlington, ON) supplemented with 10% Fetal Calf Serum (Millipore-Sigma, Oakville, ON) and antibiotics (10,000 U/ mL of\u0026nbsp;penicillin/10 mg/mL\u0026nbsp;streptomycin (ThemoFisher).\u003c/p\u003e\n\u003cp\u003ePorvac\u003csup\u003e\u0026reg;\u003c/sup\u003e subunit vaccine was kindly supplied by the Center for Genetic Engineering and Biotechnology of Camag\u0026uuml;ey (Camag\u0026uuml;ey, Cuba). The vaccine contains the chimeric E2-CD154 fusion protein, formed by the fusion of the extracellular region of the E2 glycoprotein of the CSFV \u0026ldquo;Margarita\u0026rdquo; strain and the extracellular segment of the swine CD154 molecule. The fusion protein was \u0026nbsp; expressed in HEK 293 cell line (ATCC CRL1573) using lentivirus-based gene delivery system as described elsewhere [20]. E2-CD154 fusion protein was formulated in Montanide\u003csup\u003eTM\u003c/sup\u003e ISA50 V2 (SEPPIC, La Garenne-Colombes, France) using a 60/40 proportion of aqueous/oil phase and a SD-41 homogenizer (IKA, K\u0026ouml;nigswinter, Germany). The concentration of E2-CD154 in the final emulsion was 25 \u0026mu;g/mL.\u003c/p\u003e\n\u003cp\u003eThree healthy nine-week-old crossbred Duroc/Yorkshire pigs (25\u0026ndash;30 kg) were purchased from from a non-immunized herd in Cordovanal farm (Villa Clara, Cuba). All animals were seronegative for CSFV antibodies. The animals received feed (2 kg/per animal/per day) and water \u003cem\u003ead libitum\u003c/em\u003e. They were ear tagged and housed in separate experimental paddocks at the National Parasitology Laboratory (Artemisa, Cuba), following the animal welfare regulations and standards established by the Animal Health Department, Ministry of Agriculture, Cuba. Vaccination experiments were conducted according to the animal welfare regulations and standards following the EU Directive 2010/63/EU and Good Clinical Practices [23]. The protocols were approved and supervised by the Ethical and Animal Welfare Committee of CIGB.\u003c/p\u003e\n\u003cp\u003eThe Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e vaccine (50 \u0026mu;g of E2-CD154 in 2 ml of Montanide\u003csup\u003eTM\u003c/sup\u003e 50V2 emulsion) was injected to the three pigs intramuscularly in the neck posterior to the ear using 18 gauge 1-inch needles. A two-dose immunization schedule was followed on days 0 and 21, and serum was collected 15 days after the second immunization from the ocular plexus. The serum samples were aliquoted and stored at -20\u0026deg;C in 1.5 mL aliquots until use.\u003c/p\u003e\n\u003cp\u003eThe commercial IDEXX CSFV Ab Test (IDEXX Laboratories, Inc, Maine, USA) was used to detect the presence of antibodies against E2 protein of CSFV in the sera after vaccination. The assay was conducted according to the manufacturer\u0026acute;s instructions. The test sample is positive if the blocking percentage of the sample is higher than 40.\u003c/p\u003e\n\u003cp\u003eThe serum samples were tested for the ability to neutralize the CSFV isolates in a Neutralizing Peroxidase-Linked Assay (NPLA). The NPLA assay for the Margarita strain was conducted at CIGB as previously described [16]. The neutralization assay for CSFV Brescia, Paderbom, 2016/Pinillos, Diepholz, Congenital Tremor, Kanagawa and the pestiviruses (BDV and BVDV) was performed at the National Centre for Foreign Animal Disease (NCFAD), a WOAH reference laboratory for CSF, according to the NCFAD NPLA protocol. Neutralization against CSFV Alfort/187 strain was evaluated in both laboratories.\u003c/p\u003e\n\u003cp\u003eThe three pigs immunized with Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e exhibited strong antibody responses against CSFV E2 protein by 15 days booster vaccination (31 days post-primary vaccination) as assessed with the competitive IDEXX ELISA (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Anti-E2 Antibody titers in Porvac\u003c/strong\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003cstrong\u003e-immunized pigs\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"345\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.277456647398843%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"75.72254335260115%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eIDEXX diagnostic kit\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.85496183206107%\" valign=\"top\"\u003e\n \u003cp\u003e% Blocking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"51.14503816793893%\" valign=\"top\"\u003e\n \u003cp\u003eResult\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.277456647398843%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.994219653179194%\" valign=\"top\"\u003e\n \u003cp\u003e92.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.72832369942196%\" valign=\"top\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.277456647398843%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.994219653179194%\" valign=\"top\"\u003e\n \u003cp\u003e94.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.72832369942196%\" valign=\"top\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.277456647398843%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.994219653179194%\" valign=\"top\"\u003e\n \u003cp\u003e95.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.72832369942196%\" valign=\"top\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAnti-E2 antibodies were measured with IDEXX CSFV Ab Test kit. Blocking % higher than 40 is regarded as positive.\u003c/p\u003e\n\u003cp\u003eAs reported before, high levels of neutralizing antibody (nAb) titers against the homologous CSFV Margarita strain were elicited days after the booster vaccination with Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e (Table 3). In the NPLA experiments performed at the NCFAD, the sera from animals immunized with Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e were also able to neutralize CSFV isolates belong to the six sub-genotypes representing the three main genotypes of CSFV (Table 3). The nAb titers against these viruses were above the protection threshold of 1:50 [21]. Even for the CSFV Paderbom strain, for which the lowest neutralizing titers observed, nAb titers were equal or higher than 1:320.\u003c/p\u003e\n\u003cp\u003eThe absence of serotypes in CSFV due to the high degree of cross-neutralization or even cross protection among isolates from different genotypes has been documented [7, 8, 12, 17, 32]. The degree of cross-neutralization between CSFV isolates can be more related to the immune status of the individual animals and the immunogenic properties of the vaccine administered than to the antigenic variation among the E2 protein of the different viruses [17]. However, the antigenic differences among CSFV isolates seems to significantly impair the efficacy of vaccines and differences in the nAb titers between the homologous virus and other more genetically distant isolates can be observed [17]. Moreover, escape mutant variants has been described in virus under strong selective antibody pressure in cell culture [10] or in the field [9, 20]. Therefore, the capacity of CSF subunit vaccines to protect against genetically distant isolates must be always assessed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Neutralizing antibody titers against diverse CSFV strains and other pestiviruses\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.506410256410255%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.71153846153846%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.81410256410256%\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eSera #\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.301282051282051%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.506410256410255%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.71153846153846%\"\u003e\n \u003cp\u003eStrains\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003eSub-genotype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.576923076923077%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.378205128205128%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.85897435897436%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.301282051282051%\"\u003e\n \u003cp\u003eBack titer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.506410256410255%\" rowspan=\"9\"\u003e\n \u003cp\u003e\u003cstrong\u003eCSFV isolates\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.71153846153846%\"\u003e\n \u003cp\u003eAlfort 187 (NCFAD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e(1.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.576923076923077%\"\u003e\n \u003cp\u003e\u0026gt;1280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.378205128205128%\"\u003e\n \u003cp\u003e\u0026gt;1280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.85897435897436%\"\u003e\n \u003cp\u003e\u0026gt;1280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.301282051282051%\"\u003e\n \u003cp\u003e179\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.608445297504797%\"\u003e\n \u003cp\u003eAlfort 187 (CIGB)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.961612284069098%\"\u003e\n \u003cp\u003e(1.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.667946257197697%\"\u003e\n \u003cp\u003e1600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.62763915547025%\"\u003e\n \u003cp\u003e3200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.203454894433781%\"\u003e\n \u003cp\u003e3200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.930902111324377%\"\u003e\n \u003cp\u003e213\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.608445297504797%\"\u003e\n \u003cp\u003eBrescia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.961612284069098%\"\u003e\n \u003cp\u003e(1.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.667946257197697%\"\u003e\n \u003cp\u003e640\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.62763915547025%\"\u003e\n \u003cp\u003e\u0026gt;1280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.203454894433781%\"\u003e\n \u003cp\u003e\u0026gt;1280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.930902111324377%\"\u003e\n \u003cp\u003e381\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.608445297504797%\"\u003e\n \u003cp\u003eMargarita\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.961612284069098%\"\u003e\n \u003cp\u003e(1.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.667946257197697%\"\u003e\n \u003cp\u003e9600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.62763915547025%\"\u003e\n \u003cp\u003e12800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.203454894433781%\"\u003e\n \u003cp\u003e12800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.930902111324377%\"\u003e\n \u003cp\u003e263\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.608445297504797%\"\u003e\n \u003cp\u003ePaderbom\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.961612284069098%\"\u003e\n \u003cp\u003e(2.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.667946257197697%\"\u003e\n \u003cp\u003e320\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.62763915547025%\"\u003e\n \u003cp\u003e640\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.203454894433781%\"\u003e\n \u003cp\u003e640\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.930902111324377%\"\u003e\n \u003cp\u003e256\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.608445297504797%\"\u003e\n \u003cp\u003e2016/Pinillos*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.961612284069098%\"\u003e\n \u003cp\u003e(2.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.667946257197697%\"\u003e\n \u003cp\u003e\u0026gt;1280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.62763915547025%\"\u003e\n \u003cp\u003e\u0026gt;1280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.203454894433781%\"\u003e\n \u003cp\u003e\u0026gt;1280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.930902111324377%\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.608445297504797%\"\u003e\n \u003cp\u003eDiepholz\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.961612284069098%\"\u003e\n \u003cp\u003e(2.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.667946257197697%\"\u003e\n \u003cp\u003e\u0026gt;1280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.62763915547025%\"\u003e\n \u003cp\u003e\u0026gt;1280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.203454894433781%\"\u003e\n \u003cp\u003e\u0026gt;1280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.930902111324377%\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.608445297504797%\"\u003e\n \u003cp\u003eCongenital Tremor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.961612284069098%\"\u003e\n \u003cp\u003e(3.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.667946257197697%\"\u003e\n \u003cp\u003e\u0026gt;1280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.62763915547025%\"\u003e\n \u003cp\u003e\u0026gt;1280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.203454894433781%\"\u003e\n \u003cp\u003e\u0026gt;1280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.930902111324377%\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.608445297504797%\"\u003e\n \u003cp\u003eKanagawa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.961612284069098%\"\u003e\n \u003cp\u003e(3.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.667946257197697%\"\u003e\n \u003cp\u003e640\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.62763915547025%\"\u003e\n \u003cp\u003e\u0026gt;1280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.203454894433781%\"\u003e\n \u003cp\u003e\u0026gt;1280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.930902111324377%\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.506410256410255%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eOther pestiviruses\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.71153846153846%\"\u003e\n \u003cp\u003eBDV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.576923076923077%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.378205128205128%\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.85897435897436%\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.301282051282051%\"\u003e\n \u003cp\u003e56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.608445297504797%\"\u003e\n \u003cp\u003eBVDV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.961612284069098%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.667946257197697%\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.62763915547025%\"\u003e\n \u003cp\u003e320\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.203454894433781%\"\u003e\n \u003cp\u003e640\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.930902111324377%\"\u003e\n \u003cp\u003e230\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe back titer is an internal control run in parallel for each virus.* CSFV 2016/Pinillos is field isolate from 2016 CSF outbreak in Colombia [15]\u003c/p\u003e\n\u003cp\u003eThe antibodies elicited by Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e also cross neutralized other pestiviruses, such as BVDV and BDV (Table 3). This was expected, because the presence of common epitopes among different pestivirus species is well-documented [3, 7, 11, 13]. Previous infection with BVDV primed the immune system of pigs to a challenge with CSFV, resulting in an anamnestic response [18]. Cross-protection among pestiviruses has also been documented [1, 17].\u003c/p\u003e\n\u003cp\u003eThese results suggest that Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e not only provides protection against CSFV but could also protect against related pestiviruses that can occasionally infect pigs. The nAb titers induced by Porvac\u003csup\u003e\u0026reg;\u0026nbsp;\u003c/sup\u003ewere higher against BVDV as compared to BDV. BDV and BVDV display more genetic and antigenic variability than CSFV. Therefore the extent of Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e-induced cross-reactivity against the viruses cannot be accurately estimated with a single isolate [8, 11].\u003c/p\u003e\n\u003cp\u003eIn summary, the\u0026nbsp;experimental\u0026nbsp;corroboration of the ability of the sera from animals vaccinated with Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e to \u003cem\u003ein vitro\u003c/em\u003e neutralize seven additional CSFV isolates belong to genotypes 1, 2 and 3 of CSFV supports its use, not only in the Caribbean, but also in other geographic regions, where circulating CSFV isolates are more genetically distant from the vaccine strain.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank all researchers and technicians from the National Parasitology Laboratory (Artemisa, Cuba) for their unconditional support in handling and caring for the animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by\u0026nbsp;\u003c/em\u003eYusmel Sordo-Puga\u003cem\u003e,\u0026nbsp;\u003c/em\u003eElaine Santana-Rodr\u0026iacute;guez, Danny P\u0026eacute;rez-P\u0026eacute;rez, \u003cem\u003e\u0026nbsp;\u003c/em\u003eMary Karla M\u0026eacute;ndez-Orta, Tal\u0026iacute;a Sardina-Gonz\u0026aacute;lez, Milagros Vargas-Hern\u0026aacute;ndez\u003cem\u003e\u0026nbsp;and Aruna Ambagala. The first draft of the manuscript was written by Carlos A. Duarte and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/em\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBeckenhauer WH, Brown A, Lidolph A, Norden C (1961) Immunization of swine against hog cholera with a bovine enterovirus. 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Int J Sci Res Biol Sci 9:30\u0026ndash;34\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSheffy BE, Coggins L, Baker JA (1961) Protection of pigs against hog cholera with BVD virus of cattle. Proceedings of the US Livestock Sanitary Association 65:347\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSheffy BE, Coggins L, Baker JA (1962) Relationship between hog cholera virus and virus diarrhea virus of cattle. Proceedings of the Society for Experimental Biology and Medicine 109:349\u0026ndash;352\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSordo-Puga Y, Su\u0026aacute;rez-Pedroso M, Naranjo-Vald\u0026eacute;z P, P\u0026eacute;rez-P\u0026eacute;rez D, Santana-Rodr\u0026iacute;guez E, Sardinas-Gonzalez T, Mendez-Orta MK, Duarte-Cano CA, Estrada-Garcia MP, Rodr\u0026iacute;guez-Molt\u0026oacute; MP (2021) Porvac(\u0026reg;) Subunit Vaccine E2-CD154 Induces Remarkable Rapid Protection against Classical Swine Fever Virus. 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Dtsch Tierarztl Wochenschr 108:252\u0026ndash;256\u003c/span\u003e\u003c/li\u003e\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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4535562/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4535562/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe classical swine fever is endemic and a major health problem for the swine industry in Cuba. The current efforts to control the disease include vaccination with Porvac®, a subunit marker vaccine. Although the efficacy of Porvac\u003csup\u003e®\u003c/sup\u003e against subgenotype 1.4 has been extensively documented, little is known about the abilty of the antibodies induced by this vaccine to neutralize other genotypes. Sera collected frm three pigs vaccinated with Porvac\u003csup\u003e®\u003c/sup\u003e were able to efficiently neutralize CSFV strains belonging to genotypes 1, 2 and 3. Porvac\u003csup\u003e®\u003c/sup\u003e-induced antibodies also neutralized bovine viral diarrhea virus and border disease virus. The results suggest that Porvac\u003csup\u003e®\u003c/sup\u003e marker vaccine could be used for controlling CSF globally.\u003c/p\u003e","manuscriptTitle":"Porvac® subunit vaccine induces neutralizing antibodies against all three main classical swine fever virus genotypes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-21 12:48:55","doi":"10.21203/rs.3.rs-4535562/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2024-09-01T01:36:43+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-07-02T01:35:04+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-07T11:47:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-07T07:26:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Virology","date":"2024-06-05T13:02:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a6beb0cb-aa42-4258-b86e-70285a35640e","owner":[],"postedDate":"June 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-23T16:00:36+00:00","versionOfRecord":{"articleIdentity":"rs-4535562","link":"https://doi.org/10.1007/s00705-024-06198-x","journal":{"identity":"archives-of-virology","isVorOnly":false,"title":"Archives of Virology"},"publishedOn":"2024-12-17 15:57:15","publishedOnDateReadable":"December 17th, 2024"},"versionCreatedAt":"2024-06-21 12:48:55","video":"","vorDoi":"10.1007/s00705-024-06198-x","vorDoiUrl":"https://doi.org/10.1007/s00705-024-06198-x","workflowStages":[]},"version":"v1","identity":"rs-4535562","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4535562","identity":"rs-4535562","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}