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Antigenic differentiation of classical swine fever vaccinal strain PAV-250 from other strains, including field strains from Mexico
Vaccine 25 (2007) 7120–7124
Antigenic differentiation of classical swine fever vaccinal strain PAV-250 from other strains, including field strains from Mexico Susana Mendoza a,∗ , Pablo Correa-Giron a,b , Edgar Aguilera a , Germ´an Colmenares b , Oscar Torres a , Tonatiuh Cruz a , Andres Romero a , Eliseo Hernandez-Baumgarten a , Abel Cipri´an a a
Coordinaci´on de Estudios de Posgrado, Facultad de Estudios Superiores-Cuautitl´an (Graduate Studies Coordination, Faculty of Superior Studies-Cuautitl´an), Universidad Nacional Aut´onoma de M´exico, Av. Primero de Mayo S/N, Campo I, Cuautitl´an Izcalli, C.P. 54700, Apartado Postal 222, Cuautitl´an Izcalli, Estado de M´exico, Mexico b CENID-Microbiolog´ıa Animal, Instituto Nacional de Investigaciones Forestales, Agr´ıcolas y Pecuarias (INIFAP), Carretera M´exico-Toluca, Km. 151/2 Cuajimalpa, Palo Alto, M´exico, D.F., C.P. 05110, Mexico Received 27 February 2007; received in revised form 5 July 2007; accepted 26 July 2007 Available online 14 August 2007
Abstract Twenty-nine classical swine fever virus (CSFv) strains were grown in the PK15 or SK6 cell lines. Antigenic differentiation studies were performed using monoclonal antibodies (McAbs), produced at Lelystad (CDI-DLO), The Netherlands. The monoclonals which were classified numerically as monoclonals 2–13. Epitope map patterns that resulted from the reactivity with McAbs were found to be unrelated to the pathogenicity of the viruses studied. Antigenic determinants were recognized by McAbs 5 and 8, were not detected in some Mexican strains; however, sites for McAb 6 were absent in all strains. The PAV-250 vaccine strain was recognized by all MAbs, except by MAb 6. Furthermore, the Chinese C-S vaccine strain was found to be very similar to the GPE− vaccine. None of the studied Mexican vaccines or field strains was found to be similar to the PAV-250 vaccine strain. © 2007 Elsevier Ltd. All rights reserved. Keywords: CSF virus; Monoclonal antibodies; PAV-250 vaccinal strain
1. Introduction Classical swine fever virus (CSFv) is a highly contagious disease of pigs affecting nervous, respiratory, digestive and reproductive systems, producing vascular lesions in endothelium and reticulo-endothelial cells. It is characterized by the presence of generalized haemorrhages and infarcts in the internal organs [1,2]. Bovine viral diarrhoea virus (BVDv), border disease virus (BDv) and CSFv belong to the pestivirus genus of the Flaviviridae family. Those viruses are both structurally and antigenically related [3–5].
∗ Corresponding author. Tel.: +52 52 56 23 20 58; fax: +52 52 56 23 20 58. E-mail address: [email protected] (S. Mendoza).
0264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2007.07.045
Immunologically, CSFv was initially considered as a monotypic virus. It was not until the development and availability of monoclonal antibodies (McAbs) that antigenic differences were detected, although differences have been previously reported regarding pathogenicity and immunogenicity [6]. Monoclonal antibodies obviate the great difficulties found in distinguishing between the various pestiviruses and allow the clear differentiation of CSFv isolates. McAbs have been recommended for CSFv identification and classification [7], and as means to differentiate between CSFv and BVDv [8]. Previously, complex cross-neutralization tests and pig inoculations were required to distinguish between the various pestiviruses. The need to distinguish between BVDv and CSFv arose during the development and production of tissue culture vaccines against CSFv. Culture contamination with BVDv from fetal calf serum was also a problem
S. Mendoza et al. / Vaccine 25 (2007) 7120–7124
for vaccine production and for diagnostic laboratories since this virus, as well as CSFv, react with polyclonal sera. Both cytopathogenic and non-cytopathogenic strains of the three viruses are included in this genus, although the CSFv generally is non-cytopathogenic [9]. Contamination of cell cultures with BVDv is a serious problem to vaccine producers since BVDv has produced outbreaks with signs that are similar to CSFv in piglets born from vaccinated sows [8,9]. Pestiviruses contain three proteins in their envelope: Erns , E1 and E2. These proteins either simply, or jointly give rise to 13 epitopes (numbered 1–13) that have been found to cluster in four regions: A–D [10]. Nishimori et al. [11] developed a different panel of monoclonal antibodies (McAbs II and IV–VI) that recognized regions A2, A3 or D, B and C. Another panel of McAbs was developed in Germany for use on tissue sections [12]. Those monoclonals were highly specific, as demonstrated by direct immunofluorescence and biotin–avidin techniques. In another study, eight McAbs were prepared from the Alfort 187 “strain” [13]. Those McAbs recognized conserved epitopes, and it was demonstrated that they were directed against major E1 envelope glycoprotein. Studies with McAbs in several countries revealed that the various isolates have conserved regions, probably those involved in adherence to the target cells, as well as variable regions that permit strain differentiation. However, the variable regions seem to have little or no bearing on pathogenicity. Field isolates are obtained from individual animals or outbreaks generally contain a mixture of several biotypes. M´exico is currently engaged in a swine fever control and eradication program. For this purpose, the country has been divided into three regions: region 1: control with intensive vaccination; region 2: eradication with vaccination (instead of eradication without vaccination); region 3: disease free phase. Because phases 1 and 2 require intensive vaccination, this restricted use of vaccine prompted the research team to perform a cooperative study with the Lelystad Institute to determine the antigenic composition of the vaccine currently in use and compare it with ones used in the recent past. We also compared the vaccinal strains with the field strains to ascertain that all are CSFv, which, therefore, would allow us to detect any field outbreaks caused by vaccine strains, or by other pestiviruses. The PAV-250 strain [2], prepared in the PK15 cell line, is a live modified viral vaccine against CSFv, that has been used in M´exico from 1979 to date (Jim´enez, personal communication). During the first 4 months of 2002, 100,645 doses of CSF were sold; 95.2% of them were the PAV-250 strain and 4.8% corresponded to the PAV-1 vaccine. This last strain, prepared in suspended cultures of pig red bone marrow, was used between 1977 and 2002. The Minnesota strain was produced in pig kidney cell cultures, and was used in M´exico prior to 1971–1995. The Chinese strain was commercialized around 1971 and used until 1997. This strain was produced in rabbits. The Japanese
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strain (GPE) appeared in the commercial literature from about 1987 and was produced in guinea pig cell cultures [14]. All those vaccines were available prior to, and during the beginning of the campaign, when outbreaks of the disease were present. The possibility of low pathogenicity wild strains should be considered. In the present study, identification of the viruses as CSFv, antigenic differences between Mexican CSFv vaccines, strains isolated from field clinical cases and reference strains were analyzed.
2. Materials and methods 2.1. Viral stains Twenty-nine strains of CSFv were supplied by the Virology Laboratory, Central Veterinary Institute (CDI-DLO)1 of Lelystad, The Netherlands. The strains were identified by the country origin as follows: (a) Germany: high virulence (H) Behring. (b) U.S.A.: BAI (H); Cornell (H); Ames (H); 331, medium virulence (M); New Lederle (H). (c) France: Alfort (H); Alfort 2.3.1 (M); Alfort 2.3.2 (M). (d) Japan: ALD (H) and GPE, low virulence (L). (e) The Netherlands: Brescia 1.1.1 (H); Brescia 2.1.1 (H); Baker A 1.2.1 (H); Henken (L); Cedipest (L). Field strains included Jongen (H) and Wild Boar (H). (f) Poland: field strains Spruit 2 (H); Jongerbreur (H). (g) Mexico: vaccinal strains included Minnesota (L); PAV-1 (L); China C-S (L) and PAV-250 (L). Field strains were VC-55 (H); VC126 (H); VC-039 (ND); VC-127 (ND). The pathogenic reference strain ALD (H) and field strains that were isolated and identified between 1996 and 1998 were furnished by one of us (Correa). Field strains and the ALD reference strains were adapted to the PK15 cell line and lyophilized for transport from Mexico to the Lelystad Institute. 2.2. Cell lines The CSF strains from the Lelystad Institute were grown in SK6 and/or PK15 cell lines [15]. The Mexican strains were titrated in SK6 and/or PK15 cells. 2.3. Viral titrations To standardize tests, all CSF strains were titrated in Leighton tube cultures of PK15 cells that contained glass cover slips. Four tubes were used for each virus dilution. Each strain was diluted 10-fold and mixed with 200,000 PK15 cells/ml. After incubation for 1 h with gentle stirring,
1 From the “Centro Nacional de Servicios de Diagn´ ostico en Salud Animal (CENASA), SAGARPA”, Santa Ana Tecamac, Mexico.
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the screw cap tubes were each inoculated with 1 ml of the various virus-cell dilutions. Screw caps were left slightly loose, and the tubes were incubated for 4 days at 37◦ in a CO2 incubator. At the end of the incubation period, each cover slip was washed with PBS, fixed with cold acetone and stained with a polyclonal antibody conjugated with fluorescein isothiocyanate. Titers were determined by the Reed and Muench method [16]. 2.4. Immunoperoxidase assay using monoclonal antibodies This study employed McAbs provided by the CDI-DLO, Lelystad. Each CSF strain was evaluated by infecting monolayer cell cultures and then, after 3 days, applying the 12 MAbs conjugated to horseradish peroxidase. The McAbs were developed against the Brescia strain [10,17] and numbered as 2–13. To determine the optimal virus dilution, 10-fold dilutions of each strain were made and four wells of a 96-well microplate were filled with 20 l of each virus dilution. Twenty microliters of a PK15 cell suspension was then added and plates were maintained for 3 days in a 5% CO2
incubator. The plates were then fixed, washed and incubated with the McAbs for 1 h; they were developed with 3-amino9-ethil-carbazole (AEC) and observed, using an inverted microscope [10,17,18]. Tests were considered positive when the cytoplasm of infected cell monolayers stained brown by the reaction product of the enzyme. When a monolayer was only partially stained, it was considered as a weak reaction. Three different technicians tested each CSFv strain consecutively and the results were then compared.
3. Results 3.1. Pestivirus identification Monoclonals 2–4, 7 and 9–11, wich recognize epitopes clustered in domains A1 and A2, identified all strains tested, while recognition of epitopes on domains A3 and D was variable (MAbs 12, 13 (W)). Common clusters were in all of the eight Mexican “strains” as CSFv. None of them reacted either with BVDv or with BDv specific MAbs, confirming both the
Table 1 Antigenic analysis of 29 CSFv “strains” using 12 monoclonal antibodies directed against epitopes of the Brescia strain
The shaded areas represent strains whose epitopes are recognized by the monoclonal antibodies, while the unshaded areas represent strains that are not recognized. (W) Weak reaction; (H) highly pathogenic strains; (M) medium pathogenicity strains; (L) low pathogenicity strains; (ND) strains whose pathogenicity was not determined.
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identity of the Mexican strains included in this study. The weak reaction of vaccine strains Minnesota and PAV-1 to monoclonal 13 is taken to identify a partly different epitope. 3.2. Monoclonal antibody differentiation of the CSF strains Results obtained with MAbs 2–13, which cover the antigenic spectrum of the Brescia strain, are shown in Table 1. Monoclonal 5 failed to recognize most European strains, except the Brescia 1.1.1, Brescia 2.1.1 2 and Baker A strains. It did not identify any of the Japanese or US viruses, but it react with three of the Mexican strains (PAV-250, VC-55 and VC-126). It weakly recognized the China C-S vaccine strain. Monoclonal 6 reacted with one US strain (Cornell), and weakly with another one (Ames). It identified the same ones from The Netherlands as monoclonal 5, and also the Wild Boar strain and the two Poland strains (Spruit 2 and Jongerbreur). Monoclonal 8 identified only two US strains (Cornell and 331), but all strains from The Netherlands. It also reacted strongly with the Mexican strains China C-S, PAV-250 and VC-127, but weakly with Minnesota and PAV-1. Monoclonal 12 recognized the German strain (Behring) and all of the US strains. Two French strains (Alfort and Alfort 2.3.1) were not recognized; however, there was a weak reaction with one French strain (Alfort 2.3.2). Mab 12 reacted strongly with Japanese strain ALD, but it failed to react with strain GPE. Four strains from The Netherlands (Brescia 1.1.1, Brescia 2.1.1, Baker A 1.2.1 and CEDIPEST) gave strong reactions, but the two polish strains (Spruit 2 and Jongerbreur) were negative. On the other hand this monoclonal reacted with all the Mexican strains. Monoclonal 13 reacted with the majority of the strains, except for two from the US (Cornell and 331) and one from The Netherlands (Jongen). However, there was a weak reaction with three Dutch strains (Henken, CEDIPEST and Wild Boar); and two Mexican strains (Minnesota and PAV-1). The PAV-250 vicinal strain’s antigenic determinants were recognized by all MAbs, except for Mab 6, which corresponds to subdomain B. This strain was distinctive from the other studied strains.
4. Discussion This study compared field and vaccinal strains of CSFv used in M´exico using an immunoassay that employed a panel of 13 peroxidase-labeled McAbs. The failure of McAbs directed to conserved epitopes of BVDv and BDv indicated the lack of contamination by these, or other pestiviruses [10,19]. The antigenic determinants of the A domain, the E2 viral glycoprotein (gp 51–54), are recognized as the most important ones for viral neutralization. Those domains were found in all of the studied strains, which confirm their identity as CSFv. The A1 subdomain of the A region, which is
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also a conserved neutralization site located in the same glycoprotein, was present in all the studied strains, as shown in Table 1 [15,17]. The McAbs used in this study were not able to differentiate between strains of high, medium or low pathogenicity. Results similar to those reported here were obtained in studies done in The Netherlands [10,19]. Results of studies carried out in Japan, and elsewhere, [11,12] are difficult to compare to ours since different terminology and different sets of monoclonals were used. In order to compare the various strains using the McAbs from different laboratories, studies would be required to be carried out in the same laboratory, using the various sets of McAbs. To assist the National Eradication Campaign in Mexico, the initial goal was to distinguish the pathogenic field strains isolated in Mexico from the vaccinal strains, using McAbs in a diagnostic tests that would differentiate them. 4.1. Strains The low pathogenicity strains (PAV-1 and PAV-250) were derived from the same source, but they had different reactivity to Mabs 5 and 6 (Table 1). This minor antigenic variation was independent of the number of passages required to adapt them to grow in cell cultures [10]. The PAV-250 vaccinal strain was the only strain studied that was not recognized by MAb 6 (domain B), but was recognized by the other MAbs, and we take this to mean that it has not been transmitted to non-vaccinated pigs, as it is not represented in the field strains. The Mexican field strains included in this study revealed heterologous reactions in their secondary epitopes that were distantly related to the conserved neutralization sites. This was not surprising since they had been isolated at different times, often with a separation of several years. This variation was restricted to secondary neutralization sites of CSFv. On the other hand the pathogenic strain ALD, which served as the source of the GPE− vaccine [11], kept most of its original antigenic identity. The reactivity of Mexican strains to MAb 12, directed to subdomain A3, was found in all strains, except the Chinese C-S strain. Epitope reactivity with domain B (MAb6), was not observed in the Mexican strains; epitopes from region C (MAbs 5 and 8) were also absent in some of these strains, e.g., PAV-1 and Minnesota. Only the PAV-250 vaccine has been used in phases 1 and 2 of the Mexican CSFv eradication campaign. This vaccine was shown to be superior to the other classical swine vaccines available in M´exico [2]. Pigs vaccinated with PAV-250, and challenged on postvaccination day 14 with virulent ALD-CSFv, did not transmit the challenge virus to susceptible pigs. Notwithstanding, there was low level shedding of virulent virus from the vaccinated pigs following challenge; however, virus shed was at levels below the infectious dose [20]. Since the McAbs used in this study were developed against the Brescia strain, the failure of some epitopes to be recognized in the Mexican vaccines and field strains suggests
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that the Mexican viruses differ from the Brescia strain. However, the Minnesota and PAV-1 vaccinal strains gave similar patterns. Patterns also were similar with other swine fever strains, but none of the studied Mexican field strains were found similar to the PAV-250 vaccinal strain. Similar findings were made in regard to the other strains examined. Therefore, PAV-250 may be differentiated from other strains using the panel of Mabs employed in this study. With the methods used in this study antigenic variation between CSFv strains/isolates could be detected; however the origin of the isolates could not be determined. In conclusion, the technology described here allows the detection of antigenic variants of CSFv if they occur in vaccinated pigs. The method also would detect the rare cases of BVD infection in pigs against vaccinated CSF.
Acknowledgements We thank DVM David Trujillo and Gabino S´anchez for their technical support, DVM Renate M. Thummler Blum and Dr.Leland Carmichel (Prof. Emeritus) for her help in the preparation of this manuscript. This study was partially financed by CONACYT 1082-PB.
References [1] Van Oirschot JT. Classical swine fever. In: Straw BE, Mengeling WL, D’Allaire S, Taylor DJ, editors. Diseases of swine. 7th ed. IA, USA: Iowa, State University Press; 1999. p. 159–72. [2] Correa-Gir´on P. Character´ısticas m´as importances de la vacuna PAV-250 y de las vacunas contra la Fiebre Porcina Cl´asica (FPC) usadas en M´exico. In: Morilla GA editor. La Fiebre Porcina Cl´asica en las Am´ericas. Symposium International Puebla-1998, INIFAP, SAGARPA, IICA, FUPPUE, M´exico, 2000. p. 301–23. [3] Terpstra C. Detection of border disease antigen in tissues of affected sheep and in cell cultures by immunofluorescence. Res Vet Sci 1978;25:350–5. [4] Westaway EG, Brinton MA, Gaidamovich SYA, Horzinek MC, Igarashi A, K¨aa¨ ri¨anen L, et al. Togaviridae. Intervirology 1985;24:125–39. [5] Francki R.I.B, Fauquet C.M, Knudson O.L, Brown F. Classification and nomenclature of viruses. Sixth Rep Int Committee Taxon Viruses 1995;4(24).
[6] Laude H. Nonarbo-togaviridae: Comparative hydrodynamic properties of the pestivirus genus. Arch Virol 1979;62:347–52. [7] Edwards S, Moening V, Wensvoort G. The development of an international reference panel of monoclonal antibodies for the differentiation of hog cholera virus from other pestivirus. Vet Microbiol 1991;29:101–8. [8] Tepstra C, Wensvoort G. Natural infections of pigs with bovine viral diarrhea virus associated with signs resembling swine fever. Res Vet Sci 1988;45:139–42. [9] Wenvoort G, Terpstra C. Bovine viral diarrhea infections in piglets from sows vaccinated against swine fever with contaminated vaccine. Res Vet Sci 1988;45:134–8. [10] Wensvoort G. Topographical and functional mapping of epitopes of hog cholera virus with monoclonal antibodies. J Gen Virol 1989;70:2865–76. [11] Nishimori T, Yamada S, Shimizu M. Production of monoclonal antibodies against classical swine fever and their use for antigenic characterization of Japanese isolates. J Vet Med Sci 1996;58: 707–10. [12] Zhou Y, Moening V, Coulibaly D, Dahle J, Liess B. Differentiation of hog cholera and bovine virus diarrhea. Zentralbl Vet 1989;36: 76–82. [13] Weiland G, Moening V, Coulibaly Z, Dahle J, Leder L, Liess B. Identification of conserved epitopes on a Hog Cholera virus protein. Arch Virol 1990;111:213–25. [14] Sasahara J, Kumagai T, Shimizu Y, Furuchi S. Field experiments of Hog Cholerae live vaccine prepared in guinea pig kidney cell culture. Natl Inst Annu Health Quart 1969;9:83. [15] Wensvoort G, Terpstra C, De Kluijver EP, Kragten C, Warnaar JC. Antigenic differentiation of pestivirus strains with monoclonal antibodies against hog cholera virus. Vet Microbiol 1989;21:9–20. [16] FAO. Organizaci´on de la Naciones Unidas para la Agricultura y la Alimentaci´on. Manual de T´ecnicas de Diagn´ostico Virol´ogico. Red de Cooperaci´on T´ecnica. 1a Ed. Santiago de Chile. Organizaci´on de las Naciones Unidas para la Agricultura y la Alimentaci´on; 1993. [17] Van Rijn PA, Bossers A, Wensvoort G, Moormann RJM. Classical swine fever virus (CSFV) envelope glycoprotein E2 containing one structural antigenic unit protects pigs from lethal CSFV challenge. J Gen Virol 1996;77:2737–45. [18] Terpstra C, Bloemraad M, Gielkens ALJ. The neutralizing peroxidaselinked assay for detection of antibody against swine fever virus. Vet Microbiol 1984;9:113–20. [19] Wensvoort G, Boonstra J, Bodzinga BG. Immunoaffinity purification and characterization of the envelope protein El of hog cholera virus. J Gen Virol 1990;71:531–40. [20] Gonz´alez C, Pijo´an C, Cipri´an A, Correa P, Mendoza S. Airborne transmission of Hog Cholera virus in susceptible pigs and the effect of vaccination with the PAV-250 HCV strain on the airborne transmisi´on of HVC. J Vet Med Sci 2001;63:991–6.
Antigenic differentiation of classical swine fever vaccinal strain PAV-250 from other strains, including field strains from Mexico Susana Mendoza a,∗ , Pablo Correa-Giron a,b , Edgar Aguilera a , Germ´an Colmenares b , Oscar Torres a , Tonatiuh Cruz a , Andres Romero a , Eliseo Hernandez-Baumgarten a , Abel Cipri´an a a
Coordinaci´on de Estudios de Posgrado, Facultad de Estudios Superiores-Cuautitl´an (Graduate Studies Coordination, Faculty of Superior Studies-Cuautitl´an), Universidad Nacional Aut´onoma de M´exico, Av. Primero de Mayo S/N, Campo I, Cuautitl´an Izcalli, C.P. 54700, Apartado Postal 222, Cuautitl´an Izcalli, Estado de M´exico, Mexico b CENID-Microbiolog´ıa Animal, Instituto Nacional de Investigaciones Forestales, Agr´ıcolas y Pecuarias (INIFAP), Carretera M´exico-Toluca, Km. 151/2 Cuajimalpa, Palo Alto, M´exico, D.F., C.P. 05110, Mexico Received 27 February 2007; received in revised form 5 July 2007; accepted 26 July 2007 Available online 14 August 2007
Abstract Twenty-nine classical swine fever virus (CSFv) strains were grown in the PK15 or SK6 cell lines. Antigenic differentiation studies were performed using monoclonal antibodies (McAbs), produced at Lelystad (CDI-DLO), The Netherlands. The monoclonals which were classified numerically as monoclonals 2–13. Epitope map patterns that resulted from the reactivity with McAbs were found to be unrelated to the pathogenicity of the viruses studied. Antigenic determinants were recognized by McAbs 5 and 8, were not detected in some Mexican strains; however, sites for McAb 6 were absent in all strains. The PAV-250 vaccine strain was recognized by all MAbs, except by MAb 6. Furthermore, the Chinese C-S vaccine strain was found to be very similar to the GPE− vaccine. None of the studied Mexican vaccines or field strains was found to be similar to the PAV-250 vaccine strain. © 2007 Elsevier Ltd. All rights reserved. Keywords: CSF virus; Monoclonal antibodies; PAV-250 vaccinal strain
1. Introduction Classical swine fever virus (CSFv) is a highly contagious disease of pigs affecting nervous, respiratory, digestive and reproductive systems, producing vascular lesions in endothelium and reticulo-endothelial cells. It is characterized by the presence of generalized haemorrhages and infarcts in the internal organs [1,2]. Bovine viral diarrhoea virus (BVDv), border disease virus (BDv) and CSFv belong to the pestivirus genus of the Flaviviridae family. Those viruses are both structurally and antigenically related [3–5].
∗ Corresponding author. Tel.: +52 52 56 23 20 58; fax: +52 52 56 23 20 58. E-mail address: [email protected] (S. Mendoza).
0264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2007.07.045
Immunologically, CSFv was initially considered as a monotypic virus. It was not until the development and availability of monoclonal antibodies (McAbs) that antigenic differences were detected, although differences have been previously reported regarding pathogenicity and immunogenicity [6]. Monoclonal antibodies obviate the great difficulties found in distinguishing between the various pestiviruses and allow the clear differentiation of CSFv isolates. McAbs have been recommended for CSFv identification and classification [7], and as means to differentiate between CSFv and BVDv [8]. Previously, complex cross-neutralization tests and pig inoculations were required to distinguish between the various pestiviruses. The need to distinguish between BVDv and CSFv arose during the development and production of tissue culture vaccines against CSFv. Culture contamination with BVDv from fetal calf serum was also a problem
S. Mendoza et al. / Vaccine 25 (2007) 7120–7124
for vaccine production and for diagnostic laboratories since this virus, as well as CSFv, react with polyclonal sera. Both cytopathogenic and non-cytopathogenic strains of the three viruses are included in this genus, although the CSFv generally is non-cytopathogenic [9]. Contamination of cell cultures with BVDv is a serious problem to vaccine producers since BVDv has produced outbreaks with signs that are similar to CSFv in piglets born from vaccinated sows [8,9]. Pestiviruses contain three proteins in their envelope: Erns , E1 and E2. These proteins either simply, or jointly give rise to 13 epitopes (numbered 1–13) that have been found to cluster in four regions: A–D [10]. Nishimori et al. [11] developed a different panel of monoclonal antibodies (McAbs II and IV–VI) that recognized regions A2, A3 or D, B and C. Another panel of McAbs was developed in Germany for use on tissue sections [12]. Those monoclonals were highly specific, as demonstrated by direct immunofluorescence and biotin–avidin techniques. In another study, eight McAbs were prepared from the Alfort 187 “strain” [13]. Those McAbs recognized conserved epitopes, and it was demonstrated that they were directed against major E1 envelope glycoprotein. Studies with McAbs in several countries revealed that the various isolates have conserved regions, probably those involved in adherence to the target cells, as well as variable regions that permit strain differentiation. However, the variable regions seem to have little or no bearing on pathogenicity. Field isolates are obtained from individual animals or outbreaks generally contain a mixture of several biotypes. M´exico is currently engaged in a swine fever control and eradication program. For this purpose, the country has been divided into three regions: region 1: control with intensive vaccination; region 2: eradication with vaccination (instead of eradication without vaccination); region 3: disease free phase. Because phases 1 and 2 require intensive vaccination, this restricted use of vaccine prompted the research team to perform a cooperative study with the Lelystad Institute to determine the antigenic composition of the vaccine currently in use and compare it with ones used in the recent past. We also compared the vaccinal strains with the field strains to ascertain that all are CSFv, which, therefore, would allow us to detect any field outbreaks caused by vaccine strains, or by other pestiviruses. The PAV-250 strain [2], prepared in the PK15 cell line, is a live modified viral vaccine against CSFv, that has been used in M´exico from 1979 to date (Jim´enez, personal communication). During the first 4 months of 2002, 100,645 doses of CSF were sold; 95.2% of them were the PAV-250 strain and 4.8% corresponded to the PAV-1 vaccine. This last strain, prepared in suspended cultures of pig red bone marrow, was used between 1977 and 2002. The Minnesota strain was produced in pig kidney cell cultures, and was used in M´exico prior to 1971–1995. The Chinese strain was commercialized around 1971 and used until 1997. This strain was produced in rabbits. The Japanese
7121
strain (GPE) appeared in the commercial literature from about 1987 and was produced in guinea pig cell cultures [14]. All those vaccines were available prior to, and during the beginning of the campaign, when outbreaks of the disease were present. The possibility of low pathogenicity wild strains should be considered. In the present study, identification of the viruses as CSFv, antigenic differences between Mexican CSFv vaccines, strains isolated from field clinical cases and reference strains were analyzed.
2. Materials and methods 2.1. Viral stains Twenty-nine strains of CSFv were supplied by the Virology Laboratory, Central Veterinary Institute (CDI-DLO)1 of Lelystad, The Netherlands. The strains were identified by the country origin as follows: (a) Germany: high virulence (H) Behring. (b) U.S.A.: BAI (H); Cornell (H); Ames (H); 331, medium virulence (M); New Lederle (H). (c) France: Alfort (H); Alfort 2.3.1 (M); Alfort 2.3.2 (M). (d) Japan: ALD (H) and GPE, low virulence (L). (e) The Netherlands: Brescia 1.1.1 (H); Brescia 2.1.1 (H); Baker A 1.2.1 (H); Henken (L); Cedipest (L). Field strains included Jongen (H) and Wild Boar (H). (f) Poland: field strains Spruit 2 (H); Jongerbreur (H). (g) Mexico: vaccinal strains included Minnesota (L); PAV-1 (L); China C-S (L) and PAV-250 (L). Field strains were VC-55 (H); VC126 (H); VC-039 (ND); VC-127 (ND). The pathogenic reference strain ALD (H) and field strains that were isolated and identified between 1996 and 1998 were furnished by one of us (Correa). Field strains and the ALD reference strains were adapted to the PK15 cell line and lyophilized for transport from Mexico to the Lelystad Institute. 2.2. Cell lines The CSF strains from the Lelystad Institute were grown in SK6 and/or PK15 cell lines [15]. The Mexican strains were titrated in SK6 and/or PK15 cells. 2.3. Viral titrations To standardize tests, all CSF strains were titrated in Leighton tube cultures of PK15 cells that contained glass cover slips. Four tubes were used for each virus dilution. Each strain was diluted 10-fold and mixed with 200,000 PK15 cells/ml. After incubation for 1 h with gentle stirring,
1 From the “Centro Nacional de Servicios de Diagn´ ostico en Salud Animal (CENASA), SAGARPA”, Santa Ana Tecamac, Mexico.
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the screw cap tubes were each inoculated with 1 ml of the various virus-cell dilutions. Screw caps were left slightly loose, and the tubes were incubated for 4 days at 37◦ in a CO2 incubator. At the end of the incubation period, each cover slip was washed with PBS, fixed with cold acetone and stained with a polyclonal antibody conjugated with fluorescein isothiocyanate. Titers were determined by the Reed and Muench method [16]. 2.4. Immunoperoxidase assay using monoclonal antibodies This study employed McAbs provided by the CDI-DLO, Lelystad. Each CSF strain was evaluated by infecting monolayer cell cultures and then, after 3 days, applying the 12 MAbs conjugated to horseradish peroxidase. The McAbs were developed against the Brescia strain [10,17] and numbered as 2–13. To determine the optimal virus dilution, 10-fold dilutions of each strain were made and four wells of a 96-well microplate were filled with 20 l of each virus dilution. Twenty microliters of a PK15 cell suspension was then added and plates were maintained for 3 days in a 5% CO2
incubator. The plates were then fixed, washed and incubated with the McAbs for 1 h; they were developed with 3-amino9-ethil-carbazole (AEC) and observed, using an inverted microscope [10,17,18]. Tests were considered positive when the cytoplasm of infected cell monolayers stained brown by the reaction product of the enzyme. When a monolayer was only partially stained, it was considered as a weak reaction. Three different technicians tested each CSFv strain consecutively and the results were then compared.
3. Results 3.1. Pestivirus identification Monoclonals 2–4, 7 and 9–11, wich recognize epitopes clustered in domains A1 and A2, identified all strains tested, while recognition of epitopes on domains A3 and D was variable (MAbs 12, 13 (W)). Common clusters were in all of the eight Mexican “strains” as CSFv. None of them reacted either with BVDv or with BDv specific MAbs, confirming both the
Table 1 Antigenic analysis of 29 CSFv “strains” using 12 monoclonal antibodies directed against epitopes of the Brescia strain
The shaded areas represent strains whose epitopes are recognized by the monoclonal antibodies, while the unshaded areas represent strains that are not recognized. (W) Weak reaction; (H) highly pathogenic strains; (M) medium pathogenicity strains; (L) low pathogenicity strains; (ND) strains whose pathogenicity was not determined.
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identity of the Mexican strains included in this study. The weak reaction of vaccine strains Minnesota and PAV-1 to monoclonal 13 is taken to identify a partly different epitope. 3.2. Monoclonal antibody differentiation of the CSF strains Results obtained with MAbs 2–13, which cover the antigenic spectrum of the Brescia strain, are shown in Table 1. Monoclonal 5 failed to recognize most European strains, except the Brescia 1.1.1, Brescia 2.1.1 2 and Baker A strains. It did not identify any of the Japanese or US viruses, but it react with three of the Mexican strains (PAV-250, VC-55 and VC-126). It weakly recognized the China C-S vaccine strain. Monoclonal 6 reacted with one US strain (Cornell), and weakly with another one (Ames). It identified the same ones from The Netherlands as monoclonal 5, and also the Wild Boar strain and the two Poland strains (Spruit 2 and Jongerbreur). Monoclonal 8 identified only two US strains (Cornell and 331), but all strains from The Netherlands. It also reacted strongly with the Mexican strains China C-S, PAV-250 and VC-127, but weakly with Minnesota and PAV-1. Monoclonal 12 recognized the German strain (Behring) and all of the US strains. Two French strains (Alfort and Alfort 2.3.1) were not recognized; however, there was a weak reaction with one French strain (Alfort 2.3.2). Mab 12 reacted strongly with Japanese strain ALD, but it failed to react with strain GPE. Four strains from The Netherlands (Brescia 1.1.1, Brescia 2.1.1, Baker A 1.2.1 and CEDIPEST) gave strong reactions, but the two polish strains (Spruit 2 and Jongerbreur) were negative. On the other hand this monoclonal reacted with all the Mexican strains. Monoclonal 13 reacted with the majority of the strains, except for two from the US (Cornell and 331) and one from The Netherlands (Jongen). However, there was a weak reaction with three Dutch strains (Henken, CEDIPEST and Wild Boar); and two Mexican strains (Minnesota and PAV-1). The PAV-250 vicinal strain’s antigenic determinants were recognized by all MAbs, except for Mab 6, which corresponds to subdomain B. This strain was distinctive from the other studied strains.
4. Discussion This study compared field and vaccinal strains of CSFv used in M´exico using an immunoassay that employed a panel of 13 peroxidase-labeled McAbs. The failure of McAbs directed to conserved epitopes of BVDv and BDv indicated the lack of contamination by these, or other pestiviruses [10,19]. The antigenic determinants of the A domain, the E2 viral glycoprotein (gp 51–54), are recognized as the most important ones for viral neutralization. Those domains were found in all of the studied strains, which confirm their identity as CSFv. The A1 subdomain of the A region, which is
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also a conserved neutralization site located in the same glycoprotein, was present in all the studied strains, as shown in Table 1 [15,17]. The McAbs used in this study were not able to differentiate between strains of high, medium or low pathogenicity. Results similar to those reported here were obtained in studies done in The Netherlands [10,19]. Results of studies carried out in Japan, and elsewhere, [11,12] are difficult to compare to ours since different terminology and different sets of monoclonals were used. In order to compare the various strains using the McAbs from different laboratories, studies would be required to be carried out in the same laboratory, using the various sets of McAbs. To assist the National Eradication Campaign in Mexico, the initial goal was to distinguish the pathogenic field strains isolated in Mexico from the vaccinal strains, using McAbs in a diagnostic tests that would differentiate them. 4.1. Strains The low pathogenicity strains (PAV-1 and PAV-250) were derived from the same source, but they had different reactivity to Mabs 5 and 6 (Table 1). This minor antigenic variation was independent of the number of passages required to adapt them to grow in cell cultures [10]. The PAV-250 vaccinal strain was the only strain studied that was not recognized by MAb 6 (domain B), but was recognized by the other MAbs, and we take this to mean that it has not been transmitted to non-vaccinated pigs, as it is not represented in the field strains. The Mexican field strains included in this study revealed heterologous reactions in their secondary epitopes that were distantly related to the conserved neutralization sites. This was not surprising since they had been isolated at different times, often with a separation of several years. This variation was restricted to secondary neutralization sites of CSFv. On the other hand the pathogenic strain ALD, which served as the source of the GPE− vaccine [11], kept most of its original antigenic identity. The reactivity of Mexican strains to MAb 12, directed to subdomain A3, was found in all strains, except the Chinese C-S strain. Epitope reactivity with domain B (MAb6), was not observed in the Mexican strains; epitopes from region C (MAbs 5 and 8) were also absent in some of these strains, e.g., PAV-1 and Minnesota. Only the PAV-250 vaccine has been used in phases 1 and 2 of the Mexican CSFv eradication campaign. This vaccine was shown to be superior to the other classical swine vaccines available in M´exico [2]. Pigs vaccinated with PAV-250, and challenged on postvaccination day 14 with virulent ALD-CSFv, did not transmit the challenge virus to susceptible pigs. Notwithstanding, there was low level shedding of virulent virus from the vaccinated pigs following challenge; however, virus shed was at levels below the infectious dose [20]. Since the McAbs used in this study were developed against the Brescia strain, the failure of some epitopes to be recognized in the Mexican vaccines and field strains suggests
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that the Mexican viruses differ from the Brescia strain. However, the Minnesota and PAV-1 vaccinal strains gave similar patterns. Patterns also were similar with other swine fever strains, but none of the studied Mexican field strains were found similar to the PAV-250 vaccinal strain. Similar findings were made in regard to the other strains examined. Therefore, PAV-250 may be differentiated from other strains using the panel of Mabs employed in this study. With the methods used in this study antigenic variation between CSFv strains/isolates could be detected; however the origin of the isolates could not be determined. In conclusion, the technology described here allows the detection of antigenic variants of CSFv if they occur in vaccinated pigs. The method also would detect the rare cases of BVD infection in pigs against vaccinated CSF.
Acknowledgements We thank DVM David Trujillo and Gabino S´anchez for their technical support, DVM Renate M. Thummler Blum and Dr.Leland Carmichel (Prof. Emeritus) for her help in the preparation of this manuscript. This study was partially financed by CONACYT 1082-PB.
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