Antigenic differentiation of classical swine fever viruses in China by monoclonal antibody

Virus Research 142(2009)169–174Contents lists available at ScienceDirectVirusResearchj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /v i r u s r esAntigenic differentiation of classical swine fever viruses in China by monoclonal antibodiesYan Zhu a ,Zixue Shi a ,Trevor W.Drew b ,Qin Wang c ,Huaji Qiu d ,Huanchen Guo a ,Changchun Tu a ,∗aInstitute of Veterinary Sciences,Academy of Military Medical Sciences,1068Qinglong Road,Changchun 130062,China b Veterinary Laboratories Agency,Weybridge,Surrey KT153NB,UK cChina Institute of Veterinary Drug Control,Beijing 100081,China dDivision of Swine Infectious Diseases,National Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150001,Chinaa r t i c l e i n f o Article history:Received 25November 2008Received in revised form 12February 2009Accepted 13February 2009Available online 26February 2009Keywords:CSFV mAbsAntigenic differentiationa b s t r a c tThe phylogenetic diversity of classical swine fever virus (CSFV)in China has been extensively studied previously,with the report of the classification of Chinese CSFVs into four subgroups within two of the established genotypes,but the antigenic differences amongst Chinese CSF viruses still remain unknown.To address this issue,21CSFV field strains isolated in China between 1996and 2006were grown in cell culture and characterized in comparison with two Chinese reference strains:a virulent strain Shimen and a vaccine strain CSF lapinized virus (hog cholera lapinized virus in China,HCLV),by indirect immunoflu-orescence assay (IFA)with a panel of 28monoclonal antibodies (mAbs)against four pestiviruses,CSFV,bovine viral diarrhoea virus-1(BVDV-1),bovine viral diarrhoea virus-2(BVDV-2)and border disease virus (BDV).All 23CSFV strains reacted only with CSFV-specific mAbs,not with those raised against BVDV-1,BVDV-2and BDV.Of the former mAbs,those directed against CSFV E2protein recognized more isolates than those directed against E rns and NS2/3.Of nine CSFV E2-specific mAbs used,WH303and WH302reacted with all 23strains,confirming their value in differentiating CSFV from other pestiviruses.Fur-thermore,different strains had different patterns of reactivity with CSFV-specific mAbs,and mAbs other than WH303and WH302did not recognize all strains.This study provides the first evidence for the existence of antigenic differences among Chinese CSFVs.©2009Elsevier B.V.All rights reserved.1.IntroductionClassical swine fever (CSF)is a highly contagious febrile dis-ease of domestic and wild pigs (Moennig,2000).Outbreaks can cause heavy losses to the swine industry and hamper international trade of animals and animal products;hence,it is notifiable to the World Organisation for Animal Health (OIE,2006)and to the Euro-pean Union (Anon.,1982).The etiological agent,CSFV,is a small enveloped virus with a positive,single-stranded RNA genome.This pathogen,together with BVDV-1,BVDV-2and BDV,form the genus Pestivirus of the family Flaviviridae (Heinz et al.,2004).These four viruses are both structurally and antigenically related (Terpstra,1978;Westaway et al.,1985).Serum antibodies induced by infec-tion with one species usually cross-react with antibodies induced by other members within the same genus,thereby making the dif-ferentiation of one species from another by polyclonal antibodies very difficult (Darbyshire,1960;Wensvoort et al.,1989a,b ).This∗Corresponding author.Tel.:+8643187960009;fax:+8643187960009.E-mail address:changchun tu@ (C.Tu).characteristic also makes a clear identification of serotypes of CSFVs impossible.The diversity of CSFV can be demonstrated by phylogenetic anal-ysis and monoclonal antibody analysis,two important approaches in understanding the epidemiology of CSF.Phylogenetic analysis is a particularly valuable tool for revealing genetic interrelations and tracing the likely epidemiological origins of a particular virus fol-lowing an outbreak.In the last decade,the genetic diversity of CSFV has been extensively studied by this method,resulting in the classi-fication of CSFVs into 3major groups and 10subgroups (Paton et al.,2000).The accurate antigenic analysis of viruses has traditionally involved the use of panels of mAbs,which is a sensitive and valuable method for demonstrating the degree of antigenic diversity among viruses.Serologically,CSFV is considered monotypic,generally dis-playing only small differences within the species.From the time when mAbs against pestiviruses were first described in 1986(Peters et al.,1986;Wensvoort et al.,1986),mAb-associated antigenic dif-ferentiation has been the primary means by which CSFV isolates from different parts of the world have been antigenically character-ized (Edwards and Sands,1990;Kosmidou et al.,1995;Nishimori et al.,1996;Mendoza et al.,2007).At a more fundamental level,this0168-1702/$–see front matter ©2009Elsevier B.V.All rights reserved.doi:10.1016/j.virusres.2009.02.011170Y.Zhu et al./Virus Research142(2009)169–174technique has also been used to differentiate CSFV from other pes-tiviruses(Wensvoort,1989;Zhou et al.,1989;Nishimori et al.,1996). Edwards et al.(1991)developed a panel of30mAbs as part of a study to demonstrate the differing reactivity patterns among pestiviruses, which could be used to differentiatedfield CSFV isolates,both from vaccine strains and from other pestiviruses.This work resulted in the technique becoming a standard method for confirmatory diag-nosis of CSF.Another panel of mAbs,developed by Nishimori et al. (1996),could classify20CSF viruses into6antigenic types.Our previous phylogenetic study of isolates of CSFV revealed4 genetic subgroups(1.1,2.1,2.2and2.3)prevalent in China(Tu et al.,2001),but the antigenic diversity among Chinese CSFV isolates remained unknown.In this study we describe thefirst characteri-zation offield CSFV strains isolated in China,using an established panel of mAbs against pestiviruses.2.Materials and methods2.1.Viral strains,clinical specimens and mAbsThe Chinese virulent CSFV reference strain Shimen and currently used vaccine strain HCLV were obtained from China Institute of Veterinary Drug Control.Field strains of CSFV were obtained by iso-lation from spleens and kidneys of clinically suspect CSF pigs and were randomly dependent on their availability during outbreaks. The specimens collected between1996and1999,from10differ-ent provinces of China,were described in the previous study(Tu et al.,2001).The specimens collected between2005and2006in this study were from four provinces,three of which were different from the previous study,making a total representation of thirteen provinces throughout the country(Table1).The specimens were first subjected to RT-PCR gene amplification,followed by sequenc-ing based on procedures previously published(Tu et al.,2001). All positive tissue specimens were then subjected to virus isola-tion.The mAbs used in this study were culture supernatants of hybridoma cells,which were prepared and provided by the Vet-erinary Laboratories Agency,Weybridge,UK.2.2.Viral isolationTen-fold diluted suspensions of suspected CSF tissues were added to porcine kidney(PK-15)cell monolayers and incubated at 37◦C for72h in Minimum Essential Medium(MEM)supplementedTable1Chinesefield CSFV strains.Strain Provincial origin Year Genotype Accession No.GD6(96)Guangdong1996 2.2FJ157195GD16(98)Guangdong1998 2.3FJ157193GZ1(99)Guizhou1999 2.1FJ157198GZ2(99)Guizhou1999 1.1FJ157199 HaN1(99)Hainan1999 2.3FJ157200 HeN1(98)Henan1998 2.1FJ157204 HeN10(06)Henan2006 2.1FJ157205 HeN11(06)Henan2006 2.1FJ157206 HeN12(06)Henan2006 2.1FJ157207 HeN13(06)Henan2006 2.1FJ157208 HuN5(05)Hunan2005 2.1FJ157209 HLJ4(06)Heilongjiang2006 2.1FJ157210HLJ5(06)Heilongjiang2006 2.1FJ157211JL15(06)Jilin2006 2.1FJ157212JL16(06)Jilin2006 1.1FJ157213LN2(99)Liaoning1999 2.1FJ157201 NMG7(99)Inner Mongolia1999 2.2FJ157202NX2(99)Ningxia1999 2.1FJ157203SD1(98)Shandong1998 2.2FJ157194YN4(96)Yunnan1996 2.1FJ157196ZJ2(99)Zhejiang1999 2.2FJ157197with10%new-born calf serum(NBCS)known to be free of BVDV virus and antibody(Hyclone,Logan,UT,USA).Three consecutive blind passages were performed for each specimen supernatant. Successful isolations were confirmed at the third passage by RT-PCR and IFA using pig anti-CSFV hyperimmune serum(prepared in our laboratory)and FITC-conjugated rabbit anti-pig IgG(Sigma,St. Louis,MO,USA).2.3.Antigenic differentiation by mAb staining and indirectimmunofluorescence assay(IFA)Ninety-six well microtitre plates(Costar,NY,USA)were seeded with PK-15cells in MEM+10%NBCS and cultivated at37◦C in5% CO2overnight to70–80%confluence.The cultures were then inoc-ulated with20-fold dilutedfield CSFV isolates at passage3,with rows left uninfected,to act as negative controls,and incubated at 37◦C in a5%CO2atmosphere.Each plate was infected with only one strain.The virulent strain Shimen(100TCID50/well)was cultured in PK-15cells in a similar fashion,while the HCLV was cultured in primary newborn(1–3days old)calf testicle cells since HCLV is not porcine cell line-adapted.After72h,the plates werefixed in80% cold acetone in PBS,then washed and incubated with mAbs at a 50-fold dilution.To ensure accuracy in antigenic differentiation,all mAbs were applied simultaneously to one plate,with two replicate infected wells(experimental)and one uninfected well(negative control)for each mAb.Following washes with PBS,FITC-conjugated goat anti-mouse IgG(Sigma,St.Louis,MO,USA)was added to the plates,followed by a1-h incubation in a37◦C humid box.After 3washes with PBS,50%glycerol in PBS was added to each well. Two infected wells were similarly treated,but using pig anti-CSFV hyperimmune serum and a porcine FITC conjugate,as a positive control to confirm viral growth.Fluorescence was observed using an Axioskop-40fluorescence inverted microscope(Zeiss,Germany).2.4.Phylogenetic analysisSequence comparisons and phylogenetic analyses of all iso-lated viruses and reference strains were based on190bp of the E2gene,using the methodology and,for specimens col-lected between1996and1999,the data,as published previously (Tu et al.,2001).The phylogenetic analyses were conducted using the neighbour-joining algorithm of MEGA software version 3.1(/)and the maximum parsi-mony method of PHYLIP software version 3.63(Seattle,WA, USA).The tree was estimated statistically by1000replicates of the bootstrap value and visualized by the TREEVIEW program (/rod/treeview.html).3.Results and discussion3.1.Virus isolationIn our study,63clinically suspect CSF isolates showed positive amplification of CSFV E2gene in RT-PCR,but only21of them per-mitted successful viral isolation,resulting in a harvest of21field strains representing13provinces and spanning the decade from 1996to2006(see Table1).About two-thirds of specimens failed viral isolation,possibly because the long storage at−20◦C would have greatly decreased the amount of viable virus and therefore the likelihood of isolation.In addition,many specimens had been freeze–thawed more than three times.After isolation,eachfield strain at passage3grew successfully in PK-15cells,with extensive staining by IFA using pig anti-CSFV hyperimmune serum,as well as a strong signal in RT-PCR.Therefore,the third passages offield strains in cell cultures were used for antigenic differentiation.172Y.Zhu et al./Virus Research142(2009)169–1743.2.Antigenic differentiationIn the present study,we have shown the antigenic differentia-tion of Chinese CSFVs by using28mAbs respectively against BVDV, BDV and CSFV.All21Chinesefield CSFV strains from13provinces, as well as two reference strains,were recognized by CSFV-specific mAbs but not by mAbs specific to ruminant pestiviruses(4mAbs against BVDV-1,3mAbs against BVDV-2and2mAbs against BDV: see footnote in Table2.).Table2reveals antigenic differentiation of Chinese CSFVs and summarizes their reactive patterns with19 CSFV-specific mAbs.In the positive reactions,each CSFV strain showed different levels of intensity of staining with different mAbs,which could be divided into strong(+++),moderate(++)and weak(+)reactions based on the intensity of immunofluorescence staining.Among these CSFV-specific mAbs,anti-E rns mAbs WH178and WH179detected no strains and mAbs WH174,WH180and WH 186detected only one strain.The other14mAbs reacted varyingly, providing unique reaction patterns with all23CSFV strains,indi-cating antigenic variation among the epitopes that they recognize. The anti-E2mAbs recognized more CSFV strains than the anti-E rns and anti-NS2/3mAbs.Glycoprotein E2is the major immunogenic protein of CSFV with antigenic epitopes located in four distinct anti-genic domains on E2protein(Wensvoort,1989).Of the9anti-E2 mAbs,WH303and WH302recognized all23strains,indicating that their epitopes on the E2protein are highly conserved and present in all CSFV strains.This confirms the conclusion of Edwards et al. (1991)that these two mAbs are valuable for specific and sensitive confirmation of pestivirus isolates as CSFV.A study by Lin et al. (2000)showed that the epitope recognized by mAb WH303isCSFV-Fig.1.Phylogenetic analysis of21Chinesefield strains with reference strains by comparison of190bp of the E2gene,using the methodology published previously(Tu et al.,2001).Twenty-five reference sequences were retrieved from GenBank and are:Shimen(U72047),HCLV(U72048),Alfort(J04358),Breseia(M31768),C1W(L36164),C2W (L36165),C3D(L36166),C4D(L36167),C6W(L36168),N5W(L36169),S7D2(L36171),S7D(L36170),S8D(L36172),ALD(D49532),GPE-(D49533),CAP(X96550),Weybridge (X71780),Riems(U45477),KoreaYI990(AF521710),Korea96940(AF521711),Korea88039(DQ656345),JJ9811(AF521708),TWN/93(AY571088),Taiwan(U03290),RBR88 (AF241618).Y.Zhu et al./Virus Research142(2009)169–174173specific and mapped to829–837aa of E2protein(TAVSPTTLR).This epitope is highly conserved and has not shown any genetic variation in the comparison of all CSFV E2sequences of group1and2viruses available to date from GenBank(data not shown).This epitope of WH303is a key antigenic site,since it is a determinant not only for neutralization(Liu et al.,2006),but it has also been shown that engineered changes of this site alter virulence(Risatti et al.,2006). Interestingly a CSF genotype3.3virus,Thai strain CBR/93(Sakoda et al.,1999)did not efficiently bind WH303and has the antigenic motif TAVSSTTLER,rather than TAVSPTTLER at that binding site and this isolate exhibits a somewhat different disease presentation(Everett et al.,submitted for publication).Thisfinding indicates that fur-ther study is warranted to explore the sequence diversity of the WH303epitope among genotype3viruses which are present in some countries in Asia(Sakoda et al.,1999).Anti-E2mAbs WH187,WH310,WH306and WH211detected 69.5–86.9%of strains,indicating that their epitopes are present in most Chinese CSFV strains.In contrast,the epitopes recognized by the remaining three anti-E2mAbs(WH220,WH308and WH304) may be rare or absent from the majority of Chinese strains.This is in contrast to past studies using these mAbs,where the majority of CSFV isolates were recognised(Edwards and Sands,1990).The anti-E rns mAbs and anti-NS2/3mAb detected a quite few field strains which reacted with only one or two anti-E rns mAbs. Of the anti-E rns mAbs,WH217recognized six strains.Interestingly, the reference strains Shimen and HCLV reacted with a broader spec-trum of anti-E rns mAbs,showing a unique antigenic type which may have potential use in differentiation fromfield viruses.Nishimori et al.(1996)reported that the application of20mAbs raised against CSFV virulent strain ALD or vaccine strain GPE—could classify20 Japanese CSFVs into6antigenic types and discriminate between vaccine strains andfield isolates.Edwards and Sands,1990used19CSFV-specific mAbs in a peroxidase-linked immune binding assay to determine the reac-tion patterns of CSFV isolates from Europe,Brazil,USA,Japan and Malaysia.Six of these mAbs reacted with all CSFV isolates,while the rest13recognized some isolates with a link between geographical location and reactivity pattern.In the present study,however,the21field strains were isolated from specimens collected between1996 and2006from13provinces and the antigenic differentiation did not show geographical reactivity patterns as observed by Edwards and Sands,1990.This probably reflects the endemic nature of the disease in China and the regular movements of pigs and pig meat that occur between provinces.3.3.Phylogenetic typing and mAb typingThere is only one serotype of CSFV but three major genetic groups do exist(Edwards et al.,1991;Paton et al.,2000).In order to assess the correlation between mAb typing and genetic typing, a phylogenetic analysis of the21field strains was performed,also comparing them with reference strains representing3major groups (Paton et al.,2000).Results showed that allfield CSFVs could be classified within four subgroups(subgroups1.1,2.1,2.2,and2.3: see Fig.1),which represent current genotypes circulating in China (Tu et al.,2001).No group3viruses were found.We found that some field strains placed distantly in mAb typing(Table2),although they grouped closely by genotype(e.g.,GZ-2/99and JL-16/06in sub-group1.1,GD16/98and HaN1/99in subgroup2.3,GD6/96,ZJ2/99 and SD1/98in subgroup2.2,HeN1/98and JL15/06in subgroup 2.1;see Fig.1).In contrast,JL-16/06and YN-4/96had the same mAb spectrum recognizing them,but belong to different genetic groups.While some isolates of the same genotype showed the same mAb spectrum(e.g.,HLJ4/06,HLJ5/06,HeN11/06,HeN12/06 and HeN13/06were grouped subgroup2.1and had the same mAb reactivity pattern),the diversity of reactivity among the isolates,taken as a whole,lead us to conclude that there is no correlation between mAb typing and genetic typing.Therefore these two meth-ods should be considered as two independent approaches to the analysis of CSFV diversity.In the published literature,a relatively small number offield CSFVs have been characterized antigenetically using different pan-els of mAbs.It is difficult to compare these studies,because a panel of well-defined standard mAbs was not used.In contrast, genetic typing can allow a standardised approach,using the same regions on the viral genome to compare CSFVs isolated in differ-ent geographic regions or countries,also with the ability to trace the putative origin of isolates by their degree of genetic similarity. Nowadays,field CSFVs are usually subjected to genetic typing upon their detection in outbreaks and hundreds of CSFVs have been phy-logenetically analyzed,resulting in a global view of CSFV diversity. We conclude that monoclonal antibody typing is an ideal method to study the antigenic diversity among CSFV isolates within a country or region,but genetic typing is more suitable forfield epidemiology studies,and therefore more widely used.AcknowledgementsThis work was supported by the following grants to CT:two Chinese National Scientific and Technological Supporting Programs (Nos.2006BAD06A03and2006BAD06A18),and the National973 Program(No.2005CB523200).TD was funded by the UK govern-ment in his OIE role,under Defra Project SV3000.ReferencesAnon.,1982.Council Directive82/894/EEC of21December1982on the notification of animal diseases within the Community.Official Journal L378,0058-62. 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