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Restriction endonuclease and monoclonal antibody analysis of Brazilian isolates of bovine herpesviruses types 1 and 5

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Restriction endonuclease and monoclonal antibody analysis of Brazilian isolates of bovine herpesviruses types 1 and 5
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  Restriction endonuclease and monoclonalantibody analysis of Brazilian isolatesof bovine herpesviruses types 1 and 5 R.C.F. D’Arce a , R.S. Almeida a , T.C. Silva b , A.C. Franco b ,F. Spilki b , P.M. Roehe b , C.W. Arns a,* a  Departamento de Microbiologia e Imunologia, Instituto de Biologia, UniversidadeEstadual de Campinas, Caixa Postal 6109, CEP 13081-970 Campinas, SP, Brazil b  DM/ICBS-UFRGS & CPVDF-FEPAGRO, Estrada do Conde 6000,CEP 92990-000 Eldorado do Sul, RS, Brazil Received 28 May 2001; received in revised form 10 June 2002; accepted 10 June 2002 Abstract Twelve Brazilian isolates and threereferencestrains of bovine herpesviruses(BHVs)weresubjectedto restriction endonuclease analysis (REA) and monoclonal antibody (MAb) analysis. Viral DNAwascleaved with  Bam HI,  Bst  EII,  Eco RI,  Hin dIII and  Pst  I. The monoclonal antibody panel allowed thedifferentiation between types 1 and 5 viruses, while REA with  Bst  EII and  Hin dIII showed thedistinction between BHV-1 and -5 subtypes. Typical 1.1 and 1.2a patterns were observed with twoisolates from respiratory disease. An isolate from semen of a clinically healthy bull displayed 1.2bprofile, whereas another displayed a clear 5a pattern, which was never reported before. Seven out of nine Brazilian type 5 (BHV-5) isolates displayed REA patterns similar to the Australian BHV-5 strainN569 (BHV-5a), and differing from the Argentinean A663 strain (BHV-5b) virus. Another two BHV-5isolates, which displayed an unusual MAb pattern of reactivity, showed a  Bst  EII profile different fromboth reference strains of BHV-5. These two viruses were considered BHV-5 ‘‘non-a/non-b’’ subtype. # 2002 Elsevier Science B.V. All rights reserved. Keywords:  Bovine herpesvirus type 1 (BHV-1); Bovine herpesvirus type 5 (BHV-5); Restriction endonucleaseanalysis; Monoclonal antibodies 1. Introduction Bovine herpesvirus type 1 (BHV-1), the cause of infectious bovine rhinotracheitis/ infectious pustular vulvovaginitis (IBR/IPV), is a member of the family  Herpesviridae , Veterinary Microbiology 88 (2002) 315–324 * Corresponding author. Tel.:  þ 55-19-3788-6267; fax:  þ 55-19-3788-6276. E-mail address:  arns@obelix.unicamp.br (C.W. Arns).0378-1135/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved.PII: S0378-1135(02)00126-8  subfamily  Alphaherpesvirinae  associated with a number of different clinical syndromes incattle. In contrast, bovine herpesvirus type 5 (BHV-5), previously designated as BHV-1.3,is the agent of bovine herpesvirus encephalitis (Bulach and Studdert, 1990; Roizman et al.,1992). BHV-1 and -5 are closely related, with an overall genomic similarity of about 85%(Engels et al., 1986). BHV-1 is acknowledged worldwide as a major pathogen for cattle(Weiblen et al., 1996), causing syndromes that vary from mild respiratory disease toreproductive disorders and, occasionally, encephalitis (d’Offay et al., 1993; Roels et al.,2000), with high morbidity and low lethality rates. On the other hand, disease caused byBHV-5 is characterized by a non-purulent meningoencephalitis, usually affecting cattle upto 8 months of age (Bulach and Studdert, 1990), sometimes affecting older animals, withlow morbidity and high lethality. BHV-5 disease appears to be very restricted in itsgeographical distribution, being for as still undetermined reasons, more often detected inthe southern than in the northern hemisphere.The differentiation between BHV-1 and -5 has largely relied upon the clinico-epide-miological characteristics of outbreaks, usually followed by restriction endonucleaseanalysis (REA) of viral DNA (Sreenivasa et al., 1996; Pidone et al., 1999) sometimesaccompanied by antigenic analyses with monoclonal antibodies (Metzler et al., 1985,1986). REA profiles have additionally led to the subtyping of viruses apparently belongingtothe sametype,butassociatedwithspecificclinicalsyndromes(Engelsetal.,1981;Misraet al., 1983). Thus, subtype 1 (BHV-1.1) has been proposed to group classical respiratoryBHV-1 strains; subtype 2a (BHV-1.2a) refers to viruses associated with IBR/IPV andabortions, whereas, subtype 2b (BHV-1.2b) has been applied to group isolates that causevulvovaginitis or balanopostitis, but are not usually associated with abortions (Miller,1991).The lack ofinformation on bovineherpesviruses in Brazil, allied to a growingnumber of herpesviruses isolated from cases of encephalitis in certain cattle farming regions in thecountry, has prompted the present study with the aim to compare the genomes of BrazilianBHV-1 and -5 isolates, and to examine the correlation between REA patterns, monoclonalantibody profiles of reactivity and clinical signs observed in affected animals. 2. Materials and methods 2.1. Viruses and cells The strains Los Angeles (LA), N569 and A663,representativesof BHV-1.1, -5a and -5b,respectively (Metzler et al., 1985, 1986; Roizman et al., 1992) were used as referenceviruses for REA. Oxford BHV-1.2b reference strain (Edwards et al., 1990) and the BHV-5EVI 088/95 Brazilian strain were utilized for monoclonal antibody (MAb) production.Putative Brazilian BHV-1 and -5 strains were obtained by isolation from specimens of affected animals submitted to necropsy. Viruses were isolated from ours and otherlaboratories throughout the country (Table 1). All viruses were cultured in Madin–Darbybovine kidney (MDBK) cells grown in Eagle’s minimum essential medium (Cultilab 1 )containing 6% heat inactivated fetal calf serum, free of BHV-1 and -5 viruses andantibodies. 316  R.C.F. D’Arce et al./Veterinary Microbiology 88 (2002) 315–324  2.2. Extraction of viral DNA Viruses were inoculated in roller bottles (850 cm 2 ) with nearly confluent, overnightgrown MDBK monolayers, at a multiplicity of infection of 0.01–0.1 and incubated at37  8 C. When cytopatic effect reached 80–90% (36–48 h), the culture medium was clarifiedby low speed centrifugation and the supernatant centrifuged at 100,000  g  for 1 h. Theviral pellet was re-suspended in TE (10 mM Tris, 1 mM EDTA; pH 7.6). The viralsuspension was treated with 0.5% sodium dodecyl sulfate and 0.1 mg/ml proteinase K for1 h at 37  8 C. After digestion, the viral DNA was extracted once with an equal volume of phenol:chloroform (1:1), once with chloroform:isoamyl alcohol (24:1) and precipitatedwith cold ethanol. 2.3. Restriction endonuclease analysis Viral DNA was cleaved with restriction enzymes  Bam HI,  Bst  EII,  Eco RI,  Hin dIII and Pst  I under the conditions recommended by the manufacturer (Life Technologies 1 ). Thedigestion products were separated by electrophoresis in 0.6% agarose gels at 30 V usingTAE electrophoresis buffer (4 mM Tris-acetate, 1 mM EDTA; pH 8.0). The gels werestained with ethidium bromide (1  m g/ml) and photographed under UV light. 2.4. Monoclonal antibody analysis Viruses were examined in an immunoperoxidase monolayer assay (IPMA) on infectedcell monolayers with a panel of monoclonal antibodies to BHV-1 and -5 antigens. TheseMAbs were provinient of previous studies in our laboratory. MAbs 2G5, 5A5, 7F12 and11H6 were prepared against Oxford BHV-1.2b reference strain, isolated from a IBR case. Table 1BHV-1 and -5 isolates and proposed designations based on REAViral strain Clinical signs Origin (state/country) ClassificationLos Angeles a Rhinotracheitis CA/USA BHV-1.1EVI 123/98 Rhinotracheitis MS/Brazil BHV-1.1SV 265/96 Rhinotracheitis RS/Brazil BHV-1.2aOO9 Asymptomatic b RS/Brazil BHV-1.2bV175 Asymptomatic b RS/Brazil BHV-5aA663 a Encephalitis BA/Argentina BHV-5bAA 05 Encephalitis PR/Brazil BHV-5aSV 136/88 Encephalitis RS/Brazil BHV-5aEVI 88/95 Encephalitis MS/Brazil BHV-5aEVI 340/96 Encephalitis MS/Brazil BHV-5aISO 99/292 Encephalitis PA/Brazil BHV-5aP 96/169 Encephalitis RJ/Brazil BHV-5aISO 97/45 Encephalitis RJ/Brazil BHV-5 c ISO 97/87 Encephalitis MG/Brazil BHV-5 ca Reference strains. b Clinically healthy bull. c ‘‘Non-a/non-b’’ subtype.  R.C.F. D’Arce et al./Veterinary Microbiology 88 (2002) 315–324  317  The BHV-5 EVI 088/95 Brazilian strain, isolated from a bovine encephalitis case, wasutilized to produce the MAbs BBD10, 2B6, 2C3, 2C5, 3A2, 3B12, 3D2, 3D11, 5E2 and6A2. This isolate presented a reactivity pattern compatible to BHV-5, when tested aganistprevious MAbs produced (Roehe et al., 1997). All the MAbs have their neutralizingcapacity tested by virus neutralization. 3. Results The clinical patterns of isolates examined in this study are given in Table 1. All werefrom the cases of rhinotracheitis or encephalitis, apart from the two isolates from clinicallyhealthy bull semen.The REA patterns obtained after digestion of each isolate with five restriction enzymesused,areasshowninFig.1.Allenzymesallowedthedifferentiationbetweentypes1and5.However,  Bam HI,  Eco RI and  Pst  I enzymes gave rise to patterns that did not show a cleardistinction between subtypes (Fig. 1). Within type 1, isolates could be further subtypedaccording to REA profiles observed with  Hin dIII (Figs. 1, 2 and 4). Subtyping of BHV-5was possiblewith  Bst  EII, the only enzyme that gave rise to clearly distinguishable patternsbetween type 5 strains (Figs. 1, 3 and 4).The monoclonal antibody profile of reactivity of Brazilian BHV strains examined in thisstudy is summarized in Table 2. Three MAbs (5A5, 7F12 and 11H6) reacted just to BHV-1strains. One MAb (BBD10) recognized all BHV-5 strains tested, but also reacted with LABHV-1 strain. Finally, the other five MAbs (2C5, 3A2, 3D2, 6A2 and 2G5) recognized all Fig. 1. Restriction endonuclease digestion patterns of BHV-1 and -5 strains. M, molecular weight marker; lane1, BHV-1.1 LA; lane 2, BHV-1.2a SV265/96; lane 3, BHV-5b A663; lane 4, BHV-5a SV136/88. The enzymesutilized are shown below in the figure. Arrows indicate the differences between BHV subtypes.318  R.C.F. D’Arce et al./Veterinary Microbiology 88 (2002) 315–324  Fig. 2. Differences found in  Hin dIII cleavage patterns of BHV-1 subtypes. M, molecular weight marker; lane 1,BHV-1.1 LA; lane 2, BHV-1.2a SV265/96; lane 3, BHV-1.2b 009. Arrows indicate the differences between BHVsubtypes.Fig. 3. (A)  Bst  EII cleavage patterns of BHV-5a subtypes. M, molecular weight marker; lane 1, EVI 340/96; lane2, V175; lane 3, N569. (B) Differences found in  Bst  EII cleavage patterns of BHV-5a and ‘‘non-a/non-b’’subtypes. M, molecular weight marker; lane 1, BHV-5a AA 05; lane 2, BHV-5a SV 136/88; lane 3, BHV-5‘‘non-a/non-b’’ ISO 97/45; lane 4, BHV-5 ‘‘non-a/non-b’’ ISO 97/87; lane 5, BHV-5a P96/169; lane 6, BHV-5aISO 99/292. Arrows indicate the differences between BHV subtypes.  R.C.F. D’Arce et al./Veterinary Microbiology 88 (2002) 315–324  319
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