latte是什么意思Expression of the E2envelope glycoprotein of bovine viral diarrhoea virus (BVDV)elicits virus-type specific neutralising antibodies
R.L.Toth a,1,P.F.Nettleton b ,M.A.McCrae a,*
a
Department of Biological Sciences,University of Warwick,Coventry,CV47AL,UK b Moredun Rearch Institute,International Rearch Centre,Pentlands Science Park,Bush Loan,Penicuik,Midlothian,EH260PZ,UK
Received 14May 1998;accepted 21October 1998Abstract幼稚英文
A region of genome from the NADL strain of BVDV corresponding to the coding quence for the E2glycoprotein has been molecularly cloned using RT-PCR.The viral cDNA quence was ud to construct vaccinia virus recombinants that expresd either the entire E2coding quence or fragments of it.The recombinants were ud to immuni mice of three H-2haplotypes to investigate their ability to elicit a neutralising antibody respon against BVDV .Sera from mice immunid with the recombinant expressing full length E2contained high levels of virus neutralising antibodies that in additi
on to giving neutralisation of the homologous NADL strain were also able to neutrali the Oregon C24V reference strain.The ra failed to give any neutralisation of the Osloss reference strain providing evidence for the division of BVDV isolates into at least two distinct E2rotypes.The results were confirmed in gnotobiotic lambs.Expression of E2fragments revealed the prence of at least two distinct neutralising epitopes,one of which was localid within the carboxy terminal 90amino acids of the protein.#1999Elvier Science B.V .All rights rerved.
vehicle读音Keywords :Bovine viral diarrhoea virus;Pestivirus;Vaccinia virus recombinants;Vaccine
Veterinary Microbiology 65(1999)展春园
87±101
*Corresponding author.Fax:+44-1203-524203;e-mail:malcolm@dna.bio.warwick.ac.uk 1Prent address:Department of Virology,Scottish Crop Rearch Institute,Invergowrie,Dundee.DD25DA.0378-1135/99/$±e front matter #1999Elvier Science B.V .All rights rerved.PII:S 0378-1135(98)00291-0
88R.L.Toth et al./Veterinary Microbiology65(1999)87±101
1.Introduction
Bovine viral diarrhoea virus(BVDV)is a member of the genus Pestivirus within the family Flaviviridae.The other members of the genus are classical swine fever virus (CSFV)and border dia virus(BDV)of sheep.There is a clo antigenic relationship between members of the genus,particularly BVDV and BDV(Darbyshire,1960;Plant et al.,1973).They cau a varied pattern of dias in their natural hosts,wild and domestic ruminants and pigs,and are of very significant economic importance in agriculture worldwide.The clinical signs produced by BVDV infection span a wide range,from sub-clinical through to a condition known as mucosal dia which is fatal in a majority of the cas.The virus exists in nature as two parate biotypes,classified as cytopathic(cp)and non-cytopathic(ncp)on the basis of their growth characteristics in cell culture.Thes
企业所得税怎么征收e two biotypes have been shown to be important in the aetiology of mucosal dia since it is when an animal persistently infected with the ncp virus is superinfected with an antigenically related cp isolate that the dia develops(Bolin et al.,1984;Brownlie et al.,1984).
Current control measures for BVDV are focud on preventing foetal infection.If infection with an ncp virus occurs early in gestation it can result in the birth of immunotolerant persistently infected animals which shed virus throughout their lives and are the main vehicle for maintaining the infection in cattle herds.The identification of such persistently infected animals and their culling from herds is one reasonably effective method for controlling the impact of this infection although there is still a need for more nsitive methods for detecting such animals.A number of live attenuated BVDV vaccines are available and in widespread u,although a variety of adver reactions have been reported and the current generation of live vaccines are unsafe for u in pregnant and persistently infected cattle(Bulgin,1985;Roeder and Harkness,1986;Donis and Krejci,1993).An attenuated-temperature nsitive mutant of BVDV has been isolated for u as a vaccine and this appeared to have a good degree of safety in pregnant cows (Lobmann et al.,1986)but its efficacy in preventing foetal infection has not yet been reported.Only one vaccine has been reported to protect the bovine fetus from BVDV (Brownlie et al.,1995).This is an inactivated vaccine containing a single strain of vir
us which provides relatively short-lived immunity.Further improvements in vaccine design must be predicated on a better understanding of both the antigenicity of the viral proteins and the immune correlates of protection from dia.
The application of recombinant DNA technology to pestivirus in recent years has resulted in rapid progress being made in their molecular characterisation(Collett,1992; Thiel et al.,1996).Thus the genome of BVDV has been shown to be a single-stranded positive n RNA of approximately12.5kb which encodes a single polyprotein that is proteolytically procesd during virus replication to produce a collection of viral structural and non-structural proteins(Collett,1992;Meyers and Thiel,1996).The location of the cleavage point generating the amino terminal end of E2for the NADL strain has been precily mapped as being between amino acids692and693of the single polyprotein(Rumenapf et al.,1993).The position for the carboxy terminus is more complex.Recently,it has been found that E2exists in two forms,the shorter of which is found in virus particles has its carboxy terminus at amino acid1066in strain NADL
R.L.Toth et al./Veterinary Microbiology65(1999)87±10189 (Elbers et al.,1996).The cond form,which in the related pestivirus CSFV,has been shown to be prent only in infected cells,and has the small p7protein still attached at its carboxy end,although the preci location of its carboxy tdldl
冲上云霄2歌曲erminus remains to be determined(Elbers et al.,1996).
The immunological characterisation of the virus has been somewhat slower.The E2major envelope glycoprotein is the most variable and immunodominant of the pestivirus proteins.It has been shown to elicit virus-neutralising antibodies but the exact locations and relationships of the various epitopes on the protein are not yet clearly understood(Bolin et al.,1988;Collett et al.,1989;Paton et al.,1992).It has been obrved that specific antigenic changes in the E2glycoprotein occur during a ries of alternate cattle sheep transmissions which correlated with the host from which the virus was isolated,suggesting that some virus adaptation does occur(Paton et al.,1997). Cross-neutralisation studies using ra raid against the whole virus has shown significant levels of cross-neutralisation making the division of BVDV isolates into virus rotypes problematic.Gene quence data from E2coding regions of reprentative strains have enabled pestivirus to be divided into six groups including BVDV Type I and BVDV Type II groups.Furthermore,BVDV Type I can be subdivided into subtypes BVDV Ia and Ib(van Rijn et al.,1997).In order to extend the immunological characterisation of E2and work towards achieving a better definition of its possible role in conferring protective immunity,this study has undertaken the molecular cloning of the relevant region of the BVDV genome and its expression through the construction of vaccinia virus recombinants that express either the entire E2coding quence or fragments of it.
2.Materials and methods
2.1.Cells and virus
The cytopathic NADL,Oregon C24V and Osloss reference strains of BVDV were propagated in,and titred by plaque assay on,MDBK cells using conventional methods and validated by a PCR genotyping assay(Vilcek et al.,1994).Wild-type vaccinia virus (strain WR )and vaccinia virus recombinants were propagated in CV-1cells and recombinants were initially lected for on TKÀcells(HuTK-143B)as previously described(Heath et al.,1997).All batches of foetal calf rum ud for virus propagation were screened to be free of both BVDV and anti-BVDV antibody as previously described (Dutia et al.,1990).
2.2.Antibodies
Anti-BVDV rum was raid in a calf hyperimmunid with purified BVDV(NADL strain)(Fenton et al.,1991).Three monoclonal antibodies(MAbs),WB166,WB214and WB158(Paton et al.,1992)against E2were kindly provided by D.Paton,Central Veterinary Laboratory,Weybridge,UK.
90R.L.Toth et al./Veterinary Microbiology65(1999)87±101
2.3.cDNA cloning of the E2coding quence
Cytoplasmic RNA was prepared from MDBK cells infected with the NADL strain of BVDV using the guanidinium isothiocyanate extraction method(Ausubel et al.,1990). cDNA encompassing the E2coding region was generated by RT-PCR as previously described(Xu et al.,1990)using the primer pair shown in Fig.1(A).The amplified cDNA was digested with Bgl II and Bam HI,ligated into the Bam HI site of the vaccinia shuttle plasmid pGS-62(Mackett et al.,1984)and transformed li(MC1061). Ampicillin-resistant bacterial colonies were then screened for plasmids carrying the E2 cDNA in the correct orientation with respect to the vaccinia virus p7.5promoter by restriction enzyme digestion.Three fragments of the E2cDNA encompassing different regions of the coding quence were generated by restriction enzyme digestion and sub-cloned into the vaccinia shuttle plasmid pSC11-30R2(Heath et al.,1997)which is designed to allow the expression of fragments of coding quence in vaccinia virus recombinants.
2.4.Isolation of vaccinia virus recombinants
合法的This was done by the transfection of shuttle plasmid constructs into CV-1cells infected with wild-type vaccinia virus(strain WR ),and harvesting of the progeny virus at maximum cytopathic effect.Recomb
inant virus were lected by plating on TKÀcells in the prence of BUdR(Mackett et al.,1984).Potential virus recombinants were then screened using PCR to confirm the prence of the BVDV gene.This was done by propagating picked virus plaques in CV-1cells;when significant cytoplasmic effect was evident,a cytoplasmic fraction was prepared and nucleic acid extract made from this (Johnson and McCrae,1989).A standard PCR(Saiki et al.,1988)was then carried out using one primer located in the p7.5promoter of vaccinia virus and the cond BVDV-specific primer located within the E2gene.All virus recombinants were plaque-purified three times before being grown into stocks.
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2.5.Radio-immunoprecipitation assay of E2expression
transactionImmunoprecipitation was carried out as previously described(McCrae and McCorquo-dale,1987)on cytoplasmic extracts prepared from radio-labelled CV-1cells infected with either wild type vaccinia virus or a recombinant expressing BVDV E2.35SÀmethionine labelling of virus infected cells was carried out for3h beginning4h after infection with an moi of5±10.The products of immunoprecipitation were analyd on7±13%linear gradient polyacrylamide gels as previously described(McCrae and McCorquodale, 1987).
2.6.Immunisation of mice and gnotobiotic lambs
C57BL/6(H-2b)and Balb/c(H-2d)mice were obtained from Bantin and Kingman Labs and C3H/He-mg(H-2k)mice from the breeding colony maintained at the Department of
Fig.1.RT-PCR amplification of the E2coding region of the BVDV genome.Panel A is a diagram showing the location of the E2gene within the BVDV genome and the quence of the primers ud in the RT-PCR reaction.The cross hatched region at either end of the genome map are the 5H and 3H non-coding regions.Each of the primers ud consisted of two regions,a 5H half who quence was not complementary to BVDV but contained a number of restriction enzyme recognition quences for u in subquent cDNA manipulation and a 3H half that was complementary to the BVDV strain NADL quence over the region who nucleotide co-ordinates are given and which rved to actually prime cDNA synthesis.Panel B shows a 1.5%agaro gel on which the product of RT-PCR reaction has been fractionated,stained with ethidium bromide and visualid under UV light.Track M shows the size markers with the position of the 500bp and 1Kbp bands being indicated by arrows.Track 1is a positive control for the PCR reaction in which the expected product is $500bp.Track 2shows the product of a RT-PCR reaction carried out on cytoplasmic RNA extracted from MDBK cells infected with the NADL strain of BVDV .R.L.Toth et al./Veterinary Microbiology 65(1999)87±10191
