鸭肿头败血症病毒的生物学特性

:20091016:(IRT0848),:(1964),,蒋文灿1,2,盛敏1(1.,625014;2.,625014):为了进一步明确近年来在四川广汉华阳等养鸭地区流行的鸭肿头败血症(Duckswollenheadsepticemia)的病原特征,对分离的鸭肿头败血症病毒(Duckswollenheadsepticemiavirus,DSHSV)XD株NF株进行培养特性毒力形态理化性质与鸭瘟病毒抗原相关性等方面的生物学特性研究结果证明:该病毒能在鸭胚上繁殖并传代,不能在鸡胚鸭胚成纤维细胞鸡胚成纤维细胞上繁殖鸭胚半数致死量为10-5.78/0.2mL病毒粒子呈球形,大小65~70nm,无囊膜核酸类型为dsRNA基因组大于19kb病毒不能凝集鸡鸭鹅兔绵羊红细胞对氯仿不敏感,对热有一定抵抗力,对酸碱有较强抵抗力与鸭瘟病毒无抗原相关性:鸭;鸭肿头败血症病毒;生物学特性:S855.3:A:10054545(2010)10130706BiologicalspecialityofduckswollenheadsepticemiavirusJIANGWencan1,2,SHENGMin1(1.CollegeofAnimalMedicine,SichuanAgriculturalUniversity,Ya!an,Sichun625014,China;2.KeyLaboratoryMedicineofAnimalDiseaseandHumanHealthofSichuanProvince,Ya!an,Sichun625014,china)Abstract:InordertothoroughlyunderstandthepathogenycharacteristicofduckswollenheadsepticemiadiseaserecentlyoccurredintheGuanghanandHuayangofsichuanprovince,Ducksollenheadsepticemiavirus(DSHSV)XDandNFhasstudiedontheculturecharacristic,virulence,form.physicalandchemicalcharacters,antigenrelativitywithduckplaguevirus.Theresultprovedthattheviruscouldbeculturedinduckembryo,butcould'tgrowinchickembro,DEF(duckembrofibroblast)andCEF(chickembrofibroblast).TheDELD50oftheviruswas10-5.78/0.2mL.Theviruswasround,noenvelope,about6570nmindiameterbyelectronicmicroscope.ThenucleicacidoftheviruswasdoublestrandRNA.Thewholegenomeoftheviruswasbiggerthan19kb.ItcouldnothemagglutinateCRC(cockredcell),DRC(duckredcell),GRC(gooseredcell),RRC(rabbitredcell),SRC(sheepredcell),andhasnosensitivitytochloroform.It'svirulencehadnoobviouschangewith0.25%trypsinaseintwohours.Italsocouldresistheat,acidandalkalienvironment.Therewasnoantigenrelativitybetweenduckswollenheadsepticemiavirusandduckplaguevirus.Keywords:duck;duckswollenheadsepticemiavirus;biologicalspeciality2000,,,,,90%~100%,70%~90%,,,,,,,,,,(Duckswollenheadsepticemia,DSHS)[1~3],,1材料与方法1.1(Duckswollen13072010103010ChinJVetSciOct.2010Vol.30No.10headsepticemiavirus,DSHSV)XDNF,,1.2(,);(,);(2.5kg)1.3DMEM(Hyclone,:AKA11263F);L(SIGMA);();(1∀250)(AMRESCO,,:1291B74);RNaseA(SIGAMA)S1Nuclease();EcoT14digestDNAMarker()1.4DSHSVXDNF,910,0.2mL/24h,3,24~96h,20,1.5,DSHSVXDNF,0.2mL/,37#30min,,-70#3,,201.6DSHSVXDNF110,,3(1mL/),1.7DELD50DSHSVXD10,10-3~10-8,6,4,0.2mL3,ReedMuenchDELD501.812000r/min10min,(),4#;12000r/min10min,,0.01mol/LPBS(pH7.2),SephadexG150,PBS,10mL/h,280nm,,1/40,12000r/min15min,,,,,1(2%,pH6.8)1min,,H6001.91.9.1药物抑制法DSHSVXD10,5(BUDR:DNA)100mg/L[4],4BUDRXDBUDRDPV+BUDRDHV+BUDR3,1.9.2酶消化法[57],K50mg/L,SDS1%,37#30min,/(1∀1)1,/(24∀1),,1/105mol/LNaCl,2.5,-20#2h-80#15min,4#,12000r/min15min,,70%,37#,TE(10mmol/LtrisHCl,pH8.0,1mmol/LEDTA),S1Nuclease(2g/L)RNaseA(0.2g/L),37#30min,,EcoT14digestDNAMarkers0.5∃TBE,0.8%,1~5V/cm,3h,1.102,1mLDSHSVXD11mL,21mLPBS,,10min,10000r/min5min,,10,4,0.2mL/,3/d,,DELD501.118,XDDSHSV1mL,45#1h56#10min56#20min56#30min60#30min80#5min100#1min,1,10,4,3/d,1.12,pH6.8pH7.0pH72pH7.4pH7.6PBS,1%,[5]37#4#330min1,,21.13,0.25%,37#2h,1013082010103010ChinJVetSciOct.2010Vol.30No.10,4,,3/d,1.14,,0.1mol/LHCl0.1mol/LNaOHpH3589,37#60min,5.6%NaHCO3(0.1mol/LHCl)pH7.2,5,3/d,1.151.15.1病毒的纯化[8],10000r/min10min,(),4#,12000r/min10min,001mol/LPBS(pH7.4),,PEGNaCl(75%PEG6000,10%NaCl),,4#,12000r/min10min,001mol/LPBS(pH7.2),4#,12000r/min15min,1.15.2鸭抗XD鸭抗NF鸭抗DPV制备,0.5mL;7d1,:1mL;2mL;3mL17d,,56#30min-20#1.15.3兔抗XD兔抗DPV制备[9]3,1.15.1,,-2mL,10~20mg,21,1mL,31,,56#30min-20#1.15.4交叉琼脂扩散试验[1012]DSHSVXDNFDPV3,3000r/min30min,1/40,XDNFDPV,1%PEG7,3mm,4mm;,(202-12-22-32-42-5)XDNF,XDDPV,NFDPV,37#24~48h1.15.5DSHSV与DPV的交叉中和试验[13]XDDPV2,2,11000DELD50XD,21000DELD50,37#1h3,0.4mL/,3/d,,,R[13]2R%0.8,,0.25R0.8,R0.252试验结果2.12.1.1病毒的鸭胚培养特性DSHSVXDNF,48h,,(1),,72h,9,7d,48h,14,7d,,16,,48h,,()(1),20,161.,,;.2.1.2病毒的鸡胚培养特性DSHSVXDNF,,;,,2.2DSHSVXDNF13092010103010ChinJVetSciOct.2010Vol.30No.1020,2.3DSHSVXDNF13d,;107,,,8d,,,,;;,;,,,;,;,2.4DELD501ReedMuenchXDDELD5010-5.78/0.2mL表1不同稀释度DSHSVXD株致死鸭胚数10-310-410-510-610-710-84/43/43/42/41/40/4:,2.5,,65~70nm(2)2(∃50000)2.62.6.1药物抑制法2XD+BUDRDELD50=10-5.66/0.2mL,XDDELD50=10-6/0.2mL,2.19,100,BUDR,,BUDR,BUDR,;DPV+BUDR,DHV+BUDR,BUDRDNA,RNA2.6.2酶消化法RNase3,S1Nuclease4,S1,RNADNAMaker,19kb表2BUDR对XD病毒繁殖的抑制作用XD10-310-410-510-610-710-8XD+BUDR4/44/43/41/41/40/4XD4/43/43/42/42/40/4BUDR(100g/L)0/4DPV+BUDR0/4DHV+BUDR4/413102010103010ChinJVetSciOct.2010Vol.30No.102.73ReedMuench,DELD50=10-5.78/0.2mL,DELD50=10-5.26/0.2mLDELD50/DELD50=0.3,XD,XD表3DSHSVXD毒株对氯仿的敏感性XD10-310-410-510-610-710-8XD+4/43/43/42/41/40/4XD+PBS3/43/42/42/41/40/42.84ReedMuench,XD45#1hDELD50=10-5.38,DELD50=10-5.78DELD50/DELD50=2.51100,DSHSVXD45#1h,56#30min,60#30min80#10min100#1min表4病毒耐热性测定XD10-310-410-510-610-745#1h3/43/43/42/40/44/43/43/42/41/456#10min5/556#20min5/556#30min5/560#30min0/580#10min0/5100#1min0/52.92.105ReedMuench,XD+DELD50=10-5.52,XD+DELD50=10-5.26DELD50/DELD50=0.55,0.25%2h,,表5鸭肿头败血症病毒对胰蛋白酶敏感性XD10-310-410-510-610-710-8XD+4/43/42/42/41/40/4XD+PBS3/43/42/42/41/40/42.11DSHSVpH3pH5pH8pH937#1h,,72h,DSHSV2.122.12.1交叉琼脂扩散试验6XDNF,DSHSV表6DSHSV与DPV交叉琼脂扩散试验DPVXDNFDPV-+--XD--++NF--++:+,2.12.2交叉中和试验7R=0022,表7DSHSV与DPV交叉中和效价DPVDSHSV12822643讨论3.1,,,DSHSV,,,9,7d,,,,16,48h,20,,DSHSV,,,