Canine Coronavirus Detection in Feces from Diarrhea and Healthy. Dogs by Nested2PCR

Transcription

1 PCR 3 1, 133, 1,2 (1., ;2.,210012) Canine Coronavirus Detection in Feces from Diarrhea and Healthy Dogs by Nested2PCR WAN G Yu2yan 1, LU Cheng2ping 133, WEN Hai 1,2 (1. Key L ab of A nimal disease Diagnostic and I mmunology Ministry of A griculture, N anjing A gric Univ, N anjing , China; 2. N anj ing Police Dogs Research Institute, N anjing , China) Abstract : 112 faecal samples of dogs collected from August 2003J anuary 2004 were tested for t he presence of Cani ne coronavi rus (CCV) using a nested2pcr wit h conserved primers for t he M gene. 18 out of 43 diarrhea feces f rom dogs housed singly of Nanjing city were CCV positively, and the CCV detectable rate were po sitively correlated wit h seasons, it was higher in winter t han t hat of in summer and aut umn. 58 out of 69 feces f rom heat hy dogs trained in group s were posi2 tive for CCV, and t he positive ratio of samples of dog schools from Shenyang (34/ 39) was higher t han t hat of f rom Nanjing (24/ 30). Sequence analysis of 4 CCV M gene fragment s, two from pos2 itive diarrhea samples of Nanjing and two from heat hy samples of Shenyang, showed t he presence of the CCVs wit h high similarity (94. 8 %96. 7 %) to t he CCV of giant panda f rom China in t he GenBank. All t he CCVs detected in this survey were type confirmed by a PCR gene typing. Key words :Canine coronavirus ; type CCV ; nested2pcr. : , 43, 30, 39,PCR (CCV),CCV % (18/ 43),CCV, %(58/ 69), CCV (87. 2 %) (80. 0 %) 2,,4 M 212bp GenBank CCV ( ), CCV CCV PCR, CCV : ; CCV ; PCR :S :A : (2005) (Cani ne coronavi rus, CCoV),CCoV ( T ransmissible g ast roenteritis V i rus, T GEV) ( Feli ne Coronavi ruses, FCoV) ( H um an Coronavi rus 229 E, HCoV 229 E) I [1 ] RNA,, [2 ] [3 ] 2003 SA RS CoV, [4 ] CCV, [57 ] Erles [ 8 ], (Canine in2 fectious respiractry syndrome diseases, CIRD),SHCoV2OC43 : ,: :((2003) D21 ) ; :(19762),,,, 3 3 :(19452),,,,. Correspondirg anthor. Tel : ,E2mail edu. cn

2 42 20 (BCoV), OC43 BCoV,CCV CCV, CCV CCV,,, PCR,, PCR, PCR, PCR,,, PCR,, PCR, CCV [9,10 ], CCV Pratelli [11 ] PCR, 112, CCV,CoV CCV21271 A72 ( ) Baljer 10 % MEM,CCV % MEM 1. 2 [11 ] M () : CCV1 :5 2TCC A GA TAT GTA ATG TTG GC23 CCV2 : 5 2TCT GTT GA G TAA TCA CCA GCT23 CCV3 :5 2GGT GTC ACT CTA ACA TTG CTT23 CCV1a :5 2GT G CT T CCT GAA GGT ACA23 PCR : PCR CCV1 CCV2,409bpM,PCR, 100 PCR, CCV2 CCV3 212bp CCV PCR : 100 PCR,I CCV,CCV2 CCV1a 239bp 1. 3 RNA cd NA CCV 1271 A72,80 % 3,,p H mol/ L PBS 5,5000r/ min 4 20min, 220 RNA 200L 1mL Trizol (32ZOL) (MDBio ), RNA cdna R T : RNA 5L,5 4L, 10 mmol/ L dn TPs 2L,(30p mol/l ) 0. 5L, M2ML V ( Pro2 mega ) R T 1L,RNasin ( ) 0. 5L,dd H2 O 20L, 42 1h.,95 5min PCR CCV 1271 PCR,PCR : DNA 5L, 10 PCR 2. 5L, 25mmol/ L MgCl2 2L, 10mMdN TPs 1L, 0. 5 l (30p mol/l), TaqDNA (5U/L ) 0. 5L, dd H2O 25L :94 30s 55 30s 72 1min, 35, 72 5min 1 TCID TCID50 CCV 1271, , 30, 39 (1 2) PCR 1. 6 N14 N17 S1 S24 PCR, GenBank CCV, DNAstar CCV,PCR 1 TCID50CCV 1271 PCR 409bp,PCR 212bp (1) 43 18, % (18/ 43) (1,2),

3 ,:PCR 43 1 A CCV 1271 PCR Fig. 1 Nested2PCR of CCV ,Products of the first PCR; 2, Products of the second PCR;3,Marker. CCV ( P < 0. 05) ; CCV, ;;, (2) ( ),,,, %(24/ 25) ;87. 2 %(34/ 39),CCV,( P < 0. 05) (2) 2 PCR Fig. 2 The second products of the nested2pcr for some samples. 1,Marker ;2,CCV 1271 ;36, Tested feces. 2 CCV 1 CCV Table 1 The presence of CCV in different symptom, age and sampling time feces Total no. of samples tested No. of samples positive for CCV Positive rate ( %) Symptom Diarrhea Bloody stool Age (mont h) > Sampling time Tab. 2 The presence of CCV in feces of diarrhea and heath dogs Groups Area Sampling time Age ( month) Samples Positive amples ( %) State Total Nanjing Nanjing Shengyang < (41. 9) 24 (80. 0) 11 (82. 3) 23 (88. 5) 76 (68. 76) Diarrhea/ housed Healt h/ grouped Healt h/ grouped 2. 2 CCV PCR CCV2 CCV1a,, I CCV PCR GenBank CCV,,CCV DNAstar, CCV NJ 14 CCV NJ 17 CCV SY1 CCV SY24 CCV Gen2 Bank I, CCV (A Y436635), %, T GEV ( AJ ) ( %) FCV UCD1 ( %) FCV ( ) (3,4), CCV M, ( CCV1271 CCV NJ 17 CCV S24 Gen2 Bank,A Y A Y A Y702644) 3 CCV Ten2 nant,ccv 76 %100 % [ 12 ] Pratelli, CCV % [13 ],CCV % [ 14 ] CCV, (1994) [15 ] EL ISA CCV, CCV,CCV S

4 CCV M Fig. 3 Nucleotide identity and divergence of CCV M gene (212bp) from dog feces with some known group coronaviruses 4 CCV M (212bp) Fig. 4 Phylogenetic tree of partial CCV M gene nucleotide sequence (212bp) f rom Nanjing and Shenyang feces. CCV PCR [9 ], CCV [ 10 ], CCV PCR,112, CCV %85. 5 %,, Randall [16 ], CCV,,,,CCV,, Naylor CCV, CCV %40. 8 % [17 ], Pratelli [ 18,19 ], CCV, I FCoV, FCoV2like CCV,FCoV, I CCV ;CCV II FCoV,, CCV, M FCoV2like CCV CCV1a [18 ] Pratelli CCV PCR, I CCV,CCV, I CCV %, % [19 ],CCV (41. 9 %), CCV,CPV (CAV),CCV,,CCV [20 ], CCV, CCV M CCV, %, CCV CCV,, CCV [21,22 ] CCV, CCV CoV,, :,! [1 ] Cavanagh D, Prian D A, Briton P, et al. Nidovirales : a new order comprising coronaviridae and arteriviridae [ J ]. Virol, 1997,1452 : Arch. [2 ] Lai S M C, Cavanagh D. The molevular biology of coronavir2 uses[j ]. Adv. Virus Res, 1997, 48 : [ 3 ] Herrevegh A A, Smeenk I, Horzinek M C, et al. Feline coro2 navirus type II strain and originate from a double recombination between feline coronavirus type I and ca2 nine coronavirus[j ]. J Virol, 1998,72 : [ 4 ] Michael J S, James R B, Heat her A2M. Evidence from t he ev2 olutionary anylysis of nucleotide sequences for a recombinant history of SARS2CoV. Infection, Genetics and Evolution. 2003, Available online at www. sciencedirect. com. [ 5 ] Pratelli A, Martella V, Pistello M, et al. Identification of coronaviruses in dogs that segregate separately from the canine coronavirus genotype [J ]. J Virol Met h, 2003, 107 : [ 6 ] Wesley R D. The S gene of canine coronavirus, st rain UCD21, is more closely related to S gene of transmissible gastroenteritis virus than to that of feline infectious peritoritis virus [J ]. Vi2

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