Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92999
標題: A Survey of Avian Polyomavirus (APV) Infection and Viral Genetic Characteristics in Taiwan during 2012-2013
2012-2013年間台灣鳥多瘤病毒之盛行率調查及病毒核酸序列分析
作者: Yi-Hsin Lai 賴奕欣
賴奕欣
關鍵字: 鳥多瘤病毒;病毒核酸序列;臺灣;avian polyomavirus;viral genetic characteristic;Taiwan
引用: 蔡錦文。鸚鵡圖鑑、自然珍藏系列26。貓頭鷹出版社。2005。 蔡信雄。1987。自然及人工感染小鸚哥病之病理學研究。台灣畜牧獸醫學會會報 50: 1–14。 An K, Smiley SA, Gillock ET, Reeves WM Consigli RA. Avian polyomavirus major capsid protein VP1 interacts with the minor capsid proteins and is transported into the cell nucleus but does not assemble into capsid–like particles when expressed in the baculovirus system. Virus Res 1999; 64: 173–185. Arroube AS, Halami MY, Johne R, Dorrestein GM. Mortality due to polyomavirus infection in two nightjars (Caprimulgus europaeus). J Avian Med Surg 2009; 23: 136–140. Baker TS and Rayment I, 1987, Papovaviridae. In Nermut MV and Steven AC eds., Manual of Animal Virus Research. Elsevier Science Publisher B. V., Amsterdam, pp. 335–348. Bernier G, Morin M, Marsolais G. A generalized inclusion body disease in the budgerigar (Melopsittacus undulatus) caused by a papovavirus–like agent. Avian Dis 1981/10/01 ed, 1981; 1083–1092. Bernier G, Morin M, Marsolais G. Papovavirus induced feather abnormalities and skin lesions in the budgerigar: clinical and pathological findings. Can Vet J 1984; 25: 307–310. Bert E, Tomassone L, Peccati C, Navarrete MG, Sola SC. Detection of beak and feather disease virus (BFDV) and avian polyomavirus (APV) DNA in psittacine birds in Italy. J Vet Med B Infect Dis Vet Public Health 2005; 52: 64–68. Bustin SA. Absolute quantification of mRNA using real–time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 2000; 25 (2): 169–193. Coles BH, 2005, Chapter 1: Species and Natural History. In: Harcourt-Brown N and Chitty J eds. BSAVA Manual of Psittacine Birds 2nd edition. British Small Animal Veterinary Association publishing, pp.1–6. Crandall KA, Perez–Losada M, Christensen RG, McClellan DA, Viscidi RP. Phylogenomics and molecular evolution of polyomaviruses. Adv Exp Med Biol 2006; 577: 46–59. Davis RB, Bozeman LH, Gaudry D,Fletcher OJ, Lukert PD, Dykstra MJ. A viral disease of fledgling budgerigars. Avian Dis 1981; 25: 179–183. Doneley B, 2011, Avian Medicine and Surgery in Practice: Companion and Aviary Birds. Manson Publishing, pp.111–113. Dykstra MJ and Bozeman LH. A light and electron microscopic examination of budgerigar fledgling disease virus in tissue and in cell culture. Avian Pathol 1982; 11: 11–28. Enders F, Gravendyck M, Gerlach H, Kaleta EF. Fatal avian polyomavirus infection during quarantine in adult wild–caught red–faced lovebirds (Agapornis pullaria). Avian Dis 1997; 41: 496–498. Fattaey A, Lenz L and Consigli RA. Production and characterization of monoclonal antibodies to budgerigar fledgling disease virus major capsid protein VP1. Avian Dis 1992; 36: 543–553. Fitzgerald SD, Reed WM and Fulton RM. Development and application of an immunohistochemical staining technique to detect avian polyomaviral antigen in tissue sections. J Vet Diagn Invest 1995; 7: 444–450. Garcia A, Latimer KS, Niagro FD, Norton TM, Campagnoli RP, Harmon BG, Howerth EW, Ritchie BW. Diagnosis of polyomavirus infection in seedcrackers (Pyrenestes sp.) and blue bills (Spermophaga haematina) using DNA in situ hybridization. Avian Pathol 1994; 23: 525–537. Garcia A, Latimer KS, Niagro FD, Ritchie BW, Campagnoli RP. Diagnosis of polyomavirus–induced hepatic necrosis in psittacine birds using DNA probes. J Vet Diagn Invest 1994; 6: 308–314. Gilardi KV, Lowenstine LJ, Gilardi JD, Munn CA. A survey for selected viral, chlamydial, and parasitic diseases in wild dusky–headed parakeets (Aratinga weddellii) and tui parakeets (Brotogeris sanctithomae) in Peru. J Wildl Dis 1995; 31: 523–528. Gough JF. Outbreaks of budgerigar fledgling disease in three aviaries in Ontario. Can Vet J 1989; 30: 672–674. Halami MY; Dorrestein GM; Couteel P; Heckel G; Muller H and Johne R. Whole–genome characterization of a novel polyomavirus detected in fatally diseased canary birds. J Gen Virol 2010: 3016–3022. Harcourt–Brown NH, 2009, 7: Psittacine Birds. In: Tully T, Dorrestein G and Jones A eds. Handbook of Avian Medicine 2nd edition. Elsevier Ltd, pp.138–141. Haynes JI, 2nd, Consigli RA. Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1. J Virol 1992; 66: 4551–4555. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996; 6(10): 986–994. Herrera I, Khan SR, Kaleta EF, Muller H, Dolz G, Neumann U. Serological status for Chlamydophila psittaci, Newcastle disease virus, avian polyoma virus, and Pacheco disease virus in scarlet macaws (Ara macao) kept in captivity in Costa Rica. J Vet Med B Infect Dis Vet Public Health 2001; 48: 721–726. Hsu CM, Ko CY and Tsaia HJ. Detection and sequence analysis of avian polyomavirus and psittacine beak and feather disease virus from psittacine birds in Taiwan. Avian Dis 2006; 50: 348–353. Johne R, Buck CB, Allander T,Atwood WJ, Garcea RL, Imperiale MJ, Major EO, Ramqvist T, Norkin LC. Taxonomical developments in the family Polyomaviridae. Arch Virol 2011; 156: 1627–1634. Johne R, Enderlein D, Nieper H, Muller H. Novel polyomavirus detected in the feces of a chimpanzee by nested broad–spectrum PCR. J Virol 2005; 79 (6): 3883–3887. Johne R, Jungmann A, Muller H. Agnoprotein 1a and agnoprotein 1b of avian polyomavirus are apoptotic inducers. J Gen Virol 2000; 81: 1183–1190. Johne R and Muller H. Avian polymavirus in wild birds: genome analysis of isolates from Falconiformes and Psittaciformes. Arch Virol 1998; 143: 1501–1512. Johne R and Muller H. Avian polyomavirus agnoprotein 1a is incorporated into the virus particle as a fourth structural protein, VP4. J Gen Virol 2001; 82: 909–918. Johne R and Muller H. Nuclear localization of avian polyomavirus structural protein VP1 is a prerequisite for the formation of virus–like particles. J Virol 2004; 78: 930–937. Johne R and Muller H. The genome of goose hemorrhagic polyomavirus, a new member of the proposed subgenus Avipolyomavirus. Virology 2003; 308: 291–302. Johne R and Muller H. Polyomaviruses of birds: etiologic agents of inflammatory diseases in a tumor virus family. J Virol 2007; 81: 11554–11559. Johne R, Paul G, Enderlein D, Stahl T, Grund C, Muller H. Avian polyomavirus mutants with deletions in the VP4–encoding region show deficiencies in capsid assembly and virus release, and have reduced infectivity in chicken. J Gen Virol 2007; 88: 823–830. Johne R, Wittig W, Fernandez–de–Luco D, Hofle U, Muller H. Characterization of Two Novel Polyomaviruses of Birds by Using Multiply Primed Rolling–Circle Amplification of Their Genomes. J Virol 2006; 80: 3523–3531. Joseph L, Toon A, Schirtzinger EE, Wright TF, Schodde R. A rivised nomenclature and classification for family–group taxa of parrots (Psittaciformes). Zootaxa 2012; 3205: 26–40. Katoh H, Ohya K and Fukushi H. Development of novel real–time PCR assays for detecting DNA virus infections in psittaciform birds. J Virol Methods 2008; 154: 92–98. Katoh H, Ohya K, Une Y, Yamagushi T, Fukushi H. Molecular characterization of avian polyomavirus isolated from psittacine birds based on the whole genome sequence analysis. Vet Microbiol 2009; 138: 69–77. Kingston RS. Budgerigar fledgling disease (papovavirus) in pet birds. J Vet Diagn Invest 1992; 4: 455–458. Krumbholz A, Bininda–Emonds OR, Wutzler P, Zell R. Phylogenetics, evolution, and medical importance of polyomaviruses. Infect Genet Evol 2009; 9: 784–799. Lafferty SL, Fudge AM, Schmidt RE, Wilson VG, Phalen DN. Avian polyomavirus infection and disease in a green aracaris (Pteroglossus viridis). Avian Dis 1999; 43: 577–585. Latimer KS, Niagro FD, Campagnoli RP, Ritchie BW, Pesti DA, Steffens WL. Diagnosis of concurrent avian polyomavirus and psittacine beak and feather disease virus infections using DNA probes. J Assoc Avi Vet 1993; 7: 141–146. Latimer KS, Niagro FD, Steffens WL, 3rd, Ritchie BW, Campagnoli RP. Polyomavirus encephalopathy in a Ducorps' cockatoo (Cacatua ducorpsii) with psittacine beak and feather disease. J Vet Diagn Invest 1996; 8: 291–295. Lehn H and Muller H. Cloning and characterization of budgerigar fledgling disease virus, an avian polyomavirus. Virol 1986; 151: 362–370. Li H, Soderbarg K, Houshmand H, You ZY, Magnusson G. Effect on polyomavirus T–antigen function of mutations in a conserved leucine–rich segment of the Dna J domain. J Virol 2001; 75: 2253–2261. Li J, Liu Q, Muller H, Hobom G. Avian polyomavirus expression patterns of bicistronic late mRNAs. Virol 2009; 388: 42–48. Liddington RC, Yan Y, Moulai J, Sahli R, Benjamin TL, Harrison SC. Structure of simian virus 40 at 3.8–A resolution. Nature 1991; 354: 278–284. Lierz M, 2005, Chapter 13: Systemic Infectious Diseases. In: Harcourt–Brown N and Chitty J eds. BSAVA Manual of Psittacine Birds 2nd edition. British Small Animal Veterinary Association publishing, pp.160–161. Literak I, Smid B, Dubska L, Bryndza L, Valicek L. An outbreak of the polyomavirus infection in budgerigars and cockatiels in Slovakia, including a genome analysis of an avian polyomavirus isolate. Avian Dis 2006; 50: 120–123. Liu Q and Hobom G. Recombinant expression of late genes agno–2a and agno–2b of avian polyomavirus BFDV. Virus Genes 1999; 19: 183–187. Liu Q and Hobom G. Agnoprotein–1a of avian polyomavirus budgerigar fledgling disease virus: identification of phosphorylation sites and functional importance in the virus life–cycle. J Gen Virol 2000; 81: 359–367. Liu Q and Hobom G. Evidence for translation of VP3 of avian polyomavirus BFDV by leaky ribosomal scanning. Arch Virol 2000; 145: 407–416. Luo D, Muller H, Tang XB, Hobom G. Expression and DNA binding of budgerigar fledgling disease virus large T antigen. J Gen Virol 1994; 75 (Pt 6): 1267–1280. Luo D, Muller H, Tang XB, Hobom G. Early and late pre–mRNA processing of budgerigar fledgling disease virus 1: identification of viral RNA 5' and 3' ends and internal splice junctions. J Gen Virol 1995; 76 (Pt 1): 161–166. Manarolla G, Liandris E, Pisoni G, Moroni P, Piccinini R, Rampin T. Mycobacterium genavense and avian polyomavirus co–infection in a European goldfinch (Carduelis carduelis). Avian Pathol 2007; 36: 423–426. Muller U, Zentgraf H, Eicken I, Keller W. Higher order structure of simian virus 40 chromatin. Science 1978; 201: 406–415. Norkin LC, Allander T, Atwood WJ, Buck CB, Garcea RL, Imperiale MJ, Johne R, Major EO, Pipas JM and Ramqvist T, 2012, Family Polyomaviridae. In: Virus Taxonomy. Ninth Report of the International Committee on Taxonomy of Viruses, pp. 279–290. Ogawa H, Yamaguchi T and Fukushi H. Duplex shuttle PCR for differential diagnosis of budgerigar fledgling disease and psittacine beak and feather disease. Micro Immun 2005; 49: 227–237. Ogawa H, Chahota R, Hagino T, Ohya K, Yamaguchi T, Fukushi H. A survey of avian polyomavirus (APV) infection in imported and domestic bred psittacine birds in Japan. J Vet Med Sci 2006; 68: 743–745. Ozmen O and Dorrestein GM. Avian polyomavirus infection in budgerigars (Melopsittacus undulatus) in Turkey. Turk J Vet Anim Sci 2004; 28: 409–414. Perez–Losada M, Christensen RG, McClellan DA, Adams BJ, Viscidi RP, Demma JC, Crandall KA. Comparing phylogenetic codivergence between polyomaviruses and their hosts. J Virol 2006; 80: 5663–5669. Phalen DN, Wilson VG and Graham DL. Polymerase chain reaction assay for avian polyomavirus. J Clin Microbiol 1991; 29: 1030–1037. Phalen DN, Wilson VG and Graham DL. Organ distribution of avian polyomavirus DNA and virus–neutralizing antibody titers in healthy adult budgerigars. Am J Vet Res 1993; 54: 2040–2047. Phalen DN, Wilson VG and Graham DL. Characterization of the avian polyomavirus–associated glomerulopathy of nestling parrots. Avian Dis 1996; 40: 140–149. Phalen DN, Wilaon VG and Graham DL. Prevalence of neutralizing antibody and virus shedding in psittacine birds infected with avian polyomavirus. J Avi Med Surg 1997; 11: 98–104. Phalen DN, Wilson VG, Gaskin JM, Derr JN, Graham DL. Genetic diversity in twenty variants of the avian polyomavirus. Avian Dis 1999; 43: 207–218. Phalen DN, Radabaugh CS, Dahlhausen RD, Styles DK. Viremia, virus shedding, and antibody response during natural avian polyomavirus infection in parrots. J Am Vet Med Assoc 2000; 217: 32–36. Phalen DN. Virus neutralization assays used in exotic bird medicine. Seminars in Avian and Exotic Pet medicine 2002; 11: 19–24. Potti J, Blanco G, Lemus JA, Canal D. Infectious offspring: how birds acquire and transmit an avian polyomavirus in the wild. PLoS One 2007; 2: e1276. Rahaus M, Wolff MH. A survey to detect subclinical polyomavirus infections of captive psittacine birds in Germany. Vet Micro 2005; 105: 73–76. Raidal SR, Cross GM, Tomaszewski E, Graham DL. A serologic survey for avian polyomavirus and Pacheco's disease virus in Australian cockatoos. Avian Pathol 1998; 27: 263–268. Raidal SR, 2011, Avian Circovirus and Polyomavirus Diseases. In: Miller RE and Fowler M, eds. Fowler's Zoo and Wild Animal Medicine Current Therapy. No.7. Elsevier Saunders, pp. 297–303. Ramis A, Latimer KS, Niagro FD, Campagnoli RP, Ritchie BW, Pesti D. Diagnosis of psittacine beak and feather disease (PBFD) viral infection, avian polyomavirus infection, adenovirus infection and herpesvirus infection in psittacine tissues using DNA in situ hybridization. Avian Pathol 1994; 23: 643–657. Ramis A, Latimer KS, Gibert X, Campagnoli R. A concurrent outbreak of psittacine beak and feather disease virus, and avian polyomavirus infection in budgerigars (Melopsittacus undulatus). Avian Pathol 1998; 27: 43–50. Randall CJ, Lees S and Inglis DM. Papovavirus–like infection in budgerigars (Melopsittacus undulatus). Avian Pathol 1987; 16: 623–633. Rector A, Tachezy R and Ranst MV. A Sequence–Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling–Circle Amplification. J Virol 2004; 78: 4993–4998. Rihs HP, Peters R and Hobom G. Nuclear localization of budgerigar fledgling disease virus capsid protein VP2 is conferred by residues 308–317. FEBS Lett 1991; 291: 6–8. Ritchie BW, Harrison GJ and Harrison LR, 1994, Chapter 32: Viruses. In: Avian Medicine: Principles and Application. Zoological Education Network publishing, pp. 888–894. Ritchie BW, Niagro FD, Latimer KS, Davis RB, Pesti D, Lukert PD. Avian Polyomavirus: An overview. J Assoc Avi Vet 1991; 5: 147–153. Ritchie BW, Niagro FD, Latimer KS,Pritchard N, Greenacre C, Campagnoli RP, Lukert PD. Antibody response and local reactions to adjuvanted avian polyomavirus vaccines in psittacine birds. J Assoc Avi Vet 1994; 8: 21–26. Ritchie BW, Niagro FD, Latimer KS, Pritchard N, Campagnoli RP, Lukert PD. An inactivated avian polyomavirus vaccine is safe and immunogenic in various Psittaciformes. Vaccine 1996; 14: 1103–1107. Rodgers RE, Chang D, Cai X, Consigli RA. Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly. J Virol 1994; 68: 3386–3390. Rodgers RED and Consigli RA. Characterization of a calcium binding domain in the VP1 protein of the avian polyomavirus, budgerigar fledgling disease virus. Virus Research 1996; 44: 123–135. Rossi G, Ceccherelli R, Piersigilli A, Tarantino C. Sertoli cell tumor associated with polyomavirus infection in a Gouldian finch (Erythrura gouldiae). Avian Dis 2003; 47: 240–243. Rossi G, Taccini E and Tarantino C. Outbreak of avian polyomavirus infection with high mortality in recently captured crimson's seedcrackers (Pyrenestes sanguineus). J Wild Dis 2005; 41: 236–240. Rott O, Kroger M, Muller H, Hobom G. The genome of budgerigar fledgling disease virus, an avian polyomavirus. Virol 1988; 165:74–86. Roy P, Dhillon AS, Lauerman L, Shivaprasad HL. Detection of avian polyomavirus infection by polymerase chain reaction using formalin–fixed, paraffin–embedded tissues. Avian Dis 2004; 48: 400–404. Schmidt M, Muller H, Schmidt MF, Rott R. Myristoylation of budgerigar fledgling disease virus capsid protein VP2. J Virol 1989; 63: 429–431. Shahidur Rahman Khan M, Johne R, Beck I, Pawlita M, Kaleta EF, Muller H. Development of a blocking enzyme–linked immunosorbent assay for the detection of avian polyomavirus–specific antibodies. J Virol Methods 2000; 89: 39–48. Shen PS, Enderlein D, Nelson CD, Carter WS, Kawano M, Xing L, Swenson RD, Olson NH, Baker TS, Cheng GH, Atwood WJ, Johne R, Belnap DM. The structure of avian polyomavirus reveals variably sized capsids, non–conserved inter–capsomere interactions, and a possible location of the minor capsid protein VP4. Virol 2011; 411: 142–152. Stoll R, Luo D, Kouwenhoven B, Hobom G and Muller H. Molecular and biological characteristics of avian polyomaviruses: isolates from different species of birds indicate that avian polyomaviruses form a distinct subgenus within the polyomavirus genus. J Gen Virol 1993; 74: 229–237. Stoll R, Hobom G, Muller H. Host restriction in the productive cycle of avian polyomavirus budgerigar fledgling disease virus type 3 depends on a single amino acid change in the common region of structural proteins VP2/VP3. J Gen Virol 1994; 75 (Pt 9): 2261–2269. Sundsfjord A, Johansen T, Flegstad T, Moens U, Viland P, Subramano S, Traavik T. At least two types of control regions can be found among natually occurring BK virus strains. J Virol 1990: 3864–3871. Tomasek O, Kubicek O and Tukac V. Unusual fatal avian polyomavirus infection in nestling cockatiels (Nymphicus hollandicus) detected by nested polyomerase chain reaction. Vet Med 2005; 52: 193–201. Tomasek O, Kubicek O and Tukac V. Comparison of three template preparation methods for routine detection of beak and feather disease virus and avian polyomavirus with single and nested polymerase chain reaction in clinical specimens. Avian Pathol 2008; 37: 145–149. van den Brand JM, Manvell R, Paul G, Kik MJ, Dorrestein GM.. Reovirus infections associated with high mortality in psittaciformes in The Netherlands. Avian Pathol 2007; 36: 293–299. Villiers EM, Fauquet C, Broker TR, Bernard HU, Hausen HZ. Classification of papillomaviruses. Virol 2004; 324: 17–27. Wainright PO, Lukert PD, Davis RB,Villegas P. Serological evaluation of some psittaciformes for budgerigar fledgling disease virus. Avian Dis1987; 31: 673–676. Yu ZG, Zhou LQ, Chu KH, Li CP, Anh VV. Phylogenetic analysis of polyomaviruses based on their complete genomes. Natural Computation 2008 ICNC '08 Fourth International Conference 2008; 5: 80–84. Zhuang Q, Chen J, Mushtaq MH, Liu S, Hou G, Li J, Huang B, Jiang W. Prevalence and genetic characterization of avian polyomavirus and psittacine beak and feather disease virus isolated from budgerigars in Mainland China. Arch Virol 2012; 157: 53–61. Zielonka A, Gedvilaite A, Reetz J, Rosler U, Muller H, Johne R. Serological cross–reactions between four polyomaviruses of birds using virus–like particles expressed in yeast. J Gen Virol 2012; 93: 2658–2667.
摘要: 
鳥多瘤病毒可造成多種類鸚鵡之急性、致死性及高傳染性的疾病,近年來在世界各國針對APV所做的流行病學研究均以PCR作為診斷工具,而本研究亦以PCR做檢測。收集的材料來自於臨床病例、鸚鵡繁殖場的糞便拭子及死亡鳥隻檢體,總計有443個樣本並可分類22個屬別以及41個種別。除了以PCR方法檢測病毒核酸,在剖檢檢查可見感染APV的鳥隻有肝臟腫大出血、脾臟腫大且有多發局部性壞死灶等特徵性的肉眼病變。另外,於組織病理學檢查,可見感染細胞呈現細胞核腫大並有特徵性的嗜鹼性核內包涵體,其中以脾臟及腎小管上皮細胞較容易觀察之。本實驗所設計用以診斷APV感染之PCR 引子對,其目標增幅產物為VP1基因片段,大小約為272 bp,此檢測方法亦具有良好的敏感性與特異性。以此PCR試驗檢測結果APV總陽性率為37.47% (166/443),顯示APV持續存在於台灣的事實。另外雙重基因定序設計針對病毒之VP 2/3及t/T antigen之基因片段作分析,得知本研究中所分離出20株病毒株針對此兩段部分基因的定序比對相似度高達98.3%以上,表示此20株台灣株針對此部分基因片段具有高度保留特性。進一步針對此兩段基因片段與來自於世界各國已發表之病毒株做比對,其結果亦不具有顯著性差異。然而,傳統型PCR有可能形成非特異性的產物而且其敏感性仍須再提高,這些傳統型PCR所包含的缺點可藉由發展新式定量型即時PCR來彌補。本研究所設計的即時PCR試驗是利用SYBR Green螢光系統來做標定,並使用與傳統型PCR相同之診斷引子對。此即時PCR可偵測到的病毒最低濃度為101 病毒之複製單位,此外藉由帶有其他種鸚鵡病原核酸材料包括鸚鵡喙羽病病毒、鸚鵡披衣菌、大腸桿菌以及沙門氏菌,並未有任何產物被增幅出來,顯示此試驗具有良好的特異性。即時PCR試驗除了有良好的特異性與敏感性,其所具備的再現性以及降低檢測過程中檢體之間汙染的風險,因此適合發展為快速檢測APV感染之臨床診斷試驗。

The avian polomavirus (APV) is an acute, lethal, and highly contagious viral disease in most parrot species. Recently, the worldwide epidemiological studies of APV infection in psittacine birds were performed by PCR. In this study, we collected fecal and tissue samples from clinical cases and a breeding aviary. For APV prevalence, 443 samples from 22 genera and 41 species of psittaciform birds were checked by PCR assay. Confirmed by necropsy exam which indicated hepatomegaly with hemorrhage and swollen spleen in the sick birds. In further histopathologic exam, the characteristic basophilic intranuclear inclusion bodies could be found in karyomegalic cells in spleen, renal tubular epithelium, and liver. Based on the sequence of the VP1 gene of APV from Genbank, we designed a PCR assay with high sensitivity for APV detection. The total positive rate was 37.47% (166/443) indicating APV persistently existing in Taiwan. Multiple gene sequencing assays were performed at combined region of VP2 and VP3 and the gene encoding region of t/T antigens. The similarity of 20 isolates of VP2/3 and t/T antigen coding regions from Taiwan ranged from 98.3–100.0%. This investigation revealed that APV isolated from Taiwan was highly conserved. The sequencing results of 20 isolates in this study also indicated that there was no significant difference by comparing with worldwide sequences in the tested gene regions. However, the public conventional PCR assays for APV detection might amplify non–specific products and the sensitivity of the assays should be improved. These drawbacks could be avoided by developing the quantitative real–time PCR (qPCR) assay, which showed improved rapidity, sensitivity, reproducibility, and reduced the risks of contamination. In the current study, the qPCR assay based on SYBR Green Ⅰ fluorescence and the primer set designed at the conserved region of VP1 gene was developed. The detection limit of the qPCR assay reached 1×101 copies of viral DNA, and the assay was highly specific without amplifying any product from other avian viral and bacterial genomes (including PBFD, Chlamydia, E. coli and Salmonella Typhimurium). This newly developed qPCR is a suitable tool for rapid clinical investigations
URI: http://hdl.handle.net/11455/92999
其他識別: U0005-0707201417124600
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