ACTA VETERINARIA ET ZOOTECHNICA SINICA

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Isolation and Rapid Detection of Avian Borna Virus by a Reverse Transcription Loop-mediated Isothermal Amplification Assay for Outbreaks in Psittacine Birds

TIAN Chun-jian1, WANG Hong1, LUO Qiong1, LIN Zhi-xiong1, ZHAO Yin1, LUO Chang-bao1, YU Hai-qiong1, LIU Zhi-ling1, CHEN Ru1, TANG Yi2, ZHOU Xiao-ming1, CHANG Yan-lei2, WU Xiao-wei1, ZHU Dao-zhong1   

  1. (1. Guangdong Inspection and Quarantine Technology Center, Guangzhou 510623, China; 2. Guangzhou Deaou BioTechnology Co. Ltd, Guangzhou 510631, China)
  • Received:2012-05-25 Online:2012-11-26 Published:2012-11-26

Abstract:  In this study an avian bornavirus(ABV) strain was isolated from sick parrots with proventricular dilatation disease(PDD). The virus grew in swine testicular (ST) cell monolayer with granulating, shrinking, rounding and falling off although classical Borna disease virus strains replicate very efficiently in cultured mammalian cells in which persistent, noncytolytic infections was readily established. Viruses were successfully isolated and demonstrated by reverse transcription-PCR analysis from the proventricular glands of parrot “glass 363” and “color” with confirmed PDD. The 351 bp product of the expected size bands of matrix protein (M) gene was cloned, the sequence and phylogenetic tree analysis showed that the isolated virus belonging to genotype ABV5. Five sets of M gene RT-LAMP primers ID1, ID6, ID19, ID30 and ID37 were designed using DNAStar and PrimerExplorer V5.0 (network) and later three set reactions showed positive color reaction with specific electrophoretic bands. The amplification curves of of real-time RT-LAMP using fluorescent indicator calcein were shown in 36 (ID30), 38 (ID37) and 49 (ID19) minutes, respectively and an amplification peak in 60 minutes. Meanwhile the three amplification curves of turbidimetric determination were shown in 56, 58 and 65 minutes. According to the results of clinical and related samples detected by RT-LAMP and RT-PCR, proventriculus were the ABV positive while large intestine, small intestine, duodenum, heart, liver, spleen, lung, kidney, gizzard, proventriculus contents , pancreas, crop, brain, and muscle were negative. The virus was positive in 10-1 to 10-5 of ST cell culture material by RT-LAMP detection while it was negative after 10-3 by RT-PCR detection. There was also no RT-LAMP positive response to Newcastle disease, avian flu, bursal disease, leukemia subgroup J, encephalomyelitis. The findings provide new technological tools for the PDD control of parrots and BDV public health research.

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