Construction of recombinant porcine reproductive and respiratory syndrome virus expressing porcine circovirus type Ⅱ(PRRSV-PCV2) antigen and study of PRRSV transcription mechanisms with this novel viral vector

ZHENG Hai-hong; SUN Zhi; ZHU Xing-quan; YUAN Shi-shan

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Journal of Microbes and Infections ›› 2008, Vol. 3 ›› Issue (3) : 147-153.

论著

Construction of recombinant porcine reproductive and respiratory syndrome virus expressing porcine circovirus type Ⅱ(PRRSV-PCV2) antigen and study of PRRSV transcription mechanisms with this novel viral vector

ZHENG Hai-hong;SUN Zhi;ZHU Xing-quan;YUAN Shi-shan

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Abstract

Objective The goal of the current study is to develop infectious porcine reproductive and respiratory syndrome virus(PRRSV) cDNA clones as expression vectors for porcine circovirus type Ⅱ(PCV2) ORF2 antigens and to use this new vector to dissect the replication and transcription abilities of PRRSV.Methods ORF2 from PCV2 was inserted into our previously established infectious clone of PRRSV,designated pAPRRS,at three locations:1) between ORF1b and ORF2a;2) between ORF5 and ORF6;and 3) between ORF6 and ORF7.The three full length mutant clones were then transfected into MARC-145 cells, resulting in three recombinant viruses. The genetic instability of recombinant virus was further examined via PRRSV’s mRNA2-specific RT-PCR sequence analysis of sgmRNA2, which enabled us to determine the origin of the sequence at the junction site. Results Our results demonstrated that the sgmRNA2 of this recombinant PRRSV-PCV2 has one TRS derived from the PRRSV ORF2 TRS, and two TRS segments were derived from the sequence of PCV2. One TRS was derived from the PRRSV ORF2 noncanonical TRS located at the downstream of PRRSV ORF2 initiation cordon. Conclusion Two recombinant PRRSV – PCV viruses are stable for more than ten passages, which makes them useful as marker vaccines. The most common reason for genetic instability of the PRRSV-PCV recombinant virus is an insertion of a sequence that resembles TRS and a subsequent change of the RNA structure for PRRSV ORF2 TRS and its flanking sequence. PRRSV can use a PCV2 sequence as its own TRS, which is useful for dissecting the mechanism behind PRRSV replication and transcription.

Key words

Pocine reproductive and respiratory syndrome virus / Porcine circovirus type Ⅱ / Express vector / Transcription-regulation sequence

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ZHENG Hai-hong;SUN Zhi;ZHU Xing-quan;YUAN Shi-shan. Construction of recombinant porcine reproductive and respiratory syndrome virus expressing porcine circovirus type Ⅱ(PRRSV-PCV2) antigen and study of PRRSV transcription mechanisms with this novel viral vector[J]. Journal of Microbes and Infections. 2008, 3(3): 147-153