猪繁殖与呼吸综合征病毒类NADC30株CHsx1401感染性克隆的构建

doi:10.11843/j.issn.0366-6964.2018.03.022

Abstract

Abstract:

Porcine reproductive and respiratory syndrome virus (PRRSV) remains the most economically important pathogen impacting swine industry worldwide. Recently, the novel NADC30-like strains of PRRSV resulting in the re-emerging of clinically PRRS outbreaks has been characterized in China. In order to provide a basis for research on the mechanisms in relation to the genetic variation and evolution of PRRSV NADC30-like strain, CHsx1401, as well as the differential pathogenesis between this virus and other PRRSV strains, a full-length cDNA clone of NADC30-like CHsx1401 was generated. Four fragments of the whole genome of CHsx1401 were amplified by RT-PCR, and each was cloned into pJET1.2 blunt, and each clone was then ligated into the modified low-copy-number vector pWSK29 to generate the full-length cDNA clone, plasmid pWSK-CHsx1401. To distinguish the rescued virus from its parental virus, a restriction enzyme site AscⅠ was introduced between fragment C and D through synonymous mutation (T11582G) as a genetic marker. Subsequently, the full-length cDNA clone was transfected into MARC-145 cells using Lipofectamine LTX and plus reagents to rescue the virus. As the results showed, the full-length cDNA clone (plasmid pWSK-CHsx1401) was constructed successfully, cytopathic effect (CPE) typical of PRRSV in MARC-145 cells could be observed at the second passage. The virus could be rescued from the plasmid pWSK-CHsx1401 by IFA, RT-PCR and sequencing. The rescued virus could be passaged stably in MARC-145 cells, and showed a similar growth curve to its parental isolate in vitro, with slightly lower titers at some time points than CHsx1401 in MARC-145 cells. Our results indicate that the full-length cDNA clone of the PRRSV NADC30-like CHsx1401 is infectious and viable virus can be rescued. The rescued virus (RvCHsx1401) exhibited similar growth ability to its parental virus in vitro. Our present study provides a platform for further research on the molecular mechanisms in relation to the variation and evolution of PRRSV NADC30-like and its differential pathogenesis from other strains of PRRSV.

CLC Number:

S852.659.6

BIAN Ting, ZHOU Lei, SONG Jiang-wei, GE Xin-na, GUO Xin, HAN Jun, YANG Han-chun. Construction of an Infectious cDNA Clone of Porcine Reproductive and Respiratory Syndrome Virus NADC30-like CHsx1401[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(3): 637-643.

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Construction of an Infectious cDNA Clone of Porcine Reproductive and Respiratory Syndrome Virus NADC30-like CHsx1401

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