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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References

[1] NEUMANN E J, KLIEBENSTEIN J B, JOHNSON C D, et al. Assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the United States[J]. J Am Vet Med Assoc, 2005, 227(3):385-392.
[2] PEJSAK Z, STADEJEK T, MARKOWSKA-DANIEL I. Clinical signs and economic losses caused by porcine reproductive and respiratory syndrome virus in a large breeding farm[J]. Vet Microbiol, 1997, 55(1-4):317-322.
[3] CAVANAGH D. Nidovirales:a new order comprising Coronaviridae and Arteriviridae[J]. Arch Virol, 1997, 142(3):629-633.
[4] CONZELMANN K K, VISSER N, VAN WOENSEL P, et al. Molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group[J]. Virology, 1993, 193(1):329-339.
[5] MEULENBERG J J M, PETERSEN DEN BESTEN A, DE KLUYVER E, et al. Molecular characterization of Lelystad virus[J]. Vet Microbiol, 1997, 55(1-4):197-202.
[6] JOHNSON C R, GRIGGS T F, GNANANDARAJAH J, et al. Novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative ORF5 present in all arteriviruses[J]. J Gen Virol, 2011, 92(5):1107-1116.
[7] SNIJDER E J, MEULENBERG J J. The molecular biology of arteriviruses[J]. J Gen Virol, 1998, 79(Pt 5):961-979.
[8] MARDASSI H, MOUNIR S, DEA S. Identification of major differences in the nucleocapsid protein genes of a Québec strain and European strains of porcine reproductive and respiratory syndrome virus[J]. J Gen Virol, 1994, 75(Pt 3):681-685.
[9] MENG X J, PAUL P S, HALBUR P G, et al. Phylogenetic analyses of the putative M (ORF 6) and N (ORF 7) genes of porcine reproductive and respiratory syndrome virus (PRRSV):implication for the existence of two genotypes of PRRSV in the U.S.A. and Europe[J]. Arch Virol, 1995, 140(4):745-755.
[10] NELSON E A, CHRISTOPHER-HENNINGS J, DREW T, et al. Differentiation of U.S. and European isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies[J]. J Clin Microbiol, 1993, 31(12):3184-3189.
[11] MURTAUGH M P, ELAM M R, KAKACH L T. Comparison of the structural protein coding sequences of the VR-2332 and Lelystad virus strains of the PRRS virus[J]. Arch Virol, 1995, 140(8):1451-1460.
[12] NELSEN C J, MURTAUGH M P, FAABERG K S. Porcine reproductive and respiratory syndrome virus comparison:divergent evolution on two continents[J]. J Virol, 1999, 73(1):270-280.
[13] HAN J, WANG Y, FAABERG K S. Complete genome analysis of RFLP 184 isolates of porcine reproductive and respiratory syndrome virus[J]. Virus Res, 2006, 122(1-2):175-182.
[14] KARNIYCHUK U U, GELDHOF M, VANHEE M, et al. Pathogenesis and antigenic characterization of a new East European subtype 3 porcine reproductive and respiratory syndrome virus isolate[J]. BMC Vet Res, 2010, 6:30.
[15] TIAN K G, YU X L, ZHAO T Z, et al. Emergence of fatal PRRSV variants:unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark[J]. PLoS One, 2007, 2(6):e526.
[16] WANG X, MARTHALER D, ROVIRA A, et al. Emergence of a virulent porcine reproductive and respiratory syndrome virus in vaccinated herds in the United States[J]. Virus Res, 2015, 210:34-41.
[17] ZHOU L, YANG H C. Porcine reproductive and respiratory syndrome in China[J]. Virus Res, 2010, 154(1-2):31-37.
[18] JI G B, LI Y Y, TAN F F, et al. Complete genome sequence of an NADC30-like strain of porcine reproductive and respiratory syndrome virus in China[J]. Genome Announc, 2016, 4(2):e00303-16.
[19] ZHOU L, WANG Z C, DING Y P, et al. NADC30-like strain of porcine reproductive and respiratory syndrome virus, China[J]. Emerg Infect Dis, 2015, 21(12):2256-2257.
[20] LI C, ZHUANG J, WANG J, et al. Outbreak investigation of NADC30-Like PRRSV in South-East China[J]. Transbound Emerg Dis, 2016, 63(5):474-479.
[21] SUN Z, WANG J, BAI X F, et al. Pathogenicity comparison between highly pathogenic and NADC30-like porcine reproductive and respiratory syndrome virus[J]. Arch Virol, 2016, 161(8):2257-2261.
[22] BROCKMEIER S L, LOVING C L, VORWALD A C, et al. Genomic sequence and virulence comparison of four Type 2 porcine reproductive and respiratory syndrome virus strains[J]. Virus Res, 2012, 169(1):212-221.
[23] ZHAO K, YE C, CHANG X B, et al. Importation and recombination are responsible for the latest emergence of highly pathogenic porcine reproductive and respiratory syndrome virus in China[J]. J Virol, 2015, 89(20):10712-10716.
[24] ZHOU L, ZHANG J L, ZENG J W, et al. The 30-amino-acid deletion in the Nsp2 of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in China is not related to its virulence[J]. J Virol, 2009, 83(10):5156-5167.
[25] FANG Y, ROWLAND R R, ROOF M, et al. A full-length cDNA infectious clone of North American type 1 porcine reproductive and respiratory syndrome virus:expression of green fluorescent protein in the Nsp2 region[J]. J Virol, 2006, 80(23):11447-11455.
[26] LI Y, ZHOU L, ZHANG J L, et al. Nsp9 and Nsp10 contribute to the fatal virulence of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in China[J]. PLoS Pathog, 2014, 10(7):e1004216.
[27] 杨汉春, 周磊. 猪繁殖与呼吸综合征病毒的遗传变异与演化[J]. 生命科学, 2016, 28(3):325-336.
YANG H C, ZHOU L. Genetic variation and evolution of porcine reproductive and respiratory syndrome virus[J]. Chinese Bulletin of Life Sciences, 2016, 28(3):325-336. (in Chinese)
[28] 杨汉春, 周磊. 2016年猪病流行情况与2017年流行趋势及防控对策[J]. 猪业科学, 2017, 34(2):36-37.
YANG H C, ZHOU L. Swine disease epidemic situation in 2016 and the epidemic trend, prevention and control countermeasures in 2017[J]. Swine Industry Science, 2017, 34(2):36-37. (in Chinese)
[29] ZHOU L, YANG B N, XU L, et al. Efficacy evaluation of three modified-live virus vaccines against a strain of porcine reproductive and respiratory syndrome virus NADC30-like[J]. Vet Microbiol, 2017, 207:108-116.
[30] BIAN T, SUN Y F, HAO M, et al. A recombinant type 2 porcine reproductive and respiratory syndrome virus between NADC30-like and a MLV-like:genetic characterization and pathogenicity for piglets[J]. Infect Genet Evol, 2017, 54:279-286.

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

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