Abstract：Porcine reproductive and respiratory syndrome (PRRS), one of the leading outbreaks on a world scale in swine farms, is caused by Porcine reproductive and respiratory syndrome virus (PRRSV). This study aimed to construct reverse genetic system of highly pathogenic PRRSV (HP-PRRSV) strain HN07-1. The whole genome of HN07-1 was first obtained by a method in which the genome was segmently cloned and sequenced. Based on the reverse genetics technique, fragments of the whole genome were cloned into pcDNA3.1(+) vector and full-length infectious clone of HN07-1 strain, designated as pcDNA3.2-rHN07-1, were constructed. Besides, the recombinant plasmid pcDNA3.2-rHN07-1-EGFP was constructed by inserting EGFP between PRRSV ORF1b and ORF2a. The recombinant viruses were rescued by transfecting the constructed plasmids into Marc-145 cells and identified by immunofluorescence assay (IFA) and Western blot. The results showed that the full-length of HN07-1 genome was 15 345 nt. The typical cytopathic effect (CPE) was observed at 48 h of culture after one blind passage on Marc-145 cells. The results of IFA and Western blot indicated that PRRSV N protein expression of the rescued viruses and parent strain was confirmed. Moreover, the rescued viruses exhibit similar growth dynamic characteristics with its parent strain. Taken together, these results indicate that the infectious clones can be used for reverse genetic operation, which lays a foundation for the research of pathogenesis and vaccine development of PRRSV.
[1] 申梁, 谭文杰. 2015. 冠状病毒反向遗传操作技术及其应用进展[J]. 中国生物工程杂志, 35(02): 84-91. (Shen L, Tan W J.2015. Progress on the technique and its application of reverse genetics for coronaviruses[J]. China Biotechnology, 35(02): 84-91.) [2] 孙玉章, 倪雪霞, 李金明, 等. 2016. 动物RNA病毒反向遗传学操作技术及应用进展[J]. 中国动物检疫, 33(5): 68-71. (Sun Y Z, Ni X X, Li J M, et al.2016. Development and application of the reverse genetics technology for animal RNA viruses[J]. China Animal Health Inspection, 33(5): 68-71.) [3] 唐娜, 杨慧, 刘磊, 等. 2020. 表达EGFP重组猪繁殖与呼吸综合征病毒的构建与鉴定[J]. 中国动物传染病学报, 28(4): 39-46. (Tang N, Yang H, Liu L, et al.2020. Construction and characterization of recombinant porcine reproductive and respiratory syndrome virus expressing EGFP[J]. Chinese Journal of Animal Infectious Diseases, 28(4): 39-46.) [4] Ansari I H, Kwon B, Osorio F A, et al.2006. Influence of N-linked glycosylation of porcine reproductive and respiratory syndrome virus GP5 on virus infectivity, antigenicity, and ability to induce neutralizing antibodies.[J]. Journal of Virology, 80(8): 3994-4004. [5] Gorbalenya A E, Enjuanes L, Ziebuhr J, et al.2006. Nidovirales: Evolving the largest RNA virus genome[J]. Virus Res, 117(1): 17-37. [6] Jiang Y, Li G, Yu L, et al.2020. Genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) from 1996 to 2017 in China[J]. Frontiers in Microbiology, 11: 618. [7] Lee C, Calvert J G, Welch S K, et al.2005. A DNA-launched reverse genetics system for porcine reproductive and respiratory syndrome virus reveals that homodimerization of the nucleocapsid protein is essential for virus infectivity[J]. Virology, 331(1): 47-62. [8] Li Y, Firth A E, Brierley I, et al.2019. Programmed-2/-1 ribosomal frameshifting in simarteriviruses: An evolutionarily conserved mechanism[J]. Journal of Virology, 93(16): e00370-19. [9] Li Y, Zhou L, Zhang J, et al.2014. Nsp9 and Nsp10 contribute to the fatal virulence of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in China[J]. PLoS Pathog, 10(7): e1004216. [10] Meulenberg J J M, Hulst M M, de Meijer E J, et al.1993. Lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (PEARS), is related to LDV and EAV[J]. Virology, 192(1): 62-72. [11] Montaner-Tarbes S, Del P H, Montoya M, et al.2019. Key gaps in the knowledge of the porcine respiratory reproductive syndrome virus (PRRSV)[J]. Frontiers in Veterinary Science, 6: 38. [12] Neumann E J, Kliebenstein J B, Johnson C D, et al.2005. Assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the United States[J]. Journal of the American Veterinary Medical Association, 227(3): 385-392. [13] Nielsen H S, Liu G, Nielsen J, et al.2003. Generation of an infectious clone of VR-2332, a highly virulent North American-type isolate of porcine reproductive and respiratory syndrome virus[J]. Journal of Virology, 77(6): 3702-3711. [14] Nishi T, Onozato H, Ohashi S, et al.2016. Construction and characterization of a full-length infectious cDNA clone of foot-and-mouth disease virus strain O/JPN/2010 isolated in Japan in 2010[J]. Research in Veterinary Science, 106: 165-169. [15] Snijder E J, Meulenberg J J M.1998. The molecular biology of arteriviruses.[J]. The Journal of General Virology, 79(5): 961-979. [16] Tian D, Zheng H, Zhang R, et al.2011. Chimeric porcine reproductive and respiratory syndrome viruses reveal full function of genotype 1 envelope proteins in the backbone of genotype 2[J]. Virology, 412(1): 1-8. [17] Tian K, Yu X, Zhao T, et al.2007. Emergence of fatal PRRSV variants: Unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark[J]. PLOS ONE, 2(6): e526. [18] Wang L, Hou J, Gao L, et al.2014. Attenuation of highly pathogenic porcine reproductive and respiratory syndrome virus by inserting an additional transcription unit[J]. Vaccine, 32(43): 5740-5748. [19] Pei Y, Hodgins D C, Wu J, et al.2009. Porcine reproductive and respiratory syndrome virus as a vector: Immunogenicity of green fluorescent protein and porcine circovirus type 2 capsid expressed from dedicated subgenomic RNAs[J]. Virology, 389(1-2):91-99 [20] Yoo D, Welch S K, Lee C, et al.2004. Infectious cDNA clones of porcine reproductive and respiratory syndrome virus and their potential as vaccine vectors[J]. Veterinary Immunology and Immunopathology, 102(3): 143-154. [21] Zhou L, Zhang J, Zeng J, et al.2009. 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]. Journal of Virology, 83(10): 5156-5167.
