Isolation Of PRRSV Variant With Partial Deletion In ORF2a Or NSP2 And Study On Its Growth Characteristics

Porcine reproductive and respiratory syndrome(PRRS), which is commonly known as blue ear disease, is caused by porcine reproductive and respiratory syndrome virus(PRRSV). The disease is characterized by serious reproductive dysfunction, preterm birth and high rates of miscarriage in sows, and severe respiratory system disease in all age pigs. Since the outbreak in 1987, the disease has caused great economic losses to the swine industry of the world. The highly pathogenic PRRS(HP-PRRS) was first outbreak in 2006, with high fever, high morbidity and high mortality as the main feature, resulting in the death of millions of pigs. Due to the variation is fast and easy to restructure, so large-scale field PRRSV may be more prone to mutation, resulting in a variety of strains, causing difficulties for the prevention and control of the PRRS of serious largescale farms. Therefore, a timely monitoring of PRRSV in large-scale farms is of great scientific importance for the prevalence and control of PRRS.In order to study the infection of PRRSV in large-scale fields, the study has collected 754 serums and 21 clinical samples of pigs from an immunized large-scale farm. By the ELISA test, it shows that 91.9% of the samples are positive for PRRS antibody, indicating that the pig farm had done well in vaccination. Detected by RT-PCR, we identified 104 samples as positive for PRRSV. In order to reduce the interference of vaccine strain to the wild-type PRRSV, NSP2 and GP5 sequence of individual positive samples are sequenced and analyzed and by porcine alveolar macrophages(PAM) we separate PRRSV wild type, so as to exclude vaccine strains(vaccine strain fail to passage on PAM cells). By the above steps, we isolate 34 wild PRRSVs, which accounts for 4.51% of the positive rate. These samples are sequenced separately, after compare the samples, we discover 14 new wild strain among the 34 samples, and these 14 strains are named Henan-A1-A14, and the sequence information was submitted to Gen Bank.By homology analysis we find that, although these 14 strains are isolated from the same field, but they are quite different in their genomes, generally they have about 96% homology, and the mean difference is 3.44% while the biggest difference between the strains is 4.4%. The 14 strains are sequenced for comparative analysis, and it indicate that by analyzing the results of the gene NSP2, comparing to CH-1a, CH2004 and VR2332 and BJ-4 these classic strains, Henan-A1 to A13 which 13 strains exist 1 + 29 amino acid deletion(position 481 aa and position 533-561aa) in NSP2, which molecular characteristic is consistent with the molecular characteristic of HP-PRRSV Hu N4, JXA1 and strains TJ. In addition to a 29 deletion in position 533-561 of HenanA14, there is another 48 amino acids miss in position 472-520 aa. The characteristics above indicate that 14 isolated strains belong to the HP-PRRSV; a comparison analysis for genome sequences of 14 virus and other 62 representatives PRRSV strains, phylogenetic analysis show that 76 strains can be grouped into six subgroups. The 14 strains are distinct distribution difference. A1 and the strain JXA1 isolated in 2006 are highly homologous; A2 and A5-A8 these five strains are closer in genetic relationship; A3, A4 and A9-A14 are closer among the independent branch which located relative far. Through the analysis we find that the strains has two significantly different evolutionary directions, it might be derived from the two strains, which evolved alone in the farm.We find that all these 14 PRRSV isolates can replicate in the PAM cells, but they are quite different in reproduce speed in the cell. In the study, we also find that there are 5 strains fail to proliferate on MARC-145 cell, which is an important feature different from other HP-PRRSV, which indicates that the cell tropism has changed, and may hinder the development of future vaccines and virus isolation work. In this study we use 5 neutralizing serum of Hu N4 strain to evaluate on the neutralizing titers of 14 isolate strains separately in PAM cells, and the result shows that 5 serum can barely neutralize Henan-A3, A4, A10 and A12, reflecting these 4 strains in large differences of neutralizing epitopes with Hu N4 strain. Also there is a big difference in the serum’s neutralizing titers to Henan-A8, A9, A11, A13 and A14 five strains compared to Hu N4, cross-neutralization test results show that most of the strains currently isolated have a larger variation in neutralizing antigen.By sequence alignment we discover 3 special strains, including Henan-A10 and A11 in ORF2 a come out a significant mutation, the codon TTA mutated to a stop codon TAA, leading to early termination of the C-terminus, which encodes the protein size reduced to 246 amino acids, shorter 10 amino acids than other strains. By comparison we also find that, compare with the classical strain CH-1a, VR2332, in addition to a 29 amino acids continuous deletion in NSP2 area of Henan-A14, there is another 48 amino acids successive miss in position 472-520 aa of NSP2. To study the influence of these two deletions of the virus, this study use infectious clone Hu N4-F112 as the skeleton, by site-directed mutagenesis PCR terminate the infectious clone encoding ORF2a’s termination codon of Hu N4-F112 in advance, build and rescue recombinant virus Hu N4-F112-ΔGP2, then divide them on Marc-145 cells and analyze their growth characteristics and the neutralizing antigen. The results show that the recombinant virus Hu N4-F112-ΔGP2’s growth rule and neutralization titers for the serum are not significant changed. We use fusion PCR to delete 48 amino acids in its NSP2 region to build and rescue another strain of recombinant virus Hu N4-F112-D48, and then analysis the changes on their growth characteristics and neutralizing antigens. The results show that the deletion of 48 amino acids in NSP2 area does not affect virus propagation on Marc-145 cells, which deletion of 48 amino acids has also no impact on virus neutralization antigen, so this area is a non-essential region NSP2 protein.In this study, by a continued monitoring of PRRSV in large-scale pig farms, we have isolated 14 HP-PRRSVs. The sequence alignments show that these strains are quite different with HPPRRSV representative strains, and two distinct evolutionary directions were found among these 14 strains themselves. The difference between strains suggested that the PRRSV in the same pig farm has a great diversity. In addition, the study finds that many current virus strains cell tropism has changed, and deletion was found in GP2 a protein which was proved to be not essential. The results will help to analysis the genetic evolution of the PRRSV in closed herds, discover the molecular characteristics of variant strains and popular features, understand PRRSV better, and improve the surveillance capability.