Influence of the amino acid residues at 70 in M protein of porcine reproductive and respiratory syndrome virus on viral neutralization susceptibility to the serum antibody

Abstract

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) is mainly responsible for the significant economic losses in pig industry in the world. The adaptive immune responses of the host act as an important source of selective pressure in the evolutionary process of the virus. In the previous study, we confirmed that the amino acid (aa) residues at 102 and 104 sites in GP5 played an important role in escaping from the neutralizing antibodies (NAbs) against highly pathogenic PRRSV (HP-PRRSV). In this study, we further analyzed the aa mutants affecting neutralization susceptibility of NAbs in other structure proteins in NAbs resistant variants.

Methods: Based on the different aa residues of the structural proteins between the resistant virus BB20s and the parent virus BB, 12 recombinant PRRSV strains containing these aa residue substitutions were constructed using reverse genetic techniques. The neutralizing antibody (NA) titers of the recombinant strains were tested on MARC-145 and porcine alveolar macrophages (PAMs). And the NAbs binding abilities of parent and rescued viruses were tested by using ELISA method.

Results: By using the neutralization assay, it was revealed that the NA titer of N4 serum with rBB/Ms was significantly lower than that with rBB. Meanwhile, NA titer of the serum with rBB20s/M was significantly higher than that with rBB20s. The ELISA binding results showed that rBB/Ms had higher binding inability to N4 than did rBB. And alignment of M protein revealed that the variant aa residue lysine (K) at 70 was also existed in field type 2 and vaccine PRRSV strains.

Conclusions: The aa residue at 70 in M protein of PRRSV played an important role in regulating neutralization susceptibility to the porcine serum NAbs. It may be helpful for monitoring the antigen variant strains in the field and developing new vaccine against PRRSV in the future.

Keywords: 70; M protein; Neutralizing antibody; PRRSV.

Fig. 1

The construction strategy of infectious cDNA clones of the recombinant PRRSVs using BB and BB20s strains. ^a The rBB/Ms-R and rBB20s/M-R were two revertant viruses and constructed from the full-length infectious cDNA clone pCMV-BB/Ms and pCMV-BB20s/M by using the site-directed mutagenesis, respectively

Fig. 2

Characterization of the recombinant PRRSV strains rBB, rBB/GP2s, rBB/GP3s, rBB/GP4s, rBB/Ms and revertant virus rBB/Ms-R. a Plaque morphology assay. b Virus neutralization assays of recombinant viruses by using the N4 serum. c The growth kinetics. The data are represented as the means ± s.d. of three independent experiments, and significant differences are shown (*P < 0.05) by using one-way analysis of variance (ANOVA)

Fig. 3

Characterization of the recombinant PRRSV strains rBB20s, rBB20s/GP2, rBB20s/GP3, rBB20s/GP4, rBB20s/M and revertant virus rBB20s/M-R. a Plaque morphology assay. b Virus neutralization assays of recombinant viruses by using the N4 serum. c The growth kinetics. The data are represented as the means ± s.d. of three independent experiments, and significant differences are shown (*P < 0.05 and **P < 0.01) by using one-way analysis of variance (ANOVA)

Fig. 4

Binding analysis of the resistant and recombinant mutant strains. A binding ELISA was performed using plates coated with each of the indicated viruses in triplicate. Serial dilutions of N4 serum were added to the wells, followed by the addition of a secondary anti-pig antibody conjugated to HRP. OD values were read at 450 nm. The negative control was wells that coated with ultracentrifuged non-infected MARC-145 cell lysates. The consistent concentration of ultracentrifugal PRRSV viruses that coated on the plate were identified by using Western Blot of N proteins. a rBB and related mutant viruses. b rBB20s and related mutant viruses

Fig. 5

Virus-neutralization test of resistant variants on PAMs. The growth kinetics of rBB and related viruses (a), rBB20s and related viruses (c). Virus neutralization assays of rBB and related viruses (b), rBB20s and related viruses (d). The data are represented as the means ± s.d. of three independent experiments, and significant differences are shown (*P < 0.05 and **P < 0.01) by using one-way analysis of variance (ANOVA)

Fig. 6

Alignment of partial M (a) and GP2 (b) aa sequences of BB20s with wild-type PRRSV isolates. The 70 aa site in M and 198 aa site in GP2 of BB20s and the same sites of the field PRRSV isolates are indicated by black boxes