The Unique Glycosylation at Position 986 on the E2 Glycoprotein of Classical Swine Fever Virus Is Responsible for Viral Attenuation and Protection against Lethal Challenge

Abstract

Classical swine fever (CSF) is an economically important disease of pigs caused by classical swine fever virus (CSFV). The live attenuated vaccine C-strain (also called HCLV strain) against CSF was produced by multiple passages of a highly virulent strain in rabbits. However, the molecular determinants for its attenuation and protection remain unclear. In this study, we identified a unique glycosylation at position 986 (⁹⁸⁶NYT⁹⁸⁸) on the E2 glycoprotein Domain IV of C-strain but not (⁹⁸⁶NYA⁹⁸⁸) the highly virulent CSFV Shimen strain. We evaluated the infectivity, virulence, and protective efficacy of the C-strain-based mutant rHCLV-T988A lacking the glycosylation and Shimen strain mutant rShimen-A988T acquiring an additional glycosylation at position 986. rShimen-A988T showed a significantly decreased viral replication ability in SK6 cells, while rHCLV-T988A exhibited a growth kinetics indistinguishable from that of C-strain. Removal of the C-strain glycosylation site does not affect viral replication in rabbits and the attenuated phenotype in pigs. However, rShimen-A988T was attenuated and protected the pigs from a lethal challenge at 14 days postinoculation. In contrast, the rHCLV-T988A-inoculated pigs showed transient fever, a few clinical signs, and pathological changes in the spleens upon challenge with the Shimen strain. Mechanistic investigations revealed that the unique glycosylation at position 986 influences viral spreading, alters the formation of E2 homodimers, and leads to increased production of neutralizing antibodies. Collectively, our data for the first time demonstrate that the unique glycosylation at position 986 on the E2 glycoprotein is responsible for viral attenuation and protection. IMPORTANCE Viral glycoproteins involve in infectivity, virulence, and host immune responses. Deglycosylation on the E^rns, E1, or E2 glycoprotein of highly virulent classical swine fever virus (CSFV) attenuated viral virulence in pigs, indicating that the glycosylation contributes to the pathogenicity of the highly virulent strain. However, the effects of the glycosylation on the C-strain E2 glycoprotein on viral infectivity in cells, viral attenuation, and protection in pigs have not been elucidated. This study demonstrates the unique glycosylation at position 986 on the C-strain E2 glycoprotein. C-strain mutant removing the glycosylation at the site provides only partial protection against CSFV challenge. Remarkably, the addition of the glycan to E2 of the highly virulent Shimen strain attenuates the viral virulence and confers complete protection against the lethal challenge in pigs. Our findings provide a new insight into the contribution of the glycosylation to the virus attenuation and protection.

Keywords: C-strain; attenuation; classical swine fever virus; glycosylation; protection.

FIG 1

Identification of the unique glycosylation at position 986 (⁹⁸⁶NYT⁹⁸⁸) on the E2 glycoprotein of the C-strain. (A) Analysis of the E2 glycoprotein of highly virulent CSFV Shimen strain (E2^Shimen) or C-strain (E2^HCLV) by Western blotting using the anti-E2 MAb WH303. (B) A multiple alignment of the 20 amino acids surrounding the N-glycosylation at position 986 is present for several CSFV strains of known virulence. (C) Mobility of E2^ShimenA988T harboring the mutation A988T and E2^HCLVT988A containing the mutation T988A analyzed by Western blotting. (D) Analysis of the E2^HCLV and E2^Shimen treated or not with endoglycosidase H by Western blotting.

FIG 2

Generation and characterization of the CSFV mutants rHCLV-T988A and rShimen-A988T. (A) Amino acid sequence of the parental viruses and the mutant viruses around the ⁹⁸⁶NYT⁹⁸⁸ motif and E2 glycosylation status of parental viruses and mutants. C-strain-based rHCLV-T988A lacks the glycosylation by mutation T988A in the motif ⁹⁸⁶NYT⁹⁸⁸ and the Shimen strain mutant rShimen-A988T acquires the glycosylation at position 986 by mutation A988T in its motif ⁹⁸⁶NYA⁹⁸⁸. (B) In vitro growth characteristics of glycosylation mutants and parental C-strain and the highly virulent Shimen strain in SK6 cells. The error bars represent the standard deviations for three replicates. *, P < 0.05 (Student's t test).

FIG 3

Virus spreading in cell culture infected by the mutant rShimen-A988T and the parental virus. (A) PK-15, SK6 cells, or swine macrophages were inoculated at an MOI of 0.001 with the parent and mutant viruses as indicated. The cell monolayers were overlaid with DMEM containing 1% Bacto agar and incubated at 37°C in the presence of 5% CO₂. After 48-h incubation, the cells were fixed with cold acetone, and viral E2 was examined by immunofluorescence using the anti-E2 MAb WH303. Representative focus sizes of PK-15 cells infected by the viruses are shown. (B) The infected cells per focus were counted for 30 independent foci (n = 30). Error bars represent the standard deviations. **, P < 0.01 (Student's t test).

FIG 4

Western blotting of SK6 cell lysates infected with the mutant virus rShimen-A988T and the parental virus Shimen strain. (A) Three-dimensional structure of E2 homodimers of CSFV (Model of structure of BVDV glycoprotein E2, pH 8). The boxed region in the top diagram is enlarged below, showing the dimer interface. The glycosylation at position 986 (N986) in the dimer interface and Cys at 983 (C983) are indicated. (B) Detection of E2 homodimers and E1-E2 heterodimers with the anti-E2 MAb WH303. SDS-PAGE was performed under the nonreducing conditions. GAPDH was used as an internal control. This experiment was done three times.

FIG 5

The Shimen strain mutant with the A988T mutation is attenuated in pigs. (A) Mortality of the pigs inoculated with 10⁴ TCID₅₀ of parental virus Shimen strain, rHCLV-T988A, or rShimen-A988T, respectively. The pigs in group 1 were inoculated intramuscularly (i.m.) with 10⁴ TCID₅₀ of rHCLV-T988A, the pigs in group 2 were inoculated i.m. with 10⁴ TCID₅₀ of rShimen-A988T, and the pigs in group 3 were infected with the Shimen strain as positive control to assess the pathogenicity. (B) Rectal temperatures of the pigs inoculated with different viruses are shown at the indicated days postinoculation. (C) The viral genome copy numbers in the blood of the pigs inoculated with mutants or the parental virus were tested by RT-qPCR at the indicated days postinoculation. (D) Representative pathological changes of various organs from the pigs inoculated with the parental virus Shimen strain, rHCLV-T988A, or rShimen-A988T.

FIG 6

The Shimen strain with the A988T mutation provides complete protection of pigs from lethal CSFV challenge. At 14 days postinoculation, the pigs in Group A (rHCLV-T988A), Group B (rShimen-A988T), and Group C (DMEM) were challenged with 10⁵ TCID₅₀ of the Shimen strain. (A) Rectal temperatures of the pigs challenged with the highly virulent Shimen strain were shown at the indicated days postchallenge (dpc). (B) The viral genome copy numbers in the blood samples of the pigs inoculated with mutants or parental virus were determined by RT-qPCR at the indicated dpc. (C) Representative pathological changes in various organs from the inoculated pigs following challenge with the Shimen strain.

FIG 7

The CSFV Shimen strain with the A988T mutation is more efficient to be neutralized than the parental virus by the sera. (A) Anti-E2 antibodies in the sera from the pigs inoculated with different viruses were determined at the indicated days postinoculation and postchallenge. (B) Neutralizing antibody titers against the Shimen strain. The sera were collected from the pigs inoculated with different viruses after the challenge of highly virulent CSFV Shimen strain. (C) Comparison of neutralizing ability against rShimen-A988T and parental virus using the sera collected from the pigs inoculated with the C-strain followed by challenging with the Shimen strain. Bar, 400 μm. (D) Neutralizing titers of the same sera (n = 3) to neutralize each of the viruses. **, P < 0.01 (Student's t test).

FIG 8

A schematic model of the effects of the unique glycosylation at position 986 on CSFV replication, antigenicity, and pathogenicity. The addition of the unique glycosylation at position 986 to E2 of the highly virulent Shimen strain can influence virus replication, increase neutralizing antibodies, and attenuate the virus in pigs. The dotted line indicates the previous findings on deglycosylation, and the full line indicates the findings on additional glycosylation in the present study.