scholarly journals 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

2021 ◽  
Author(s):  
Yongfeng Li ◽  
Mengqi Yuan ◽  
Yuying Han ◽  
Libao Xie ◽  
Yuteng Ma ◽  
...  
Keyword(s):  
Viral Replication ◽  
Classical Swine Fever Virus ◽  
Virulent Strain ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Lethal Challenge ◽  
E2 Glycoprotein ◽  
Viral Virulence ◽  
Viral Attenuation ◽  
Highly Virulent

Classical swine fever (CSF) is an economically important disease of pigs caused by the 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 ( 986 NYT 988 ) on the E2 glycoprotein Domain IV of C-strain but not ( 986 NYA 988 ) 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 post-inoculation. 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 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.

scholarly journals Analysis of Virus Population Profiles within Pigs Infected with Virulent Classical Swine Fever Viruses: Evidence for Bottlenecks in Transmission but Absence of Tissue-Specific Virus Variants

2020 ◽  
Vol 94 (19) ◽  
Author(s):  
Camille Melissa Johnston ◽  
Ulrik Fahnøe ◽  
Louise Lohse ◽  
Jens Bukh ◽  
Graham J. Belsham ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Population Change ◽  
Virulent Strain ◽  
Severe Disease ◽  
Clinical Signs ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Host Cells ◽  
Virus Population ◽  
Highly Virulent

ABSTRACT Classical swine fever virus (CSFV) contains a specific motif within the E2 glycoprotein that differs between strains of different virulence. In the highly virulent CSFV strain Koslov, this motif comprises residues S763/L764 in the polyprotein. However, L763/P764 represent the predominant alleles in published CSFV genomes. In this study, changes were introduced into the CSFV strain Koslov (here called vKos_SL) to generate modified CSFVs with substitutions at residues 763 and/or 764 (vKos_LL, vKos_SP, and vKos_LP). The properties of these mutant viruses, in comparison to those of vKos_SL, were determined in pigs. Each of the viruses was virulent and induced typical clinical signs of CSF, but the vKos_LP strain produced them significantly earlier. Full-length CSFV cDNA amplicons (12.3 kb) derived from sera of infected pigs were deep sequenced and cloned to reveal the individual haplotypes that contributed to the single-nucleotide polymorphism (SNP) profiles observed in the virus population. The SNP profiles for vKos_SL and vKos_LL displayed low-level heterogeneity across the entire genome, whereas vKos_SP and vKos_LP displayed limited diversity with a few high-frequency SNPs. This indicated that vKos_SL and vKos_LL exhibited a higher level of fitness in the host and more stability at the consensus level, whereas several consensus changes were observed in the vKos_SP and vKos_LP sequences, pointing to adaptation. For each virus, only a subset of the variants present within the virus inoculums were maintained in the infected pigs. No clear tissue-dependent quasispecies differentiation occurred within inoculated pigs; however, clear evidence for transmission bottlenecks to contact animals was observed, with subsequent loss of sequence diversity. IMPORTANCE The surface-exposed E2 protein of classical swine fever virus is required for its interaction with host cells. A short motif within this protein varies between strains of different virulence. The importance of two particular amino acid residues in determining the properties of a highly virulent strain of the virus has been analyzed. Each of the different viruses tested proved highly virulent, but one of them produced earlier, but not more severe, disease. By analyzing the virus genomes present within infected pigs, it was found that the viruses which replicated within inoculated animals were only a subset of those within the virus inoculum. Furthermore, following contact transmission, it was shown that a very restricted set of viruses had transferred between animals. There were no significant differences in the virus populations present in various tissues of the infected animals. These results indicate mechanisms of virus population change during transmission between animals.

scholarly journals P108 and T109 on E2 Glycoprotein Domain I Are Critical for the Adaptation of Classical Swine Fever Virus to Rabbits but Not for Virulence in Pigs

2020 ◽  
Vol 94 (17) ◽  
Author(s):  
Libao Xie ◽  
Yuying Han ◽  
Yuteng Ma ◽  
Mengqi Yuan ◽  
Weike Li ◽  
...  
Keyword(s):  
Viral Entry ◽  
Classical Swine Fever Virus ◽  
Molecular Basis ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
E2 Glycoprotein ◽  
Viral Adaptation ◽  
Glycoprotein E2 ◽  
Domain I ◽  
Highly Virulent

ABSTRACT The classical swine fever virus (CSFV) live attenuated vaccine C-strain is adaptive to rabbits and attenuated in pigs, in contrast with the highly virulent CSFV Shimen strain. Previously, we demonstrated that P108 and T109 on the E2 glycoprotein (E2P108-T109) in domain I (E2DomainI) rather than R132, S133, and D191 in domain II (E2DomainII) determine C-strain’s adaptation to rabbits (ATR) (Y. Li, L. Xie, L. Zhang, X. Wang, C. Li, et al., Virology 519:197–206, 2018). However, it remains elusive whether these critical amino acids affect the ATR of the Shimen strain and virulence in pigs. In this study, three chimeric viruses harboring E2P108-T109, E2DomainI, or E2DomainII of C-strain based on the non-rabbit-adaptive Shimen mutant vSM-HCLVErns carrying the Erns glycoprotein of C-strain were generated and evaluated. We found that E2P108-T109 or E2DomainI but not E2DomainII of C-strain renders vSM-HCLVErns adaptive to rabbits, suggesting that E2P108-T109 in combination with the Erns glycoprotein (E2P108-T109-Erns) confers ATR on the Shimen strain, creating new rabbit-adaptive CSFVs. Mechanistically, E2P108-T109-Erns of C-strain mediates viral entry during infection in rabbit spleen lymphocytes, which are target cells of C-strain. Notably, pig experiments showed that E2P108-T109-Erns of C-strain does not affect virulence compared with the Shimen strain. Conversely, the substitution of E2DomainII and Erns of C-strain attenuates the Shimen strain in pigs, indicating that the molecular basis of the CSFV ATR and that of virulence in pigs do not overlap. Our findings provide new insights into the mechanism of adaptation of CSFV to rabbits and the molecular basis of CSFV adaptation and attenuation. IMPORTANCE Historically, live attenuated vaccines produced by blind passage usually undergo adaptation in cell cultures or nonsusceptible hosts and attenuation in natural hosts, with a classical example being the classical swine fever virus (CSFV) lapinized vaccine C-strain, which was developed by hundreds of passages in rabbits. However, the mechanism of viral adaptation to nonsusceptible hosts and the molecular basis for viral adaptation and attenuation remain largely unknown. In this study, we demonstrated that P108 and T109 on the E2 glycoprotein together with the Erns glycoprotein of the rabbit-adaptive C-strain confer adaptation to rabbits on the highly virulent CSFV Shimen strain by affecting viral entry during infection but do not attenuate the Shimen strain in pigs. Our results provide vital information on the different molecular bases of CSFV adaptation to rabbits and attenuation in pigs.

scholarly journals The E2 Glycoprotein of Classical Swine Fever Virus Is a Virulence Determinant in Swine

2005 ◽  
Vol 79 (6) ◽  
pp. 3787-3796 ◽  
Author(s):  
G. R. Risatti ◽  
M. V. Borca ◽  
G. F. Kutish ◽  
Z. Lu ◽  
L. G. Holinka ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Vaccine Virus ◽  
Fever Virus ◽  
Genetic Determinants ◽  
Highly Pathogenic ◽  
Virus Shedding ◽  
E2 Glycoprotein ◽  
Viral Virulence

ABSTRACT To identify genetic determinants of classical swine fever virus (CSFV) virulence and host range, chimeras of the highly pathogenic Brescia strain and the attenuated vaccine strain CS were constructed and evaluated for viral virulence in swine. Upon initial screening, only chimeras 138.8v and 337.14v, the only chimeras containing the E2 glycoprotein of CS, were attenuated in swine despite exhibiting unaltered growth characteristics in primary porcine macrophage cell cultures. Additional viral chimeras were constructed to confirm the role of E2 in virulence. Chimeric virus 319.1v, which contained only the CS E2 glycoprotein in the Brescia background, was markedly attenuated in pigs, exhibiting significantly decreased virus replication in tonsils, a transient viremia, limited generalization of infection, and decreased virus shedding. Chimeras encoding all Brescia structural proteins in a CS genetic background remained attenuated, indicating that additional mutations outside the structural region are important for CS vaccine virus attenuation. These results demonstrate that CS E2 alone is sufficient for attenuating Brescia, indicating a significant role for the CSFV E2 glycoprotein in swine virulence.

scholarly journals Mutations in the carboxyl terminal region of E2 glycoprotein of classical swine fever virus are responsible for viral attenuation in swine

Virology ◽  
2007 ◽  
Vol 364 (2) ◽  
pp. 371-382 ◽  
Author(s):  
G.R. Risatti ◽  
L.G. Holinka ◽  
I. Fernandez Sainz ◽  
C. Carrillo ◽  
G.F. Kutish ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Terminal Region ◽  
Fever Virus ◽  
E2 Glycoprotein ◽  
Carboxyl Terminal ◽  
Viral Attenuation

scholarly journals N-Linked Glycosylation Status of Classical Swine Fever Virus Strain Brescia E2 Glycoprotein Influences Virulence in Swine

2006 ◽  
Vol 81 (2) ◽  
pp. 924-933 ◽  
Author(s):  
G. R. Risatti ◽  
L. G. Holinka ◽  
I. Fernandez Sainz ◽  
C. Carrillo ◽  
Z. Lu ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Infectious Clone ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Site Directed Mutagenesis ◽  
Target Cells ◽  
Virus Attachment ◽  
E2 Glycoprotein ◽  
Glycosylation Sites ◽  
Viral Virulence

ABSTRACT E2 is one of the three envelope glycoproteins of classical swine fever virus (CSFV). Previous studies indicate that E2 is involved in several functions, including virus attachment and entry to target cells, production of antibodies, induction of protective immune response in swine, and virulence. Here, we have investigated the role of E2 glycosylation of the highly virulent CSFV strain Brescia in infection of the natural host. Seven putative glycosylation sites in E2 were modified by site-directed mutagenesis of a CSFV Brescia infectious clone (BICv). A panel of virus mutants was obtained and used to investigate whether the removal of putative glycosylation sites in the E2 glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all putative glycosylation sites in E2 but restored when mutation N185A reverted to wild-type asparagine produced viable virus that was attenuated in swine. Single mutations of each of the E2 glycosylation sites showed that amino acid N116 (N1v virus) was responsible for BICv attenuation. N1v efficiently protected swine from challenge with virulent BICv at 3 and 28 days postinfection, suggesting that glycosylation of E2 could be modified for development of classical swine fever live attenuated vaccines.

scholarly journals A Novel E2 Glycoprotein Subunit Marker Vaccine Produced in Plant Is Able to Prevent Classical Swine Fever Virus Vertical Transmission after Double Vaccination

Vaccines ◽  
2021 ◽  
Vol 9 (5) ◽  
pp. 418
Author(s):  
Youngmin Park ◽  
Yeonsu Oh ◽  
Miaomiao Wang ◽  
Llilianne Ganges ◽  
José Alejandro Bohórquez ◽  
...  
Keyword(s):  
Vertical Transmission ◽  
Classical Swine Fever Virus ◽  
Neutralizing Antibodies ◽  
Subunit Vaccine ◽  
Vaccine Dose ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Tissue Samples ◽  
Marker Vaccine ◽  
E2 Glycoprotein

The efficacy of a novel subunit vaccine candidate, based in the CSFV E2 glycoprotein produced in plants to prevent classical swine fever virus (CSFV) vertical transmission, was evaluated. A Nicotiana benthamiana tissue culture system was used to obtain a stable production of the E2-glycoprotein fused to the porcine Fc region of IgG. Ten pregnant sows were divided into three groups: Groups 1 and 2 (four sows each) were vaccinated with either 100 μg/dose or 300 μg/dose of the subunit vaccine at 64 days of pregnancy. Group 3 (two sows) was injected with PBS. Groups 1 and 2 were boosted with the same vaccine dose. At 10 days post second vaccination, the sows in Groups 2 and 3 were challenged with a highly virulent CSFV strain. The vaccinated sows remained clinically healthy and seroconverted rapidly, showing efficient neutralizing antibodies. The fetuses from vaccinated sows did not show gross lesions, and all analyzed tissue samples tested negative for CSFV replication. However, fetuses of non-vaccinated sows had high CSFV replication in tested tissue samples. The results suggested that in vaccinated sows, the plant produced E2 marker vaccine induced the protective immunogenicity at challenge, leading to protection from vertical transmission to fetuses.

scholarly journals Classical swine fever virus employs the PERK- and IRE1-dependent autophagy for viral replication in cultured cells.

Virulence ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 130-149
Author(s):  
Erpeng Zhu ◽  
Huawei Wu ◽  
Wenxian Chen ◽  
Yuwei Qin ◽  
Jiameng Liu ◽  
...  
Keyword(s):  
Viral Replication ◽  
Classical Swine Fever Virus ◽  
Cultured Cells ◽  
Classical Swine Fever ◽  
Fever Virus
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