scholarly journals A Polyuridine Insertion in the 3′ Untranslated Region of Classical Swine Fever Virus Activates Immunity and Reduces Viral Virulence in Piglets

2019 ◽  
Vol 94 (2) ◽  
Author(s):  
Miaomiao Wang ◽  
Matthias Liniger ◽  
Sara Muñoz-González ◽  
José Alejandro Bohórquez ◽  
Yoandry Hinojosa ◽  
...  
Keyword(s):  
Immune Responses ◽  
Classical Swine Fever Virus ◽  
Untranslated Region ◽  
Severe Disease ◽  
Classical Swine Fever ◽  
Viral Disease ◽  
Fever Virus ◽  
Diagnostic Tools ◽  
Virulence Determinants ◽  
Viral Virulence

ABSTRACT Low-virulence classical swine fever virus (CSFV) strains make CSF eradication particularly difficult. Few data are available on the molecular determinants of CSFV virulence. The aim of the present study was to assess a possible role for CSFV virulence of a unique, uninterrupted 36-uridine (poly-U) sequence found in the 3′ untranslated region (3′ UTR) of the low-virulence CSFV isolate Pinar de Rio (PdR). To this end, a pair of cDNA-derived viruses based on the PdR backbone were generated, one carrying the long poly-U insertion in the 3′ UTR (vPdR-36U) and the other harboring the standard 5 uridines at this position (vPdR-5U). Two groups of 20 5-day-old piglets were infected with vPdR-36U and vPdR-5U. Ten contact piglets were added to each group. Disease progression, virus replication, and immune responses were monitored for 5 weeks. The vPdR-5U virus was significantly more virulent than the vPdR-36U virus, with more severe disease, higher mortality, and significantly higher viral loads in serum and body secretions, despite similar replication characteristics in cell culture. The two viruses were transmitted to all contact piglets. Ninety percent of the piglets infected with vPdR-36U seroconverted, while only one vPdR-5U-infected piglet developed antibodies. The vPdR-5U-infected piglets showed only transient alpha interferon (IFN-α) responses in serum after 1 week of infection, while the vPdR-36U-infected piglets showed sustained IFN-α levels during the first 2 weeks. Taken together, these data show that the 3′ UTR poly-U insertion acquired by the PdR isolate reduces viral virulence and activates the innate and humoral immune responses without affecting viral transmission. IMPORTANCE Classical swine fever (CSF), a highly contagious viral disease of pigs, is still endemic in some countries of Asia and Central and South America. Considering that the 3′ untranslated region (3′ UTR) plays an important role in flavivirus replication, the present study showed for the first time that a long polyuridine sequence acquired in the 3′ UTR by an endemic CSFV isolate can activate immunity, control viral replication, and modulate disease in piglets. Our findings provide new avenues for the development of novel vaccines against infections with CSF virus and other flaviviruses. Knowledge of molecular virulence determinants is also relevant for future development of rapid and efficient diagnostic tools for the prediction of the virulence of field isolates and for efficient CSF control.

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 Immunogenicity in Swine of Orally Administered Recombinant Lactobacillus plantarum Expressing Classical Swine Fever Virus E2 Protein in Conjunction with Thymosin α-1 as an Adjuvant

2015 ◽  
Vol 81 (11) ◽  
pp. 3745-3752 ◽  
Author(s):  
Yi-Gang Xu ◽  
Xue-Ting Guan ◽  
Zhong-Mei Liu ◽  
Chang-Yong Tian ◽  
Li-Chun Cui
Keyword(s):  
Immune Responses ◽  
Lactobacillus Plantarum ◽  
Classical Swine Fever Virus ◽  
Oral Vaccine ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Economic Losses ◽  
E2 Protein ◽  
Content Type ◽  
Cellular Immune

ABSTRACTClassical swine fever, caused by classical swine fever virus (CSFV), is a highly contagious disease that results in enormous economic losses in pig industries. The E2 protein is one of the main structural proteins of CSFV and is capable of inducing CSFV-neutralizing antibodies and cytotoxic T lymphocyte (CTL) activitiesin vivo. Thymosin α-1 (Tα1), an immune-modifier peptide, plays a very important role in the cellular immune response. In this study, genetically engineeredLactobacillus plantarumbacteria expressing CSFV E2 protein alone (L. plantarum/pYG-E2) and in combination with Tα1 (L. plantarum/pYG-E2-Tα1) were developed, and the immunogenicity of each as an oral vaccine to induce protective immunity against CSFV in pigs was evaluated. The results showed that recombinantL. plantarum/pYG-E2 andL. plantarum/pYG-E2-Tα1 were both able to effectively induce protective immune responses in pigs against CSFV infection by eliciting immunoglobulin A (IgA)-based mucosal, immunoglobulin G (IgG)-based humoral, and CTL-based cellular immune responses via oral vaccination. Significant differences (P< 0.05) in the levels of immune responses were observed betweenL. plantarum/pYG-E2-Tα1 andL. plantarum/pYG-E2, suggesting a better immunogenicity ofL. plantarum/pYG-E2-Tα1 as a result of the Tα1 molecular adjuvant that can enhance immune responsiveness and augment specific lymphocyte functions. Our data suggest that the recombinantLactobacillusmicroecological agent expressing CSFV E2 protein combined with Tα1 as an adjuvant provides a promising strategy for vaccine development against CSFV.

Induction of immune responses in mice and pigs by oral administration of classical swine fever virus E2 protein expressed in rice calli

2014 ◽  
Vol 159 (12) ◽  
pp. 3219-3230 ◽  
Author(s):  
Myunghwan Jung ◽  
Yun Ji Shin ◽  
Ju Kim ◽  
Seung-Bin Cha ◽  
Won-Jung Lee ◽  
...  
Keyword(s):  
Immune Responses ◽  
Oral Administration ◽  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
E2 Protein ◽  
Rice Calli

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 The Laminin Receptor Is a Cellular Attachment Receptor for Classical Swine Fever Virus

2015 ◽  
Vol 89 (9) ◽  
pp. 4894-4906 ◽  
Author(s):  
Jianing Chen ◽  
Wen-Rui He ◽  
Liang Shen ◽  
Hong Dong ◽  
Jiahui Yu ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Viral Disease ◽  
Fever Virus ◽  
Laminin Receptor ◽  
Cellular Receptor ◽  
Small Interfering Rnas ◽  
Virus Attachment ◽  
Cellular Attachment

ABSTRACTClassical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious, economically important viral disease in many countries. The Ernsand E2 envelope glycoproteins are responsible for the binding to and entry into the host cell by CSFV. To date, only one cellular receptor, heparan sulfate (HS), has been identified as being involved in CSFV attachment. HS is also present on the surface of various cells that are nonpermissive to CSFV. Hence, there must be another receptor(s) that has been unidentified to date. In this study, we used a set of small interfering RNAs (siRNAs) against a number of porcine cell membrane protein genes to screen cellular proteins involved in CSFV infection. This approach resulted in the identification of several proteins, and of these, the laminin receptor (LamR) has been demonstrated to be a cellular receptor for several viruses. Confocal analysis showed that LamR is colocalized with CSFV virions on the membrane, and a coimmunoprecipitation assay indicated that LamR interacts with the CSFV Ernsprotein. In inhibition assays, anti-LamR antibodies, soluble laminin, or LamR protein significantly inhibited CSFV infection in a dose-dependent manner. Transduction of PK-15 cells with a recombinant lentivirus expressing LamR yielded higher viral titers. Moreover, an attachment assay demonstrated that LamR functions during virus attachment. We also demonstrate that LamR acts as an alternative attachment receptor, especially in SK6 cells. These results indicate that LamR is a cellular attachment receptor for CSFV.IMPORTANCEClassical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an economically important viral disease affecting the pig industry in many countries. To date, only heparan sulfate (HS) has been identified to be an attachment receptor for CSFV. Here, using RNA interference screening with small interfering RNAs (siRNAs) against a number of porcine membrane protein genes, we identified the laminin receptor (LamR) to be another attachment receptor. We demonstrate the involvement of LamR together with HS in virus attachment, and we elucidate the relationship between LamR and HS. LamR also serves as an attachment receptor for many viral pathogens, including dengue virus, a fatal human flavivirus. The study will help to enhance our understanding of the life cycle of flaviviruses and the development of antiviral strategies for flaviviruses.

scholarly journals Generation and immunogenicity analysis of recombinant classical swine fever virus glycoprotein E2 and Erns expressed in baculovirus expression system

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Qiang Wei ◽  
Yilin Bai ◽  
Yapeng Song ◽  
Yunchao Liu ◽  
Wei Yu ◽  
...  
Keyword(s):  
Immune Responses ◽  
Classical Swine Fever Virus ◽  
Expression System ◽  
Classical Swine Fever ◽  
Neutralizing Antibody ◽  
Baculovirus Expression System ◽  
Viral Envelope ◽  
Fever Virus ◽  
Complete Protection ◽  
Glycoprotein E2

AbstractClassical swine fever (CSF) caused by the classical swine fever virus (CSFV) is a highly contagious swine disease resulting in large economical losses worldwide. The viral envelope glycoprotein E2 and Erns are major targets for eliciting antibodies against CSFV in infected animals. In this report, the glycoprotein E2 and Erns were expressed using the baculovirus system and their protective immunity in rabbits were tested. Twenty CSFV seronegative rabbits were randomly divided into five groups. Each rabbit was intramuscularly immunized with CSFV-E2, CSFV-Erns, or their combination (CSFV-E2 + Erns). Besides, a commercial CSFV vaccine (C-strain) and PBS were used as positive or negative controls, respectively. Four weeks after the second immunization, all the rabbits were challenged with 100 RID50 of CSFV C-strain. High levels of CSFV E2-specific antibody, neutralizing antibody and cellular immune responses to CSFV were elicited in the rabbits inoculated with C-strain, CSFV-E2, and CSFV-E2 + Erns. And the rabbits inoculated with the three vaccines received complete protection against CSFV C-strain. However, no neutralizing antibody was detected in the Erns vaccinated rabbits and the rabbits exhibited fever typical of CSFV, suggesting the Erns alone is not able to induce a protective immune response. Taken together, while the Erns could not confer protection against CSFV, E2 and E2 + Erns could not only elicit humoral and cell-mediated immune responses but also confer complete protection against CSFV C-strain in rabbits.

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