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Characterization of essential domains and plasticity of the classical Swine Fever virus Core protein.

ABSTRACT: Pestiviruses are pathogens of cloven-hoofed animals, belonging to the Flaviviridae. The pestiviral particle consists of a lipid membrane containing the three envelope glycoproteins Erns, E1, and E2 and a nucleocapsid of unknown symmetry, which is composed of the Core protein and the viral positive-sense RNA genome. The positively charged pestiviral Core protein consists of 86 to 89 amino acids. To analyze the organization of essential domains, N- and C-terminal truncations, as well as internal deletions, were introduced into the Core coding sequence in the context of an infectious cDNA clone of classical swine fever virus strain Alfort. Amino acids 179 to 180, 194 to 198, and 208 to 212 proved to be of special importance for the generation of progeny virus. The results of transcomplementation of a series of C-terminally truncated Core molecules indicate the importance of Ala255 at the C terminus. The plasticity of Core protein was examined by the construction of concatemeric arrays of Core coding regions and the insertion of up to three yellow fluorescent protein (YFP) genes between two Core genes. Even a Core fusion protein with more than 10-fold-increased molecular mass was integrated into the viral particle and supported the production of infectious progeny virus. The unexpected plasticity of Core protein brings into question the formation of a regular icosahedric particle and supports the idea of a histone-like protein-RNA interaction. All viruses with a duplicated Core gene were unstable and reverted to the wild-type sequence. Interestingly, a nonviable YFP-Core construct was rescued by a mutation within the C-terminal domain of the nonstructural protein NS3.

SUBMITTER: Riedel C

PROVIDER: S-EPMC2953163 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Characterization of essential domains and plasticity of the classical Swine Fever virus Core protein.

Riedel Christiane C Lamp Benjamin B Heimann Manuela M Rümenapf Till T

Journal of virology 20100811 21

Pestiviruses are pathogens of cloven-hoofed animals, belonging to the Flaviviridae. The pestiviral particle consists of a lipid membrane containing the three envelope glycoproteins Erns, E1, and E2 and a nucleocapsid of unknown symmetry, which is composed of the Core protein and the viral positive-sense RNA genome. The positively charged pestiviral Core protein consists of 86 to 89 amino acids. To analyze the organization of essential domains, N- and C-terminal truncations, as well as internal d ...[more]

PMID: 20702631

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Project description:Recently moderate-virulence classical swine fever virus (CSFV) strains have been proven capable of generating postnatal persistent infection (PI), defined by the maintenance of viremia and the inability to generate CSFV-specific immune responses in animals. These animals also showed a type I interferon blockade in the absence of clinical signs. In this study, we assessed the infection generated in 7-week-old CSFV PI wild boars after infection with the African swine fever virus (ASFV). The wild boars were divided in two groups and were infected with ASFV. Group A comprised boars who were CSFV PI in a subclinical form and Group B comprised pestivirus-free wild boars. Some relevant parameters related to CSFV replication and the immune response of CSFV PI animals were studied. Additionally, serum soluble factors such as IFN-α, TNF-α, IL-6, IL-10, IFN-γ and sCD163 were analysed before and after ASFV infection to assess their role in disease progression.After ASFV infection, only the CSFV PI wild boars showed progressive acute haemorrhagic disease; however, the survival rates following ASFV infection was similar in both experimental groups. Notwithstanding, the CSFV RNA load of CSFV PI animals remained unaltered over the study; likewise, the ASFV DNA load detected after infection was similar between groups. Interestingly, systemic type I FN-α and IL-10 levels in sera were almost undetectable in CSFV PI animals, yet detectable in Group B, while detectable levels of IFN-γ were found in both groups. Finally, the flow cytometry analysis showed an increase in myelomonocytic cells (CD172a+) and a decrease in CD4+ T cells in the PBMCs from CSFV PI animals after ASFV infection.Our results showed that the immune response plays a role in the progression of disease in CSFV subclinically infected wild boars after ASFV infection, and the immune response comprised the systemic type I interferon blockade. ASFV does not produce any interference with CSFV replication, or vice versa. ASFV infection could be a trigger factor for the disease progression in CSFV PI animals, as their survival after ASFV was similar to that of the pestivirus-free ASFV-infected group. This fact suggests a high resistance in CSFV PI animals even against a virus like ASFV; this may mean that there are relevant implications for CSF control in endemic countries. The diagnosis of ASFV and CSFV co-infection in endemic countries cannot be ruled out and need to be studied in greater depth.

Project description:Two groups with three wild boars each were used: Group A (animals 1 to 3) served as the control, and Group B (animals 4 to 6) was postnatally persistently infected with the Cat01 strain of CSFV (primary virus). The animals, six weeks old and clinically healthy, were inoculated with the virulent strain Margarita (secondary virus). For exclusive detection of the Margarita strain, a specific qRT-PCR assay was designed, which proved not to have cross-reactivity with the Cat01 strain. The wild boars persistently infected with CSFV were protected from superinfection by the virulent CSFV Margarita strain, as evidenced by the absence of clinical signs and the absence of Margarita RNA detection in serum, swabs and tissue samples. Additionally, in PBMCs, a well-known target for CSFV viral replication, only the primary infecting virus RNA (Cat01 strain) could be detected, even after the isolation in ST cells, demonstrating SIE at the tissue level in vivo. Furthermore, the data analysis of the Margarita qRT-PCR, by means of calculated ΔCt values, supported that PBMCs from persistently infected animals were substantially protected from superinfection after in vitro inoculation with the Margarita virus strain, while this virus was able to infect naive PBMCs efficiently. In parallel, IFN-α values were undetectable in the sera from animals in Group B after inoculation with the CSFV Margarita strain. Furthermore, these animals were unable to elicit adaptive humoral (no E2-specific or neutralising antibodies) or cellular immune responses (in terms of IFN-γ-producing cells) after inoculation with the second virus. Finally, a sequence analysis could not detect CSFV Margarita RNA in the samples tested from Group B. Our results suggested that the SIE phenomenon might be involved in the evolution and phylogeny of the virus, as well as in CSFV control by vaccination. To the best of our knowledge, this study was one of the first showing efficient suppression of superinfection in animals, especially in the absence of IFN-α, which might be associated with the lack of innate immune mechanisms.

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Characterization of essential domains and plasticity of the classical Swine Fever virus Core protein.

Publications

Characterization of essential domains and plasticity of the classical Swine Fever virus Core protein.

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