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Characterization of helper virus-independent cytopathogenic classical swine fever virus generated by an in vivo RNA recombination system.

ABSTRACT: Molecular analyses revealed that most cytopathogenic (cp) pestivirus strains evolve from noncytopathogenic (noncp) viruses by nonhomologous RNA recombination. In contrast to bovine viral diarrhea virus (BVDV), cp classical swine fever virus (CSFV) field isolates were rarely detected and always represented helper virus-dependent subgenomes. To investigate RNA recombination in more detail, we recently established an in vivo system allowing the efficient generation of recombinant cp BVDV strains in cell culture after transfecting a synthetic subgenomic and nonreplicatable transcript into cells being infected with noncp BVDV (A. Gallei, A. Pankraz, H.-J. Thiel, and P. Becher, J. Virol. 78:6271-6281, 2004). Using an analogous approach, the first helper virus-independent cp CSFV strain (CP G1) has now been generated by RNA recombination. Accordingly, this study demonstrates the applicability of RNA recombination for designing new viral RNA genomes. The genomic RNA of CP G1 has a calculated size of 18.139 kb, almost 6 kb larger than all previously described CSFV genomes. It contains cellular sequences encoding a polyubiquitin fragment directly upstream of the nonstructural protein NS3 coding gene together with a duplication of viral sequences. CP G1 induces a cytopathic effect on different tissue culture cell lines from pigs and cattle. Subsequent analyses addressed growth kinetics, expression of NS3, and genetic stability of CP G1.

SUBMITTER: Gallei A

PROVIDER: S-EPMC546568 | biostudies-literature | 2005 Feb

REPOSITORIES: biostudies-literature

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Characterization of helper virus-independent cytopathogenic classical swine fever virus generated by an in vivo RNA recombination system.

Gallei Andreas A Rümenapf Till T Thiel Heinz-Jürgen HJ Becher Paul P

Journal of virology 20050201 4

Molecular analyses revealed that most cytopathogenic (cp) pestivirus strains evolve from noncytopathogenic (noncp) viruses by nonhomologous RNA recombination. In contrast to bovine viral diarrhea virus (BVDV), cp classical swine fever virus (CSFV) field isolates were rarely detected and always represented helper virus-dependent subgenomes. To investigate RNA recombination in more detail, we recently established an in vivo system allowing the efficient generation of recombinant cp BVDV strains in ...[more]

PMID: 15681445

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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.

Project description:For members of the Flaviviridae, it is known that, besides the structural proteins, nonstructural (NS) proteins also play a critical role in virion formation. Pestiviruses, such as bovine viral diarrhea virus (BVDV), rely on uncleaved NS2-3 for virion formation, while its cleavage product, NS3, is selectively active in RNA replication. This dogma was recently challenged by the selection of gain-of-function mutations in NS2 and NS3 which allowed virion formation in the absence of uncleaved NS2-3 in BVDV type 1 (BVDV-1) variants encoding either a ubiquitin (Ubi) (NS2-Ubi-NS3) or an internal ribosome entry site (IRES) (NS2-IRES-NS3) between NS2 and NS3. To determine whether the ability to adapt to NS2-3-independent virion morphogenesis is conserved among pestiviruses, we studied the corresponding NS2 and NS3 mutations (2/T444-V and 3/M132-A) in classical swine fever virus (CSFV). We observed that these mutations were capable of restoring low-level NS2-3-independent virion formation only for CSFV NS2-Ubi-NS3. Interestingly, a second NS2 mutation (V439-D), identified by selection, was essential for high-titer virion production. Similar to previous findings for BVDV-1, these mutations in NS2 and NS3 allowed for low-titer virion production only in CSFV NS2-IRES-NS3. For efficient virion morphogenesis, additional exchanges in NS4A (A48-T) and NS5B (D280-G) were required, indicating that these proteins cooperate in NS2-3-independent virion formation. Interestingly, both NS5B mutations, selected independently for NS2-IRES-NS3 variants of BVDV-1 and CSFV, are located in the fingertip region of the viral RNA-dependent RNA polymerase, classifying this structural element as a novel determinant for pestiviral NS2-3-independent virion formation. Together, these findings will stimulate further mechanistic studies on the genome packaging of pestiviruses.IMPORTANCE For Flaviviridae members, the nonstructural proteins are essential for virion formation and thus exert a dual role in RNA replication and virion morphogenesis. However, it remains unclear how these proteins are functionalized for either process. In wild-type pestiviruses, the NS3/4A complex is selectively active in RNA replication, while NS2-3/4A is essential for virion formation. Mutations recently identified in BVDV-1 rendered NS3/4A capable of supporting NS2-3-independent virion morphogenesis. A comparison of NS3/4A complexes incapable/capable of supporting virion morphogenesis revealed that changes in NS3/NS4A surface interactions are decisive for the gain of function. However, so far, the role of the NS2 mutations as well as the accessory mutations additionally required in the NS2-IRES-NS3 virus variant has not been clarified. To unravel the course of genome packaging, the additional sets of mutations obtained for a second pestivirus species (CSFV) are of significant importance to develop mechanistic models for this complex process.

Project description:Classical swine fever (CSF) is an infectious disease caused by Classical swine fever virus (CSFV), which is characterized by depression, high fever, extensive skin bleeding, leukopenia, anorexia, alternating constipation, and diarrhea. Hemorrhagic infarction of the spleen is the main characteristic pathological change following CSFV infection. Large-scale outbreaks of CSF are rare in China and are mainly distributed regionally. The clinical symptoms of CSF are not obvious, and show variation from typical to atypical symptoms, which makes diagnosis based on clinical symptoms and pathology challenging. In recent years, the incidence of CSF-immunized pig farms in China has increased and new CSFV gene subtypes have appeared, posing new challenges to the prevention and control of CSF in China. Changes in metabolites caused by viral infection reflect the pathogenic process. Metabonomics can reveal the trace metabolites of organisms; however, plasma metabonomics of CSFV-infected pigs have rarely been investigated. Therefore, we used an established pig CSFV infection model to study changes in plasma metabolites. The results showed significant differences in forty-five plasma metabolites at different time periods after CSFV infection in pigs, with an increase in twenty-five metabolites and a decrease in twenty metabolites. These changed metabolites were mainly attributed to the tricarboxylic acid cycle, amino acid cycle, sugar metabolism, and fat metabolism. Thirteen metabolic pathways changed significantly in CSFV-infected pigs, including tricarboxylic acid cycle, inositol phosphate metabolism, glycine, serine and threonine metabolism,lysine degradation, alanine, aspartate and glutamic acid metabolism, pantothenate and CoA biosynthesis, β-alanine metabolism, lysine degradation, arginine and proline metabolism, glycerolipid metabolism, phenylalanine metabolism, arachidonic acid metabolism, linoleic acid metabolism. Among these, changes in fatty acid biosynthesis and metabolism occurred at all time periods post-infection. These results indicate that CSFV infection in pigs could seriously alter metabolic pathways.

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Characterization of helper virus-independent cytopathogenic classical swine fever virus generated by an in vivo RNA recombination system.

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Characterization of helper virus-independent cytopathogenic classical swine fever virus generated by an in vivo RNA recombination system.

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