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Improved strategy for phylogenetic analysis of classical swine fever virus based on full-length E2 encoding sequences.

ABSTRACT: Molecular epidemiology has proven to be an essential tool in the control of classical swine fever (CSF) and its use has significantly increased during the past two decades. Phylogenetic analysis is a prerequisite for virus tracing and thus allows implementing more effective control measures. So far, fragments of the 5´NTR (150 nucleotides, nt) and the E2 gene (190 nt) have frequently been used for phylogenetic analyses. The short sequence lengths represent a limiting factor for differentiation of closely related isolates and also for confidence levels of proposed CSFV groups and subgroups. In this study, we used a set of 33 CSFV isolates in order to determine the nucleotide sequences of a 3508-3510 nt region within the 5´ terminal third of the viral genome. Including 22 additional sequences from GenBank database different regions of the genome, comprising the formerly used short 5´NTR and E2 fragments as well as the genomic regions encoding the individual viral proteins Npro, C, Erns, E1, and E2, were compared with respect to variability and suitability for phylogenetic analysis. Full-length E2 encoding sequences (1119 nt) proved to be most suitable for reliable and statistically significant phylogeny and analyses revealed results as good as obtained with the much longer entire 5´NTR-E2 sequences. This strategy is therefore recommended by the EU and OIE Reference Laboratory for CSF as it provides a solid and improved basis for CSFV molecular epidemiology. Finally, the power of this method is illustrated by the phylogenetic analysis of closely related CSFV isolates from a recent outbreak in Lithuania.

SUBMITTER: Postel A

PROVIDER: S-EPMC3403906 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Improved strategy for phylogenetic analysis of classical swine fever virus based on full-length E2 encoding sequences.

Postel Alexander A Schmeiser Stefanie S Bernau Jennifer J Meindl-Boehmer Alexandra A Pridotkas Gediminas G Dirbakova Zuzana Z Mojzis Miroslav M Becher Paul P

Veterinary research 20120607

Molecular epidemiology has proven to be an essential tool in the control of classical swine fever (CSF) and its use has significantly increased during the past two decades. Phylogenetic analysis is a prerequisite for virus tracing and thus allows implementing more effective control measures. So far, fragments of the 5´NTR (150 nucleotides, nt) and the E2 gene (190 nt) have frequently been used for phylogenetic analyses. The short sequence lengths represent a limiting factor for differentiation o ...[more]

PMID: 22676246

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Project description:Vaccination is an effective method to control the spread of classical swine fever virus (CSFV), which is a major cause of economic losses to the swine industry. Although serological detection assays are commonly used to assess immune status, current methods for monitoring of antibodies (Abs) are time-consuming, expensive, and require cell culture and virus manipulation. To address these problems, the E2 protein of CSFV was expressed in transgenic rice seeds as a labeled antigen for the development of an immunochromatographic test strip (ICTS) for rapid, precise, and cost-effective detection of Abs. The ICTS has a reasonable sensitivity of 1:128,000 for detection of serum Abs against CSFV and no cross-reactivity with Abs of other porcine viruses. The similarity of the results between the proposed ICTS and a commercial enzyme-linked immunosorbent assay was 94.1% (128/136) for detection of serum Abs from immunized animals and 92.3% (72/78) for detection of maternally derived Abs. The proposed assay was successfully used to monitor Abs against E2 of both pigs and rabbits immunized with a live attenuated vaccine or an E2 subunit vaccine. The results confirmed that the ICTS can be applied to detect Ab levels in animals with different immunological backgrounds. The ICTS based on plant-derived E2 is a relatively inexpensive, rapid, and accurate assay for detection of Abs against CSFV and avoids the risk of contamination by animal products. IMPORTANCE The E2 protein of classical swine fever virus (CSFV) was expressed in transgenic rice endosperms as a diagnostic antigen for use with a rapid colloidal gold assay for the detection of antibodies (Abs) against CSFV. This improved test was used to monitor Abs against the E2 protein in both pigs and rabbits immunized with a live attenuated vaccine or E2 subunit vaccine. The assay successfully detected Ab levels in serum samples from piglets with different immunological backgrounds. In contrast to current E2 protein-based diagnostic methods using Escherichia coli or insect cells as expression systems, plant-derived E2 avoids the limitations of low immunogenicity of eukaryotic expression systems and potential contamination of fetal bovine serum with bovine viral diarrhea virus in cell culture.

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.

Dataset Information

Improved strategy for phylogenetic analysis of classical swine fever virus based on full-length E2 encoding sequences.

Publications

Improved strategy for phylogenetic analysis of classical swine fever virus based on full-length E2 encoding sequences.

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