Isolation and Cultivation of a New Isolate of BTV-25 and Presumptive Evidence for a Potential Persistent Infection in Healthy Goats

Abstract

Recently, several so-called "atypical" Bluetongue virus (BTV) serotypes were discovered, including BTV-25 (Toggenburg virus), in Switzerland. Most "atypical" BTV were identified in small ruminants without clinical signs. In 2018, two goats from a holding in Germany tested positive for BTV-25 genome by RT-qPCR prior to export. After experimental inoculation of the two goats with the BTV-25 positive field blood samples for generation of reference materials, viremia could be observed in one animal. For the first time, the BTV-25-related virus was isolated in cell culture from EDTA-blood and the full genome of isolate "BTV-25-GER2018" could be generated. BTV-25-GER2018 was only incompletely neutralized by ELISA-positive sera. We could monitor the BTV-25 occurrence in the respective affected goat flock of approximately 120 goats over several years. EDTA blood samples were screened with RT-qPCR using a newly developed BTV-25 specific assay. For serological surveillance, serum samples were screened using a commercial cELISA. BTV-25-GER2018 was detected over 4.5 years in the goat flock with intermittent PCR-positivity in some animals, and with or without concomitantly detected antibodies since 2015. We could demonstrate the viral persistence of BTV-25-GER2018 in goats for up to 4.5 years, and the first BTV-25 isolate is now available for further characterization.

Keywords: BTV; Bluetongue virus; atypical BTV; goats; persistent infection; serotype 25.

Figure 1

Phylogenetic analyses of the BTV-25-GER2018 genome. The phylogenetic trees of each of the 10 segments were created with MegaX using the genetic distinction model Tamura–Nei and tree-built method UPGMA including BTV strains representing the known BTV serotypes published [6]. We performed a bootstrap analysis with 1000 replications. The colors of the phylogenetic trees of segment 2 (Seg-2) and segment 6 (Seg-6) represent the different nucleotype groups [32] and trees were modified with FigTrees. For easier identification of the different nucleotypes A-N, the unrooted tree layout was chosen for Seg-2 and Seg-6. The arrows point at the BTV-25-GER2018 sequence.

Figure 1

Phylogenetic analyses of the BTV-25-GER2018 genome. The phylogenetic trees of each of the 10 segments were created with MegaX using the genetic distinction model Tamura–Nei and tree-built method UPGMA including BTV strains representing the known BTV serotypes published [6]. We performed a bootstrap analysis with 1000 replications. The colors of the phylogenetic trees of segment 2 (Seg-2) and segment 6 (Seg-6) represent the different nucleotype groups [32] and trees were modified with FigTrees. For easier identification of the different nucleotypes A-N, the unrooted tree layout was chosen for Seg-2 and Seg-6. The arrows point at the BTV-25-GER2018 sequence.

Figure 1

Phylogenetic analyses of the BTV-25-GER2018 genome. The phylogenetic trees of each of the 10 segments were created with MegaX using the genetic distinction model Tamura–Nei and tree-built method UPGMA including BTV strains representing the known BTV serotypes published [6]. We performed a bootstrap analysis with 1000 replications. The colors of the phylogenetic trees of segment 2 (Seg-2) and segment 6 (Seg-6) represent the different nucleotype groups [32] and trees were modified with FigTrees. For easier identification of the different nucleotypes A-N, the unrooted tree layout was chosen for Seg-2 and Seg-6. The arrows point at the BTV-25-GER2018 sequence.

Figure 1

Phylogenetic analyses of the BTV-25-GER2018 genome. The phylogenetic trees of each of the 10 segments were created with MegaX using the genetic distinction model Tamura–Nei and tree-built method UPGMA including BTV strains representing the known BTV serotypes published [6]. We performed a bootstrap analysis with 1000 replications. The colors of the phylogenetic trees of segment 2 (Seg-2) and segment 6 (Seg-6) represent the different nucleotype groups [32] and trees were modified with FigTrees. For easier identification of the different nucleotypes A-N, the unrooted tree layout was chosen for Seg-2 and Seg-6. The arrows point at the BTV-25-GER2018 sequence.

Figure 2

ELISA and PCR results of the 5 bleeding time points of the goat herd. The median values of the (A) reactivities of the ID.Vet cELISA in percent of negative control (≥50% is negative according to the manufacturer) and (B) the Cq values of the BTV-25 Mix13 Cq-values during the 5 bleeding time points of the goat flock are shown. The box and whisker plots show the median (broad central line), the interquartile range (box), the range of values (bars) and outliers (points).

Figure 3

Comparison of BTV-8 and BTV-25-GER2018 virus neutralization. Virus neutralization results of (A) methodical control with BTV-8 virus and BTV-8 positive serum (1:10, 1:80 and 1:1280) (B) cELISA positive serum of a BTV-25-GER2018 field infected goat (1:10, 1:80 and 1:280).