New Emergence of the Novel Pestivirus Linda Virus in a Pig Farm in Carinthia, Austria

Abstract

Linda virus (LindaV) was first identified in a pig farm in Styria, Austria in 2015 and associated with congenital tremor (CT) type A-II in newborn piglets. Since then, only one more LindaV affected farm was retrospectively discovered 10 km away from the initially affected farm. Here, we report the recent outbreak of a novel LindaV strain in a farrow-to-finish farm in the federal state Carinthia, Austria. No connection between this farm and the previously affected farms could be discovered. The outbreak was characterized by severe CT cases in several litters and high preweaning mortality. A herd visit two months after the onset of clinical symptoms followed by a diagnostic workup revealed the presence of several viremic six-week-old nursery pigs. These animals shed large amounts of virus via feces and saliva, implying an important epidemiological role for within- and between-herd virus transmission. The novel LindaV strain was isolated and genetically characterized. The findings underline a low prevalence of LindaV in the Austrian pig population and highlight the threat when introduced into a pig herd. Furthermore, the results urge the need to better understand the routes of persistence and transmission of this enigmatic pestivirus in the pig population.

Keywords: Austria; Bungowannah virus; Flaviviridae; Linda virus; atypical porcine pestivirus; congenital tremor; emerging disease; novel Linda virus strain; pestivirus; viruses.

Figure 1

Locations of the farms where Linda virus (LindaV) strains Austria1, Austria2, and the novel strain Austria3 were isolated. Stars indicate the locations of the farms. Different brown shades mark regions with a high (dark brown) and a low (light brown) pig density. LindaV: Linda virus. (Modified from: https://www.statistik.at/atlas/?mapid=them_lw_as2010_viehbetriebe&layerid=layer1&sublayerid=sublayer0&languageid=0 (accessed on 18 August 2021). © Statistics Austria—Cartography and GIS, created 1 September 2018).

Figure 2

Results of the serum virus neutralization (SVN) assay (ND₅₀/mL), the LindaV-specific RT-qPCR assay from serum, plasma and PBMCs (GE/mL), and virus isolation from PBMCs of six-week-old nursery pigs (P1–P15). Virus particles represent successful virus isolation through co-culturing of PBMCs with SK-6 cells. Neutralizing antibody titers are given as the reciprocal ND₅₀ value and error bars indicate positive and negative standard deviations. ND₅₀, 50% neutralization dose; GE, genome equivalents; PBMC, peripheral blood mononuclear cell.

Figure 3

Detection of LindaV by immunohistochemistry in the cytoplasm of neurons (arrowheads) in the brainstem of a CT piglet. Primary antibody: pestivirus E2-specific monoclonal antibody 6A5; counterstain: hematoxylin, magnification 400×, insert 600×.

Figure 4

Phylogenetic analysis of selected pestivirus species and sequence analysis of the full-genomic sequences of the so far known Linda virus strains. (A) An unrooted phylogenetic tree was constructed based on the full-genomic sequences of LindaV strain Austria1, LindaV strain Austria2, LindaV strain Austria3, Bungowannah virus, Phocoena pestivirus isolate NS170386, Dongyang pangolin pestivirus isolate DYAJ, CSFV strain Alfort_187, and APPV strain AUT-2016_C using the neighbor-joining algorithm and bootstrapping with 1000 replicates. Bootstrap values are shown in percentage at each node. (B) Sequence identities between the different coding regions of the viral genome of LindaV strains Austria1, Austria2, and Austria3 are given in percentage. The coding regions for the nonstructural proteins are highlighted in dark green and coding regions for the structural proteins are in light green. DYPV, Dongyang pangolin pestivirus; PhoPeV, Phocoena pestivirus; BungoV, Bungowannah virus; APPV, atypical porcine pestivirus; CSFV, classical swine fever virus; UTR, untranslated region; C, Core; E, envelope glycoprotein; NS, nonstructural protein.