The entry of BVDV into bovine cells was studied using CRIB cells (cells resistant to infection with bovine viral diarrhea virus [BVDV]) that have evolved from MDBK cells by a spontaneous loss of susceptibility to BVDV. Recently, larger genetic deletions were reported but no correlation of the affected genes and the resistance to BVDV infection could be established. The metalloprotease ADAM17 was reported as an essential attachment factor for the related classical swine fever virus (CSFV). To assess whether ADAM17 might be involved in the resistance of CRIB-1 cells to pestiviruses, we analyzed its expression in CRIB-1 and MDBK cells. While ADAM17 protein was detectable in MBDK cells, it was absent from CRIB-1 cells. No functional full-length ADAM17 mRNA could be detected in CRIB cells and genetic analysis revealed the presence of two defective alleles. Transcomplementation of functional ADAM17 derived from MDBK cells in CRIB-1 cells resulted in a nearly complete reversion of their resistance to pestiviral infection. Our results demonstrate that ADAM17 is a key cellular factor for the pestivirus resistance of CRIB-1 cells and establishes its essential role for a broader range of pestiviruses.

The novel pestivirus species known as lateral-shaking inducing neuro-degenerative agent (LINDA) virus emerged in 2015 in a piglet-producing farm in Austria. Affected piglets showed strong congenital tremor as a result of severe lesions in the central nervous system. Here, we report the results of a controlled animal infection experiment. Post-weaning piglets were infected with LINDA to determine the susceptibility of pigs, the clinical consequences of infection and the humoral immune response against LINDA. No clinically overt disease signs were observed in the piglets. Viremia was hardly detectable, but LINDA was present in the spleen and several lymphatic organs until the end of the experiment on day 28 post-infection. Oronasal virus shedding together with the infection of one sentinel animal provided additional evidence for the successful replication and spread of LINDA in the piglets. Starting on day 14 post-infection, all infected animals showed a strong humoral immune response with high titers of neutralizing antibodies against LINDA. No cross-neutralizing activity of these sera with other pestiviral species was observed. According to these data, following postnatal infection, LINDA is a rather benign virus that can be controlled by the pig’s immune system. However, further studies are needed to investigate the effects of LINDA on the fetus after intrauterine infection.

During the annual hunt in a privately owned Austrian game population in fall 2019 and 2020, 64 red deer (Cervus elaphus), 5 fallow deer (Dama dama), 6 mouflon (Ovis gmelini musimon), and 95 wild boars (Sus scrofa) were shot and sampled for PCR testing. Pools of spleen, lung, and tonsillar swabs were screened for specific nucleic acids of porcine circoviruses. Wild ruminants were additionally tested for herpesviruses and pestiviruses, and wild boars were screened for pseudorabies virus (PrV) and porcine lymphotropic herpesviruses (PLHV-1-3). PCV2 was detectable in 5% (3 of 64) of red deer and 75% (71 of 95) of wild boar samples. In addition, 24 wild boar samples (25%) but none of the ruminants tested positive for PCV3 specific nucleic acids. Herpesviruses were detected in 15 (20%) ruminant samples. Sequence analyses showed the closest relationships to fallow deer herpesvirus and elk gammaherpesvirus. In wild boars, PLHV-1 was detectable in 10 (11%), PLHV-2 in 44 (46%), and PLHV-3 in 66 (69%) of animals, including 36 double and 3 triple infections. No pestiviruses were detectable in any ruminant samples, and all wild boar samples were negative in PrV-PCR. Our data demonstrate a high prevalence of PCV2 and PLHVs in an Austrian game population, confirm the presence of PCV3 in Austrian wild boars, and indicate a low risk of spillover of notifiable animal diseases into the domestic animal population.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most relevant porcine pathogens worldwide. Active control of the disease relies on modified live virus vaccines (MLVs), as most inactivated vaccines provide very limited protection. Neutralizing antibodies occur late in infection; therefore, CD8⁺ T cells are considered important correlates of protection and are a frequent focus of investigation. Our aim was to identify viral peptides naturally bound by the class I major histocompatibility complex (MHC-I) and to confirm their ability to stimulate CD8⁺ T cells. For this purpose, we immunoprecipitated MHC-I/peptide complexes of PRRSV (strain AUT15-33) -infected cells (SLA-I Lr-Hp 35.0/24 mod) to isolate the viral epitopes and analyzed them with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Furthermore, we employed these identified peptides to stimulate peripheral blood mononuclear cells (PBMCs) of previously PRRSV-infected pigs and measured the PRRSV-specific CD8⁺ T-cell response with an intracellular cytokine staining (ICS). Our data revealed that PRRSV non-structural proteins (NSPs), encoded in open reading frame 1a and 1b (ORF1), present the major source of MHC-I-presented peptides. Additionally, we show that our identified epitopes are able to trigger IFNγ responses in vitro. These findings are a basis for understanding the proteasomal degradation of PRRSV proteins, the cellular ability to display them via MHC-I, and their potential to restimulate CD8⁺ T cells.

A novel pestivirus species, termed Lateral-shaking Inducing Neuro-Degenerative Agent virus (LindaV), was discovered in a piglet-producing farm in Austria in 2015 related to severe congenital tremor cases. Since the initial outbreak LindaV has not been found anywhere else. In this study, we determined the seroprevalence of LindaV infections in the domestic pig population of Austria. A fluorophore labeled infectious cDNA clone of LindaV (mCherry-LindaV) was generated and used in serum virus neutralization (SVN) assays for the detection of LindaV specific neutralizing antibodies in porcine serum samples. In total, 637 sera from sows and gilts from five federal states of Austria, collected between the years 2015 and 2020, were analyzed. We identified a single serum showing a high neutralizing antibody titer, that originated from a farm (Farm S2) in the proximity of the initially affected farm. The analysis of 57 additional sera from Farm S2 revealed a wider spread of LindaV in this pig herd. Furthermore, a second LindaV strain originating from this farm could be isolated in cell culture and was further characterized at the genetic level. Possible transmission routes and virus reservoir hosts of this emerging porcine virus need to be addressed in future studies.

The role of bovine CD46 in the host cell entry of BVDV has been established for more than a decade. By generating novel MDBK CD46 knock-out clones, we confirm previously reported data on the CD46 motives important for BVDV binding and the importance of the G479R exchange within BVDV E^rns to gain independence of bovine CD46 during entry. The comparison of different knock-out genotypes revealed a high variability of cellular susceptibility for a BVDV encoding the G479R exchange. These data highlight the effect of clonal selection of knock-outs on virus susceptibility, which should be considered when planning knock-out experiments.