1383-1392. 10.1080/22221751.2019.1664940. 31526243<\/bibtext>\r\n <\/blist>\r\n <\/ref>\r\n \r\n Por Alexandra Kiesler; Lucas Schwartz; Christiane Riedel; Sandra Hügler; Ren\u00e9 Brunthaler; Catherine Dimmel; Angelica Auer; Marianne Za (2023)

AN0155712847;[b0fe]01feb.22;2022Mar17.09:01;v2.2.500<\/anid>\r\nNew occurrence of the new Pestivirus Linda virus in a pig farm in Carinthia, Austria\u00a0<\/title>\r\n<p>Linda virus (LindaV) was first identified in a pig farm in Styria, Austria in 2015 and has been associated with type A-II congenital tremor (CT) in newborn piglets. Since then, only one LindaV-affected farm within 10 km of the initially affected farm has been retrospectively discovered. Here, we report the recent outbreak of a new LindaV strain on a full-cycle farm in the federal state of Carinthia, Austria. No connection could be discovered between this farm and previously affected farms. The outbreak was characterized by severe cases of CT in multiple litters and high pre-weaning mortality. A visit to the farm two months after the onset of clinical symptoms, followed by a diagnostic study, revealed the presence of several viremic piglets at six weeks of age. These animals shed large amounts of virus through feces and saliva, implying an important epidemiological role for virus transmission within and between herds. The new strain LindaV was isolated and genetically characterized. The findings underscore a low prevalence of LindaV in the Austrian swine population and highlight the threat when introduced into a herd of pigs. Furthermore, the results call attention to the need to better understand the persistence and transmission routes of this enigmatic pestivirus in the swine population.<\/p>\r\n</p><p>Keywords: emerging disease; flaviviridae; pestivirus; atypical swine pestivirus; Bungowannah virus; congenital tremor; lindavirus; new strain of virus Linda; virus; Austria</\/p>\r\n<hd>1. Introduction<\/hd>\r\n<p>the genero<emph>Pestivirus<\/emph> within the family<emph>Flaviviridae<\/emph> includes economically important pathogens of pigs. In addition to OIE-listed classical swine fever virus (CSFV,<emph>Pestivirus C<\/emph>) [[<reflink idref='\"bib1\"'>1<\/reflink>]], Bungowannah virus (BungoV,<emph>Pestivirus F<\/emph>), atypical swine pestivirus (APPV,<emph>pestivirus K<\/emph>) and Linda virus (LindaV,<emph>pestivirus L<\/emph>) were detected in pigs [[<reflink idref='\"bib2\"'>2<\/reflink>], [<reflink idref='\"bib4\"'>4<\/reflink>]]. BungoV emerged in 2003 and is responsible for reproductive disorders, stillborn and mummified piglets and sudden death in weaning age piglets [[<reflink idref='\"bib2\"'>2<\/reflink>]]. A single BungoV outbreak has been detected and contained in Australia, but the virus has yet to be eradicated [[<reflink idref='\"bib5\"'>5<\/reflink>]]. The spread of BungoV has been considered a threat to global swine health, but to date there is no evidence that BungoV has established itself in other regions of the world [[<reflink idref='\"bib6\"'>6<\/reflink>], [<reflink idref='\"bib8\"'>8<\/reflink>]]. In contrast, APPV occurs worldwide and is responsible for type A-II congenital tremor (CT) in piglets that causes moderate economic losses [[<reflink idref='\"bib10\"'>10<\/reflink>], [<reflink idref='\"bib12\"'>12<\/reflink>], [<reflink idref='\"bib14\"'>14<\/reflink>]].<\/p>\r\n<p>LindaV was discovered as a new \"lateral jerk-inducing neurodegenerative agent\" on a piglet farm in Styria, Austria, in 2015 [[<reflink idref='\"bib4\"'>4<\/reflink>]]. Piglets infected in utero showed severe lateral tremors throughout the body, resulting in an inability to suck milk, leading to a high pre-weaning mortality [[<reflink idref='\"bib4\"'>4<\/reflink>]]. CT type A-II was confirmed during histopathological examinations of sick piglets with the presence of severe hypomyelination in the spinal cord and viral antigen in central nervous system (CNS) tissues [[<reflink idref='\"bib4\"'>4<\/reflink>]]. Experimental LindaV infections in immunocompetent piglets have been found to result in transient viremia and rapid seroconversion [[<reflink idref='\"bib15\"'>15<\/reflink>]]. However, the virus persisted in lymphoid tissues and was still detectable after 21 days. LindaV has so far been detected in the index case and at a farm 10 km away as a result of a nationwide LindaV neutralizing antibody test [[<reflink idref='\"bib16\"'>16<\/reflink>]]. This study revealed a very low seroprevalence of 0.15% (based on the number of sera tested) and 0.7% (at farm level) in Austria [[<reflink idref='\"bib16\"'>16<\/reflink>]]. A new genetically related LindaV strain (LindaV Austria2 strain) could be isolated from a serum sample in 2016 [[<reflink idref='\"bib16\"'>16<\/reflink>]]. Interestingly, no clinical signs of CT had ever been present in pigs on that farm, according to the farmer and the attending veterinarian.<\/p>\r\n<p>In this study, we report the isolation of a new LindaV strain (LindaV strain Austria3) causing a clinically relevant disease in a closed-loop farm in Carinthia, Austria, in 2020\/2021.<\/p>\r\n<hd>2. Materials and Methods<\/hd>\r\n<p><\/p>\r\n<hd>2.1. Farm Description<\/hd>\r\n<p>A full cycle commercial farm in Carinthia, Austria, produces piglets with 60 Large White and Landrace cross sows in a continuous farrowing cycle. A Pietrain boar is used for semen production, natural insemination and sexual stimulation of sows, which are artificially inseminated. Piglets are weaned at 28 days of age. Piglets are routinely vaccinated against<emph>Mycoplasma hyopneumoniae<\/emph> and against porcine circovirus 2 (PCV2) using inactivated vaccines (<emph>M. hyopneumoniae<\/emph> in the first week of life with Suvaxyn<sups>MH-One, Zoetis\u0dsterreich GmbH, Vienna, Austria; PCV2 at 21 days old with Suvaxyn Circus, Louvain-la-Neuve, Belgium).<\/p>\r\n<p>Prior to the onset of reproductive disorders, new gilts were purchased in March 2020 from a commercial gilt producer and introduced into the herd after an eight-week isolation phase. Gilts and sows are immunized against parvovirus and erysipelas by a combined inactivated vaccine (Parvoruvac, Ceva Sant\u00e9 Animale, Libourne, France) in the isolation unit and at weaning, respectively. Sows and gilts are vaccinated against Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) using a modified live virus vaccine (Porcilis PRRS, Intervet GesmbH, Vienna, Austria) at five-month intervals. Vaccination against swine influenza A virus using a trivalent vaccine was introduced after the onset of reproductive disorders.<\/p>\r\n<hd>2.2. Diagnostic samples<\/hd>\r\n<p>Samples were obtained by veterinarians from the University Pig Clinic (Vienna University of Veterinary Medicine) for diagnostic purposes during a farm visit. Therefore, ethical approval was not required for this study. A one-day-old non-viable piglet and an eight-week-old weanling piglet with paralysis of both hind legs were humanely sacrificed under general anesthesia (anesthesia: 1.3 mg/kg azaperone and 10 mg/kg azaperone hydrochloride ). euthanasia: T61 (5.0 mg\/mL of tetracaine hydrochloride, 50 mg\/mL of mebezonium iodide and 200 mg\/mL of embutramide; 1 mL\/10 kg)). The following tissues were collected from euthanized piglets: parotid gland, tonsil, lymph node (inguinal, tracheobronchial and mesentery), heart, lung, liver, pancreas, duodenum, jejunum, ileum, cecum, colon, kidney, genital tract, urinary bladder, thymus , spleen, cerebellum, medulla oblongata, spinal ganglion, spinal cord, sciatic nerve and umbilical cord (newborn piglets only). EDTA-treated whole blood and blood samples were collected from two sows with previous CT litters, 15 piglets weaned and 2 piglets euthanized as mentioned above (blood sample only from 8-week-old euthanized weaned piglets treated with EDTA). Pooled fecal samples were collected from the gestation pen, farrowing crates and table. Semen and a saliva sample were obtained from the boar. Additional saliva swabs were collected from 12 sows and gilts and 15 weaned piglets (pooled swab sample from weaned piglets). A pooled sample of placenta from newly farrowed sows and tails from newborn piglets was also obtained.<\/p>\r\n</p><p>In addition, the veterinary diagnostic laboratory of the federal state of Carinthia kindly provided paraffin-embedded tissue samples (brain, liver, spleen, kidney, gastrointestinal tract, lung and heart) from a CT-affected piglet. These tissue samples were obtained during the acute congenital tremor phase in December 2020.<\/p>\r\n<hd>23. Pathological Examination and Immunohistochemistry<\/hd>\r\n<p>A complete necropsy was performed on the eight-week-old weaned pig, euthanized due to paralysis of both hind legs. Samples of the brain, spinal cord, peripheral nerves, liver, spleen, kidneys, gastrointestinal tract, lung, lymph nodes, tonsils, thymus, pancreas and salivary glands were fixed in 4% buffered formalin and embedded in paraffin. Two µm-thick sections were cut and stained with hematoxylin and eosin (HE). Selected brain and spinal cord samples were stained with a combination of luxol fast blue and HE to assess myelin content. In addition, paraffin-embedded samples (brain, liver, spleen, kidney, gastrointestinal tract, lung and heart) from a piglet affected by CT, which was confirmed LindaV positive by RT-PCR, were stained with HE. /p >\r\n</p><p>CNS samples from both pigs were evaluated by immunohistochemistry using an anti-pestivirus E2 primary antibody (mouse monoclonal antibody (mAb) 6A5, 1:100 dilution) for the detection of LindaV. The stains were performed automatically in an autostainer (Lab Vision AS 360, Thermo Fisher Scientific, Waltham, MA, USA). Antigenic retrieval was performed by pronase digestion. After application of the primary antibody, a polymer detection system (UltraVision LP Large Volume Detection System; Thermo Fisher Scientific, Waltham, MA, USA) was used, which consists of a universal formulation of secondary antibody conjugated to an enzyme-labelled polymer. . The polymeric complex was then visualized with diaminobenzidine (Labvision/Thermo Fisher Scientific, Waltham, MA, USA). Sections were counterstained with hematoxylin. Formalin-fixed and paraffin-embedded spinal ganglia from pigs from the previous LindaV outbreak in 2015 served as positive and negative controls, respectively.<\/p>\r\n<hd>2.4. Isolation of peripheral blood mononuclear cells (PBMC)<\/hd>\r\n<p>EDTA-treated blood was centrifuged for 10 min at 2000<emph>g<\/emph> and 4 \u00b0C. Plasma was collected from the supernatant and stored at -0°C. The buffy coat was carefully removed (approximate volume of 1 ml), transferred to a centrifuge tube containing 5 ml of erythrocyte lysis buffer (EL buffer, QIAGEN, Hilden, Germany) and shaken vigorously. The mixture was incubated for 10 min at 4°C, followed by a centrifugation step for 10 min at 1000°C<emph>g<\/emph> and 4 \u00b0C. The supernatant was removed and the procedure was repeated twice. Finally, the PBMC pellet was resuspended in 1 mL of Dulbecco's modified Eagle medium (DMEM, Biowest, Nuaill\u00e9, France).<\/p>\r\n<hd>2.5. Cell culture<\/hd>\r\n<p>SK-6 cells [[<reflink idref='\"bib17\"'>17<\/reflink>]] in DMEM (Biowest) supplemented with 10% heat-inactivated fetal calf serum (FCS, Corning, Tewksbury, MA, USA; tested negative for pestivirus), 100 U\/mL of penicillin and 100 \u00b5g\/mL of streptomycin. Cells were maintained at 37 °C and 5% CO<subs>2<\/subs>.<\/p>\r\n<hd>2.6. Indirect immunofluorescence assay<\/hd>\r\n<p>The indirect immunofluorescence assay was performed as previously described [[<reflink idref='\"bib4\"'>4<\/reflink>]]. Briefly, SK-6 cells were fixed with 4% paraformaldehyde in PBS for 20 min at 4°C and permeabilized with 1% Triton-X 100 (Merck, Darmstadt, Germany) in PBS for 5 min at room temperature. The cross-reactive mAb 6A5 (anti-BVDV E2) was used as the primary antibody and a Cy3-conjugated goat anti-mouse IgG (Dianova, Hamburg, Germany) was used as the secondary antibody.<\/p>\r\ n<hd>2.7. Virus Isolation<\/hd>\r\n<p>A total volume of 50 µl of PBMC resuspended in DMEM (as described in Section 2.4) was co-cultured with 5 µl 10<sups>4 <\/sups> SK-6 cells in a 24-well cell culture plate (STARLAB, Hamburg, Germany). Cells and cell culture supernatant were passaged 72 h after co-cultivation. Successful infection of SK-6 cells was detected by an indirect immunofluorescence assay (as described in Section 2.6) and a LindaV-specific RT-qPCR assay (as described in Section 2.9).<\/p> \r \n<hd>2.8. Serum virus neutralization assay (SVN)<\/hd>\r\n<p>The SVN assay was performed as previously described [[<reflink idref='\"bib16\"'>16<\/reflink>]]. Briefly, sera were heat-inactivated at 56°C for 30 min and a 5-fold serial dilution (1/5 initial dilution, 1/390.625 final dilution) was prepared in DMEM (Biowest) in a culture dish. 96-well cell. (STARLAB). A piece of LindaV labeled with mCherry (1.78 \u00d7 10<sups>5<\/sup> TCID<subs>50<\/subs>\/mL) diluted to 100 TCID<subs>50<\/subs>\/50 \u00b5L, are added to serum dilutions and incubated for 2 h. A total number of 1 \u00d7 10<sups>4 SK-6 cells were added to the serum/virus mixture and then incubated for 72h. Serum controls, cell controls, positive and negative reference antisera, and a virus back-titration were included. Cells were fixed with 4% paraformaldehyde and evaluated under a fluorescence microscope (Olympus IX70 fluorescence microscope; OLYMPUS, Hamburg, Germany).<\/p>\r\n<p>The 50% neutralization dose (ND<subs>50<\/subs>\/mL) was calculated based on the Spearman-Kaerber method and expressed as the reciprocal value (1\/ND<subs>50<\/subs>\/mL) of serum dilution.<\/p>\r\n<hd>2.9. LindaV-specific RNA extraction and RT-qPCR assay<\/hd>\r\n<p>Total RNA extraction was performed with the QIAamp Viral RNA Mini Kit (QIAGEN) according to the manufacturer's instructions. A total volume of 140 µl of serum, plasma or semen was used directly for RNA extraction. Saliva swab samples were wetted with 1 mL of PBS, mixed by vortexing, then centrifuged and 140 5L of supernatant used for RNA extraction. Each 100 mg tissue sample was mixed with 1 ml of PBS in a 2 ml microcentrifuge tube containing stainless steel beads and homogenized at a rate of 30/sec for 3 min using a TissueLyser II (QIAGEN). After centrifugation, a volume of 140 \u00b5L of supernatant from the homogenate was used for extraction. PBMCs were lysed by three freeze/thaw cycles, cell debris was removed by centrifugation, and 140 µl of supernatant was used for RNA extraction.<\/p>\r\n</p><p>For LindaV RNA detection, a LindaV-specific RT-qPCR assay was performed on a Rotor-Gene Q cycler (QIAGEN) using the Luna Universal Probe One-Step RT-qPCR kit (NEB, Ipswich, Massachusetts, USA). ) as previously described [[<reflink idref='\"bib15\"'>15<\/reflink>]]. A beta-actin RT-qPCR assay was used as an internal control as previously described [[<reflink idref='\"bib16\"'>16<\/reflink>]] under the same cycling conditions as the LindaV RT-qPCR assay. Saliva swab samples were spiked with a plasmid containing the enhanced green fluorescent protein (EGFP) coding sequence prior to RNA extraction to exclude inhibitory factors in RT-qPCR performance, since beta-actin RT-qPCR produces inconsistent results on analysis of this sample material Oligonucleotides EGFP-F (5'-GACCACTACCAGCAGAACAC-3'), EGFP-R (5'-GAACTCCAGCAGGACCATG-3'), and EGFP probe -HEX (5'-HEX- AGCACCCAGTCCGCCCTGAGCA-BHQ-1-3') for the amplification of a 132 bp fragment of the EGFP sequence, as described by Hoffmann et al. [[<reflink idref='\"bib18\"'>18<\/reflink>]], under the same cycling conditions as the LindaV RT-qPCR assay.<\/p>\r\n<p>A ten-fold dilution series of a plasmid containing the target sequence for the LindaV RT-qPCR assay was included in each series to obtain a standard curve for quantification of viral load in samples as described previously [[<reflink idref='\"bib15\"'>15<\/reflink>]]. Briefly, the Rotor-Gene Q software (version 2.3.4.; QIAGEN) estimated the number of copies per reaction based on the number of DNA copies of the input plasmid and converted it into the respective equivalent genomes of each sample ( GE\/ mL , GE\ /g or GE\/swab). As we used a DNA standard and not an RNA standard in our RT-qPCR assay, the efficiency of the reverse transcription step could not be evaluated, which could lead to small biases in the copy number calculations.< \/p >\r\n<hd>2.10. RT-PCR, Sanger sequencing and sequence analysis<\/hd>\r\n<p>A pan-pestivirus RT-PCR amplifying a fragment of the NS5B coding region was performed using the OneTaq One-Step RT-PCR Kit (NEB) and oligonucleotides as previously described [[<reflink idref='\"bib4\"'>4<\/reflink>]]. The following cycling conditions were used: 48°C for 20 min, 94°C for 1 min, followed by 45 cycles of 94°C for 15 s, 50°C for 30 s, 68°C for 1:10 min, and a final elongation at 68°C. \u00b0C for 5 min. The amplicon of approximately 800 bp in length was purified using Quantum Prep PCR Kleen Spin Columns (Bio-Rad, Hercules, CA, USA) and the sequence of the purified PCR product was determined by Sanger sequencing (Eurofins Genomics, Ebersberg, Germany ) . ). The obtained sequence was analyzed using the Basic Local Alignment Nucleotide Search Tool (BLASTN) (https:\/\/blast.ncbi.nlm.nih.gov\/Blast.cgi?PROGRAM=blastn&PAGE%5fTYPE=BlastSearch&LINK%5fLOC=blasthome, accessed June 4, 2021).<\/p>\r\n<p>The complete genome of the new LindaV Austria3 strain (GenBank accession number: OK086026) was obtained by a two-step RT-PCR approach with the generation of overlapping PCR fragments using oligonucleotides and cycling conditions as previously described [[<reflink idref='\"bib16\"'>16<\/reflink>]]. PCR products were purified using Quantum Prep PCR Kleen Spin Columns (Bio-Rad) and sequences determined by Sanger sequencing (Eurofins Genomics). For determination of the complete genomic sequence of the new LindaV strain, a consensus sequence lacking only the 5' and 3' ends was generated and compared with available LindaV sequences (GenBank accession numbers: KY436034.1 (LindaV strain Austria1) and MZ027894 .1 (Strain LindaV Austria2)). Sequence analysis was performed with DNA Strider 3.0 software [[<reflink idref='\"bib19\"'>19<\/reflink>]] and CLC Sequence Viewer 7.7.1 (CLC Bio\/QIAGEN Digital Insights, Aarhus, Denmark).<hd>2.11. Phylogenetic analysis<\/hd>\r\n<p>Phylogenetic analysis was performed using CLC Sequence Viewer 7.7.1 (CLC bio\/QIAGEN Digital Insights, Aarhus, Denmark) based on complete genomic sequences. For the construction of the phylogenetic tree, the algorithm of union of neighbors and bootstrap with 1000 repetitions was used. The following pestivirus sequences were used for analysis: Linda Austria1 virus strain (KY436034.1,<emph>Pestivirus L<\/emph>), cepa do virus Linda Austria2 (MZ027894.1), cepa do virus Linda Austria3 (OK086026), Bungowannah virus (EF100713.2,<emph>Pestivirus F<\/emph>), Dongyang pangolin pestivirus isolate DYAJ1 (MK636874.1,<emph>Pestivirus P<\/emph>), Phocoena pestivirus isolate NS170386 (MK910229.1,<emph>Pestivirus M<\/emph>), pestivirus porcino atípico 1 cepa AUT-2016_C (KX778724.1,<emph>pestivirus K<\/emph>) and classical swine fever virus strain Alfort_187 (X87939.1,<emph>Pestivírus C<\/emph>).<\/p>\r\n<hd>3. Results<\/hd>\r\n<p><\/p>\r\n<hd>3.1. Description of the new essay LindaV<\/hd>\r\n<p>In autumn 2020, a full-cycle farm in the federal state of Carinthia in southern Austria (Figure 1) reported cases of reproductive disorders (miscarriages, neonatal deaths, stillbirths and mummified piglets) in several sows and gilts. Symptoms began in October 2020, when four sows farrowed four weeks before the estimated farrowing date. These piglets were born alive but died shortly after birth. The next seven litters showed known symptoms of parvovirus: mummies at different stages of development, stillbirths and piglets born alive. The episode of reproductive failure was followed by the appearance of severe CT in a total of 20 litters and was associated with a high pre-weaning mortality of 80-90% in December 2020 (Video S1). The last litters affected by CT occurred in early January 2021. During the CT episode on the farm, approximately 10–20% of the piglets were born weak and the rate of return to estrus was 10%. The number of piglets weaned per sow per year dropped from an average of 28 to an average of 22 due to reproductive disorders and piglet mortality. Pooled organ samples from five piglets affected by CT were sent to the University of Veterinary Medicine in Vienna in mid-January 2021. Analysis of the organ samples using pan-pestivirus RT-PCR in the NS5B region yielded a positive result. In the diagnostic laboratory of the Institute of Virology at the University of Veterinary Medicine in Vienna, PRRSV and porcine parvovirus (PPV) were excluded by PCR as differential diagnoses, and PCV2 qPCR results were below the level of quantification. Sequencing of the NS5B pestiviral amplicon of approximately 800 bp in length and analysis of the consensus sequence using the Basic Site Alignment Nucleotide Search Tool (BLASTN) revealed 98.3% sequence identity for the Austria1 strain of LindaV found in 2015 (GenBank accession number: KY436034.1). Pooled organ samples were retested using LindaV-specific RT-qPCR in the 5'-UTR region, which determined a very high viral load of 3.9' 10<sups>8<\/sups>GE\/g.<\/p>\r\n<p>Subsequently, a farm visit was carried out in February 2021 to diagnose the disease outbreak. At this time, no clinical symptoms associated with LindaV infection were observed and reproduction data approached pre-outbreak levels. Two sows, which farrowed the day before the farm visit, received homogeneous litters of clinically healthy newborn piglets. All other sows and suckling piglets in the farrowing barn appeared clinically healthy. A batch of six-week-old weanling piglets that survived the TC phase presented as a heterogeneous group with weak and weak piglets. An eight-week old breeding pig suffering from paralysis of both hind legs was observed and humanely euthanized. All sows and the neck appeared clinically healthy, while a mild dry cough was observed in finishing pigs.<\/p>\r\n<hd>3.2. Diagnostic job<\/hd>\r\n<p><\/p>\r\n<hd>3.2.1. Serum virus neutralization assay<\/hd>\r\n<p>Sera from 15 six-week-old weaned piglets (P1-P15), plasma from an eight-week-old weaned piglet, sera from two sows with previous CT litters, and serum from a one-day-old non-viable piglet were analyzed. parsed in the SVN test (as described in Section 2.8).<\/p>\r\n</p><p>High titers of neutralizing antibodies \u22651\/193.2 ND<subs>50<\/subs>\/ml were detected in nine weaned piglets (P1, P5, P7\u2013P9 and P12\u2013P15). Intermediate to low titers of neutralizing antibodies between 1/17.2 ND<subs>50<\/subs>\/mL e 1\/86,4 ND<subs>50<\/subs>\/mL were found in four animals (P2, P6, P10 and P11). Two newly weaned piglets (P3 and P4) did not show neutralizing activity (Figure 2). A high neutralizing activity of 1\/10,640 ND<subs>50<\/subs>\/mL was determined in the plasma of an eight week old weaned piglet sacrificed for paralysis of both hind legs. Two sows with previous CT litters showed strong neutralizing activity against LindaV (both 1/2180 ND<subs>50<\/subs>\/mL). No neutralizing activity was detected in a one day old non-viable piglet.<\/p>\r\n<hd>3.2.2. LindaV-specific RT-qPCR assay<\/hd>\r\n<p>LindaV-specific RT-qPCR assay and RT-qPCR assays for beta-actin and eGFP internal control genes were performed as described in Section 2.9. All samples tested in the LindaV RT-qPCR assay were RT-qPCR positive for the internal control genes beta-actin and eGFP.<\/p>\r\n</p><p>LindaV-specific RT-qPCR assay results of six-week-old weaned piglets (P1-P15; serum, plasma, and PBMC) are shown in Figure 2 and described in detail in Supplementary Table S1. Analysis of a saliva swab sample and a stool sample from six-week-old weaned piglets (P1-P15) confirmed viral shedding, showing a high viral load of 1.17-10<sups>6<\/sups> GE\/swab (pooled saliva swab sample) and 2.65 \u00d7<sups>6<\/sups> GE\/g (combined fecal sample) (Supplementary Table S1).<\/p>\r\n<p>Furthermore, tissue samples from different organs and blood samples (plasma and PBMC) from an eight-week-old weaned pig sacrificed due to paralysis of both hind legs were analyzed in the RT-qPCR assay. LindaV RNA was detected in lymphoid organs (inguinal lymph nodes and tonsils) and tissues of the CNS (cerebellum and medulla oblongata) and peripheral nervous system (PNS; spinal ganglion). Viral loads ranged from 2.25 \u00d7 10<sups>5<\/sups> GE\/g a 5,36 \u00d7 10<sups>6<\/sups> GE\/g (Supplementary Table S2). All other tissue samples were negative by RT-qPCR and viremia was not detected in this animal.<\/p>\r\n<p>LindaV RNA was not detectable in any of the samples obtained from sows and gilts (feces, saliva swab samples and blood samples). All samples obtained from the boar, newborn piglets and the combined placental sample were negative by LindaV-specific RT-qPCR.<\/p>\r\n<hd>3.2.3. Virus Isolation<\/hd>\r\n<p>Virus isolation was performed by co-cultivating PBMC with susceptible SK-6 cells (as described in Section 2.7). Virus isolation was successful in 7 out of 15 weaned piglets (P1-P4, P6, P10 and P11) (Figure 2 and Supplementary Table S1). No virus could be isolated from the PBMC of the sows and the day-old euthanized piglet.<\/p>\r\n<hd>3.2.4. Histopathology and Immunohistochemistry<\/hd>\r\n<p>Paraffin-embedded samples from the cerebellum and brainstem of a CT piglet were available for histological and immunohistochemical evaluation (as described in Section 2.3). HE staining showed no lesions, but LindaV could be detected in the cytoplasm of neurons by immunohistochemistry (Figure 3).<\/p>\r\n</p><p>In addition, tissue samples from the eight-week-old weaned piglet were analyzed. Immunohistochemistry for LindaV detection did not show a positive result in any of the tissues. In determining the cause of the paralysis of both hindlimbs, randomly distributed foci of neuronal necrosis were detected in the gray matter and white matter degeneration of the caudal cervical spinal cord with very mild reactive changes and scattered small hemorrhages. The etiology of these lesions could not be revealed. There were scattered multifocal perivascular lymphoplasmacellular infiltrates in the brain and spinal cord.<\/p>\r\n<hd>3.3. Genetic characterization of the new strain LindaV Austria3<\/hd>\r\n<p>The complete genome sequence of the new strain LindaV Austria3 (OK086026) was obtained using a two-step RT-PCR approach and Sanger sequencing. We chose the sample with the highest viral load in the RT-qPCR assay (3.16 \u00d7 10<sups>8<\/sups> GE\/mL; PBMC from the P3 weanling pig) for this sequencing attempt. A consensus sequence of 12,568 nt was determined and compared to sequences from the LindaV Austria1 strain (KY436034.1) and the LindaV Austria2 strain (MZ027894.1) (Figure 4). Interestingly, the LindaV Austria2 strain and the LindaV Austria3 strain were found to be slightly more closely related than the index case, the LindaV Austria1 strain; overall nucleic acid sequence identity was 98.9% compared to 98.5%, respectively. Sequence identities between the different coding regions of the LindaV genome are shown in Figure 4B. The divergence in nucleic acid sequence resulted in a comparable overall amino acid identity of 98.3% between LindaV Austria1 strain and LindaV Austria2 strain, as well as LindaV Austria1 strain and LindaV Austria3 strain. In contrast, the total amino acid identity was 99.1% when comparing the LindaV Austria2 strain to the LindaV Austria3 strain.<\/p>\r\n<hd>4. Discussion</\/hd>\r\n<p>The discovery of LindaV in Styria, Austria, in 2015 expanded the group of pestiviruses that infect swine host species and revealed the much-sought-after European relative of BungoV from Australia [[<reflink idref='\"bib4\"'>4<\/reflink>]]. Despite a high level of attention following the first LindaV outbreak, no signs of viral spread or the existence of this virus in other herds were observed [[<reflink idref='\"bib8\"'>8<\/reflink>], [<reflink idref='\"bib12\"'>12<\/reflink>]]. However, LindaV was found on a farm 10 km from the index case by retrospective serosurveillance without clinical indications [[<reflink idref='\"bib16\"'>16<\/reflink>]]. In this article, we describe a new outbreak of LindaV that occurred six years later in another province of Austria. As in the index case, CT and high pre-weaning mortality in piglets occurred after reproductive disorders in sows. Furthermore, LindaV was detectable in six-week-old pigs that underwent CT as newborn piglets, suggesting a chronic or persistent infection in these animals.<\/p>\r\n<p>Intrauterine infections with pestivirus in early pregnancy may cause immune tolerance [[<reflink idref='\"bib21\"'>21<\/reflink>], [<reflink idref='\"bib23\"'>23<\/reflink>], [<reflink idref='\"bib25\"'>25<\/reflink>]]. Immunotolerant animals become persistently infected (PI) and shed large amounts of virus throughout their lives. After intrauterine infection of the fetus with BungoV, a prolonged high-level viremia above 10<sups>6<\/sups> GE\/mL were observed up to 75 days of age [[<reflink idref='\"bib25\"'>25<\/reflink>]]. Although there is still a lack of studies evaluating the outcome of fetal infection with LindaV, high-level viremia of up to 3 \u00d7 10<sups>8 <\/sups> GE\/mL together with the lack of humoral immune response against LindaV in individual animals (P3 and P4 piglets) indicate the occurrence of such chronically, possibly persistently infected animals. In addition, these six-week-old animals shed large amounts of virus via the fecal and oral routes, which supports this hypothesis.<\/p>\r\n<p>High titers of neutralizing antibodies were found in some viremic piglets, which may have been acquired passively by ingestion of colostrum. Unfortunately, in our test it was not possible to discriminate between maternally derived and actively acquired antibodies. Maternally derived antibody titers can last a long time after pestivirus infection. Maternal antibodies against BVDV were detected around 190 days [[<reflink idref='\"bib26\"'>26<\/reflink>]] and against VPSC for a period of seven weeks [[<reflink idref='\"bib27\"'>27<\/reflink>]]. Therefore, the presence of maternal antibodies cannot be excluded.<\/p>\r\n<p>PBMC have been shown to represent a reliable sample for detecting BVDV cell-associated viremia [[<reflink idref='\"bib28\"'>28<\/reflink>]] e CSFV [[<reflink idref='\"bib29\"'>29<\/reflink>]]. In this study, we detected high LindaV RNA viral loads in PBMC and isolated the virus by co-cultivation with SK-6 cells. Several highly viremic animals showed a 10-fold higher viral load in PBMCs compared to serum and plasma, suggesting that the PBMC assay represents a more sensitive detection method for the viremic phase of LindaV infections. PBMC virus isolation was successful from 2.58 \u00d7 10<sups>5<\/sups> GE\/mL, although a high neutralizing antibody titer of 1\/434 ND<subs>50<\/subs>\/mL were observed in one animal (P1). In this case, attempts to isolate the virus from serum or plasma may lead to false negative results due to circulating neutralizing antibodies.<\/p>\r\n<p>In the eight-week-old weanling pig suffering from paralysis of both hind legs, acute focally extensive gray and white matter lesions were present in the caudal cervical spinal cord with only a mild reaction. Neither the distribution nor the type of lesions was representative of a viral infection. Therefore, it was thought that the lesions were probably of ischemic or traumatic origin. Unrelated to these lesions, scattered multifocal perivascular lymphoplasmacellular infiltrates were detected in the brain and spinal cord, which may be due to LindaV infection, consistent with viral RNA detected in CNS structures by RT-qPCR. Negative results in immunohistochemical analysis may indicate a lower sensitivity of this technique compared to the RT-qPCR assay.<\/p>\r\n</p><p>There is a high sequence identity between the three currently known LindaV strains (Austria1–Austria3). We found no evidence of transmission or epidemiological links between the cases in Styria and the new outbreak in Carinthia. Therefore, we must assume a low but constant prevalence of LindaV in southern Austria. The purchase of new gilts from a commercial gilt producer and the outbreak of the disease approximately six months after introduction into the pig herd could be a possible route of transmission. Investigations into the presence of LindaV at the gold production facility are ongoing. However, gilt farms in Austria often sell pigs to multiple pig farms and a single disease outbreak would be unlikely given the low seroprevalence of LindaV infections in the Austrian pig population [[<reflink idref='\"bib16\"'>16<\/reflink>]]. As our seroprevalence study was unable to detect reservoirs in domestic swine herds, it is reasonable to postulate a reservoir host in nature. Wild boar and ruminant species may represent the LindaV reservoir. Because pestiviruses have also been described in the recent past in some non-biungulate animals [[<reflink idref='\"bib30\"'>30<\/reflink>], [<reflink idref='\"bib32\"'>32<\/reflink>]], other wildlife reservoirs such as rodents or bats cannot be excluded. To prevent further introduction of the virus into the domestic swine population, identification of reservoir hosts is essential. Constant vigilance should also be established in the at-risk region of southern Austria to prevent spread through clinically inconspicuous long-term viremic animals.<\/p>\r\n<hd>5. Patents<\/hd>\r\n<p>The authors B.L., L.S. and T. R. are the inventors of a patent on the LindaV pestivirus (PCT\/EP2017\/084453; Isolation of a new pestivirus that causes congenital tremor).<\/p>\r\n<hd>Cifras<\/hd>\r\n<p>Graph: Figure 1 Location of farms where Linda virus strains Austria1, Austria2 and the new Austria3 (LindaV) were isolated. The stars indicate the location of the farms. Different shades of brown mark regions with high (dark brown) and low (light brown) density of pigs. Linda V: Linda virus. (Modified from: (Accessed 18 Aug 2021). \u00a9 Statistics Austria\u2014Cartography and GIS, Created 1 Sep 2018.)<\/p>\r\n</p><p>Graph: Figure 2 Results of serum virus neutralization (SVN) assay (ND50/mL), LindaV-specific RT-qPCR assay of serum, plasma, and PBMC (GE/mL), and viral isolation from six um PBMCs week-old weaned piglets (P1-P15). Viral particles represent successful viral isolation via co-cultivation 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.<\/p>\r\n</p><p>Graph: Figure 3 Detection of LindaV by immunohistochemistry in the cytoplasm of neurons (arrowheads) in the brainstem of a CT piglet. Primary antibody: monoclonal antibody 6A5 specific for pestivirus E2; contrast: hematoxylin, magnification 400\u00d7, insertion 600\u00d7.<\/p>\r\n</p><p>Graph: Figure 4 Phylogenetic analysis of selected pestivirus species and sequence analysis of complete genomic sequences of Linda virus strains known to date. (A) A rootless phylogenetic tree was constructed based on the complete 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 neighbor join algorithm and starting with 1000 replicas. Bootstrap values ​​are displayed in percentage on each node. (B) Sequence identities between the different coding regions of the viral genome of LindaV Austria1, Austria2 and Austria3 strains are given in percentage. Non-structural protein coding regions are highlighted in dark green and structural protein coding regions are highlighted in light green. DYPV, Dongyang pangolin pestivirus; PhoPeV, Phocoena pestivirus; BungoV, Bungowannah virus; APPV, porcine atypical pestivirus; CSFV, classical swine fever virus; UTR, untranslated region; C, Nucleus; E, envelope glycoprotein; NS, non-structural protein.<\/p>\r\n<hd>Author Contributions<\/hd>\r\n<p>Conceptualization, L.S., C.R., B.L. and T.R.; methodology, A.K., C.R., S.H., R.B., B.L. and T.R.; validation, A.K., L.S., B.L. and T.R.; formal analysis, A.K., L.S., A.L. and B.L.; research, A.K., L.S., C.R., S.H., R.B., K.D., A.A., M.Z., M.M. and K.S.; Resources, A.L., B.L. and T.R.; data curation, A.K. and LS; writing — preparing the original draft, A.K., L.S., S.H. and T.R.; writing - proofreading and editing, all authors; display, A.K. and S.H.; supervision, L.S., C.R., A.L., B.L. and T.R.; project management, T.R.; acquisition of funds, T.R. All authors have read and accepted the published version of the manuscript.<\/p>\r\n<hd>Financing<\/hd>\r\n<p>This research was funded by Boehringer Ingelheim Vetmedica GmbH (Binger Strasse 173, 55216 Ingelheim am Rhein, Germany, contract number: 396806).<\/p>\r\n<hd>Institutional Review Board Statement<\/hd>\r\n<p>Ethical review and approval was waived for this study as samples were collected by licensed veterinarians as part of a farm visit for diagnostic purposes.<\/p>\r\n<hd>Declaration of informed consent<\/hd>\r\n<p>Not applicable.<\/p>\r\n<hd>Declaration of Data Availability<\/hd>\r\n<p>All data analyzed or generated during this study are included in the manuscript.<\/p>\r\n<hd>Conflicts of interest<\/hd>\r\n<p>The authors declare no conflict of interest. Funders had no role in study design; in the collection, analysis or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.<\/p>\r\n<hd>Thanks<\/hd>\r\n<p>We thank the breeders and the veterinarian in charge of the herd for their cooperation and Kati Szakmary-Br\u00e4ndle, Hann-Wei Chen and Petra Kodajova for their excellent technical assistance. Open access funding from the Vienna University of Veterinary Medicine.<\/p>\r\n<hd>Supplementary Materials<\/hd>\r\n<p>The following support information can be downloaded from: https:\/\/<ulink href="%5C%22http:%5C/%5C/www.mdpi.com%5C/article%5C/10.3390%5C/v14020326%5C/s1,%5C%22">www.mdpi.com\/article\/10.3390\/v14020326\/s1,<\/ulink> Video S1: Piglets affected with congenital tremor (CT), Supplementary table S1: Results of serum virus neutralization assay (SVN) (1 / NORTH DAKOTA<subs>50<\/subs>\/mL), RT-qPCR assay (GE\/mL; GE\/g; GE\/swab) and virus isolation from six-week-old weaned piglets, Supplementary Table S2: Results of SVN attempt (1\/ND<subs>50<\/subs>\/mL), RT-qPCR assay (GE\/mL, GE\/g) and virus isolation from an eight-week-old weaned piglet with paralysis of both hind legs.< \ / p>\r\n<ref>\r\n<title>Footnotes <\/title>\r\n<blist>\r\n<bibl idref='\"ref1\"'>1<\/biblia>\r\n<bibtext>Editor's Note: MDPI remains neutral regarding jurisdictional claims on published maps and institutional affiliations.<\/bibtext>\r\n <\/blist>\r\n <\/ref>\r\n<ref>\r\n<title>References <\/title>\r\n<blist>\r\n<bibtext>OIE Terrestrial Animal Health Code Available online: https:\/\/<ulink href="%5C%22http:%5C/%5C/www.oie.int%5C/en%5C/what-we-do%5C/standards%5C/codes-and-manuals%5C/terrestrial-code-online-access%5C/(accessed%5C%22">www.oie.int\/en\/what-we-do\/standards\/codes-and-manuals\/terrestrial-code-online-access\/(acessado<\/ulink> em 9 de outubro de 2021)< \ /bibtext>\r\n <\/blist>\r\n<blist>\r\n<bibl idref='\"ref2\"'>2<\/biblia>\r\n<bibtext>Kirkland P.D., Frost M.J., Finlaison D.S., King K.R., Ridpath J.F., Gu X. 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An evolutionary divergent pestivirus that systematically lacks the gene Npro infects a species of whale. Emerging microbes infect. 2019; 8:1383-1392. 10.1080\/22221751.2019.1664940. 31526243<\/bibtext>\r\n <\/blist>\r\n <\/ref>\r\n<aug>\r\n<p>By Alexandra Kiesler; Lucas Negro; Christiane Riedel; Sandra Huegler; Ren\u00e9 Brunthaler; Catalina Dimmel; Angelica Auer; Marianne Zaruba; Marlene M\u00f6tz; Kerstin Seitz; other cargo; Benjamin Lamp e Till R\u00fcmenapf<\/p>\r\n</p><p>Reported by the author; Author; Author; Author; Author; Author; Author; Author; Author; Author; Author; Author; Author<\/p>\r\n <\/August>\r\n<nolink bibid='\"bib10\"' firstref='\"ref8\"' nlid='\"nl1\"'><\/no connection>\r\n<nolink bibid='\"bib12\"' firstref='\"ref9\"' nlid='\"nl2\"'><\/no connection>\r\n<nolink bibid='\"bib14\"' firstref='\"ref10\"' nlid='\"nl3\"'><\/no connection>\r\n<nolink bibid='\"bib15\"' firstref='\"ref14\"' nlid='\"nl4\"'><\/no connection>\r\n<nolink bibid='\"bib16\"' firstref='\"ref15\"' nlid='\"nl5\"'><\/no connection>\r\n</nolink></nolink></nolink></nolink></nolink></p></aug></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></blist></bibtext></bibl></blist></bibtext></bibl></blist></bibtext></bibl></blist></bibtext></bibl></blist></bibtext></bibl></blist></bibtext></bibl></blist></bibtext></bibl></blist></bibtext></bibl></blist></ulink></bibtext></blist>

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