Neurotropism and Encephalitis of a Novel Pegivirus with Experimental Evidence Across Avian Species

Neurotropism and Encephalitis of a Novel Pegivirus with Experimental Evidence Across Avian Species | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Neurotropism and Encephalitis of a Novel Pegivirus with Experimental Evidence Across Avian Species Miguel Matos, Ivana Bilic, Nicolas Viloux, Barbara Jaskulska, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6888535/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Pegiviruses have traditionally been regarded as non-pathogenic viruses with controversial clinical significance. Here, we describe a novel avian pegivirus (partridge pegivirus, ParPgV) associated with field outbreaks of encephalitis in red-legged partridges ( Alectoris rufa ) in France. Next-generation sequencing identified ParPgV in brain tissues, and full-length genomic characterization revealed two distinct ParPgV strains linked to the outbreaks, confirming their phylogenetic relationship to avian-origin pegiviruses. Histopathology and electron microscopy revealed encephalitic lesions, neuronal degeneration, and virus-like particles within neurons. Field surveillance demonstrated widespread vertical transmission across multiple red-legged partridge flocks. Experimental inoculation of red-legged partridges, grey partridges, and specific-pathogen-free chickens demonstrated viral neurotropism and systemic distribution. Infected red-legged partridges developed cerebellar atrophy detectable by MRI, in the course of transient clinical signs. Detection of negative-strand RNA replication intermediates confirmed active viral replication in neural tissues and lymphoid organs, across the different experimental hosts, and red-legged partridge embryonated eggs. RNAscope in situ hybridization and immunohistochemistry further confirmed the presence of viral RNA and antigen, respectively, in neural and lymphoid tissues. These findings provide the first experimental evidence linking a pegivirus to encephalitis and suggest that pegiviruses may possess underappreciated neuropathogenic potential. Biological sciences/Microbiology/Virology/Viral pathogenesis Biological sciences/Microbiology/Virology/Virus–host interactions Health sciences/Pathogenesis/Infection Biological sciences/Microbiology/Pathogens Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Pegiviruses belong to the genus Pegivirus within the family Flaviviridae and are currently classified into 11 recognized species based on amino acid sequence divergence in conserved regions of NS3 and NS5B 1 , 2 . These viruses are enveloped, spherical particles measuring 50–100 nm in diameter, with a positive-sense single-stranded RNA genome ranging from 8.9 to 11.3 kb 1 . Pegiviruses have been identified in a broad range of mammalian hosts, including humans, non-human primates, horses, pigs, rodents, and bats 3 , 4 . More recently, the first non-mammalian pegiviruses were reported in diseased geese and metagenomic studies of healthy wild birds in China, Australia, and New Zealand, although their clinical relevance remains unclear 5 – 9 . In addition, the extent to which pegiviruses can transmit across different host species remains poorly understood 3 . Historically referred to as GB viruses or hepatitis G viruses (HGV), pegiviruses establish persistent infections, as reflected in their name (pe, persistent; g, GB or G) 10 . Initially, members of Pegivirus hominis and Pegivirus equi were implicated in human and equine hepatitis (Theiler’s disease), respectively 10 , 11 . However, subsequent studies failed to substantiate these associations, and pegiviruses are largely considered non-pathogenic 4 , 12 , 13 . Nonetheless, human pegivirus (HPgV) infection has been linked to an increased risk of developing non-Hodgkin’s lymphoma and has also been associated with encephalitis 3 . In support of this, HPgV RNA and viral antigen have been detected in post-mortem brain tissue of patients with encephalitis, and in vitro studies have demonstrated viral replication in human astrocytes and microglia 14 , 15 . The primary cellular targets of pegiviruses remain uncertain, representing a major obstacle to advancing mechanistic studies on infection and pathogenesis, particularly due to the absence of a robust in vitro culture system. Despite this limitation, pegiviruses are well recognized as lymphotropic viruses, with previous studies highlighting the bone marrow and spleen as key target organs 8 , 16 , 17 . Additionally, HPgV has been shown to exert immunomodulatory effects that enhance survival in patients co-infected with HIV, hepatitis C virus (HCV), and Ebola virus 3 . In this study, we investigated outbreaks of viral encephalitis in red-legged partridges ( Alectoris rufa ) from affected flocks in France. Initial diagnostic efforts targeting known avian neurotropic viruses — including avian encephalomyelitis virus, avian influenza virus, different arboviruses, and Marek’s disease virus — yielded negative results, prompting a non-targeted virological approach. This led to the identification of a novel pegivirus, designated partridge pegivirus (ParPgV), in the brain tissue of affected birds. Its consistent detection in encephalitis cases prompted the hypothesis that ParPgV was the causative agent of the outbreaks. To investigate this, we conducted comprehensive genomic characterization and performed in vivo studies in both homologous and heterologous hosts, providing key insights into its viral dynamics, infection kinetics, and host range. These findings strongly support the role of ParPgV as the etiological agent of the observed clinical signs. Materials and Methods Farm background and flock management Since 2017, sporadic neurological signs have been observed in red-legged partridge ( Alectoris rufa ) flocks on two geographically proximate farms in France. These events primarily involved three breeder flocks (designated A, B, and C), composed of birds aged 20–30 weeks, housed in pairs within cage systems. Flock sizes ranged from approximately 13,000 to 30,000 birds. All flocks were routinely vaccinated against Newcastle disease virus (NDV) and managed under standard photostimulation protocols to induce egg laying, which typically began in early February. Histological Analysis Tissue samples were preserved in a 4% neutral buffered formaldehyde solution (SAV LP GmbH, Flintsbach, Germany). After fixation, the samples were rinsed in water and subjected to dehydration and subsequently embedded in paraffin. Thin sections, 4 µm in thickness, were prepared from the paraffin blocks using a microtome (Microm HM 360; Microm Laborgeräte GmbH, Walldorf, Germany). Sections were stained with hematoxylin and eosin (H&E) for histological examination. Transmission Electron Microscopy (TEM) Brain samples preserved in 4% neutral buffered formaldehyde were used for transmission electron microscopy (TEM). The samples were sectioned into 1-mm³ pieces in 0.1 M phosphate buffer (Sigma-Aldrich, Vienna, Austria) at pH 7.2 and maintained at 4°C for 3 hours. The following preparation steps underwent the usual procedure with post fixation steps in a buffered 2,5% Karnovsky solution (pH 7,3) and in a cold phosphate buffered 1% osmium tetroxide solution (pH 7,3, 4°C, Agar Scientific, UK), with dehydration in a graded ethanol series followed by treatment with propylene oxide (Merck, Darmstadt, Germany) and with embedding in EPON 812 resin (Serva, Heidelberg, Germany). Ultrathin sections were stained with 1% methanolic uranyl acetate (Ted Pella, USA) and lead citrate after Reynolds (Merck, Germany), and examined using a Zeiss TEM 906 electron microscope (Zeiss, Oberkochen, Germany) operating at 80 kV. Nucleic acid extraction and construction of an NGS library Two next-generation sequencing (NGS) experiments were performed: the first on brain samples from clinically affected red-legged partridges during field outbreaks, and the second on partridge embryos. Total RNA was extracted from brain and embryo homogenates with the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), followed by genomic DNA removal using RNase-free DNase I (Qiagen) (unless otherwise stated, all reagents and kits were used according to the manufacturers’ protocols). Ribosomal RNA was depleted prior to library preparation using the NEBNext rRNA Depletion Kit v2 (New England Biolabs, Frankfurt am Main, Germany). Sequencing libraries were constructed with the NEBNext® Ultra™ II RNA Library Prep Kit for Illumina (New England Biolabs) and sequenced on the Illumina NextSeq 2000 platform in paired-end 150 bp mode (PE150) at the Vienna BioCenter Core Facilities GmbH (Next Generation Sequencing Facility, Vienna, Austria). Analysis of NGS-data obtained from NextSeq In the first NGS experiment, three brain samples, representing each flock, were processed in a single sequencing run. Raw reads were imported into CLC Genomics Workbench 23 ( https://digitalinsights.qiagen.com ), trimmed to remove low-quality bases and adapter sequences, and assembled de novo using the Microbial Genomics Module (default settings). Assembled contigs were screened for candidate viruses using a local BLASTN search against a database of complete viral genomes downloaded from NCBI on July 11, 2022 (-evalue 1e-05 -max_target_seqs 1). For each contig, only the top match was retained. In the second experiment, two partridge embryo samples were sequenced in separate runs. Reads were trimmed using Cutadapt (v4.9) with parameters -a AGATCGGAAGAGC -A AGATCGGAAGAGC -m 20, then imported into CLC Genomics Workbench 24 and assembled de novo as above. Contigs were screened by BLASTN against a custom database of avian pegivirus sequences and the complete genome of the ParPgV-A strain initially derived in this study. Matching contigs were used in the reconstruction of the partial genome sequence of ParPgV-C. Establishing the complete genome sequence The complete genomic sequence was established using a modified Sequence-Independent Single Primer Amplification (SISPA) protocol 18 . Briefly, total RNA from outbreak brain homogenates was reverse-transcribed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) and 2 µM SISPA-N primer (primers and thermal cycling conditions are listed in Supplementary Table 1.). cDNA was amplified with LongAmp® Taq 2X Master Mix (New England Biolabs) and 2 µM SISPA primer, and purified with AMPure Beads XP (Beckman Coulter, Krefeld, Germany) at a bead-to-cDNA ratio of 0.8:1. Pegivirus-specific primers (Supplementary Table 1) were designed based on NGS-derived contigs and aligned to avian pegivirus sequences from GenBank. PCRs were performed with LongAmp® Taq 2X Master Mix and 0.4 µM primers. Amplicons of expected sizes were excised and purified from agarose gels using the QIAquick Gel Extraction Kit (Qiagen). Purified fragments were cloned into the TOPO-TA vector (Invitrogen, Thermo Fisher Scientific) following the manufacturer’s instructions. From each PCR, at least three independent positive clones were sequenced by Sanger method (LGC Genomics, Berlin, Germany) using M13 and pegivirus-specific primers (Supplementary Table 1). Consensus sequences from ≥ 3 independent clones were assembled using Accelrys Gene version 2.5 (Accelrys, San Diego, CA, USA). Complete genome sequences of ParPgV-A and ParPgV-C strains were submitted to the NCBI database under accession numbers PV472371 and PV472372, respectively. Genome and Phylogenetic analyses Predicted proteins within the viral polyprotein were identified using HMMER Hmmscan search against the Pfam protein database (HmmerWeb version 2.43) and by comparison to the Goose Pegivirus-1 polyprotein. Complete polyprotein sequences of all known avian-origin pegiviruses were aligned using the MUSCLE method within the MegAlign Pro module of Lasergene v18.0.1 software (DNASTAR, Madison, WI, USA). Signal peptide prediction and cleavage site identification were performed using SignalP 6.0 0 (SignalP 6.0 - DTU Health Tech - Bioinformatic Services) 19 with the organism parameter set to "other." Phylogenetic analyses were conducted based on the NS3 and NS5B regions of the viral polyprotein. A total of 27 pegivirus polyprotein sequences from mammalian and avian origins were downloaded from the NCBI protein database. Amino acid alignments were generated using MUSCLE within MegAlign Pro. Conserved blocks were selected using GBlocks ( https://ngphylogeny.fr/tools/tool/276/form ). Phylogenetic trees were inferred using the Maximum Likelihood method (RAxML) with 100 bootstrap replicates. Distance matrices (NS3 region; 544 amino acids) were calculated using uncorrected pairwise distances and global gap removal in MegAlign Pro. Detection of ParPgV RNA by real-time qRT-PCR Total RNA was extracted from organ tissues with the RNeasy Mini Kit (Qiagen). Real-time RT-PCR assays targeting the conserved 5′-UTR region of the ParPgV genome were performed using the AriaMx Real-Time PCR System and the Brilliant III Ultra-Fast QRT-PCR Master Mix (Agilent Technologies, Vienna, Austria). Each 20 µL reaction contained 2 µL RNA template, 0.5 µM primers, and 0.2 µM probe (primer sequences and PCR conditions are provided in Supplementary Table 1). Fluorescence signals were analyzed with Agilent AriaMx software (version 1.7) with manual threshold settings. A Ct cutoff of 38 was applied. No-template controls were included to monitor contamination. Absolute quantification of viral load in animal samples was performed using a standard curve generated from in vitro transcribed 5′-UTR RNA cloned into a pCR®4-TOPO vector (Invitrogen). RNA transcription was carried out with a MAXIscript T7 kit (Thermo Fisher Scientific) after PCR amplification using the OneStep RT-PCR Kit (Qiagen) and specific primers (Supplementary Table 1). DNA templates were removed with the TURBO DNA-free Kit (Thermo Fisher Scientific). Viral RNA copies/µL were calculated based on RNA concentration and molecular weight. Detection of ParPgV-specific RNA by RNAscope Custom RNAscope probes were designed and provided by Bio-Techne (Dublin, Ireland) to target the highly conserved region at the 5′ end and the NS3 regions of the ParPgV genome, based on the consensus sequences of ParPgV-A nucleotide 2-949 (5’-UTR, signal sequence, and partial E1region) and ParPgV-C nucleotide 5450–6368 (NS4A-NS5A region) (catalogue no. 1333321-C1 and 1331411-C1, respectively). Probes targeting the messenger RNA (mRNA) of the widely expressed housekeeping gene peptidyl-prolyl-isomerase-B in red-legged partridges ( Alectoris rufa - PPIB; cat. no. 1331421-C1) and chickens ( Gallus gallus - PPIB; cat. no. 453371) served as positive controls, while a probe targeting bacterial dihydropicolinate reductase (DapB; cat. no. 310043) was used as a negative control. Detection of viral nucleic acid was performed using in-situ hybridization (ISH) with the manual RNAscope 2.5 High Definition RED assay (Bio-Techne), following the manufacturer’s protocol. Briefly, deparaffinized brain sections were pre-treated with 1× Target Retrieval solution and RNAscope® Protease Plus solution before hybridization with the target probe. Post-hybridization, the tissue underwent a series of amplification steps using pre-amplifier and amplifier solutions, followed by the application of a chromogenic substrate. Slides were counterstained with hematoxylin. As positive controls, tissue sections from field outbreaks were used, while negative controls included tissue sections from ParPgV-negative specific-pathogen-free (SPF) chickens. Signal scoring was performed according to the manufacturer’s guidelines. Recombinant protein production and generation of a polyclonal anti-ParPgV envelope glycoprotein antibody The partial E2 envelope glycoprotein (amino acids 414–570) of the ParPgV-A strain was expressed in the Bac-to-Bac Baculovirus system (Invitrogen). The coding region was cloned into the pFastBacHT-A vector using the NEBuilder HiFi DNA Assembly Kit (New England Biolabs). PCR amplification used Q5 High-Fidelity DNA Polymerase (New England Biolabs) with conditions listed in Supplementary Table 1. Recombinant protein was solubilized in lysis buffer (8 M urea, 50 mM NaH2PO4 pH 7.4, 0.5 M NaCl, 20 µg/mL DNase I, 1 mM MgCl2, 1 mM PMSF) and purified under denaturing conditions via His-tag affinity chromatography (His GraviTrap™ TALON®; Cytiva, Marlborough, MA, USA). Protein expression and purification were verified by Coomassie-stained SDS-PAGE and Western blotting using anti-polyHis-tag (Sigma Aldrich) and anti-mouse-HRP secondary antibodies (BioRad Laboratories) (Supplementary Fig. 1). Approximately 0.5 mg of purified E2 protein was delivered to Davids Biotechnologie GmbH (Regensburg, Germany) for rabbit immunization and polyclonal antibody production. Detection of ParPgV antigen by Immunohistochemistry For immunohistochemistry, 4-µm sections of formalin-fixed paraffin-embedded (FFPE) samples were prepared using a microtome (Microm HM 360) and mounted on positively charged glass slides (Superfrost Plus; Menzel-Gläser, Braunschweig, Germany). Tissue samples from partridges and SPF chickens, including samples from healthy birds and those infected with heterologous agents such as fowl adenovirus or Histomonas meleagridis , were included to evaluate the specificity of the polyclonal serum. Slides were dewaxed, rehydrated, and subjected to antigen retrieval in citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked using a 1.5% hydrogen peroxide solution in methanol for 30 minutes. To prevent nonspecific binding, the sections were incubated with a blocking solution consisting of a 1:10 dilution of normal goat serum (Vector Laboratories, Burlingame, USA) combined with 2% bovine serum albumin (Roche Diagnostics GmbH, Mannheim, Germany) for 60 minutes at room temperature in a humidified chamber. Primary antibody incubation was carried out overnight at 4°C using rabbit polyclonal anti-E2 serum at dilutions of 1:100, 1:500, and 1:1000. Additional sections were incubated with phosphate-buffered saline (PBS) instead of the primary antibody and used as negative controls. After washing with PBS, the sections were incubated with a 1:400 dilution of biotinylated anti-rabbit IgG (Vector Laboratories) for 30 minutes, followed by treatment with the Vectastain ABC Kit (Vector Laboratories) for 60 minutes. Signal detection used the DAB Substrate Kit for peroxidase (Vector Laboratories). The sections were counterstained with Mayer’s hematoxylin (Merck, Darmstadt, Germany), dehydrated, and mounted under coverslips with Neomount (VWR, Vienna, Austria). Field sampling of red-legged partridge flocks for detection of ParPgV A field screening for ParPgV was conducted across eight red-legged partridge flocks, from eight different farms, including both farms with a documented history of encephalitis and those without prior outbreaks. From each flock, 30 cloacal swabs were collected, comprising samples from 15 female and 15 male birds. Additionally, 30 embryonated eggs were collected from each flock. The yolk and allantoic fluid were extracted from 15 eggs for screening, while the remaining 15 eggs were incubated for 14 days to allow embryo collection for further analysis. Samples were pooled into groups of five for each type of material, and all pools were tested using real-time RT-PCR. Experimental in vivo investigations of ParPgV in partridge and chicken hosts Inoculum preparation Due to unsuccessful attempts to culture the virus in vitro (data not shown), an experimental inoculum was prepared using a filtrate from a ParPgV PCR-positive brain sample collected from flock A. For this purpose, brain tissue was suspended in PBS at a concentration of 20% (wt/vol), supplemented with 1 mg/ml streptomycin and 100,000 IU/ml penicillin. The suspension was homogenized using a T25 Ultra Turrax® (IKA, Staufen, Germany) at 20,000 rpm. The resulting homogenate was clarified by centrifugation at 2000 × g for 10 minutes, and the supernatant was passed through a 0.2 µm filter (Filtropur S 0.2, Sarstedt, Nümbrecht, Germany) to obtain the final filtrate (10 2.760 viral genome copies/µl). Following the first in vivo experiment, PCR analysis targeting the 260bp-ParPgV NS4A gene (Supplementary Table 1), followed by Sanger sequencing, identified two distinct ParPgV strains — designated ParPgV-A and ParPgV-C (the strain prevalent in flock C) — in the inoculum. As ParPgV-C was the predominant strain detected in the brains of inoculated grey partridges and was also highly prevalent in field monitoring samples, it was selected for further studies. Consequently, the RLP and SPF in vivo experiments were conducted using a ParPgV-C inoculum (10 2.752 viral genome copies/µl), derived from a ParPgV PCR-positive brain sample from flock C, prepared following the same protocol as ParPgV-A. Birds, experimental inoculation, and sampling Three separate consecutive experiments were conducted, each involving distinct groups of birds: (GP) grey partridges ( Perdix perdix ), (RLP) red-legged partridges ( Alectoris rufa ), and (SPF) SPF chickens ( Gallus gallus ). Grey partridges were sourced as 5-month-old birds from a local supplier in Austria (Mühlböck, Natternbach, Austria). In contrast, embryonated eggs of red-legged partridges (Gibovendeé, Les Herbiers, France) and SPF chickens (VALO BioMedia GmbH, Osterholz-Scharmbeck, Germany) were incubated at the facilities of the Clinic for Poultry Medicine at the University of Veterinary Medicine Vienna, Austria. Upon hatching, birds were housed under controlled conditions with feed and water ad libitum . All procedures were approved by the Ethics and Animal Welfare Committee of the University of Veterinary Medicine, Vienna, in accordance with the University’s guidelines for Good Scientific Practice and authorized by the Austrian Federal Ministry of Education, Science, and Research (ref BMBWF 2022 − 0.713.294, and Extension 2023 − 0.430.929), in accordance with current legislation. Details of the experimental design are summarized in Table 1 . Birds were intravenously inoculated with either ParPgV filtrate or PBS as a control. Clinical signs, which included weakness, reluctance to move, lack of avoidance to capture, and closed eyes, were monitored daily throughout the study. At predetermined timepoints, birds were humanely culled, and tissue samples were collected for histopathological and molecular analyses. In the grey partridge experiment, tissues included the brain, spleen, liver, and kidney. In the red-legged partridge experiment, additional samples from the bone marrow and caecal tonsils were collected. For the SPF chicken experiment, the thymus and bursa of Fabricius were also sampled in addition to the previously mentioned organs. All collected tissues were preserved in 4% neutral buffered formaldehyde solution (SAV LP GmbH) for histopathological analysis and stored at -80°C for virological and molecular investigations. Additionally, at different time points, tracheal and cloacal swabs were collected in the grey partridge and red-legged partridge experiments, while only cloacal swabs were collected in the SPF chicken experiment. Table 1 Overview of in vivo experimental infections with ParPgV in grey partridges, red-legged partridges, and SPF chickens. Study ID Host Age at Inoculation Group ID No. Birds Inoculum (ml)/bird Route of inoculation Sequential Culling (dpi): no. birds 7 10 14 21 GP Grey partridge ( Perdix perdix ) 5-months old GP-VG 7 0.2 ParPgV-A filtrate iv n.d. n.d. 2 3 GP-CG 3 0.2 PBS iv n.d. n.d. 1 1 RLP Red-legged partridge ( Alectoris rufa ) 5-months old RLP-VG 14 0.2 ParPgV-C filtrate iv 4 4 3 3 RLP-CG 6 0.2 PBS iv 2 2 1 1 SPF SPF chicken ( Gallus gallus ) 4-weeks old SPF-VG 12 0.2 ParPgV-C filtrate iv 4 n.d. 4 4 SPF-CG 8 0.2 PBS iv 2 n.d. 3 3 Abbreviations: dpi, days post-inoculation; iv, intravenous; PBS, phosphate-buffered saline; n.d., not done. Magnetic resonance imaging (MRI) Before culling, birds from the RLP and SPF experiments were transported under deep sedation to the diagnostic imaging unit. Magnetic resonance imaging (MRI) of the head was performed on two ParPgV-inoculated birds and one control bird per time point using a 1.5 Tesla scanner (Magentom Espree, Siemens Healthineers, Erlangen, Germany). MRI scans were performed at the time points indicated in Table 1 using a 70 mm medium-sized loop coil. Standardized imaging protocols were applied to ensure consistency across all groups and time points, including T2-weighted constructive interference in steady-state (CISS) 3D (TR 6.9 ms, TE 2.97 ms), T1-weighted turbo spin echo (TSE) transverse (TR 907 ms, TE 16 ms, slice thickness 5 mm), T2-weighted turbo inversion recovery (TIR) transverse (TR 4770 ms, TE 65 ms, slice thickness 0.8 mm), and T1-weighted gradient echo (GRE) turbo flash 3D (TR 1720 ms, TE 5.92 ms). The imaging data were anonymized and independently assessed by a European Board-certified radiologist, who performed subjective scoring of pathological changes. Detection of ParPgV negative-strand RNA by RT-PCR Detection of the negative-strand RNA was adapted from Lin et al . 20 with modifications. A 260-bp fragment of the NS4A region was amplified using OneStep RT-PCR Kit (Qiagen) and cloned into a pCR®4-TOPO vector (Invitrogen). Primers and thermal cycling conditions are provided in Supplementary Table 1. Positive and negative RNA strands were generated by in vitro transcription from T3 and T7 priming sites, respectively, using the MAXIScript T3 and T7 kits (Thermo Fisher Scientific). DNA templates were removed with the TURBO DNA-free Kit. To detect negative-strand RNA, cDNA synthesis was performed using the OmniScript RT Kit (Qiagen) and a chimeric primer combining an oligonucleotide tag with a virus-specific sequence. PCR amplification was conducted with HotStarTaq Master Mix Kit (Qiagen) using the oligonucleotide tag as the forward primer and a ParPgV-specific reverse primer (Supplementary Table 1). A 257-bp PCR product indicated the presence of negative-strand RNA. Data Analysis and Statistics Data were recorded in Excel (Office 365) and analyzed in Python (version 3.13). Data processing and statistical analyses were performed using pandas, scipy, and matplotlib/seaborn libraries. Normality of continuous variables was assessed using the Shapiro–Wilk test. Comparisons of viral load in organs of experimentally inoculated red-legged partridges were conducted using unpaired t-tests. Statistical significance was defined as P < 0.05. Results Outbreak Description and Clinical observations Prior to 2021, clinical signs suggestive of CNS involvement, including apathy, torticollis, ataxia, and prostration (Supplementary Video 1), were generally transient, sporadic, and subtle, and were often overlooked by farm personnel. Affected birds were commonly culled upon detection, which may have limited onward disease transmission within the flock. These clinical occurrences were not initially considered economically relevant. This perception changed in 2021, when a more severe and prolonged outbreak occurred during the laying period on one of the affected farms. At the time, the breeder flock consisted of 6,550 pairs of red-legged partridges. The onset of the outbreak coincided with the beginning of the laying cycle in February/March and was characterized by persistent and recurring neurological signs. Farmers reported culling 10 to 20 birds per week during the first weeks of the season. However, by April, due to the substantial impact on flock productivity and in the absence of evidence for vertical transmission, culling was halted, as affected birds continued to lay eggs despite exhibiting clinical signs. It is estimated that at least 2% of birds were affected over the 16-week laying period, with egg production being reduced by approximately 4.6 eggs per hen in 2021 compared to other years. No bacterial pathogens were isolated during routine diagnostic investigations conducted at the time of the outbreak. Gross, Histopathological, and Ultrastructural Evidence of Viral Encephalitis in Affected Partridges At necropsy, affected partridges from the field outbreaks did not exhibit gross lesions consistent with the observed clinical signs. However, histopathological examination of the brain revealed widespread inflammatory lesions affecting all regions, including both white and, predominantly, grey matter (Fig. 1 a). These lesions were characterized by foci of gliosis frequently associated with neuronal degeneration, necrosis, or, in some cases, vacuolation of the neuropil. Consistent perivascular mononuclear infiltrates, composed mainly of lymphocytes and macrophages, were observed. Transmission electron microscopy (TEM) revealed the presence of viral particles, 70–90 nm in diameter, within neurons in both the cerebrum and cerebellum (Fig. 1 b-d). In the cerebrum, these particles were detected in the perikaryon of granular and pyramidal cells (Fig. 1 b-c), whereas, in the cerebellum, they were localized within cerebellar granular cells (Fig. 1 d). The viral particles were found free in the cytoplasm or associated with transitional vesicles of the endoplasmic reticulum and Golgi complex. Additionally, viral particles were identified in the cytoplasm of endothelial cells lining the cerebral capillaries (Fig. 1 b, insert b2). Morphologically, the particles displayed a spherical shape with a dense, rounded core surrounded by a diffuse outer layer. Discovery of a novel Pegivirus by NGS Brain samples from field outbreaks were screened by PCR for avian influenza virus, avian encephalomyelitis virus, Marek’s disease virus, and arboviruses of the genus Orthoflavivirus . All tests were negative (data not shown). To further investigate the aetiology of the outbreaks, RNA extracted from brain samples of all three outbreaks was subjected to next-generation sequencing (NGS). Metagenomic profiling using de novo assembled contigs identified 887 viral contigs, including three short contigs mapping to Goose pegivirus 2 (GPgV-2) and other avian-origin pegiviruses. The presence of a novel pegivirus was confirmed by conventional PCR targeting the 5'-UTR and NS4A regions, followed by Sanger sequencing of the PCR products, which indicated the presence of different strains among the samples. To obtain the complete genome sequence of one strain, designated ParPgV-A, a set of primers covering the pegivirus genome was designed (Supplementary Table 1, Supplementary Fig. 2a), resulting in a 10,127-bp final genome sequence. For the ParPgV-C strain, total RNA from infected partridge embryos was subjected to NGS, followed by PCR and Sanger sequencing to close gaps (Supplementary Table 1, Supplementary Fig. 2b). This yielded a final genome sequence of 10,687 bp, slightly larger than that of ParPgV-A. Both ParPgV genomes consist of short 5'- and 3'-untranslated regions (UTRs) and a single open reading frame (ORF). The longer UTRs observed in ParPgV-C suggest that the ParPgV-A genome may be incomplete. Similar to other pegiviruses, both ParPgV strains encode a single polyprotein of 3,258 amino acids. Sequence comparison with known pegiviruses suggested that the polyprotein encodes a signal peptide (S), two structural proteins (E1 and E2) at the N-terminal region, followed by six non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (Fig. 2 a). A putative signalase cleavage site was identified between amino acids 19 and 20. HMMER searches against the Pfam database identified protein domains conserved in other Flaviviridae, corroborating the predicted organization of the viral polyprotein (Table 2 ). Phylogenetic analyses based on the NS3 and NS5B regions positioned both ParPgV strains on a distinct branch within the avian pegivirus clade, closely related to several strains of avian origin (Fig. 2 b). In contrast to mammalian pegiviruses, which segregate into two clades, all avian-origin pegiviruses formed a third distinct clade. The percent identity matrix showed 97.98% amino acid identity between the two ParPgV strains and 59.38–77.39% identity to other avian-origin pegiviruses (Supplementary Table 2). Based on the proposed pegivirus species demarcation criterion of 69% amino acid identity within the NS3 region 2 , ParPgV belongs to the same species as Goose pegivirus 2 (GPgV-2), Fernbird pegivirus 1, Monifringilla taczanowskii pegivirus, and Pin virus. Furthermore, based on this demarcation criterion, the analysis indicated the existence of two additional species within the avian pegivirus clade. One represented by the Goose pegivirus 1 (GPgV-1) and the other one consisting of Leucosticte brandti pegivirus and Passer montanus pegivirus (Fig. 2 b). Table 2 Predicted viral proteins of ParPgV-A and ParPgV-C, with Pfam domain matches and genome positions. ParPgV variant protein description Pfam match (Family Id; accession; description; polyprotein start-aa_end-aa) amino acid position (polyprotein) nucleotide position (genome) A E1 envelope glycoprotein HCV_env; PF01539; Hepatitis C virus envelope glycoprotein E1; start-28_end-210 20–211 369:942 C HCV_env; PF01539; Hepatitis C virus envelope glycoprotein E1; aa28_aa209 605:1178 A E2 envelope glycoprotein GBV-C_env; PF12786; GB virus C genotype envelope; aa390_aa586 212–679 945:2346 C GBV-C_env; PF12786; GB virus C genotype envelope; aa361_aa586 1181:2582 A X additional glycoprotein n.d. 680–750 2349:2559 C 2585:2795 A NS2 non-structural protein NS2 HCV_NS2; PF01538; Hepatitis C virus non-structural protein NS2; aa768_aa963 751–986 2562:3177 C 2798:3503 A NS3 protease and helicase activity Peptidase_S29; PF02907; Hepatitis C virus NS3 protease; aa1015_aa1163 Flavi_DEAD; PF07652; Flavivirus DEAD domain; aa1181_aa1323 987–1613 3270:5148 C Peptidase_S29; PF02907; Hepatitis C virus NS3 protease; aa1015_aa1163 Flavi_DEAD; PF07652; Flavivirus DEAD domain; aa1181_aa1323 DEAD; PF00270; DEAD/DEAH box helicase; aa1168_aa1319 3506:5384 A NS4A non-structural protein NS4A n.d. 1614–1679 5151:5346 C 5387:5582 A NS4B non-structural protein NS4B HCV_NS4b; PF01001; Hepatitis C virus non-structural protein NS4b; aa1686_aa1875 1680–1925 5349:6084 C 5585:6320 A NS5A non-structural protein NS5A HCV_NS5a_1a; PF08300; Hepatitis C virus non-structural 5a zinc finger domain; aa1960_aa2010 1926–2694 6087:8391 C 6323:8627 A NS5B RNA-dependent RNA polymerase RdRP_3; PF00998; Viral RNA-dependent RNA polymerase; aa2697_aa3187 RdRP_1; PF00680; Viral RNA-dependent RNA polymerase; aa2737_aa3103 2695–3258 8394:10164 C RdRP_3; PF00998; Viral RNA-dependent RNA polymerase; aa2697_aa3186 RdRP_1; PF00680; Viral RNA-dependent RNA polymerase; aa2738_aa3077 8630:10319 Abbreviations: n.d., not determined; Pfam, Protein family database. Prevalence of ParPgV in Red-Legged Partridge Farms ParPgV was detected in cloacal swabs from all investigated farms (Fig. 3a). All pooled samples from female birds, except one from Farm G, tested positive. In males, all samples from Farms A and F were positive, while negative pools were observed in Farms B and E, and two of three pools were negative in Farms C, D, G, and H. Female samples showed higher positivity rates than male samples. In embryonated eggs, all pooled yolk sac (YS) samples tested positive for ParPgV (Fig. 3b). Allantoic fluid (AF) samples from Farms A, B, E, G, and H tested positive, while negative pools were detected in Farms C, D, and F. Embryo samples were positive across all farms, with some negative pools identified in Farms D, F, and H. Viral loads were highest in embryos, followed by YS and AF. Pathogenicity and tissue distribution of ParPgV in experimentally inoculated birds Grey partridges Of the seven inoculated grey partridges (P4–P10), two (P5 and P7) died shortly after inoculation due to unrelated causes. Clinical signs in the remaining birds were limited to transient reduction of flying behaviour observed at 4 dpi (Supplementary Video 2). Histological examination revealed mild kidney, liver, and spleen alterations (Fig. 4 a). Kidney congestion was observed in all inoculated birds, with some cases exhibiting lymphoid infiltration. Liver congestion was noted in most birds, except one. Splenic congestion ranged from mild to severe, with occasional lymphocytic infiltration and encapsulation of connective tissue. No significant histological changes were detected in other organs. Post-mortem analysis detected viral RNA in the brain and spleen of all birds, with viral load values remaining stable between 14 and 21 dpi (Fig. 4 b), ranging from 3.43 to 5.24 log 10 viral copies/reaction in the brain and 3.35 to 4.28 log 10 in the spleen. In the kidney, viral RNA was detected in 1 of 2 birds at 14 dpi and in 2 of 3 birds at 21 dpi, with viral loads reaching 2.80 log 10 viral copies/reaction at the latter time point. Similarly, no viral RNA was detected in the liver at 14 dpi, whereas 2 of 3 birds were positive at 21 dpi, with viral loads reaching 4.60 log 10 viral copies/reaction (Fig. 4 b). Real-time RT-qPCR analysis of swabs and whole blood revealed sporadic viral detection (Fig. 4 c). Tracheal swabs tested positive only at 21 dpi in P8 and P10, while cloacal swabs showed positivity at 10 dpi in P4 and at 21 dpi in P10. In blood, all birds except P9 were positive at 7 dpi, whereas at 14 dpi only P4 and P8 remained positive. By 21 dpi, ParPgV RNA was detected solely in P8. Red-legged partridges Prior to inoculation, whole blood and cloacal swabs from all red-legged partridges revealed low levels of ParPgV RNA (Fig. 5 a, 0 dpi), confirming previous investigations on farm samples. Following inoculation with the ParPgV inoculum, birds exhibited decreased activity and reduced flying behaviour between 5 and 8 dpi, with no other signs being observed. The viral RNA load in blood, tracheal, and cloacal swabs increased until 10 dpi, subsequently declining, with lower levels detected at 21 dpi. At this final time point, no viral RNA was detected in blood samples. Viral RNA was consistently detected in all examined organs throughout the experiment, including the brain, liver, kidney, spleen, bone marrow, and caecal tonsils (Fig. 5 b). Following ParPgV inoculation, viral load significantly increased in the liver, kidney, bone marrow, and caecal tonsils compared to PBS-inoculated controls (RLP-CG). In the brain, viral load remained stable until the final time point, when a non-significant decline was observed. In contrast, viral load in the spleen remained unaffected by inoculation. Histological examination revealed no observable lesions until 21 dpi. At this final time point, all ParPgV-inoculated birds displayed encephalitic lesions, both in the cerebrum and cerebellum, characterized by gliosis, neuronal degeneration or necrosis, and perivascular mononuclear cell infiltrations (Fig. 5 c-d). Magnetic resonance imaging (MRI) showed a widening of the subarachnoid space in the caudal cranial fossa on fluid-sensitive sequences (T2W-CISS and T2-TIR) at 21 dpi compared to the control group and the prior time points, indicative of cerebellar atrophy (Fig. 5 e). No other parenchymal abnormalities or alterations in signal intensity were detected. SPF chickens Birds were inoculated at 4 weeks of age, and no clinical signs were observed throughout the study. However, viral RNA was detected in all investigated organs at 7 days post-inoculation (dpi), with the highest loads found in the liver, kidney, caecal tonsils, and bursa of Fabricius (Fig. 6 a). By 14 dpi, viral RNA was detected only in the caecal tonsils, and by 21 dpi, it was restricted to the bone marrow, with all other organs testing negative. Prior to inoculation, blood and cloacal swabs from SPF chickens tested negative for ParPgV by PCR (Fig. 6 b). Following inoculation, viremia peaked at 7 dpi, with four birds testing positive for viral RNA in blood. At 14 dpi, only one bird remained positive, with a low viral load, and by 21 dpi, all birds were negative. All cloacal swabs remained negative throughout the study (Fig. 6 b). No histological lesions were observed in any of the organs examined at any time point, and MRI analysis revealed no detectable abnormalities. Immunohistochemistry (IHC) and RNAscope in-situ hybridization investigations To assess the neurotropism of ParPgV in infected birds, IHC and RNAscope in-situ hybridization were performed on brain samples from both field outbreaks and the three in vivo experiments (Fig. 7 ). To investigate systemic distribution, spleen, caecal tonsils, liver, and kidney tissues from experimentally inoculated red-legged partridges were similarly analysed (Fig. 8 ). A primary antibody dilution of 1:500 yielded optimal signal intensity in IHC and was standardized across all samples. In the brain, contrary to investigations in negative birds (Fig. 7 a, b), IHC confirmed the presence of ParPgV antigen, particularly in perivascular regions and areas of inflammatory infiltrates in naturally infected partridges (Fig. 7 c). RNAscope detected viral RNA within neuronal and glial cells of the cerebrum (Fig. 7 d). Experimentally inoculated grey and red-legged partridges showed diffuse immunoreactivity throughout the neuropil (Fig. 7 e, g). RNAscope also revealed strong signals in Purkinje cells and scattered glial cells (Fig. 7 f, h), extending into the cerebellum of red-legged partridges, possibly suggesting viral involvement in motor coordination centers. These findings correlate with the observed neurological signs in infected birds. In SPF chickens, brain sections from PCR-positive individuals showed only faint signals, consistent with lower viral loads. In the spleen, viral antigen was localized to periarteriolar lymphoid sheaths (PALS) and reticuloendothelial cells (Fig. 8 a), with RNAscope confirming viral RNA in the same regions (Fig. 8 b). In caecal tonsils, both antigen and RNA were detected in the follicular epithelium and scattered mononuclear cells (Fig. 8 c, d). In the liver, viral antigen and RNA were found in hepatocytes and sinusoidal lining cells (Fig. 8 e, f). In the kidney, both signals were observed in tubular epithelial cells and glomerular structures (Fig. 8 g, h). ParPgV negative-strand RNA replication Detection of the negative-strand RNA, an intermediate of viral replication, was assessed by strand-specific RT-PCR across different tissues and species (Supplementary Table 3). In the brain, negative-strand RNA was consistently detected in inoculated red-legged partridges (RLP-VG group) and grey partridges (GP). Interestingly, control red-legged partridges (RLP-CG group), which were vertically infected in ovo , also tested positive. Additionally, in red-legged partridge embryonated eggs, negative-strand RNA was detected in both the embryos and the yolk sac. In SPF chickens, investigation of brain tissue did not yield conclusive results due to the presence of low viral load (see Fig. 6 a). Nonetheless, positive results were obtained from the liver, kidney, spleen, bone marrow, and caecal tonsils in SPF chickens. Discussion Pegiviruses were initially suspected to cause hepatitis due to their association with non-A–E hepatitis in humans and genomic similarities to hepatitis C virus (HCV) 4 . However, extensive studies have since shown that pegiviruses are not directly linked to acute or chronic hepatitis 3 . Most available data come from human pegivirus (HPgV-1), which is widely considered non-pathogenic, though its immunomodulatory effects have gained interest for their potential benefits in co-infections 3 . Recently, however, HPgV-1 has been implicated in fatal leukocytic encephalitis, with evidence of lymphocytic infiltration and gliosis in brain tissue 14 . Further supporting its neurotropism, studies have demonstrated HPgV-1 replication in astrocytes and microglia, highlighting its ability to infect neural cells 15 . In agreement with this, the actual study provides the first experimental evidence that pegiviruses can cause encephalitis, challenging the prevailing assumption that members of the Pegivirus genus are non-pathogenic. A key finding of this work was the detection of encephalitis in red-legged partridges during field outbreaks, which included histopathological lesions consistent with viral infection and electron microscopy confirmation of virus-like particles in affected neurons. Despite repeated attempts to propagate the virus in primary liver chicken embryo cells and via yolk sac inoculation of chicken embryos, viral titers declined over subsequent passages (data not shown). This aligns with the broader challenge of establishing efficient in vitro systems for pegiviruses, which often demonstrate narrow tropism and can be notoriously difficult to culture 4 . In agreement with this, a goose pegivirus (GPgV) was passaged eight times in primary goose embryonic fibroblasts, reaching high viral loads, but failed to replicate in other five different cell types 8 . Notwithstanding these limitations, next-generation sequencing of clinical material revealed novel pegiviral sequences, confirming the presence of an avian pegivirus as a likely etiological agent. Detailed sequence analysis revealed the presence of potentially more than one pegivirus strain in the outbreak samples. This was confirmed by obtaining the complete genome sequences of two distinct strains, designated as ParPgV-A and ParPgV-C. Among these, the ParPgV-C strain appears to be more prevalent in red-legged partridges, as it was detected in both embryos and outbreak samples. Phylogenetic analysis supports the previously proposed existence of a third clade within the genus Pegivirus, comprising all reported avian origin pegiviruses 6 , 8 . Moreover, based on the species demarcation threshold of 69% amino acid identity within the NS3 region, our findings confirm the presence of three distinct species within this third Pegivirus clade 2 . The ParPgV strains identified in this study cluster within the species that includes the majority of currently reported avian pegiviruses. However, no distinct phylogenetic clustering was observed correlating with clinical presentation. This may be because most avian pegiviruses reported to date have been identified through metagenomic surveys of asymptomatic birds, whereas the ParPgV genomes described here are among the few sequences present to date associated with clinical disease. An initial experiment in grey partridges was conducted as an exploratory model, partly because red-legged partridges are native to southern Europe and were less accessible in Austria. Despite this limitation, the study confirmed not only the neurotropic and lymphotropic nature of ParPgV but also its propensity for persistence. Stable viral loads were detected in the brain and spleen until the end of the trial, whereas viral RNA in blood and swabs fluctuated or was only intermittently positive. However, no histopathological lesions were evident in the brain of grey partridges. By contrast, in the subsequent experiment involving red-legged partridges — the original host of ParPgV — encephalitic lesions were observed upon histopathological examination, and cerebellar atrophy was confirmed by MRI, although clinical signs were limited to subtle behavioural changes such as reduced activity and flight responsiveness. The cerebellar atrophy noted in superinfected birds suggests a progressive, degenerative process likely associated with chronic infection and might explain the ataxia and prostration observed in affected birds in the field. Such findings parallel recent reports implicating HPgV-1 in neurological disease, in which a leukoencephalitis patient presented progressive lesions in the white matter by MRI investigation, with involvement of the brainstem and cervical cord 14 . Additionally, the present findings resemble outcomes in other experimental neurotropic flavivirus models, where infection-induced neuronal loss and persistent inflammation lead to long-term neuropathological changes 21 , 22 . Nonetheless, these results differ from those in a rhesus monkey pegivirus model, which did not demonstrate increased viral RNA loads in neural tissues 16 . The combined RNAscope ISH and IHC findings reinforce the hypothesis that ParPgV exhibits a strong neurotropic and systemic infection pattern. The presence of viral RNA and antigen in the cerebrum and cerebellum supports the notion that ParPgV actively replicates in neural tissues, contributing to encephalitic lesions and cerebellar atrophy observed in infected birds. Additionally, viral localization in immune-associated tissues such as the spleen and caecal tonsils suggests that the virus persists in lymphoid organs, potentially modulating immune responses. These findings were further corroborated by strand-specific RT-PCR detection of the negative-strand RNA replication intermediate, confirming active viral replication in the brain tissues of inoculated red-legged partridges and grey partridges. Moreover, negative-strand RNA was identified in the liver, kidney, spleen, bone marrow, and caecal tonsils of SPF chickens, and in the embryos and yolk sacs of red-legged partridge embryonated eggs, providing evidence of systemic and in ovo viral replication. The detection of PgV RNA and antigen in renal and hepatic tissues further highlights the systemic nature of the infection, raising questions about the potential for viral shedding and long-term persistence. These findings are consistent with other neurotropic flavivirus infections, where persistent viral replication in neural and immune tissues leads to chronic pathology and progressive neurological impairment 23 – 26 . Further research is needed to clarify whether viral persistence in these organs contributes to long-term disease progression and whether immune modulation plays a role in maintaining viral reservoirs in infected hosts. Interestingly, although red-legged partridges developed clear neurological lesions in this study, they displayed only mild outward signs of disease, implying that ParPgV could cause a subclinical or progressive form of disease, as it has been reported in mice experimentally inoculated with West Nile virus 21 . This possibility may have implications for both wild and farmed partridge populations, as clinically apparent signs might be infrequent or overlooked, especially in flocks where routine culling removes symptomatic individuals. A critical limitation of the red-legged partridge experiment lies in the fact that these birds already tested positive for ParPgV at the time of inoculation, likely through vertical transmission. Screening of multiple partridge flocks failed to identify birds free of ParPgV; moreover, females consistently harboured higher viral loads than males. This sex-associated difference in viral load, along with the temporal association of clinical outbreaks with the onset of egg laying, raises the hypothesis that physiological or hormonal changes linked to reproduction, such as immunomodulation during the laying period, may favour viral replication or persistence in females. In agreement with this, embryonated eggs from all flocks were also positive, strongly indicating that vertical transmission occurs regularly in these populations. Vertical mother-to-child transmission of HPgV has been reported in humans 27 – 30 , however, avian pegiviruses have just been recently identified 5 – 9 and in ovo transmission was unknown until now. The widespread distribution of ParPgV in red-legged partridges, together with the predominantly mild clinical presentation observed in experimentally inoculated birds, suggests a well-adapted host-virus relationship. This observation raises important questions regarding whether in ovo infection influences host immune responses and predisposes birds to future neurological disease. If birds become infected as embryos, they may develop tolerance, permitting lifelong viral persistence 31 . Because all study birds were ParPgV-positive prior to the experimental challenge, it remains unclear whether any observed lesions were specifically induced by superinfection or reflected exacerbation of a pre-existing infection. The final stage of this investigation employed SPF chickens as a potential laboratory model. Although the infection was successfully established in these chickens — evidenced by viral RNA in numerous organs — they showed neither clinical manifestations nor significant histopathological changes, and MRI findings were unremarkable. This is in contrast to red-legged partridges, where viral RNA persisted in multiple tissues, including the brain and spleen. The differences in disease expression may stem from variations in immune responses or viral replication dynamics among host species. Furthermore, the possibility that congenital infection shapes immune responses and facilitates later neurological disease in red-legged partridges remains a key question. Host-restricted pathogenicity of other flaviviruses, such as Usutu virus, western equine encephalomyelitis, and St. Louis encephalitis viruses, underscores how some avian species can harbour high viral loads subclinically 32 – 35 . Additional research is needed to determine if immune factors restrict ParPgV neuroinvasion in chickens. In conclusion, the identification of an avian pegivirus associated with encephalitis has significant implications for both wildlife health and the broader understanding of pegivirus evolution. Historically, pegiviruses have been regarded as benign, persistent viruses with limited pathogenic potential​. However, our findings, along with recent reports of HPgV-1 in human CNS infections, suggest that pegiviruses may have underappreciated neuropathogenic capabilities. Further studies are needed to elucidate the molecular mechanisms of ParPgV neurotropism and its potential interactions with the host immune system. The role of vertical transmission in viral persistence should also be explored, particularly in the context of population dynamics in farmed and wild partridge populations. Additionally, the development of a reliable in vitro culture system for ParPgV would be invaluable for future studies to further unravel pathogenesis of such new viruses. Altogether, this study provides the first experimental evidence linking a pegivirus to encephalitis. While red-legged partridges appear to tolerate persistent infection with limited clinical manifestations, the histopathological and MRI findings indicate that ParPgV can cause significant neurological disease. These findings challenge the notion of pegivirus non-pathogenicity and highlight the need for further research into their role in avian and mammalian hosts. Declarations Acknowledgments The authors extend their gratitude to Attila Sandor, Bibiane Pollak, Patricia Wernsdorf, and Vesna Stanisavljevic for their valuable technical assistance. Author contributions Field outbreak documentation, sample collection, and procurement of embryonated red-legged partridge eggs: N.V.; Histopathology: M.M., D.L., O.A., and F.G.; Recombinant protein expression and polyclonal antibody production: I.B. and B.J.; Immunohistochemistry and RNAscope: M.M., I.B., and D.L.; Transmission electron microscopy: S.R.; MRI investigation: Y.V. and E.L.; In vivo experiments: M.M.; Nucleic acid extraction, NGS library preparation, and detection of negative-strand RNA: I.B. and B.J.; qPCR: M.M., B.J., and F.G.; NGS data analysis, genome assembly, and phylogenetic analyses: I.B. and N.P.; Conceptualization and project administration: M.M., I.B., and M.H.; Writing—original draft: M.M. and I.B. All authors reviewed and approved the final manuscript and are accountable for their contributions. Competing interests The authors declare no competing interests. References Simmonds, P. et al. ICTV virus taxonomy profile: Flaviviridae . Journal of General Virology 98 , 2–3 (2017). Smith, D. B. et al. Proposed update to the taxonomy of the genera Hepacivirus and Pegivirus within the Flaviviridae family. Journal of General Virology 97 , 2894–2907 (2016). Stapleton, J. T. Human Pegivirus Type 1: A Common Human Virus That Is Beneficial in Immune-Mediated Disease? Front Immunol 13 , 1–17 (2022). Yu, Y., Wan, Z., Wang, J. H., Yang, X. & Zhang, C. Review of human pegivirus: Prevalence, transmission, pathogenesis, and clinical implication. Virulence 13 , 323–340 (2022). Grimwood, R. M. et al. From islands to infectomes: host-specific viral diversity among birds across remote islands. BMC Ecol Evol 24 , 1–18 (2024). Zhu, W. et al. Novel pegiviruses infecting wild birds and rodents. Virol Sin 37 , 208–214 (2022). Zhen, W. et al. Emergence of a novel pegivirus species in southwest China showing a high rate of coinfection with parvovirus and circovirus in geese. Poult Sci 100 , 1–6 (2021). Wu, Z. et al. The First Nonmammalian Pegivirus Demonstrates Efficient In Vitro Replication and High Lymphotropism. J Virol 94 , 1–18 (2020). Chang, W. S., Rose, K. & Holmes, E. C. Meta-transcriptomic analysis of the virome and microbiome of the invasive Indian myna ( Acridotheres tristis ) in Australia. One Health 13 , 1–11 (2021). Stapleton, J. T., Foung, S., Muerhoff, A. S., Bukh, J. & Simmonds, P. The GB viruses: A review and proposed classification of GBV-A, GBV-C (HGV), and GBV-D in genus Pegivirus within the family Flaviviridae . Journal of General Virology 92 , 233–246 (2011). Chandriani, S. et al. Identification of a previously undescribed divergent virus from the Flaviviridae family in an outbreak of equine serum hepatitis. Proc Natl Acad Sci U S A 110 , 1407–1415 (2013). Tomlinson, J. E. et al. Viral testing of 18 consecutive cases of equine serum hepatitis: A prospective study (2014-2018). J Vet Intern Med 33 , 251–257 (2019). Tomlinson, J. E. et al. Viral testing of 10 cases of Theiler’s disease and 37 in-contact horses in the absence of equine biologic product administration: A prospective study (2014-2018). J Vet Intern Med 33 , 258–265 (2019). Balcom, E. F. et al. Human pegivirus-1 associated leukoencephalitis: Clinical and molecular features. Ann Neurol 84 , 781–787 (2018). Doan, M. A. L. et al. Infection of Glia by Human Pegivirus Suppresses Peroxisomal and Antiviral Signaling Pathways. J Virol 95 , 1–15 (2021). Bailey, A. L. et al. Durable sequence stability and bone marrow tropism in a macaque model of human pegivirus infection. Sci Transl Med 7 , 1–20 (2015). Chivero, E. T. & Stapleton, J. T. Tropism of human pegivirus (Formerly known as GB virus C/hepatitis G virus) and host immunomodulation: Insights into a highly successful viral infection. Journal of General Virology 96 , 1521–1532 (2015). Chrzastek, K. et al. Use of Sequence-Independent, Single-Primer-Amplification (SISPA) for rapid detection, identification, and characterization of avian RNA viruses. Virology 509 , 159–166 (2017). Teufel, F. et al. SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat Biotechnol 40 , 1023–1025 (2022). Lin, L., Fevery, J. & Yap, S. H. A novel strand-specific RT-PCR for detection of hepatitis C virus negative-strand RNA (replicative intermediate): evidence of absence or very low level of HCV replication in peripheral blood mononuclear cells. J Virol Methods 100 , 97–105 (2002). Appler, K. K. et al. Persistence of west Nile virus in the central nervous system and periphery of mice. PLoS One 5 , 1–12 (2010). Turtle, L., Griffiths, M. J. & Solomon, T. Encephalitis caused by flaviviruses. QJM: An International Journal of Medicine 105 , 219–223 (2012). de Vries, L. & Harding, A. T. Mechanisms of Neuroinvasion and Neuropathogenesis by Pathologic Flaviviruses. Viruses 15 , 1–20 (2023). Maximova, O. A. & Pletnev, A. G. Flaviviruses and the Central Nervous System: Revisiting Neuropathological Concepts. Annu. Rev. Virol. 5 , 255–272 (2018). Cody, S. G., Adam, A., Siniavin, A., Kang, S. S. & Wang, T. Flaviviruses—Induced Neurological Sequelae. Pathogens 14 , 1–19 (2025). Blackhurst, B. M. & Funk, K. E. Molecular and Cellular Mechanisms Underlying Neurologic Manifestations of Mosquito-Borne Flavivirus Infections. Viruses 15 , 1–19 (2023). Supapol, W. B. et al. Reduced mother-to-child transmission of HIV associated with infant but not maternal GB virus C infection. Journal of Infectious Diseases 197 , 1369–1377 (2008). Santos, L. M. et al. Prevalence and vertical transmission of human pegivirus among pregnant women infected with HIV. International Journal of Gynecology and Obstetrics 138 , 113–118 (2017). Lin, H.-H. et al. Mother-to-Infant Transmission of GB Virus C/Hepatitis G Virus: The Role of High-Titered Maternal Viremia and Mode of Delivery and Ding-Shinn Chen. J Infect Dis 177 , 1202–1206 (1998). Zanetti, A. R. et al. Multicenter trial on mother-to-infant transmission of GBV-C virus. J Med Virol 54 , 107–112 (1998). He, S. et al. High-frequency and activation of CD4+CD25+ T cells maintain persistent immunotolerance induced by congenital ALV-J infection. Vet Res 52 , 1–15 (2021). Chvala, S. et al. Limited pathogenicity of Usutu virus for the domestic chicken ( Gallus domesticus ). Avian Pathology 34 , 392–395 (2005). Chvala, S. et al. Limited pathogenicity of usutu virus for the domestic goose ( Anser anser f. domestica ) following experimental inoculation. Journal of Veterinary Medicine Series B: Infectious Diseases and Veterinary Public Health 53 , 171–175 (2006). Kuchinsky, S. C. et al. North American House Sparrows Are Competent for Usutu Virus Transmission. mSphere 7 , 1–12 (2022). Reisen, W. K., Chiles, R. E., Martinez, V. M., Fang, Y. & Green, E. N. Experimental Infection of California Birds with Western Equine Encephalomyelitis and St. Louis Encephalitis Viruses. J. Med. Entomol 40 , 968–982 (2003). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryTable1Listofprimersprobesandcyclingconditions.xlsx Supplementary Table 1. List of primers, probes, and PCR cycling conditions used for the amplification and detection of partridge pegivirus (ParPgV). SupplementaryTable2DistancematrixNS3.xlsx Supplementary Table 2. Percent amino acid identity matrix for the NS3 region (544 amino acids) among partridge pegivirus strains and related avian-origin pegiviruses. SupplementaryTable3.DetectionofParPgVnegativestrandRNA.docx Supplementary Table 3. Detection of ParPgV negative-strand RNA in tissues from experimentally inoculated birds and embryonated eggs. SupplementaryFigs12.pdf Supplementary Figs 1 and 2 VideoS1RedleggedpartridgesfieldoutbreakParPgV.mp4 Video S1. Neurological signs in a red-legged partridge from the field outbreak VideoS2Greypartridges.mp4 Video S2. Altered behavior in grey partridges inoculated with ParPgV compared to negative controls nrreportingsummaryMatosfinal.pdf Reporting Summary Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6888535","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":479523590,"identity":"3e676218-26b2-45af-92ad-0eb019439598","order_by":0,"name":"Miguel 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Vienna","correspondingAuthor":false,"prefix":"","firstName":"Ivana","middleName":"","lastName":"Bilic","suffix":""},{"id":479523592,"identity":"c3eb2e00-38d7-4e76-bfa7-bf8e3f3529c5","order_by":2,"name":"Nicolas Viloux","email":"","orcid":"","institution":"Labovet Conseil","correspondingAuthor":false,"prefix":"","firstName":"Nicolas","middleName":"","lastName":"Viloux","suffix":""},{"id":479523593,"identity":"662229ef-a900-46ac-9599-b221db4533df","order_by":3,"name":"Barbara Jaskulska","email":"","orcid":"","institution":"University of Veterinary Medicine Vienna","correspondingAuthor":false,"prefix":"","firstName":"Barbara","middleName":"","lastName":"Jaskulska","suffix":""},{"id":479523594,"identity":"9c0d59d3-bc47-43e0-93f4-34a83dd9470a","order_by":4,"name":"Fatou Geissler","email":"","orcid":"","institution":"University of Veterinary Medicine Vienna","correspondingAuthor":false,"prefix":"","firstName":"Fatou","middleName":"","lastName":"Geissler","suffix":""},{"id":479523595,"identity":"61ae4284-6bfa-4036-a8ba-77c7106a1207","order_by":5,"name":"Yasamin Vali","email":"","orcid":"","institution":"University of Veterinary Medicine Vienna","correspondingAuthor":false,"prefix":"","firstName":"Yasamin","middleName":"","lastName":"Vali","suffix":""},{"id":479523596,"identity":"dd6d43aa-0c44-482a-8539-79f86a9223f9","order_by":6,"name":"Eberhard Ludewig","email":"","orcid":"","institution":"University of Veterinary Medicine Vienna","correspondingAuthor":false,"prefix":"","firstName":"Eberhard","middleName":"","lastName":"Ludewig","suffix":""},{"id":479523597,"identity":"05be5a27-029e-46c5-9909-00aaaac4f77d","order_by":7,"name":"Olivier Albaric","email":"","orcid":"","institution":"Independent Pathologist","correspondingAuthor":false,"prefix":"","firstName":"Olivier","middleName":"","lastName":"Albaric","suffix":""},{"id":479523598,"identity":"4d7eaac5-c6e6-4308-9c74-1a085c9c725d","order_by":8,"name":"Susanne Richter","email":"","orcid":"","institution":"Austrian Agency for Health and Food Safety","correspondingAuthor":false,"prefix":"","firstName":"Susanne","middleName":"","lastName":"Richter","suffix":""},{"id":479523599,"identity":"33e21a2b-550d-48ba-aa6b-8026e8414c86","order_by":9,"name":"Dieter Liebhart","email":"","orcid":"https://orcid.org/0000-0003-2412-1248","institution":"University of Veterinary Medicine Vienna","correspondingAuthor":false,"prefix":"","firstName":"Dieter","middleName":"","lastName":"Liebhart","suffix":""},{"id":479523600,"identity":"97dcd584-0fa6-4272-9e92-1f830d455ca2","order_by":10,"name":"Nicola Palmieri","email":"","orcid":"","institution":"University of Veterinary Medicine","correspondingAuthor":false,"prefix":"","firstName":"Nicola","middleName":"","lastName":"Palmieri","suffix":""},{"id":479523601,"identity":"1ad491d2-b6ce-43a6-b4f2-6901f6a2902f","order_by":11,"name":"Michael Hess","email":"","orcid":"","institution":"Clinic for Poultry and Fish Medicine","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Hess","suffix":""}],"badges":[],"createdAt":"2025-06-13 13:10:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6888535/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6888535/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86135929,"identity":"b49243f5-97f7-471a-bc3c-24858491c582","added_by":"auto","created_at":"2025-07-07 07:46:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":681321,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathological and ultrastructural findings in the brain of red-legged partridges with encephalitis. \u003c/strong\u003e(a) Histopathological changes in the brain. Hematoxylin and eosin (H\u0026amp;E) staining reveals marked perivascular cuffing composed of mononuclear inflammatory cells (lymphocytes and macrophages) (arrow). Diffuse microgliosis and scattered microglial nodules are evident within the neuropil (*), along with neuronal degeneration and reactive astrocytosis (scale bar = 100 µm). (b–d) Ultrastructural evidence of virus particles in the cerebrum and cerebellum. (b) Cerebrum – granular layer, granular cell (GC). Virus particles are observed in transport vesicles near the Golgi complex (inset b1) and in close association with the luminal wall of a capillary (inset b2). (c) Cerebrum – pyramidal layer. Clusters of virus particles are located in the cytoplasm of a pyramidal cell, associated with rough endoplasmic reticulum (Nissl bodies). (d) Cerebellum – granular layer, cerebellar granular cell (CGC). Virus particles are visible in transport vesicles adjacent to the smooth endoplasmic reticulum (inset d1). Annotations: C = capillary, Cl = capillary lumen, G = Golgi complex, N = nucleus, M = mitochondrion, R = ribosomes, rER = rough endoplasmic reticulum, sER = smooth endoplasmic reticulum, Vp = virus particles. Scale bars: (b) 2.5 µm (main image), 100 nm (insets); (c) 100 nm; (d) 1 µm (main image), 100 nm (inset).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6888535/v1/27e297b73e1a1f7c9ffada29.png"},{"id":86135931,"identity":"cfc045c8-9da4-453a-8503-8236e616ec1d","added_by":"auto","created_at":"2025-07-07 07:46:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":244167,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenomic features of novel partridge pegivirus (ParPgV).\u003c/strong\u003e (a) Schematic representation of the ParPgV genome and polyprotein organization.\u003cstrong\u003e \u003c/strong\u003e(b) Phylogenetic trees based on the NS3 and NS5B regions of the viral polyprotein, inferred using the Maximum Likelihood method (RAxML) from amino acid alignments of conserved sites (544 and 424 positions for NS3 and NS5B, respectively). The analysis included 27 pegivirus polyprotein sequences. Sequences from NCBI GenBank are labelled with accession numbers and names; ParPgV strains are highlighted in bold red. Pegivirus species as defined by ICTV are indicated. Branch support was assessed by bootstrap analysis with 100 replicates; bootstrap values are shown at the corresponding nodes. Trees are drawn to scale, with branch lengths proportional to the number of substitutions per site. Evolutionary analyses were performed using the MegAlign Pro module of Lasergene v18.0 (DNASTAR, Madison, WI, USA).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6888535/v1/2cf426d5cebd5c37cf827ab9.png"},{"id":86135924,"identity":"16139bf4-0597-41c8-ba9e-2c055026acac","added_by":"auto","created_at":"2025-07-07 07:46:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":78395,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eField screening for pegivirus across eight farms (A–H).\u003c/strong\u003e Viral load is presented as ΔC\u003csub\u003eT\u003c/sub\u003e values (38 - C\u003csub\u003eT\u003c/sub\u003e of the sample), with higher ΔC\u003csub\u003eT\u003c/sub\u003e values indicating higher viral loads. Each farm was screened using 15 males and 15 females, as well as 30 embryonated eggs for allantoic fluid (AF), yolk sac (YS), and embryo. (a) Comparison of viral load between male and female birds across farms. Females show higher viral loads than males, with greater variability observed in some farms. (b) Comparison of viral load in embryonated eggs across different sample types (AF, YS, and embryo). Embryos consistently exhibit higher viral loads across farms, while AF and YS show more variable results. Error bars represent standard deviations.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6888535/v1/d601151bb6aa915d334a4911.png"},{"id":86135928,"identity":"f3a90fca-9c85-4b08-852f-9de23a35772c","added_by":"auto","created_at":"2025-07-07 07:46:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":381211,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathological changes and viral load dynamics in grey partridges (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePerdix perdix\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) experimentally inoculated with ParPgV. \u003c/strong\u003e(a) Histopathological analysis of kidney and spleen tissues. Kidney section at 14 days post-inoculation (dpi) showing marked congestion and a focal area of lymphocytic infiltration (*). Spleen section at 21 dpi exhibiting congestion and areas of lymphocytic infiltration (black box). Hematoxylin and eosin (H\u0026amp;E) staining. Scale bar = 100 µm. (b) Viral load in brain, spleen, liver, and kidney from individual partridges (P4 and P9 at 14 dpi; P6, P8, and P10 at 21 dpi), expressed as viral copies per reaction (log\u003csub\u003e10\u003c/sub\u003e scale). (c) Heatmap representation of viral load in blood, cloacal swabs, and tracheal swabs across different time points (0, 4, 7, 10, 14, and 21 dpi). Blood was collected weekly; swabs were collected at each indicated time point.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6888535/v1/886aa5bdd6014975d57d6fc7.png"},{"id":86135944,"identity":"3c7a0d16-801c-4acc-8efd-a112883ee1bb","added_by":"auto","created_at":"2025-07-07 07:46:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":472952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eViral load dynamics, histopathological lesions, and MRI findings in red-legged partridges (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAlectoris rufa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) experimentally inoculated with ParPgV.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Violin plot representing viral load in blood, tracheal swabs, and cloacal swabs collected at 0, 7, 10, 14, and 21 days post-inoculation (dpi), except for tracheal swabs, which were not taken at day 0, expressed as viral copies per reaction (log\u003csub\u003e10\u003c/sub\u003e). Blood samples show a wide range of viral loads over time, with peak levels around 14 dpi. Tracheal and cloacal swabs also show variation, with higher viral loads observed at 10 dpi. Each violin shows the distribution of data, with dashed lines indicating the median and interquartile range. (b) Viral load in brain, liver, kidney, spleen, bone marrow, and caecal tonsils at 7, 10, 14, and 21 dpi (mean ± standard deviation). Statistically significant differences (p \u0026lt; 0.05) in viral load between ParPgV-inoculated partridges at certain time points compared to control birds (RLP-CG, inoculated with PBS) are highlighted above the bars. (c) Cerebrum at 21 dpi showing moderate to prominent perivascular cuffing (black arrow) with lymphocytes and histiocytes, multifocal gliosis, and neuronal degeneration (*). H\u0026amp;E staining; scale bars: 100 µm. (d) Cerebellum displaying severe inflammatory infiltrates affecting molecular and granular layers, with neuronal degeneration and necrosis (box), and degenerated Purkinje neurons with vacuolated cytoplasm, nuclear condensation, and surrounding reactive glial cells (right). H\u0026amp;E staining; scale bars: 100 and 50 µm, respectively. (e) T2-weighted CISS sagittal MRI images. The cerebellum is indicated by asterisks (*). In ParPgV-inoculated birds at 21 dpi (right), widening of the subarachnoid space at the cerebellar level is visible (red arrows) in comparison to the control bird (left), suggestive of inflammation or cerebrospinal fluid accumulation.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6888535/v1/02a3ac158dd6f01bde14f372.png"},{"id":86135934,"identity":"0c55bb8b-32d0-4ee0-9b3c-a98397dbf350","added_by":"auto","created_at":"2025-07-07 07:46:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":60784,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eViral load dynamics in specific pathogen-free (SPF) chickens following experimental inoculation.\u003c/strong\u003e Viral load is expressed as viral copies per reaction (Log\u003csub\u003e10\u003c/sub\u003e). (a) Box plot showing viral load detected in brain, spleen, liver, kidney, bone marrow, caecal tonsil, bursa of Fabricius, and thymus at 7, 14, and 21 days post-inoculation (dpi). Highest viral loads were detected in the liver, kidney, and caecal tonsil at 7 dpi. The caecal tonsil and bone marrow showed persistent viral presence at later time points. (b) Strip plot representing individual viral loads detected in blood samples collected at 0, 7, 14, and 21 dpi. Peak viremia was observed at 7 dpi, followed by a decline at 14 and 21 dpi.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6888535/v1/88c44aa8ed859831158758ea.png"},{"id":86135950,"identity":"fa72bf78-adfd-400c-9004-1071bbcc56d3","added_by":"auto","created_at":"2025-07-07 07:46:37","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1029937,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of ParPgV antigen and RNA in brain tissue from field and experimentally infected partridges using immunohistochemistry (IHC) and RNAscope in-situ hybridization.\u003c/strong\u003e Brain sections from negative control birds show no ParPgV staining by IHC (a) and no RNA hybridization signal by RNAscope (b). Strong immunoreactivity was observed in the cerebrum of naturally infected red-legged partridges (c), experimentally inoculated grey partridges (e), and red-legged partridges (g). Corresponding RNAscope signals localized ParPgV RNA to neuronal cell bodies and glial cells in the cerebrum of naturally infected red-legged partridges (d), experimentally inoculated grey (f), and red-legged partridges (h). Counterstaining with hematoxylin was omitted in panel (c). Scale bars: 100 µm.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6888535/v1/aee4d9cf29fe27af2664ba03.png"},{"id":86135936,"identity":"296c4c7d-69af-4ece-911c-1c2b3d95533b","added_by":"auto","created_at":"2025-07-07 07:46:37","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1341819,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of ParPgV antigen and RNA in systemic organs of experimentally inoculated red-legged partridges.\u003c/strong\u003e ParPgV antigen was detected by IHC in the spleen (a), particularly within periarteriolar lymphoid sheaths, and in caecal tonsils (c), liver (e), and kidney (g). RNAscope hybridization confirmed the presence of viral RNA in the same tissues: lymphoid follicles and reticuloendothelial cells in the spleen (b), follicular epithelium and mononuclear cells in the caecal tonsils (d), hepatocytes and sinusoidal lining cells in the liver (f), and tubular epithelial cells and glomerular structures in the kidney (h). Scale bars: 100 µm.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6888535/v1/b5db835511eea9a113ec6c1d.png"},{"id":86138949,"identity":"66231159-33d5-4347-b4fe-68448e32b1ca","added_by":"auto","created_at":"2025-07-07 08:10:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6206186,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6888535/v1/8f8cbd70-838a-43cb-a237-2b8100cfcbe3.pdf"},{"id":86135923,"identity":"21fae532-e1e6-4161-9a8e-993219514dd9","added_by":"auto","created_at":"2025-07-07 07:46:36","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16102,"visible":true,"origin":"","legend":"Supplementary Table 1. List of primers, probes, and PCR cycling conditions used for the amplification and detection of partridge pegivirus (ParPgV).","description":"","filename":"SupplementaryTable1Listofprimersprobesandcyclingconditions.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6888535/v1/ec0a5827e8590d4787959eda.xlsx"},{"id":86136861,"identity":"90f89cfb-4361-444c-8369-cf5f62fe8809","added_by":"auto","created_at":"2025-07-07 07:54:37","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17445,"visible":true,"origin":"","legend":"Supplementary Table 2. Percent amino acid identity matrix for the NS3 region (544 amino acids) among partridge pegivirus strains and related avian-origin pegiviruses.","description":"","filename":"SupplementaryTable2DistancematrixNS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6888535/v1/165d85f60d1e37dd396529e6.xlsx"},{"id":86135926,"identity":"d8d4cdf7-28be-4498-89cf-5886808b7916","added_by":"auto","created_at":"2025-07-07 07:46:36","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":16252,"visible":true,"origin":"","legend":"Supplementary Table 3. Detection of ParPgV negative-strand RNA in tissues from experimentally inoculated birds and embryonated eggs.","description":"","filename":"SupplementaryTable3.DetectionofParPgVnegativestrandRNA.docx","url":"https://assets-eu.researchsquare.com/files/rs-6888535/v1/b6524f3532005fd7034e17e8.docx"},{"id":86136871,"identity":"8c030019-c05a-4cbf-ae42-85888697fb97","added_by":"auto","created_at":"2025-07-07 07:54:37","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":292496,"visible":true,"origin":"","legend":"Supplementary Figs 1 and 2","description":"","filename":"SupplementaryFigs12.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6888535/v1/030a72414a110223a5e6ce23.pdf"},{"id":86135957,"identity":"6f588152-3402-44af-9388-48377cae0640","added_by":"auto","created_at":"2025-07-07 07:46:37","extension":"mp4","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":29894825,"visible":true,"origin":"","legend":"\u003cp\u003eVideo S1. Neurological signs in a red-legged partridge from the field outbreak\u003c/p\u003e","description":"","filename":"VideoS1RedleggedpartridgesfieldoutbreakParPgV.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6888535/v1/909db20d1be3ad77c09410dc.mp4"},{"id":86136867,"identity":"6667d46c-f242-4978-b9ab-54650feb5784","added_by":"auto","created_at":"2025-07-07 07:54:37","extension":"mp4","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":16508954,"visible":true,"origin":"","legend":"\u003cp\u003eVideo S2. Altered behavior in grey partridges inoculated with ParPgV compared to negative controls\u003c/p\u003e","description":"","filename":"VideoS2Greypartridges.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6888535/v1/c37e38b9c8801094dce606c1.mp4"},{"id":86135952,"identity":"60ac492e-4ec1-4304-87e9-553ac302eb56","added_by":"auto","created_at":"2025-07-07 07:46:37","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":587443,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"nrreportingsummaryMatosfinal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6888535/v1/4bc0ce8c13dc9fe69547b42e.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Neurotropism and Encephalitis of a Novel Pegivirus with Experimental Evidence Across Avian Species","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePegiviruses belong to the genus \u003cem\u003ePegivirus\u003c/em\u003e within the family \u003cem\u003eFlaviviridae\u003c/em\u003e and are currently classified into 11 recognized species based on amino acid sequence divergence in conserved regions of NS3 and NS5B\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. These viruses are enveloped, spherical particles measuring 50\u0026ndash;100 nm in diameter, with a positive-sense single-stranded RNA genome ranging from 8.9 to 11.3 kb\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Pegiviruses have been identified in a broad range of mammalian hosts, including humans, non-human primates, horses, pigs, rodents, and bats\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. More recently, the first non-mammalian pegiviruses were reported in diseased geese and metagenomic studies of healthy wild birds in China, Australia, and New Zealand, although their clinical relevance remains unclear\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In addition, the extent to which pegiviruses can transmit across different host species remains poorly understood\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHistorically referred to as GB viruses or hepatitis G viruses (HGV), pegiviruses establish persistent infections, as reflected in their name (pe, persistent; g, GB or G)\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Initially, members of \u003cem\u003ePegivirus hominis\u003c/em\u003e and \u003cem\u003ePegivirus equi\u003c/em\u003e were implicated in human and equine hepatitis (Theiler\u0026rsquo;s disease), respectively\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, subsequent studies failed to substantiate these associations, and pegiviruses are largely considered non-pathogenic\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Nonetheless, human pegivirus (HPgV) infection has been linked to an increased risk of developing non-Hodgkin\u0026rsquo;s lymphoma and has also been associated with encephalitis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In support of this, HPgV RNA and viral antigen have been detected in post-mortem brain tissue of patients with encephalitis, and \u003cem\u003ein vitro\u003c/em\u003e studies have demonstrated viral replication in human astrocytes and microglia\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The primary cellular targets of pegiviruses remain uncertain, representing a major obstacle to advancing mechanistic studies on infection and pathogenesis, particularly due to the absence of a robust \u003cem\u003ein vitro\u003c/em\u003e culture system. Despite this limitation, pegiviruses are well recognized as lymphotropic viruses, with previous studies highlighting the bone marrow and spleen as key target organs\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Additionally, HPgV has been shown to exert immunomodulatory effects that enhance survival in patients co-infected with HIV, hepatitis C virus (HCV), and Ebola virus\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we investigated outbreaks of viral encephalitis in red-legged partridges (\u003cem\u003eAlectoris rufa\u003c/em\u003e) from affected flocks in France. Initial diagnostic efforts targeting known avian neurotropic viruses \u0026mdash; including avian encephalomyelitis virus, avian influenza virus, different arboviruses, and Marek\u0026rsquo;s disease virus \u0026mdash; yielded negative results, prompting a non-targeted virological approach. This led to the identification of a novel pegivirus, designated partridge pegivirus (ParPgV), in the brain tissue of affected birds. Its consistent detection in encephalitis cases prompted the hypothesis that ParPgV was the causative agent of the outbreaks. To investigate this, we conducted comprehensive genomic characterization and performed \u003cem\u003ein vivo\u003c/em\u003e studies in both homologous and heterologous hosts, providing key insights into its viral dynamics, infection kinetics, and host range. These findings strongly support the role of ParPgV as the etiological agent of the observed clinical signs.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFarm background and flock management\u003c/h2\u003e \u003cp\u003eSince 2017, sporadic neurological signs have been observed in red-legged partridge (\u003cem\u003eAlectoris rufa\u003c/em\u003e) flocks on two geographically proximate farms in France. These events primarily involved three breeder flocks (designated A, B, and C), composed of birds aged 20\u0026ndash;30 weeks, housed in pairs within cage systems. Flock sizes ranged from approximately 13,000 to 30,000 birds. All flocks were routinely vaccinated against Newcastle disease virus (NDV) and managed under standard photostimulation protocols to induce egg laying, which typically began in early February.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistological Analysis\u003c/h3\u003e\n\u003cp\u003eTissue samples were preserved in a 4% neutral buffered formaldehyde solution (SAV LP GmbH, Flintsbach, Germany). After fixation, the samples were rinsed in water and subjected to dehydration and subsequently embedded in paraffin. Thin sections, 4 \u0026micro;m in thickness, were prepared from the paraffin blocks using a microtome (Microm HM 360; Microm Laborger\u0026auml;te GmbH, Walldorf, Germany). Sections were stained with hematoxylin and eosin (H\u0026amp;E) for histological examination.\u003c/p\u003e\n\u003ch3\u003eTransmission Electron Microscopy (TEM)\u003c/h3\u003e\n\u003cp\u003eBrain samples preserved in 4% neutral buffered formaldehyde were used for transmission electron microscopy (TEM). The samples were sectioned into 1-mm\u0026sup3; pieces in 0.1 M phosphate buffer (Sigma-Aldrich, Vienna, Austria) at pH 7.2 and maintained at 4\u0026deg;C for 3 hours. The following preparation steps underwent the usual procedure with post fixation steps in a buffered 2,5% Karnovsky solution (pH 7,3) and in a cold phosphate buffered 1% osmium tetroxide solution (pH 7,3, 4\u0026deg;C, Agar Scientific, UK), with dehydration in a graded ethanol series followed by treatment with propylene oxide (Merck, Darmstadt, Germany) and with embedding in EPON 812 resin (Serva, Heidelberg, Germany). Ultrathin sections were stained with 1% methanolic uranyl acetate (Ted Pella, USA) and lead citrate after Reynolds (Merck, Germany), and examined using a Zeiss TEM 906 electron microscope (Zeiss, Oberkochen, Germany) operating at 80 kV.\u003c/p\u003e\n\u003ch3\u003eNucleic acid extraction and construction of an NGS library\u003c/h3\u003e\n\u003cp\u003eTwo next-generation sequencing (NGS) experiments were performed: the first on brain samples from clinically affected red-legged partridges during field outbreaks, and the second on partridge embryos. Total RNA was extracted from brain and embryo homogenates with the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), followed by genomic DNA removal using RNase-free DNase I (Qiagen) (unless otherwise stated, all reagents and kits were used according to the manufacturers\u0026rsquo; protocols). Ribosomal RNA was depleted prior to library preparation using the NEBNext rRNA Depletion Kit v2 (New England Biolabs, Frankfurt am Main, Germany). Sequencing libraries were constructed with the NEBNext\u0026reg; Ultra\u0026trade; II RNA Library Prep Kit for Illumina (New England Biolabs) and sequenced on the Illumina NextSeq 2000 platform in paired-end 150 bp mode (PE150) at the Vienna BioCenter Core Facilities GmbH (Next Generation Sequencing Facility, Vienna, Austria).\u003c/p\u003e\n\u003ch3\u003eAnalysis of NGS-data obtained from NextSeq\u003c/h3\u003e\n\u003cp\u003eIn the first NGS experiment, three brain samples, representing each flock, were processed in a single sequencing run. Raw reads were imported into CLC Genomics Workbench 23 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://digitalinsights.qiagen.com\u003c/span\u003e\u003cspan address=\"https://digitalinsights.qiagen.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), trimmed to remove low-quality bases and adapter sequences, and assembled de novo using the Microbial Genomics Module (default settings). Assembled contigs were screened for candidate viruses using a local BLASTN search against a database of complete viral genomes downloaded from NCBI on July 11, 2022 (-evalue 1e-05 -max_target_seqs 1). For each contig, only the top match was retained. In the second experiment, two partridge embryo samples were sequenced in separate runs. Reads were trimmed using Cutadapt (v4.9) with parameters -a AGATCGGAAGAGC -A AGATCGGAAGAGC -m 20, then imported into CLC Genomics Workbench 24 and assembled de novo as above. Contigs were screened by BLASTN against a custom database of avian pegivirus sequences and the complete genome of the ParPgV-A strain initially derived in this study. Matching contigs were used in the reconstruction of the partial genome sequence of ParPgV-C.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEstablishing the complete genome sequence\u003c/h2\u003e \u003cp\u003eThe complete genomic sequence was established using a modified Sequence-Independent Single Primer Amplification (SISPA) protocol\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Briefly, total RNA from outbreak brain homogenates was reverse-transcribed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) and 2 \u0026micro;M SISPA-N primer (primers and thermal cycling conditions are listed in Supplementary Table\u0026nbsp;1.). cDNA was amplified with LongAmp\u0026reg; Taq 2X Master Mix (New England Biolabs) and 2 \u0026micro;M SISPA primer, and purified with AMPure Beads XP (Beckman Coulter, Krefeld, Germany) at a bead-to-cDNA ratio of 0.8:1.\u003c/p\u003e \u003cp\u003ePegivirus-specific primers (Supplementary Table\u0026nbsp;1) were designed based on NGS-derived contigs and aligned to avian pegivirus sequences from GenBank. PCRs were performed with LongAmp\u0026reg; Taq 2X Master Mix and 0.4 \u0026micro;M primers. Amplicons of expected sizes were excised and purified from agarose gels using the QIAquick Gel Extraction Kit (Qiagen). Purified fragments were cloned into the TOPO-TA vector (Invitrogen, Thermo Fisher Scientific) following the manufacturer\u0026rsquo;s instructions. From each PCR, at least three independent positive clones were sequenced by Sanger method (LGC Genomics, Berlin, Germany) using M13 and pegivirus-specific primers (Supplementary Table\u0026nbsp;1). Consensus sequences from \u0026ge;\u0026thinsp;3 independent clones were assembled using Accelrys Gene version 2.5 (Accelrys, San Diego, CA, USA). Complete genome sequences of ParPgV-A and ParPgV-C strains were submitted to the NCBI database under accession numbers PV472371 and PV472372, respectively.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGenome and Phylogenetic analyses\u003c/h3\u003e\n\u003cp\u003ePredicted proteins within the viral polyprotein were identified using HMMER Hmmscan search against the Pfam protein database (HmmerWeb version 2.43) and by comparison to the Goose Pegivirus-1 polyprotein. Complete polyprotein sequences of all known avian-origin pegiviruses were aligned using the MUSCLE method within the MegAlign Pro module of Lasergene v18.0.1 software (DNASTAR, Madison, WI, USA). Signal peptide prediction and cleavage site identification were performed using SignalP 6.0 0 (SignalP 6.0 - DTU Health Tech - Bioinformatic Services)\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e with the organism parameter set to \"other.\" Phylogenetic analyses were conducted based on the NS3 and NS5B regions of the viral polyprotein. A total of 27 pegivirus polyprotein sequences from mammalian and avian origins were downloaded from the NCBI protein database. Amino acid alignments were generated using MUSCLE within MegAlign Pro. Conserved blocks were selected using GBlocks (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ngphylogeny.fr/tools/tool/276/form\u003c/span\u003e\u003cspan address=\"https://ngphylogeny.fr/tools/tool/276/form\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Phylogenetic trees were inferred using the Maximum Likelihood method (RAxML) with 100 bootstrap replicates. Distance matrices (NS3 region; 544 amino acids) were calculated using uncorrected pairwise distances and global gap removal in MegAlign Pro.\u003c/p\u003e\n\u003ch3\u003eDetection of ParPgV RNA by real-time qRT-PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from organ tissues with the RNeasy Mini Kit (Qiagen). Real-time RT-PCR assays targeting the conserved 5\u0026prime;-UTR region of the ParPgV genome were performed using the AriaMx Real-Time PCR System and the Brilliant III Ultra-Fast QRT-PCR Master Mix (Agilent Technologies, Vienna, Austria). Each 20 \u0026micro;L reaction contained 2 \u0026micro;L RNA template, 0.5 \u0026micro;M primers, and 0.2 \u0026micro;M probe (primer sequences and PCR conditions are provided in Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eFluorescence signals were analyzed with Agilent AriaMx software (version 1.7) with manual threshold settings. A Ct cutoff of 38 was applied. No-template controls were included to monitor contamination. Absolute quantification of viral load in animal samples was performed using a standard curve generated from \u003cem\u003ein vitro\u003c/em\u003e transcribed 5\u0026prime;-UTR RNA cloned into a pCR\u0026reg;4-TOPO vector (Invitrogen). RNA transcription was carried out with a MAXIscript T7 kit (Thermo Fisher Scientific) after PCR amplification using the OneStep RT-PCR Kit (Qiagen) and specific primers (Supplementary Table\u0026nbsp;1). DNA templates were removed with the TURBO DNA-free Kit (Thermo Fisher Scientific). Viral RNA copies/\u0026micro;L were calculated based on RNA concentration and molecular weight.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDetection of ParPgV-specific RNA by RNAscope\u003c/h2\u003e \u003cp\u003eCustom RNAscope probes were designed and provided by Bio-Techne (Dublin, Ireland) to target the highly conserved region at the 5\u0026prime; end and the NS3 regions of the ParPgV genome, based on the consensus sequences of ParPgV-A nucleotide 2-949 (5\u0026rsquo;-UTR, signal sequence, and partial E1region) and ParPgV-C nucleotide 5450\u0026ndash;6368 (NS4A-NS5A region) (catalogue no. 1333321-C1 and 1331411-C1, respectively). Probes targeting the messenger RNA (mRNA) of the widely expressed housekeeping gene peptidyl-prolyl-isomerase-B in red-legged partridges (\u003cem\u003eAlectoris rufa\u003c/em\u003e - PPIB; cat. no. 1331421-C1) and chickens (\u003cem\u003eGallus gallus\u003c/em\u003e - PPIB; cat. no. 453371) served as positive controls, while a probe targeting bacterial dihydropicolinate reductase (DapB; cat. no. 310043) was used as a negative control. Detection of viral nucleic acid was performed using \u003cem\u003ein-situ\u003c/em\u003e hybridization (ISH) with the manual RNAscope 2.5 High Definition RED assay (Bio-Techne), following the manufacturer\u0026rsquo;s protocol. Briefly, deparaffinized brain sections were pre-treated with 1\u0026times; Target Retrieval solution and RNAscope\u0026reg; Protease Plus solution before hybridization with the target probe. Post-hybridization, the tissue underwent a series of amplification steps using pre-amplifier and amplifier solutions, followed by the application of a chromogenic substrate. Slides were counterstained with hematoxylin. As positive controls, tissue sections from field outbreaks were used, while negative controls included tissue sections from ParPgV-negative specific-pathogen-free (SPF) chickens. Signal scoring was performed according to the manufacturer\u0026rsquo;s guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRecombinant protein production and generation of a polyclonal anti-ParPgV envelope glycoprotein antibody\u003c/h2\u003e \u003cp\u003eThe partial E2 envelope glycoprotein (amino acids 414\u0026ndash;570) of the ParPgV-A strain was expressed in the Bac-to-Bac Baculovirus system (Invitrogen). The coding region was cloned into the pFastBacHT-A vector using the NEBuilder HiFi DNA Assembly Kit (New England Biolabs). PCR amplification used Q5 High-Fidelity DNA Polymerase (New England Biolabs) with conditions listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003eRecombinant protein was solubilized in lysis buffer (8 M urea, 50 mM NaH2PO4 pH 7.4, 0.5 M NaCl, 20 \u0026micro;g/mL DNase I, 1 mM MgCl2, 1 mM PMSF) and purified under denaturing conditions via His-tag affinity chromatography (His GraviTrap\u0026trade; TALON\u0026reg;; Cytiva, Marlborough, MA, USA). Protein expression and purification were verified by Coomassie-stained SDS-PAGE and Western blotting using anti-polyHis-tag (Sigma Aldrich) and anti-mouse-HRP secondary antibodies (BioRad Laboratories) (Supplementary Fig.\u0026nbsp;1). Approximately 0.5 mg of purified E2 protein was delivered to Davids Biotechnologie GmbH (Regensburg, Germany) for rabbit immunization and polyclonal antibody production.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDetection of ParPgV antigen by Immunohistochemistry\u003c/h2\u003e \u003cp\u003eFor immunohistochemistry, 4-\u0026micro;m sections of formalin-fixed paraffin-embedded (FFPE) samples were prepared using a microtome (Microm HM 360) and mounted on positively charged glass slides (Superfrost Plus; Menzel-Gl\u0026auml;ser, Braunschweig, Germany). Tissue samples from partridges and SPF chickens, including samples from healthy birds and those infected with heterologous agents such as fowl adenovirus or \u003cem\u003eHistomonas meleagridis\u003c/em\u003e, were included to evaluate the specificity of the polyclonal serum. Slides were dewaxed, rehydrated, and subjected to antigen retrieval in citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked using a 1.5% hydrogen peroxide solution in methanol for 30 minutes. To prevent nonspecific binding, the sections were incubated with a blocking solution consisting of a 1:10 dilution of normal goat serum (Vector Laboratories, Burlingame, USA) combined with 2% bovine serum albumin (Roche Diagnostics GmbH, Mannheim, Germany) for 60 minutes at room temperature in a humidified chamber. Primary antibody incubation was carried out overnight at 4\u0026deg;C using rabbit polyclonal anti-E2 serum at dilutions of 1:100, 1:500, and 1:1000. Additional sections were incubated with phosphate-buffered saline (PBS) instead of the primary antibody and used as negative controls. After washing with PBS, the sections were incubated with a 1:400 dilution of biotinylated anti-rabbit IgG (Vector Laboratories) for 30 minutes, followed by treatment with the Vectastain ABC Kit (Vector Laboratories) for 60 minutes. Signal detection used the DAB Substrate Kit for peroxidase (Vector Laboratories). The sections were counterstained with Mayer\u0026rsquo;s hematoxylin (Merck, Darmstadt, Germany), dehydrated, and mounted under coverslips with Neomount (VWR, Vienna, Austria).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eField sampling of red-legged partridge flocks for detection of ParPgV\u003c/h2\u003e \u003cp\u003eA field screening for ParPgV was conducted across eight red-legged partridge flocks, from eight different farms, including both farms with a documented history of encephalitis and those without prior outbreaks. From each flock, 30 cloacal swabs were collected, comprising samples from 15 female and 15 male birds. Additionally, 30 embryonated eggs were collected from each flock. The yolk and allantoic fluid were extracted from 15 eggs for screening, while the remaining 15 eggs were incubated for 14 days to allow embryo collection for further analysis. Samples were pooled into groups of five for each type of material, and all pools were tested using real-time RT-PCR.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExperimental\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003einvestigations of ParPgV in partridge and chicken hosts\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eInoculum preparation\u003c/h2\u003e \u003cp\u003eDue to unsuccessful attempts to culture the virus \u003cem\u003ein vitro\u003c/em\u003e (data not shown), an experimental inoculum was prepared using a filtrate from a ParPgV PCR-positive brain sample collected from flock A. For this purpose, brain tissue was suspended in PBS at a concentration of 20% (wt/vol), supplemented with 1 mg/ml streptomycin and 100,000 IU/ml penicillin. The suspension was homogenized using a T25 Ultra Turrax\u0026reg; (IKA, Staufen, Germany) at 20,000 rpm. The resulting homogenate was clarified by centrifugation at 2000 \u0026times; g for 10 minutes, and the supernatant was passed through a 0.2 \u0026micro;m filter (Filtropur S 0.2, Sarstedt, N\u0026uuml;mbrecht, Germany) to obtain the final filtrate (10\u003csup\u003e2.760\u003c/sup\u003e viral genome copies/\u0026micro;l).\u003c/p\u003e \u003cp\u003eFollowing the first \u003cem\u003ein vivo\u003c/em\u003e experiment, PCR analysis targeting the 260bp-ParPgV NS4A gene (Supplementary Table\u0026nbsp;1), followed by Sanger sequencing, identified two distinct ParPgV strains \u0026mdash; designated ParPgV-A and ParPgV-C (the strain prevalent in flock C) \u0026mdash; in the inoculum. As ParPgV-C was the predominant strain detected in the brains of inoculated grey partridges and was also highly prevalent in field monitoring samples, it was selected for further studies. Consequently, the RLP and SPF \u003cem\u003ein vivo\u003c/em\u003e experiments were conducted using a ParPgV-C inoculum (10\u003csup\u003e2.752\u003c/sup\u003e viral genome copies/\u0026micro;l), derived from a ParPgV PCR-positive brain sample from flock C, prepared following the same protocol as ParPgV-A.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBirds, experimental inoculation, and sampling\u003c/h2\u003e \u003cp\u003eThree separate consecutive experiments were conducted, each involving distinct groups of birds: (GP) grey partridges (\u003cem\u003ePerdix perdix\u003c/em\u003e), (RLP) red-legged partridges (\u003cem\u003eAlectoris rufa\u003c/em\u003e), and (SPF) SPF chickens (\u003cem\u003eGallus gallus\u003c/em\u003e). Grey partridges were sourced as 5-month-old birds from a local supplier in Austria (M\u0026uuml;hlb\u0026ouml;ck, Natternbach, Austria). In contrast, embryonated eggs of red-legged partridges (Gibovende\u0026eacute;, Les Herbiers, France) and SPF chickens (VALO BioMedia GmbH, Osterholz-Scharmbeck, Germany) were incubated at the facilities of the Clinic for Poultry Medicine at the University of Veterinary Medicine Vienna, Austria. Upon hatching, birds were housed under controlled conditions with feed and water \u003cem\u003ead libitum\u003c/em\u003e. All procedures were approved by the Ethics and Animal Welfare Committee of the University of Veterinary Medicine, Vienna, in accordance with the University\u0026rsquo;s guidelines for Good Scientific Practice and authorized by the Austrian Federal Ministry of Education, Science, and Research (ref BMBWF 2022\u0026thinsp;\u0026minus;\u0026thinsp;0.713.294, and Extension 2023\u0026thinsp;\u0026minus;\u0026thinsp;0.430.929), in accordance with current legislation.\u003c/p\u003e \u003cp\u003eDetails of the experimental design are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Birds were intravenously inoculated with either ParPgV filtrate or PBS as a control. Clinical signs, which included weakness, reluctance to move, lack of avoidance to capture, and closed eyes, were monitored daily throughout the study. At predetermined timepoints, birds were humanely culled, and tissue samples were collected for histopathological and molecular analyses. In the grey partridge experiment, tissues included the brain, spleen, liver, and kidney. In the red-legged partridge experiment, additional samples from the bone marrow and caecal tonsils were collected. For the SPF chicken experiment, the thymus and bursa of Fabricius were also sampled in addition to the previously mentioned organs. All collected tissues were preserved in 4% neutral buffered formaldehyde solution (SAV LP GmbH) for histopathological analysis and stored at -80\u0026deg;C for virological and molecular investigations. Additionally, at different time points, tracheal and cloacal swabs were collected in the grey partridge and red-legged partridge experiments, while only cloacal swabs were collected in the SPF chicken experiment.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOverview of \u003cem\u003ein vivo\u003c/em\u003e experimental infections with ParPgV in grey partridges, red-legged partridges, and SPF chickens.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStudy ID\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eHost\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAge at Inoculation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGroup ID\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNo. Birds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eInoculum (ml)/bird\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRoute of inoculation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c11\" namest=\"c8\"\u003e \u003cp\u003eSequential Culling (dpi):\u003c/p\u003e \u003cp\u003eno. birds\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGrey partridge\u003c/p\u003e \u003cp\u003e(\u003cem\u003ePerdix perdix\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e5-months old\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGP-VG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2 ParPgV-A filtrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eiv\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGP-CG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2 PBS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eiv\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRLP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRed-legged partridge (\u003cem\u003eAlectoris rufa\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e5-months old\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRLP-VG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2 ParPgV-C filtrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eiv\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRLP-CG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2 PBS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eiv\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSPF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSPF chicken\u003c/p\u003e \u003cp\u003e(\u003cem\u003eGallus gallus\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e4-weeks old\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSPF-VG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2 ParPgV-C filtrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eiv\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSPF-CG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2 PBS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eiv\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003eAbbreviations: dpi, days post-inoculation; iv, intravenous; PBS, phosphate-buffered saline; n.d., not done.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMagnetic resonance imaging (MRI)\u003c/h2\u003e \u003cp\u003eBefore culling, birds from the RLP and SPF experiments were transported under deep sedation to the diagnostic imaging unit. Magnetic resonance imaging (MRI) of the head was performed on two ParPgV-inoculated birds and one control bird per time point using a 1.5 Tesla scanner (Magentom Espree, Siemens Healthineers, Erlangen, Germany). MRI scans were performed at the time points indicated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e using a 70 mm medium-sized loop coil. Standardized imaging protocols were applied to ensure consistency across all groups and time points, including T2-weighted constructive interference in steady-state (CISS) 3D (TR 6.9 ms, TE 2.97 ms), T1-weighted turbo spin echo (TSE) transverse (TR 907 ms, TE 16 ms, slice thickness 5 mm), T2-weighted turbo inversion recovery (TIR) transverse (TR 4770 ms, TE 65 ms, slice thickness 0.8 mm), and T1-weighted gradient echo (GRE) turbo flash 3D (TR 1720 ms, TE 5.92 ms). The imaging data were anonymized and independently assessed by a European Board-certified radiologist, who performed subjective scoring of pathological changes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eDetection of ParPgV negative-strand RNA by RT-PCR\u003c/h2\u003e \u003cp\u003eDetection of the negative-strand RNA was adapted from Lin \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e with modifications. A 260-bp fragment of the NS4A region was amplified using OneStep RT-PCR Kit (Qiagen) and cloned into a pCR\u0026reg;4-TOPO vector (Invitrogen). Primers and thermal cycling conditions are provided in Supplementary Table\u0026nbsp;1. Positive and negative RNA strands were generated by \u003cem\u003ein vitro\u003c/em\u003e transcription from T3 and T7 priming sites, respectively, using the MAXIScript T3 and T7 kits (Thermo Fisher Scientific). DNA templates were removed with the TURBO DNA-free Kit.\u003c/p\u003e \u003cp\u003eTo detect negative-strand RNA, cDNA synthesis was performed using the OmniScript RT Kit (Qiagen) and a chimeric primer combining an oligonucleotide tag with a virus-specific sequence. PCR amplification was conducted with HotStarTaq Master Mix Kit (Qiagen) using the oligonucleotide tag as the forward primer and a ParPgV-specific reverse primer (Supplementary Table\u0026nbsp;1). A 257-bp PCR product indicated the presence of negative-strand RNA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis and Statistics\u003c/h2\u003e \u003cp\u003eData were recorded in Excel (Office 365) and analyzed in Python (version 3.13). Data processing and statistical analyses were performed using pandas, scipy, and matplotlib/seaborn libraries. Normality of continuous variables was assessed using the Shapiro\u0026ndash;Wilk test. Comparisons of viral load in organs of experimentally inoculated red-legged partridges were conducted using unpaired t-tests. Statistical significance was defined as \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eOutbreak Description and Clinical observations\u003c/h2\u003e\n \u003cp\u003ePrior to 2021, clinical signs suggestive of CNS involvement, including apathy, torticollis, ataxia, and prostration (Supplementary Video 1), were generally transient, sporadic, and subtle, and were often overlooked by farm personnel. Affected birds were commonly culled upon detection, which may have limited onward disease transmission within the flock. These clinical occurrences were not initially considered economically relevant. This perception changed in 2021, when a more severe and prolonged outbreak occurred during the laying period on one of the affected farms. At the time, the breeder flock consisted of 6,550 pairs of red-legged partridges. The onset of the outbreak coincided with the beginning of the laying cycle in February/March and was characterized by persistent and recurring neurological signs. Farmers reported culling 10 to 20 birds per week during the first weeks of the season. However, by April, due to the substantial impact on flock productivity and in the absence of evidence for vertical transmission, culling was halted, as affected birds continued to lay eggs despite exhibiting clinical signs. It is estimated that at least 2% of birds were affected over the 16-week laying period, with egg production being reduced by approximately 4.6 eggs per hen in 2021 compared to other years. No bacterial pathogens were isolated during routine diagnostic investigations conducted at the time of the outbreak.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eGross, Histopathological, and Ultrastructural Evidence of Viral Encephalitis in Affected Partridges\u003c/h2\u003e\n \u003cp\u003eAt necropsy, affected partridges from the field outbreaks did not exhibit gross lesions consistent with the observed clinical signs. However, histopathological examination of the brain revealed widespread inflammatory lesions affecting all regions, including both white and, predominantly, grey matter (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). These lesions were characterized by foci of gliosis frequently associated with neuronal degeneration, necrosis, or, in some cases, vacuolation of the neuropil. Consistent perivascular mononuclear infiltrates, composed mainly of lymphocytes and macrophages, were observed.\u003c/p\u003e\n \u003cp\u003eTransmission electron microscopy (TEM) revealed the presence of viral particles, 70\u0026ndash;90 nm in diameter, within neurons in both the cerebrum and cerebellum (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb-d). In the cerebrum, these particles were detected in the perikaryon of granular and pyramidal cells (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb-c), whereas, in the cerebellum, they were localized within cerebellar granular cells (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed). The viral particles were found free in the cytoplasm or associated with transitional vesicles of the endoplasmic reticulum and Golgi complex. Additionally, viral particles were identified in the cytoplasm of endothelial cells lining the cerebral capillaries (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb, insert b2). Morphologically, the particles displayed a spherical shape with a dense, rounded core surrounded by a diffuse outer layer.\u003c/p\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003eDiscovery of a novel Pegivirus by NGS\u003c/h2\u003e\n \u003cp\u003eBrain samples from field outbreaks were screened by PCR for avian influenza virus, avian encephalomyelitis virus, Marek\u0026rsquo;s disease virus, and arboviruses of the genus \u003cem\u003eOrthoflavivirus\u003c/em\u003e. All tests were negative (data not shown). To further investigate the aetiology of the outbreaks, RNA extracted from brain samples of all three outbreaks was subjected to next-generation sequencing (NGS). Metagenomic profiling using de novo assembled contigs identified 887 viral contigs, including three short contigs mapping to \u003cem\u003eGoose pegivirus 2\u003c/em\u003e (GPgV-2) and other avian-origin pegiviruses. The presence of a novel pegivirus was confirmed by conventional PCR targeting the 5\u0026apos;-UTR and NS4A regions, followed by Sanger sequencing of the PCR products, which indicated the presence of different strains among the samples. To obtain the complete genome sequence of one strain, designated ParPgV-A, a set of primers covering the pegivirus genome was designed (Supplementary Table 1, Supplementary Fig. 2a), resulting in a 10,127-bp final genome sequence. For the ParPgV-C strain, total RNA from infected partridge embryos was subjected to NGS, followed by PCR and Sanger sequencing to close gaps (Supplementary Table 1, Supplementary Fig. 2b). This yielded a final genome sequence of 10,687 bp, slightly larger than that of ParPgV-A. Both ParPgV genomes consist of short 5\u0026apos;- and 3\u0026apos;-untranslated regions (UTRs) and a single open reading frame (ORF). The longer UTRs observed in ParPgV-C suggest that the ParPgV-A genome may be incomplete. Similar to other pegiviruses, both ParPgV strains encode a single polyprotein of 3,258 amino acids. Sequence comparison with known pegiviruses suggested that the polyprotein encodes a signal peptide (S), two structural proteins (E1 and E2) at the N-terminal region, followed by six non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). A putative signalase cleavage site was identified between amino acids 19 and 20. HMMER searches against the Pfam database identified protein domains conserved in other Flaviviridae, corroborating the predicted organization of the viral polyprotein (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Phylogenetic analyses based on the NS3 and NS5B regions positioned both ParPgV strains on a distinct branch within the avian pegivirus clade, closely related to several strains of avian origin (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). In contrast to mammalian pegiviruses, which segregate into two clades, all avian-origin pegiviruses formed a third distinct clade. The percent identity matrix showed 97.98% amino acid identity between the two ParPgV strains and 59.38\u0026ndash;77.39% identity to other avian-origin pegiviruses (Supplementary Table\u0026nbsp;2). Based on the proposed pegivirus species demarcation criterion of 69% amino acid identity within the NS3 region\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, ParPgV belongs to the same species as Goose pegivirus 2 (GPgV-2), Fernbird pegivirus 1, \u003cem\u003eMonifringilla taczanowskii\u003c/em\u003e pegivirus, and Pin virus. Furthermore, based on this demarcation criterion, the analysis indicated the existence of two additional species within the avian pegivirus clade. One represented by the Goose pegivirus 1 (GPgV-1) and the other one consisting of \u003cem\u003eLeucosticte brandti\u003c/em\u003e pegivirus and \u003cem\u003ePasser montanus\u003c/em\u003e pegivirus (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePredicted viral proteins of ParPgV-A and ParPgV-C, with Pfam domain matches and genome positions.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParPgV variant\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eprotein\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003edescription\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePfam match (Family Id; accession; description; polyprotein start-aa_end-aa)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eamino acid position (polyprotein)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003enucleotide position (genome)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eE1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eenvelope glycoprotein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHCV_env; PF01539; Hepatitis C virus envelope glycoprotein E1; start-28_end-210\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e20\u0026ndash;211\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e369:942\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHCV_env; PF01539; Hepatitis C virus envelope glycoprotein E1; aa28_aa209\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e605:1178\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eE2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eenvelope glycoprotein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGBV-C_env; PF12786; GB virus C genotype envelope; aa390_aa586\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e212\u0026ndash;679\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e945:2346\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGBV-C_env; PF12786; GB virus C genotype envelope; aa361_aa586\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1181:2582\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eadditional glycoprotein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e680\u0026ndash;750\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2349:2559\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2585:2795\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eNS2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003enon-structural protein NS2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eHCV_NS2; PF01538; Hepatitis C virus non-structural protein NS2; aa768_aa963\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e751\u0026ndash;986\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2562:3177\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2798:3503\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eNS3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eprotease and helicase activity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePeptidase_S29; PF02907; Hepatitis C virus NS3 protease; aa1015_aa1163\u003c/p\u003e\n \u003cp\u003eFlavi_DEAD; PF07652; Flavivirus DEAD domain; aa1181_aa1323\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e987\u0026ndash;1613\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3270:5148\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePeptidase_S29; PF02907; Hepatitis C virus NS3 protease; aa1015_aa1163\u003c/p\u003e\n \u003cp\u003eFlavi_DEAD; PF07652; Flavivirus DEAD domain; aa1181_aa1323\u003c/p\u003e\n \u003cp\u003eDEAD; PF00270; DEAD/DEAH box helicase; aa1168_aa1319\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3506:5384\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eNS4A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003enon-structural protein NS4A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e1614\u0026ndash;1679\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5151:5346\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5387:5582\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eNS4B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003enon-structural protein NS4B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eHCV_NS4b; PF01001; Hepatitis C virus non-structural protein NS4b; aa1686_aa1875\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e1680\u0026ndash;1925\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5349:6084\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5585:6320\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eNS5A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003enon-structural protein NS5A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eHCV_NS5a_1a; PF08300; Hepatitis C virus non-structural 5a zinc finger domain; aa1960_aa2010\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e1926\u0026ndash;2694\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6087:8391\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6323:8627\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eNS5B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eRNA-dependent RNA polymerase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRdRP_3; PF00998; Viral RNA-dependent RNA polymerase; aa2697_aa3187\u003c/p\u003e\n \u003cp\u003eRdRP_1; PF00680; Viral RNA-dependent RNA polymerase; aa2737_aa3103\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e2695\u0026ndash;3258\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8394:10164\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRdRP_3; PF00998; Viral RNA-dependent RNA polymerase; aa2697_aa3186\u003c/p\u003e\n \u003cp\u003eRdRP_1; PF00680; Viral RNA-dependent RNA polymerase; aa2738_aa3077\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8630:10319\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003eAbbreviations: n.d., not determined; Pfam, Protein family database.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003ePrevalence of ParPgV in Red-Legged Partridge Farms\u003c/h2\u003e\n \u003cp\u003eParPgV was detected in cloacal swabs from all investigated farms (Fig.\u0026nbsp;3a). All pooled samples from female birds, except one from Farm G, tested positive. In males, all samples from Farms A and F were positive, while negative pools were observed in Farms B and E, and two of three pools were negative in Farms C, D, G, and H. Female samples showed higher positivity rates than male samples.\u003c/p\u003e\n \u003cp\u003eIn embryonated eggs, all pooled yolk sac (YS) samples tested positive for ParPgV (Fig. 3b). Allantoic fluid (AF) samples from Farms A, B, E, G, and H tested positive, while negative pools were detected in Farms C, D, and F. Embryo samples were positive across all farms, with some negative pools identified in Farms D, F, and H. Viral loads were highest in embryos, followed by YS and AF.\u003c/p\u003e\n \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n \u003ch2\u003ePathogenicity and tissue distribution of ParPgV in experimentally inoculated birds\u003c/h2\u003e\n \u003cdiv id=\"Sec27\" class=\"Section4\"\u003e\n \u003ch2\u003eGrey partridges\u003c/h2\u003e\n \u003cp\u003eOf the seven inoculated grey partridges (P4\u0026ndash;P10), two (P5 and P7) died shortly after inoculation due to unrelated causes. Clinical signs in the remaining birds were limited to transient reduction of flying behaviour observed at 4 dpi (Supplementary Video 2). Histological examination revealed mild kidney, liver, and spleen alterations (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). Kidney congestion was observed in all inoculated birds, with some cases exhibiting lymphoid infiltration. Liver congestion was noted in most birds, except one. Splenic congestion ranged from mild to severe, with occasional lymphocytic infiltration and encapsulation of connective tissue. No significant histological changes were detected in other organs.\u003c/p\u003e\n \u003cp\u003ePost-mortem analysis detected viral RNA in the brain and spleen of all birds, with viral load values remaining stable between 14 and 21 dpi (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb), ranging from 3.43 to 5.24 log\u003csub\u003e10\u003c/sub\u003e viral copies/reaction in the brain and 3.35 to 4.28 log\u003csub\u003e10\u003c/sub\u003e in the spleen. In the kidney, viral RNA was detected in 1 of 2 birds at 14 dpi and in 2 of 3 birds at 21 dpi, with viral loads reaching 2.80 log\u003csub\u003e10\u003c/sub\u003e viral copies/reaction at the latter time point. Similarly, no viral RNA was detected in the liver at 14 dpi, whereas 2 of 3 birds were positive at 21 dpi, with viral loads reaching 4.60 log\u003csub\u003e10\u003c/sub\u003e viral copies/reaction (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e\n \u003cp\u003eReal-time RT-qPCR analysis of swabs and whole blood revealed sporadic viral detection (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec). Tracheal swabs tested positive only at 21 dpi in P8 and P10, while cloacal swabs showed positivity at 10 dpi in P4 and at 21 dpi in P10. In blood, all birds except P9 were positive at 7 dpi, whereas at 14 dpi only P4 and P8 remained positive. By 21 dpi, ParPgV RNA was detected solely in P8.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\n \u003ch2\u003eRed-legged partridges\u003c/h2\u003e\n \u003cp\u003ePrior to inoculation, whole blood and cloacal swabs from all red-legged partridges revealed low levels of ParPgV RNA (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, 0 dpi), confirming previous investigations on farm samples. Following inoculation with the ParPgV inoculum, birds exhibited decreased activity and reduced flying behaviour between 5 and 8 dpi, with no other signs being observed. The viral RNA load in blood, tracheal, and cloacal swabs increased until 10 dpi, subsequently declining, with lower levels detected at 21 dpi. At this final time point, no viral RNA was detected in blood samples. Viral RNA was consistently detected in all examined organs throughout the experiment, including the brain, liver, kidney, spleen, bone marrow, and caecal tonsils (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb). Following ParPgV inoculation, viral load significantly increased in the liver, kidney, bone marrow, and caecal tonsils compared to PBS-inoculated controls (RLP-CG). In the brain, viral load remained stable until the final time point, when a non-significant decline was observed. In contrast, viral load in the spleen remained unaffected by inoculation. Histological examination revealed no observable lesions until 21 dpi. At this final time point, all ParPgV-inoculated birds displayed encephalitic lesions, both in the cerebrum and cerebellum, characterized by gliosis, neuronal degeneration or necrosis, and perivascular mononuclear cell infiltrations (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec-d). Magnetic resonance imaging (MRI) showed a widening of the subarachnoid space in the caudal cranial fossa on fluid-sensitive sequences (T2W-CISS and T2-TIR) at 21 dpi compared to the control group and the prior time points, indicative of cerebellar atrophy (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee). No other parenchymal abnormalities or alterations in signal intensity were detected.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\n \u003ch2\u003eSPF chickens\u003c/h2\u003e\n \u003cp\u003eBirds were inoculated at 4 weeks of age, and no clinical signs were observed throughout the study. However, viral RNA was detected in all investigated organs at 7 days post-inoculation (dpi), with the highest loads found in the liver, kidney, caecal tonsils, and bursa of Fabricius (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). By 14 dpi, viral RNA was detected only in the caecal tonsils, and by 21 dpi, it was restricted to the bone marrow, with all other organs testing negative.\u003c/p\u003e\n \u003cp\u003ePrior to inoculation, blood and cloacal swabs from SPF chickens tested negative for ParPgV by PCR (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb). Following inoculation, viremia peaked at 7 dpi, with four birds testing positive for viral RNA in blood. At 14 dpi, only one bird remained positive, with a low viral load, and by 21 dpi, all birds were negative. All cloacal swabs remained negative throughout the study (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e\n \u003cp\u003eNo histological lesions were observed in any of the organs examined at any time point, and MRI analysis revealed no detectable abnormalities.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eImmunohistochemistry (IHC) and RNAscope\u003c/strong\u003e \u003cstrong\u003ein-situ\u003c/strong\u003e \u003cstrong\u003ehybridization investigations\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTo assess the neurotropism of ParPgV in infected birds, IHC and RNAscope \u003cem\u003ein-situ\u003c/em\u003e hybridization were performed on brain samples from both field outbreaks and the three \u003cem\u003ein vivo\u003c/em\u003e experiments (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). To investigate systemic distribution, spleen, caecal tonsils, liver, and kidney tissues from experimentally inoculated red-legged partridges were similarly analysed (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). A primary antibody dilution of 1:500 yielded optimal signal intensity in IHC and was standardized across all samples.\u003c/p\u003e\n \u003cp\u003eIn the brain, contrary to investigations in negative birds (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea, b), IHC confirmed the presence of ParPgV antigen, particularly in perivascular regions and areas of inflammatory infiltrates in naturally infected partridges (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ec). RNAscope detected viral RNA within neuronal and glial cells of the cerebrum (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ed). Experimentally inoculated grey and red-legged partridges showed diffuse immunoreactivity throughout the neuropil (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ee, g). RNAscope also revealed strong signals in Purkinje cells and scattered glial cells (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ef, h), extending into the cerebellum of red-legged partridges, possibly suggesting viral involvement in motor coordination centers. These findings correlate with the observed neurological signs in infected birds. In SPF chickens, brain sections from PCR-positive individuals showed only faint signals, consistent with lower viral loads.\u003c/p\u003e\n \u003cp\u003eIn the spleen, viral antigen was localized to periarteriolar lymphoid sheaths (PALS) and reticuloendothelial cells (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ea), with RNAscope confirming viral RNA in the same regions (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eb). In caecal tonsils, both antigen and RNA were detected in the follicular epithelium and scattered mononuclear cells (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ec, d). In the liver, viral antigen and RNA were found in hepatocytes and sinusoidal lining cells (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ee, f). In the kidney, both signals were observed in tubular epithelial cells and glomerular structures (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eg, h).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eParPgV negative-strand RNA replication\u003c/h3\u003e\n\u003cp\u003eDetection of the negative-strand RNA, an intermediate of viral replication, was assessed by strand-specific RT-PCR across different tissues and species (Supplementary Table 3). In the brain, negative-strand RNA was consistently detected in inoculated red-legged partridges (RLP-VG group) and grey partridges (GP). Interestingly, control red-legged partridges (RLP-CG group), which were vertically infected \u003cem\u003ein ovo\u003c/em\u003e, also tested positive. Additionally, in red-legged partridge embryonated eggs, negative-strand RNA was detected in both the embryos and the yolk sac. In SPF chickens, investigation of brain tissue did not yield conclusive results due to the presence of low viral load (see Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). Nonetheless, positive results were obtained from the liver, kidney, spleen, bone marrow, and caecal tonsils in SPF chickens.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePegiviruses were initially suspected to cause hepatitis due to their association with non-A\u0026ndash;E hepatitis in humans and genomic similarities to hepatitis C virus (HCV)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. However, extensive studies have since shown that pegiviruses are not directly linked to acute or chronic hepatitis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Most available data come from human pegivirus (HPgV-1), which is widely considered non-pathogenic, though its immunomodulatory effects have gained interest for their potential benefits in co-infections\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Recently, however, HPgV-1 has been implicated in fatal leukocytic encephalitis, with evidence of lymphocytic infiltration and gliosis in brain tissue\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Further supporting its neurotropism, studies have demonstrated HPgV-1 replication in astrocytes and microglia, highlighting its ability to infect neural cells\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In agreement with this, the actual study provides the first experimental evidence that pegiviruses can cause encephalitis, challenging the prevailing assumption that members of the Pegivirus genus are non-pathogenic.\u003c/p\u003e \u003cp\u003eA key finding of this work was the detection of encephalitis in red-legged partridges during field outbreaks, which included histopathological lesions consistent with viral infection and electron microscopy confirmation of virus-like particles in affected neurons. Despite repeated attempts to propagate the virus in primary liver chicken embryo cells and via yolk sac inoculation of chicken embryos, viral titers declined over subsequent passages (data not shown). This aligns with the broader challenge of establishing efficient \u003cem\u003ein vitro\u003c/em\u003e systems for pegiviruses, which often demonstrate narrow tropism and can be notoriously difficult to culture\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In agreement with this, a goose pegivirus (GPgV) was passaged eight times in primary goose embryonic fibroblasts, reaching high viral loads, but failed to replicate in other five different cell types\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Notwithstanding these limitations, next-generation sequencing of clinical material revealed novel pegiviral sequences, confirming the presence of an avian pegivirus as a likely etiological agent.\u003c/p\u003e \u003cp\u003eDetailed sequence analysis revealed the presence of potentially more than one pegivirus strain in the outbreak samples. This was confirmed by obtaining the complete genome sequences of two distinct strains, designated as ParPgV-A and ParPgV-C. Among these, the ParPgV-C strain appears to be more prevalent in red-legged partridges, as it was detected in both embryos and outbreak samples. Phylogenetic analysis supports the previously proposed existence of a third clade within the genus Pegivirus, comprising all reported avian origin pegiviruses\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Moreover, based on the species demarcation threshold of 69% amino acid identity within the NS3 region, our findings confirm the presence of three distinct species within this third Pegivirus clade\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The ParPgV strains identified in this study cluster within the species that includes the majority of currently reported avian pegiviruses. However, no distinct phylogenetic clustering was observed correlating with clinical presentation. This may be because most avian pegiviruses reported to date have been identified through metagenomic surveys of asymptomatic birds, whereas the ParPgV genomes described here are among the few sequences present to date associated with clinical disease.\u003c/p\u003e \u003cp\u003eAn initial experiment in grey partridges was conducted as an exploratory model, partly because red-legged partridges are native to southern Europe and were less accessible in Austria. Despite this limitation, the study confirmed not only the neurotropic and lymphotropic nature of ParPgV but also its propensity for persistence. Stable viral loads were detected in the brain and spleen until the end of the trial, whereas viral RNA in blood and swabs fluctuated or was only intermittently positive. However, no histopathological lesions were evident in the brain of grey partridges. By contrast, in the subsequent experiment involving red-legged partridges \u0026mdash; the original host of ParPgV \u0026mdash; encephalitic lesions were observed upon histopathological examination, and cerebellar atrophy was confirmed by MRI, although clinical signs were limited to subtle behavioural changes such as reduced activity and flight responsiveness. The cerebellar atrophy noted in superinfected birds suggests a progressive, degenerative process likely associated with chronic infection and might explain the ataxia and prostration observed in affected birds in the field. Such findings parallel recent reports implicating HPgV-1 in neurological disease, in which a leukoencephalitis patient presented progressive lesions in the white matter by MRI investigation, with involvement of the brainstem and cervical cord\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Additionally, the present findings resemble outcomes in other experimental neurotropic flavivirus models, where infection-induced neuronal loss and persistent inflammation lead to long-term neuropathological changes\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Nonetheless, these results differ from those in a rhesus monkey pegivirus model, which did not demonstrate increased viral RNA loads in neural tissues\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe combined RNAscope ISH and IHC findings reinforce the hypothesis that ParPgV exhibits a strong neurotropic and systemic infection pattern. The presence of viral RNA and antigen in the cerebrum and cerebellum supports the notion that ParPgV actively replicates in neural tissues, contributing to encephalitic lesions and cerebellar atrophy observed in infected birds. Additionally, viral localization in immune-associated tissues such as the spleen and caecal tonsils suggests that the virus persists in lymphoid organs, potentially modulating immune responses. These findings were further corroborated by strand-specific RT-PCR detection of the negative-strand RNA replication intermediate, confirming active viral replication in the brain tissues of inoculated red-legged partridges and grey partridges. Moreover, negative-strand RNA was identified in the liver, kidney, spleen, bone marrow, and caecal tonsils of SPF chickens, and in the embryos and yolk sacs of red-legged partridge embryonated eggs, providing evidence of systemic and \u003cem\u003ein ovo\u003c/em\u003e viral replication. The detection of PgV RNA and antigen in renal and hepatic tissues further highlights the systemic nature of the infection, raising questions about the potential for viral shedding and long-term persistence. These findings are consistent with other neurotropic flavivirus infections, where persistent viral replication in neural and immune tissues leads to chronic pathology and progressive neurological impairment\u003csup\u003e\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Further research is needed to clarify whether viral persistence in these organs contributes to long-term disease progression and whether immune modulation plays a role in maintaining viral reservoirs in infected hosts.\u003c/p\u003e \u003cp\u003eInterestingly, although red-legged partridges developed clear neurological lesions in this study, they displayed only mild outward signs of disease, implying that ParPgV could cause a subclinical or progressive form of disease, as it has been reported in mice experimentally inoculated with West Nile virus\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. This possibility may have implications for both wild and farmed partridge populations, as clinically apparent signs might be infrequent or overlooked, especially in flocks where routine culling removes symptomatic individuals.\u003c/p\u003e \u003cp\u003eA critical limitation of the red-legged partridge experiment lies in the fact that these birds already tested positive for ParPgV at the time of inoculation, likely through vertical transmission. Screening of multiple partridge flocks failed to identify birds free of ParPgV; moreover, females consistently harboured higher viral loads than males. This sex-associated difference in viral load, along with the temporal association of clinical outbreaks with the onset of egg laying, raises the hypothesis that physiological or hormonal changes linked to reproduction, such as immunomodulation during the laying period, may favour viral replication or persistence in females. In agreement with this, embryonated eggs from all flocks were also positive, strongly indicating that vertical transmission occurs regularly in these populations. Vertical mother-to-child transmission of HPgV has been reported in humans\u003csup\u003e\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, however, avian pegiviruses have just been recently identified\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003ein ovo\u003c/em\u003e transmission was unknown until now. The widespread distribution of ParPgV in red-legged partridges, together with the predominantly mild clinical presentation observed in experimentally inoculated birds, suggests a well-adapted host-virus relationship. This observation raises important questions regarding whether \u003cem\u003ein ovo\u003c/em\u003e infection influences host immune responses and predisposes birds to future neurological disease. If birds become infected as embryos, they may develop tolerance, permitting lifelong viral persistence\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Because all study birds were ParPgV-positive prior to the experimental challenge, it remains unclear whether any observed lesions were specifically induced by superinfection or reflected exacerbation of a pre-existing infection.\u003c/p\u003e \u003cp\u003eThe final stage of this investigation employed SPF chickens as a potential laboratory model. Although the infection was successfully established in these chickens \u0026mdash; evidenced by viral RNA in numerous organs \u0026mdash; they showed neither clinical manifestations nor significant histopathological changes, and MRI findings were unremarkable. This is in contrast to red-legged partridges, where viral RNA persisted in multiple tissues, including the brain and spleen. The differences in disease expression may stem from variations in immune responses or viral replication dynamics among host species. Furthermore, the possibility that congenital infection shapes immune responses and facilitates later neurological disease in red-legged partridges remains a key question. Host-restricted pathogenicity of other flaviviruses, such as Usutu virus, western equine encephalomyelitis, and St. Louis encephalitis viruses, underscores how some avian species can harbour high viral loads subclinically\u003csup\u003e\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Additional research is needed to determine if immune factors restrict ParPgV neuroinvasion in chickens.\u003c/p\u003e \u003cp\u003eIn conclusion, the identification of an avian pegivirus associated with encephalitis has significant implications for both wildlife health and the broader understanding of pegivirus evolution. Historically, pegiviruses have been regarded as benign, persistent viruses with limited pathogenic potential​. However, our findings, along with recent reports of HPgV-1 in human CNS infections, suggest that pegiviruses may have underappreciated neuropathogenic capabilities. Further studies are needed to elucidate the molecular mechanisms of ParPgV neurotropism and its potential interactions with the host immune system. The role of vertical transmission in viral persistence should also be explored, particularly in the context of population dynamics in farmed and wild partridge populations. Additionally, the development of a reliable \u003cem\u003ein vitro\u003c/em\u003e culture system for ParPgV would be invaluable for future studies to further unravel pathogenesis of such new viruses.\u003c/p\u003e \u003cp\u003eAltogether, this study provides the first experimental evidence linking a pegivirus to encephalitis. While red-legged partridges appear to tolerate persistent infection with limited clinical manifestations, the histopathological and MRI findings indicate that ParPgV can cause significant neurological disease. These findings challenge the notion of pegivirus non-pathogenicity and highlight the need for further research into their role in avian and mammalian hosts.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors extend their gratitude to Attila Sandor, Bibiane Pollak, Patricia Wernsdorf, and Vesna Stanisavljevic for their valuable technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eField outbreak documentation, sample collection, and procurement of embryonated red-legged partridge eggs: N.V.; Histopathology: M.M., D.L., O.A., and F.G.; Recombinant protein expression and polyclonal antibody production: I.B. and B.J.; Immunohistochemistry and RNAscope: M.M., I.B., and D.L.; Transmission electron microscopy: S.R.; MRI investigation: Y.V. and E.L.; \u003cem\u003eIn vivo\u003c/em\u003e experiments: M.M.; Nucleic acid extraction, NGS library preparation, and detection of negative-strand RNA: I.B. and B.J.; qPCR: M.M., B.J., and F.G.; NGS data analysis, genome assembly, and phylogenetic analyses: I.B. and N.P.; Conceptualization and project administration: M.M., I.B., and M.H.; Writing—original draft: M.M. and I.B. All authors reviewed and approved the final manuscript and are accountable for their contributions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSimmonds, P. \u003cem\u003eet al.\u003c/em\u003e ICTV virus taxonomy profile: \u003cem\u003eFlaviviridae\u003c/em\u003e. \u003cem\u003eJournal of General Virology\u003c/em\u003e \u003cstrong\u003e98\u003c/strong\u003e, 2\u0026ndash;3 (2017).\u003c/li\u003e\n\u003cli\u003eSmith, D. B. \u003cem\u003eet al.\u003c/em\u003e Proposed update to the taxonomy of the genera Hepacivirus and Pegivirus within the \u003cem\u003eFlaviviridae\u003c/em\u003e family. \u003cem\u003eJournal of General Virology\u003c/em\u003e \u003cstrong\u003e97\u003c/strong\u003e, 2894\u0026ndash;2907 (2016).\u003c/li\u003e\n\u003cli\u003eStapleton, J. T. Human Pegivirus Type 1: A Common Human Virus That Is Beneficial in Immune-Mediated Disease? \u003cem\u003eFront Immunol\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 1\u0026ndash;17 (2022).\u003c/li\u003e\n\u003cli\u003eYu, Y., Wan, Z., Wang, J. H., Yang, X. \u0026amp; Zhang, C. Review of human pegivirus: Prevalence, transmission, pathogenesis, and clinical implication. \u003cem\u003eVirulence\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 323\u0026ndash;340 (2022).\u003c/li\u003e\n\u003cli\u003eGrimwood, R. M. \u003cem\u003eet al.\u003c/em\u003e From islands to infectomes: host-specific viral diversity among birds across remote islands. \u003cem\u003eBMC Ecol Evol\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 1\u0026ndash;18 (2024).\u003c/li\u003e\n\u003cli\u003eZhu, W. \u003cem\u003eet al.\u003c/em\u003e Novel pegiviruses infecting wild birds and rodents. \u003cem\u003eVirol Sin\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 208\u0026ndash;214 (2022).\u003c/li\u003e\n\u003cli\u003eZhen, W. \u003cem\u003eet al.\u003c/em\u003e Emergence of a novel pegivirus species in southwest China showing a high rate of coinfection with parvovirus and circovirus in geese. \u003cem\u003ePoult Sci\u003c/em\u003e \u003cstrong\u003e100\u003c/strong\u003e, 1\u0026ndash;6 (2021).\u003c/li\u003e\n\u003cli\u003eWu, Z. \u003cem\u003eet al.\u003c/em\u003e The First Nonmammalian Pegivirus Demonstrates Efficient In Vitro Replication and High Lymphotropism. \u003cem\u003eJ Virol\u003c/em\u003e \u003cstrong\u003e94\u003c/strong\u003e, 1\u0026ndash;18 (2020).\u003c/li\u003e\n\u003cli\u003eChang, W. S., Rose, K. \u0026amp; Holmes, E. C. Meta-transcriptomic analysis of the virome and microbiome of the invasive Indian myna (\u003cem\u003eAcridotheres tristis\u003c/em\u003e) in Australia. \u003cem\u003eOne Health\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 1\u0026ndash;11 (2021).\u003c/li\u003e\n\u003cli\u003eStapleton, J. T., Foung, S., Muerhoff, A. S., Bukh, J. \u0026amp; Simmonds, P. The GB viruses: A review and proposed classification of GBV-A, GBV-C (HGV), and GBV-D in genus Pegivirus within the family \u003cem\u003eFlaviviridae\u003c/em\u003e. \u003cem\u003eJournal of General Virology\u003c/em\u003e \u003cstrong\u003e92\u003c/strong\u003e, 233\u0026ndash;246 (2011).\u003c/li\u003e\n\u003cli\u003eChandriani, S. \u003cem\u003eet al.\u003c/em\u003e Identification of a previously undescribed divergent virus from the Flaviviridae family in an outbreak of equine serum hepatitis. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e110\u003c/strong\u003e, 1407\u0026ndash;1415 (2013).\u003c/li\u003e\n\u003cli\u003eTomlinson, J. E. \u003cem\u003eet al.\u003c/em\u003e Viral testing of 18 consecutive cases of equine serum hepatitis: A prospective study (2014-2018). \u003cem\u003eJ Vet Intern Med\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 251\u0026ndash;257 (2019).\u003c/li\u003e\n\u003cli\u003eTomlinson, J. E. \u003cem\u003eet al.\u003c/em\u003e Viral testing of 10 cases of Theiler\u0026rsquo;s disease and 37 in-contact horses in the absence of equine biologic product administration: A prospective study (2014-2018). \u003cem\u003eJ Vet Intern Med\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 258\u0026ndash;265 (2019).\u003c/li\u003e\n\u003cli\u003eBalcom, E. F. \u003cem\u003eet al.\u003c/em\u003e Human pegivirus-1 associated leukoencephalitis: Clinical and molecular features. \u003cem\u003eAnn Neurol\u003c/em\u003e \u003cstrong\u003e84\u003c/strong\u003e, 781\u0026ndash;787 (2018).\u003c/li\u003e\n\u003cli\u003eDoan, M. A. L. \u003cem\u003eet al.\u003c/em\u003e Infection of Glia by Human Pegivirus Suppresses Peroxisomal and Antiviral Signaling Pathways. \u003cem\u003eJ Virol\u003c/em\u003e \u003cstrong\u003e95\u003c/strong\u003e, 1\u0026ndash;15 (2021).\u003c/li\u003e\n\u003cli\u003eBailey, A. L. \u003cem\u003eet al.\u003c/em\u003e Durable sequence stability and bone marrow tropism in a macaque model of human pegivirus infection. \u003cem\u003eSci Transl Med\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 1\u0026ndash;20 (2015).\u003c/li\u003e\n\u003cli\u003eChivero, E. T. \u0026amp; Stapleton, J. T. Tropism of human pegivirus (Formerly known as GB virus C/hepatitis G virus) and host immunomodulation: Insights into a highly successful viral infection. \u003cem\u003eJournal of General Virology\u003c/em\u003e \u003cstrong\u003e96\u003c/strong\u003e, 1521\u0026ndash;1532 (2015).\u003c/li\u003e\n\u003cli\u003eChrzastek, K. \u003cem\u003eet al.\u003c/em\u003e Use of Sequence-Independent, Single-Primer-Amplification (SISPA) for rapid detection, identification, and characterization of avian RNA viruses. \u003cem\u003eVirology\u003c/em\u003e \u003cstrong\u003e509\u003c/strong\u003e, 159\u0026ndash;166 (2017).\u003c/li\u003e\n\u003cli\u003eTeufel, F. \u003cem\u003eet al.\u003c/em\u003e SignalP 6.0 predicts all five types of signal peptides using protein language models. \u003cem\u003eNat Biotechnol\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 1023\u0026ndash;1025 (2022).\u003c/li\u003e\n\u003cli\u003eLin, L., Fevery, J. \u0026amp; Yap, S. H. A novel strand-specific RT-PCR for detection of hepatitis C virus negative-strand RNA (replicative intermediate): evidence of absence or very low level of HCV replication in peripheral blood mononuclear cells. \u003cem\u003eJ Virol Methods\u003c/em\u003e \u003cstrong\u003e100\u003c/strong\u003e, 97\u0026ndash;105 (2002).\u003c/li\u003e\n\u003cli\u003eAppler, K. K. \u003cem\u003eet al.\u003c/em\u003e Persistence of west Nile virus in the central nervous system and periphery of mice. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 1\u0026ndash;12 (2010).\u003c/li\u003e\n\u003cli\u003eTurtle, L., Griffiths, M. J. \u0026amp; Solomon, T. Encephalitis caused by flaviviruses. \u003cem\u003eQJM: An International Journal of Medicine\u003c/em\u003e \u003cstrong\u003e105\u003c/strong\u003e, 219\u0026ndash;223 (2012).\u003c/li\u003e\n\u003cli\u003ede Vries, L. \u0026amp; Harding, A. T. Mechanisms of Neuroinvasion and Neuropathogenesis by Pathologic Flaviviruses. \u003cem\u003eViruses\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 1\u0026ndash;20 (2023).\u003c/li\u003e\n\u003cli\u003eMaximova, O. A. \u0026amp; Pletnev, A. G. Flaviviruses and the Central Nervous System: Revisiting Neuropathological Concepts. \u003cem\u003eAnnu. Rev. Virol.\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 255\u0026ndash;272 (2018).\u003c/li\u003e\n\u003cli\u003eCody, S. G., Adam, A., Siniavin, A., Kang, S. S. \u0026amp; Wang, T. Flaviviruses\u0026mdash;Induced Neurological Sequelae. \u003cem\u003ePathogens\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1\u0026ndash;19 (2025).\u003c/li\u003e\n\u003cli\u003eBlackhurst, B. M. \u0026amp; Funk, K. E. Molecular and Cellular Mechanisms Underlying Neurologic Manifestations of Mosquito-Borne Flavivirus Infections. \u003cem\u003eViruses\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 1\u0026ndash;19 (2023).\u003c/li\u003e\n\u003cli\u003eSupapol, W. B. \u003cem\u003eet al.\u003c/em\u003e Reduced mother-to-child transmission of HIV associated with infant but not maternal GB virus C infection. \u003cem\u003eJournal of Infectious Diseases\u003c/em\u003e \u003cstrong\u003e197\u003c/strong\u003e, 1369\u0026ndash;1377 (2008).\u003c/li\u003e\n\u003cli\u003eSantos, L. M. \u003cem\u003eet al.\u003c/em\u003e Prevalence and vertical transmission of human pegivirus among pregnant women infected with HIV. \u003cem\u003eInternational Journal of Gynecology and Obstetrics\u003c/em\u003e \u003cstrong\u003e138\u003c/strong\u003e, 113\u0026ndash;118 (2017).\u003c/li\u003e\n\u003cli\u003eLin, H.-H. \u003cem\u003eet al.\u003c/em\u003e Mother-to-Infant Transmission of GB Virus C/Hepatitis G Virus: The Role of High-Titered Maternal Viremia and Mode of Delivery and Ding-Shinn Chen. \u003cem\u003eJ Infect Dis\u003c/em\u003e \u003cstrong\u003e177\u003c/strong\u003e, 1202\u0026ndash;1206 (1998).\u003c/li\u003e\n\u003cli\u003eZanetti, A. R. \u003cem\u003eet al.\u003c/em\u003e Multicenter trial on mother-to-infant transmission of GBV-C virus. \u003cem\u003eJ Med Virol\u003c/em\u003e \u003cstrong\u003e54\u003c/strong\u003e, 107\u0026ndash;112 (1998).\u003c/li\u003e\n\u003cli\u003eHe, S. \u003cem\u003eet al.\u003c/em\u003e High-frequency and activation of CD4+CD25+ T cells maintain persistent immunotolerance induced by congenital ALV-J infection. \u003cem\u003eVet Res\u003c/em\u003e \u003cstrong\u003e52\u003c/strong\u003e, 1\u0026ndash;15 (2021).\u003c/li\u003e\n\u003cli\u003eChvala, S. \u003cem\u003eet al.\u003c/em\u003e Limited pathogenicity of Usutu virus for the domestic chicken (\u003cem\u003eGallus domesticus\u003c/em\u003e). \u003cem\u003eAvian Pathology\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 392\u0026ndash;395 (2005).\u003c/li\u003e\n\u003cli\u003eChvala, S. \u003cem\u003eet al.\u003c/em\u003e Limited pathogenicity of usutu virus for the domestic goose (\u003cem\u003eAnser anser f. domestica\u003c/em\u003e) following experimental inoculation. \u003cem\u003eJournal of Veterinary Medicine Series B: Infectious Diseases and Veterinary Public Health\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 171\u0026ndash;175 (2006).\u003c/li\u003e\n\u003cli\u003eKuchinsky, S. C. \u003cem\u003eet al.\u003c/em\u003e North American House Sparrows Are Competent for Usutu Virus Transmission. \u003cem\u003emSphere\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 1\u0026ndash;12 (2022).\u003c/li\u003e\n\u003cli\u003eReisen, W. K., Chiles, R. E., Martinez, V. M., Fang, Y. \u0026amp; Green, E. N. Experimental Infection of California Birds with Western Equine Encephalomyelitis and St. Louis Encephalitis Viruses. \u003cem\u003eJ. Med. Entomol\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 968\u0026ndash;982 (2003).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6888535/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6888535/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePegiviruses have traditionally been regarded as non-pathogenic viruses with controversial clinical significance. Here, we describe a novel avian pegivirus (partridge pegivirus, ParPgV) associated with field outbreaks of encephalitis in red-legged partridges (\u003cem\u003eAlectoris rufa\u003c/em\u003e) in France. Next-generation sequencing identified ParPgV in brain tissues, and full-length genomic characterization revealed two distinct ParPgV strains linked to the outbreaks, confirming their phylogenetic relationship to avian-origin pegiviruses. Histopathology and electron microscopy revealed encephalitic lesions, neuronal degeneration, and virus-like particles within neurons. Field surveillance demonstrated widespread vertical transmission across multiple red-legged partridge flocks. Experimental inoculation of red-legged partridges, grey partridges, and specific-pathogen-free chickens demonstrated viral neurotropism and systemic distribution. Infected red-legged partridges developed cerebellar atrophy detectable by MRI, in the course of transient clinical signs. Detection of negative-strand RNA replication intermediates confirmed active viral replication in neural tissues and lymphoid organs, across the different experimental hosts, and red-legged partridge embryonated eggs. RNAscope in situ hybridization and immunohistochemistry further confirmed the presence of viral RNA and antigen, respectively, in neural and lymphoid tissues. These findings provide the first experimental evidence linking a pegivirus to encephalitis and suggest that pegiviruses may possess underappreciated neuropathogenic potential.\u003c/p\u003e","manuscriptTitle":"Neurotropism and Encephalitis of a Novel Pegivirus with Experimental Evidence Across Avian Species","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-07 07:46:32","doi":"10.21203/rs.3.rs-6888535/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7fda6286-1575-4c32-b49a-b9dc1f972c72","owner":[],"postedDate":"July 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":50917457,"name":"Biological sciences/Microbiology/Virology/Viral pathogenesis"},{"id":50917458,"name":"Biological sciences/Microbiology/Virology/Virus\u0026#x2013;host interactions"},{"id":50917459,"name":"Health sciences/Pathogenesis/Infection"},{"id":50917460,"name":"Biological sciences/Microbiology/Pathogens"}],"tags":[],"updatedAt":"2026-05-14T09:30:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-07 07:46:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6888535","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6888535","identity":"rs-6888535","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}