First Report: Molecular detection and characterization of Parrot Bornavirus 4 (PaBV-4) in captive psittacines in India

First Report: Molecular detection and characterization of Parrot Bornavirus 4 (PaBV-4) in captive psittacines in India | 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 First Report: Molecular detection and characterization of Parrot Bornavirus 4 (PaBV-4) in captive psittacines in India Pankaj Deka, Sangeeta Das, Ritam Hazarika, Parikshit Kakati, Bhaskar Choudhury, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6968515/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Avian bornaviruses are a recently described genetically diverse group consisting of 15 separate viruses within five different viral species belonging to the genus Orthobornavirus within the family Bornaviridae . Amongst the avian bornaviruses discovered, the parrot bornaviruses (PaBV) belonging to species Orthobornavirus alphapsittaciforme possess the highest veterinary relevance and is considered to be a major threat to psittacine aviculture. Since the discovery of PaBV in psittacine birds suffering from proventricular dilatation disease (PDD) in 2008, PaBV infections have been reported worldwide. In India, to assess whether avian bornaviruses circulates in parrots, 83 psittacines from 13 different species including birds with suspected PDD based on clinical examination results (n=33), cage mates of PDD-suspected birds without any clinical signs (n=26) and dead birds with previous clinic suspicious for PDD (n=24) were tested for PaBV. Cloacal swabs were collected from live birds and tissues were collected from dead birds and investigated for the presence of PaBV-RNA using reverse transcription polymerase chain reaction (RT-PCR). PaBV infection was detected in 44 birds (53.01%) belonging to 9 psittaciform species. Eighteen of the group of PDD-suspected birds (54.54%), 21 dead birds (87.50%), and 5 clinically healthy cage mates (19.23%) were positive for PaBV-RNA. Sequence analysis of the matrix (M) gene revealed infection by PaBV-4, belonging to the species Orthobornavirus alphapsittaciforme . To the best of our knowledge, there is no publication describing the circulation of Orthobornavirus alphapsittaciforme , PaBv-4 in captive psittacines in India. This study highlights the major impact on conservation projects including endangered/ vulnerable/ near threatened species as these birds rely on captive breeding for their survival. Therefore, there is an urgent need to recognize and understand the factors that might play a critical role in recent expansion of emergent avian pathogens and how they continue to spread and thrive. Biological sciences/Molecular biology Health sciences/Diseases Avian bornaviruses Orthobornavirus PaBV-4 Orthobornavirus alphapsittaciforme proventricular dilatation disease (PDD) parrots Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Bornaviruses (Order: Mononegavirales , Family: Bornaviridae ) have long been recognized in humans, mammals, birds, and reptiles. Currently, the family includes three genera: Orthobornavirus, Carbovirus, and Cultevirus [ 1 ]. Bornaviruses are enveloped, 80 to 100 nm in diameter, with a non-segmented, linear, single-stranded negative-sense RNA genome having around 9000 nucleotides (nt) in length [ 2 ]. Orthobornavirus genome encodes six proteins, namely nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), the large RNA-dependent RNA polymerase (L), and an accessory protein p10 (X) [ 3 – 4 ]. All avian bornaviruses belong to the genus Orthobornavirus and are currently classified as 15 separate viruses within five different viral species. Avian bornaviruses discovered in parrots (Psittaciformes) include parrot bornavirus 1 to 8 (PaBV-1 to PaBV-8), belonging to species Orthobornavirus alphapsittaciforme (PaBV-1 to 4, PaBV-7 and − 8) and Orthobornavirus betapsittaciforme (PaBV-5 and − 6). Additionally, avian bornaviruses discovered in passerine birds (Passeriformes) include canary bornavirus 1 to 3 (CnBV-1 to CnBV-3) and munia bornavirus 1 (MuBV 1), belonging to the species Orthobornavirus serini and estrildid finch bornavirus 1 (EsBV-1), belonging to Orthobornavirus estrildidae. Avian bornaviruses discovered in aquatic birds (Anseriformes and Charadriiformes) belong to the species Orthobornavirus avisaquaticae, which includes aquatic bird bornavirus 1 and 2 (ABBV- 1 and ABBV- 2 ) [ 5 , 6 , 1 ]. Avian bornavirus infections have been reported in more than 50 species of psittacine birds, including endangered species. Studies report the detection of PaBVs in captive psittacine populations worldwide. More frequently, infections with psittacine bornaviruses PaBV-2 and PaBV-4 are detected [ 7 – 11 ]. In contrast, the association of PaBVs in free-ranging indigenous and wild psittacine populations is rarely reported [ 12 – 13 ]. Unlike psittacine bornaviruses, CnBV-1, CnBV-2, and CnBV-3 have been described in a narrow host range exclusively within captive populations of common canaries [ 14 , 15 , 9 ]. In contrast to all other known avian bornaviruses, ABBV- 1 and ABBV- 2 have been found almost exclusively in wild bird populations [ 16 – 17 ] Bornaviruses establish a non-cytolytic infection and remain highly cell-associated throughout the infectious cycle, leading to long-lasting and persistent infections [ 18 – 20 ]. The routes and mechanisms involved in the transmission of avian bornaviruses are poorly understood. However, oral ingestion of contaminated feed or water was assumed to be the most likely transmission route of avian bornaviruses. Infectious viruses have been detected in cloacal and pharyngeal swabs of infected birds [ 21 , 15 ]. Some studies report the possibility of vertical transmission of avian bornaviruses due to the detection of viral RNA in embryonated eggs originating from infected psittacines [ 22 , 23 ], canaries [ 21 ], or Canada geese [ 24 ]. Clinical manifestations of bornavirus-induced diseases may vary both in severity and type across individual birds, with proventricular dilatation disease (PDD) being the most characteristic form. Typical PDD-like gastrointestinal tract signs are proventricular dilatation, regurgitation, passage of undigested food, and proventricular and intestinal stasis. The affected bird progressively loses body weight and becomes emaciated due to impaired digestion [ 25 – 27 ]. Additionally, the infected birds manifest neurological symptoms like incoordination, ataxia, tremors, and proprioceptive defects [ 25 , 28 ]. The course of the disease varies from sudden death before clinical signs, acute to chronic fatal condition, or even lifelong healthy carrier status. The incubation period is highly variable, ranging from three weeks to more than nine months in experimental studies [ 29 – 32 , 25 ]. Direct viral RNA detection by reverse-transcriptase polymerase chain reaction (RT-PCR) or (semi-)quantitative TaqMan-based real-time RT-PCR (RT-qPCR) assays is used for diagnosis of avian bornavirus infection [ 21 , 33 – 36 ]. The primary embryonic cultures, which include duck embryo fibroblast (DEF), chicken embryo fibroblast (CEF), and quail embryo fibroblasts (QEF), are recommended for the initial virus isolation [ 33 , 37 ]. Besides, immortalized avian cell lines, namely, CEC-32, QM-7, QT-6 (quail origin), DF-1, LMH (chicken origin), and immortalized mammalian cell lines, namely, Vero, MDCK, and C6, are known to support avian bornavirus replication [ 37 ]. However, avian bornaviruses lack distinct cytopathic effect (CPE) and therefore, rely on RT-PCR or indirect methods – immunofluorescence and western blotting to detect viral antigens [ 38 , 33 , 39 ]. In the present study, captive psittacines were tested for PaBV using molecular methods. RESULTS Molecular detection of PaBV Conventional RT-PCR RT-PCR amplification of the M gene confirmed the presence of PaBV infection in 44 out of 83 birds (53.01%). The positive birds belonged to 9 of the 13 different psittacine species included in the investigation. Eighteen of the group of PDD-suspected birds (54.55%), 21 dead birds (87.50%), and 5 clinically healthy cage mates (19.23%) were positive for PaBV-RNA (Table 1). A 350 bp fragment covering a part of the M gene of PaBV was successfully amplified (Figure 1). The positive samples from 9 psittaciform species included African grey parrot ( Psittacus erithacus ), Blue-throated conure ( Pyrrhura cruuentata ), Rosenberg’s lorikeet ( Trichoglossus rosenbergii ), Red-and- blue lory ( Eos histrio ), Blue streaked lory ( Eos reticulate ), Yellow- collared macaw ( Primolius auricollis ), Splendid grass parakeet ( Neophema splendida ), Blue- crowned hanging parrot ( Loriculus galgulus ), and Perfect lorikeet ( Trichoglosus euteles ). Among them, there were two endangered (EN), three vulnerable (V), one near threatened (NT), and only four least concerned (LC) species (Table 2) according to the Red List of IUCN (www.iucnredlist.org). Table 1: Detection of PaBV by RT-PCR Category of the birds Nature of the samples Number of samples tested Test Result Number positive (%) PDD suspected live birds Cloacal swab 33 18 (54.54) PDD suspected dead birds Brain 24 21 (87.50) Proventriculus 24 14 (58.33) Healthy cage mates of PDD-suspected birds Cloacal swab 26 5 (19.23) Total number of samples 107 58 (54.20) Phylogenetic analysis Partial genome sequences of the conventional RT-PCR products encoding protein M were obtained from representative PCR-positive birds belonging to 9 species out of 13 species included in the study. The sequences were submitted to the GenBank under the accession numbers ON960231, ON995435 to ON995442. During multiple sequence alignment (MSA), it was found that the sequences recovered under this study were 98.29 to 100% identical to each other (Figure 2). Phylogenetic analysis classified our sequences as PaBV-4, belonging to the species Orthobornavirus alphapsittaciforme (Figure 3), and revealed that the sequences were closely related to sequences of Japan, South Korea, the USA, Canada, and Israel, indicating a close genetic relationship. However, the analysis did not reveal any association of individual clusters to host species, geographical origin and time of sampling. In this study, the PaBV-4 sequences As/20/284/PaBV-4/01 (Accession no. ON995442) and As/20/284/PaBV-4/02 (Accession no. ON960231) recovered from African grey parrot ( Psittacus erithacus ) and Blue-throated conure ( Pyrrhura cruuentata ) respectively presented 100% identity with sequences from Japan ( Ara ararauna , accession no. LC486412), South Korea ( Pyrrheea molinae , accession no. MZ310194) and USA ( Ara ararauna , accession no. JN014950), 99.71% identity with sequence from Canada (Pheasant; accession no. KM508811), 99.43% identity with sequence from Japan ( Aratinga jandaya ; accession no. LC486417), 98.57% identity with sequence from Canada (African grey; accession no. GQ496351) and 98.29% identity with sequence from Israel ( Cacateca deucorpsii ; accession no. FJ002330). The PaBV-4 sequence As/20/284/PaBV-4/09 (Accession no. ON995439) from Rosenberg’s lorikeet ( Trichoglossus rosenbergii) , As/20/284/PaBV-4/10 (Accession no. ON995440) from Red-and- blue lory ( Eos histrio) , As/20/284/PaBV-4/03 (Accession no. ON995435) from Blue streaked lory ( Eos reticulate), As/20/284/PaBV-4/04 (Accession no. ON995436) from Yellow- collared macaw ( Primolius auricollis) , As/20/284/PaBV-4/08 (Accession no. ON995438) from Splendid grass parakeet ( Neophema splendid) , As/20/284/PaBV-4/07 (Accession no. ON995437) from Blue- crowned hanging parrot ( Loriculus galgulus) , and As/20/284/PaBV-4/13 (Accession no. ON005441) from Perfect lorikeet ( Trichoglossus euteles) presented 98.29 to 99.43% identity with LC486412, MZ310194 and JN014950, 98.29 to 99.14% identity with KM508811, 97.71 to 98.86% identity with LC486416, 97.43 to 98.86% identity with GQ496351 and 97.71 to 98.57% identity with FJ002330. DISCUSSION Avian bornaviruses can potentially infect a wide range of avian species, including parrots, canaries, and other birds. Several studies report the ability of avian bornaviruses to infect the vast majority of parrot species [ 40 – 42 , 8 ]. The present study is the first published report to identify and phylogenetically characterize PaBV-4 in various captive psittacine species in India. Avian bornavirus infections with PaBV-2 and PaBV-4 have been detected in captive psittacine populations worldwide [ 42 – 47 , 21 , 8 ]. However, the evidence for the presence of avian bornaviruses in free-ranging and wild psittacines is very limited. The role of parrot bornaviruses as the causative agent of PDD and other bornavirus-induced diseases in psittacines has been confirmed in previous studies. Although there is a wide variety of clinical symptoms associated with bornavirus-induced disorders in psittacines, PDD is the most distinctive form [ 47 , 48 , 26 , 49 ]. According to study reports, the course of the disease is highly variable, ranging from peracute to chronic, and in some cases the affected birds may die suddenly without showing any clinical signs. Although the majority of infected birds die as the disease progresses chronically, a considerable portion may stay clinically healthy for months or years, or even become lifelong healthy carriers [ 29 , 50 , 31 , 25 , 49 ]. Accordingly, birds included in this study were classified into 3 categories: birds with typical PDD-like signs, cage mates of PDD-like birds without any clinical signs, and dead birds. In the present investigation, 18 of the group of PDD-suspected birds (54.55%), 21 dead birds (87.50%), and 5 clinically healthy cage mates (19.23%) were positive for PaBV-RNA by RT-PCR amplification of the M gene. An experimental study by [ 51 ] demonstrated age at the time of infection as a crucial factor for the development of immunopathogenesis in PaBV-4 infected cockatiels which showed experimentally infected adults showed PDD-like signs and nestlings exhibited inflammatory lesions without clinical signs. Based on the published work on avian bornaviruses, scanty information is available on the pathogenesis of PaBV infections. The routes and mechanisms of natural avian bornavirus transmission are poorly understood. One probable reason for these may be attributed to a lack of systematic prevalence studies of avian bornavirus infections. RT-PCR and real-time PCR are the current recommended diagnostic assays for direct avian bornavirus detection [ 10 , 52 ]. Avian bornaviruses can be detected from cloacal swabs, pharyngeal swabs, whole blood samples, urine samples, and multiple tissues of infected birds. However, cloacal swabs usually contain higher amounts of viral RNA among the antemortem samples, and the brain has the highest virus load in postmortem samples when compared to other organs [ 53 , 54 , 55 , 21 , 33 , 27 ]. Several studies reported PaBV-4 as the circulating avian bornavirus genotype in Europe, North America, and Brazil [ 14 , 22 , 56 , 42 , 46 , 8 , 57 ]. However, there is, to the best of our knowledge, no publication describing the circulation of the PaBV-4 genotype in captive psittacines in India. Among the avian bornaviruses, PaBV-4 is the most widely distributed bornavirus of psittacines [ 9 ]. The PaBV-4 sequences obtained from captive psittacines under this study were 98.29 to 100% identical among themselves. When compared to PaBV-4 sequences previously deposited in GenBank, the sequences recovered in this study showed greater similarity (97.71 to 100%) to sequences from Japan, South Korea, the USA, Canada, and Israel, but lacked individual clusters to host species, geographical origin and time of sampling. This lacking association might reflect extensive illegal trading, smuggling and global distribution of various genetic variants of captive psittacines [ 9 ]. Infected captive birds can potentially introduce diseases into wild psittacine population, threatening their breeding and survival. In this study, there were two endangered (EN), three vulnerable (V), one near threatened (NT), and only four least concerned (LC) species according to the Red List of IUCN positive for PaBV-4 infection. The spread of avain bornaviruses might have devastating impacts on endangered parrot species, here, Psittacus erithacus and Eos histrio , further jeopardizing their survival. MATERIALS AND METHODS Birds sampling Samples of 83 psittacines were collected from 5 aviaries (pet owners/ breeders) from Assam, Kolkata, and Bangalore, and examined for the presence of PaBVs. The birds belonged to 13 different genera of the order Psittaciformes (Table 3 ). Considering PDD to be the most characteristic manifestation of bornavirus-induced diseases[ 25 ], the birds included in this study were classified into 3 categories - birds with typical PDD-like signs (n = 33) based on clinical examination having a history of severe weight loss, emaciation (Fig. 4 A), gastrointestinal signs, such as crop stasis, inappetence and undigested feed in feces (Fig. 4 B) and neurological signs, such as ataxia, tremor, seizures and inability to perch; cage mates of PDD-like birds without any clinical signs (n = 26); and dead birds with clinical or postmortem suspicion of PDD-like signs (n = 24). Fifty-nine cloacal swabs were obtained from PDD suspected as well as healthy cage mates’ live birds using sterile cotton swabs and subsequently preserved in tubes containing phosphate-buffered saline (PBS). Organ samples (brain, n = 24 and proventriculus, n = 24) were collected from dead birds and stored at − 80°C until diagnostic tests for the detection of viral RNA. All methods were carried out in accordance with ARRIVE guidelines. Table 3 Different species of psittacine birds for sampling Sr. No. Family Genus/ Species of birds Common name Number of birds screened 1 Psittacidae Psittacus erithacus African grey parrot 5 2 Psittacidae Pyrrhura cruuentata Blue- throated conure 4 3 Psittacidae Eos reticulata Blue streaked lory 7 4 Psittacidae Primolius auricollis Yellow- collared macaw 6 5 Psittacidae Eclectus roratus Eclectus parrot 8 6 Psittaculidae Trichogloss us moluccanus Swainson’ s lory 7 7 Psittacidae Loriculus galgulus Blue- crowned hanging parrot 7 8 Psittacidae Neophema splendida Splendid grass parakeet 6 9 Psittaculidae Trichoglossus rosenbergii Rosenberg ’s lorikeet 5 10 Psittaculidae Eos histrio Red-and- blue lory 6 11 Psittaculidae Platycercus eximius Eastern rosella 8 12 Psittacidae Eclectus roratus roratus Grand eclectus parrot 6 13 Psittaculidae Trichoglossus euteles Perfect lorikeet 8 Total 83 Molecular analyses The presence of the PaBV-RNA in cloacal swabs and/ or tissue samples was detected by RT-PCR targeting the M gene of PaBV [ 33 ]. RNA extraction and RT-PCR amplification of M-gene of avian bornavirus From collected 107 samples (cloacal swab, n = 59; brain, n = 24; proventriculus, n = 24) preserved at -80 0 C, viral RNA was extracted using the Qiagen RNeasy Extraction Kit (Hilden, Germany) according to the manufacturer’s instructions. The extracted RNA was subjected to two steps RT-PCR using Revert Aid (M-Mulv). Previously described primers, M Forward (5´-GGTAATTGTTCCTGGATGG-3´) and M Reverse (5´-ACACCAATGTTCCGAAGACG-3´) were used to amplify a 350 bp partial M gene of avian bornavirus [ 33 ]. For cDNA synthesis, the condition was 25°C for 10 mins, 37°C for 120 mins, 85°C for 5 mins, followed by hold at 4°C. The RT-PCR cycle program was initial denaturation at 94 0 C for 2mins, 38 cycles with denaturation at 94 0 C for 30 secs, annealing at 55 0 C for 30 secs, extension at 72 0 C for 30 secs, and final extension at 72 0 C for 5 mins. The amplified RT-PCR products were analyzed by 1.5% agarose gel electrophoresis containing ethidium bromide (0.5µg mL − 1 ) and visualized in an image documentation system. The PCR products of the expected length were considered positive. RT-PCR-positive products of representative samples from birds belonging to different species were sequenced for confirmation. Sequencing was done by outsourcing at the 1st BASE DNA sequencing division, Malaysia. Sequences obtained were submitted to the Genbank database. Phylogenetic analysis Phylogenetic analysis was conducted based on partial M genes from PaBV reference sequences available in GenBank. BLAST ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ) was used to compare the sequences and to find regions of similarity between sequences obtained and the GenBank to estimate the percent identity (% id). A phylogenetic tree based on the partial M gene of PaBV was constructed using the Neighbour-Joining method and the evolutionary distance model Maximum Composite Likelihood and bootstrap with 1000 replicates using MEGA X software. CONCLUSION In conclusion, our investigation revealed, this is the first published report that detects and phylogenetically characterizes PaBV-4 in captive psittacines in India. This study highlights the urgent need of future investigations of avian bornavirus infection status in the wild psittacine population as well, especially of the endangered and near threatened species. Based on the results of our study, a better understanding of the epidemiology of PaBV infections, establishment of routine diagnostics for appropriate control measures and development of a health standard for breeding psittacines in captivity is essential, as most of these species rely on captive breeding for their survival. Declarations Author Contribution All authors contributed to the conception and design. P. Deka and S. Das performed conceptualization. S. Das wrote the first draft of the manuscript, and all authors commented on previous versions of the manuscript. P. Deka, P. Kakati and B. Choudhury did sampling. R. Hazarika, S. Doloi, A, Kasheef, S. M. Gogoi, S. Das, R. K. Sharma, S. Islam, M. Nath, M. Sharma, and I. Deka checked the data and edited the draft. All authors read and approved the final manuscript. Ethical approval All authors certified that the present work was carried after approval of Institute Animal Ethic Committee (IAEC) and as per the guidelines set by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Animal Welfare Division, Government of India. The ethical approval for the present study was accorded by the IAEC vide Approval No. 770/GO/Re/S/03/CPCSEA/FVSc/AAU/IAEC/19-20/801 Dated 23-12-2019. Consent to participate All authors participated voluntarily in the research. Consent for publication All authors read and approved the final manuscript. Conflict of interest The authors declare that they have no competing interests. Availability of data and materials All data generated or analysed during this study are provided within the manuscript. The sequences were submitted to the NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/) under the accession numbers ON960231, ON995435 to ON995442. References Rubbenstroth, D.; Briese, T.; Durrwald, R.; Horie, M.; Hyndman, T.H.; Kuhn, J.H.; Nowotny, N.; Payne, S.; Stenglein, M.D.; Tomonaga, K.; et al. ICTV Virus Taxonomy Profile: Bornaviridae. J. Gen. Virol. 2021, 102, 001613. http://doi.org/10.1099/jgv.0.001613 Tizard, I.; Shivaprasad, H.L.; Guo, J.; Hameed, S.; Ball, J.; Payne, S. The pathogenesis of proventricular dilatation disease. Anim. Health Res. Rev. 2017, 17, 110–126. http://doi.org/10.1017/S1466252316000189 Briese, T.; Schneemann, A.; Lewis, A.J.; Park, Y.S.; Kim, S.; Ludwig, H.; Lipkin, W.I. Genomic organization of Borna disease virus. Proc. Natl. Acad. Sci. USA 1994, 91, 4362–4366. http://doi.org/10.1073/pnas.91.10.4362 Tomonaga, K.; Kobayashi, T.; Ikuta, K. Molecular and cellular biology of Borna disease virus infection. Microbes Infect. 2002, 4, 491–500. http://doi.org/10.1016/S1286-4579(02)01564-2 Kuhn,J.H.;Dürrwald,R.; Bào,Y.; Briese, T.; Carbone, K.; Clawson, A.N.; DeRisi, J.L.; Garten, W.; Jahrling, P.B.; Kolodziejek, J.; et al. Taxonomic reorganization of the family Bornaviridae. Arch. Virol. 2015, 160, 621–632. http://doi.org/10.1007/s00705-014-2276-z Amarasinghe, G.K.; Ayllón, M.A.; Bào, Y.; Basler, C.F.; Bavari, S.; Blasdell, K.R.; Briese, T.; Brown, P.A.; Bukreyev, A.; Balkema-Buschmann, A.; et al. Taxonomy of the order Mononegavirales: Update 2019. Arch. Virol. 2019, 164, 1967–1980. http://doi.org/10.1007/s00705-019-04247-4 Kistler, A.L.; Gancz, A.; Clubb, S.; Skewes-Cox, P.; Fischer, K.; Sorber, K.; Chiu, C.Y.; Lublin, A.; Mechani, S.; Farnoushi, Y.; et al. Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: Identification of a candidate etiologic agent. Virol. J. 2008, 5, 88. http://doi.org/10.1186/1743-422X-5-88 Philadelpho, N.A.; Rubbenstroth, D.; Guimarães, M.B.; Ferreira, A.J.P. Survey of bornaviruses in pet psittacines in Brazil reveals a novel parrot bornavirus. Vet. Microbiol. 2014, 174, 584–590. http://doi.org/10.1016/j.vetmic.2014.10.020 Rubbenstroth, D.; Schmidt, V.; Rinder, M.; Legler, M.; Twietmeyer, S.; Schwemmer, P.; Corman, V.M. Phylogenetic Analysis Supports Horizontal Transmission as a Driving Force of the Spread of Avian Bornaviruses. PLoS ONE 2016, 11, e0160936. http://doi.org/10.1371/journal.pone.0160936 Rossi, G., Dahlhausen, R. D., Galosi, L., Orosz, S. E. Avian Ganglioneuritis in Clinical Practice. Vet Clin North Am Exot Anim Pract. 2017, 21(1) , 33-67. https://doi.org/10.1016/j.cvex.2017.08.009 Boatright-Horowitz S. L. (2020). Avian Bornaviral Ganglioneuritis: Current Debates and Unanswered Questions. Vet Med Int. , 2020, 6563723. https://doi.org/10.1155/2020/6563723 Villanueva, I.; Gray, P.; Mirhosseini, N.; Payne, S.; Hoppes, S.; Honkavuori, K.S.; Briese, T.; Turner, D.; Tizard, I. The diagnosis of proventricular dilatation disease: Use of a Western blot assay to detect antibodies against avian Borna virus. Vet. Microbiol . 2009, 143, 196–201. http://doi.org/10.1016/j.vetmic.2009.11.041 Kessler, S.; Heenemann, K.; Krause, T.; Twietmeyer, S.; Fuchs, J.; Lierz, M.; Corman, V.M.; Vahlenkamp, T.M.; Rubbenstroth, D. Monitoring of free-ranging and captive Psittacula populations in Western Europe for avian bornaviruses, circoviruses and polyomaviruses . Avian Pathol. 2019, epub ahead of print. http://doi.org/10.1080/03079457.2019.1681359 Weissenböck, H., Bakonyi, T., Sekulin, K., Ehrensperger, F., Doneley, R. J., Dürrwald, R., Hoop, R., Erdélyi, K., Gál, J., Kolodziejek, J., Nowotny, N. (2009). Avian bornaviruses in psittacine birds from Europe and Australia with proventricular dilatation disease. Emerg Infect Dis. 2009, 15(9), 1453–1459. https://doi.org/10.3201/eid1509.090353 Rubbenstroth, D.; Rinder, M.; Stein, M.; Höper, D.; Kaspers, B.; Brosinski, K.; Horie, M.; Schmidt, V.; Legler, M.; Korbel, R.; et al. Avian bornaviruses are widely distributed in canary birds ( Serinus canaria f. domestica). Vet. Microbiol . 2013, 165, 287–295. http://doi.org/10.1016/j.vetmic.2013.03.024 Murray, M.; Guo, J.; Tizard, I.; Jennings, S.; Shivaprasad, H.L.; Payne, S.; Ellis, J.C.; Van Wettere, A.J.; O’Brien, K.M. Aquatic Bird Bornavirus-Associated Disease in Free-Living Canada Geese ( Branta canadensis ) in the Northeastern USA . J. Wildl. Dis . 2017, 53, 607–611. http://doi.org/10.7589/2016-07-160 Swieton, E.; Dziadek, K.; ´ Smietanka, K. Avian Bornaviruses in Wild Aquatic Birds of the Anseriformes Order in Poland. Pathogens 2022, 11, 98. http://doi.org/10.3390/pathogens11010098 Ludwig, T.H.; Becht, H.; Groh, L. Borna disease (BD), a slow virus infection biological properties of the virus. Med Microbiol Immunol. 1973;158(4):275–89. https://doi.org/10.1007/BF02121414 Duchala, C.S.; Carbone, K.M.; Narayan, O. Preliminary Studies on the Biology of Borna Disease Virus. J. Gen. Virol . 1989, 70 Pt 12, 3507–3511. http://doi.org/10.1099/0022-1317-70-12-3507 Matsumoto, Y.; Hayashi, Y.;Omori, H.; Honda, T.; Daito, T.; Horie, M.; Ikuta, K., Fujino, K., Nakamura, S., Schneider, U.,et al. Bornavirus closely associates and segregates with host chromosomes to ensure persistent Intranuclear infection. Cell Host Microbe . 2012;11(5):492–503 https://doi.org/10.1016/j.chom.2012.04.009 Rubbenstroth, D.; Rinder, M.; Kaspers, B.; Staeheli, P. Efficient isolation of avian bornaviruses (ABV) from naturally infected psittacine birds and identification of a new ABV genotype from a salmon-crested cockatoo ( Cacatua moluccensis ). Vet. Microbiol . 2012, 161, 36–42. http://doi.org/10.1016/j.vetmic.2012.07.004 Lierz, M., Hafez, H.M., Honkavuori, K.S., Gruber, A.D., Olias, P., Abdelwhab, E.M., Kohls, A., Lipkin, W.I., Briese, T., Hauck, R. Anatomical distribution of avian bornavirus in parrots, its occurrence in clinically healthy birds and ABV-antibody detection. Avian Pathol . 2009, 38 (6):491-96. https://doi.org/10.1080/03079450903349238 Kerski, A.; de Kloet, A.H.; de Kloet, S.R. Vertical transmission of avian bornavirus in Psittaciformes: Avian bornavirus RNA and anti-avian bornavirus antibodies in eggs, embryos, and hatchlings obtained from infected sun conures ( Aratinga solstitialis ). Avian Dis . 2012, 56, 471–478. http://doi.org/10.1637/9879-080511-Reg.1 Delnatte, P.; Nagy, E.; Ojkic, D.; Crawshaw, G.; Smith, D.A. Investigation into the possibility of vertical transmission of avian bornavirus in free-ranging Canada geese ( Branta canadensis ). Avian Pathol. 2014, 43, 301–304. http://doi.org/10.1080/03079457.2014.921279 Rubbenstroth, D.; Brosinski, K.; Rinder, M.; Olbert, M.; Kaspers, B.; Korbel, R.; Staeheli, P. No contact transmission of avian bornavirus in experimentally infected cockatiels ( Nymphicus hollandicus ) and domestic canaries ( Serinus canaria forma domestica). Vet. Microbiol . 2014, 172, 146–156. http://doi.org/10.1016/j.vetmic.2014.05.011 Piepenbring, A.K.; Enderlein, D.; Herzog, S.; Al-Ibadi, B.; Heffels-Redmann, U.; Heckmann, J.; Lange-Herbst, H.; Herden, C.; Lierz, M. Parrot Bornavirus (PaBV)-2 isolate causes different disease patterns in cockatiels than PaBV-4. Avian Pathol . 2016, 45, 156–168. http://doi.org/10.1080/03079457.2015.1137867 De Araújo, J.L.; Rech, R.R.; Heatley, J.J.; Guo, J.; Giaretta, P.R.; Tizard, I.; Rodrigues-Hoffmann, A. From nerves to brain to gastrointestinal tract: A time-based study of parrot bornavirus 2 (PaBV-2) pathogenesis in cockatiels (Nymphicus hollandicus). PLoS One 2017, 12, e0187797. https://doi.org/10.1371/journal.pone.0187797 Fluck, A.; Enderlein, D.; Piepenbring, A.; Heffels-Redmann, U.; Herzog, S.; Pieper, K.; Herden, C.; Lierz, M. Correlation of avian bornavirus-specific antibodies and viral ribonucleic acid shedding with neurological signs and feather-damaging behaviour in psittacine birds. Vet. Rec. 2019, 184, 476.https://doi.org/10.1136/vr.104860 DeKloet, S.R.; Dorrestein, G.M. Presence of Avian Bornavirus RNA and Anti-Avian Bornavirus Antibodies in Apparently Healthy Macaws. Avian Dis. 2009, 53, 568–573.https://doi.org/10.1637/8828-040209-Reg.1 Payne, S.; Shivaprasad, H.L.; Mirhosseini, N.; Gray, P.; Hoppes, S.; Weissenböck, H.; Tizard, I. Unusual and severe lesions of proventricular dilatation disease in cockatiels (Nymphicus hollandicus) acting as healthy carriers of avian bornavirus (ABV) and subsequently infected with a virulent strain of ABV. Avian Pathol. 2011, 40, 15–22.https://doi.org/10.1080/03079457.2010.536978 Piepenbring et al., 2012; Piepenbring, A.K.; Enderlein, D.; Herzog, S.; Kaleta, E.F.; Heffels-Redmann, U.; Ressmeyer, S.; Herden, C.; Lierz, M. Pathogenesis of Avian Bornavirus in Experimentally Infected Cockatiels. Emerg. Infect. Dis. 2012, 18, 234–241.https://doi.org/10.3201/eid1802.111525 Heffels-Redmann, U.; Enderlein, D.; Herzog, S.; Piepenbring, A.; Bürkle, M.; Neumann, D.; Herden, C.; Lierz, M. Follow-Up Investigations on Different Courses of Natural Avian Bornavirus Infections in Psittacines. Avian Dis. 2012, 56, 153–159.https://doi.org/10.1637/9844-062811-Reg.1 Guo, J.; Payne, S.; Zhang, S.; Turner, D.; Tizard, I.; Suchodolski, P. Avian Bornaviruses: Diagnosis, Isolation, and Genotyping. Curr. Protoc. Microbiol. 2014, 34, 15I.1.1–15I.1.33.https://doi.org/10.1002/9780471729259.mc15i01s34 Delnatte, P.; Mak, M.; Ojkic, D.; Raghav, R.; DeLay, J.; Smith, D.A. Detection of Avian bornavirus in multiple tissues of infected psittacine birds using real-time reverse transcription polymerase chain reaction. J. Vet. Diagn. Investig. 2014, 26, 266–271. https://doi.org/10.1177/1040638713519641 Olbert, M.; Römer-Oberdörfer, A.; Herden, C.; Malberg, S.; Runge, S.; Staeheli, P.; Rubbenstroth, D. Viral vector vaccines expressing nucleoprotein and phosphoprotein genes of avian bornaviruses ameliorate homologous challenge infections in cockatiels and common canaries. Sci. Rep. 2016, 6, 36840.https://doi.org/10.1038/srep36840 Sigrist, B.; Geers, J.; Albini, S.; Rubbenstroth, D.; Wolfrum, N. A New Multiplex Real-Time RT-PCR for Simultaneous Detection and Differentiation of Avian Bornaviruses. Viruses 2021, 13, 1358.https://doi.org/10.3390/v13071358 Pham, P.H.; Leacy, A.; Deng, L.; Nagy, E.; Susta, L. Isolation of Ontario aquatic bird bornavirus 1 and characterization of its replication in immortalized avian cell lines. Virol. J. 2020, 17, 16.https://doi.org/10.1186/s12985-020-1286-6 Herzog, S.; Enderlein, D.; Heffels-Redmann, U.; Piepenbring, A.; Neumann, D.; Kaleta, E.F.; Müller, H.; Lierz, M.; Herden, C. Indirect Immunofluorescence Assay for Intra Vitam Diagnosis of Avian Bornavirus Infection in Psittacine Birds. J. Clin. Microbiol. 2010, 48, 2282–2284. https://doi.org/10.1128/JCM.00145-10 Maclachlan. N.; Dubovi, E.J. Bornaviridae. In: Fenner’s Veterinary Virology. London: Academic Press; 2017. p. 381–7. Honkavuori, K.S.; Shivaprasad, H.L.; Williams, B.L.; Quan, P.-L.; Hornig, M.; Street, C.; Palacios, G.; Hutchison, S.K.; Franca, M.;Egholm, M.; et al. Novel Borna Virus in Psittacine Birds with Proventricular Dilatation Disease. Emerg. Infect. Dis. 2008, 14, 1883–1886.https://doi.org/10.3201/eid1412.080984 Horie, M.; Ueda, K.; Ueda, A.; Honda, T.; Tomonaga, K. Detection of Avian bornavirus 5 RNA in Eclectus roratus with feather picking disorder. Microbiol. Immunol. 2012, 56, 346–349.https://doi.org/10.1111/j.1348-0421.2012.00436.x Last, R.D.; Weissenböck, H.; Nedorost, N.; Shivaprasad, H. Avian bornavirus genotype 4 recovered from naturally infected psittacine birds with proventricular dilatation disease in South Africa. J. South Afr. Vet. Assoc. 2012, 83, 4.https://doi.org/10.4102/jsava.v83i1.938 Kistler, A.L.; Gancz, A.; Clubb, S.; Skewes-Cox, P.; Fischer, K.; Sorber, K.; Chiu, C.Y.; Lublin, A.; Mechani, S.; Farnoushi, Y.; et al. Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: Identification of a candidate etiologic agent. Virol. J. 2008, 5, 88. https://doi.org/10.1186/1743-422X-5-88 Gray, P.; Hoppes, S.; Suchodolski, P.; Mirhosseini, N.; Payne, S.; Villanueva, I.; Shivaprasad, H.; Honkavuori, K.S.; Briese, T.; Reddy, S.M.; et al. Use of Avian Bornavirus Isolates to Induce Proventricular Dilatation Disease in Conures. Emerg. Infect. Dis . 2010, 16, 473–479.https://doi.org/10.3201/eid1603.091257 Heffels-Redmann, U.; Enderlein, D.; Herzog, S.; Herden, C.; Piepenbring, A.; Neumann, D.; Muller, H.; Capelli, S.; Müller, H.; Oberhäuser, K.; et al. Occurrence of avian bornavirus infection in captive psittacines in various European countries and its association with proventricular dilatation disease. Avian Pathol. 2011, 40, 419–426.https://doi.org/10.1080/03079457.2011.589825 Donatti, R.V.; Resende, M.; Junior, F.F.; Marques, M.V.R.; Ecco, R.; Shivaprasad, H.L.; De Resende, J.S.; Martins, N.R.D.S. Fatal Proventricular Dilatation Disease in Captive Native Psittacines in Brazil. Avian Dis. 2014, 58, 187–193.https://doi.org/10.1637/10588-061013-Case.1 Mirhosseini, N.; Gray, P.L.; Hoppes, S.; Tizard, I.; Shivaprasad, H.L.; Payne, S. Proventricular Dilatation Disease in Cockatiels (Nymphicus hollandicus) After Infection with a Genotype 2 Avian Bornavirus. J. Avian Med. Surg. 2011, 25, 199–204.https://doi.org/10.1647/2010-030.1 Payne, S.; Shivaprasad, H.L.; Mirhosseini, N.; Gray, P.; Hoppes, S.; Weissenböck, H.; Tizard, I. Unusual and severe lesions of proventricular dilatation disease in cockatiels (Nymphicus hollandicus) acting as healthy carriers of avian bornavirus (ABV) and subsequently infected with a virulent strain of ABV. Avian Pathol. 2011, 40, 15–22.https://doi.org/10.1080/03079457.2010.536978 Hameed, S.S.; Guo, J.; Tizard, I.; Shivaprasad, H.L.; Payne, S. Studies on immunity and immunopathogenesis of parrot bornaviral disease in cockatiels. Virology 2018, 515, 81–91.https://doi.org/10.1016/j.virol.2017.12.007 Staeheli, P.; Rinder, M.; Kaspers, B. Avian Bornavirus Associated with Fatal Disease in Psittacine Birds. J. Virol . 2010, 84, 6269–6275. https://doi.org/10.1128/JVI.02567-09 Gartner, A.M.; Link, J.; Bücking, B.; Enderlein, D.; Herzog, S.; Petzold, J.; Malberg, S.; Herden, C.; Lierz, M. Age-dependent development and clinical characteristics of an experimental parrot bornavirus-4 (PaBV-4) infection in cockatiels (Nymphicus hollandicus). Avian Pathol . 2021, 50, 138–150.https://doi.org/10.1080/03079457.2020.1852177 Rubbenstroth, D. Avian Bornavirus Research—A Comprehensive Review. Viruses 2022, 14, 1513. https://doi.org/10.3390/v14071513 Raghav, R.; Taylor, M.; DeLay, J.; Ojkic, D.; Pearl, D.L.; Kistler, A.L.; DeRisi, J.L.; Ganem, D.; Smith, D.A.Avian Bornavirus isPresent in Many Tissues of Psittacine Birds with Histopathologic Evidence of Proventricular Dilatation Disease. J. Vet. Diagn. Investig. 2010, 22, 495–508.https://doi.org/10.1177/104063871002200402 Heatley, J. J & Villalobos, A.R. Avian bornavirus in the urine of infected birds. Vet Med (Auckl) . 2012, 3, 19-23. https://doi.org/10.2147/VMRR.S31336 Delnatte, P.; Nagy, E.; Ojkic, D.; Leishman, D.; Crawshaw, G.; Elias, K.; Smith, D.A. Avian bornavirus in free-ranging waterfowl: Prevalence of antibodies and cloacal shedding of viral RNA J Wildl Dis . 2014, 50 (3), 512–523. https://doi.org/10.7589/2013-08-218 Rinder, M.; Ackermann, A.; Kempf, H.; Kaspers, B.; Korbel, R.; Staeheli, P. Broad Tissue and Cell Tropism of Avian Bornavirus in Parrots with Proventricular Dilatation Disease . J. Virol . 2009, 83, 5401–5407. https://doi.org/10.1128/JVI.00133-09 Silva, A. S. G., Raso, T. F., Costa, E. A., Gómez, S. Y. M., Martins, N. R. D. S. (2020) Parrot bornavirus in naturally infected Brazilian captive parrots: Challenges in viral spread control. PLoS ONE . 15 (6), e0232342. https://doi.org/10.1371/journal.pone.0232342 Table Table 2 is available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files Table2.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 14 Oct, 2025 Reviews received at journal 07 Oct, 2025 Reviews received at journal 04 Oct, 2025 Reviewers agreed at journal 28 Sep, 2025 Reviewers agreed at journal 28 Sep, 2025 Reviewers invited by journal 27 Sep, 2025 Editor assigned by journal 19 Sep, 2025 Editor invited by journal 01 Jul, 2025 Submission checks completed at journal 01 Jul, 2025 First submitted to journal 24 Jun, 2025 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6968515","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":526163809,"identity":"a27eec6f-9d9a-4f98-9443-3c1fd0080078","order_by":0,"name":"Pankaj Deka","email":"","orcid":"","institution":"Assam Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Pankaj","middleName":"","lastName":"Deka","suffix":""},{"id":526163811,"identity":"afba1da1-846c-4212-a096-89f65f7f4987","order_by":1,"name":"Sangeeta Das","email":"data:image/png;base64,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","orcid":"","institution":"Assam Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Sangeeta","middleName":"","lastName":"Das","suffix":""},{"id":526163813,"identity":"e8597c59-9eaa-4a34-983d-5f4c368a77b2","order_by":2,"name":"Ritam Hazarika","email":"","orcid":"","institution":"Assam Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ritam","middleName":"","lastName":"Hazarika","suffix":""},{"id":526163817,"identity":"fc7401b0-17f2-49e5-8053-c8557cd55a98","order_by":3,"name":"Parikshit Kakati","email":"","orcid":"","institution":"Greens Zoological Rescue and Rehabilitation Centre, Jamnagar, Gujarat-361142","correspondingAuthor":false,"prefix":"","firstName":"Parikshit","middleName":"","lastName":"Kakati","suffix":""},{"id":526163823,"identity":"8359b04c-e602-42aa-b992-6cdc724c96f2","order_by":4,"name":"Bhaskar Choudhury","email":"","orcid":"","institution":"Wildlife Trust of India","correspondingAuthor":false,"prefix":"","firstName":"Bhaskar","middleName":"","lastName":"Choudhury","suffix":""},{"id":526163825,"identity":"1c4b1fe7-1413-435c-af10-95acb1403b67","order_by":5,"name":"Suchanda Doloi","email":"","orcid":"","institution":"Assam Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Suchanda","middleName":"","lastName":"Doloi","suffix":""},{"id":526163826,"identity":"e126e722-f9a1-4b8f-b1da-3cf263d8b28a","order_by":6,"name":"Abdul Kasheef","email":"","orcid":"","institution":"Assam Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Abdul","middleName":"","lastName":"Kasheef","suffix":""},{"id":526163828,"identity":"e5011916-2e6f-49b9-a896-9daac2a5f1e4","order_by":7,"name":"Sophia M Gogoi","email":"","orcid":"","institution":"Assam Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Sophia","middleName":"M","lastName":"Gogoi","suffix":""},{"id":526163829,"identity":"83c51a3b-d770-414d-9133-315734841450","order_by":8,"name":"Sutopa Das","email":"","orcid":"","institution":"Assam Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Sutopa","middleName":"","lastName":"Das","suffix":""},{"id":526163830,"identity":"3e6be35a-0027-473b-9a48-0e1d75b92dd1","order_by":9,"name":"Rajeev Kumar Sharma","email":"","orcid":"","institution":"Assam Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Rajeev","middleName":"Kumar","lastName":"Sharma","suffix":""},{"id":526163831,"identity":"81a8ade6-dccd-4fd9-a480-09feea36e261","order_by":10,"name":"Saidul Islam","email":"","orcid":"","institution":"Assam Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Saidul","middleName":"","lastName":"Islam","suffix":""},{"id":526163832,"identity":"fe432768-f165-486a-a6d5-9b23be1b8cee","order_by":11,"name":"Mrinal Nath","email":"","orcid":"","institution":"Assam Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Mrinal","middleName":"","lastName":"Nath","suffix":""},{"id":526163833,"identity":"717ee621-2939-43ed-acc5-61824e3d79c2","order_by":12,"name":"Mihir Sarma","email":"","orcid":"","institution":"Assam Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Mihir","middleName":"","lastName":"Sarma","suffix":""},{"id":526163834,"identity":"b567f170-dc47-40c5-9a24-96a495d959af","order_by":13,"name":"Ilakshy Deka","email":"","orcid":"","institution":"Krishi Vigyan Kendra, Assam Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ilakshy","middleName":"","lastName":"Deka","suffix":""}],"badges":[],"createdAt":"2025-06-24 19:23:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6968515/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6968515/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":93121928,"identity":"6e86f519-d5a5-435d-8ddc-79d19b5dfefd","added_by":"auto","created_at":"2025-10-09 09:44:22","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1424357,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscriptjj30.06.2025Revision3.docx","url":"https://assets-eu.researchsquare.com/files/rs-6968515/v1/a2dd6d27364649048998c10e.docx"},{"id":93121923,"identity":"f9a62a2e-c857-43c0-95cc-08be7a5787e6","added_by":"auto","created_at":"2025-10-09 09:44:22","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":14070,"visible":true,"origin":"","legend":"","description":"","filename":"c7df5c2f90374853b40a90e0b0b9129d.json","url":"https://assets-eu.researchsquare.com/files/rs-6968515/v1/9a03f30bfe7ff498115b96ad.json"},{"id":93122256,"identity":"987e24d3-59d0-4b28-8ae3-1a5460063b7b","added_by":"auto","created_at":"2025-10-09 09:52:22","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":21545,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1Gel.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6968515/v1/67eeafeab85df85c71b8889f.pdf"},{"id":93123698,"identity":"2ebf5a74-9cd9-44ec-898e-e520a5ca2e85","added_by":"auto","created_at":"2025-10-09 10:08:26","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":144532,"visible":true,"origin":"","legend":"","description":"","filename":"c7df5c2f90374853b40a90e0b0b9129d1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-6968515/v1/893a71353648901700beea4d.xml"},{"id":93121927,"identity":"43e8a856-09c6-4589-86db-ca2682f98af2","added_by":"auto","created_at":"2025-10-09 09:44:22","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":97568,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6968515/v1/ff5dfec42aab9cc89ecbf103.png"},{"id":93121929,"identity":"a29a1fc9-9b07-4989-b1b3-d561371a0cce","added_by":"auto","created_at":"2025-10-09 09:44:22","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":471480,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6968515/v1/7db06bc6064ab704bf226ed8.png"},{"id":93121932,"identity":"4a5fc64c-cba5-4d4f-af7f-fa173501d439","added_by":"auto","created_at":"2025-10-09 09:44:22","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":28823,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6968515/v1/5d519178a7a723a94e3622a1.png"},{"id":93122259,"identity":"89bf8022-3d74-4712-b41b-e3bf274a9311","added_by":"auto","created_at":"2025-10-09 09:52:22","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":663718,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6968515/v1/737b5cab258fb9e55b8317cc.jpeg"},{"id":93123041,"identity":"8331db02-bb10-4e02-afb5-5e30d7246040","added_by":"auto","created_at":"2025-10-09 10:00:22","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":13341,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6968515/v1/c2f02de26ffafab6e435f101.png"},{"id":93123696,"identity":"093c0c3b-f0bb-4f22-bc28-9d63c6c484eb","added_by":"auto","created_at":"2025-10-09 10:08:26","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":101797,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6968515/v1/7d80bd70c3b59c7b1fa7618e.png"},{"id":93122257,"identity":"38f323b0-ae3e-469f-8288-9b17440177d8","added_by":"auto","created_at":"2025-10-09 09:52:22","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":26994,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6968515/v1/0910d06f27ca9759f29d24f3.png"},{"id":93123042,"identity":"0d1de320-2b1f-4056-b5cf-8a6e839afbbe","added_by":"auto","created_at":"2025-10-09 10:00:22","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":245585,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6968515/v1/1d9eb4e131b673d7452f808e.png"},{"id":93121938,"identity":"ed28441c-a5a4-43bc-ab08-a04c49776e44","added_by":"auto","created_at":"2025-10-09 09:44:22","extension":"xml","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":143577,"visible":true,"origin":"","legend":"","description":"","filename":"c7df5c2f90374853b40a90e0b0b9129d1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-6968515/v1/bac235d657f9c9d5cdeb955d.xml"},{"id":93121939,"identity":"60d90981-a930-46b1-953b-9a8fcb5d25ab","added_by":"auto","created_at":"2025-10-09 09:44:22","extension":"html","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":159334,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-6968515/v1/bafbef66ad28a834c2e91e64.html"},{"id":93122255,"identity":"6c09ace4-3ed3-4727-9acb-e02ce0b8ac7c","added_by":"auto","created_at":"2025-10-09 09:52:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":98721,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of avian PaBV through RT-PCR with \u003cem\u003eM\u003c/em\u003e gene-specific primers. Marker (M): DNA ladder; Lane 2-10 show 350 bp \u003cem\u003eM \u003c/em\u003egene amplification product of test samples (cloacal swabs, brain, and proventriculus) from PaBV infected birds, and Lane 1, negative control\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6968515/v1/fbc8c3e821349504acb20638.png"},{"id":93121924,"identity":"21b3b7f4-1c7b-4ab1-9819-c67360d6810a","added_by":"auto","created_at":"2025-10-09 09:44:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":455320,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple sequence alignment (MSA) of partial \u003cem\u003eM\u003c/em\u003egene (350bp) of PaBV-4 strains from captive psittacines in India recovered under this study with reference to PaBV-4 strain from Blue-and-yellow macaw (\u003cem\u003eAra ararauna\u003c/em\u003e) with Accession number LC486412\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6968515/v1/09fab3d4936b0593ace52fff.png"},{"id":93121925,"identity":"4d67b199-1a6f-41fc-93c3-1753a822eced","added_by":"auto","created_at":"2025-10-09 09:44:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":143110,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree constructed based on the partial \u003cem\u003eM \u003c/em\u003egene sequence of the PaBVs from captive psittacines in India. The tree was constructed using the Neighbour-Joining algorithm of MEGA X (1000 bootstrap repetition) and evolutionary distances were computed using the Maximum Composite Likelihood method. Sequences are identified by GenBank accession numbers and the abbreviated name of the virus. Sequences emphasized in red were generated during this study.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6968515/v1/8a24bd732ac473e91cf2951d.png"},{"id":93121933,"identity":"4e7a2ce0-6e8a-4e7f-a4c9-24dc9759a81e","added_by":"auto","created_at":"2025-10-09 09:44:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":347124,"visible":true,"origin":"","legend":"\u003cp\u003eClinical signs of avian bornavirus-induced diseases in infected birds (A) Emaciation and prominent keel bone (B) Shedding of undigested feeds in the feces\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6968515/v1/bb72d5e46ebcbb67d667ea13.png"},{"id":93123954,"identity":"def78321-54a4-4ee7-afc0-ec116c3df307","added_by":"auto","created_at":"2025-10-09 10:08:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1696266,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6968515/v1/af39e8e4-3aec-4295-ab72-f42edadb7123.pdf"},{"id":93121921,"identity":"60869d33-905c-4990-8d12-b2c9e7f9e91b","added_by":"auto","created_at":"2025-10-09 09:44:22","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18062,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6968515/v1/b1ac63be610d766c0a008bb7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"First Report: Molecular detection and characterization of Parrot Bornavirus 4 (PaBV-4) in captive psittacines in India","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eBornaviruses (Order: \u003cem\u003eMononegavirales\u003c/em\u003e, Family: \u003cem\u003eBornaviridae\u003c/em\u003e) have long been recognized in humans, mammals, birds, and reptiles. Currently, the family includes three genera: Orthobornavirus, Carbovirus, and Cultevirus [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Bornaviruses are enveloped, 80 to 100 nm in diameter, with a non-segmented, linear, single-stranded negative-sense RNA genome having around 9000 nucleotides (nt) in length [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Orthobornavirus genome encodes six proteins, namely nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), the large RNA-dependent RNA polymerase (L), and an accessory protein p10 (X) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. All avian bornaviruses belong to the genus Orthobornavirus and are currently classified as 15 separate viruses within five different viral species. Avian bornaviruses discovered in parrots (Psittaciformes) include parrot bornavirus 1 to 8 (PaBV-1 to PaBV-8), belonging to species Orthobornavirus alphapsittaciforme (PaBV-1 to 4, PaBV-7 and \u0026minus;\u0026thinsp;8) and Orthobornavirus betapsittaciforme (PaBV-5 and \u0026minus;\u0026thinsp;6). Additionally, avian bornaviruses discovered in passerine birds (Passeriformes) include canary bornavirus 1 to 3 (CnBV-1 to CnBV-3) and munia bornavirus 1 (MuBV 1), belonging to the species Orthobornavirus serini and estrildid finch bornavirus 1 (EsBV-1), belonging to Orthobornavirus estrildidae. Avian bornaviruses discovered in aquatic birds (Anseriformes and Charadriiformes) belong to the species Orthobornavirus avisaquaticae, which includes aquatic bird bornavirus 1 and 2 (ABBV-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and ABBV-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAvian bornavirus infections have been reported in more than 50 species of psittacine birds, including endangered species. Studies report the detection of PaBVs in captive psittacine populations worldwide. More frequently, infections with psittacine bornaviruses PaBV-2 and PaBV-4 are detected [\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In contrast, the association of PaBVs in free-ranging indigenous and wild psittacine populations is rarely reported [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Unlike psittacine bornaviruses, CnBV-1, CnBV-2, and CnBV-3 have been described in a narrow host range exclusively within captive populations of common canaries [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In contrast to all other known avian bornaviruses, ABBV-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and ABBV-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e have been found almost exclusively in wild bird populations [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eBornaviruses establish a non-cytolytic infection and remain highly cell-associated throughout the infectious cycle, leading to long-lasting and persistent infections [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The routes and mechanisms involved in the transmission of avian bornaviruses are poorly understood. However, oral ingestion of contaminated feed or water was assumed to be the most likely transmission route of avian bornaviruses. Infectious viruses have been detected in cloacal and pharyngeal swabs of infected birds [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Some studies report the possibility of vertical transmission of avian bornaviruses due to the detection of viral RNA in embryonated eggs originating from infected psittacines [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], canaries [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], or Canada geese [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eClinical manifestations of bornavirus-induced diseases may vary both in severity and type across individual birds, with proventricular dilatation disease (PDD) being the most characteristic form. Typical PDD-like gastrointestinal tract signs are proventricular dilatation, regurgitation, passage of undigested food, and proventricular and intestinal stasis. The affected bird progressively loses body weight and becomes emaciated due to impaired digestion [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Additionally, the infected birds manifest neurological symptoms like incoordination, ataxia, tremors, and proprioceptive defects [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The course of the disease varies from sudden death before clinical signs, acute to chronic fatal condition, or even lifelong healthy carrier status. The incubation period is highly variable, ranging from three weeks to more than nine months in experimental studies [\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDirect viral RNA detection by reverse-transcriptase polymerase chain reaction (RT-PCR) or (semi-)quantitative TaqMan-based real-time RT-PCR (RT-qPCR) assays is used for diagnosis of avian bornavirus infection [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The primary embryonic cultures, which include duck embryo fibroblast (DEF), chicken embryo fibroblast (CEF), and quail embryo fibroblasts (QEF), are recommended for the initial virus isolation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Besides, immortalized avian cell lines, namely, CEC-32, QM-7, QT-6 (quail origin), DF-1, LMH (chicken origin), and immortalized mammalian cell lines, namely, Vero, MDCK, and C6, are known to support avian bornavirus replication [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, avian bornaviruses lack distinct cytopathic effect (CPE) and therefore, rely on RT-PCR or indirect methods \u0026ndash; immunofluorescence and western blotting to detect viral antigens [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn the present study, captive psittacines were tested for PaBV using molecular methods.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eMolecular detection of PaBV\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConventional RT-PCR\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRT-PCR amplification of the \u003cem\u003eM\u003c/em\u003e gene confirmed the presence of PaBV infection in 44 out of 83 birds (53.01%). The positive birds belonged to 9 of the 13 different psittacine species included in the investigation. \u0026nbsp;Eighteen of the group of PDD-suspected birds (54.55%), 21 dead birds (87.50%), and 5 clinically healthy cage mates (19.23%) were positive for PaBV-RNA (Table 1). A 350 bp fragment covering a part of the \u003cem\u003eM\u003c/em\u003e gene of PaBV was successfully amplified (Figure 1). The positive samples from 9 psittaciform species included African grey parrot (\u003cem\u003ePsittacus erithacus\u003c/em\u003e), Blue-throated conure (\u003cem\u003ePyrrhura cruuentata\u003c/em\u003e), Rosenberg\u0026rsquo;s lorikeet (\u003cem\u003eTrichoglossus rosenbergii\u003c/em\u003e), Red-and- blue lory (\u003cem\u003eEos histrio\u003c/em\u003e), Blue streaked lory (\u003cem\u003eEos reticulate\u003c/em\u003e), Yellow- collared macaw (\u003cem\u003ePrimolius auricollis\u003c/em\u003e), Splendid grass parakeet (\u003cem\u003eNeophema splendida\u003c/em\u003e), Blue- crowned hanging parrot (\u003cem\u003eLoriculus galgulus\u003c/em\u003e), and Perfect lorikeet (\u003cem\u003eTrichoglosus euteles\u003c/em\u003e). Among them, there were two endangered (EN), three vulnerable (V), one near threatened (NT), and only four least concerned (LC) species (Table 2) according to the Red List of IUCN (www.iucnredlist.org).\u003c/p\u003e\n\u003cp\u003eTable 1: Detection of PaBV by RT-PCR\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 27px;\"\u003e\n \u003cp\u003eCategory of the birds\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003eNature of the samples\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003eNumber of samples tested\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003eTest Result\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003eNumber positive (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 27px;\"\u003e\n \u003cp\u003ePDD suspected live birds\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003eCloacal swab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e18 (54.54)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 27px;\"\u003e\n \u003cp\u003ePDD suspected dead birds\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003eBrain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e21 (87.50)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003eProventriculus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e14 (58.33)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 27px;\"\u003e\n \u003cp\u003eHealthy cage mates of PDD-suspected birds\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003eCloacal swab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e5 (19.23)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003eTotal number of samples\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e107\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e58 (54.20)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePhylogenetic analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Partial genome sequences of the conventional RT-PCR products encoding protein M were obtained from representative PCR-positive birds belonging to 9 species out of 13 species included in the study. The sequences were submitted to the GenBank under the accession numbers ON960231, ON995435 to ON995442. During multiple sequence alignment (MSA), it was found that the sequences recovered under this study were 98.29 to 100% identical to each other (Figure 2). Phylogenetic analysis classified our sequences as PaBV-4, belonging to the species Orthobornavirus alphapsittaciforme (Figure 3), and revealed that the sequences were closely related to sequences of\u003cem\u003e\u0026nbsp;\u003c/em\u003eJapan, South Korea, the USA, Canada, and Israel, indicating a close genetic relationship. However, the analysis did not reveal any association of individual clusters to host species, geographical origin and time of sampling. In this study, the PaBV-4 sequences As/20/284/PaBV-4/01 (Accession no. ON995442) \u0026nbsp;and As/20/284/PaBV-4/02 (Accession no. ON960231) recovered from African grey parrot (\u003cem\u003ePsittacus erithacus\u003c/em\u003e) and Blue-throated conure (\u003cem\u003ePyrrhura cruuentata\u003c/em\u003e) respectively presented 100% identity with sequences from Japan (\u003cem\u003eAra ararauna\u003c/em\u003e, accession no. LC486412), South Korea (\u003cem\u003ePyrrheea molinae\u003c/em\u003e, accession no. MZ310194) and USA (\u003cem\u003eAra ararauna\u003c/em\u003e, accession no. JN014950), 99.71% identity with sequence from Canada (Pheasant; accession no. KM508811), 99.43% identity with sequence from Japan (\u003cem\u003eAratinga jandaya\u003c/em\u003e; accession no. LC486417), 98.57% identity with sequence from Canada (African grey; accession no. GQ496351) and 98.29% identity with sequence from Israel (\u003cem\u003eCacateca deucorpsii\u003c/em\u003e; accession no. FJ002330). The PaBV-4 sequence As/20/284/PaBV-4/09 (Accession no. ON995439) from Rosenberg\u0026rsquo;s lorikeet (\u003cem\u003eTrichoglossus rosenbergii)\u003c/em\u003e, As/20/284/PaBV-4/10 (Accession no. ON995440) from Red-and- blue lory (\u003cem\u003eEos histrio)\u003c/em\u003e, As/20/284/PaBV-4/03 (Accession no. ON995435) from Blue streaked lory (\u003cem\u003eEos reticulate),\u0026nbsp;\u003c/em\u003eAs/20/284/PaBV-4/04 (Accession no. ON995436) from Yellow- collared macaw (\u003cem\u003ePrimolius auricollis)\u003c/em\u003e, As/20/284/PaBV-4/08 (Accession no. ON995438) from Splendid grass parakeet (\u003cem\u003eNeophema splendid)\u003c/em\u003e, As/20/284/PaBV-4/07 (Accession no. ON995437) from Blue- crowned hanging parrot (\u003cem\u003eLoriculus galgulus)\u003c/em\u003e, and As/20/284/PaBV-4/13 (Accession no. ON005441) from Perfect lorikeet (\u003cem\u003eTrichoglossus euteles)\u003c/em\u003e presented 98.29 to 99.43% identity with LC486412, MZ310194 and JN014950, 98.29 to 99.14% identity with KM508811, 97.71 to 98.86% identity with LC486416, 97.43 to 98.86% identity with GQ496351 and 97.71 to 98.57% identity with FJ002330.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAvian bornaviruses can potentially infect a wide range of avian species, including parrots, canaries, and other birds. Several studies report the ability of avian bornaviruses to infect the vast majority of parrot species [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The present study is the first published report to identify and phylogenetically characterize PaBV-4 in various captive psittacine species in India. Avian bornavirus infections with PaBV-2 and PaBV-4 have been detected in captive psittacine populations worldwide [\u003cspan additionalcitationids=\"CR43 CR44 CR45 CR46\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, the evidence for the presence of avian bornaviruses in free-ranging and wild psittacines is very limited.\u003c/p\u003e\u003cp\u003eThe role of parrot bornaviruses as the causative agent of PDD and other bornavirus-induced diseases in psittacines has been confirmed in previous studies. Although there is a wide variety of clinical symptoms associated with bornavirus-induced disorders in psittacines, PDD is the most distinctive form [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. According to study reports, the course of the disease is highly variable, ranging from peracute to chronic, and in some cases the affected birds may die suddenly without showing any clinical signs. Although the majority of infected birds die as the disease progresses chronically, a considerable portion may stay clinically healthy for months or years, or even become lifelong healthy carriers [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Accordingly, birds included in this study were classified into 3 categories: birds with typical PDD-like signs, cage mates of PDD-like birds without any clinical signs, and dead birds. In the present investigation, 18 of the group of PDD-suspected birds (54.55%), 21 dead birds (87.50%), and 5 clinically healthy cage mates (19.23%) were positive for PaBV-RNA by RT-PCR amplification of the \u003cem\u003eM\u003c/em\u003e gene. An experimental study by [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] demonstrated age at the time of infection as a crucial factor for the development of immunopathogenesis in PaBV-4 infected cockatiels which showed experimentally infected adults showed PDD-like signs and nestlings exhibited inflammatory lesions without clinical signs. Based on the published work on avian bornaviruses, scanty information is available on the pathogenesis of PaBV infections. The routes and mechanisms of natural avian bornavirus transmission are poorly understood. One probable reason for these may be attributed to a lack of systematic prevalence studies of avian bornavirus infections.\u003c/p\u003e\u003cp\u003eRT-PCR and real-time PCR are the current recommended diagnostic assays for direct avian bornavirus detection [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Avian bornaviruses can be detected from cloacal swabs, pharyngeal swabs, whole blood samples, urine samples, and multiple tissues of infected birds. However, cloacal swabs usually contain higher amounts of viral RNA among the antemortem samples, and the brain has the highest virus load in postmortem samples when compared to other organs [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSeveral studies reported PaBV-4 as the circulating avian bornavirus genotype in Europe, North America, and Brazil [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. However, there is, to the best of our knowledge, no publication describing the circulation of the PaBV-4 genotype in captive psittacines in India. Among the avian bornaviruses, PaBV-4 is the most widely distributed bornavirus of psittacines [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The PaBV-4 sequences obtained from captive psittacines under this study were 98.29 to 100% identical among themselves. When compared to PaBV-4 sequences previously deposited in GenBank, the sequences recovered in this study showed greater similarity (97.71 to 100%) to sequences from Japan, South Korea, the USA, Canada, and Israel, but lacked individual clusters to host species, geographical origin and time of sampling. This lacking association might reflect extensive illegal trading, smuggling and global distribution of various genetic variants of captive psittacines [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eInfected captive birds can potentially introduce diseases into wild psittacine population, threatening their breeding and survival. In this study, there were two endangered (EN), three vulnerable (V), one near threatened (NT), and only four least concerned (LC) species according to the Red List of IUCN positive for PaBV-4 infection. The spread of avain bornaviruses might have devastating impacts on endangered parrot species, here, \u003cem\u003ePsittacus erithacus\u003c/em\u003e and \u003cem\u003eEos histrio\u003c/em\u003e, further jeopardizing their survival.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cb\u003eBirds sampling\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSamples of 83 psittacines were collected from 5 aviaries (pet owners/ breeders) from Assam, Kolkata, and Bangalore, and examined for the presence of PaBVs. The birds belonged to 13 different genera of the order \u003cem\u003ePsittaciformes\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Considering PDD to be the most characteristic manifestation of bornavirus-induced diseases[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], the birds included in this study were classified into 3 categories - birds with typical PDD-like signs (n\u0026thinsp;=\u0026thinsp;33) based on clinical examination having a history of severe weight loss, emaciation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), gastrointestinal signs, such as crop stasis, inappetence and undigested feed in feces (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) and neurological signs, such as ataxia, tremor, seizures and inability to perch; cage mates of PDD-like birds without any clinical signs (n\u0026thinsp;=\u0026thinsp;26); and dead birds with clinical or postmortem suspicion of PDD-like signs (n\u0026thinsp;=\u0026thinsp;24). Fifty-nine cloacal swabs were obtained from PDD suspected as well as healthy cage mates\u0026rsquo; live birds using sterile cotton swabs and subsequently preserved in tubes containing phosphate-buffered saline (PBS). Organ samples (brain, n\u0026thinsp;=\u0026thinsp;24 and proventriculus, n\u0026thinsp;=\u0026thinsp;24) were collected from dead birds and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until diagnostic tests for the detection of viral RNA. All methods were carried out in accordance with ARRIVE guidelines.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDifferent species of psittacine birds for sampling\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\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=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSr. No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFamily\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGenus/ Species of birds\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCommon name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNumber of birds screened\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePsittacidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePsittacus erithacus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAfrican grey parrot\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePsittacidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePyrrhura cruuentata\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBlue- throated conure\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePsittacidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eEos reticulata\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBlue streaked lory\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePsittacidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePrimolius auricollis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eYellow- collared macaw\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePsittacidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eEclectus roratus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEclectus parrot\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePsittaculidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eTrichogloss us moluccanus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSwainson\u0026rsquo; s lory\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePsittacidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eLoriculus galgulus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBlue- crowned hanging parrot\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePsittacidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eNeophema splendida\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSplendid grass parakeet\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePsittaculidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eTrichoglossus rosenbergii\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRosenberg \u0026rsquo;s lorikeet\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePsittaculidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eEos histrio\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRed-and- blue lory\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePsittaculidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePlatycercus eximius\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEastern rosella\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePsittacidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eEclectus roratus roratus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGrand eclectus parrot\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePsittaculidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eTrichoglossus euteles\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePerfect lorikeet\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e83\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular analyses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe presence of the PaBV-RNA in cloacal swabs and/ or tissue samples was detected by RT-PCR targeting the \u003cem\u003eM\u003c/em\u003e gene of PaBV [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA extraction and RT-PCR amplification of M-gene of avian bornavirus\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFrom collected 107 samples (cloacal swab, n\u0026thinsp;=\u0026thinsp;59; brain, n\u0026thinsp;=\u0026thinsp;24; proventriculus, n\u0026thinsp;=\u0026thinsp;24) preserved at -80\u003csup\u003e0\u003c/sup\u003eC, viral RNA was extracted using the Qiagen RNeasy Extraction Kit (Hilden, Germany) according to the manufacturer\u0026rsquo;s instructions. The extracted RNA was subjected to two steps RT-PCR using Revert Aid (M-Mulv). Previously described primers, M Forward (5\u0026acute;-GGTAATTGTTCCTGGATGG-3\u0026acute;) and M Reverse (5\u0026acute;-ACACCAATGTTCCGAAGACG-3\u0026acute;) were used to amplify a 350 bp partial \u003cem\u003eM\u003c/em\u003e gene of avian bornavirus [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. For cDNA synthesis, the condition was 25\u0026deg;C for 10 mins, 37\u0026deg;C for 120 mins, 85\u0026deg;C for 5 mins, followed by hold at 4\u0026deg;C. The RT-PCR cycle program was initial denaturation at 94\u003csup\u003e0\u003c/sup\u003e C for 2mins, 38 cycles with denaturation at 94\u003csup\u003e0\u003c/sup\u003e C for 30 secs, annealing at 55\u003csup\u003e0\u003c/sup\u003e C for 30 secs, extension at 72\u003csup\u003e0\u003c/sup\u003e C for 30 secs, and final extension at 72\u003csup\u003e0\u003c/sup\u003e C for 5 mins. The amplified RT-PCR products were analyzed by 1.5% agarose gel electrophoresis containing ethidium bromide (0.5\u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and visualized in an image documentation system. The PCR products of the expected length were considered positive. RT-PCR-positive products of representative samples from birds belonging to different species were sequenced for confirmation. Sequencing was done by outsourcing at the 1st BASE DNA sequencing division, Malaysia. Sequences obtained were submitted to the Genbank database.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePhylogenetic analysis was conducted based on partial \u003cem\u003eM\u003c/em\u003e genes from PaBV reference sequences available in GenBank. BLAST (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to compare the sequences and to find regions of similarity between sequences obtained and the GenBank to estimate the percent identity (% id). A phylogenetic tree based on the partial \u003cem\u003eM\u003c/em\u003e gene of PaBV was constructed using the Neighbour-Joining method and the evolutionary distance model Maximum Composite Likelihood and bootstrap with 1000 replicates using MEGA X software.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn conclusion, our investigation revealed, this is the first published report that detects and phylogenetically characterizes PaBV-4 in captive psittacines in India. This study highlights the urgent need of future investigations of avian bornavirus infection status in the wild psittacine population as well, especially of the endangered and near threatened species. Based on the results of our study, a better understanding of the epidemiology of PaBV infections, establishment of routine diagnostics for appropriate control measures and development of a health standard for breeding psittacines in captivity is essential, as most of these species rely on captive breeding for their survival.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the conception and design. P. Deka and S. Das performed conceptualization. S. Das wrote the first draft of the manuscript, and all authors commented on previous versions of the manuscript. P. Deka, P. Kakati and B. Choudhury did sampling. R. Hazarika, S. Doloi, A, Kasheef, S. M. Gogoi, S. Das, R. K. Sharma, S. Islam, M. Nath, M. Sharma, and I. Deka checked the data and edited the draft. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors certified that the present work was carried after approval of Institute Animal Ethic Committee (IAEC) and as per the guidelines set by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Animal Welfare Division, Government of India. The ethical approval for the present study was accorded by the IAEC vide Approval No. 770/GO/Re/S/03/CPCSEA/FVSc/AAU/IAEC/19-20/801 Dated 23-12-2019.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors participated voluntarily in the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are provided within the manuscript. The sequences were submitted to the NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/) under the accession numbers ON960231, ON995435 to ON995442.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRubbenstroth, D.; Briese, T.; Durrwald, R.; Horie, M.; Hyndman, T.H.; Kuhn, J.H.; Nowotny, N.; Payne, S.; Stenglein, M.D.; Tomonaga, K.; et al. ICTV Virus Taxonomy Profile: Bornaviridae. J. Gen. Virol. 2021, 102, 001613. http://doi.org/10.1099/jgv.0.001613\u003c/li\u003e\n\u003cli\u003eTizard, I.; Shivaprasad, H.L.; Guo, J.; Hameed, S.; Ball, J.; Payne, S. The pathogenesis of proventricular dilatation disease. Anim. Health Res. Rev. 2017, 17, 110\u0026ndash;126. http://doi.org/10.1017/S1466252316000189\u003c/li\u003e\n\u003cli\u003eBriese, T.; Schneemann, A.; Lewis, A.J.; Park, Y.S.; Kim, S.; Ludwig, H.; Lipkin, W.I. Genomic organization of Borna disease virus. Proc. Natl. Acad. Sci. USA 1994, 91, 4362\u0026ndash;4366. http://doi.org/10.1073/pnas.91.10.4362\u003c/li\u003e\n\u003cli\u003eTomonaga, K.; Kobayashi, T.; Ikuta, K. Molecular and cellular biology of Borna disease virus infection. Microbes Infect. 2002, 4, 491\u0026ndash;500. http://doi.org/10.1016/S1286-4579(02)01564-2\u003c/li\u003e\n\u003cli\u003eKuhn,J.H.;D\u0026uuml;rrwald,R.; B\u0026agrave;o,Y.; Briese, T.; Carbone, K.; Clawson, A.N.; DeRisi, J.L.; Garten, W.; Jahrling, P.B.; Kolodziejek, J.; et al. Taxonomic reorganization of the family Bornaviridae. Arch. Virol. 2015, 160, 621\u0026ndash;632. http://doi.org/10.1007/s00705-014-2276-z\u003c/li\u003e\n\u003cli\u003eAmarasinghe, G.K.; Ayll\u0026oacute;n, M.A.; B\u0026agrave;o, Y.; Basler, C.F.; Bavari, S.; Blasdell, K.R.; Briese, T.; Brown, P.A.; Bukreyev, A.; Balkema-Buschmann, A.; et al. Taxonomy of the order Mononegavirales: Update 2019. Arch. Virol. 2019, 164, 1967\u0026ndash;1980. http://doi.org/10.1007/s00705-019-04247-4\u003c/li\u003e\n\u003cli\u003eKistler, A.L.; Gancz, A.; Clubb, S.; Skewes-Cox, P.; Fischer, K.; Sorber, K.; Chiu, C.Y.; Lublin, A.; Mechani, S.; Farnoushi, Y.; et al. Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: Identification of a candidate etiologic agent. Virol. J. 2008, 5, 88. http://doi.org/10.1186/1743-422X-5-88\u003c/li\u003e\n\u003cli\u003ePhiladelpho, N.A.; Rubbenstroth, D.; Guimar\u0026atilde;es, M.B.; Ferreira, A.J.P. Survey of bornaviruses in pet psittacines in Brazil reveals a novel parrot bornavirus. Vet. Microbiol. 2014, 174, 584\u0026ndash;590. http://doi.org/10.1016/j.vetmic.2014.10.020 \u003c/li\u003e\n\u003cli\u003eRubbenstroth, D.; Schmidt, V.; Rinder, M.; Legler, M.; Twietmeyer, S.; Schwemmer, P.; Corman, V.M. Phylogenetic Analysis Supports Horizontal Transmission as a Driving Force of the Spread of Avian Bornaviruses. PLoS ONE 2016, 11, e0160936. http://doi.org/10.1371/journal.pone.0160936\u003c/li\u003e\n\u003cli\u003eRossi, G., Dahlhausen, R. D., Galosi, L., Orosz, S. E. Avian Ganglioneuritis in Clinical Practice. \u003cem\u003eVet Clin North Am Exot Anim Pract.\u003c/em\u003e 2017, \u003cem\u003e21(1)\u003c/em\u003e, 33-67. https://doi.org/10.1016/j.cvex.2017.08.009\u003c/li\u003e\n\u003cli\u003eBoatright-Horowitz S. L. (2020). Avian Bornaviral Ganglioneuritis: Current Debates and Unanswered Questions. \u003cem\u003eVet Med Int.\u003c/em\u003e, 2020, 6563723. https://doi.org/10.1155/2020/6563723\u003c/li\u003e\n\u003cli\u003eVillanueva, I.; Gray, P.; Mirhosseini, N.; Payne, S.; Hoppes, S.; Honkavuori, K.S.; Briese, T.; Turner, D.; Tizard, I. The diagnosis of proventricular dilatation disease: Use of a Western blot assay to detect antibodies against avian Borna virus. \u003cem\u003eVet. Microbiol\u003c/em\u003e. 2009, 143, 196\u0026ndash;201. http://doi.org/10.1016/j.vetmic.2009.11.041\u003c/li\u003e\n\u003cli\u003eKessler, S.; Heenemann, K.; Krause, T.; Twietmeyer, S.; Fuchs, J.; Lierz, M.; Corman, V.M.; Vahlenkamp, T.M.; Rubbenstroth, D. Monitoring of free-ranging and captive Psittacula populations in Western Europe for avian bornaviruses, circoviruses and polyomaviruses\u003cem\u003e. Avian Pathol. 2019, epub ahead of print.\u003c/em\u003ehttp://doi.org/10.1080/03079457.2019.1681359\u003c/li\u003e\n\u003cli\u003eWeissenb\u0026ouml;ck, H., Bakonyi, T., Sekulin, K., Ehrensperger, F., Doneley, R. J., D\u0026uuml;rrwald, R., Hoop, R., Erd\u0026eacute;lyi, K., G\u0026aacute;l, J., Kolodziejek, J., Nowotny, N. (2009). Avian bornaviruses in psittacine birds from Europe and Australia with proventricular dilatation disease. \u003cem\u003eEmerg Infect Dis. \u003c/em\u003e2009, 15(9), 1453\u0026ndash;1459. https://doi.org/10.3201/eid1509.090353\u003c/li\u003e\n\u003cli\u003eRubbenstroth, D.; Rinder, M.; Stein, M.; H\u0026ouml;per, D.; Kaspers, B.; Brosinski, K.; Horie, M.; Schmidt, V.; Legler, M.; Korbel, R.; et al. Avian bornaviruses are widely distributed in canary birds (\u003cem\u003eSerinus canaria f. domestica). Vet. Microbiol\u003c/em\u003e. 2013, 165, 287\u0026ndash;295. http://doi.org/10.1016/j.vetmic.2013.03.024\u003c/li\u003e\n\u003cli\u003eMurray, M.; Guo, J.; Tizard, I.; Jennings, S.; Shivaprasad, H.L.; Payne, S.; Ellis, J.C.; Van Wettere, A.J.; O\u0026rsquo;Brien, K.M. Aquatic Bird Bornavirus-Associated Disease in Free-Living Canada Geese (\u003cem\u003eBranta canadensis\u003c/em\u003e) in the Northeastern USA\u003cem\u003e. J. Wildl. Dis\u003c/em\u003e. 2017, 53, 607\u0026ndash;611. http://doi.org/10.7589/2016-07-160\u003c/li\u003e\n\u003cli\u003eSwieton, E.; Dziadek, K.; \u0026acute; Smietanka, K. Avian Bornaviruses in Wild Aquatic Birds of the Anseriformes Order in Poland. \u003cem\u003ePathogens\u003c/em\u003e 2022, 11, 98. http://doi.org/10.3390/pathogens11010098\u003c/li\u003e\n\u003cli\u003eLudwig, T.H.; Becht, H.; Groh, L. Borna disease (BD), a slow virus infection biological properties of the virus. Med Microbiol Immunol. 1973;158(4):275\u0026ndash;89. https://doi.org/10.1007/BF02121414\u003c/li\u003e\n\u003cli\u003eDuchala, C.S.; Carbone, K.M.; Narayan, O. Preliminary Studies on the Biology of Borna Disease Virus. \u003cem\u003eJ. Gen. Virol\u003c/em\u003e. 1989, 70 Pt 12, 3507\u0026ndash;3511. http://doi.org/10.1099/0022-1317-70-12-3507\u003c/li\u003e\n\u003cli\u003eMatsumoto, Y.; Hayashi, Y.;Omori, H.; Honda, T.; Daito, T.; Horie, M.; Ikuta, K., Fujino, K., Nakamura, S., Schneider, U.,et al. Bornavirus closely associates and segregates with host chromosomes to ensure persistent Intranuclear infection. \u003cem\u003eCell Host Microbe\u003c/em\u003e. 2012;11(5):492\u0026ndash;503 https://doi.org/10.1016/j.chom.2012.04.009\u003c/li\u003e\n\u003cli\u003eRubbenstroth, D.; Rinder, M.; Kaspers, B.; Staeheli, P. Efficient isolation of avian bornaviruses (ABV) from naturally infected psittacine birds and identification of a new ABV genotype from a salmon-crested cockatoo (\u003cem\u003eCacatua moluccensis\u003c/em\u003e). \u003cem\u003eVet. Microbiol\u003c/em\u003e. 2012, 161, 36\u0026ndash;42. http://doi.org/10.1016/j.vetmic.2012.07.004\u003c/li\u003e\n\u003cli\u003eLierz, M., Hafez, H.M., Honkavuori, K.S., Gruber, A.D., Olias, P., Abdelwhab, E.M., Kohls, A., Lipkin, W.I., Briese, T., Hauck, R. Anatomical distribution of avian bornavirus in parrots, its occurrence in clinically healthy birds and ABV-antibody detection. \u003cem\u003eAvian Pathol\u003c/em\u003e. 2009, \u003cem\u003e38\u003c/em\u003e(6):491-96. https://doi.org/10.1080/03079450903349238\u003c/li\u003e\n\u003cli\u003eKerski, A.; de Kloet, A.H.; de Kloet, S.R. Vertical transmission of avian bornavirus in Psittaciformes: Avian bornavirus RNA and anti-avian bornavirus antibodies in eggs, embryos, and hatchlings obtained from infected sun conures (\u003cem\u003eAratinga solstitialis\u003c/em\u003e). \u003cem\u003eAvian Dis\u003c/em\u003e. 2012, 56, 471\u0026ndash;478. http://doi.org/10.1637/9879-080511-Reg.1\u003c/li\u003e\n\u003cli\u003eDelnatte, P.; Nagy, E.; Ojkic, D.; Crawshaw, G.; Smith, D.A. Investigation into the possibility of vertical transmission of avian bornavirus in free-ranging Canada geese (\u003cem\u003eBranta canadensis\u003c/em\u003e). \u003cem\u003eAvian Pathol.\u003c/em\u003e 2014, 43, 301\u0026ndash;304. http://doi.org/10.1080/03079457.2014.921279\u003c/li\u003e\n\u003cli\u003eRubbenstroth, D.; Brosinski, K.; Rinder, M.; Olbert, M.; Kaspers, B.; Korbel, R.; Staeheli, P. No contact transmission of avian bornavirus in experimentally infected cockatiels (\u003cem\u003eNymphicus hollandicus\u003c/em\u003e) and domestic canaries (\u003cem\u003eSerinus canaria \u003c/em\u003eforma domestica). \u003cem\u003eVet. Microbiol\u003c/em\u003e. 2014, 172, 146\u0026ndash;156. http://doi.org/10.1016/j.vetmic.2014.05.011\u003c/li\u003e\n\u003cli\u003ePiepenbring, A.K.; Enderlein, D.; Herzog, S.; Al-Ibadi, B.; Heffels-Redmann, U.; Heckmann, J.; Lange-Herbst, H.; Herden, C.; Lierz, M. Parrot Bornavirus (PaBV)-2 isolate causes different disease patterns in cockatiels than PaBV-4. \u003cem\u003eAvian Pathol\u003c/em\u003e. 2016, 45, 156\u0026ndash;168. http://doi.org/10.1080/03079457.2015.1137867\u003c/li\u003e\n\u003cli\u003eDe Ara\u0026uacute;jo, J.L.; Rech, R.R.; Heatley, J.J.; Guo, J.; Giaretta, P.R.; Tizard, I.; Rodrigues-Hoffmann, A. From nerves to brain to gastrointestinal tract: A time-based study of parrot bornavirus 2 (PaBV-2) pathogenesis in cockatiels (Nymphicus hollandicus). \u003cem\u003ePLoS One \u003c/em\u003e2017, 12, e0187797. https://doi.org/10.1371/journal.pone.0187797\u003c/li\u003e\n\u003cli\u003eFluck, A.; Enderlein, D.; Piepenbring, A.; Heffels-Redmann, U.; Herzog, S.; Pieper, K.; Herden, C.; Lierz, M. Correlation of avian bornavirus-specific antibodies and viral ribonucleic acid shedding with neurological signs and feather-damaging behaviour in psittacine birds. \u003cem\u003eVet. Rec. \u003c/em\u003e2019, 184, 476.https://doi.org/10.1136/vr.104860\u003c/li\u003e\n\u003cli\u003eDeKloet, S.R.; Dorrestein, G.M. Presence of Avian Bornavirus RNA and Anti-Avian Bornavirus Antibodies in Apparently Healthy Macaws. \u003cem\u003eAvian Dis.\u003c/em\u003e 2009, 53, 568\u0026ndash;573.https://doi.org/10.1637/8828-040209-Reg.1\u003c/li\u003e\n\u003cli\u003ePayne, S.; Shivaprasad, H.L.; Mirhosseini, N.; Gray, P.; Hoppes, S.; Weissenb\u0026ouml;ck, H.; Tizard, I. Unusual and severe lesions of proventricular dilatation disease in cockatiels (Nymphicus hollandicus) acting as healthy carriers of avian bornavirus (ABV) and subsequently infected with a virulent strain of ABV. \u003cem\u003eAvian Pathol.\u003c/em\u003e 2011, 40, 15\u0026ndash;22.https://doi.org/10.1080/03079457.2010.536978\u003c/li\u003e\n\u003cli\u003ePiepenbring et al., 2012; Piepenbring, A.K.; Enderlein, D.; Herzog, S.; Kaleta, E.F.; Heffels-Redmann, U.; Ressmeyer, S.; Herden, C.; Lierz, M. Pathogenesis of Avian Bornavirus in Experimentally Infected Cockatiels. \u003cem\u003eEmerg. Infect. Dis.\u003c/em\u003e 2012, 18, 234\u0026ndash;241.https://doi.org/10.3201/eid1802.111525\u003c/li\u003e\n\u003cli\u003eHeffels-Redmann, U.; Enderlein, D.; Herzog, S.; Piepenbring, A.; B\u0026uuml;rkle, M.; Neumann, D.; Herden, C.; Lierz, M. Follow-Up Investigations on Different Courses of Natural Avian Bornavirus Infections in Psittacines. \u003cem\u003eAvian Dis.\u003c/em\u003e 2012, 56, 153\u0026ndash;159.https://doi.org/10.1637/9844-062811-Reg.1\u003c/li\u003e\n\u003cli\u003eGuo, J.; Payne, S.; Zhang, S.; Turner, D.; Tizard, I.; Suchodolski, P. Avian Bornaviruses: Diagnosis, Isolation, and Genotyping. \u003cem\u003eCurr. Protoc. Microbiol. \u003c/em\u003e2014, 34, 15I.1.1\u0026ndash;15I.1.33.https://doi.org/10.1002/9780471729259.mc15i01s34\u003c/li\u003e\n\u003cli\u003eDelnatte, P.; Mak, M.; Ojkic, D.; Raghav, R.; DeLay, J.; Smith, D.A. Detection of Avian bornavirus in multiple tissues of infected psittacine birds using real-time reverse transcription polymerase chain reaction. \u003cem\u003eJ. Vet. Diagn.\u003c/em\u003e Investig. 2014, 26, 266\u0026ndash;271. https://doi.org/10.1177/1040638713519641\u003c/li\u003e\n\u003cli\u003eOlbert, M.; R\u0026ouml;mer-Oberd\u0026ouml;rfer, A.; Herden, C.; Malberg, S.; Runge, S.; Staeheli, P.; Rubbenstroth, D. Viral vector vaccines expressing nucleoprotein and phosphoprotein genes of avian bornaviruses ameliorate homologous challenge infections in cockatiels and common canaries. \u003cem\u003eSci. Rep.\u003c/em\u003e 2016, 6, 36840.https://doi.org/10.1038/srep36840\u003c/li\u003e\n\u003cli\u003eSigrist, B.; Geers, J.; Albini, S.; Rubbenstroth, D.; Wolfrum, N. A New Multiplex Real-Time RT-PCR for Simultaneous Detection and Differentiation of Avian Bornaviruses. \u003cem\u003eViruses \u003c/em\u003e2021, 13, 1358.https://doi.org/10.3390/v13071358\u003c/li\u003e\n\u003cli\u003ePham, P.H.; Leacy, A.; Deng, L.; Nagy, E.; Susta, L. Isolation of Ontario aquatic bird bornavirus 1 and characterization of its replication in immortalized avian cell lines. \u003cem\u003eVirol. J. \u003c/em\u003e2020, 17, 16.https://doi.org/10.1186/s12985-020-1286-6\u003c/li\u003e\n\u003cli\u003eHerzog, S.; Enderlein, D.; Heffels-Redmann, U.; Piepenbring, A.; Neumann, D.; Kaleta, E.F.; M\u0026uuml;ller, H.; Lierz, M.; Herden, C. Indirect Immunofluorescence Assay for Intra Vitam Diagnosis of Avian Bornavirus Infection in Psittacine Birds. \u003cem\u003eJ. Clin. Microbiol.\u003c/em\u003e 2010, 48, 2282\u0026ndash;2284. https://doi.org/10.1128/JCM.00145-10\u003c/li\u003e\n\u003cli\u003eMaclachlan. N.; Dubovi, E.J. Bornaviridae. In: Fenner\u0026rsquo;s Veterinary Virology. London: Academic Press; 2017. p. 381\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eHonkavuori, K.S.; Shivaprasad, H.L.; Williams, B.L.; Quan, P.-L.; Hornig, M.; Street, C.; Palacios, G.; Hutchison, S.K.; Franca, M.;Egholm, M.; et al. Novel Borna Virus in Psittacine Birds with Proventricular Dilatation Disease.\u003cem\u003eEmerg. Infect. Dis.\u003c/em\u003e 2008, 14, 1883\u0026ndash;1886.https://doi.org/10.3201/eid1412.080984\u003c/li\u003e\n\u003cli\u003eHorie, M.; Ueda, K.; Ueda, A.; Honda, T.; Tomonaga, K. Detection of Avian bornavirus 5 RNA in Eclectus roratus with feather picking disorder. \u003cem\u003eMicrobiol. Immunol.\u003c/em\u003e 2012, 56, 346\u0026ndash;349.https://doi.org/10.1111/j.1348-0421.2012.00436.x\u003c/li\u003e\n\u003cli\u003eLast, R.D.; Weissenb\u0026ouml;ck, H.; Nedorost, N.; Shivaprasad, H. Avian bornavirus genotype 4 recovered from naturally infected psittacine birds with proventricular dilatation disease in South Africa. \u003cem\u003eJ. South Afr. Vet. Assoc. \u003c/em\u003e2012, 83, 4.https://doi.org/10.4102/jsava.v83i1.938\u003c/li\u003e\n\u003cli\u003eKistler, A.L.; Gancz, A.; Clubb, S.; Skewes-Cox, P.; Fischer, K.; Sorber, K.; Chiu, C.Y.; Lublin, A.; Mechani, S.; Farnoushi, Y.; et al. Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: Identification of a candidate etiologic agent. \u003cem\u003eVirol. J.\u003c/em\u003e 2008, 5, 88. https://doi.org/10.1186/1743-422X-5-88\u003c/li\u003e\n\u003cli\u003eGray, P.; Hoppes, S.; Suchodolski, P.; Mirhosseini, N.; Payne, S.; Villanueva, I.; Shivaprasad, H.; Honkavuori, K.S.; Briese, T.; Reddy, S.M.; et al. Use of Avian Bornavirus Isolates to Induce Proventricular Dilatation Disease in Conures. \u003cem\u003eEmerg. Infect. Dis\u003c/em\u003e. 2010, 16, 473\u0026ndash;479.https://doi.org/10.3201/eid1603.091257\u003c/li\u003e\n\u003cli\u003eHeffels-Redmann, U.; Enderlein, D.; Herzog, S.; Herden, C.; Piepenbring, A.; Neumann, D.; Muller, H.; Capelli, S.; M\u0026uuml;ller, H.; Oberh\u0026auml;user, K.; et al. Occurrence of avian bornavirus infection in captive psittacines in various European countries and its association with proventricular dilatation disease. \u003cem\u003eAvian Pathol.\u003c/em\u003e 2011, 40, 419\u0026ndash;426.https://doi.org/10.1080/03079457.2011.589825\u003c/li\u003e\n\u003cli\u003eDonatti, R.V.; Resende, M.; Junior, F.F.; Marques, M.V.R.; Ecco, R.; Shivaprasad, H.L.; De Resende, J.S.; Martins, N.R.D.S. Fatal Proventricular Dilatation Disease in Captive Native Psittacines in Brazil. \u003cem\u003eAvian Dis.\u003c/em\u003e 2014, 58, 187\u0026ndash;193.https://doi.org/10.1637/10588-061013-Case.1\u003c/li\u003e\n\u003cli\u003eMirhosseini, N.; Gray, P.L.; Hoppes, S.; Tizard, I.; Shivaprasad, H.L.; Payne, S. Proventricular Dilatation Disease in Cockatiels (Nymphicus hollandicus) After Infection with a Genotype 2 Avian Bornavirus.\u003cem\u003e J. Avian Med. Surg.\u003c/em\u003e 2011, 25, 199\u0026ndash;204.https://doi.org/10.1647/2010-030.1\u003c/li\u003e\n\u003cli\u003ePayne, S.; Shivaprasad, H.L.; Mirhosseini, N.; Gray, P.; Hoppes, S.; Weissenb\u0026ouml;ck, H.; Tizard, I. Unusual and severe lesions of proventricular dilatation disease in cockatiels (Nymphicus hollandicus) acting as healthy carriers of avian bornavirus (ABV) and subsequently infected with a virulent strain of ABV. \u003cem\u003eAvian Pathol.\u003c/em\u003e 2011, 40, 15\u0026ndash;22.https://doi.org/10.1080/03079457.2010.536978\u003c/li\u003e\n\u003cli\u003eHameed, S.S.; Guo, J.; Tizard, I.; Shivaprasad, H.L.; Payne, S. Studies on immunity and immunopathogenesis of parrot bornaviral disease in cockatiels. \u003cem\u003eVirology \u003c/em\u003e2018, 515, 81\u0026ndash;91.https://doi.org/10.1016/j.virol.2017.12.007\u003c/li\u003e\n\u003cli\u003eStaeheli, P.; Rinder, M.; Kaspers, B. Avian Bornavirus Associated with Fatal Disease in Psittacine Birds.\u003cem\u003e J. Virol\u003c/em\u003e. 2010, 84, 6269\u0026ndash;6275. https://doi.org/10.1128/JVI.02567-09\u003c/li\u003e\n\u003cli\u003eGartner, A.M.; Link, J.; B\u0026uuml;cking, B.; Enderlein, D.; Herzog, S.; Petzold, J.; Malberg, S.; Herden, C.; Lierz, M. Age-dependent development and clinical characteristics of an experimental parrot bornavirus-4 (PaBV-4) infection in cockatiels (Nymphicus hollandicus). \u003cem\u003eAvian Pathol\u003c/em\u003e. 2021, 50, 138\u0026ndash;150.https://doi.org/10.1080/03079457.2020.1852177\u003c/li\u003e\n\u003cli\u003eRubbenstroth, D. Avian Bornavirus Research\u0026mdash;A Comprehensive Review. \u003cem\u003eViruses \u003c/em\u003e2022, 14, 1513. https://doi.org/10.3390/v14071513\u003c/li\u003e\n\u003cli\u003eRaghav, R.; Taylor, M.; DeLay, J.; Ojkic, D.; Pearl, D.L.; Kistler, A.L.; DeRisi, J.L.; Ganem, D.; Smith, D.A.Avian Bornavirus isPresent in Many Tissues of Psittacine Birds with Histopathologic Evidence of Proventricular Dilatation Disease. \u003cem\u003eJ. Vet. Diagn.\u003c/em\u003eInvestig. 2010, 22, 495\u0026ndash;508.https://doi.org/10.1177/104063871002200402\u003c/li\u003e\n\u003cli\u003eHeatley, J. J \u0026amp; Villalobos, A.R. Avian bornavirus in the urine of infected birds. \u003cem\u003eVet Med (Auckl)\u003c/em\u003e. 2012, 3, 19-23. https://doi.org/10.2147/VMRR.S31336\u003c/li\u003e\n\u003cli\u003eDelnatte, P.; Nagy, E.; Ojkic, D.; Leishman, D.; Crawshaw, G.; Elias, K.; Smith, D.A. Avian bornavirus in free-ranging waterfowl: Prevalence of antibodies and cloacal shedding of viral RNA\u003cem\u003e J Wildl Dis\u003c/em\u003e. 2014, \u003cem\u003e50\u003c/em\u003e(3), 512\u0026ndash;523. https://doi.org/10.7589/2013-08-218\u003c/li\u003e\n\u003cli\u003eRinder, M.; Ackermann, A.; Kempf, H.; Kaspers, B.; Korbel, R.; Staeheli, P. Broad Tissue and Cell Tropism of Avian Bornavirus in Parrots with Proventricular Dilatation Disease\u003cem\u003e. J. Virol\u003c/em\u003e. 2009, 83, 5401\u0026ndash;5407. https://doi.org/10.1128/JVI.00133-09\u003c/li\u003e\n\u003cli\u003eSilva, A. S. G., Raso, T. F., Costa, E. A., G\u0026oacute;mez, S. Y. M., Martins, N. R. D. S. (2020) Parrot bornavirus in naturally infected Brazilian captive parrots: Challenges in viral spread control. \u003cem\u003ePLoS ONE\u003c/em\u003e. \u003cem\u003e15\u003c/em\u003e(6), e0232342. https://doi.org/10.1371/journal.pone.0232342\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 2 is available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Avian bornaviruses, Orthobornavirus, PaBV-4, Orthobornavirus alphapsittaciforme, proventricular dilatation disease (PDD), parrots","lastPublishedDoi":"10.21203/rs.3.rs-6968515/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6968515/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAvian bornaviruses are a recently described genetically diverse group consisting of 15 separate viruses within five different viral species belonging to the genus \u003cem\u003eOrthobornavirus\u003c/em\u003e within the family \u003cem\u003eBornaviridae\u003c/em\u003e. Amongst the avian bornaviruses discovered, the parrot bornaviruses (PaBV) belonging to species \u003cem\u003eOrthobornavirus alphapsittaciforme\u003c/em\u003e possess the highest veterinary relevance and is considered to be a major threat to psittacine aviculture. Since the discovery of PaBV in psittacine birds suffering from proventricular dilatation disease (PDD) in 2008, PaBV infections have been reported worldwide. In India, to assess whether avian bornaviruses circulates in parrots, 83 psittacines from 13 different species including birds with suspected PDD based on clinical examination results (n=33), cage mates of PDD-suspected birds without any clinical signs (n=26) and dead birds with previous clinic suspicious for PDD (n=24) were tested for PaBV. Cloacal swabs were collected from live birds and tissues were collected from dead birds and investigated for the presence of PaBV-RNA using reverse transcription polymerase chain reaction (RT-PCR). PaBV infection was detected in 44 birds (53.01%) belonging to 9 psittaciform species. Eighteen\u003cem\u003e \u003c/em\u003eof the group of PDD-suspected birds (54.54%), 21 dead birds (87.50%), and 5 clinically healthy cage mates (19.23%) were positive for PaBV-RNA. Sequence analysis of the \u003cem\u003ematrix (M)\u003c/em\u003e gene revealed infection by PaBV-4, belonging to the species \u003cem\u003eOrthobornavirus alphapsittaciforme\u003c/em\u003e. To the best of our knowledge, there is no publication describing the circulation of \u003cem\u003eOrthobornavirus alphapsittaciforme\u003c/em\u003e, PaBv-4 in captive psittacines in India. This study highlights the major impact on conservation projects including endangered/ vulnerable/ near threatened species as these birds rely on captive breeding for their survival. Therefore, there is an urgent need to recognize and understand the factors that might play a critical role in recent expansion of emergent avian pathogens and how they continue to spread and thrive.\u003c/p\u003e","manuscriptTitle":"First Report: Molecular detection and characterization of Parrot Bornavirus 4 (PaBV-4) in captive psittacines in India","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-09 09:44:17","doi":"10.21203/rs.3.rs-6968515/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-14T12:37:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-07T20:22:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-04T13:10:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"262977900702050011320778585110692392659","date":"2025-09-28T20:19:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"215996048023178022741976241782166172984","date":"2025-09-28T09:21:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-27T15:17:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-19T10:15:05+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-01T11:18:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-01T06:18:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-24T19:14:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b21b885a-3fa1-40cc-b0ce-40050cc735cc","owner":[],"postedDate":"October 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":55925185,"name":"Biological sciences/Molecular biology"},{"id":55925186,"name":"Health sciences/Diseases"}],"tags":[],"updatedAt":"2026-04-22T18:23:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-09 09:44:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6968515","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6968515","identity":"rs-6968515","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}