Surveillance of Parrot Bornavirus in Taiwan Captive Psittaciformes

Parrot bornavirus (PaBV) is an infectious disease linked with proventricular dilatation disease (PDD) with severe digestive and neurological symptoms affecting psittacine birds. Despite its detection in 2008, PaBV prevalence in Taiwan remains unexplored. Taiwan is one of the leading psittacine bird breeders; hence, understanding the distribution of PaBV aids preventive measures in controlling spread, early disease recognition, epidemiology, and transmission dynamics. Here, we aimed to detect the prevalence rate of PaBV and assess its genetic variation in Taiwan. Among 124 psittacine birds tested, fifty-seven were PaBV-positive, a prevalence rate of 45.97%. Most of the PaBV infections were adult psittacine birds, with five birds surviving the infection, resulting in a low survival rate (8.77%). A year of parrot bornavirus surveillance presented a seasonal pattern, with peak PaBV infection rates occurring in the spring season (68%) and the least in the summer season (25%), indicating the occurrence of PaBV infections linked to seasonal factors. Histopathology reveals severe meningoencephalitis in the cerebellum and dilated cardiomyopathy of the heart in psittacine birds who suffered from PDD. Three brain samples underwent X/P gene sequencing, revealing PaBV-2 and PaBV-4 viral genotypes through phylogenetic analyses. This underscores the necessity for ongoing PaBV surveillance and further investigation into its pathophysiology and transmission routes.

In Taiwan, psittacine birds are the main pet birds and the majority of psittacine bird owners have more than one psittacine bird.Taiwan is also one of the leading exporters of parrots in global trade and has the largest number of naturalized or breeding psittacine birds [31,32].With the great population of psittacine birds and the global lead in breeding, it is essential to understand the presence of infectious-disease-causing agents such as PaBV.This will provide insight into initial evidence of the potential risk that PaBV carries, especially in Taiwan's psittacine birds.
In Taiwan, there has been scarce available information regarding PaBV infection cases.Moreover, due to the high economic value of psittacine birds which are favored as companion pets in Taiwan, it is essential to further investigate the infectious virus, thus highlighting the importance of PaBV investigation.Hence, the presence and surveillance of PaBV across Taiwan are of significant importance in PaBV investigations and prevention.In this study, we screened one hundred twenty-four feces of captive psittacine birds collected for a year to report the prevalence rate of PaBV infection cases in Taiwan.Likewise, the sequencing of the PaBV X/P gene from a brain sample revealed the genotypes present in Taiwan.Furthermore, histopathology and radiograph images provide some insight into the nature of the proventriculus, cerebellum, and heart due to PaBV infection.This study underscores the critical need for ongoing surveillance and investigation into PaBV infection cases in Taiwan, particularly given the economic significance and popularity of psittacine birds as companion pets.By understanding the prevalence rates, genotypes, and pathological effects of PaBV, we can better inform prevention strategies and safeguard both psittacine birds of Taiwan.

Sample Collection
One hundred twenty-four psittacine feces samples used in this study were freshly collected by the veterinarian at Xing Yu Animal Hospital, Linyuan District, Kaohsiung City, Taiwan, or directly from the private psittacine owner's or breeder's house where the psittacine bird is housed and following the permission of the psittacine owners.For psittacine birds housed together, they were separated first and fresh feces were collected immediately after dropping.Clinal organs such as the brain, lung, pancreas, stomach, intestine, liver, eye, kidney, heart, muscle, and proventriculus from three psittacine birds that suffered from PaBV were donated for necropsy and RT-qPCR detection in different organ tissue.All the samples were placed in an ice bath right after collection and stored in a −80 • C Ultra-Low-Temperature Freezer (MDF-DU302VX-PA, TwinGuard, PHCBI, Gunma, Japan).The collection and sample handling guidelines were followed and approved by NPUST-IACUC.The sample collection started in June of 2022 and lasted until May 2023.Several psittacine birds with severe clinical symptoms of proventricular dilation disease (PDD) were screened through X-ray scans (E7239X, Rotanode TM , Toshiba, Otawara, Japan) for their proventriculus size.Feces or clinical organ samples were prepared as 20% homogenates with 1x Phosphate-Buffered Solution (PBS), incubated for 30 min, and centrifuged (Centrisart A-14C, Sartorius, Göttingen, Germany) for 12,000× g for 15 min at 4 • C, and then the clarified supernatants were collected for RNA extraction and the excess was stored at −80

RNA Extraction
The RNA was extracted from the supernatant samples of 1XPBS homogenized feces of 124 psittacine birds following the manufacturer's instructions of R.T.U REzol TM C&T (Protech, Taipei, Taiwan, KP200CT) with slight modification.An amount of 500 µL of the feces supernatant was mixed with 500 µL of REzol TM C&T in 1.5 mL sterile microcentrifuge tubes (JetBiofil, Guangzhou, China, CFT100015), shaken vigorously for 15 s, and incubated for 5 min, and then 100 µL of chloroform was added, shaken for 15 s, incubated for 2 min, and centrifuged for 12,000× g for 15 min at 4 • C. A 500 µL colorless upper aqueous phase was collected and transferred in a new microcentrifuge tube, added with 500 µL of isopropanol (Honeywell Burdick & Jackson, Ulsan, Republic of Korea, AH323-4) with gentle mixing, incubated for 10 min, and centrifuged for 12, 000× g for 10 min at 4 • C, and isopropanol supernatant was discarded.The RNA precipitate pellets were washed with 1 mL of 75% ethanol (Echo, Miaoli, Taiwan) by vortex-mixing for 2 s and centrifuged for 5 min at 12,000× g for 4 • C, ethanol supernatant was discarded, and RNA pellets were air-dried for 15 min or more if required.Dried RNA pellets were dissolved in 50 µL of UltraPure DEPC (Protech, Taipei, Taiwan, PT-P560-500) and quantified using NanoDrop2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).

Reverse Transcription-Quantitative Polymerase Chain Reaction
The 124 freshly collected feces samples were extracted for RNA immediately and tested for PaBV through RT-qPCR.Clinical organ samples such as the brain, kidney, eyes, stomach, lung, intestine, liver, pancreas, and heart from three psittacine birds donated to our lab were also subjected to RT-qPCR.The RT-qPCR detection for parrot bornavirus (PaBV) was performed in a StepOne™ Real-Time PCR System Thermal Cycling Block (Applied Biosystems, Thermo Fisher Scientific, Marsiling, Singapore) using the qPCRBIO Probe 1-Step Go Hi-ROX kit (PCR Biosystems, London, UK, PB25.42) following the manufacturer's instructions with slight modification.Each reaction contained 5 µL of 2x qPCRBIO Probe 1-Step Go Mix, 0.4 µL of reverse and forward primers each with concentrations of 10 µM, 0.2 µL of the probe at 10 µM concentration, 1.5 µL of UltraPure DEPC water (Protech, Taipei, Taiwan, PT-P560-500), and 2.5 µL of RNA extracted from feces samples in a total reaction volume of 10 µL.The thermal cycle was as follows: 45 • C for 10 min for reverse transcription, 95 • C for 2 min for polymerase activation, and 40 cycles of 95 • C for 5 s and 60 • C for 30 s for denaturation and annealing/extension.The primers and the probe (Genomics BioSci & Tech, New Taipei City, Taiwan) used for RT-qPCR detection were as follows: for the forward primer, BornaPCA3 (5 ′ -GATCCGCAGACAGYACGT-3 ′ ) was used; for the reverse primer; BornaPCA6 (5 ′ -GAGATCATGGANGGRTTCTT-3 ′ ) was used; and for the probe, BornaP_Fam (5 ′ -FAM-CGAATWCCCAGGGAGGCYCT-BHQ1-3 ′ ) was used, specific for PaBV-1, PaBV-2, PaBV-4, and PaBV-7 detection with a product size of 125 bp [30].

Gel Electrophoresis
The RT-qPCR products underwent gel electrophoresis to further confirm the detection of parrot bornavirus.An amount of 1.5 g of Biotechnology Grade Agarose I (VWR Chemicals, Solon, OH, USA, 97062-250) was dissolved in 100 mL of 0.5x Tris-borate-EDTA (TBE) solution, microwave-heated (RE-0711, Sampo, Taoyuan, Taiwan) for 2 min, shaken for 10 s, microwaved-heated for 2 min, shaken for 10 s, added with 5 µL of EtB"Out" Nucleic Acid Staining Solution 2.0 (Yeastern Biotech, Taipei City, Taiwan, FYD007-200P), shaken for 10 s to mix, and placed in a mold to solidify for 15 min.A Mupid ® -2plus Submarine Electrophoresis System (Advance, Tokyo, Japan) was used for gel electrophoresis with a run time of 5 min for half voltage followed by 25 min for full voltage with 0.5x TBE running buffer, enough to submerge and cover the gel.The RT-qPCR products were added with 1 µL of 6x DNA Loading Dye (GeneMarkbiolab, Taichung, Taiwan, DL02), and 100 bp of DNA Ladder (Bio-Van, New Taipei City, Taiwan, M0100) was used.The gel band was viewed through a UV Transilluminator (MUV21-312, Major Science, Taoyuan City, Taiwan) compound with UV Imager (CI-01, Major Science, Taoyuan City, Taiwan), visualized with Major Science 1D Analysis software version 1.0 at 24x integration, and the gel band image was captured using a mono camera (MTV-12V6HE-R, Mintron, New Taipei City, Taiwan) with an 8.5-51 mm F1.2 lens (SSL85051M, Avenir Lens, Seiko, Tokyo, Japan).

Donated clinical cerebellum and heart samples of deceased psittacine birds due to proventriculus dilatation disease were fixed with 10% phosphate buffer formalin solution
Viruses 2024, 16, 805 5 of 19 and embedded in paraffin.A hematoxylin-eosin (H&E) stain was used for every 5 µm thick tissue sample.The samples were observed under a light microscope (Eclipse E200, Nikon, Shonan, Japan), and images were obtained using a Nikon DS-L2 camera (Nikon, Shonan, Japan).

Transmission Electron Microscope
1x PBS-homogenized brain samples were centrifuged for 10 min at 4000× g at 4 • C followed by 12,000× g at 4 • C for 30 min.The supernatant was passed through a 0.22 µm Millex ® -GV filter (Merck Millipore, Carrigtwohill, Ireland, PR05099).Then, the virus was concentrated through polyethylene glycol 6000 (Alfa Aesar, Karlsruhe, Germany, 10212393) with 10% w/v, centrifuged at 3500× g for 5 min, and then 1.45 g of NaCl (Showa Chemical, Tokyo, Japan, KGE-343A) at 1 M was added for every 20 mL of virus supernatant.It was incubated overnight with shaking at 4 • C. Afterwards, it was centrifuged in 10,000× g for 30 min, the supernatant was discarded, and pellets were dissolved with 1x PBS.The virus solution was allowed to settle in a formvar/carbon 200-mesh copper grid (Ted Pella, Redding, CA, USA, 01800-F) for 30 s and coated with 2% aqueous uranyl acetate solution (Spi-Chem, Structure Probe, West Chester, PA, USA, 02624-AB) for 45 s.Excess stain was removed and the specimen was examined using an H-7500 transmission electron microscope (Hitachi, Tokyo, Japan).

Phylogenetic Analysis
Alignments of X/P gene sequences were performed using the MUSCLE algorithm at 1000 bootstrap replicates of MEGA 11.0.13software.Using available Orthobornavirus sequences, phylogenetic analyses were performed through the maximum likelihood algorithm and GTR + I model at 1000 bootstrap replicates.

Results
One hundred twenty-four (124) fecal samples were tested for PaBV, of which 2 were from Taipei City, 2 were from Taoyuan City, 9 were from Taichung City, 1 was from Changhua County, 3 were from Yunlin County, 1 was from Nantou County, 7 were from Chiayi City, 3 was from Chiayi County, 16 were from Tainan City, 48 were from Kaohsiung City, and 32 were from Pingtung County (Figure 1).Altogether, the feces-sampled psittacine birds were privately owned or breeding birds and were mostly contained in their appropriate cages.Not long after testing positive with PaBV RT-qPCR detection, the birds passed away (around 1 week-2 months).Most PaBV-positive birds were observed with digestive symptoms (loss of appetite, weight loss, emaciation, undigested seeds shedding in feces, diarrhea, proventriculus dilation, and delayed crop emptying) and neurological symptoms (incoordination, seizure, tremor, lameness, and retinitis).A total of 124 psittacine birds were subjected to PaBV detection, and 57 (57/124; 45.97%) were positive for PaBV.The C T values ranged from 19.93 to 31.81.PaBV-positive cases were as follows: 2 from Taoyuan City (2/2; 100%), 1 from Taichung City (1/9; 11.11%), 2 from Yunlin County (2/3; 66.67%), 6 from Chiayi City (6/7; 85.71%), 1 from Chiayi County (1/1; 100%), 14 from Tainan City (14/16; 87.5%), 17 from Kaohsiung City (17/48; 35.42%), and 14 from Pingtung County (14/32; 43.75%) (Figure 1).Only five out of the fifty-seven PaBV-positive birds (5/57; 8.77%) survived PaBV infection, with observed proventricular dilatation, of which there were 2 from Tainan, 1 from Kaohsiung, and 2 from Pingtung, suggesting a high fatality rate (52/57; 91.23%) and mortality rate (52/124; 41.94%).Sample collection started from June of 2022 to May of 2023.The highest PaBV-positive rate was observed in April of 2023 (21/25; 84%) followed by October of 2022 and February of 2023 (2/3; 66.67% and 5/7; 71.4%), while zero positive cases were observed in January of 2023 (Figure 2).During the summer season (June to August), there were 25% (8/32) PaBV-positive cases, with 32% (8/25) during the fall season (September to November) and 41% (7/17) during the winter season (December to February), and an increasing trend was observed up to 68% (34/50) in the spring season (March to May).Most psittacine birds were companion birds, spending most of their time indoors or housed outdoors.These findings underscore the significant impact of PaBV infection on psittacine birds, highlighting the importance of continued monitoring and preventive measures to mitigate the high fatality and mortality rates associated with the disease.1).Only five out of the fiftyseven PaBV-positive birds (5/57; 8.77%) survived PaBV infection, with observed proventricular dilatation, of which there were 2 from Tainan, 1 from Kaohsiung, and 2 from Pingtung, suggesting a high fatality rate (52/57; 91.23%) and mortality rate (52/124; 41.94%).Sample collection started from June of 2022 to May of 2023.The highest PaBV-positive rate was observed in April of 2023 (21/25; 84%) followed by October of 2022 and February of 2023 (2/3; 66.67% and 5/7; 71.4%), while zero positive cases were observed in January of 2023 (Figure 2).During the summer season (June to August), there were 25% (8/32) PaBVpositive cases, with 32% (8/25) during the fall season (September to November) and 41% (7/17) during the winter season (December to February), and an increasing trend was observed up to 68% (34/50) in the spring season (March to May).Most psittacine birds were companion birds, spending most of their time indoors or housed outdoors.These findings underscore the significant impact of PaBV infection on psittacine birds, highlighting the importance of continued monitoring and preventive measures to mitigate the high fatality and mortality rates associated with the disease.PaBV-infection cases were found to be highest with Psittacus erithacus psittacine bird species (11/57; 19%) followed by Nymphicus hollandicus (8/57; 14%) and Aratinga solstitialis (8/57; 14%) (Table 1).The rest of the PaBV-positive rates per psittacine species were as follows: 7% (4/57) for each species of Myiopsitta monachus, Diopsittaca nobilis, and Amazona ochrocephala; 4% (2/57) for each species of Cacatua ophthalmica, Eclectus roratus, Ara severus, Pionites leucogaster, Amazona aestiva, Agapornis sp., and Psittacula krameri; and 2% (1/57) for each species of Poicephalus gulielmi, Pyrrhura molinae, Melopsittacus undulatus, and Cacatua moluccensis.PaBV-infection cases were found to be highest with Psittacus erithacus psittacine bird species (11/57; 19%) followed by Nymphicus hollandicus (8/57; 14%) and Aratinga solstitialis (8/57; 14%) (Table 1).The rest of the PaBV-positive rates per psittacine species were as follows: 7% (4/57) for each species of Myiopsitta monachus, Diopsittaca nobilis, and Amazona ochrocephala; 4% (2/57) for each species of Cacatua ophthalmica, Eclectus roratus, Ara severus, Pionites leucogaster, Amazona aestiva, Agapornis sp., and Psittacula krameri; and 2% (1/57) for each species of Poicephalus gulielmi, Pyrrhura molinae, Melopsittacus undulatus, and Cacatua moluccensis.The ambiguity surrounding the age and sex of the psittacine birds often reflects on their adoption, mostly without prior information.Most of the PDD-suffering birds were adults, 1 year old and up.Based on thirty-one (31) identified sexes of the psittacine birds, 64.52% (20/31) were male and 35.48% (11/31) were female (Table 2).Mutual symptoms were weight loss, crop stasis, maldigestion, and proventriculus dilation followed by depression and regurgitation.Seizure was only observed in one identified male bird (case no.2302013), while both ataxia and tremor were observed in 25% (5/20) of the males, and for females, and 18.18% (2/11) ataxia and 45.45% (5/11) tremor symptoms were observed.Additionally, psittacine birds with obvious and severe PDD clinical symptoms were subjected to X-ray scans to screen digestive organs, specifically proventriculus dilatation, observed in Figure 3A, while Figure 3B displays a proventriculus of a healthy psittacine bird.The proventriculus diameter could determine the short-term survival of psittacine birds with gastric implications.The normal range of the proventricular diameter-to-dorsoventral keel height ratio score was 0.200-0.476[33,34], among the PDD-suffering birds a range from the proventricular diameter-to-dorsoventral keel height recorded exceeded the normal range, from 0.681-0.981implying their poor survival.The elevated presence of lymphocytes in the cerebellum as shown in Figure 4A uncovers that PaBV-positive birds had a severe and diffused meningoencephalitis in contrast with a PaBV-negative cerebellum of a psittacine bird in Figure 4B.In Figure 4C, the psittacine bird suffered from PDD/AGN and PaBV-positive birds demonstrate dilated cardiomyopathy in the heart and extensive degeneration to necrosis of the myocardium.A transmission electron image of the PaBV is observed in Figure 5A, where the size ranges from 82.9 nm to 113 nm.Parrot bornaviruses are membrane viruses that allow them to enlarge or contract in size.The shedding of undigested seeds and diarrhea symptoms in feces were observed among PDD-suffering psittacine birds as a clinical symptom of gastric implications or digestive disorder (Figure 5B).At the same time, physiological symptoms such as tilting of the head, twitching of the eyes, and loss of balance were observed (Figure 5C,F).The twitching of the eyes and the rousing of the feathers indicate the discomfort of the bird (Figure 5C).Typical loss of balance is due to ataxia or loss of muscle, limiting the bird from grabbing and climbing.A 125 bp RT-qPCR product for PaBV detection was further confirmed in gel electrophoresis for gel band viewing.Figure 5D displays the gel band image for the lung, intestine, brain, eye, proventriculus, stomach, heart, kidney, liver, pancreas, muscle, and feces samples.Likewise, a clinical symptom of proventriculus dilation causing thinning of the gastrointestinal wall with undigested seeds was observed in Figure 5E of a PaBV-positive bird after suffering from PDD.The proventriculus measured about 6 cm in length over one-third to the overall length of the bird (about 18 cm) and had a 0.915 proventricular diameter-to-dorsoventral keel height ratio, exceeding the The elevated presence of lymphocytes in the cerebellum as shown in Figure 4A uncovers that PaBV-positive birds had a severe and diffused meningoencephalitis in contrast with a PaBV-negative cerebellum of a psittacine bird in Figure 4B.In Figure 4C, the psittacine bird suffered from PDD/AGN and PaBV-positive birds demonstrate dilated cardiomyopathy in the heart and extensive degeneration to necrosis of the myocardium.A transmission electron image of the PaBV is observed in Figure 5A, where the size ranges from 82.9 nm to 113 nm.Parrot bornaviruses are membrane viruses that allow them to enlarge or contract in size.The shedding of undigested seeds and diarrhea symptoms in feces were observed among PDD-suffering psittacine birds as a clinical symptom of gastric implications or digestive disorder (Figure 5B).At the same time, physiological symptoms such as tilting of the head, twitching of the eyes, and loss of balance were observed (Figure 5C,F).The twitching of the eyes and the rousing of the feathers indicate the discomfort of the bird (Figure 5C).Typical loss of balance is due to ataxia or loss of muscle, limiting the bird from grabbing and climbing.A 125 bp RT-qPCR product for PaBV detection was further confirmed in gel electrophoresis for gel band viewing.Figure 5D displays the gel band image for the lung, intestine, brain, eye, proventriculus, stomach, heart, kidney, liver, pancreas, muscle, and feces samples.Likewise, a clinical symptom of proventriculus dilation causing thinning of the gastrointestinal wall with undigested seeds was observed in Figure 5E of a PaBV-positive bird after suffering from PDD.The proventriculus measured about 6 cm in length over one-third to the overall length of the bird (about 18 cm) and had a 0.915 proventricular diameter-to-dorsoventral keel height ratio, exceeding the normal range for the proventricular diameter-to-dorsoventral keel height score.The diverse clinical manifestations observed in PaBV-positive psittacine birds, ranging from severe meningoencephalitis to gastrointestinal complications, underscore the multifaceted nature of the disease and the need for comprehensive veterinary care and management strategies.For further analysis, the three brain samples (Bird cases 2208023, 2301024, and 2302010) were amplified for their X/P gene with 682 bp in length.Prior to X/P gene amplifications, the brain samples were tested for PaBV through RT-qPCR with C T values of 18.59, 12.49, and 5.15 (Table 3 and Figure 5D).The organs were collected several days after the decease of the psittacine birds affecting viral RNA load and C T values in different organs.The brain has consistently low C T values reflecting high viral load and a much more suitable source for PCR amplification of the X/P gene.Therefore, it was selected for X/P gene amplification.NCBI BLASTn search provided the homology analysis, which revealed that two of the Taiwan strains (NPUSTIAVT-PaBV/Brain-2208023 and NPUSTIAVT-PaBV/Brain-2302010) were PaBV-4, with both originating from Changzhi, Pingtung, and isolated from Diopsittaca nobilis (red-shouldered macaw), and one (NPUSTIAVT-PaBV/Brain-2301024) PaBV-2 was isolated from Nymphicus hollandicus (cockatiel) in Kangshan, Kaohsiung.The sequences were submitted and deposited in GenBank with accession numbers PP529446-PP529448.
A phylogenetic tree was applied, using our sequenced X/P gene and all the available X/P gene sequences (682 nucleotides) of Orthobornavirus alphapsittaciformes, Orthobornavirus betapsittaciformes, and other genotypes of Orthobornavirus, with one representative per species isolates in each country.The analysis conclusively shows that our X/P gene sequence, Taiwan strain NPUSTIAVT-PaBV/Brain-2301024, belongs to the PaBV-2 genotype, and Taiwan strains NPUSTIAVT-PaBV/Brain-2302010 and NPUSTIAVT-PaBV/Brain-2208023 belong to the PaBV-4 genotype (Figure 6).

Discussion
Since the first report of avian bornaviruses in 2008, there has been an increase in the number of countries reporting the presence of PaBV, a novel infectious viral agent causing

Discussion
Since the first report of avian bornaviruses in 2008, there has been an increase in the number of countries reporting the presence of PaBV, a novel infectious viral agent causing proventricular dilatation disease affecting psittacine birds [1,11,14,[16][17][18][19]21,[35][36][37][38][39][40][41].In Taiwan, the presence of PaBV has been known through the submitted sequences of the matrix protein (M) gene (MK736729.1-MK736782.1),nucleoprotein (N) gene (MK770085.1-MK770118.1),and PaBV-2 (OM933586.1),PaBV-4 (OM939725.1),and PaBV-5 (OM777141.1)complete genome in GenBank [42].To further elucidate the PaBV presence in Taiwan, we screened feces samples from 124 psittacine birds collected for a year (June 2022 to May 2023), and demonstrated that 57 were PaBV-positive, with a prevalence of 45.97% and a low survival rate of 8.77%.Feces samples were collected freshly and tested for RT-qPCR immediately to secure the presence of PaBV in the feces and reduce false negative results.The majority of the tested feces were collected in a local animal hospital where the psittacine owners brought them for either regular health evaluation or veterinary care concerns due to diarrhea or loss of appetite.This may influence the prevalence rate determined in this study, which may require further surveillance of parrot bornavirus in Taiwan.
In the detection of PaBV through RT-qPCR assay, C T < 21 is strongly positive [30]; however, viral RNA present in the feces samples were observed to have a higher C T , carried out with the impurities in feces samples [11].In the RT-qPCR detection, C T s higher than 21 were also observed as a possible outcome [20,30].Gel electrophoresis of the RT-qPCR product may be suitable in securing and further confirming detection results to reduce false-negative or -positive results.Tissue samples such as the proventriculus, kidney, colon, cerebrum, and cerebellum have the most viral RNA, much more suitable for RT-qPCR detection [20].However, viral RNA extraction and PCR detection should be performed promptly to minimize false results due to the unstable nature of the virus as single-strand negative-sense RNA.Delays can lead to degradation of the RNA, potentially yielding inaccurate results.Due to the sampling limitations, it is crucial to conduct further surveillance studies of PaBV in Taiwan.This would help in understanding the transmission dynamics and potential impact of PaBV on parrot populations, especially in Taiwan.Enhanced surveillance can provide valuable insights for disease management and prevention strategies, safeguarding both companion and wildlife psittacine birds.
Young psittacine birds are the most vulnerable to PaBV infection or PDD [43,44]; however, they may have no clinical signs but are the probable source of seroconversion and PaBV-RNA shedding [45].In most cases, psittacine birds are housed together with other birds.Viral transmission sources to housed birds may come from newly introduced young bird/s in the enclosure, a potential carrier of PaBV.Then, PaBV shedding in the enclosure infects adult birds and possibly introduces the PaBV to the brood or clutch [44,46].Adult psittacine birds appeared to have more clinical symptoms [43] and sex appeared to have no significant influence on PaBV infection.The age and sex of psittacine birds are often unknown to owners since most were adopted without any preexisting information.Moreover, among the tested PaBV-positive psittacine birds in Taiwan, it was more common in Psittacus erithacus or the African grey parrots (19%).Similar PaBV host species cases were reported in the United States, Japan, South Korea, China, Portugal, Thailand, Germany, Canada, Austria, Hungary, Switzerland, and Israel [10,12,14,[25][26][27][28].
PDD affects the overall digestive health of the psittacine bird such as body weight, crop emptying, and seed digestion, alongside neurological problems such as severe meningoencephalitis [27,43,47].Proventriculus dilation displayed thinning of the gastrointestinal wall and a build-up of undigested seeds that may cause pain and discomfort to psittacine birds, leading to a loss of appetite and further causing malnutrition, weight loss, depression, loss of balance, and tremors.Through radiograph scans, the proventricular diameter-todorsoventral keel height is determined, from 0.681 to 0.981, indicating the poor survival of the psittacine birds.The highly enlarged proventriculus and thinning wall are clinical signs of neurogenic atrophy.Histopathology of the cerebellum in deceased psittacine birds due to PaBV infection elucidates severe diffuse meningoencephalitis due to accumulated lymphocytes.The PaBV infection could cause PDD due to gastrointestinal crisis; however, neurological damage was more likely to appear in PaBV infection.Recently, it has become more appropriate to address this neurological crisis as avian ganglioneuritis (AGN) [48,49].Probable mechanisms of PaBV leading to PDD/AGN were attributed to CD8+ T-cells, causing injury to neurons and ganglia recruiting CD4 T-cells, facilitating the antibody-mediated phagocytosis of axons [48].This leads to persistent damage in the nervous system, suggest-ing an autoimmune response to gangliosides [48].Histopathology of the heart showed a dilated cardiomyopathy or myocardium.This highlights the etiological implications of PaBV in neurological disorders other than PDD [38].
The spring season (68%) has the highest cases of PaBV infection while the summer season (28%) has the least, which might suggest that PaBV has a cycle transmission with regard to the season.Low PaBV infection during the summer season could be due to dry air, while the fall season could potentially allow the spread of PaBV in the environment, most probably due to dry feces particles in the air or fecal-oronasal route [11], viral trace contamination of waters, and residues of PaBV that are already present in the surroundings [1].An increase in moisture or humidity during the winter season could contribute to viral persistence, spread, and incubation leading to contamination in feeds or drinking water, resulting in high cases of PaBV in the spring season and permitting a continuous incubation life cycle of PaBV [1].The presence of PaBV in the environment and horizontal transmission to companion psittacine birds is still a challenge to understand [8].Potential viral reservoirs such as wild free-ranging psittacine birds or other potential host carriers have not been fully illustrated, similar to PaBV host species which are most prevalent, and surveillance reports were carried out in captive, breeding, or companion psittacine birds [1,2,39,42,44,50,51].In Taiwan, all psittacine birds were introduced, mostly kept as captive, companion, or breeding birds.Certainly, preventive measures targeting both horizontal and vertical transmission are vital for successfully managing PaBV infection cases [8,17,44,46,52,53], particularly in Taiwan.
Furthermore, our X/P gene sequences confirmed the presence of PaBV-2 and PaBV-4 genotypes in Taiwan strains.The two identified PaBV-4 X/P genes have 95.89% similarity from each other.Among our sequenced X/P genes, Germany strains were found to be consistently similar to our sequences.Taiwan strain NTUCL7 PaBV-4 has 95.89% sequence similarity with NPUSTIAVT-PaBV/Brain-2208023 and 99.7% sequence similarity with NPUSTIAVT-PaBV/Brian-2302010, while Taiwan strain NTUCL51 PaBV-2 has 97.80% sequence similarity with NPUSTIAVT-PaBV/Brain-2301024.Recently, phylogeographic analysis revealed that the South American ancestor could be the origin of PaBV-1, -2, and -8 genotypes [42].This highlights the importance of further surveillance of Orthobornavirus for captive Psittaciformes, similar to Taiwan.
Moreover, avian bornavirus, specifically parrot bornavirus, causes severe neurological and digestive disorders or death in infected birds [14,16,36,54], a threat to psittacine birds of Taiwan.Additionally, it could disrupt local ecosystems affecting unique and vulnerable bird species and have economic implications for industries reliant on bird populations, such as agriculture, bird breeding farms, and tourism.
The findings of this study highlight the importance of the ongoing monitoring of Parrot bornavirus in Taiwan's psittacine populations to monitor new strains or potential recombinants, as demonstrated in a recent report [2].

Figure 1 .
Figure 1.The map shows the geographical location of PaBV-positive (red dots) and PaBV-negat (black dots) psittacine birds in Taiwan; map was sourced from the National Land Surveying a Mapping Center (NLSC).

Figure 1 .
Figure 1.The map shows the geographical location of PaBV-positive (red dots) and PaBV-negative (black dots) psittacine birds in Taiwan; map was sourced from the National Land Surveying and Mapping Center (NLSC).

Figure 2 .
Figure 2. PaBV detection cases per month (June 2022 to May 2023); PaBV-positive case are in red while PaBV-negative cases are in black.

Figure 2 .
Figure 2. PaBV detection cases per month (June 2022 to May 2023); PaBV-positive case are in red while PaBV-negative cases are in black.

21 Figure 3 .
Figure 3. (A) X-ray scan of a proventriculus dilation (PD), 31.4 mm, and intestine, 6.4 mm and 6.1 mm; and (B) a normal proventriculus.Two radiographic images clearly illustrate the difference in the proventriculus diameter of a PDD-suffering psittacine bird compared to a healthy psittacine bird.

Figure 3 .
Figure 3. (A) X-ray scan of a proventriculus dilation (PD), 31.4 mm, and intestine, 6.4 mm and 6.1 mm; and (B) a normal proventriculus.Two radiographic images clearly illustrate the difference in the proventriculus diameter of a PDD-suffering psittacine bird compared to a healthy psittacine bird.

Viruses 2024 ,Figure 4 .
Figure 4. (A) Histopathological image at 10× and 40× magnification of the cerebellum with severe and diffuse lymphocytes indicating severe meningoencephalitis of PaBV-infected psittacine bird suffering from PDD/AGN (a: lymphocytes and b: red blood cells), and (B) histopathological image of the cerebellum of psittacine birds negative to PaBV infection or any signs of digestive or neurological disorders.(C) Histopathological image at 10× and 40× magnification of the heart from a PaBVpositive psittacine bird suffering from PDD/AGN with extensive degeneration to necrosis of the myocardium and dilated cardiomyopathy can be observed.

Figure 4 .
Figure 4. (A) Histopathological image at 10× and 40× magnification of the cerebellum with severe and diffuse lymphocytes indicating severe meningoencephalitis of PaBV-infected psittacine bird suffering from PDD/AGN (a: lymphocytes and b: red blood cells), and (B) histopathological image of the cerebellum of psittacine birds negative to PaBV infection or any signs of digestive or neurological disorders.(C) Histopathological image at 10× and 40× magnification of the heart from a PaBV-positive psittacine bird suffering from PDD/AGN with extensive degeneration to necrosis of the myocardium and dilated cardiomyopathy can be observed.

Figure 5 .
Figure 5. (A) Transmission electron microscopy image of the PaBV at 200,000× magnification, with a diameter of 82.9 nm and 113 nm.(B) Feces droppings with shedding of undigested seeds (red Figure 5. (A) Transmission electron microscopy image of the PaBV at 200,000× magnification, with a diameter of 82.9 nm and 113 nm.(B) Feces droppings with shedding of undigested seeds (red arrow).(C) Red-shouldered macaw (Diopsittaca nobilis) suffering from proventriculus dilation and showing neurological signs; discomfort can be seen in the twitching of the eyes.(D) Gel band image of the RT-qPCR product, 125 bp, for PaBV detection in lung, intestine, brain, eye, proventriculus, stomach, heart, kidney, liver, pancreas, muscle, and feces samples from case number 2301024.(E) Dilated proventriculus with a thin wall containing undigested seeds, about 6 cm in length, of a red-shouldered macaw (Diopsittaca nobilis) enduring proventriculus dilatation disease.(F) Photo of an African grey parrot (Psittacus erithacus) unable to balance right before it fell due to loss of control of the left leg (red arrow).

Table 1 .
List of PaBV-positive rates in different psittacine species.

Table 2 .
Summary of information for PaBV-positive cases.C T values were obtained from feces samples.

Table 3 .
Summary of the C T values in different organs for PaBV detection.The information is unknown as the organ is not tested or the organ is unavailable.