Immunogenicity of a recombinant hemagglutinin neuraminidase-Porcine rubulavirus produced by Escherichia coli of Porcine rubulavirus gives protective immunity of litter after challenge.

Porcine rubulavirus (PRV) is a contagious virus that affects the Mexican swine industry. This work aimed to evaluate the immunogenicity of an recombinant hemagglutinin neuraminidase-Porcine rubulavirus (rHN-PorPV) candidate vaccine on pregnant sows, and the protective efficacy afforded to their 7-day-old suckling piglets against PRV lethal challenge. Three sows were immunized with rHN-PorPV formulated with immune-stimulating complex (ISCOMs) and two sows with rHN-PorPV protein alone as well as a mock-immunized pregnant sow (negative control). Quantitative ELISA detected a high concentration of anti-rHN-PorPV Immunoglobulin G (IgG) antibodies in sow sera after the second dose of vaccine administered on day 14 until farrowing, showing viral-neutralizing and cross-neutralization activity against different variants of PRV. Sera samples from piglets of immunized sows (with or without adjuvant), showed high concentrations of IgG antibodies. As expected, piglets from the negative control sow (n=5), exhibited severe signs of disease and 100% of mortality after PRV challenge study. Conversely, 75% and 87.5% of the piglets born from the rHN-PorPV and the rHN-PorPV-ISCOMs-immunized sows (n=8), survived, respectively, showing milder PRV clinical signs. Our data indicate that rHN-PorPV candidate vaccine produced in Escherichia coli induces efficient humoral response in pregnant sows and that the maternally derived immunity provides high protection to suckling piglets against PRV lethal challenge.

Characterization of Pipistrellus pygmaeus Bat Virome from Sweden.

Increasing amounts of data indicate that bats harbor a higher viral diversity relative to other mammalian orders, and they have been recognized as potential reservoirs for pathogenic viruses, such as the Hendra, Nipah, Marburg, and SARS-CoV viruses. Here, we present the first viral metagenomic analysis of Pipistrellus pygmaeus from Uppsala, Sweden. Total RNA was extracted from the saliva and feces of individual bats and analyzed using Illumina sequencing. The results identified sequences related to 51 different viral families, including vertebrate, invertebrate, and plant viruses. These viral families include Coronaviridae, Picornaviridae, Dicistroviridae, Astroviridae, Hepeviridae, Reoviridae, Botourmiaviridae, Lispviridae, Totiviridae, Botoumiaviridae, Parvoviridae, Retroviridae, Adenoviridae, and Partitiviridae, as well as different unclassified viruses. We further characterized three near full-length genome sequences of bat coronaviruses. A phylogenetic analysis showed that these belonged to alphacoronaviruses with the closest similarity (78-99% at the protein level) to Danish and Finnish bat coronaviruses detected in Pipistrellus and Myotis bats. In addition, the full-length and the near full-length genomes of picornavirus were characterized. These showed the closest similarity (88-94% at the protein level) to bat picornaviruses identified in Chinese bats. Altogether, the results of this study show that Swedish Pipistrellus bats harbor a great diversity of viruses, some of which are closely related to mammalian viruses. This study expands our knowledge on the bat population virome and improves our understanding of the evolution and transmission of viruses among bats and to other species.

The order of events on Avian coronavirus life cycle shapes the order of quasispecies evolution during host switches.

To understand the population genetics events during coronavirus host switches, the Beaudette strain of Avian coronavirus (AvCoV) adapted to BHK-21 cells was passaged 15 times in VERO cells, the virus load and the variants at each passage being determined by RT-qPCR and genome-length deep sequencing. From BHK-21 P2 to VERO P3, a trend for the extinction of variants was followed by stability up to VERO P11 and both the emergence and the rise in frequency in some variants, while the virus loads were stable up to VERO P12. At the spillover from BHK-21 to VERO cells, variants that both emerged, showed a rise in frequency or were extinguished were detected on the spike, while variants at the M gene showed the same pattern only at VERO passage 13. Furthermore, nsps 3-5, 9 and 15 variants were detected at lower passages compared to the consensus sequences, with those at nsp3 being detected in the spectra also at higher passages. This suggests that quasispecies coronavirus evolution in spillovers follows the virus life cycle, starting with the evolution of the receptor binding proteins, followed by the replicase and then proteins involved in virion assembly, keeping the general fitness of the mutant spectrum stable.

Crossing the Line: Seroprevalence and Risk Factors for Transboundary Animal Diseases Along the Tanzania-Zambia Border.

Transboundary pathogens pose a threat to livelihood security in countries such as Zambia and Tanzania. This study aimed to investigate the seroprevalence of peste des petits ruminants virus (PPRV), foot and mouth disease virus (FMDV), sheep and goat pox virus (SGPV), Rift Valley fever virus (RVFV) and Brucella spp. in sheep and goats along the Tanzania-Zambia border. Another aim was to assess the association between certain predictor variables and seroprevalence, focusing on trade and proximity to an international border, to a town and to the Tanzania-Zambia highway. During September-October 2018, 486 serum samples from small ruminants in Zambia and 491 in Tanzania were collected and analyzed using enzyme-linked immunosorbent assays (ELISA). A questionnaire focused on management strategies was administered to each household. The animal-level seroprevalence in Zambia was 0.21% [95% confidence interval (CI) (0.01-1.14) for PPRV, 1.03% (95% CI 0.33-2.39) for FMDV, 0% (95% CI 0-0.76) for SGPV, 2.26% (95% CI 1.14-4.01) for RVFV and 1.65% (95% CI 0.71-3.22) for Brucella spp.]. In Tanzania, animal-level seroprevalence was 2.85% (95% CI 1.57-4.74) for PPRV, 16.9% (95% CI 13.7-20.5) for FMDV, 0.20% (95% CI 0.01-1.13) for SGPV, 3.26% (95% CI 1.87-5.24) for RVFV and 20.0% (95% CI 14.5-26.5) for Brucella spp. For PPRV (OR 6.83, 95% CI 1.37-34.0, p = 0.019) and FMDV (OR 5.68, 95% CI 1.58-20.3, p = 0.008), herds situated more than 30 km from an international border were more likely to be seropositive, while being located 10-30 km (OR 4.43, 95% CI 1.22-16.1 p = 0.024) from a border was identified as a risk factor for Brucella spp. For FMDV (OR 79.2, 95% CI 4.52-1388.9, p = 0.003), being situated within 30 km from a town was associated with seropositivity. Furthermore, contact with wild ruminants (OR 18.2, 95% CI 1.36-244), and the presence of sheep in the household (OR 5.20, 95% CI 1.00-26.9, p = 0.049), was associated with seropositivity for PPRV, and FMDV. No significant associations between trade or distance to the Tan-Zam highway and seroprevalence were found. We recommend that the impact of trade and proximity to borders, towns and roads should be further evaluated in larger studies, ideally incorporating aspects such as temporal trade fluctuations.

Gizzard erosions in broiler chickens in Sweden caused by fowl adenovirus serotype 1 (FAdV-1): investigation of outbreaks, including whole-genome sequencing of an isolate.

The present paper describes the investigation of the first outbreaks of adenoviral gizzard erosions (AGE) in Sweden, in five broiler flocks. The investigation included whole viral genome sequencing and investigation of genomic organization and sequence relationships with other adenoviruses. All five flocks had a history of decreased growth and uneven size of birds since 9-10 days of age. Macroscopically, lesions consistent with AGE (detached koilin layers, discolouration, bleeding, erosions) were identified in gizzards in all five flocks. In four flocks histology was performed, and degeneration and inflammation of the koilin layer and gizzard mucosa were identified in all four. In one flock, intranuclear inclusion bodies typical for fowl adenovirus (FAdV) were detected in trapped epithelial cells in the koilin layer. In four flocks in situ hybridization was performed, and cells positive for FAdV serotype 1 (FAdV-1) were demonstrated in the koilin layer and gizzard mucosa. FAdV species A (FAdV-A) was detected in gizzard, liver, caecal tonsils and bursa of Fabricius by polymerase chain reaction (PCR) and sequencing. Ten out of ten examined parent flocks of the affected chickens were seropositive for FAdV, indicating former or on-going infection. However, FAdV was not detected in embryos from seropositive parent flocks and thus vertical transmission was not demonstrated. The entire nucleotide sequence of one sample was determined and found to be 43,856 base pairs (bp) in length. The genome sequence and organization were found to be similar to that of the reference apathogenic avian adenovirus "chicken embryo lethal orphan" (CELO). RESEARCH HIGHLIGHTSAGE in Swedish broilers: necropsy, histopathology, ISH, PCR, whole-genome sequencing.Whole FAdV-genome analysed: 43,856 bp, found to be most similar to CELO (U46933.1).Multiple point mutations, site insertions and deletions identified compared to CELO.Paper adds knowledge about European disease situation and pathogenic FAdV-strains.

Investigation of the Virome and Characterization of Issyk-Kul Virus from Swedish Myotis brandtii Bats.

Bats are reservoirs for many different viruses, including some that can be transmitted to and cause disease in humans and/or animals. However, less is known about the bat-borne viruses circulating in Northern European countries such as in Sweden. In this study, saliva from Myotis brandtii bats, collected from south-central Sweden, was analyzed for viruses. The metagenomic analysis identified viral sequences belonging to different viral families, including, e.g., Nairoviridae, Retroviridae, Poxviridae, Herpesviridae and Siphoviridae. Interestingly, through the data analysis, the near-complete genome of Issyk-Kul virus (ISKV), a zoonotic virus within the Nairoviridae family, was obtained, showing 95-99% protein sequence identity to previously described ISKVs. This virus is believed to infect humans via an intermediate tick host or through contact with bat excrete. ISKV has previously been found in bats in Europe, but not previously in the Nordic region. In addition, near full-length genomes of two novel viruses belonging to Picornavirales order and Tymoviridae family were characterized. Taken together, our study has not only identified novel viruses, but also the presence of a zoonotic virus not previously known to circulate in this region. Thus, the results from these types of studies can help us to better understand the diversity of viruses circulating in bat populations, as well as identify viruses with zoonotic potential that could possibly be transmitted to humans.

Oncolytic virus purification with periodic counter-current chromatography.

Virus-based biologicals are one of the most promising biopharmaceuticals of the 21st century medicine and play a significant role in the development of innovative therapeutic, prophylactic, and clinical applications. Oncolytic virus manufacturing scale can range from 5 L in research and development up to 50 L for clinical studies and reach hundreds of liters for commercial scale. The inherent productivity and high integration potential of periodic counter-current chromatography (PCC) offer a transversal solution to decrease equipment footprint and the reduction of several non-value-added unit operations. We report on the design of an efficient PCC process applied to the intermediate purification of oncolytic adenovirus. The developed ion-exchange chromatographic purification method was carried out using a four-column setup for three different scenarios: (i) variation in the feedstock, (ii) potential use of a post-load washing step to improve virus recovery, and (iii) stability during extended operation. Obtained virus recoveries (57%-86%) and impurity reductions (>80% DNA, and >70% total protein) match or overcome batch purification. Regarding process stability and automation, our results show that not only the dynamic control strategy used is able to suppress perturbations in the sample inlet but also allows for unattended operation in the case of ion exchange capture.

Molecular Diversity of Hard Tick Species from Selected Areas of a Wildlife-Livestock Interface Ecosystem at Mikumi National Park, Morogoro Region, Tanzania.

Ticks are one of the most important arthropod vectors and reservoirs as they harbor a wide variety of viruses, bacteria, fungi, protozoa, and nematodes, which can cause diseases in human and livestock. Due to their impact on human, livestock, and wild animal health, increased knowledge of ticks is needed. So far, the published data on the molecular diversity between hard ticks species collected in Tanzania is scarce. The objective of this study was to determine the genetic diversity between hard tick species collected in the wildlife-livestock interface ecosystem at Mikumi National Park, Tanzania using the mitochondrion 16S rRNA gene sequences. Adult ticks were collected from cattle (632 ticks), goats (187 ticks), and environment (28 ticks) in the wards which lie at the border of Mikumi National Park. Morphological identification of ticks was performed to genus level. To identify ticks to species level, molecular analysis based on mitochondrion 16S rRNA gene was performed. Ticks representing the two genera (Hyalomma and Rhipicephalus) were identified using morphological characters. Six species were confirmed based on mitochondrion 16S rRNA gene, including Rhipicephalus microplus, Rhipicephalus evertsi, Hyalomma rufipes, Hyalomma truncatum, Hyalomma marginatum, and Hhyalomma turanicum. The presence of different clusters of tick species reflects the possible biological diversity of the hard ticks present in the study region. Further studies are however required to quantify species of hard ticks present in the study region and the country in general over a larger scale.

Genetic Relationship Between Hard Ticks (Ixodidae) Infesting Cattle from Select Areas of a Wildlife-Livestock Interface Ecosystem at Mikumi National Park, Tanzania.

Background: There has recently been a substantial increase in the number of tick species and tick-borne infectious agents in Tanzania. Owing to their impact on human, livestock, and wild animal health, increased knowledge of ticks is needed. So far, no published data on the genetic relationship between hard tick (Ixodidae) sequences collected from cattle are available in Tanzania. Methods: Ticks from cattle in the wards, which lie at the border of Mikumi National Park, were collected in the dry season, November to December 2019. Morphological identification of ticks was initially performed at the genus level. To identify ticks at the species level, molecular analysis based on the 16S rRNA gene was performed. Evolutionary relationships and genetic distances between ticks were determined using MaximumLikelihood and Kimura 2-parameter methods, respectively. Results: Based on morphology, two genera (Rhipicephalus and Hyalomma) were identified in the 630 adult ticks collected from a total of 252 cattle. Six species (Rhipicephalus microplus, Rhipicephalus evertsi, Hyalomma marginatum, Hyalomma rufipes, Hyalomma truncatum, and Hyalomma turanicum) were confirmed by BLASTn and phylogenetic analyses. Considerable mean and pairwise genetic distances were observed for Rhipicephalus and Hyalomma genera. Conclusion: The presence of different phylogenetic clusters and considerable mean and pairwise genetic distances observed reflect possible biological diversity of hard ticks present in the study area. Considering the value of the cattle in the livelihoods and economies of people and the country, the outcomes of this study will be useful in planning integrated control strategies for ticks and tick-borne diseases in Tanzania.

Field-Adapted Full Genome Sequencing of Peste-Des-Petits-Ruminants Virus Using Nanopore Sequencing.

Peste-des-petits-ruminants virus (PPRV) is currently the focus of a control and eradication program. Full genome sequencing has the opportunity to become a powerful tool in the eradication program by improving molecular epidemiology and the study of viral evolution. PPRV is prevalent in many resource-constrained areas, with long distances to laboratory facilities, which can lack the correct equipment for high-throughput sequencing. Here we present a protocol for near full or full genome sequencing of PPRV. The use of a portable miniPCR and MinION brings the laboratory to the field and in addition makes the production of a full genome possible within 24 h of sampling. The protocol has been successfully used on virus isolates from cell cultures and field isolates from tissue samples of naturally infected goats.

Complete Coding Sequence of a Pasivirus Found in Swedish Pigs.

Here, we report the complete coding sequence of a pasivirus found in the tonsil of a conventionally reared pig from a herd with respiratory disease in Sweden. The genome displays 75% to 87% and 81% to 94% nucleotide and amino acid sequence identity, respectively, to genomes of pasiviruses from other parts of the world.

Automated pH and conductivity conditioning using feedback control to support a two-step continuous purification process.

As discovery research organizations push more molecules and new modalities through their company pipelines, there comes a need to widen purification development and production bandwidth by increasing automation and throughput. Continuous processing technologies have the unique property of reducing manufacturing floor space and reducing costs. We can speed development and production by implementing automation and continuous process technologies early in discovery research. Here we describe an automated continuous instrument made up of an ÄKTA™ pcc for initial capture by protein A, an ÄKTA pure 150 retrofitted to automatically condition protein A eluate, and a second ÄKTA pure 150 built for flow-through anion exchange chromatography. The continuous instrument we have designed and built recirculates protein A eluate from the ÄKTA pcc in a closed loop while signals from the pH and conductivity meters direct addition of titrant for accurate and precise adjustments to the pH and salt concentration. The instrument is run without user intervention and can be used continuously for production or for development as a tool for screening running conditions on the anion exchange step.

Genome constellations of rotavirus a isolated from avian species in Brazil, 2008-2015.

Rotaviruses are members of the family Reoviridae and are a common cause of acute diarrhea in many mammalian and avian species. They are non-enveloped icosahedral particles and their genome comprises 11 segments of double-stranded RNA, which encodes six structural proteins (VP1-4, VP6-7) and six nonstructural proteins (NSP1-6). Genotypes are defined based upon the diversity found in these genes and viral characterization plays a central role on epidemiological studies and prevention. Here we investigate the distribution of Brazilian RVAs genotypes in 8 chicken samples collected between 2008 and 2015 from different regions by RT-PCR, partial (Sanger) nucleotide sequencing and phylogenetic analysis from all rotavirus genes. Although the identified genotypes were typical from avian host species, when analyzed together, they form novel genetic constellations: G19-P[31]-I11-R6-C6-M7-A16-N6-T8-E10-H8 and G19-P[31]-I4-R4-C4-M4-A16-N4-T4-E4-H4. This study highlights that avian rotaviruses are widespread among commercial farms in Brazil, and the co-circulation of at least two different genomic constellations indicates that may present a way bigger genetic variability, that can be increased by the possible transmission events from other birds, lack of specific preventive measures, as well as the different viral evolution mechanisms.

Extinction and emergence of genomic haplotypes during the evolution of Avian coronavirus in chicken embryos.

Avian coronavirus (AvCoV) is ubiquitously present on poultry as a multitude of virus lineages. Studies on AvCoV phenotypic traits are dependent on the isolation of field strains in chicken embryonated eggs, but the mutant spectrum on each isolate is not considered. This manuscript reports the previously unknown HTS (high throughput sequencing)-based complete genome haplotyping of AvCoV isolates after passages of two field strains in chicken embryonated eggs. For the first and third passages of strain 23/2013, virus loads were 6.699 log copies/ µL and 6 log copies/ µL and, for 38/2013, 5.699 log copies/µL and 2.699 log copies/µL of reaction, respectively. The first passage of strain 23/2013 contained no variant haplotype, while, for the third passage, five putative variant haplotypes were found, with > 99.9% full genome identity with each other and with the dominant genome. Regarding strain 38/2013, five variant haplotypes were found for the first passage, with > 99.9% full genome identity with each other and with the dominant genome, and a single variant haplotype was found. Extinction and emergence of haplotypes with polymorphisms in genes involved in receptor binding and regulation of RNA synthesis were observed, suggesting that phenotypic traits of AvCoV isolates are a result of their mutant spectrum.

Application of Viral Metagenomics for Study of Emerging and Reemerging Tick-Borne Viruses.

Ticks are important vectors for different tick-borne viruses, some of which cause diseases and death in humans, livestock, and wild animals. Tick-borne encephalitis virus, Crimean-Congo hemorrhagic fever virus, Kyasanur forest disease virus, severe fever with thrombocytopenia syndrome virus, Heartland virus, African swine fever virus, Nairobi sheep disease virus, and Louping ill virus are just a few examples of important tick-borne viruses. The majority of tick-borne viruses have RNA genomes that routinely undergo rapid genetic modifications such as point mutations during their replication. These genomic changes can influence the spread of viruses to new habitats and hosts and lead to the emergence of novel viruses that can pose a threat to public health. Therefore, investigation of the viruses circulating in ticks is important to understand their diversity, host and vector range, and evolutionary history, as well as to predict new emerging pathogens. The choice of detection method is important, as most methods detect only those viruses that have been previously well described. On the other hand, viral metagenomics is a useful tool to simultaneously identify all the viruses present in a sample, including novel variants of already known viruses or completely new viruses. This review describes tick-borne viruses, their historical background of emergence, and their reemergence in nature, and the use of viral metagenomics for viral discovery and studies of viral evolution.

Bornaviruses in naturally infected Psittacus erithacus in Portugal: insights of molecular epidemiology and ecology.

Background: The genus Orthobornavirus comprises non-segmented, negative-stranded RNA viruses able to infect humans, mammals, reptiles and various birds. Parrot bornavirus 1 to 8 (PaBV-1 to 8) causes neurological and/or gastrointestinal syndromes and death on psittacines. We aimed to identify and to produce epidemiologic knowledge about the etiologic agent associated with a death of two female Psittacus erithacus (grey parrot). Methods and Results: Both parrots were submitted for a complete standardised necropsy. Tissue samples were analysed by PCR. The findings in necropsy were compatible with bornavirus infection. Analysis revealed PaBV-4 related with genotypes detected in captive and in wild birds. The N and X proteins of PaBV-4 were more related to avian bornaviruses, while phosphoprotein was more related to variegated squirrel bornavirus 1 (VSBV-1). Within the P gene/phosphoprotein a highly conserved region between and within bornavirus species was found. Conclusions: Portugal is on the routes of the intensive world trade of psittacines. Broad screening studies are required to help understanding the role of wild birds in the emergence and spread of pathogenic bornaviruses. PaBV-4 phosphoprotein is closer to VSBV-1 associated with lethal encephalitis in humans than with some of the avian bornaviruses. The highly conserved P gene/phosphoprotein region is a good target for molecular diagnostics screenings.

First-time detection of bovine viral diarrhoea virus, BVDV-1, in cattle in Botswana.

Infectious diseases are serious constraints for improving livestock productivity. Bovine viral diarrhoea virus (BVDV) is a virus causing grave economic losses throughout the cattle producing world. Infection is often not apparent, but the virus can also cause respiratory signs, diarrhoea, reproductive problems and immunosuppression. Risk factors for disease transmission include, but are not limited to, herd size, animal trade and grazing on communal pastures. Several prevalence studies have been conducted in southern Africa, but in Botswana the occurrence is largely unknown. In this study, blood samples were obtained from 100 goats from three villages around the capital city, Gaborone. Also, 364 blood samples from cattle around Gaborone, collected as part of another study, were analysed. The detected antibody prevalence was 0% in goats and 53.6% in cattle when using a competitive enzyme-linked immunoassay. Three animals from two different herds were positive for viral nucleic acids on polymerase chain reaction. The two herds with viraemic animals had significantly higher antibody prevalence compared to the other herds. Also, two of the detected viruses were sequenced and found to be most similar to BVDV-1a. To the authors' knowledge, this is the first time that sequencing has been performed on BVDV isolated in Botswana.

Updated unified phylogenetic classification system and revised nomenclature for Newcastle disease virus.

Several Avian paramyxoviruses 1 (synonymous with Newcastle disease virus or NDV, used hereafter) classification systems have been proposed for strain identification and differentiation. These systems pioneered classification efforts; however, they were based on different approaches and lacked objective criteria for the differentiation of isolates. These differences have created discrepancies among systems, rendering discussions and comparisons across studies difficult. Although a system that used objective classification criteria was proposed by Diel and co-workers in 2012, the ample worldwide circulation and constant evolution of NDV, and utilization of only some of the criteria, led to identical naming and/or incorrect assigning of new sub/genotypes. To address these issues, an international consortium of experts was convened to undertake in-depth analyses of NDV genetic diversity. This consortium generated curated, up-to-date, complete fusion gene class I and class II datasets of all known NDV for public use, performed comprehensive phylogenetic neighbor-Joining, maximum-likelihood, Bayesian and nucleotide distance analyses, and compared these inference methods. An updated NDV classification and nomenclature system that incorporates phylogenetic topology, genetic distances, branch support, and epidemiological independence was developed. This new consensus system maintains two NDV classes and existing genotypes, identifies three new class II genotypes, and reduces the number of sub-genotypes. In order to track the ancestry of viruses, a dichotomous naming system for designating sub-genotypes was introduced. In addition, a pilot dataset and sub-trees rooting guidelines for rapid preliminary genotype identification of new isolates are provided. Guidelines for sequence dataset curation and phylogenetic inference, and a detailed comparison between the updated and previous systems are included. To increase the speed of phylogenetic inference and ensure consistency between laboratories, detailed guidelines for the use of a supercomputer are also provided. The proposed unified classification system will facilitate future studies of NDV evolution and epidemiology, and comparison of results obtained across the world.

Detection and phylogenetic analysis of parrot bornavirus 4 identified from a Swedish Blue-winged macaw (Primolius maracana) with unusual nonsuppurative myositis.

Background: The genus Orthobornavirus comprises RNA viruses infecting humans, mammals, birds and reptiles, where parrot bornavirus 1 to 8 causes fatal neurological and/or gastrointestinal syndromes in psittacines. There is, to the best of our knowledge, no publication describing avian bornaviruses in pet parrots in Sweden. We aimed to identify and to produce epidemiologic knowledge about the etiologic agent associated with a history of severe weight loss and death of a Primolius maracana.Methods and results: The results of histopathology, immunohistochemistry and real-time RT-PCR were compatible with avian bornavirus infection. Sequencing indicated infection by parrot bornavirus 4 (PaBV-4). The genotype reported shared high identity with PaBV-4 identified from pet psittacines and from wild birds in several countries. The N gene and X protein showed genotype clusters formation. P protein revealed to be more conserved within and between species of bornaviruses. Findings suggest horizontal transmission within and between avian orders and species.Conclusion: There seems to be a worldwide trading without biosafety measures, hence, further disease transmission could be avoided. For screening purposes, the P gene is a good candidate as a universal target in molecular diagnostics. Wild birds may be key pieces in the puzzle of bornavirus epidemiology.