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

Figures & data

Figure 1. Borna disease virus 1 (BoDV-1) genomic map and protein-coding mRNA transcripts. BoDV uses alternative transcription strategies, like over-lapping open reading frames (ORFs) and usage of host cellular splicing mechanisms. Abbreviations: S1–S3 initiation sites of transcription; T1–T4 termination sites of transcription; (N) nucleoprotein gene; (X) X protein gene; (P) phosphoprotein gene; (M) matrix protein gene; (G) glycoprotein gene; (L) RNA-dependent RNA-polymerase gene [1]. The genomic map is similar for all bornaviruses, as far as known, except for Queensland carbovirus and the Southwest carbovirus, which have the gene order 3ʹ-N-X-P-G-M-L-5ʹ, representing a transposition of the G and M genes [19].

Figure 2. Phylogeny shows the species and strains belonging to the Bornaviridae family, which were associated with the development of neurological and/or gastrointestinal disease and/or death of its hosts. The phylogenetic relationship between species and strains was based on a segment of the M gene. Sequences identified by GenBank® accession numbers and abbreviated name of virus. Bornaviruses marked with asterisks are not classified following the currently accepted taxonomy [4]. ABBV-1 to 2 = aquatic bird bornavirus 1 to 2, BoDV-1 to 2 = Borna disease virus 1 to 2, CnBV-1 to 3 = canary bornavirus 1 to 3, EsBV-1 = estrildid finch bornavirus 1, JCPV = jungle carpet python virus. LGSV-1 = Loveridge’s garter snake virus 1, SWCPV = southwest carpet python virus, PaBV-1 to 8 = parrot bornavirus 1 to 8, VSBV-1 = variegated squirrel bornavirus 1.

Figure 3. X-ray performed on the parrot case number B261018, using barium contrast. (a) Dorsal-ventral view and (b) side-to-side view after the introduction of barium contrast in the crop (marked with the blue arrow). (c) Dorsal-ventral view and (d) side-to-side view of the diffusion start of barium contrast in the proventriculus. (e) Dorsal-ventral view and (f) lateral-lateral view allowing the visualization of the diffusion of barium contrast throughout the proventriculus, highlighting its delineation, which revealed a severe dilatation (marked with the blue arrow).

Figure 4. The proventriculus of parrot case number B261018, at necropsy. The severe dilatation (asterisk) produced a small rupture in the proventriculus wall (arrow).

Table 1. The per cent identity between the PaBV-4 genotypes detected in Portugal and bornaviruses worldwide distributed, regarding X and P protein.

	Amino acid sequence of X to P protein (287 amino acid length) from bornaviruses worldwide distributed
	X protein (X)					Phosphoprotein (P)
	Total		Non-shared region		Shared region		Non-shared region		Total		Sequences compared
Bornaviruses ^a Worldwide distributed	86 amino acid length		18 amino acid length		68 amino acid length		133 amino acid length		201 amino acid length		Present study versus
	Id. (%) ^b	E value ^c	Id. (%) ^b	E value ^c	Id. (%) ^b	E value ^c	Id. (%) ^b	E value ^c	Id. (%) ^b	E value ^c	Present study	GenBank® Accession nº	Hosts
Avian
Psittaciform 1 orthobornavirus	74 - 95	4e^−48 - 3e^−32	81 - 100	5e^−17 - 2e^−12	91 - 99	2e^−43 - 8e^−21	50 - 82	4e^−53 - 3e^−08	92 - 98	1e^−40 - 9e^−20	B240818	KU748814.1; MK192982.1; JX065204.2; EU781967.1; FJ620690.1; JX065207.1; GU249595.2; JX065210.2; FJ169440.1; NC_030689.1	Parrot
	75 - 100	6e^−62 - 1e^−35	81 - 100	5e^−17 - 2e^−12	94 - 99	1e^−40 - 1e^−19	42 - 87	2e^−60 - 1e^−10	89 - 93	6e^−122 - 2e^−109	B261018
Psittaciform 2 orthobornavirus	51 - 53	1e^−25 - 1e^−24	81	2e^−13	69 - 77	5e^−20 - 2e^−20	38 - 41	2e^−04	72	4e^−19 - 2e^−18	B240818	AB519144.1; KT378600.1	Parrot
	53	1e^−26 - 1e^−26	81	2e^−13	74	1e^−19 - 2e^−19	55 - 60	1e^−0.7 - 1e^0.3	64	7e^−74 -1e^−72	B261018
Passeriform 1 orthobornavirus	51 - 58	1e^−27 - 7e^−23	75	2e^−12	71 - 73	3e^−21 - 1e^−19	33 - 38	5e^−05 - 1e^−0.9	72 - 73	5e^−19 - 3e^−18	B240818	NC_030690.1; KC464471.1; NC_027892.1; KC464478.1; KC595273.2; NC_024296.1	Canary
	53 - 57	1e^−29 - 2e^−25	75 - 81	2e^−13 - 2e^−12	73 - 75	2e^−20 - 1e^−18	30 - 33	5e^−04 - 1e^−2.8	63	1e^−83 -7e^−82	B261018
Passeriform 2 orthobornavirus	64	2e^−23	81	3e^−12	73	4e^−20	33	1e^0.96	75	2e^−19	B240818	KF680099.1	Estrildid
	63	1e^−34	81	3e^−12	76	2e^−19	36	1e^−5.1	65	2e^−70	B261018
Waterbird 1 orthobornavirus	56 - 57	2e^−27 - 3e^−26	75 - 81	2e^−13 - 2e^−12	64 - 69	3e^−19 - 4e^−18	33 - 37	2e^−04 - 1e^−1.1	67 - 72	2e^−16 - 5e^−16	B240818	KF578398.1; NC_029642.1; NC_030691.1; KJ756399.2	Duck Goose
	55 - 56	7e^−29 - 3e^−28	75 - 81	2e^−13 - 2e^−12	69 - 75	9e^−20 - 9e^−18	44 - 45	1e^−4.3 - 1e^−0.8	63 - 65	2e^−73 - 3e^−72	B261018
Mammalian
Mammalian 1 orthobornavirus	45 - 50	6e^−20 – 6e^−07	75	2e^−12 - 7e^−11	61 - 64	9e^−25 - 2e^−23	34 - 38	1e^−3.6- 1e^3.85	62 - 65	5e^−23 - 5e^−22	B240818	AJ311522.1; NC_030692.1; AJ311524.1; U04608.1	Horse
	46 - 49	2e^−21 - 1e^−20	75	2e^−12 - 7e^−11	65 - 67	6e^−24 - 9e^−23	37 - 44	1e^−4.6 - 1e^−0.4	56 - 58	1e^−81 - 2e^−80	B261018
Mammalian 2 orthobornavirus	49	9e^−22	75	2e^−12 - 1e^−11	69	2e^−20	36	2e^−04	70	4e^−18	B240818	KY488729.1; MF597762.1; NC_030701.1; LN713681.1	SquirrelHuman
	52	5e^−24	75	2e^−12 - 1e^−11	74	5e^−20	38	1e^−01	63	1e^−80	B261018
Reptilian
Elapid 1 orthobornavirus	37	2e^−05	53	1e^−6.2 -1e^−5.8	44	3e^−11	41	e^4.29	46	8e^−09	B240818	KM114265.1; NC_024778.1	Snake
	37	5e^−07	53	1e^−6.2 - 1e^−5.8	45	5e^−09	41	e^4.29	39	7e^−41	B261018
Queensland carbovirus	*	---	*	---	83	1e^2.1	63	1e^1.1	41	1e^1.6.	B240818	NC_039013.1	Snake
	*	---	*	---	83	1e^2.2	*	---	45	1e^1.5	B261018
Southwest carbovirus	36	1e^1.6	*	---	36	1e^1.3	36	1e^−0.1	80	1e^1.8	B240818	NC_039014.1	Snake
	36	1e^1.5	*	---	36	1e^1.2	55	1e^0.9	50	1e^0.4	B261018

^a Bornaviruses identification as established by the International Committee on Taxonomy of Viruses (ICTV) Bornaviridae Study Group.

^b The highest percent identity of all query-subject alignment given by Basic Local Alignment Search Tool (BLAST®).

^c The best (lowest) Expect value (E value) of all alignments from that database sequence given by BLAST®.

* No significant similarity found.

Figure 5. Phylogenetic relationships between PaBV-4 genotypes identified in the present study and PaBV-4 detected in wild birds as in pet parrots, regarding N gene. The evolutionary history was inferred using the Neighbor-Joining method [21]. The confidence probability was estimated using the bootstrap test (1000 replicates) and is shown above the branches [22,23]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura two-parameter method [24] and are in the units of the number of base substitutions per site. The analysis involved 60 nucleotide sequences. All positions containing gaps and missing data were eliminated. There was a total of 163 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [20]. Sequences identified by GenBank® accession numbers, abbreviation name of virus and its hosts, year and geographic origin of sampling. PaBV-4 = parrot bornavirus 4. The sequences marked with a circle were produced during this study and triangles marked nucleotide sequences identified from wild birds.

Figure 6. Phylogenetic relationships between PaBV-4 genotypes identified in the present study and bornaviruses detected in mammalian and in wild birds, based on N gene (a) and on N protein (b). The evolutionary history was inferred using the Neighbor-Joining method [21]. The confidence probability (multiplied by 100) was estimated using the bootstrap test (1000 replicates) and is shown next to the branches [22,23]. Both trees were drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Both trees were rooted with a vaccine strain (accession nº DQ680832.1). Evolutionary analyses were conducted in MEGA7 [20]. (a) The evolutionary distances were computed using the Kimura two-parameter method [24] and are in the units of the number of base substitutions per site. The analysis involved 23 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 215 positions in the final dataset. (b) The evolutionary distances were computed using the Poisson correction method [25] and are in the units of the number of amino acid substitutions per site. The analysis involved 23 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 71 positions in the final dataset. Sequences identified by GenBank® accession numbers, abbreviation name of virus and its hosts, year and geographic origin of sampling. ABBV-1 = aquatic bird bornavirus 1, ABBV-2 = aquatic bird bornavirus 2, BoDV-1 = Borna disease virus 1, PaBV-4 = parrot bornavirus 4, VSBV-1 = variegated squirrel bornavirus 1. The sequences marked with a circle were produced during this study and triangles marked nucleotide sequences identified from samples of wild birds.

Figure 7. Phylogenetic relationships between PaBV-4 genotypes identified in the present study and bornaviruses detected in mammalian and wild birds, based on P gene (a) and on P protein (b). The evolutionary history was inferred using the Neighbor-Joining method [21]. The confidence probability (multiplied by 100) was estimated using the bootstrap test (1000 replicates) and is shown next to the branches [22,23]. Both trees were drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Both trees were rooted with a vaccine strain (accession nº DQ680832.1). Evolutionary analyses were conducted in MEGA7 [20]. (a) The evolutionary distances were computed using the Kimura two-parameter method [24] and are in the units of the number of base substitutions per site. The analysis involved 20 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 386 positions in the final dataset (b). The evolutionary distances were computed using the Poisson correction method [25] and are in the units of the number of amino acid substitutions per site. The analysis involved 20 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 110 positions in the final dataset. Sequences identified by GenBank® accession numbers, abbreviation name of virus and its hosts, year and geographic origin of sampling. ABBV-1 = aquatic bird bornavirus 1, ABBV-2 = aquatic bird bornavirus 2, BoDV-1 = Borna disease virus 1, PaBV-4 = parrot bornavirus 4, VSBV-1 = variegated squirrel bornavirus 1. The sequences marked with a circle were produced during this study and triangles marked nucleotide sequences identified from samples of wild birds.

Lipkin WI, Briese T, Hornig M. Borna disease virus – fact and fantasy. Virus Res. 2011;162(1–2):1–14.

 Web of Science ®Google Scholar

Hyndman TH, Shilton CM, Stenglein MD, et al. Divergent bornaviruses from Australian carpet pythons with neurological disease date the origin of extant Bornaviridae prior to the end-Cretaceous extinction. PLoS Pathog. 2018 20;14(2):1006881.

 Web of Science ®Google Scholar

Maes P, Amarasinghe GK, Ayllón MA, et al. Taxonomy of the order Mononegavirales: update 2019. Arch Virol. 2019;164(7):1967.

 Web of Science ®Google Scholar

Saitou N, Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4(4):406.

 Web of Science ®Google Scholar

Dopazo J. Estimating errors and confidence intervals for branch lengths in phylogenetic trees by a bootstrap approach. J Mol Evol. 1994;38(3):300.

 Web of Science ®Google Scholar

Rzhetsky A, Nei MA. A simple method for estimating and testing minimum evolution trees. Mol Biol Evol. 1992;9(5):945.

 Web of Science ®Google Scholar

Kimura M. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980;16(16):111.

 Google Scholar

Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33(7):1870.

 PubMed Web of Science ®Google Scholar

Zuckerkandl E, Pauling L. Evolutionary divergence and convergence in proteins. In: Bryson V, Vogel HJ, editors. Edited in evolving genes and proteins. New York: Academic Press; 1995. p. 97.

 Google Scholar