Results

3.3.1 Sample collection and virus isolation

As a result of the avian influenza surveillance program on resident birds in Taiwan, a novel virus, AHRI33, showing hemagglutination activity was isolated from a

clinically healthy red collared dove (Streptopelia tranquebarica) in 2009. The AHRI33 virus propagated well in embryonated chicken eggs, and the harvested infective

allantoic fluid had an HA titer of 32. Moreover, we also obtained additional 13 AHRI33-like virus isolates from resident doves and pigeons in two different time periods. In 2009-2010, a research project for surveillance of AIV in resident birds was conducted. Swab samples were mainly collected from sparrows, doves, pigeons, and Chinese bulbuls, the dominant species of resident birds in Taiwan, and 11 AHRI33-like strains were isolated only from red collared doves. Since the 2015 outbreak of the clade 2.3.4.4 H5Nx highly pathogenic avian influenza (HPAI) viruses in Taiwan, dead birds found in public places have been sent to AHRI for detection of AIV. Two AHRI33-like strains were isolated. Among them, AHRI104 was obtained from a dead oriental turtle dove (Streptopelia orientalis) in 2016 and AHRI128 from a dead pigeon (Columba livia) in 2017.

3.3.2 Electron microscopy

The fine structure of the virion showed typical characteristics of a paramyxovirus.

The virion was pleomorphic, roughly spherical, and varied from 150 to 500 nm in diameter. Herringbone-like nucleocapsids were encased in a fragile envelope coated with approximately 10 nm long spikes (Fig. 3.1).

3.3.3 Intracerebral pathogenicity index

After inoculation of the AHRI33 virus into the cerebrums of one-day-old chickens, the obtained ICPI value was zero, indicating no clinical signs. This result suggested that the AHRI33 isolate was avirulent to chickens.

3.3.4 Serological characterization

As shown in Table 1, the titers of antisera against each representative APMV species were higher with the homologous virus. The AHRI33 isolate only showed titers of 1:4 to 1:16 with the reference antisera against APMV-1, -2, -3, -4, -6, -8, and -9, but it was antigenically similar to APMV-7 on the basis of HI typization (4-fold difference in HI titers). The R-value between AHRI33 and APMV-7 was then calculated based on the HI titers obtained from the cross-HI assay, and the R-value of 0.125 indicated antigenic similarity between these viruses, although the R-value was less than would be expected between viruses of the same serogroup.

3.3.5 Determination of nucleotide sequences of full-length APMV genome

The complete genome sequence of the AHRI33 isolate was characterized using a Sanger sequencing approach. Assembly of the sequences produced a contig of 16,914

nucleotides (nt), making it close in size to APMV-5 (GU206351, 17,262 nt, 348 nt longer than that of AHRI33), APMV-11 (NC_025407, 17,412 nt, 498 nt longer), and APMV-3/Netherlands (EU403085, 16,272 nt, 642 nt shorter). Other APMV genome sequences range in length from 14,880 nt for APMV-2/F8 (HQ896023) to 17,412 nt for APMV-11.

Six genes, nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F),

hemagglutinin-neuraminidase (HN), and large polymerase (L), were identified in the genome of the AHRI33 virus, showing a typical gene arrangement of APMVs

3'-leader-NP-P-M-F-HN-L-trailer-5' (Fig. 3.2), which predicted six viral proteins: NP, 461 amino acids (aa); P, 410 aa; M, 365 aa; F, 540 aa; HN, 571 aa, and L, 2226 aa. The 3’ leader sequence of the AHRI33 isolate was 55 nt in length, as conserved among most APMV species (Anderson andWang, 2011). The length of the trailer at the 5’ end was 506 nt. The first 13 nt of the leader sequence and the last 13 nt of the 5’ trailer sequence showed complete complement. The conserved sequences for the gene start (GS) and gene end (GE) of the AHRI33 virus were CCCCCNNUN and AAUNNU6-7, respectively.

The lengths of the intergenic region sequences ranged from 1 to 35 nt. The deduced amino acid sequence of the F gene cleavage site was STQQER/LFG, which lacked multiple basic residues at the C-terminus of the F2 protein and a phenylalanine residue at the N-terminus of the F1 protein.

3.3.6 Sequence comparison and phylogenetic analysis

Comparison of the AHRI33 full-length genome with those of known APMV species revealed that AHRI33 was more closely related to APMV-7 (62.4% nucleotide identity) than to the other APMVs (43.5-53.7%) (Table 3.2). The highest inter-serotypic identity was 68.1% between APMV-1 and -16, and the lowest was 42.7% between

APMV-4 and -6.

The nucleotide sequence identities of each AHRI33 gene to the corresponding genes of other APMVs ranged from 39.3% (P) to 68.0% (N) (Table 3.3). All six genes of AHRI33 were most similar to those of APMV-7, with identities of 55.7% to 68.0%.

The amino acid identities were consistent with the corresponding nucleotide identities (Table 3.3).

Phylogenetic trees were constructed based on alignment of the full-length genome of AHRI33 (Fig. 3.3), the F gene (Fig. 3.4), and the HN gene (Fig. 3.5) with

corresponding genes of representative APMV species. Phylogenetic analysis based on the full-length genome sequences (Fig. 3.3) revealed that the four AHRI33-like isolates were grouped along with APMV-7 and -11 but were distinguishable from these two species. Within this group, AHRI33-like isolates appeared to be more closely related to APMV-7 than to APMV-11. Phylogenetic analysis based on F gene sequences (Fig. 3.4) gave similar results. The other 13 AHRI33-like viruses isolated in Taiwan were highly similar to AHRI33, with 98.9% to 99.9% nucleotide identities of the fusion gene. All of them were assigned to the same cluster in the phylogenetic analyses (Fig. 3.4).

Phylogenetic analysis of amino acid sequences of the RdRp revealed that all APMVs clustered together in a group designated as subfamily Avulavirinae, when compared with the closely related Mumps virus as an outgroup (Fig. 3.6). Three main clades designated as genera Orthoavulavirus, Metaavulavirus, and Paraavulavirus were observed within the subfamily Avulavirinae. The AHRI33-like isolates characterized in this study nested within genus Metaavulavirus, and the branch length between the nearest node and the tip of the branch is 0.28.