Complete Genome Sequence of an Avian Orthoavulavirus 13 Strain Detected in Ukraine

ABSTRACT

We report the complete genome sequence of an avian orthoavulavirus 13 strain, isolated from a white-fronted goose in the Odesa region of Ukraine in 2013. The detection of avian orthoavulavirus 13 in Ukraine confirms that the geographic distribution of this virus extends beyond Asia.

ANNOUNCEMENT

Avian paramyxoviruses (APMVs) belong to the recently assigned subfamily Avulavirinae of the family Paramyxoviridae and have been shown to infect a wide variety of poultry and wild bird species (1). Currently, there are 22 confirmed serotypes of APMVs. In this study, we report the complete genome sequence of an avian orthoavulavirus 13 (APMV-13) strain that was isolated in the Azov-Black Sea region of Ukraine in 2013.

A hemagglutinating agent was isolated from allantoic fluid harvested from 9-day-old specific-pathogen-free embryonated chicken eggs inoculated with feces of a white-fronted goose (Anser albifrons) collected during active surveillance of wild birds for APMV and avian influenza virus in Prymorske, Odesa region of Ukraine (N45°32′25.8, E29°36′38.4), in 2013 (2). This isolate weakly cross-reacted with antisera to APMV-1, APMV-3, and APMV-7 in the hemagglutination inhibition assay (3–5). A frozen cryovial containing allantoic fluid was transferred to the Southeast Poultry Research Laboratory of the U.S. Department of Agriculture (USDA) in Athens, GA, for sequencing.

Viral RNA was extracted from allantoic fluid using the MagMAX AI/ND viral RNA isolation kit (Applied Biosystems, USA). Host DNA was removed using an in-house depletion protocol (6). Sequence-independent single-primer amplification (7) utilizing a barcoded random octamer primer and Klenow fragments was used to produce random amplicons that were further processed using the Nextera DNA Flex library preparation kit (Illumina, USA). The distribution size and concentration of the prepared DNA library were checked on the Agilent TapeStation 4200 using high-sensitivity D5000 screen tape. Next-generation paired-end sequencing of the generated libraries was performed on an Illumina MiSeq instrument using the 600-cycle MiSeq reagent kit v3 (Illumina, USA). A total of 2,191,672 raw paired-end reads were generated. Their quality was assessed using FastQC v0.63 (8), residual adapter sequences were trimmed using Cutadapt v1.16.6 (9), and sequence data were de novo assembled using MIRA3 version 0.0.1 (10) within a customized workflow on the Galaxy platform (11), as described previously (12, 13). The obtained contigs were subjected to a BLASTn search and aligned with the full-length APMV-13 genome (GenBank accession number KX119151) (14) to obtain a draft genome scaffold. The genome consensus was then recalled from 187,971 raw APMV-13 reads using the BWA-MEM (15). The median read depth of the APMV-13 assembly was 1,480. Twenty nucleotides missing at the 3′ terminus of the genome were sequenced using Sanger technology utilizing a single 3′ nucleotide tailing reaction, followed by a reverse transcription reaction targeted to the common polynucleotide tail, as described previously (16). The final genome consensus of the isolate designated white-fronted goose/Ukraine/Prymorske/71-15-02/2013 was 16,152 nucleotides long (100% genome coverage). The BLAST search revealed that this sequence showed the highest nucleotide identity of 99.66% to APMV-13 strain goose/Kazakhstan/5751/2013 (KU646513) (Fig. 1) (17). To date, Ukraine is the only country outside Asia (China, Japan, Kazakhstan, and South Korea) where APMV-13 has been detected (14, 17–21). The previously isolated Ukrainian APMV-13 strain white-fronted goose/Ukraine/Askania-Nova/48-15-02/2011 (KX119151) shared only 96.79% nucleotide identity with the isolate presented in this study. This may be explained by intercontinental viral transmission, as the Azov-Black Sea region of Ukraine is one of the major stopping point locations for migratory birds (2, 22–25). This complete genome sequence information of APMV-13 from Ukraine expands our knowledge and facilitates future studies on orthoavulavirus diversity and evolution.

FIG 1.

Phylogenetic analysis of all APMVs within the subfamily Avulavirinae based on the complete F gene nucleotide sequences constructed in MEGA7 with the maximum likelihood method based on the general time reversible and JTT matrix-based model, respectively. The trees with the highest log likelihoods (−39657.29) are shown. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 35 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1,526 positions in the final data set. The APMV-13 isolate used in this study is shown in magenta.

Data availability.

The complete genome sequence of the APMV-13 isolate white-fronted goose/Ukraine/Prymorske/71-15-02/2013 has been deposited in GenBank under the accession number OQ286221. Raw data were deposited in the SRA under accession number SRR23112095.