Complete genome sequence of a novel avian paramyxovirus isolated from wild birds in South Korea

A novel avian paramyxovirus (APMV), Cheonsu1510, was isolated from wild bird feces in South Korea and serologically and genetically characterized. In hemagglutination inhibition tests, antiserum against Cheonsu1510 showed low reactivity with other APMVs and vice versa. The complete genome of Cheonsu1510 comprised 15,408 nucleotides, contained six open reading frames (3’-N-P-M-F-HN-L-5’), and showed low sequence identity to other APMVs (< 63%) and a unique genomic composition. Phylogenetic analysis revealed that Cheonsu1510 was related to but distinct from APMV-1, -9, and -15. These results suggest that Cheonsu1510 represents a new APMV serotype, APMV-17. Electronic supplementary material The online version of this article (doi:10.1007/s00705-017-3588-6) contains supplementary material, which is available to authorized users.

The Ministry of Environment has conducted an AI surveillance program of migratory birds in South Korea since 2012. On January 7, 2015, a viral agent with hemagglutination activity was isolated from a single fecal sample collected from a migratory bird habitat in Cheonsu-bay, in the western region of South Korea (GPS coordinates 36°36′52.84″ N, 126°29′12.31″ E), but it was not an AI virus. In this study, we provide the first description of the serological and genomic features of this novel APMV virus, APMV/wild birds/Cheonsu1510/2015 (Cheonsu1510).
To determine the serotype of the unidentified APMV, a cross-hemagglutination-inhibition (HI) test was performed using a reference panel comprising antigens and chicken antisera against representatives of APMV-1 to APMV-9 (except APMV-5; National Veterinary Service Laboratories, USA) using the method outlined in the Terrestrial Manual of the World Organisation for Animal Health (OIE) [5]. Chicken antiserum against Cheonsu1510 was prepared by intravenously injecting 3-week-old specific pathogen-free (SPF) chickens with purified Cheonsu1510 (10 9.0 50% egg infectious dose [EID 50 ] per dose) as described previously [7].
Viruses propagated in 9-to 11-day-old SPF embryonated chicken eggs were used for total RNA extraction using TRIzol Reagent (Life Technologies, USA). The nucleotide (nt) sequence of the full viral genome was determined using a next-generation sequencing (NGS) approach. The cDNA libraries were prepared for 100-bp paired-end sequencing using a TruSeq RNA Sample Preparation Kit (Illumina, CA, USA) [3]. Briefly, mRNAs were purified and fragmented from 2 μg of total RNA using oligo (dT) magnetic beads and used as a template for cDNA synthesis through random hexamer priming. Paired-end sequencing was performed using an Illumina HiSeq2500 System (Illumina). The reads of the full-length viral genome were assembled de novo with SPAdes assembler version 3.7 [3]. To confirm the NGS results, a conventional reverse transcription polymerase chain reaction (RT-PCR) (iNtRon Biotechnology, Seoul, Korea) was performed with virus-specific primers (sequences available on request). PCR-amplified segments were purified using a QIAquick PCR Purification Kit (QIA-GEN, Hilden, Germany) and sequenced directly by the Sanger method at Macrogen, Korea. The sequence obtained by the Sanger method was 100% identical to that obtained by NGS sequencing. Nucleotide sequences were deposited in the GenBank database (accession no. MF594598).
Sequences were analyzed using BLAST (National Center for Biotechnology Information, USA) to identify related sequences and aligned using CLUSTALW2. Multiple alignments were used to infer the phylogenies, with the maximum-likelihood (ML) method implemented in MEGA 6. To obtain the ML tree topologies, 1,000 bootstrap replicates were performed for each dataset. The inferred tree topologies were inspected visually using FigTree version 1.3.1.
The antiserum titers against each representative APMV serotype were highest with the homologous viruses ( Table 1). The HI titer of antiserum against isolated Cheonsu1510 had the highest HI with the homologous virus (1:128) and reacted only weakly with APMV-1 (1:16) and -9 (1:8). The HI titers of the Cheonsu1510 isolate with antisera against the APMVs were less than 1:16 for all except APMV-9 (1:32). The Cheonsu1510 virus showed an R-value < 0.05 to other APMVs except for APMV-1 (0.063; Online Resource 1). Table 1 Results of cross-haemagglutination-inhibition tests with representative APMVs and homologous chicken antisera A cross-haemagglutination-inhibition (HI) test was performed using a reference panel comprising antigens and chicken anti-sera against representatives of APMV-1 to APMV-9 (except APMV-5; National Veterinary Service Laboratories, USA) using the method outlined in the World Organization for Animal Health (OIE) Terrestrial Manual [5] Antigen Antiserum to The complete genome of Cheonsu1510 consisted of 15,408 nt, and making it closest in size to APMV-9, at 15,438 nt (Fig. 1). The genomic organization of the virus was typical of the genus Avulavirus, with six genes (3'-leader-N-P-M-F-HN-L-trailer-5'), including intergenic regions of 10-35 nt. Theoretical amino acid (aa) lengths of the putative proteins were as follows: N, 487 aa; P, 439 aa; M, 365 aa; F, 551 aa; HN, 567 aa; L, 2203 aa. The 3' leader of Cheonsu1510 was 55 nt and was highly homologous to those of known APMV serotypes. The first 12 nt of the leader sequence (3'-UCU UUG UUU GGU -5') were identical to those of APMV-9. The 5' trailers of AMPVs had variable sizes (17-776 nt); the Cheonsu1510 5' trailer was 46 nt and was most closely related to those of APMV-9 and -15(Kr), both of which are 47 nt (Fig. 1). The first 12 nt of the 3' leader and 5' trailer sequences (5'-AGA AAC AAA CCA -3') of Cheonsu1510 were perfectly complementary to each other. The gene-start (GS) and gene-end (GE) sequences of the six genes were 3'-UGC CCA UC(C) UU-5' and 3'-AAUCU 6 -5' respectively, and were well conserved. The P gene of Cheonsu1510 contained a putative RNA editing site (3'-UUU UUC CC-5') at 2,272-2,281 in the genome, which relates to the production of additional V and W proteins through the insertion of single and double G residues, respectively [10]. The sequences were identical to those of most of the APMV groups, except for APMV-3 and -4 (3'-AAU UUC CC-5'), -11 (3'-UCU UAG UC-5'), and -14 (3'-AUU UUC CC-5'). The F protein cleavage site of Cheonsu1510 is D-R-E-G-R↓ L, which lacks multiple basic residues and resembles the aa motif of the lentogenic APMV-1. Comparison of the full genome sequences revealed that APMV-9 (63.0%), -15(Kr) (55.8%), -1 (55.7%), and -12 (51.9 %) are most closely related to Cheonsu1510, which shares less than 50% identity with representatives of other APMV groups (Online Resource 2).   As shown in Fig. 2A, a phylogenetic tree of complete genome sequences placed Cheonsu1510 on a major branch with APMV-1, -9, and -15(Kr), with a bootstrap value of 100. Within the group, Cheonsu1510 was closer to APMV-9 than APMV-1 and -15(Kr), consistent with the nt sequence identity results. The F and HN genes of Cheonsu1510 also clustered with APMV-1, -9, and -15(Kr), with a bootstrap value of 99, but clearly formed a separate phylogenetic group, especially from APMV-9 isolates from New York and Italy (Fig 2B and C). Consistently, when evolutionary distance from known serotypes of APMVs was calculated, Cheonsu1510 was relatively close to APMV-9 (0.453), -1 (0.586), and -15(Kr) (0.589; Online Resource 3).
The genome of Cheonsu1510 had several features in common with those of other APMVs: the gene order and composition (3'-leader-N-P-M-F-HN-L-trailer 5'), 3'-leader homology, existence of GE and GS signals, complementation of 3' and 5' terminal sequences (12 nt), and the existence of putative RNA editing sites in the P gene (Fig. 1). In contrast, the Cheonsu1510 genome displayed several differences from known APMVs, such as the length of the complete sequence, the transcriptional units, the untranslated region and aa sequences of the six genes, and the GE, GS and RNA-editing sequences of the P gene (Online Resource 4). Cheonsu1510 also harbors a unique cleavage site within the F protein (D-R-E-G-R↓ L). In addition, sequence identity between Cheonsu1510 and other AMPVs was relatively low (< 50%), with the exception of APMV-9, -15(Kr), and -1 at 63.0%, 55.8, and 55.7%, respectively. However, these levels of identity between distinct APMVs are not uncommon (e.g., between APMV-1 and APMV-15(Kr) (64.9%) and between APMV-12 and APMV-13 (58.2%); Online Resource 3)).
In summary, the results obtained in the present study indicate that the Cheonsu1510 isolate is sufficiently serologically and genetically different from known APMVs for it to be considered the prototype of a new APMV group, APMV-17. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.