ABSTRACT
Avian metapneumovirus (aMPV) causes a highly contagious upper respiratory and reproductive disease in chickens, turkeys, and ducks. Here, complete genome sequences of aMPV-B vaccine strains BR/1890/E1/19 (PL21, Nemovac; Boehringer Ingelheim Animal Health, Brazil) and BR/1891/E2/19 (1062; Hipraviar, France) were sequenced and compared with the pathogenic field strain VCO3/60616.
ANNOUNCEMENT
Genomic data from nontargeted next-generation sequencing (NGS) of clinical samples can simultaneously identify and pathotype viruses, predict their likelihood of causing severe disease, and provide information about the appropriate vaccines for disease control (1). During a project on improving NGS for diagnostics of avian pathogens in South American commercial chickens, all samples were preserved by spotting onto Whatman FTA cards (Millipore-Sigma) and shipped at room temperature to USDA-ARS Southeast Poultry Research Laboratory (Athens, GA), and avian metapneumovirus (aMPV; family Pneumoviridae) was commonly identified (2). The aMPVs are important respiratory pathogens consisting of subtypes A to D and two unclassified subtypes (3–8). Here, we sequenced the full genomes of the most commonly used aMPV-B vaccine strains in Brazil, BR/1890/E1/19 (PL21; Nemovac; batch 009/18) and BR/1891/E2/19 (1062; Hipraviar; batch 019/18).
Lyophilized vaccine samples were reconstituted and spotted onto FTA cards, followed by total RNA extraction using the MagMAX-96 RNA isolation kit (Thermo Fisher Scientific, USA) as recently described (9). Random amplicons were produced using sequence-independent, single-primer amplification (10), and sequencing libraries were prepared using the Nextera DNA Flex kit (Illumina, USA) according to the manufacturer’s recommendations. After quantification using the Thermo Fisher Scientific Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) and Agilent TapeStation HS kits, pooled libraries (4 nM; 8 μL each) were spiked with 5% PhiX control library v3 and sequenced in paired-end format (Illumina MiSeq reagent kit v3). The raw data were processed using a nontargeted classification and de novo assembly pipeline with Trim Galore v0.6.7 (https://github.com/FelixKrueger/TrimGalore) and MEGAHIT v1.2.9 (11). Variant sites (12) with a minor frequency of ≥10% were replaced with IUPAC codes. The assembled consensus sequences were annotated using Geneious Prime v2023.1.1 as recently described (2, 13), aligned with published sequences using MAFFT v7.490 (14), trimmed using trimAl v1.3 (15), and subjected to phylogenetic analysis using MEGA (maximum likelihood [ML] method; 1,000 bootstrap replicates) (16). Unless stated otherwise, default parameters were used for all tools.
The genome sequences of BR/1890/E1/19 and BR/1891/E2/19 are 13,483 and 13,513 nucleotides (nt) long (100% coverage based on the reference field strain VCO3/60616/86 [GenBank accession number AB548428]; identified with 411,871 and 611,583 read pairs; median coverage depths, 6,052× and 9,215×, respectively), with 43.3% GC content. Both genomes have typical metapneumovirus organization with 3′-leader (27 and 40 nt) and 5′-trailer (123 and 136 nt) regions flanking the N (1,176 nt), P (840 nt), M (765 nt), F (1,617 nt), M2-1/M2-2 (561/222 nt, respectively), SH (528 nt), G (1,245 nt), and L (6,015 nt) genes. Strains BR/1890/E1/19 and BR/1891/E2/19 are most similar to VCO3/60616 (identities, 99.79% and 99.48%, respectively) (17–19) (Fig. 1A), but the G-gene sequences phylogenetically group BR/1890/E1/19 with Vietnamese Nemovac and Hipraviar vaccine strains (20) in a separate cluster from BR/1891/E2/19 (Fig. 1B). Compared to VCO3/60616, the vaccine strains have 38 nonsynonymous substitutions (Table 1), and the SH protein coding sequence of BR/1890/E1/19 is truncated (399 nt) by an A5764T mutation. The fusion protein cleavage site (99RKKR↓F102) is conserved, as is typical of aMPV-B viruses (19, 21). The G gene of BR/1891/E2/19 contains unusual C-to-T heterogeneity (n = 26), confirmed by resequencing and indicated by IUPAC codes. These data provide evolutionary information that may facilitate the use and development of aMPV vaccines.
FIG 1.
(A) Comparative pairwise identities of the complete genome and protein coding sequences of the vaccine strains BR/1890/E1/19 and BR/1891/E2/19 reported in this paper with the pathogenic field strain VCO3/60616 and (B) their phylogenetic relatedness with other aMPV-B viruses based on G gene nucleotide sequences, including the Vietnamese attenuated Nemovac and Hipraviar vaccine strains (shown in blue). Sequence names include the GenBank accession numbers and abbreviated country of origin.
TABLE 1.
Summary of 38 nonsynonymous mutations of Brazilian aMPV-B vaccine strains compared to the reference strain VCO3/60616a
| Genomic region | Data for strain: |
||||||
|---|---|---|---|---|---|---|---|
| VCO3/60616b |
BR/1890/E1/19c |
BR/1891/E2/19d |
|||||
| Positione | Codon | Amino acid residue | Codon | Amino acid residue | Codon | Amino acid residue | |
| N | 375 | CGA | Arg | CGA | Arg | CAA | Gln |
| 1220 | AAG | Lys | GAG | Glu | AAG | Lys | |
| P | 1577 | AAA | Lys | AGA | Arg | AAA | Lys |
| M | 2414 | TAC | Tyr | CAC | His | TAC | Tyr |
| 2768 | GGT | Gly | GGT | Gly | AGT | Ser | |
| F | 3210 | ATT | Ile | TTT | Phe | ATT | Ile |
| 3391 | TTC | Phe | TCC | Ser | TCC | Ser | |
| 3479 | AAC | Asn | AAC | Asn | AAA | Lys | |
| 3912 | GAA | Glu | GAA | Glu | AAA | Lys | |
| 4150 | CAA | Gln | CTA | Leu | CAA | Gln | |
| 4441 | GTA | Val | GTA | Val | GCA | Ala | |
| 4461 | GTA | Val | GTA | Val | ATA | Ile | |
| M2 | 5139 | AGC | Ser | AGC | Ser | GGC | Gly |
| SH | 5641 | TAT | Tyr | TAT | Tyr | CAT | His |
| 5695 | GAT | Asp | GAT | Asp | AAT | Asn | |
| 5777 | AAA | Lys | TAA | Stop codon | AAA | Lys | |
| 5812 | GTA | Val | GTA | Val | ATA | Ile | |
| G | 6106 | GGA | Gly | AGA | Arg | GGA | Gly |
| 6269 | GGT | Gly | GGT | Gly | GAT | Asp | |
| 6298–6299 | TTA | Leu | TTA | Leu | YYA | Xaa | |
| 6525 | AAA | Lys | AAA | Lys | AAC | Asn | |
| 6553 | TCC | Ser | TCC | Ser | YCC | Pro/Ser | |
| 6587 | GTA | Val | GTA | Val | GYA | Ala/Val | |
| 6592 | TCA | Ser | TCA | Ser | YCA | Pro/Ser | |
| 6671–6672 | ATT | Ile | ATT | Ile | AYY | Thr/Ile | |
| 6685 | TCA | Ser | TCA | Ser | YCA | Pro/Ser | |
| 6728 | CTC | Leu | CTC | Leu | CYC | Pro/Leu | |
| 6734 | ATC | Ile | ATC | Ile | AYC | Thr/Ile | |
| 6742 | TCG | Ser | TCG | Ser | YCG | Pro/Ser | |
| 6860 | GAA | Glu | GAA | Glu | GGA | Gly | |
| 7002 | AAA | Lys | AAA | Lys | AAT | Asn | |
| L | 9573 | CTA | Leu | CAA | Gln | CTA | Leu |
| 9819 | AGA | Arg | AAA | Lys | AAA | Lys | |
| 11367 | GTG | Val | GTG | Val | ATG | Met | |
| 11949 | GGG | Gly | GGG | Gly | GAG | Glu | |
| 12308 | CAC | His | CAC | His | TAC | Tyr | |
| 12984 | GCT | Ala | ACT | Thr | GCT | Ala | |
| 13139 | AGT | Ser | AGT | Ser | GGT | Gly | |
Variation in the nucleotide codons of the Brazilian genes is indicated with bold and underline. Unusual C-to-T heterogeneity in the G gene of BR/1891/E2/19 is indicated by IUPAC codes (“Y,” in italics).
VCO3/60616 is a pathogenic field strain (GenBank accession number AB548428).
PL21; Nemovac.
1062; Hipraviar.
In aligned consensus sequence.
Data availability.
The complete genome sequences of vaccine strains BR/1890/E1/19 and BR/1891/E2/19 reported in this paper have been deposited at GenBank under accession numbers OP572408 and OP572409, respectively. The raw data were deposited in the SRA under accession numbers SRR22875041 and SRR22875042, BioSample accession numbers SAMN32373034 and SAMN32373035, and BioProject accession number PRJNA915113.
ACKNOWLEDGMENTS
This study was supported by the Agricultural Research Service (ARS) (USDA CRIS project 6040-32000-082-00-D and ARS project 6040-32000-082-001T) and by a postdoctoral appointment to the Oak Ridge Institute for Science and Education (ORISE). The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA-ARS, BASE2BIO LLC, BIAH, or ORISE.
Contributor Information
Henry M. Kariithi, Email: henry.kariithi@usda.gov.
Jelle Matthijnssens, Katholieke Universiteit Leuven.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The complete genome sequences of vaccine strains BR/1890/E1/19 and BR/1891/E2/19 reported in this paper have been deposited at GenBank under accession numbers OP572408 and OP572409, respectively. The raw data were deposited in the SRA under accession numbers SRR22875041 and SRR22875042, BioSample accession numbers SAMN32373034 and SAMN32373035, and BioProject accession number PRJNA915113.