ABSTRACT
This study presents a draft genome sequence of a Newcastle disease virus (NDV) strain (VFAR-136) isolated from a fighting cock (Gallus gallus) in the south of Peru. Strain VFAR-136 is a new report of NDV genotype VII circulating in Peru.
ANNOUNCEMENT
Newcastle disease (ND) is an avian viral disease that affects the poultry industry worldwide. ND is caused by Newcastle disease virus (NDV), recently known as Avian orthoavulavirus 1 (AOAV-1); it belongs to the genus Orthoavulavirus in the family Paramyxoviridae and has a negative-sense single-stranded RNA (−ssRNA) genome (1). According to molecular analysis, NDV strains have been classified as class I (single genotype) or class II (genotypes I to XXI). The class I strains are avirulent, while class II strains are generally lentogenic, mesogenic, or velogenic (2). In recent years, only subgenotype XIIa has been reported as circulating in Peru (3–5).
In this study, an NDV outbreak in fighting cocks (Gallus gallus) was detected in 2022 in Chincha, Peru. Viral isolation was performed in 9-day-old specific-pathogen-free (SPF) embryonated chicken eggs from a tracheal swab (6). Viral RNA (500 ng) from the isolated virus (VFAR-136) was extracted using the QIAamp viral RNA minikit (Qiagen), double-stranded cDNA was synthesized, and a library was prepared using the SQK-DCS109 kit (Oxford Nanopore Technologies). The double-stranded cDNA was prepared with SuperScript IV reverse transcriptase (Invitrogen), LongAmp Taq 2× master mix (New England), and PR2 primer (Oxford Nanopore Technologies) (7). End prep repair and dA-tailing were performed using Ultra II end prep enzyme mix (New England Biolabs). The sample was ligated to the flow cell adapter with blunt/TA ligase master mix (New England Biolabs). Purification steps were performed using AMPure XP beads (Beckman Coulter). A portion (50 ng) of the library (prepared using the SQK-DCS109 kit) was loaded into a FLO-MIN106 (R9.4) flow cell. The sample was sequenced on a MinION Mk1b device, using the Fast base-calling model, with MinKNOW software v.22.08.8. After base calling with Guppy v.6.37 using the high-accuracy model, the fastq files were analyzed using the Galaxy Web platform (https://usegalaxy.eu/) (8). Adapters were removed using Porechop (Galaxy v.0.2.4+galaxy0). The remaining reads were assembled de novo using rnaviralSPAdes (Galaxy v.3.15.4+galaxy2), Flye (Galaxy v.2.9+galaxy0), MEGAHIT (Galaxy v.1.2.9+galaxy0), and finally, WTDBG (Galaxy v.1.2.8.1). We trimmed the 5′ end because it had a low average read depth (<20×). Alignment and phylogenetic analysis were conducted using MAFFT on XSEDE v.7.490 (9) and the MEGA11 v.11.013 program (10).
Genome sequencing generated 356,400 reads, comprising 6.41 Gb with an N50 value of 1.65 kb. Assembly generated a viral genome of 11,041 nucleotides (nt) (average depth, 45.46×), with 45% G+C content. Local alignment using BLASTn showed high similarity (95%) with other NDV genotype VII strains (±95%), such as Chicken/China/SDSG01/2011 (GenBank accession no. JN400896.1), Md/CH/LGD/1/2005 (KM885167.1), Muscovy duck/China (Fujian)/FP1/02 (FJ872531.1), and BP01 (JN599167.1). Phylogenetics analysis based on the whole genome placed strain VFAR-136 within the genotype VII clade (100% bootstrap) (Fig. 1). The analysis suggested that strain VFAR-136 represents a virulent pathotype due to the presence of a polybasic amino acid motif (112RRQKR↓F117) in the F protein cleavage site, which is referred to as a key determinant of virulence (11). The genomic sequence of strain VFAR-136 will be important for the development of homolog vaccines against genotype VII strains circulating in Peru and for epidemiological surveillance studies.
FIG 1.
Phylogenetic tree constructed using the neighbor-joining method with 1,000 bootstraps. Evolutionary distances were calculated using the Tamura-Nei method. The rate variation among sites was modeled with a gamma distribution of 1. Gaps and missing data were discarded (complete deletion option). A total of 10,942 positions were analyzed. The analysis was performed using the MEGA11 program. The new NDV genotype VII isolate from the current study (VFAR-136) is indicated with a red circle. The NDV strains isolated from Peru in the other studies are marked with red triangles.
Data availability.
The whole-genome sequence of strain VFAR-136 has been deposited at GenBank under accession no. OP586782. The version described in this paper is the first version. The raw data were submitted to the SRA under accession no. PRJNA914773.
ACKNOWLEDGMENT
This study was funded by Farmacológicos Veterinarios S.A.C. (FARVET).
Contributor Information
Katherine Calderón, Email: kcalderon@farvet.com.
Jelle Matthijnssens, Katholieke Universiteit Leuven.
REFERENCES
- 1.Cattoli G, Susta L, Terregino C, Brown C. 2011. Newcastle disease: a review of field recognition and current methods of laboratory detection. J Vet Diagn Invest 23:637–656. doi: 10.1177/1040638711407887. [DOI] [PubMed] [Google Scholar]
- 2.Dimitrov KM, Abolnik C, Afonso CL, Albina E, Bahl J, Berg M, Briand FX, Brown IH, Choi KS, Chvala I, Diel DG, Durr PA, Ferreira HL, Fusaro A, Gil P, Goujgoulova GV, Grund C, Hicks JT, Joannis TM, Torchetti MK, Kolosov S, Lambrecht B, Lewis NS, Liu H, Liu H, McCullough S, Miller PJ, Monne I, Muller CP, Munir M, Reischak D, Sabra M, Samal SK, Servan de Almeida R, Shittu I, Snoeck CJ, Suarez DL, Van Borm S, Wang Z, Wong FYK. 2019. Updated unified phylogenetic classification system and revised nomenclature for Newcastle disease virus. Infect Genet Evol 74:103917. doi: 10.1016/j.meegid.2019.103917. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Diel DG, da Silva LHA, Liu H, Wang Z, Miller PJ, Afonso CL. 2012. Genetic diversity of avian paramyxovirus type 1: proposal for a unified nomenclature and classification system of Newcastle disease virus genotypes. Infect Genet Evol 12:1770–1779. doi: 10.1016/j.meegid.2012.07.012. [DOI] [PubMed] [Google Scholar]
- 4.Chumbe A, Izquierdo-Lara R, Tataje-Lavanda L, Figueroa A, Segovia K, Gonzalez R, Cribillero G, Montalvan A, Fernández-Díaz M, Icochea E. 2015. Characterization and sequencing of a genotype XII Newcastle disease virus isolated from a peacock (Pavo cristatus) in Peru. Genome Announc 3:e00792-15. doi: 10.1128/genomeA.00792-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Chumbe A, Izquierdo-Lara R, Tataje L, Gonzalez R, Cribillero G, González AE, Fernández-Díaz M, Icochea E. 2017. Pathotyping and phylogenetic characterization of Newcastle disease viruses isolated in Peru: defining two novel subgenotypes within genotype XII. Avian Dis 61:16–24. doi: 10.1637/11456-062016-Reg. [DOI] [PubMed] [Google Scholar]
- 6.OIE. 2021. Newcastle disease. OIE terrestrial manual 2021. https://www.woah.org/fileadmin/Home/fr/Health_standards/tahm/3.03.14_NEWCASTLE_DIS.pdf.
- 7.Olasz F, Tombácz D, Torma G, Csabai Z, Moldován N, Dörmő Á, Prazsák I, Mészáros I, Magyar T, Tamás V, Zádori Z, Boldogkői Z. 2020. Short and long-read sequencing survey of the dynamic transcriptomes of African swine fever virus and the host cells. Front Genet 11:758. doi: 10.3389/fgene.2020.00758. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Afgan E, Baker D, Batut B, Van Den Beek M, Bouvier D, Ech M, Chilton J, Clements D, Coraor N, Grüning BA, Guerler A, Hillman-Jackson J, Hiltemann S, Jalili V, Rasche H, Soranzo N, Goecks J, Taylor J, Nekrutenko A, Blankenberg D. 2018. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res 46:W537–W544. doi: 10.1093/nar/gky379. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Miller MA, Pfeiffer W, Schwartz T. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In 2010 Gatew Comput Environ Work GCE 2010. IEEE, Piscataway, NJ. doi: 10.1109/GCE.2010.5676129. [DOI] [Google Scholar]
- 10.Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549. doi: 10.1093/molbev/msy096. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Panda A, Huang Z, Elankumaran S, Rockemann DD, Samal SK. 2004. Role of fusion protein cleavage site in the virulence of Newcastle disease virus. Microb Pathog 36:1–10. doi: 10.1016/j.micpath.2003.07.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The whole-genome sequence of strain VFAR-136 has been deposited at GenBank under accession no. OP586782. The version described in this paper is the first version. The raw data were submitted to the SRA under accession no. PRJNA914773.