VIRUSES
crossm Complete Genome Sequence of an Avian Metapneumovirus Subtype A Strain Isolated from Chicken (Gallus gallus) in Brazil Laís S. Rizotto,b Guilherme P. Scagion,b Tereza C. Cardoso,d Raphael M. Simão,b Leonardo C. Caserta,e Julia C. Benassi,a Lara B. Keid,a,b Trícia M. F. de S. Oliveira,a,b Rodrigo M. Soares,b Clarice W. Arns,e Steven Van Borm,c Helena L. Ferreiraa,b Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of Sao Paulo (FZEA-USP), Pirassununga, Brazila; Graduate Program in Experimental Epidemiology of Zoonoses, Faculty of Veterinary Medicine and Animal Science, University of Sao Paulo (FMVZ-USP), Sao Paulo, Brazilb; Molecular Platform Unit, CODA-CERVA Veterinary and Agrochemical Research Centre, Brussels, Belgiumc; DAPSA Department, Laboratory of Animal Virology and Cell Culture, College of Veterinary Medicine, Universidade Estadual Paulista, Araçatuba, Sao Paulo, Brazild; Laboratory of Animal Virology, Institute of Biology, University of Campinas-UNICAMP, Campinas, Brazile
ABSTRACT We report here the complete genome sequence of an avian metapneumovirus (aMPV) isolated from a tracheal tissue sample of a commercial layer flock. The complete genome sequence of aMPV-A/chicken/Brazil-SP/669/2003 was obtained using MiSeq (Illumina, Inc.) sequencing. Phylogenetic analysis of the complete genome classified the isolate as avian metapneumovirus subtype A.
A
vian metapneumovirus (family Pneumoviridae, genus Metapneumovirus) (1) is the viral agent responsible for rhinotracheitis of turkeys and is associated with swollen head syndrome in chickens (2, 3). The tracheal tissue sample used in this study was obtained from a Brazilian commercial layer flock showing swollen head syndrome, previously detected by reverse transcription (RT)-PCR (4) and confirmed by real-time RT-PCR (5). Samples were collected according to international, national, and institutional guidelines for the care and use of animals. The virus was isolated in chicken embryo-related cells after three passages. The cell supernatant sample was pretreated according to a previous study (6). Afterward, RNA extraction was performed with TRIzol (Ambion, Thermo Fisher Scientific) and an RNeasy MinElute clean-up kit (Qiagen). The RNAs were quantified by spectrophotometry and Qubit (Invitrogen) fluorimetry. Both cDNA synthesis and double-stranded DNA synthesis were performed using the SuperScript IV first-strand cDNA synthesis (Invitrogen) and second-strand synthesis mRNA kits (New England Biolabs), respectively, following the manufacturers’ instructions using random hexamer primers. The libraries were prepared with the Nextera XT sample preparation kit (Illumina, Inc.) using standard Nextera XT adapters for library multiplexing. The quantification of the libraries was performed with the SYBR FAST qPCR master mix kit (Kapa Biosystems), and the sizes of the libraries were estimated with a 2100 Bioanalyzer (Agilent Genomics) before sequencing on a MiSeq platform using the MiSeq version 3 (600-cycle) reagent kit (Illumina, Inc.). The quality of the sequences was checked with FastQC version 0.10.1 (http://www .bioinformatics.babraham.ac.uk/projects/fastqc). Cutadapt version 1.3 (7) was used to trim stretches containing unidentified nucleotides (“N”) before quality trimming using Sickle version 1.210 (Q score, ⬍30; length, ⬍50 bp) (8). Cell culture contaminants were removed by mapping to Mycoplasma mycoides, M. conjunctivae, and M. genitalium Volume 5 Issue 29 e00688-17
Received 1 June 2017 Accepted 5 June 2017 Published 20 July 2017 Citation Rizotto LS, Scagion GP, Cardoso TC, Simão RM, Caserta LC, Benassi JC, Keid LB, Oliveira TMFDS, Soares RM, Arns CW, Van Borm S, Ferreira HL. 2017. Complete genome sequence of an avian metapneumovirus subtype A strain isolated from chicken (Gallus gallus) in Brazil. Genome Announc 5:e00688-17. https://doi.org/10.1128/genomeA.00688-17. Copyright © 2017 Rizotto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Helena L. Ferreira, [email protected]. L.S.R. and G.P.S. contributed equally to this article.
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Rizotto et al.
using the Burrows–Wheeler version 0.7.12 alignment tool (9). All reads (2,422,670) were mapped to the closest BLASTn hit (AY640317) using Geneious version 9.1.8 (Biomatter, Ltd.), resulting in a 14,377-bp contig (24,350 reads) with an average coverage of 473⫻. The protein-coding genes were predicted relative to the reference sequence AY640317 by GATU (10). Phylogenetic analyses of the complete genome confirmed the isolate as avian metapneumovirus subtype A. Accession number(s). The complete genome sequence of aMPV-A/chicken/BrazilSP/669/2003 has been deposited in GenBank under accession number MF093139. ACKNOWLEDGMENTS T.C.C. and C.W.A. are recipients of a fellowship from CNPq. This work was done with grants from FAPESP (2014/50696-4 and 2015/11510-5) and from CAPES (003/16) and the Newton Fund (5126-15-0).
REFERENCES 1. Adams MJ, Lefkowitz EJ, King AM, Harrach B, Harrison RL, Knowles NJ, Kropinski AM, Krupovic M, Kuhn JH, Mushegian AR, Nibert M, Sabanadzovic S, Sanfaçon H, Siddell SG, Simmonds P, Varsani A, Zerbini FM, Gorbalenya AE, Davison AJ. 2016. Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2016). Arch Virol 161: 2921–2949. https://doi.org/10.1007/s00705-016-2977-6. 2. McDougall JS, Cook JK. 1986. Turkey rhinotracheitis: preliminary investigations. Vet Rec 118:206 –207. https://doi.org/10.1136/vr.118.8.206. 3. Buys SB, Du Preez JH. 1980. A preliminary report on the isolation of virus causing sinusitis in turkeys in South Africa and attempts to attenuate the virus. Turkeys 28:36 – 46. 4. D’Arce RC, Coswig LT, Almeida RS, Trevisol IM, Monteiro MC, Rossini LI, Di Fabio J, Hafez HM, Arns CW. 2005. Subtyping of new Brazilian avian metapneumovirus isolates from chickens and turkeys by reverse transcriptase-nested-polymerase chain reaction. Avian Pathol 34: 133–136. https://doi.org/10.1080/03079450500059180. 5. Ferreira HL, Spilki FR, Santos MMABd, Almeida RSd, Arns CW. 2009. Comparative evaluation of conventional RT-PCR and real-time RT-PCR (RRT-PCR) for detection of avian metapneumovirus subtype A. Ciênc Rural 39: 1445–1451. https://doi.org/10.1590/S0103-84782009005000057.
Volume 5 Issue 29 e00688-17
6. Van Borm S, Rizotto LS, Ullmann LS, Scagion GP, Malossi CD, Simão RM, Araújo JP, Jr, Cordeiro IM, Keid LB, Oliveira TM, Soares RM, Martini MC, Orsi MA, Arns CW, Ferreira HL. 2016. Complete genome sequence of a vaccinal Newcastle disease virus strain isolated from an owl (Rhinoptynx clamator). Genome Announc 4(6):e01243-16. https://doi.org/10.1128/ genomeA.01243-16. 7. Martin M. 2011. Cutadapt removes adapter sequences from highthroughput sequencing reads. EMBnetJ 17:10 –12. https://doi.org/10 .14806/ej.17.1.200. 8. Joshi NA, Fass JN. 2011. Sickle: a sliding-window, adaptive, quality-based trimming tool for FastQ files (version 1.33). https://github.com/najoshi/ sickle. 9. Li H, Durbin R. 2009. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25:1754 –1760. https://doi .org/10.1093/bioinformatics/btp324. 10. Tcherepanov V, Ehlers A, Upton C. 2006. Genome annotation transfer utility (GATU): rapid annotation of viral genomes using a closely related reference genome. BMC Genomics 7:150. https://doi.org/10.1186/1471 -2164-7-150.
genomea.asm.org 2
crossm Complete Genome Sequence of an Avian Metapneumovirus Subtype A Strain Isolated from Chicken (Gallus gallus) in Brazil Laís S. Rizotto,b Guilherme P. Scagion,b Tereza C. Cardoso,d Raphael M. Simão,b Leonardo C. Caserta,e Julia C. Benassi,a Lara B. Keid,a,b Trícia M. F. de S. Oliveira,a,b Rodrigo M. Soares,b Clarice W. Arns,e Steven Van Borm,c Helena L. Ferreiraa,b Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of Sao Paulo (FZEA-USP), Pirassununga, Brazila; Graduate Program in Experimental Epidemiology of Zoonoses, Faculty of Veterinary Medicine and Animal Science, University of Sao Paulo (FMVZ-USP), Sao Paulo, Brazilb; Molecular Platform Unit, CODA-CERVA Veterinary and Agrochemical Research Centre, Brussels, Belgiumc; DAPSA Department, Laboratory of Animal Virology and Cell Culture, College of Veterinary Medicine, Universidade Estadual Paulista, Araçatuba, Sao Paulo, Brazild; Laboratory of Animal Virology, Institute of Biology, University of Campinas-UNICAMP, Campinas, Brazile
ABSTRACT We report here the complete genome sequence of an avian metapneumovirus (aMPV) isolated from a tracheal tissue sample of a commercial layer flock. The complete genome sequence of aMPV-A/chicken/Brazil-SP/669/2003 was obtained using MiSeq (Illumina, Inc.) sequencing. Phylogenetic analysis of the complete genome classified the isolate as avian metapneumovirus subtype A.
A
vian metapneumovirus (family Pneumoviridae, genus Metapneumovirus) (1) is the viral agent responsible for rhinotracheitis of turkeys and is associated with swollen head syndrome in chickens (2, 3). The tracheal tissue sample used in this study was obtained from a Brazilian commercial layer flock showing swollen head syndrome, previously detected by reverse transcription (RT)-PCR (4) and confirmed by real-time RT-PCR (5). Samples were collected according to international, national, and institutional guidelines for the care and use of animals. The virus was isolated in chicken embryo-related cells after three passages. The cell supernatant sample was pretreated according to a previous study (6). Afterward, RNA extraction was performed with TRIzol (Ambion, Thermo Fisher Scientific) and an RNeasy MinElute clean-up kit (Qiagen). The RNAs were quantified by spectrophotometry and Qubit (Invitrogen) fluorimetry. Both cDNA synthesis and double-stranded DNA synthesis were performed using the SuperScript IV first-strand cDNA synthesis (Invitrogen) and second-strand synthesis mRNA kits (New England Biolabs), respectively, following the manufacturers’ instructions using random hexamer primers. The libraries were prepared with the Nextera XT sample preparation kit (Illumina, Inc.) using standard Nextera XT adapters for library multiplexing. The quantification of the libraries was performed with the SYBR FAST qPCR master mix kit (Kapa Biosystems), and the sizes of the libraries were estimated with a 2100 Bioanalyzer (Agilent Genomics) before sequencing on a MiSeq platform using the MiSeq version 3 (600-cycle) reagent kit (Illumina, Inc.). The quality of the sequences was checked with FastQC version 0.10.1 (http://www .bioinformatics.babraham.ac.uk/projects/fastqc). Cutadapt version 1.3 (7) was used to trim stretches containing unidentified nucleotides (“N”) before quality trimming using Sickle version 1.210 (Q score, ⬍30; length, ⬍50 bp) (8). Cell culture contaminants were removed by mapping to Mycoplasma mycoides, M. conjunctivae, and M. genitalium Volume 5 Issue 29 e00688-17
Received 1 June 2017 Accepted 5 June 2017 Published 20 July 2017 Citation Rizotto LS, Scagion GP, Cardoso TC, Simão RM, Caserta LC, Benassi JC, Keid LB, Oliveira TMFDS, Soares RM, Arns CW, Van Borm S, Ferreira HL. 2017. Complete genome sequence of an avian metapneumovirus subtype A strain isolated from chicken (Gallus gallus) in Brazil. Genome Announc 5:e00688-17. https://doi.org/10.1128/genomeA.00688-17. Copyright © 2017 Rizotto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Helena L. Ferreira, [email protected]. L.S.R. and G.P.S. contributed equally to this article.
genomea.asm.org 1
Rizotto et al.
using the Burrows–Wheeler version 0.7.12 alignment tool (9). All reads (2,422,670) were mapped to the closest BLASTn hit (AY640317) using Geneious version 9.1.8 (Biomatter, Ltd.), resulting in a 14,377-bp contig (24,350 reads) with an average coverage of 473⫻. The protein-coding genes were predicted relative to the reference sequence AY640317 by GATU (10). Phylogenetic analyses of the complete genome confirmed the isolate as avian metapneumovirus subtype A. Accession number(s). The complete genome sequence of aMPV-A/chicken/BrazilSP/669/2003 has been deposited in GenBank under accession number MF093139. ACKNOWLEDGMENTS T.C.C. and C.W.A. are recipients of a fellowship from CNPq. This work was done with grants from FAPESP (2014/50696-4 and 2015/11510-5) and from CAPES (003/16) and the Newton Fund (5126-15-0).
REFERENCES 1. Adams MJ, Lefkowitz EJ, King AM, Harrach B, Harrison RL, Knowles NJ, Kropinski AM, Krupovic M, Kuhn JH, Mushegian AR, Nibert M, Sabanadzovic S, Sanfaçon H, Siddell SG, Simmonds P, Varsani A, Zerbini FM, Gorbalenya AE, Davison AJ. 2016. Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2016). Arch Virol 161: 2921–2949. https://doi.org/10.1007/s00705-016-2977-6. 2. McDougall JS, Cook JK. 1986. Turkey rhinotracheitis: preliminary investigations. Vet Rec 118:206 –207. https://doi.org/10.1136/vr.118.8.206. 3. Buys SB, Du Preez JH. 1980. A preliminary report on the isolation of virus causing sinusitis in turkeys in South Africa and attempts to attenuate the virus. Turkeys 28:36 – 46. 4. D’Arce RC, Coswig LT, Almeida RS, Trevisol IM, Monteiro MC, Rossini LI, Di Fabio J, Hafez HM, Arns CW. 2005. Subtyping of new Brazilian avian metapneumovirus isolates from chickens and turkeys by reverse transcriptase-nested-polymerase chain reaction. Avian Pathol 34: 133–136. https://doi.org/10.1080/03079450500059180. 5. Ferreira HL, Spilki FR, Santos MMABd, Almeida RSd, Arns CW. 2009. Comparative evaluation of conventional RT-PCR and real-time RT-PCR (RRT-PCR) for detection of avian metapneumovirus subtype A. Ciênc Rural 39: 1445–1451. https://doi.org/10.1590/S0103-84782009005000057.
Volume 5 Issue 29 e00688-17
6. Van Borm S, Rizotto LS, Ullmann LS, Scagion GP, Malossi CD, Simão RM, Araújo JP, Jr, Cordeiro IM, Keid LB, Oliveira TM, Soares RM, Martini MC, Orsi MA, Arns CW, Ferreira HL. 2016. Complete genome sequence of a vaccinal Newcastle disease virus strain isolated from an owl (Rhinoptynx clamator). Genome Announc 4(6):e01243-16. https://doi.org/10.1128/ genomeA.01243-16. 7. Martin M. 2011. Cutadapt removes adapter sequences from highthroughput sequencing reads. EMBnetJ 17:10 –12. https://doi.org/10 .14806/ej.17.1.200. 8. Joshi NA, Fass JN. 2011. Sickle: a sliding-window, adaptive, quality-based trimming tool for FastQ files (version 1.33). https://github.com/najoshi/ sickle. 9. Li H, Durbin R. 2009. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25:1754 –1760. https://doi .org/10.1093/bioinformatics/btp324. 10. Tcherepanov V, Ehlers A, Upton C. 2006. Genome annotation transfer utility (GATU): rapid annotation of viral genomes using a closely related reference genome. BMC Genomics 7:150. https://doi.org/10.1186/1471 -2164-7-150.
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