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Identification and complete genome sequencing of paramyxoviruses in mallard ducks (Anas platyrhynchos) using random access amplification and next generation sequencing technologies

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Abstract
Background: During a wildlife screening program for avian influenza A viruses (AIV) and avian paramyxoviruses (APMV) in Belgium, we isolated two hemagglutinating agents from pools of cloacal swabs of wild mallards (Anas platyrhynchos) caught in a single sampling site at two different times. AIV and APMV1 were excluded using hemagglutination inhibition (HI) testing and specific real-time RT-PCR tests. Methods: To refine the virological identification of APMV2-10 realized by HI subtyping tests and in lack of validated molecular tests for APMV2-10, random access amplification was used in combination with next generation sequencing for the sequence independent identification of the viruses and the determination of their genomes. Results: Three different APMVs were identified. From one pooled sample, the complete genome sequence (15054 nucleotides) of an APMV4 was assembled from the random sequences. From the second pooled sample, the nearly complete genome sequence of an APMV6 (genome size of 16236 nucleotides) was determined, as well as a partial sequence for an APMV4. This APMV4 was closely related but not identical to the APMV4 isolated from the first sample. Although a cross-reactivity with other APMV subtypes did not allow formal identification, the HI subtyping revealed APMV4 and APMV6 in the respective pooled samples but failed to identify the co-infecting APMV4 in the APMV6 infected pool. Conclusions: These data further contribute to the knowledge about the genetic diversity within the serotypes APMV4 and 6, and confirm the limited sensitivity of the HI subtyping test. Moreover, this study demonstrates the value of a random access nucleic acid amplification method in combination with massive parallel sequencing. Using only a moderate and economical sequencing effort, the characterization and full genome sequencing of APMVs can be obtained, including the identification of viruses in mixed infections.
Keywords
TURKEYS, PATHOGENICITY, YUCAIPA, VIRUS, INFECTION, SEROTYPE 3, AVIAN PARAMYXOVIRUS, SISPA, random amplification, next generation sequencing, avian paramyxovirus, mallard, APMV6, APMV4, INFLUENZA, CHICKENS, REVEALS

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MLA
Rosseel, Toon et al. “Identification and Complete Genome Sequencing of Paramyxoviruses in Mallard Ducks (Anas Platyrhynchos) Using Random Access Amplification and Next Generation Sequencing Technologies.” VIROLOGY JOURNAL 8 (2011): n. pag. Print.
APA
Rosseel, T., Lambrecht, B., Vandenbussche, F., van den Berg, T., & Van Borm, S. (2011). Identification and complete genome sequencing of paramyxoviruses in mallard ducks (Anas platyrhynchos) using random access amplification and next generation sequencing technologies. VIROLOGY JOURNAL, 8.
Chicago author-date
Rosseel, Toon, Benedicte Lambrecht, Frank Vandenbussche, Thierry van den Berg, and Steven Van Borm. 2011. “Identification and Complete Genome Sequencing of Paramyxoviruses in Mallard Ducks (Anas Platyrhynchos) Using Random Access Amplification and Next Generation Sequencing Technologies.” Virology Journal 8.
Chicago author-date (all authors)
Rosseel, Toon, Benedicte Lambrecht, Frank Vandenbussche, Thierry van den Berg, and Steven Van Borm. 2011. “Identification and Complete Genome Sequencing of Paramyxoviruses in Mallard Ducks (Anas Platyrhynchos) Using Random Access Amplification and Next Generation Sequencing Technologies.” Virology Journal 8.
Vancouver
1.
Rosseel T, Lambrecht B, Vandenbussche F, van den Berg T, Van Borm S. Identification and complete genome sequencing of paramyxoviruses in mallard ducks (Anas platyrhynchos) using random access amplification and next generation sequencing technologies. VIROLOGY JOURNAL. 2011;8.
IEEE
[1]
T. Rosseel, B. Lambrecht, F. Vandenbussche, T. van den Berg, and S. Van Borm, “Identification and complete genome sequencing of paramyxoviruses in mallard ducks (Anas platyrhynchos) using random access amplification and next generation sequencing technologies,” VIROLOGY JOURNAL, vol. 8, 2011.
@article{6676352,
  abstract     = {Background: During a wildlife screening program for avian influenza A viruses (AIV) and avian paramyxoviruses (APMV) in Belgium, we isolated two hemagglutinating agents from pools of cloacal swabs of wild mallards (Anas platyrhynchos) caught in a single sampling site at two different times. AIV and APMV1 were excluded using hemagglutination inhibition (HI) testing and specific real-time RT-PCR tests.
Methods: To refine the virological identification of APMV2-10 realized by HI subtyping tests and in lack of validated molecular tests for APMV2-10, random access amplification was used in combination with next generation sequencing for the sequence independent identification of the viruses and the determination of their genomes.
Results: Three different APMVs were identified. From one pooled sample, the complete genome sequence (15054 nucleotides) of an APMV4 was assembled from the random sequences. From the second pooled sample, the nearly complete genome sequence of an APMV6 (genome size of 16236 nucleotides) was determined, as well as a partial sequence for an APMV4. This APMV4 was closely related but not identical to the APMV4 isolated from the first sample. Although a cross-reactivity with other APMV subtypes did not allow formal identification, the HI subtyping revealed APMV4 and APMV6 in the respective pooled samples but failed to identify the co-infecting APMV4 in the APMV6 infected pool.
Conclusions: These data further contribute to the knowledge about the genetic diversity within the serotypes APMV4 and 6, and confirm the limited sensitivity of the HI subtyping test. Moreover, this study demonstrates the value of a random access nucleic acid amplification method in combination with massive parallel sequencing. Using only a moderate and economical sequencing effort, the characterization and full genome sequencing of APMVs can be obtained, including the identification of viruses in mixed infections.},
  articleno    = {463},
  author       = {Rosseel, Toon and Lambrecht, Benedicte and Vandenbussche, Frank and van den Berg, Thierry and Van Borm, Steven},
  issn         = {1743-422X},
  journal      = {VIROLOGY JOURNAL},
  keywords     = {TURKEYS,PATHOGENICITY,YUCAIPA,VIRUS,INFECTION,SEROTYPE 3,AVIAN PARAMYXOVIRUS,SISPA,random amplification,next generation sequencing,avian paramyxovirus,mallard,APMV6,APMV4,INFLUENZA,CHICKENS,REVEALS},
  language     = {eng},
  pages        = {11},
  title        = {Identification and complete genome sequencing of paramyxoviruses in mallard ducks (Anas platyrhynchos) using random access amplification and next generation sequencing technologies},
  url          = {http://dx.doi.org/10.1186/1743-422X-8-463},
  volume       = {8},
  year         = {2011},
}

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