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

Abstract

BackgroundDuring 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.MethodsTo 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.ResultsThree 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.ConclusionsThese 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.

Avian Paramyxoviruses: Detection by Transmission Electron Microscopy Techniques

“…It was observed the occurrence of serotype 4 (APMV-4) in free-living birds, such as duck (Alexander et al, 1979;Gough & Alexander, 1984;Stallknecht et al;Maldonado et al, 1995;Stanislawek et al, 2002;Jeon et al, 2008;Rosseel et al, 2011), goose and coot (Maldonado et al) and the serotype 5 (APMV-5) in parakeet, leading to depression, dyspnea, torticollis, vomiting, diarrhea and death (Mustaffabab Jee, 1974;Nerome et al, 1978;Gough et al, 1993).…”

“…The serotypes 6 (APMV-6) and 7 (APMV-7) were isolated from healthy free-living birds such as duck Maldonado et al;Stanislawek et al;Warke et al, 2008;Rosseel et al), goose, grey heron, blackheaded gull, coot, spoonbill, house sparrow (Maldonado et al), turkey with respiratory disease associated to egg production drop (Saif et al, 1997) and ostrich with enteritis (Wollcook et al, 1996).…”

Complete Nucleotide Sequence of Avian Paramyxovirus Type 6 Strain JL Isolated from Mallard Ducks in China

“…There are 11 serotypes (APMV-1 to APMV-11) of APMVs described (1, 2, 5) based on hemagglutination inhibition (HI). Newcastle disease induced by APMV-1 is one of the most serious diseases in poultry; APMV-2, APMV-3, and APMV-6 might also cause severe disease and a drop in egg production in turkeys (6). During an influenza virus surveillance program in Jilin province in 2011, a virus causing hemagglutination was isolated from mallard ducks (Anas poecilorhyncha).…”

“…Avian paramyxoviruses are classified in the genus Avulavirus of the subfamily Paramyxovirinae, family Paramyxoviridae (6). There are 11 serotypes (APMV-1 to APMV-11) of APMVs described (1, 2, 5) based on hemagglutination inhibition (HI).…”

Molecular characterization of Tasmanian aquabirnaviruses from 1998 to 2013

“…The random PCR method to obtain complementary DNA (cDNA) from viral dsRNA was based on the sequence independent single primer amplification (SISPA) method described by Rosseel et al (2011Rosseel et al ( , 2013. RNase-and DNase-treated nucleic acid (7 µl) was heat-denatured in the presence of 2.5 µM random primer (FR20RV-12N) at 95°C for 5 min before quenching on ice.…”