Complete Genome Sequence of Avian Paramyxovirus Strain APMV-6/red-crested pochard/Balkhash/5842/2013 from Kazakhstan

An avian paramyxovirus 6 strain was isolated during a wild bird monitoring study in Kazakhstan in 2013. The virus was isolated from a wild duck red-crested pochard (Netta rufina) in southeastern Kazakhstan. Here, we present the complete genome sequence of the virus.

rus in the family Paramyxoviridae and include 12 serotypes, APMV-1 to APMV-12 (1)(2)(3)(4). APMV-1, or Newcastle disease virus (NDV), is the most frequently isolated APMV. To date, the complete genome sequences of eight isolates of APMV-6 and partial sequences of 3 isolates are available in public databases (GenBank, updated 1 February 2015). In this study, we report the complete genome sequence of an isolate of APMV-6 in Kazakhstan determined by next-generation sequencing. This is the first report of APMV-6 from this country.
During a study in 2013 monitoring wild birds, a hemagglutinating virus was isolated from a cloacal swab sample of a redcrested pochard after inoculation into 10-day-old embryonated chicken eggs. Hemagglutination inhibition tests using reference sera specific to APMV-1 to APMV-9 revealed that the virus belongs to APMV serotype 6.
Viral RNA was extracted from infected allantoic fluid using the QIAamp viral RNA minikit (Qiagen). Pan-PMV primers (5) were used for amplification and sequencing of a short genome fragment using an ABI 3730xl DNA analyzer (Applied Biosystems). A BLAST search confirmed that the virus belongs to APMV-6, and it was designated APMV-6/red-crested pochard/Balkhash/5842/ 2013. The sample was negative for avian influenza viruses, as revealed by a duplex reverse transcription-PCR (RT-PCR) (6) and by a generic real-time RT-PCR (7).
For whole-genome sequencing, viral RNA was used as a template for double-stranded cDNA synthesis (Roche). The Covaris M220 ultrasonicator was used for DNA fragmentation. For library preparation, Illumina adaptors (Bioo Scientific) and SPRIworks fragment library cartridge II (Beckman Coulter) were used, with manual size selection afterward. The quality of the library was checked on a Bioanalyzer 2100 (Agilent Technologies). Quantity was determined via quantitative PCR (qPCR) with the Kapa library quantification kit (Kapa Biosystems). Paired-end sequencing was performed on an Illumina MiSeq instrument using the MiSeq reagent kit version 3. Raw sequence data were analyzed and assembled using the Genome Sequencer software suite (version 2.8; Roche).
Phylogenetic studies of full-length hemagglutinin-neuraminidase (HN) genes and investigations of the antigenic cross-reactivity studies grouped the APMV-6 viruses into two separate clusters (8), the first one including the reference strain duck/HongKong/18/199/1977 and the Kazakhstan strain and the second one represented by rednecked stint/Japan/8KS0813/2008 and duck/Italy/IT4524-2/2007, both with a shortened genome. The Kazakhstani isolate showed the highest identity with mallard/Jilin/127/2011 (98.8%). This might be explained by the close geographical relation of Kazakhstan to China and potential migration of wild birds through common flyways.

ACKNOWLEDGMENTS
This study was supported by the Kazakhstan governmental project 0112PK02470 "Study of ecology and genetic diversity of paramyxoviruses in wild and domestic birds in Kazakhstan" of the Ministry of Education and Science of the Republic of Kazakhstan. The whole-genome sequencing was carried out at the Friedrich-Loeffler-Institut, Insel Riems, Germany, and was supported by the EMIDA ERA-NET project "Molecular