We report the sequence and phylogenetic analysis of the entire M1,

We report the sequence and phylogenetic analysis of the entire M1, M2, and M3 genome segments of the novel duck reovirus (NDRV) NP03. and located in a common lineage in the M1 and M3 gene trees, 848318-25-2 supplier respectively. Rsum Nous rapportons ici la squence ainsi que lanalyse phylogntique des segments gnomiques entiers M1, M2 et M3 du nouveau reovirus de canard NP03 (NDRV). Lalignement entre les squences nouvellement dtermines ainsi que les squences dduites en acides amins et les squences publies des reovirus aviaires (ARV) a t effectu laide du logiciel DNASTAR. La comparaison des squences a dmontr que le gne M2 avait le plus de variabilit parmi les gnes de classe-M de DRV. Lanalyse phylogntique des gnes de la classe M des souches dARV a rvl lexistence de diffrentes lignes et regroupements parmi les DRVs. Les cinq souches de NDRV utilises dans la prsente tude se retrouvent lintrieur dune ligne qui inclut des souches dARV de poulets, alors que les souches de DRV Muscovy (MDRV) sont spares des souches de NDRV et forment une ligne gntique distincte dans larbre du gne M2. Toutefois, les souches MDRV et NDRV sont troitement lies et localises dans une ligne commune dans les arbres des gnes M1 et M3, respectivement. (Traduit par Docteur ATV Serge Messier) Infection with Muscovy duck reovirus (MDRV) has been observed to cause high morbidity and mortality rates in Muscovy ducks in Fujian Province and other regions of China since 1997. The characteristic signs of MDRV infection were described as leg weakness and liver white spots in the 1990s (1). However, MDRV isolates are usually nonpathogenic in experimentally 848318-25-2 supplier infected Pekin ducks. Since 2005 in China a number of virulent field DRV strains have been isolated from several duck species. These strains caused multiple organ hemorrhage and necrosis involving the liver (hepatorrhagia with necrotic foci), spleen (splenomegaly with necrotic foci), and kidney (nephrorrhagia). These isolated strains were designated as novel pathogen DRVs (NDRVs) to distinguish them from the previously reported MDRVs (2). Duck reoviruses have been recovered from several species of birds (3C8). They belong to the genus family together with avian reoviruses (ARVs) (chicken origin) and mammalian 848318-25-2 supplier reoviruses (MRVs), with which they share physicochemical properties and morphologic characteristics. These common traits include a double-stranded RNA genome consisting of 10 segments packaged into a nonenveloped icosahedral double-capsid shell. The genomic electropherotype of DRV consists of 10 segments classified as large (L1, L2, and L3), medium (Ml, M2, and M3), and small (S1, S2, S3, and S4) that code for the proteins of classes , , and , respectively (1,2). Cross-neutralization tests and viral nucleic acid extraction and segment separation by polyacrylamide gel electrophoresis and genome sequence analysis have been used to diagnose DRV infections. Experimental results showed considerable antigenic and genomic differences between MDRV and 848318-25-2 supplier NDRV strains (6C10). Genetic and phylogenetic analyses of both S and M segment sequences have revealed that gene mutation and reassortment of these genes occur frequently among cocirculating ARVs (11C15). Among chicken ARV and MDRV strains the genes coding for the outer capsid proteins vary considerably, whereas the genes for the inner capsid proteins and the nonstructural proteins show an intermediate degree of conservation. In contrast, relatively little is known about the genetic variation and evolution of the M-class genome segments of NDRVs. To expand the DRV sequences available in public databases and to better understand the extent of diversity and evolution of NDRV genomes, we cloned and sequenced the A, B, and NS-encoding genes of the NDRV NP03 strain isolated in Fujian Province and compared the sequences with those of other DRVs as well as chicken ARV reference strains. Viral RNA was extracted from purified virions of NDRV NP03 with the use of TRIzol LS reagent (Invitrogen, Corp., Carlsbad, California, USA) according to the manufacturers recommendations and used as a template for reverse- transcription polymerase chain reaction (RT-PCR). To determine the gene sequences encoding A, B, and NS, the M-class genome segments were amplified with use of the primers listed in Table I, according to methods previously described by Bnyai et al (16). The RT-PCR products were purified and cloned into pMD18-T vector (TaKaRa Biotechnology Company, Dalian, China) for sequencing with universal M13 forward and reverse primers. The sequence diversity of homologous M1, M2, and M3 genes between NP03 and other DRV strains and between NP03 and chicken ARV strains was analyzed with ClustalW in the DNASTAR software package (DNASTAR, Madison, Wisconsin, USA). Table I Oligonucleotide primers used to amplify and sequence.

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