| In the past two decades, many novel viral diseases have been emerging in duck populations, which have raisedserious threat and significant economic impact on Chinese duck industry. It is, therefore, important to investigate the potential novel duck viruses.A total of107samples were collected from Guangdong and Guangxi respectively. Six flocks of diseased egg-laying ducks, four flocks of recovered egg-laying ducks, one flock of broiler breeder ducks and one flock of commercial broiler ducks were covered by this research. PCR-based assays were employed to detect the presence of Tembusu virus and other viruses including bunyavirus, alphavirus, calicivirus, avian bornavirus (ABV), avian astrovirus and picornavirus. Picornaviruses were detected from one fecal sample of a broiler breeder duck and two intestinal samples of commercial broiler ducks. A picobirnavirus (PBV) was detected from one fecal sample of an egg-laying duck due to non-specific amplification of the ABV PCR assay. Sequencing and sequence analysis of the amplicons suggested that the duck-origin picornaviruses and duck-origin PBV (DuPBV) may belong to two different novel viruses.The duck picornavirus GL-12strain was subjected to full-length genome sequencing using RT-PCR and5’/3’RACE strategies. The virus was shown to have a picornavirus-like genome layout. Interestingly, the genome contained a total of up to six2As, including four2As (2A1-2A4) each having an NPGP motif, an AIGl-like2A5, and aparechovirus-like2A6. The5’UTR was predicted to possess a hepacivirus/pestivirus (HP)-like internal ribosome entry site (IRES). However, the subdomain Ⅲe consisted of a3nt stem and five unpaired bases, distinct from those found in all other HP-like IRESs. The virus was most closely related to duck hepatitis A virus (DHAV), with amino acid identities of37.7%,39%and43.7%in the P1, P2and P3regions, respectively. Phylogenetic analyses revealed that GL/12was closely related to, but highly divergent from, DHAV and Avisivirus A (AsV-A). Based on these investigations, the virus could be considered as the founding member of a novel picornavirus genusthat we tentatively named Aalivirus, with Aalivirus A as the type species.Using a PBV-specific RT-PCR assay, DuPBV were detected in70of the142samples collected from15duck flocks in Guangdong province, suggesting that DuPBV infections in egge-laying ducks were common. Phylogenetic analyses of a partial RNA-dependent RNA polymerase (RdRp) region demonstrated that the obtained264RdRp sequences belonged to PBV genogroup I. However, these sequences were highly divergent from each other, with amino acid identities ranging from56%to100%. The DuPBV4-17strain was subjected to full-length genome sequencing using RT-PCR and5’RACE strategy. The virus was shown to be composed of two genome segments, encoding capsid pretein and RdRp respectively. This genomic feature was similar to those of other PBVs. Phylogenetic analyses demonstrated that the DuPBV4-17strain was highly divergent from previously known picorbirnaviruses.A posavirus-like sequence was unexpectively detected when the genome sequence of another DuPBV strain (4-23) was determined. Thus, the posavirus-like duck virus that was presented in the sample was subjected to genome determination using the sequence available as starting point.Approximately78%of the genome of the virus was obtained. The4-23strain was shown to possess a typical genome organization of Picornavirales. Similar to posaviruses and fisavirus, the non-structural and structural regions were located in5’and3’parts of the genome respectively. Phylogenetic analyses based on3CD and capsid sequences of Picornavirales revealed that the4-23strain was related to, but highly divergent from, posaviruses and fisavirus. Thus, the virus is likely to be classified together with posaviruses and fisavirus as members of a novel family in the order Picornavirals. In addition, the virus could be identified as the founding member of a novel genus in the that we tentatively named Dusavirus, with Dusavirus A (DuV-A) as the type species.In August2013, a suspected outbreak of duck viral hepatitis (DVH) occurred in regions of intensive Jinding duck production in Northeast China. This disease caused deaths of both ducklings aged1-6weeks and adult ducks aged more than12weeks, and egg drop in affected adult ducks. The diseased ducks exhibited signs and lesions typical of DVH. Hemmoragic lesions were also observed in the overies of diseased adult ducks. Histopathologic changes could be observed in different viscera of diseased ducks, including hemorrhage, necrosis, and lymphocyte infiltration. Screening with PCR-based assays suggested that duck astrovirus1(DAstV-1) may be the causative agent. Immunological and tissue distribution analyses showed that multiple organs were infected by DAstV-1. The disease could be reproduced in Pekin duckings, Jinding ducklings and adult Jinding ducks by experimental infections. Full-length genome sequence analysis of four DAstV-1strains from diseased duckswith different ages demonstrated that these strains shared closely related relationship with reference DAstV-1(strain C-NGB)(98-99%amino acid identity). Taken together, it is concluded that DAstV-1infection can cause outbreaks of DVH in adult ducks.Comparative genome analysis revealed that accurrence of mutations at18amino acid positions in proteins of Jinding duck-origin astroviruses, which may be implicated in the pathogenicity of DAstV-1to adult ducks. Therefore, we further constructed a genomic-length cDNA clone of DAstV-1using the D17strain as parent virus. Following transfection of BHK cells with RNA transcribed in vitro, the obtained cultures were inoculated onto LMH cells and passaged. Expression of the viral proteins in the third LMH cell cultures could be confirmed by fluorescent antibody test, suggesting that infectious virus particles were rescued successfully. The present study provides a useful tool forfuture research on genomic functions and molecular pathogenesis of DAstV-1. |
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