Discoveries Of Novel Viruses In Ducks And Geese Using Universal PCR-based Assay And Metagenomics

During the past 20 years,new emerging infectious diseases continue to emerge in domestic waterfowl populations,which have caused great economic losses to Chinese waterfowl industry.Therefore,digging for novel viruses circulating in domestic waterfowl populations can help to control diseases of waterfowl.In the present study,we focused on discoveries of novel viruses in geese and Ma ducks by using pan-picornavirus and pan-calicivirus PCR-based assays and metagenomics respectively.Tissue and fecal samples were collected from geese showing enteritis between 2014 and 2015.17(27.0%)of the samples were tested positive by a pan-picornavirus RT-PCR assay.9(14.3%)of the samples were tested positive by using a pan-calicivirus RT-PCR assay.Based on pairwise comparisons and phylogenetic analyses of the sequences generated from the amplicons,the picornaviruses could be divided into four groups,which shared the highest amino acid(aa)sequence identities with Duck megrivirus(DMV;90.5-99.3%),Bat picornavirus 3(BatPV3;66.2%),Duck hepatitis A virus(DHAV;53.4%),and Simian sapelovirus 1(55.5%)respectively.While the caliciviruses belonged to 2 groups,with the highest aa identities with Nacovirus(47.6-66.7%)and Sapovirus(42.9-46.4%)respectively.Therefore,geese could be act as reservoirs of multiple picornaviruses and caliciviruses.Complete genome sequences of three goose-origin picornaviruses(HN50-k,W18 and HN56)were determined.The 8059-nt-long genome of HN50-k was organized into 5'UTR-L-P1(VP0-VP3-VP1)-P2(2AH-box/NC-2B-2Chel)-P3(3A-3BVPg-3CPro-3DPol)-3'UTR-Poly(A).Pairwise comparisons revealed that HN50-k shared the highest aa identities with Kobuvirus in P1(47.86%)and P2(42.70%),while was most closely related to Passerivirus in P3(46.83%aa identity).Phylogenetic analyses showed that HN50-k formed a separate clade in 2C and 3CD,and was genetically related to Kobuvirus in P1.Together these findings suggest that HN50-k may belong to a novel species in genus Kobuvirus.Of 42 tissue and fecal samples collected from geese,69.05%were tested positive for HN50-k,reflecting a high prevalence of HN50-k in the studied goose flocks.The genome lengths of W18 and HN56 were 9840 and 10101 nt respectively,both of which were organized into 5'UTR-P1(VP0-VP3-VP1)-P2(2A1-2A2-2A3H-box/NC-2B-2Chel)-P3(3A-3BVPg-3CPro-3DPol)-3'UTR-Poly(A).Both W18 and HN56 shared the highest aa sequence identities(>91.7%)with DMV in P2 and P3,whereas they were most closely related to DMV(68.5%aa identity)and Mesivirus-1(52.2%aa identity)in P1,respectively.The findings,together with phylogenetic analyses,suggest that W18,HN56 and DMV may belong to three different genotypes of a novel species in genus Megrivirus.90.5%of 42 tissue and fecal samples collected from geese were tested positive for megriviruses,reflecting a high prevalence of megriviruses in the studied goose flocks.A 3711-nt-long sequence in the 3' part of the picornavirus HN27 genome was determined.The virus shared the highest aa identities with DHAV in 2C(48.3%),3CD(48.7%),and P3(43.9%)regions,suggesting that HN27 may belong to a member of a novel picornavirus genus,which was tentatively named "Goalvirus".Five novel caliciviruses were detected from intestinal samples collected from Hortobagy geese,which could be divided into two groups,represented by HN50-1 and HN27-1 respectively.Complete genome sequence of HN50-1(8449 nt)and most genome sequence of HN27-1(6273 nt)were determined.Both HN50-1 and HN27-1 possessed a typical calicivirus genomic organization.HN50-1 had the highest aa identities with Nacovirus in NS(35.83-37.64%),Pro-Pol(47.39-50.46%),and VP1(30.34-32.62%)regions,while was most closely to Norovirus in VP2(19.15-20.97%).HN27-1 was most closely related to Nacovirus in Pro-Pol(45.72-47.25%),VP1(32.29-33.64%)and VP2(16.59-23.11%)regions.The findings,togther with phylogenetic analyses,HN50-1 and HN27-1 may belong to members of two novel calicivirus genera,which we tentatively named "Sanovirus," and "Hunovirus" respectively.Further analysis indicated that HN50-1 and two related strains may belong to 3 different genetypes of a same species,while HN27 and a related strain may belong to different strains of a same species.Viral metagenomics was applied to detect the RNA virome of two intestinal samples collected from Jingding ducks showing egg dropping.A total of 1672 contigs were obtained and shown to be closely related to picobirnavirus(PBV)(255 contigs),rotavirus(10 contigs),and picornavirus(2 contigs),respectively.For further analysis,the full-length and nearly full-length genome segments of duck-origin PBVs were selected,including segment 1(S1)of 19 PBV strains and segment 2(S2)of 11 PBVs.In the capsid region,the 19PBVs shared aa identities of 16.07-51.89%with each other,and 15.12-40.46%with other PBVs.In the RdRp region,the 11 PBVs shared aa identities of 47.73-70.65%w:ith each other,and the highest aa identities(45.45-70.64%)with PBVs of genegroup I(GI).The findings,together with phylogenetic analyses,suggest that the Ma duck-origin PBVs display a high degree of variation although they belong to PBV GI.Partial or nearly full-length sequences were determined for 7 genome segments of RV LH530.On the basis of sequence identity analysis,LH530 shared the highest aa identities with Rotavirus D(RVD)in VP1(94.70-95.42%),VP2(96.99-97.19%),VP3(83.28-83.44%),VP4(73.02-73.62%),VP7(62.01-62.66%),NSP2(86.00-86.03%),and NSP3(72.95-75.85%),respectively.Phylogenetic analyses showed that LH530 was most closely related to,but distinct from,chicken-origin RVD,suggesting that LH530 may represent a new RVD genotype.Taken together,the Ma ducks can be infected by multiple RNA viruses.