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Molecular Detection And Characterization Of Newly Discovered Astrovirus, Picornavirus And Calicivirus Of Waterfowl

Posted on:2015-03-22Degree:DoctorType:Dissertation
Country:ChinaCandidate:Q F LiaoFull Text:PDF
GTID:1263330431968155Subject:Prevention veterinarian
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In the past20years, a number of new diseases have emerged in waterfowl, which have raised serious threat on the Chinese waterfowl industry. It is, therefore, important to screen the potential novel viruses present in waterfowl populations for the controlling of the emerging diseases in waterfowl.155samples were collected from ducks and geese in the years2011to2012, including19fecal samples from2-month-old healthy Landes geese and18fecal samples from healthy Ma ducks in Jiangxi,90fecal samples from Pekin ducks in live-bird markets in Guangdong and Hubei, and28intestinal samples from dead-in-shell Pekin duckings in Shandong. Using an open reading frame (ORF)1b-based astrovirus-specific reverse transcription (RT)-PCR assay, astroviruses were detected in20(12.9%) of the samples. Comparative analyses based on the ORF1b amplicon sequences showed that the astroviruses could be divided into two groups (A and B) which shared low nucleotide (55-58%) and amino acid (57-59%) identities with one another. The nucleotide and amino acid identities shared by the20astroviruses with previously known avastroviruses were55-58%and57-59%respectively. Phylogenetic analysis of the partial ORFlb sequence demonstrated that the astroviruses in this study were distinct from previously known avastroviruses, suggesting the presence of two novel astroviruses in waterfowl. Group A included7isolates, all of which were detected in Pekin ducks. Group B included one strain detected in Pekin duck,3strains detected in Pekin duck embryos,2strains detected in Ma ducks, and7strains detected in Landes geese. These investigations demonstrated that astrovirus infections were geographically widespread and that at least three waterfowl species could be infected by astroviruses. The results also suggested that astroviruses in the two groups could be shed in waterfowl feces and that astrovirus in group B have a broad host range.In the partial ORFlb region, the virus strains in group B was shown to be closely related to duck astrovirus (DastV) strain CPH detected recently in our laboratory, with nucleotide and amino acid identities of95%-96%and97-99%. This result showed that they may belong to the same avastrovirus species. Thus, DAstV/YP2in the group A was selected for full-length sequencing, using sequence-independent PCR amplification, RT-PCR and5’/3’ rapid amplification of cDNA ends (RACE) strategies. Sequence analysis revealed that the complete genome of DAstV/YP2was7287nt, excluding the poly (A) tail. The polyadenylated genome was organized into three overlapping ORFs of3363(ORF1a),1545(ORF1b) and2184nt (ORF2) as well as a short5’ untranslated region (UTR) of12nt and a3’UTR of177nt. The findings indicated that DAstV/YP2possessed a typical astrovirus organization. Sequence identity and phylogenetic analyses demonstrated that the DAstV/YP2was distinct from all known avastroviruses. Genetic analysis of the complete ORF2region revealed that mean amino acid genetic distances between DAstV/YP2and previously known avastroviruses were between0.599and0.801, suggesting that DAstV/YP2may be classified as an additional species in the genus Avastrovirus of the family Astroviridae.Using sequence-independent PCR amplification, sequencing, and sequence similarity searches, a novel picomavirus (designated LY) was detected from a fecal sample collected from a Pekin duck located in Guangdong. Subsequently, the complete genome sequence of the LY isolate was determined using RT-PCR and5’/’RACE. The virus was shown to be most closely related to the genus Mergivirus of the family Picornaviridae, with average amino acid identities of32%to68%,35%to45%,51%to57%,41%to50%, and61%to63%in the P1, P2, P3, polyprotein and2C+3CD regions, respectively. The virus was thus identified as an additional species in the genus Megrivirus and tentatively named Duck megrivirus (DMV). Sequence analyses indicated that the DMV genome possessed a MeV-A-like organization and also exhibited several unique features. The polyadenylated genome comprised9700nt, the largest among known picoraviruses. A notable feature was the2A region, which had an association of two distinct, function-unknown2As (2A1and2A2) and a parechovirus-like2A3. The5’UTR contained a variant type IVB internal ribosome entry site (IRES), which possessed a long helix Ⅲ4ending with the "8"-like20-nt-long conserved structure at the top of domain III. The secondary structure model of inferred domain III of DMV-like IRES was also conserved in MeV-A, Mesivirus, Quail picornavirus, and Pigeon picornavirus B. Domain II in DMV contained the conserved internal and apical loops previously identified in groups A and C of type IV IRESs. Moreover, DMV was closely related to different megriviruses in different genomic regions. These findings suggest that recombination events involving exchange of coding and noncoding regions may have occurred during the evolution of the virus. To understand the prevalence of DMV infection, additional117duck samples collected from Guangdong, Shandong, Hubei and Hainan provinces between2011and2012were tested using the RT-PCR method. DMV-specific RNA was found in23.9%samples from domestic ducks, reflecting a high prevalence of DMV in duck populations. The positive samples were detected from four provinces, suggesting that DMV infections were geographically widespread.Using the strategy described above, a novel calicivirus was detected in a Landes goose from Jiangxi and then completely sequenced. Sequence analysis revealed that the Goose calicivirus (GoCV) genome comprised8013nt, which was organized into two ORFs, ORF1and ORF2. The two ORFs were in the same frame and separated by3nt, most similar to the case for turkey calicivirus (TuCV). The6960-nt ORF1was predicted to encode a large polyprotein which was predicted to be cleaved into nonstructural proteins Nterm, NTPase,3A-like protein, VPg and Pro-Pol as well as major structural protein VP1. The825-nt-long ORF2was predicted to encode a minor structural protein VP2. These findings indicated that GoCV possessed a TuCV-like genome organization. Comparison of GoCV with other caliciviruses showed that it shared the highest amino acid identities of62%,38%, and52%in nonstructural protein, VP1, and VP2, respectively, with TuCV. Phylogenetic analyses based on amino acid sequences of nonstructural protein and VP1both demonstrated that GoCV was most closely related to but distinct from TuCV. Thus, GoCV was identified as a novel member in the proposed genus "Nacovirus" of the family Caliciviridae.
Keywords/Search Tags:Astrovirus, Duck astrovirus, Picornavirus, Duck picornavirus, Calicivirus, Goosecalicivirus
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