key: cord-017984-w19kd6yp authors: Chen, Yi-Ning; Akin, Aydemir; Loa, Chien Chang; Ababneh, Mustafa; Cao, Jianzhong; Chen, Wan-Jung; Wu, Ching Ching; Lin, Tsang Long title: PCR Amplification and Sequencing Analysis of Full-Length Turkey Coronavirus Spike Gene date: 2015-09-10 journal: Animal Coronaviruses DOI: 10.1007/978-1-4939-3414-0_14 sha: doc_id: 17984 cord_uid: w19kd6yp Turkey coronaviral enteritis caused by turkey coronavirus (TCoV) continues to infect turkey flocks, resulting in significant economic loss. Determining and understanding genetic relationships among different TCoV isolates or strains is important for controlling the disease. Using two-step RT-PCR assays that amplify the full length of TCoV spike (S) gene, TCoV isolates can be sequenced, analyzed, and genotyped. Described in this chapter is the protocol on PCR amplification and sequencing analysis of full-length TCoV S gene. Such protocol is useful in molecular epidemiology for establishing an effective strategy to control the transmission of TCoV among turkey flocks. Turkey coronaviral enteritis caused by turkey coronavirus (TCoV) has been reported with varied severity in clinical signs in the affected turkey fl ocks from different states in the USA [ 1 , 2 ] , Canada [ 3 ] , Brazil [ 4 ] , and Europe [ 5 ] . The major clinical signs of TCoV infection include depression, ruffl ed feathers, watery diarrhea, decreased body weight gain, and uneven fl ock growth. The most striking gross lesions are markedly distended intestine with gaseous and watery content, especially in the ileum and ceca. The salient histopathologic fi ndings include shortening of the intestinal villi, increase in crypt depth, and widening of intervillous spaces [ 1 ] . TCoV belongs to species Avian coronavirus of the genus Gammacoronavirus in the family Coronaviridae . The genome of TCoV is a linear positive-sense single-stranded RNA encoding three major structural proteins, including spike (S), membrane (M), and nucleocapsid (N) protein. While M and N genes are conserved, S gene is a more common target used for coronavirus (CoV) differentiation because S gene is highly variable among different CoVs. The S gene sequences among different TCoV isolates (93-99.7 %) are relatively conserved as compared to different infectious bronchitis virus (IBV) strains, although both TCoV and IBV belong to the same species Avian coronavirus [ 6 -8 ] . Because the pair-wise comparison of S gene sequences revealed only 34 % of similarity between TCoV isolates and IBV strains while the remaining 3′-end encoding region shared over 80 % of similarity, it has been suggested that TCoV arose through a recombination of S gene from IBV [ 6 , 9 -11 ] . A protocol for PCR amplifi cation, sequencing , and sequence analysis of TCoV S gene for genotyping of TCoV isolates based on TCoV S gene sequences is highlighted in this chapter. In step 1, intestine tissues are collected from TCoV-infected turkeys and TCoV is purifi ed through ultracentrifugation on continuous sucrose gradient. In step 2, TCoV RNA is extracted from intestines and reverse transcribed to cDNA. In step 3, the full-length S gene of TCoV is amplifi ed and sequenced. In step 4, the nucleotide sequence of the full-length S gene of TCoV is assembled from 12 sequence fragments fl anking the entire length of S gene. The S gene sequences of TCoV isolates and those from other coronaviruses published in GenBank are further analyzed by alignment and phylogenetic tree and thus TCoV genotypes are determined. 4. Electrophorese the PCR product on 1 % agarose gel to confi rm the size (about 3.9 kb) and purity of the PCR product amplifi ed by the primers Sup and Sdown3. 5. Purify Sup/Sdown3 PCR product from agarose gel by Zymoclean™ gel DNA recovery kit for sequencing . 6. Obtain the nucleotide sequences of the purifi ed Sup/Sdown3 PCR product by sequencing using the 12 sequencing primers listed in Table 1 at a certifi ed genomics facility. 7. Clone the purifi ed Sup/Sdown3 PCR product into pCR ® II-TOPO ® plasmid vector and transform it into Escherichia coli strain One Shot ® TOP10F′ for storage purpose ( see Note 3 ). 1. Assemble 12 nucleotide sequences from 12 sequencing primers (Table 1 ) to obtain the full length of TCoV S gene by using DNAstar Lasergene ® software. 2. Analyze the full-length S genes from different TCoV fi eld isolates by Clustal W alignment method using DNAstar Lasergene ® software or Web-based MEGA6 program ( http:// www.megasoftware.net ). Sequence alignment can arrange the sequences of DNA and deduced amino acids to identify regions of similarity resulting from evolutionary relationships among the compared sequences. (a) Open MEGA6 program, click "Align" on the tool bar, choose "Edit/Build Alignment," check the option of "create a new alignment" in the fi rst dialog box, push "OK," and then choose either " DNA " or "Protein" sequence alignment, and the Alignment Explorer window will open. (b) There are three ways to add sequences into alignment program. For the fi rst two ways, go to "Edit" menu and click either "Insert Blank Sequence" to key in each sequence manually or "Insert Sequence From File" to import the selected sequences in Text (*.txt, *.seq) or FASTA fi le formats. For the third way, go to "Web" menu and click "Query GenBank" to import Web-based sequences by checking the "Add to Alignment" option for each interested sequence. (c) After all sequences for alignment are added, go to "Edit" menu in the Alignment Explorer window to select all sequences for alignment, and then go to "Alignment" menu to select "Align by Clustal W (Codons)" for the process of alignment. Choose Align Codons for DNA alignment to avoid introducing gaps into positions that would result in frame shifts in the real sequences. Use the default settings of Clustal W method for DNA alignment but change the Multiple Alignment Gap Opening penalty to 3 and the Multiple Alignment Gap Extension penalty to 1.8 for protein alignment. When the alignment is complete, go to "Data" menu and choose "Save Session" to save the alignment result as Aln session (*.mas) format. For further construct of phylogenetic tree, go to "Data": menu and click "Export Alignment" to choose "MEGA Format" to save the alignment result as .meg extension. 3. A phylogenetic tree can be constructed by many methods used widely and maximum likelihood (ML) method is chosen based on previous coronaviral studies. (a) ML uses a variety of substitution models to correct for multiple changes at the same site during the evolutionary history of the sequences, like Tamura 3-parameter and Kimura 2-parameter models, and the best model to use for each analysis can be calculated by MEGA6 program. Go to MEGA6 main window and choose "Models" to select "Find Best DNA /Protein Models (ML)." Choose the alignment fi le saved before for calculation. Note the preferred model to construct a phylogenetic tree based on the list of models in order of preferences. (b) Go to MEGA6 main window and choose "Phylogeny" to select "Construct/Test Maximum Likelihood Tree." Fill in the parameters of "Model/Method" and "Rates among Sites" based on the preferred parameters calculated previously in the preference dialog appearing. Select "Partial Deletion" in "Gap/Missing Data Treatment" to avoid losing too much information. Set "No. of Bootstrap Replicates" to 1000 under "Phylogeny Test" to estimate the reliability of the tree. (c) After computing, a tree explorer window will open the tree. Save the tree as a (*.mts) fi le, choose "Export Current Tree (Newick)" from "File" menu for further modifi cation in other tree drawing program, choose "Save as PDF File" from "Image" menu for graphic fi le format accepted for publishing, or choose "Save as PNG File" or "Save as Enhanced Meta File" for further process. 1. Put the turkey intestines containing TCoV in 50 mL tube. If the volume of intestines reaches 5 mL mark of 50 mL tube, add 25 mL (5 volumes) of chill sterile PBS for homogenization. It is recommended to homogenize the intestine tissue for 30 s and cool the tube in ice in cycles until the intestine tissue is homogenized thoroughly because the heat produced by the process of homogenization can damage the integrity of RNA in intestine tissues. 2. To prepare 40-60 % sucrose continuous gradient, Bio-Rad Model 395 Gradient former or equivalent gradient maker is used. Put 5 mL of 40 % sucrose and 5 mL of 60 % sucrose in two connected compartments of Gradient former, respectively. Place a stirrer bar in the compartment with 40 % sucrose (lower extreme concentration of sucrose gradient). There is a valve to control the connection between two compartments and a plastic tube leading to an ultracentrifuge tube (Beckman Coulter) from the compartment with 40 % sucrose. Put the Gradient former on top of magnetic stirrer. Open the stirrer and wait till the stirrer bar stir steady and then open the valve. Collect the mixed sucrose in an ultracentrifuge tube to get 10 mL of 40-60 % sucrose continuous gradient. Characterization of turkey coronavirus from turkey poults with acute enteritis Investigating turkey enteric coronavirus circulating in the Southeastern United States and Arkansas during Infection with a pathogenic turkey coronavirus isolate negatively affects growth performance and intestinal morphology of young turkey poults in Canada Detection of turkey coronavirus in commercial turkey poults in Brazil First full-length sequences of the S gene of European isolates reveal further diversity among turkey coronaviruses Comparison of 3′-end encoding regions of turkey coronavirus isolates from Indiana, North Carolina, and Minnesota with chicken infectious bronchitis coronavirus strains Relationship between serotypes and genotypes based on the hypervariable region of the S1 gene of infectious bronchitis virus Antigenic relationship of turkey coronavirus isolates from different geographic locations in the United States Complete nucleotide sequence of polyprotein gene 1 and genome organization of turkey coronavirus Emergence of a group 3 coronavirus through recombination Recombinational histories of avian infectious bronchitis virus and turkey coronavirus The protocol " PCR amplifi cation and sequencing analysis of fulllength turkey coronavirus spike gene" outlined in this chapter had been successfully carried out in the authors' studies on molecular diagnostics, molecular virology, immunology, and/or vaccinology of turkey coronavirus infection . Those studies were in part fi nancially supported by USDA, North Carolina Poultry Federation, and/or Indiana Department of Agriculture and technically assisted by Drs. Tom Bryan and David Hermes, Mr. Tom Hooper, and Ms. Donna Schrader for clinical investigation, virus isolation and propagation, and animal experimentation.