TITLE: Phylogenetic Analysis of Complete Bovine Coronavirus Genome Sequences
AUTHORS: Aspen M Workman, Tara  G. McDaneld, Gregory P Harhay, Subha Das, John  Dustin Loy, Benjamin  M. Hause

[DESCRIPTION]
Bovine coronavirus (BCoV) has spilled over to many species, including humans, where the host range variant coronavirus OC43 is endemic. Balance of the opposing activities of the surface spike (S) and hemagglutinin esterase (HE) glycoproteins control virion avidity which is critical for interspecies transmission and host adaptation.  Here, 78 genomes were sequenced directly from clinical samples collected between 2013 and 2022 from cattle in 12 states, primarily in the Midwestern U.S.  Relatively little genetic diversity was observed, with genomes having >98% nucleotide identity. Eleven isolates collected between 2020 and 2022 from four states (Nebraska, Colorado, California, and Wisconsin) contained a 12-nucleotide insertion in the receptor-binding domain (RBD) of the HE gene identical to one recently reported in China, and a single genome from Nebraska collected in 2020 contained a novel 12-nucleotide deletion in the HE gene RBD. Isogenic HE proteins containing either the insertion or deletion in the HE RBD maintained esterase activity and the ability to bind bovine submaxillary mucin, a substrate enriched in the receptor 9-O-acetylated-sialic acid, despite modeling that predicted structural changes in the HE R3 loop critical for receptor binding.  The emergence of BCoV with structural variants in the RBD raises the possibility of further interspecies transmission.

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SECTION: Software
1. 
 

SECTION: Software
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SECTION: Software
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SECTION: MAFFT Alignment
5. Align  using MAFFT v7.450

The sequences were aligned using the MAFFT accuracy  methods   ---globalpair as defined in the mafft manual page  below 

 


Keep in mind ...

The commands below generate files that contain both STDOUT (mafft output to the "terminal") as well as the multifasta alignment file (.afa). The STDOUT is found at the beginning of the file and the alignments follow the STDOUT
 All .afa_out files were split in a text editor into two separate .out and .afa files for downstream analysis.  
The input FASTA file headers contain description information; this header information should be stripped out to facilitate readable, compact leaf names/identifiers. Use the following command to strip out the description text.  
 thethe


SECTION: MAFFT Alignment
5.1. The input FASTA files are:

             

The spike and HE genes were extracted from the annotated genomes into the file of fasta sequences above. There are duplicated sequences within 192_HE_6_22.fasta  as well as within 192_Spike_6_14_22.fasta.  The genome sequences within 192_Genomes_Final_6_14_22.fasta are unique.

The spike and HE  FASTA sequence files were deduplicated with fassort &  fasuniq from the FAST Analysis of Sequences Toolbox. The unique sequences present in multiple genomes will be designated with a sequence identifier comprised of a concatenation of the sequence identifiers used in the multiple genomes separated by a "__" or ":". For example, the HE fasta sequence id IWT-11:SHG-6:TCG-21:TCG-23:TCG-22 represents the identical HE fasta sequences IWT-11, SHG-6, TCG-21, TCG-23 and TCG-22.


 a

  


 genomes

  




 

  







 

  

SECTION: MAFFT Alignment
5.2. Remove description text (all text after sequence id) in HE , Spike, and Genomes fasta fileps.
 
output :  
 

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SECTION: MAFFT Alignment
5.3. Align 192 BCV genomes  using the default PAM (JTT) 200 substitution matrix 

 
outputs :   


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SECTION: MAFFT Alignment
5.4. Align 192 BCV genomes  using the  PAM (JTT) 100 substitution matrix 
 
outputs :    


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SECTION: MAFFT Alignment
5.5. Align SPIKE using the default PAM (JTT) 200 substitution matrix 
 
Outputs :     


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SECTION: MAFFT Alignment
5.6. Align SPIKE  using the  PAM (JTT) 100 substitution matrix 
 
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SECTION: MAFFT Alignment
5.7. Align HE using the default PAM (JTT) 200 substitution matrix 
 
Outputs :     

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SECTION: MAFFT Alignment
5.8. Align HE using the PAM (JTT) 100 substitution matrix 
 
Output:     


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SECTION: Tree Building
6.  RaXML Tee Building


Notes  below are cut from the RaXML manual to put the command line parameters used in context
 


raxmlHPC reports 192_Genomes_Final_6_14_22.jtt_100.globalpair.SEQID_Only.afa has  3438 DNA alignment patterns 

 

Given the directions above from the RaXML manual, use 8 threads for raxmlHPC tree building

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Output :

    
  
      

Use RAxML_bipartitionsBranchLabels.192_Genomes_Final_6_14_22.jtt_100.globalpair.SEQID_Only.T8.N2000 file to create phylogenetic tree with bootstrap support values provided as branch labels.


Visualize this tree of 192 Bovine coronavirus genomes, MAFFT Global Pair Alignment, PAM 100 in the Interactive Tree of Life 


    
The pdf suitable for download is    



SECTION: MAFFT Alignment
5.9. Align  unique BCV HE sequences the PAM (JTT) 100 substitution matrix 


 

Output:   


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SECTION: Tree Building
7. Following the same approach with the 192 BCoV genomes other RaXML Trees were generated for spike and HE and are included here

  
          


 

SECTION: BLAST
4. Extract consensus HE sequence  from the MAFFT aligned 192 whole genomes (see below)  using Geneious

Discontiguous MegaBLAST input:   
 
Output:    This list of 844  subject sequences  (hits) was reduced to 714  by requiring that each hit covered at least 98.4% (1255 bp) of query HE sequence resulting in the following FASTA file : 

    c 

 This file was filtered for sequences with "bovine coronavirus" in the FASTA description field yielding :