Phylogenetic Analysis of Complete Bovine Coronavirus Genome Sequences

Aspen M Workman; Tara  G. McDaneld; Gregory P Harhay; Subha Das; John  Dustin Loy; Benjamin  M. Hause

Sep 22, 2022

Version 3

Phylogenetic Analysis of Complete Bovine Coronavirus Genome Sequences V.3

DOI

dx.doi.org/10.17504/protocols.io.kqdg3pyeql25/v3

Aspen M Workman¹,
Tara G. McDaneld¹,
Gregory P Harhay¹,
Subha Das²,
John Dustin Loy³,
Benjamin M. Hause²

¹United States Department of Agriculture (USDA) Agricultural Research Service (ARS), US Meat Animal Research Center (USMARC), State Spur 18D, Clay Center, NE 68933, USA;
²South Dakota State University, Brookings, SD 57007, USA;
³Nebraska Veterinary Diagnostic Center, School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, 4040 East Campus Loop N, Lincoln, NE 68503-0907

Gregory P Harhay

United States Department of Agriculture (USDA) Agricultural ...

DOI: dx.doi.org/10.17504/protocols.io.kqdg3pyeql25/v3

External link: https://doi.org/10.3390/v14102125

Protocol Citation: Aspen M Workman, Tara G. McDaneld, Gregory P Harhay, Subha Das, John Dustin Loy, Benjamin M. Hause 2022. Phylogenetic Analysis of Complete Bovine Coronavirus Genome Sequences. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3pyeql25/v3Version created by Gregory P Harhay

Manuscript citation:

Workman AM,  McDaneld TG,  Harhay GP,  Das S,  Loy JD,  Hause BM, Recent Emergence of Bovine Coronavirus Variants with Mutations in the Hemagglutinin-Esterase Receptor Binding Domain in U.S. Cattle. Viruses  14(10). doi: 10.3390/v14102125

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and it's working

Created: September 21, 2022

Last Modified: September 22, 2022

Protocol Integer ID: 70350

Keywords: coronavirus, hemagglutinin-esterase, spike protein, bovine respiratory disease, calf diarrhea, variant

Funders Acknowledgement:

National Institute of Food and Agriculture

Grant ID: 2019-67015-29847

USDA ARS CRIS

Grant ID: 3040-32000-036-00D

National Institutes of Allergy and Infectious Diseases

Grant ID: 1R21AI162594-01

Disclaimer

•The findings and conclusions in this presentation are those of the authors and should not be construed to represent any official USDA or U.S. Government determination or policy.
•Mention of trade names or commercial products in this presentation is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA.
•USDA is an equal opportunity provider and employer

Abstract

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.

Software

Software
MAFFT
NAME
Kazutaka Katoh
DEVELOPER

Software
RAxML
NAME
UBUNTU Linux ` 20.4
OS
Alexandros Stamatakis
DEVELOPER
https://github.com/stamatak/standard-RAxML
SOURCE LINK

Software
FAST --- Fast Analysis of Sequences Toolbox
NAME
UBUNTU Linux 22 LTS
OS
Travis Lawrence
DEVELOPER
Github
SOURCE LINK

BLAST

Extract consensus HE sequence  from the MAFFT aligned 192 whole genomes (see below)  using Geneious

Discontiguous MegaBLAST input: HE_realigned_consensus_sequence.fasta  
Discontiguous MegaBLAST of BCoV HE consensus sequence with E-value of E-20 against all BetaCoronavirus sequences  in the GenBank nt database
Output: BetaCoronaVirus_HE_BlastMatch_Bovine_HE_Consensus.txt   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 : 

    BetaCoronaVirus_HE_GE_1255_BP_BlastMatch_Bovine_HE_Consensus.fastac 

 This file was filtered for sequences with "bovine coronavirus" in the FASTA description field yielding : 
 
345_Sequences_Bovine_HE_only.fasta  

MAFFT Alignment

Align  using MAFFT v7.450

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

Note
DESCRIPTION
MAFFT is a multiple sequence alignment program for unix-like operating systems. It offers a range of multiple alignment methods.

Accuracy-oriented methods:
• L-INS-i (probably most accurate; recommended for <200 sequences; iterative refinement method incorporating local pairwise alignment information):

mafft --localpair --maxiterate 1000input [> output]

linsi input [> output]

• G-INS-i (suitable for sequences of similar lengths; recommended for <200 sequences; iterative refinement method incorporating global pairwise alignment
information):

mafft--globalpair--maxiterate1000input [> output]

ginsi input [> output]

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.  
Command
Strip out description text from multifasta alignment file (UBUNTU Linux)
sed '/^>/ s/ .*//'  input_alignment_multifasta_file.afa  >  output_alignment_multifasta_file.SEQID_Only.afa

The input FASTA files are:

 192_Spike_6_14_22.fasta  192_HE_6_14_22.fasta  192_Genomes_Final_6_14_22.fasta  345_Sequences_Bovine_HE_only.fasta  BetaCoronaVirus_HE_GE_1255_BP_BlastMatch_Bovine_HE_Consensus.fasta  HE_realigned_consensus_sequence.fasta  

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.

Command
Create a file of unique HE FASTA sequences. 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  double underscore "__" (UBUNTU Linux)
fassort -s 192_HE_6_14_22.fasta | fasuniq --concat='__'  > unique_HE_6_14_22.fasta

unique_HE_6_14_22.fasta  

Command
Create a file of unique Spike FASTA sequences. 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 double underscore "__" (UBUNTU Linux)
fassort -s 192_Spike_6_14_22.fasta | fasuniq --concat='__'  > unique_Spike_6_14_22.fasta

unique_Spike_6_14_22.fasta  

Command
fassort -s 345_Sequences_Bovine_HE_only.fasta | fasuniq --concat='__' > unique_345_Sequences_Bovine_HE_only.fasta

unique_345_Sequences_Bovine_HE_only.fasta  

Command
fassort -s BetaCoronaVirus_HE_GE_1255_BP_BlastMatch_Bovine_HE_Consensus.fasta | fasuniq --concat="__" > unique_BetaCoronaVirus_HE_GE_1255_BP_BlastMatch_Bovine_HE_Consensus.fasta

unique_BetaCoronaVirus_HE_GE_1255_BP_BlastMatch_Bovine_HE_Consensus.fasta

Remove description text (all text after sequence id) in HE , Spike, and Genomes fasta fileps.
Command
sed to strip out the description in the header from FASTA (UBUNTU Linux)
sed '/^>/ s/ .*//'  unique_HE_6_14_22.fasta  >  unique_HE_6_14_22.SEQID_Only.fasta
output :unique_HE_6_14_22.SEQID_Only.fasta  
Command
sed to strip out the description in the header from FASTA (UBUNTU Linux)
sed '/^>/ s/ .*//'  unique_HE_6_14_22.fasta  >  unique_HE_6_14_22.SEQID_Only.fasta

output: unique_Spike_6_14_22.SEQID_Only.fasta  

Command
sed to strip out the description in the header from FASTA (UBUNTU Linux)
sed '/^>/ s/ .*//'  192_Genomes_Final_6_14_22.fasta > 192_Genomes_Final_6_14_22.SEQID_Only.fasta

output: 192_Genomes_Final_6_14_22.SEQID_Only.fasta  

Command
sed '/^>/ s/ .*//' unique_345_Sequences_Bovine_HE_only.fasta  >  unique_345_Sequences_Bovine_HE_only.SEQID_Only.fasta
unique_345_Sequences_Bovine_HE_only.SEQID_Only.fasta  

Command
sed '/^>/ s/ .*//'  unique_BetaCoronaVirus_HE_GE_1255_BP_BlastMatch_Bovine_HE_Consensus.fasta  >  unique_BetaCoronaVirus_HE_GE_1255_BP_BlastMatch_Bovine_HE_Consensus_SeqID_Only.fasta

unique_BetaCoronaVirus_HE_GE_1255_BP_BlastMatch_Bovine_HE_Consensus_SeqID_Only.fasta

Align 192 BCV genomes  using the default PAM (JTT) 200 substitution matrix 

Command
nohup mafft --thread 25 --globalpair --maxiterate 1000 --jtt 200 --reorder 192_Genomes_Final_6_14_22.SEQID_Only.fasta > 192_Genomes_Final_6_14_22.SEQID_Only.jtt_200.globalpair.afa_out &
outputs : 192_Genomes_Final_6_14_22.jtt_200.globalpair.SEQID_Only.afa_out  


This  was split into  192_Genomes_Final_6_14_22.jtt_200.globalpair.SEQID_Only.afa  &  192_Genomes_Final_6_14_22.jtt_200.globalpair.SEQID_Only.out  

Align 192 BCV genomes  using the  PAM (JTT) 100 substitution matrix 
Command
nohup mafft --thread 30 --globalpair --maxiterate 1000 --jtt 100 --reorder 192_Genomes_Final_6_14_22.SEQID_Only.fasta > 192_Genomes_Final_6_14_22.jtt_100.globalpair.SEQID_Only.afa_out &
outputs :  192_Genomes_Final_6_14_22.jtt_100.globalpair.SEQID_Only.afa_out  


This  was split into  192_Genomes_Final_6_14_22.jtt_100.globalpair.SEQID_Only.afa  & 192_Genomes_Final_6_14_22.jtt_100.globalpair.SEQID_Only.out  

Align SPIKE using the default PAM (JTT) 200 substitution matrix 
Command
nohup mafft --thread 15 --globalpair --maxiterate 1000 --jtt 200 --reorder unique_Spike_6_14_22.SEQID_Only.fasta > unique_Spike_6_14_22.SEQID_Only.jtt_200.globalpair.afa_out &
Outputs :   unique_Spike_6_14_22.SEQID_Only.jtt_200.globalpair.afa_out  


This  was split into unique_Spike_6_14_22.SEQID_Only.jtt_200.globalpair.afa  &unique_Spike_6_14_22.SEQID_Only.jtt_200.globalpair.out  

Align SPIKE  using the  PAM (JTT) 100 substitution matrix 
Command
nohup mafft --thread 15 --globalpair --maxiterate 1000 --jtt 100 --reorder unique_Spike_6_14_22.SEQID_Only.fasta > unique_Spike_6_14_22.SEQID_Only.jtt_100.globalpair.afa_out &
Outputs :  unique_Spike_6_14_22.SEQID_Only.jtt_100.globalpair.afa_out  


This  was split into unique_Spike_6_14_22.SEQID_Only.jtt_100.globalpair.afa  &unique_Spike_6_14_22.SEQID_Only.jtt_100.globalpair.out  

Align HE using the default PAM (JTT) 200 substitution matrix 
Command
nohup mafft --thread 15 --globalpair --maxiterate 1000 --jtt 200 --reorder unique_HE_6_14_22.SEQID_Only.fasta > unique_HE_6_14_22.SEQID_Only.jtt_200.globalpair.afa_out
Outputs :   unique_HE_6_14_22.SEQID_Only.jtt_200.globalpair.afa_out  

This  was split into  unique_HE_6_14_22.SEQID_Only.jtt_200.globalpair.afa  &unique_HE_6_14_22.SEQID_Only.jtt_200.globalpair.out  

Align HE using the PAM (JTT) 100 substitution matrix 
Command
nohup mafft --thread 15 --globalpair --maxiterate 1000 --jtt 100 --reorder unique_HE_6_14_22.SEQID_Only.fasta > unique_HE_6_14_22.SEQID_Only.jtt_100.globalpair.afa_out
Output:unique_HE_6_14_22.SEQID_Only.jtt_100.globalpair.afa_out     


This  was split into:    unique_HE_6_14_22.SEQID_Only.jtt_100.globalpair.afa  &
unique_HE_6_14_22.SEQID_Only.jtt_100.globalpair.out 

Align  unique BCV HE sequences the PAM (JTT) 100 substitution matrix 

Command
nohup mafft --thread 25 --globalpair --maxiterate 1000 --jtt 100 --reorder  unique_345_Sequences_Bovine_HE_only.SEQID_Only.fasta> unique_345_Sequences_Bovine_HE_only.SEQID_Only.jtt_100.globalpair.afa_out &

Output: unique_345_Sequences_Bovine_HE_only.SEQID_Only.jtt_100.globalpair.afa_out  

Was split into unique_345_Sequences_Bovine_HE_only.SEQID_Only.jtt_100.globalpair.afa   &unique_345_Sequences_Bovine_HE_only.SEQID_Only.jtt_100.globalpair.out  

Command
nohup mafft --thread 15 --globalpair --maxiterate 1000 --jtt 100 --reorder unique_BetaCoronaVirus_HE_GE_1255_BP_BlastMatch_Bovine_HE_Consensus_SeqID_Only.fasta  >   unique_BetaCoronaVirus_HE_GE_1255_BP_BlastMatch_Bovine_HE_Consensus_SeqID_Only.jtt_100.globalpair.afa_out

unique_BetaCoronaVirus_HE_GE_1255_BP_BlastMatch_Bovine_HE_Consensus_SeqID_Only.jtt_100.globalpair.afa_out  
unique_BetaCoronaVirus_HE_GE_1255_BP_BlastMatch_Bovine_HE_Consensus_SeqID_Only.jtt_100.globalpair.afa  unique_BetaCoronaVirus_HE_GE_1255_BP_BlastMatch_Bovine_HE_Consensus_SeqID_Only.jtt_100.globalpair.out

Tree Building

 RaXML Tee Building

Notes  below are cut from the RaXML manual to put the command line parameters used in context
Note
"This is a how-to, which describes how RAxML  should best be used for a simple real-world biological analysis, given an example alignment named ex_al"

Now, if you want to run a full analysis, i.e., BS and ML search type:

raxmlHPC -­f a ­-x 12345 -­p 12345 ­-# 100 ­m GTRGAMMA ­-s ex_al -­n TEST

This will first conduct a BS search and once that is done a search for the best–scoring ML tree.
Such a program run will return the bootstrapped trees (RAxML_bootstrap.TEST), the best scoring ML tree(RAxML_bestTree.TEST), and the BS support values drawn on the best-scoring tree as node labels (RAxML_bipartitions.TEST) as well as, more correctly since support values refer to branches as branch labels (RAxML_bipartitionsBranchLabels.TEST).

Finally, note that by increasing the number of BS replicates via  -# you will also make the ML thorough since for ML optimization every 5th BS tree is used as a starting point to
for ML trees.

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

Note
Thus,  if  you  run  RAxML  with  32  instead  of  1  thread  this  does  not  mean  that  it  will  automatically become 32 times faster, it may actually even become slower. As I already mentioned, the parallel efficiency,  that  is,  with  how  many  threads/cores  you  can  still  execute  it  efficiently  in  parallel depends on the alignment length, or to be more precise on the number of distinct patterns in your alignment. 

This  number  is  printed  by  RAxML  to  the  terminal  and  into  the  RAxML_info.runID file"	
and look like this:

Alignment has 70 distinct alignment patterns	

As a rule of thumb I'd use one core/thread per 500 DNA site patterns, i.e., if you have less, than it's probably better to just use the sequential version. Single-gene DNA alignments with around 1000 sites can be analyzed with 2 or at most 4 threads. Thus, the more patterns your alignment has, the more threads/cores you can use efficiently.

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

Command
Run raxmlHPC command using nohup on 'nix platform to protect from premature job termination in case of remote connection loss ... and run in background with & at end of command line. Perform a 2000 boostrap search followed by a search for the best-scoring maximum likelihood tree.

-T 8  ( use 8 threads)
-f a  
-x 12345
-p 12345
-m GTRGAMMA
-N 2000
nohup raxmlHPC -T 8 -f a -x 12345 -p 12345 -N 2000 -m GTRGAMMA -s 192_Genomes_Final_6_14_22.jtt_100.globalpair.SEQID_Only.afa -n 192_Genomes_Final_6_14_22.jtt_100.globalpair.SEQID_Only.T8.N2000 >  192_Genomes_Final_6_14_22.jtt_100.globalpair.SEQID_Only.T8.N2000.out &

Output :

RAxML_bootstrap.192_Genomes_Final_6_14_22.jtt_100.globalpair.SEQID_Only.T8.N2000  192_Genomes_Final_6_14_22.jtt_100.globalpair.SEQID_Only.T8.N2000.out  
RAxML_info.192_Genomes_Final_6_14_22.jtt_100.globalpair.SEQID_Only.T8.N2000  
RAxML_bipartitionsBranchLabels.192_Genomes_Final_6_14_22.jtt_100.globalpair.SEQID_Only.T8.N2000  RAxML_bipartitions.192_Genomes_Final_6_14_22.jtt_100.globalpair.SEQID_Only.T8.N2000  RAxML_bestTree.192_Genomes_Final_6_14_22.jtt_100.globalpair.SEQID_Only.T8.N2000  

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 

RAxML tree of 192 bovine coronavirus genomes. Bootstrap support values are proportional to the line thickness of the branches. Isolates with HE deletions are denoted with a filled red square while the single isolate with an HE insertion is denoted with a filled blue square.  Tree metrics are best investigated in the IToL tree.
  
The pdf suitable for download is  RaXML tree of 192 livestock coronavirus genomes.pdf

Following the same approach with the 192 BCoV genomes other RaXML Trees were generated for spike and HE and are included here

RAxML_bipartitionsBranchLabels.unique_HE_6_14_22.SEQID_Only.jtt_100.globalpair.T8.N2000  
RAxML_bipartitionsBranchLabels.unique_HE_6_14_22.SEQID_Only.jtt_200.globalpair.T8.N2000 RAxML_bipartitionsBranchLabels.unique_Spike_6_14_22.SEQID_Only.jtt_100.globalpair.T8.N2000  RAxML_bipartitionsBranchLabels.unique_Spike_6_14_22.SEQID_Only.jtt_200.globalpair.T8.N2000  RAxML_bipartitionsBranchLabels.unique_345_Sequences_Bovine_HE_only.SEQID_Only.jtt_100.globalpair.T8.N2000  unique_BetaCoronaVirus_HE_GE_1255_BP_BlastMatch_Bovine_HE_Consensus_SeqID_Only.T8.N200.out RAxML_bipartitionsBranchLabels.unique_BetaCoronaVirus_HE_GE_1255_BP_BlastMatch_Bovine_HE_Consensus_SeqID_Only.T8.N2000

Public workspacePhylogenetic Analysis of Complete Bovine Coronavirus Genome Sequences V.3

Phylogenetic Analysis of Complete Bovine Coronavirus Genome Sequences V.3