DATA ANALYSIS PROBLEM

Ksiazek, T.G., D.V.M. Ksiazek, D. Erdman, C.S. Goldsmith, S.R. Zaki, T. Peret, S. Emery, S. Tong, C. Urbani, J.A. Comer, et al. 2003. A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med. 348:1953–1966.

Question 8.15

T. G. Ksiazek and other members of the SARS Working Group described their discovery of the SARS virus and its identification as a novel coronavirus. They identified the virus as a coronavirus through electron microscopy, and then confirmed it with a sequence analysis of PCR-amplified genomic segments from SARS patient samples.

  1. The PCR method for amplifying the coronavirus genomic sample requires the use of reverse transcriptase. Why?

  2. To amplify sequences from a new virus with an unsequenced genome, what considerations would go into the design of appropriate PCR primers?

    A sequence alignment involving a 405 bp segment of the DNA polymerase gene in four coronaviruses, BCoV, HEV, SARS CoV, and TGEV, is given on the facing page—the alignment that Ksiazek and colleagues used to generate the phylogenetic tree below (this includes coronaviruses not discussed in this problem; nt = nucleotides). In the alignment, nucleotide polymorphisms relative to the bovine coronavirus sequence (bcov) are shown in red.

  3. How many sequence differences exist between the genome segments from HEV (hev) and BCoV? How many between BCoV and SARS CoV (sars)? How many between TGEV (tgev) and SARS CoV? Which two coronaviruses are most closely related? Are your counts in general agreement with the phylogenetic tree?

295

296

     1 60

bcov.pol.seq

    

TCGTGCTATGCCAAACATACTACGTATTGTTAGTAGTCTGGTTTTGGCTCGAAAACATGA

hev.pol.seq

    

TCGTGCTATGCCAAACATACTACGTATTGTTAGTAGTCTGGTATTGGCCCGAAAACATGA

sars.pol.seq

    

CAGAGCCATGCCTAACATGCTTAGGATAATGGCCTCTCTTGTTCTTGCTCGCAAACATAA

tgev.pol.seq

    

CCGTGCTTTACCTAATATGATTAGAATGGCTTCTGCCATGATATTAGGTTCTAAGCATGT
     61 120

bcov.pol.seq

    

GGCATGTTGTTCGCAAAGCGATAGGTTTTATCGACTTGCGAATGAATGCGCACAAGTTCT

hev.pol.seq

    

GGCATGTTGTTCGCAAAGCGATAGGTTTTATCGACTTGCGAATGAATGCGCACAAGTTCT

sars.pol.seq

    

CACTTGCTGTAACTTATCACACCGTTTCTACAGGTTAGCTAACGAGTGTGCGCAAGTATT

tgev.pol.seq

    

TGGTTGTTGTACACATAATGATAGGTTCTACCGCCTCTCCAATGAGTTAGCTCAAGTACT
     121 180

bcov.pol.seq

    

GAGTGAAATTGTTATGTGTGGTGGCTGTTATTATGTTAAGCCTGGTGGCACTAGTAGTGG

hev.pol.seq

    

TAGTGAAATTGTTATGTGTGGTGGCTGTTATTATGTTAAGCCTGGTGGCACTAGTAGTGG

sars.pol.seq

    

AAGTGAGATGGTCATGTGTGGCGGCTCACTATATGTTAAACCAGGTGGAACATCATCCGG

tgev.pol.seq

    

CACAGAAGTTGTGCATTGCACAGGTGGTTTTTATTTTAAACCTGGTGGTACAACTAGCGG
     181 240

bcov.pol.seq

    

TGATGCAACTACTGCTTTTGCTAATTCAGTTTTTAACATATGTCAAGCTGTTTCAGCCAA

hev.pol.seq

    

TGATGCAACTACTGCTTTTGCTAATTCAGTCTTTAACATATGTCAAGCTGTTTCAGCCAA

sars.pol.seq

    

TGATGCTACAACTGCTTATGCTAATAGTGTCTTTAACATTTGTCAAGCTGTTACAGCCAA

tgev.pol.seq

    

TGATGGTACTACAGCATATGCTAACTCTGCTTTTAACATCTTTCAAGCTGTTTCTGCTAA
     241 300

bcov.pol.seq

    

TGTATGTGCTTTAATGTCATGCAATGGTAATAAGATTGAAGATTTGAGTATACGTGCTCT

hev.pol.seq

    

TGTATGTTCCTTAATGTCATGCAATGGCAATAAGATTGAAGATTTGAGTATACGTGCTCT

sars.pol.seq

    

TGTAAATGCACTTCTTTCAACTGATGGTAATAAGATAGCTGACAAGTATGTCCGCAATCT

tgev.pol.seq

    

TGTTAATAAGCTTTTGGGGGTTGATTCAAACGCTTGTAACAACGTTACAGTAAAATCCAT
     301 360

bcov.pol.seq

    

TCAGAAGCGCTTATACTCACATGTGTATAGAAGTGATATGGTTGATTCAACCTTTGTCAC

hev.pol.seq

    

TCAGAAGCGTTTATACTCACATGTGTATAGAAGTGATATGGTTGATTCAACCTTTGTCAC

sars.pol.seq

    

ACAACACAGGCTCTATGAGTGTCTCTATAGAAATAGGGATGTTGATCATGAATTCGTGGA

tgev.pol.seq

    

ACAACGTAAAATTTACGATAATTGTTATCGTAGTAGCAGCATTGATGAAGAATTTGTTGT
     361 406

bcov.pol.seq

    

AGAATATTATGAATTTTTAAATAAGCATTTTAGTATGATGATTTTG

hev.pol.seq

    

AGAATATTATGAATTTTTAAATAAGCATTTTAGTATGATGATTTTG

sars.pol.seq

    

TGAGTTTTACGCTTACCTGCGTAAACATTTCTCCATGATGATTCTT

tgev.pol.seq

    

TGAGTACTTTAGTTATTTGAGAAAACACTTTTCTATGATGATTTTA

[Source: Sequence alignment courtesy of Dr. Ann Palmenberg, Department of Biochemistry and the Institute for Molecular Virology, University of Wisconsin–Madison.]