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The Use of Arms PCR and RFLP Analysis in Identifying Genetic Profiles of Virulent, Attenuated or Vaccine Strains of TGEV and PRCV

  • Chih-Hung Lai
  • Mark W. Welter
  • Lisa M. Welter
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 380)

Abstract

The use of ARMS (amplification refractory mutation system) PCR coupled with RFLP (restriction fragment length polymorphism) analysis has been used to identify a unique genetic marker on the Ambico oral vaccine strain. This method was also used to characterize the genetic profiles of a number of other TGEV strains. This procedure takes advantage of the nucleotide differences between the Ambico strain, and the Miller and Purdue strains. Within the S gene there are three nucleotide differences between the Ambico strain and the published Purdue sequence. There are additional nucleotide dif-ferences in the structural and non-structural gene sequences, but we have chosen to focus on the differences contained within the S gene. The Ambico strain has a closer sequence homology to the Purdue strain than to the Miller strain. The Ambico and Purdue strains contain a six nucleotide deletion at position 1122 that is not present in the Miller published sequence or the ISU-1 strain of PRCV (based on our PCR experiments). We have designed a 5’ oligo whose sequence is homologous to a region located 80 nucleotides upstream of the TGEV and PRCV S gene initiation codons to be used in conjunction with either of two 3’ oligos whose sequences are identical with the exception of the last six nucleotides of their 3’ ends. When utilized with the appropriate PCR conditions, these oligos can differentiate between PRCV, Miller and Purdue prototype virus strains. These PCR products were then subjected to RFLP analysis using four separate restriction enzymes (BstE II, Alw26 I, Dra III, or MspA\ I). We have used this procedure to analyze six TGEV vaccine strains, intestinal derived virulent viruses, cell cultured viruses at different cell passage numbers, and field isolates of TGEV or PRCV.

Keywords

Vaccine Strain Genetic Profile RFLP Analysis amplificatIOn Refractory mutatIOn System Small Intestinal Content 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Doyle L.P., Hutchings L.M. A transmissible gastroenteritis in pigs. J. Am. Vet. Med. Assoc. 1946; 108:257–259.PubMedGoogle Scholar
  2. 2.
    Pensaert M., Callebaut P., Vergote J. Isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis. Vet.Q. 1986;8:257–261.PubMedCrossRefGoogle Scholar
  3. 3.
    Hill H.T., Biwer J.D., Wood R.D., Wesley R.D. Porcine respiratory coronavirus isolated from two U.S. swine herds. Proc. Am. Assoc. Swine Prac. 1989;333–335.Google Scholar
  4. 4.
    Paul P.S., Vaughn E.M., Halbur P.G. Characterization and pathogenicity of a new porcine respiratory coronavirus strain AR310. Proc. Int. Pig Vet. Soc. Congr. 1992; 12:92.Google Scholar
  5. 5.
    Wesley R.D., Woods R.D., Hill H.T., Biwer J.D. Evidence for a porcine respiratory coronavirus, antigenically similar to transmissible gastroenteritis virus. J. Vet. Diagn. Invest. 1990;2:312–317.PubMedCrossRefGoogle Scholar
  6. 6.
    Vaughn E.M., Halbur P.G., Paul P.S., Three new isolates of porcine respiratory coronavirus with pathogenicities and spike (S) gene deletions. J. Clin. Micro. 1994;32:1809–1812.Google Scholar
  7. 7.
    Britton P., Kottier S., Chen C.-M., Pocock D.H., Salmon H., Aynaud J.M. The use of PCR genome mapping for the characterization of TGEV strains. Adv. Exp. Med. and Biol. 1993;342:29–34.CrossRefGoogle Scholar
  8. 8.
    Sanchez CM., Gebauer F., Sune C, Mendez A., Dopazo J., Enjuanes L. Genetic evolution and tropism of transmissible gastroenteritis coronaviruses. Virology 1992;190:92–105.PubMedCrossRefGoogle Scholar
  9. 9.
    Britton P., Page K.W. Sequence of the S gene from a virulent British field isolate of transmissible gastroenteritis virus. Vir. Res. 1990;18:71–80.CrossRefGoogle Scholar
  10. 10.
    Laude H., Van Reeth K., Pensaert M. Porcine respiratory coronavirus: molecular features and virus-host interactions. Vet. Res.1993;24:125–150.PubMedGoogle Scholar
  11. 11.
    Wesley R.D., Wood R.D., Cheung A.K. Genetic basis for the pathogenesis of transmissible gastroenteritis virus. J. Virol. 1990;64:4761–4766.PubMedGoogle Scholar
  12. 12.
    Wesley R.D., Wood R.D., Cheung A.K. Genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmissible gastroenteritis virus. J.Virol. 1991;65:3369–3373.PubMedGoogle Scholar
  13. 13.
    Newton C.R., Graham A., Heptinstall L.E., Powell S.J., Summers C., Kalsheker N., Smith J.C., Markham A.F. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nuc. Acids Res. 1989;17:2503–2516.CrossRefGoogle Scholar
  14. 14.
    Huang M.M., Arnheim N., Goodman M.F. Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR. Nuc.Acids Res. 1992;20:4567–4573.CrossRefGoogle Scholar
  15. 15.
    Kwok S., Kellogg D.E., McKinney N., Spasic D., Goda L., Leverson C., Sninsky J.J. Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nuc. Acids Res. 1990;18 999–1005.CrossRefGoogle Scholar
  16. 16.
    Register K.B., Wesley R.D. Molecular characterization of attenuated vaccine strains of transmissible gastroenteritis virus. J. Vet. Diagn. Invest. 1994;6:16–22.PubMedCrossRefGoogle Scholar
  17. 17.
    Ault G.S., Ryschkewitsch C.F., Stoner G.L. Type-specific amplification of viral DNA using touchdown and hot start PCR. J. Virological Meth. 1994;46:145–156.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1995

Authors and Affiliations

  • Chih-Hung Lai
    • 1
  • Mark W. Welter
    • 2
  • Lisa M. Welter
    • 1
  1. 1.Ambico West, USC School of MedicineLos AngelesUSA
  2. 2.Ambico, Inc., Dallas CenterUSA

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