Quantitative-competitive PCR monitoring of viral load following experimental guinea pig cytomegalovirus infection

Mark R. Schleiss, Nigel Bourne, Fernando J. Bravo, Nancy J. Jensen, David I. Bernstein

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Human cytomegalovirus (HCMV) is the most common cause of congenital viral infection in the developed world, and can lead to significant morbidity. Animal models of HCMV infection are required for study of pathogenesis, because of the strict species-specificity of cytomegalovirus (CMV). Among the small animal CMV models, the guinea pig CMV (GPCMV) has unique advantages, in particular its propensity to cross the placenta, causing disease in utero. In order to develop quantitative endpoints for vaccine and antiviral therapeutic studies in the GPCMV model, a quantitative-competitive PCR (qcPCR) assay was developed, based on the GPCMV homolog of the HCMV UL83 gene, GP83. Optimal amplification of GPCMV DNA was observed using primers spanning a 248 base pair (bp) region of this gene. A 91 bp deletion of this cloned fragment was generated for use as an internal standard (IS) for PCR amplification. Standard curves based upon the fluorescent intensity of full-length external target to IS were compared with signal intensity of DNA extracted from blood and organs of experimentally infected guinea pigs in order to quantify viral load. Viral load in newborn guinea pigs infected transplacentally was determined and compared with that of pups infected with GPCMV as neonates. Viral loads were highest in pups infected as neonates. The most consistent isolation and highest quantities of viral DNA were observed in liver and spleen, although viral genome could be readily identified in brain, lung, and salivary gland. Viral load determination should be useful for monitoring outcomes following vaccine studies, as well as responses to experimental antiviral agents.

Original languageEnglish (US)
Pages (from-to)103-110
Number of pages8
JournalJournal of Virological Methods
Volume108
Issue number1
DOIs
StatePublished - Mar 2003

Fingerprint

Roseolovirus
Cytomegalovirus Infections
Viral Load
Guinea Pigs
Cytomegalovirus
Polymerase Chain Reaction
Base Pairing
Antiviral Agents
Placenta Diseases
Vaccines
Animal Models
Species Specificity
Viral Genome
DNA
Viral DNA
Virus Diseases
Salivary Glands
Genes
Spleen
Morbidity

Keywords

  • Congenital CMV infection
  • Cytomegalovirus
  • Guinea pig model
  • PCR

ASJC Scopus subject areas

  • Virology

Cite this

Quantitative-competitive PCR monitoring of viral load following experimental guinea pig cytomegalovirus infection. / Schleiss, Mark R.; Bourne, Nigel; Bravo, Fernando J.; Jensen, Nancy J.; Bernstein, David I.

In: Journal of Virological Methods, Vol. 108, No. 1, 03.2003, p. 103-110.

Research output: Contribution to journalArticle

Schleiss, Mark R. ; Bourne, Nigel ; Bravo, Fernando J. ; Jensen, Nancy J. ; Bernstein, David I. / Quantitative-competitive PCR monitoring of viral load following experimental guinea pig cytomegalovirus infection. In: Journal of Virological Methods. 2003 ; Vol. 108, No. 1. pp. 103-110.
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