Quantitative-competitive PCR monitoring of viral load following experimental guinea pig cytomegalovirus infection.
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.
Schleiss
Mark R
MR
Cincinnati Children's Hospital Medical Center, Division of Infectious Diseases, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA. schlm0@chmcc.org
Bourne
Nigel
N
Bravo
Fernando J
FJ
Jensen
Nancy J
NJ
Bernstein
David I
DI
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D003586:Cytomegalovirus Infections / Q000151:congenital / Q000209:etiology / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D004195:Disease Models, Animal; D005260:Female; D005814:Genes, Viral; D016679:Genome, Viral; D006168:Guinea Pigs; D006801:Humans; D009928:Organ Specificity; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D019211:Roseolovirus / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity*; D014773:Virology / Q000379:methods*
D017931:DNA Primers; D004279:DNA, Viral