Schleiss Mark R, Bourne Nigel, Bravo Fernando J, Jensen Nancy J, Bernstein David I
Cincinnati Children's Hospital Medical Center, Division of Infectious Diseases, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA.
J Virol Methods. 2003 Mar;108(1):103-10. doi: 10.1016/s0166-0934(02)00265-3.
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.
人巨细胞病毒（HCMV）是发达国家先天性病毒感染最常见的病因，可导致严重发病。由于巨细胞病毒（CMV）具有严格的种属特异性，因此需要HCMV感染的动物模型来研究发病机制。在小型动物CMV模型中，豚鼠巨细胞病毒（GPCMV）具有独特优势，尤其是它易于穿过胎盘，在子宫内引发疾病。为了在GPCMV模型中开发用于疫苗和抗病毒治疗研究的定量终点，基于HCMV UL83基因的GPCMV同源物GP83开发了一种定量竞争PCR（qcPCR）检测方法。使用跨越该基因248个碱基对（bp）区域的引物观察到GPCMV DNA的最佳扩增。对该克隆片段进行91 bp的缺失以用作PCR扩增的内标（IS）。将基于全长外部靶标与IS荧光强度的标准曲线与从实验感染豚鼠的血液和器官中提取的DNA的信号强度进行比较，以量化病毒载量。测定经胎盘感染的新生豚鼠的病毒载量，并与新生时感染GPCMV的幼崽的病毒载量进行比较。新生时感染的幼崽的病毒载量最高。尽管在脑、肺和唾液腺中可轻易鉴定出病毒基因组，但在肝脏和脾脏中观察到最一致的病毒分离和最高量的病毒DNA。病毒载量测定对于监测疫苗研究后的结果以及对实验性抗病毒药物的反应应该是有用的。
