Hamprecht Klaus, Witzel Simone, Maschmann Jens, Dietz Klaus, Baumeister Andrea, Mikeler Elfriede, Goelz Rangmar, Speer Christian P, Jahn Gerhard
Institute of Medical Virology and Epidemiology of Viral Diseases, University Hospital of Tübingen, Elfriede-Aulhorn-Str 6, 72076 Tübingen, Germany.
J Clin Virol. 2003 Dec;28(3):303-16. doi: 10.1016/s1386-6532(03)00074-x.
Human cytomegalovirus (HCMV) is reactivated in nearly every seropositive breastfeeding mother during lactation [Lancet 357 (2001) 513]. Conventional tissue culture (TC) and low-speed centrifugation-enhanced microtiter culture methods are not able to detect HCMV from milk during all stages of lactation.
Development of a sensitive and quantitative microculture technique to describe the dynamics of HCMV reactivation in different milk compartments during lactation.
Milk samples were collected longitudinally from seropositive breastfeeding mothers of preterm infants. Native milk samples were separated into fraction 1 (aqueous extract of milk fat), fraction 2 (cell and fat free milk whey) and fraction 3 (milk cells). Each of these fractions was screened qualitatively (TC, nPCR, pp67 late mRNA) and quantitatively (high-speed centrifugation-based microculture, quantitative PCR).
Prior to low-speed centrifugation-enhanced inoculation, virus particles were concentrated by high-speed centrifugation (60 min at 50,000 x g, 4 degrees C). Using fraction 2 we were able to describe the dynamics of viral reactivation during lactation. We present the course of the quantitative virolactia and DNAlactia and qualitative detection of HCMV pp67 late mRNA in milk whey of four mothers (three transmitters and one non-transmitter). In all these cases virolactia described an unimodal and self limited course. Peak levels of virolactia for transmitters (T1: day 44; T2: day 43; T3: day 50) were closely related the onset of viruria of the corresponding preterm infants (U1: day 39; U2a/U2b: day 44/57; U3: day 60). The courses of viral load coincidence with the courses of DNA load.
We present a rapid and highly sensitive microculture method for the quantification of cell free HCMV from milk whey and aqueous extracts from milk fat. Viral reactivation during lactation describes an unimodal course. Our findings have strong implications for quality control of any virus inactivation procedure.
人巨细胞病毒（HCMV）在几乎每一位血清反应阳性的哺乳期母亲体内都会在哺乳期重新激活[《柳叶刀》357（2001）513]。传统的组织培养（TC）和低速离心增强微量滴定培养方法无法在哺乳期的所有阶段从乳汁中检测到HCMV。
开发一种灵敏且定量的微量培养技术，以描述哺乳期不同乳汁成分中HCMV重新激活的动态变化。
从早产血清反应阳性的哺乳期母亲中纵向采集乳汁样本。天然乳汁样本被分离为组分1（乳脂肪水提取物）、组分2（无细胞和脂肪的乳清）和组分3（乳汁细胞）。对这些组分中的每一个进行定性（TC、巢式PCR、pp67晚期mRNA）和定量（基于高速离心的微量培养、定量PCR）筛选。
在低速离心增强接种之前，通过高速离心（50,000×g，4℃，60分钟）浓缩病毒颗粒。使用组分2，我们能够描述哺乳期病毒重新激活的动态变化。我们展示了四位母亲（三位传播者和一位非传播者）乳清中HCMV pp67晚期mRNA的定量病毒乳汁和DNA乳汁过程以及定性检测结果。在所有这些案例中，病毒乳汁呈现出单峰且自我限制的过程。传播者的病毒乳汁峰值水平（T1：第44天；T2：第43天；T3：第50天）与相应早产婴儿病毒尿的发作密切相关（U1：第39天；U2a/U2b：第44/57天；U3：第60天）。病毒载量过程与DNA载量过程一致。
我们提出了一种快速且高度灵敏的微量培养方法，用于定量乳清和乳脂肪水提取物中无细胞的HCMV。哺乳期的病毒重新激活呈现出单峰过程。我们的发现对任何病毒灭活程序的质量控制具有重要意义。
