PubMed · 14461595
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O LAHELLE. 1962-01-25. [Enterovirus].. https://pubmed.ncbi.nlm.nih.gov/14461595/
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Enterovirus 71 (EV71) infection may be asymptomatic or may cause diarrhea, rashes, and hand, foot, and mouth disease (HFMD). However, EV71 also has the potential to cause severe neurological disease. To date, little is known about the molecular mechanisms of host response to EV71 infection. In this report, we utilized cDNA microarray to profile the kinetics and patterns of host gene expression in EV71-infected human neural SF268 cells. We have identified 157 genes with significant changes in mRNA expression and performed hierarchical clustering to classify these genes into five different groups based on their kinetics of expression. EV71 infection led to increases in the level of mRNAs encoding chemokines, proteins involved in protein degradation, complement proteins, and proapoptotis proteins. cDNA microarray expression comparisons of EV71- and mock-infected cells also revealed the down-regulation of several genes encoding proteins involved in host RNA synthesis. Expression of interferon-regulated proteins was increased early in the infection and then decreased. Expression of proteins involved in cellular development and differentiation, some oncogenes, and transcription and translation regulators were suppressed and then stimulated late in the infection. Our findings illustrate the overall host response to EV71 infection, and will aid in understanding the host response to this virus.
The objective of the present study was the development of a diagnostic reverse transcription (RT)-PCR for the specific detection of enterovirus (EV) RNA in clinical specimens controlled by an internal control (IC) RNA. The IC RNA contains the same primer binding sites as EV RNA but has a different probe region. The IC RNA was packaged into an MS2 phage core particle (armored) and was added to the clinical sample to allow monitoring of both extraction efficiency and RT-PCR efficiency. Serial dilutions of the IC RNA were made, and the detection limit of the RT-PCR was tested in a background of EV RNA-negative cerebrospinal fluid. The sensitivity and specificity of the RT-PCR assay were tested by using all 64 known EV serotypes, several non-EV serotypes, and two Quality Control for Molecular Diagnostics (QCMD) Program EV proficiency panels from 2001 and 2002. In total, 322 clinical specimens were tested by RT-PCR, and to establish the clinical utility of the RT-PCR, a comparison of the results of viral culture and RT-PCR was done with 87 clinical specimens. The lower limit of sensitivity was reached at about 150 copies of IC RNA/ml. All 64 EV serotypes were positive, while all non-EV serotypes were negative. All culture-positive samples of the 2001 QCMD proficiency panel (according to the 50% tissue culture infective doses per milliliter) were positive by RT-PCR. Invalid results, i.e., negativity for both EV RNA and IC RNA, due to inhibition of RT-PCR were observed for 33.3% of the members of the 2002 QCMD proficiency panel and 3.1% of the clinical specimens. Inhibition of RT-PCR could be relieved by the addition of 400 ng of bovine alpha-casein per microl to both the RT reaction mixture and the PCR mixture. With this optimized protocol, the results for all samples of the 2002 QCMD proficiency panel and all clinical specimens except one fecal sample (0.3%) were valid. Evaluation of the clinical samples demonstrated that EV infection could be detected in 12 of 87 samples (13.8%) by RT-PCR, while viral culture was negative. Our data show that the RT-PCR with armored IC RNA offers a very reliable and rapid diagnostic tool for the detection of EV in clinical specimens and that the addition of bovine alpha-casein relieved inhibition of the RT-PCR for 99.7% of clinical specimens.
A method of a modified nest-type polymerase chain reaction (MN-PCR), made in one tube, was elaborated that enhances the sensitivity and cuts the risk of cross-contamination in enteroviruses (EV) detection. The method, as described in detail above, was used to detect EV RNA in 76.9% and 31.25% of examined autopsy samples (13 liquor and 16 cardiac-tissue samples, respectively). It enabled the detection of EV RNA in 6.25% of samples that used to be negative, when tested by MN-PCR in 2 tubes. MN-PCR with one tube is a reliable, sensitive and specific diagnostic tool; it can be recommended for the routine diagnostics of enterovirus infection.