Construction And Application Of Rubella Virus Ribonuclease-resistant RNA Phage-like Particles

Rubella virus(RV) is a positive-sense RNA genome virus within the family Togaviridae.RV is an enveloped RNA virus that causes a disease known as rubella. Primary infection is established in the upper respiratory mucosa and/or the nasopharyngeal lymphoid tissue,after which a systemic infection occurs.Rubella virus infection can cause quite severe complications such as subacute sclerosing panencephalitis(SSPE),which may lead to death.More important,if rubella occurs during the first trimester of pregnancy there is a 90%risk of congenital malformations in the fetus.Persistent fetal infection may result in a series of birth defects known as the congenital rubella syndrome(CRS).The real time RT-PCR technology developed recently has become a mainstream technology for the rapid,specific and sensitive detection and quantification of RNA targets of infectious disease.Central to these quantitative assays are reliable RNA positive controls and standards.The current positive controls and standards used in RV RNA-based amplification diagnostic assays comprise an attenuated or inactivated infectious rubella virus itself or a complementary DNA susceptible to DNases or an in vitro-transcribed naked RNA highly susceptible to degradation by ubiquitous RNases. Intact rubella virus as positive controls and standards is of potential serious harm to laboratory workers.During a clinical run,a naked RNA or complementary DNA as positive controls and standards in amplification assays inadvertently contaminated with RNase or DNase would cause the failure of an entire run and unnecessary loss of time and money.In addition,partial degradation of the positive controls and standards may not be detected and may lead to erroneous results.The ideal positive controls or standards would be containing an RNA target,not dependent on a RNase-free and/or DNase-free environment,stable in plasma and carrying no risk of infection to laboratory workers,because in many developing countries there are no rubella vaccination programme to all laboratory workers who are working on rubella virus.Therfore,it was considered that rubella virus ribonuclease-resistant RV RNA virus-like particles(VLPs) be constructed as positive controls and standards for quantitatively amplification assays for rubella virus by the armored technology and be applied in real-time quantitative RT-PCR assay.The present study aimed at the following purpose:1.Establishment of a real-time quantitative reverse transcription-polymerase chain reaction assay.2.Construction and expression of rubella virus ribonuclease-resistant RV RNA virus-like particles by the armored technology.3.Preclinical evaluation of rubella virus ribonuclease-resistant RV RNA virus-like particles as positive standard to apply in real-time quantitative RT-PCR assay. The first part of experiment was to establish a real-time quantitative reverse transcription-polymerase chain reaction assay,and a rubella virus specific PCR amplicon was made as extemal standard to analytically estimate this real time quantitative assay.112 clinical samples were detected by the assay to determine the specificity and sensitivity of the assay.The second part of experiment was to construct rubella virus ribonuclease-resistant RV RNA virus-like particles by the armored technology that specific encapsidation of heterologous RNA could be obtained in E.coli by linking the MS2 operator sequence to the heterologous RNA.And then test its nuclease-resistant property and its stability in plasma at 4℃and at 37℃.The third part of experiment was to evaluate a real time quantitative reverse transcription-polymerase chain reaction for rubella virus(RV) using RV armored RNA standard constructed in the laboratory.112 clinical samples were detected by the assay to determine the specificity and sensitivity of the assay using RV armored RNA standard,and then compare the specificity and sensitivity with those of the assay using RV cDNA standard.The main methods were shown as follows:1.A real-time quantitative reverse transcription-polymerase chain reaction assay was established,and a rubella virus specific PCR amplicon was made as external standard to analytically estimate this real time quantitative assay.112 clinical samples were detected by the assay to determine the specificity and sensitivity of the assay.2.Rubella virus ribonuclease-resistant RV RNA virus-like particles(VLPs) were constructed as positive controls and standards for quantitatively amplification assays for rubella virus by the armored technology that specific encapsidation of heterologous RNA could be obtained in E.coli by linking the MS2 operator sequence to the heterologous RNA.3.RNA packaged within RV RNA VLPs was identified by PCR both after and without reverse transcription.4.A real-time quantitative reverse transcription-polymerase chain reaction assay was applied for quantitative analysis of RV RNA VLPs.5.A real time fluorescence quantitative RT-PCR was performed on the three dilutions of the RV RNA VLPs treated with RNase(lmg/L) placed at room temperature for 60min and those of the RV RNA VLPs without RNase treatment in parallel.Each of the samples was assayed repeatedly for six times to test the property of resistance to RNase digestion of RV RNA VLPs.6.RV RNA VLPs were incubated with the normal human ethylene diamine tetraacetic acid(EDTA)-anticoagulated plasma both at 4℃and 37℃for 60 days and 30 days,respectively.RV RNA VLPs taken respectively at the seven time points were assayed in duplicate by quantitative RT-PCR to test its stability in plasma.An inactivated RV TR06 strain,and RV Armored RNA were added to normal human plasma,respectively and incubated individually at 37℃for30 days. Samples taken at each time point were stored at -70℃and then assayed in duplicate by real time fluorescence quantitative RT-PCR simultaneously to compare the both stability.7.RV armored RNA was used as positive standard for a real time quantitative RT-PCR for rubella virus.112 clinical samples were detected by the assay to determine the specificity and sensitivity of the assay.8.The PCR efficiency,the detecting limit,the reproducibility,the specificity and sensitivity of the real time quantitative RT-PCR for rubella virus with RV armored RNA standard were compared to those of the assay with RV cDNA standard.The main results were shown as follows:1.A real-time quantitative reverse transcription-polymerase chain reaction assay was built,and a rubella virus(RV) specific PCR amplicon was made as external standard to analytically estimate this real time quantitative assay.The detecting limit of the established real time RT-PCR assay was 1.0×10~3 copies/ml.The PCR amplification efficiency was 1.89.The real time RT-PCR assay was performed in triplicate on a series of dilutions from 1×10~8 to 1×10~3 of RV amplicon standard in order to examine its reproducibility.The coefficient of variation(CV) range of the Ct was from 3.29%～4.36%.When the quantitative RT-PCR assay was applied to detect the RV RNA in 112 clinical samples,the specificity and sensitivity of the assay was 88.9%, 81.3%,respectively,comparison to gold standard.2.RV RNA virus-like particles(VLPs) were shown to be constructed successfully by sequence analysis.3.A series of dilutions from 1×10~7 to 1×10~4 of RV RNA VLPs were amplified by PCR both after and without reverse transcription.The amplified products after reverse transcription were positive,while the amplified products without reverse transcription were negative,which showed the RV RNA VLPs were truly RNA,but not DNA or DNA contamination.4.A series of dilutions from 1×10~6 to 1×10~4 of RV RNA VLPs were quantified in duplicate by a quantitative RT-PCR,and the concentration determinations were averaged.The quantification results of RV RNA VLPs calculated according to the standard curve were 2.0×10~5 copies/μl,2.8×10~4 copies/μl,2.6×10~3 copies/μl, respectively.The original concentration of RV RNA VLPs calculated was 2.45×10~9 copies/μl.5.RV RNA VLPs serially diluted to a concentration range from 2.45×10~5 to 2.45×10~3 copies/μl treated with RNase and those without RNase treatment were assayed repeatedly for six times by quantitative RT-PCR.The comparison of threshold cycle(Ct) values between RNase treatment group and the RNase non-treatment group showed that there was no significant difference in Ct determinations of each of dilutions of RV RNA VLPs between the two groups.This showed RV RNA VLPs possessed the property of resistance to RNase digestion.6.RV Armored RNA added to normal human plasma to a final concentration of 2.45×10~4 copies/μl were incubated at 4℃over 60 days.Samples taken from eight time point were assayed in duplicate,and the concentration determinations were averaged.The mean for all of the samples was 2.54×10~4 copies/μl,and the coefficient of variation was 4.66%.This showed that RV Armored RNA were stable in human plasma at 4℃over 60 days.7.RV Armored RNA added to normal human plasma to a final concentration of 2.45×10~4 copies/μl were incubated at 37℃over 30 days.Samples taken from seven time point were assayed in duplicate,and the concentration determinations were averaged.The mean for all of the samples was 2.50×10~4 copies/μl,and the coefficient of variation was 3.46%.This showed that RV Armored RNA were stable in human plasma at 37℃over 30 days.8.An attenuated RV TR06 strain,and RV Armored RNA added to normal human plasma to a final concentration at approximately 5.0×10~3 and 2.45×10~3 copies/μl, respectively,and incubated individually at 37℃over 30days.Samples taken from six time point were assayed in duplicate,and the concentration determinations were averaged.The mean for all the RV Armored RNA samples was 2.54×10~3 copies/μl, and the coefficient of variation was 4.11%.Therefore the RV Armored RNA was stable over the time course.But the RV TR06 copy number(concentration range:1.05×10~3 copies/μl～5.0×10~3 copies/μl)declined by 79%over 30days compared to the original input. 9.The detecting limit of the established real time RT-PCR assay using RV Armored RNA as external standard was 2.45×10~2 copies/ml.The PCR amplification efficiency was 1.97.The real time RT-PCR assay was performed in triplicate on a series of dilutions of RV Armored standard in order to examine its reproducibility.The coefficient of variation(CV) range of the Ct was from 0.98%～1.03%.When the quantitative RT-PCR assay was applied to detect the RV RNA in 112 clinical samples, the specificity and sensitivity of the assay was 98.1%,91.1%,respectively, comparison to gold standard.10.Compared to those of the real time quantitative RT-PCR assay for rubella virus with RV cDNA standard,the PCR efficiency of the assay with RV armored RNA standard was as same as that of of the assay with RV cDNA standard(p＞0.05),the detecting limit was lower(p＜0.05),the reproducibility was better(p＜0.001),the specificity(p＜0.01) and sensitivity(p＜0.05) were both higher.The results suggest that:1.The real-time quantitative reverse transcription-polymerase chain reaction assay is a rapid,specific and sensitive assay.2.RV Armored RNA can be constructed successfully by the armored technology.3.RV Armored RNA is resistant to RNase digestion.4.RV Armored RNA is stable in human plasma.5.Compared to those of the real time quantitative RT-PCR assay for rubella virus with RV cDNA standard,the PCR efficiency of the assay with RV armored RNA standard was the same,the detecting limit was lower,the reproducibility was better.6.The specificity and sensitivity of the real time quantitative RT-PCR assay with RV armored RNA standard were both higher than those of the assay with RV cDNA standard.