MS2 maturase and coat protein genes were amplified by using primers S-MC and A-MC (Table 1) from the pMS₂₇ plasmid (kindly provided by D. S. Peabody) containing nucleotides (nt) 81 to 1749 of the MS2 bacteriophage gene (GenBank accession no. V00642). Sense and reverse primers contained BamHI and HindIII restriction sites (underlined), respectively. These primers correspond to nt 81 to 101 and nt 1721 to 1741 of the MS2 phage genome sequence. The 1.7-kb PCR-amplified DNA fragments were gel purified, digested with BamHI and HindIII, and then ligated to a linearized pET28b (Novagen) vector to generate the recombinant plasmid pET-MC. This plasmid was transformed into competent E. coli DH5α cells according to the manufacturer's instructions. The pET-MC plasmids in positive clones that could replicate in LB agar in the presence of 100 μg of kanamycin ml⁻¹ were isolated by using a Takara MiniBEST plasmid purification kit (TaKaRa). The DNA insert was sequenced with vector-specific primers using an automated fluorescent DNA sequencer (model 3730XL; Applied Biosystems). The resulting sequences were identified by a search of the NCBI databases for homologous sequences using BLAST.

An exogenous chimeric sequence 2,248 bp in length comprising the following sequences was inserted into a pACYCDuet-1 plasmid (p15A-type replication origin; Novagen): M-300 (nt 17∼373, 357 bp from avian influenza virus matrix gene; GenBank accession no. DQ864720), SARS-CoV1 (nt 15224 to 15618, 395 bp from SARS-CoV; GenBank accession no. AY864806), SARS-CoV2 (nt 18038 to 18340, 303 bp from SARS-CoV; GenBank accession no. AY864806), SARS-CoV3 (nt 328110 to 28692, 583 bp from SARS-CoV; GenBank accession no. AY864806), a pac site (19 bp), HCV (nt 18 to 310, 293 bp from HCV 5′UTR; GenBank accession no. AF139594), and HA300 (nt 295 to 611, 317 bp from H5N1 avian influenza virus; GenBank accession no. DQ864720). The target sequence included the forward and reverse primer sites, flanking regions, and probe-binding sites previously published or described. A 19-mer pac site was placed between SARS-CoV3 and HCV (Fig. 1). We spliced the six target DNA sequences using overlapping extensions (10). During the first-round PCR, these six small fragments were amplified as follows. SARS-CoV1 was amplified from a pBSSR-V6 plasmid (kindly provided by the Chinese Academy of Medical Sciences and Peking Union Medical College, Institute of Basic Medical Sciences), containing nt 13785 to 16051 of the SARS-CoV gene, using the primers S-SARS1 and LAP-SARS1. SARS-CoV2 was amplified from a pNCCL-SARS plasmid (constructed by our laboratory), containing nt 18038 to 18340 of the SARS-CoV gene, using the primers LAP-SARS2 and A-SARS2. SARS-CoV3 was amplified from a pBSSR7-8 plasmid (kindly provided by the Chinese Academy of Medical Sciences and Peking Union Medical College, Institute of Basic Medical Sciences), containing nt 27730 to 29212 of the SARS-CoV gene, using the primers LAP-SARS3 and A-SARS3. The HCV fragment was amplified from a pNCCL-HCV plasmid (constructed by our laboratory), containing nt 18 to 310 of the HCV gene, using the primers S-HCV and HCV-LAP1(underlined for 19mer pac site). HA300 was amplified from a pNCCL-H5N1 plasmid (constructed by our laboratory) using the primers HA300LAP and A-HA300. M300 was amplified from a pNCCL-H5N1 plasmid (constructed by our laboratory) using the primers M300-S′ and M+SLAP1. The PCR products from the first-round amplifications were gel purified and used, together with outside primers, in the overlap extension PCR. In the second-round PCR, amplified SARS-CoV1 plus SARS-CoV2 and HCV plus HA300 were amplified using the primers pairs S-SARS1-LAP-SARS2⁺ and FIVELAP2-OverlapA′, respectively. The PCR products from the second round were gel purified. The third-round PCR amplified SARS-CoV1 plus SARS-CoV2 plus SARS-CoV3 using the primers M+SLAP2 and FIVELAP1. The PCR products from the third round were gel purified. The fourth-round PCR amplified SARS-CoV1 plus SARS-CoV2 plus SARS-CoV3 plus HCV plus HA300 using primers M+SLAP2 and OverlapA′. The fifth-round PCR amplified M300 plus SARS-CoV1 plus SARS-CoV2 plus SARS-CoV3 plus HCV plus HA300 using the primers M300-S′ and OverlapA′. Sense and reverse primers contained FseI and PacI restriction sites (underlined in Table 1), respectively. The fifth-round PCR products were gel purified. The purified fragments were cloned into a pGEM-T Easy vector (Promega) and then excised from the resulting recombinant plasmid with the FseI and PacI restriction enzymes. Simultaneously, the pACYCDuet-1 plasmid was digested with FseI and PacI, and the resulting fragments were ligated into the linearized pACYCDuet-1 plasmid to produce a new donor plasmid (pACYC-3V). pACYC-3V plasmids in positive clones, which could replicate on LB agar in the presence of 100 μg of chloramphenicol ml⁻¹, were confirmed by PCR and sequencing.

In order to compare armored RNA particles and armored L-RNA particles, we constructed pET-MS2-3V according to routine armored RNA technology (15). The DNAs encoding the maturase; the coat protein; the pac site; and the exogenous chimeric sequence (1,900 bp) containing SARS-CoV1, SARS-CoV2, SARS-CoV3, HCV, and HA300 were cloned downstream of the inducible T7 promoter of pET28b.

Both pET-MC and pACYC-3V plasmids were cotransformed into E. coli strain BL21(DE3). The 3V armored L-RNA was expressed as described previously (16). The cells were harvested by centrifugation and then washed three times with phosphate-buffered saline. The cells were pelleted and then resuspended in 20 ml of sonication buffer (5 mM MgSO₄, 0.1 M NaCl, 50 mM Tris [pH 8.0]). The cells were sonicated (Branson Sonifier 350) by using a small sonication probe operating at 50% duty cycle (unit 5 power) for five pulses. The sonicate was placed on ice for 1 min, and then the sonication step was repeated another five times. The sonicate was centrifuged in order to pellet the cell debris. A total of 20 ml of supernatant was then incubated with 1,000 U of E. coli RNase 1 and 200 U of bovine pancreatic DNase 1 at 37°C for 40 min in order to eliminate E. coli RNA and DNA. After nuclease treatment, 5 μl of supernatant was electrophoresed on an agarose gel in TAE buffer and stained with ethidium bromide to assay for armored L-RNA. A CsCl gradient was then performed as the standard method (16). In order to compare the densities of 3V armored L-RNA and 3V armored RNA particles, the 3V armored RNA from pET-MS2-3V was expressed as described previously (16). Each RNA was loaded on separate gradients. After ultracentrifugation, the ultracentrifugation tube was stabilized in an upright position. An 18-gauge needle was slowly inserted into the bottom of the tube, and 0.5-ml fractions were collected and weighed in order to determine the density of the CsCl. The optical density of each fraction at 260 nm was measured in order to quantify the 3V armored L-RNA and 3V armored RNA particles. A 5-μl portion of each fraction was electrophoresed on an agarose gel in TAE buffer and stained with ethidium bromide in order to determine the fractions containing armored L-RNA and those containing armored RNA. The fractions were then pooled and dialyzed against sonication buffer in order to remove CsCl. The dialysate was collected and stored at 4°C.

RNA was extracted from 140 μl of the purified armored L-RNA by using a QIAamp viral RNA minikit (Qiagen) according to the manufacturer's instructions. The extracted RNA was eluted in 60 μl of diethyl pyrocarbonate-treated H₂O and then used as a template for RT-PCR and RNA electrophoresis.

RT of the 3V armored L-RNA was carried out by using the Overlap-A downstream primer; this primer corresponds to nt 581 to 605 of the H5N1 gene. PCR was carried out with the primers M300RT-S and HA300RT-A in order to amplify the full length of the 3V L-RNA. RT-PCR was conducted in separate (two-step) reactions by using an Eppendorf PCR system Autorisierter thermal cycler (Eppendorf). For the RT step, each reaction mixture (20 μl) contained 4 μl of first-strand buffer (Invitrogen), 1 μl of 10 mM deoxynucleoside triphosphates, 1 μl of RNaseOUT, 1 μl of 0.1 mM dithiothreitol, 1 μl of reverse primer (Overlap-A), 1 μl of SuperScript III reverse transcriptase (Invitrogen), 5 μl of RNA, and sterile distilled water to 20 μl. The reaction mixture was incubated initially at 55°C for 50 min and then at 70°C for 15 min. An aliquot (5 μl) of the resulting cDNA was amplified by PCR using a 25-μl mixture that contained 1× PCR buffer (Promega), 1.5 mM MgCl₂, 300 μM deoxynucleoside triphosphate, 1.5 μM concentrations of each primer, and 1.25 U of Taq DNA polymerase (Promega). After an initial incubation at 95°C for 3 min, 40 cycles of the following temperature conditions were used: 95°C for 30 s, 56°C for 30 s, and 72°C for 140 s. A final extension at 72°C for 10 min was performed. Several controls, including a negative control with no template, a positive control with DNA from pACYC-3V, and a negative control with supernatant of the virus-like particles without RT, were tested simultaneously. PCR products (5 μl) were analyzed by electrophoresis on agarose gels containing ethidium bromide.

In order to increase the sensitivity of the RT-PCR, a second amplification was performed in a 25-μl reaction mixture containing 5 μl of the amplification product under the same PCR conditions.

The identity of the amplification products was confirmed by agarose gel electrophoresis. The strands of full-length 3V PCR product were cloned into pGEM-T Easy vectors (Promega), and then the recombinant AT clones were sent to Beijing Sunbiotech Co. to be sequenced using T7 and SP6 primers. The sequences obtained were compared to the target sequences.

In order to determine the nature of the RNA packaged into 3V armored RNA, RT of the RNA was carried out with three downstream primers: Overlap-A, HCV-LAP1, and A-SARS3. PCR was carried out with the primers S-SARS1 and HA300RT-A in order to amplify the full length of the 3V RNA and with the primer pairs S-SARS1 and HCV-LAP1, S-SARS1 and A-SARS3, and S-SARS1 and A-SARS2 to amplify the SARS-CoV1+SARS-CoV2+SARS-CoV3+HCV sequence, the SARS-CoV1+SARS-CoV2+SARS-CoV3 sequence, and the SARS-CoV1+SARS-CoV2 sequence, respectively.

The armored L-RNA was examined for stability in newborn calf serum. Initially, the purified 3V armored L-RNA preparation was quantified, in duplicate, with an HCV RNA PCR fluorescence quantitative diagnostic kit (Shanghai Kehua Bio-Engineering Co., Ltd.). The quantified 3V armored L-RNA was serially diluted 10-fold with the newborn calf serum to obtain 10,000 and 1,000,000 copies/ml. For each stability study, a single batch was separated into aliquots in individual time point samples of 100 μl. The samples were then incubated at 4°C, 37°C, or room temperature. 3V armored L-RNA plasma samples were removed at each time point and were stored at −80°C until completion of the experiment. All samples were quantified by using an HCV RNA RT-PCR fluorescence quantitative diagnostic kit (Shanghai Kehua) and LightCycler thermal cycler (Roche). The data were then analyzed by using LightCycler software (Roche).

Initially, the purified 3V armored L-RNA preparation was quantified, in duplicate, using a HCV RNA PCR fluorescence quantitative diagnostic kit (Shanghai Kehua). The quantified 3V armored L-RNA was diluted with newborn calf serum, and 500-μl aliquots were prepared by 10-fold serial dilution to obtain samples containing 10⁶ to 10² copies/ml.

In order to calibrate the chimeric armored RNA, the National Reference material for HCV RNA (GBW09151, 2.26 × 10³ IU ml⁻¹ to 4.22 × 10⁷ IU ml⁻¹) was used; these RNA values were assigned using the WHO HCV International Standard (NIBSC 96/790). This material is HCV gene type I. The reference material was redissolved in 300 μl of diethyl pyrocarbonate-H₂O when used.

Three different commercially available reagent kits were used for the calibration. The HCV RNA PCR-fluorescence quantitative diagnostic kit (Shanghai Kehua) was used for the HCV RNA tests, the SARS virus RNA PCR fluorescence quantitative diagnostic kit (Shenzhen PG Bio-Technology Co., Ltd.) was used for the SARS-Cov2 RNA tests, and the AIV-H5 virus RNA PCR fluorescence quantitative diagnostic kit (Shenzhen PG) was used for the HA300 RNA tests. The tests were carried out using a LightCycler thermal cycler (Roche). First, we used an international standard to calibrate the chimeric armored RNA. We isolated template RNA from the international standard and the diluted chimeric armored RNA. Newborn calf serum was used as a negative control. All RNA templates were assayed in a single run using the HCV RNA PCR fluorescence quantitative diagnostic kit. We then used the calibrated chimeric armored RNA to prepare calibrators of the SARS-CoV2 and HA300 real-time RT-PCR assays. Samples were assayed in three replicates in a 25-μl final volume containing 12.5 μl of extracted RNA and 12.5 μl of the master mix supplied with the respective kits. The thermal cycling conditions used for the three different kits are given in Table 2.

RNA was isolated from purified armored L-RNA particles. The majority of the 3V L-RNA packaged was approximately 2,200 bases in length, as detected by ethidium bromide staining (Fig. 2).

Armored RNA and armored L-RNA particles form narrow bands at 1.37 and 1.36 g ml⁻¹, respectively, when sedimented to their buoyant density in CsCl density gradients. This compares favorably with the previously reported value of 1.35 g ml⁻¹ (15).

The size of the RT-PCR amplification products of the RNA extracted from 3V armored L-RNA was full length (2,248 bp), whereas the size of RNA extracted from 3V armored RNA was between 1,000 and 2,000 bp (Fig. 3). The sequencing result demonstrated that the size was 1,200 bp.

The armored L-RNA was completely resistant to DNase and RNase treatment under conditions in which naked DNA and RNA are both degraded rapidly (data not shown).

The 3V armored L-RNA in newborn calf serum incubated at 4, 37, and 25°C was stable over 2 months (Fig. 4).

In order to evaluate the 3V armored L-RNA as a calibrator for multiple virus assays, we used the National Reference HCV RNA assigned the HCV International Standard (NIBSC 96/790) to calibrate the serially diluted chimeric armored L-RNA. The concentrations of the chimeric armored L-RNA for the five samples (10⁶, 10⁵, 10⁴, 10³, and 10²) were 1.354 × 10⁷ IU ml⁻¹, 5.740 ×10⁵ IU ml⁻¹, 6.580 × 10⁴ IU ml⁻¹, 5.428 × 10³ IU ml⁻¹, and 9.613 × 10² IU ml⁻¹, respectively (Fig. 5).