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In this study, we screened various viral encephalitis pathogens based on their domestic and international incidences and epidemiological trends. Ultimately, 12 species were selected: HSV-1, HSV-2, VZV, EBV, and HCMV in the family Herpesviridae; JEV and WNV in the family Flaviviridae; EV71, CA6, CA10, and CA16 in the genus Enterovirus; and mumps virus in the family Paramyxoviridae.
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The complete workflow of this study is shown in detail in Figure 1, including (i) primer design, (ii) DNA/RNA extraction, (iii) real-time reverse transcription-polymerase chain reaction(RT-PCR) for concentration estimation, (iv) cDNA synthesis, (v) multiplex PCR amplification, (vi) library preparation, (vii) sequencing, (viii) data analysis.
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Primers for the selected pathogens were designed and validated against their target genes (Table 1), and the specificity of the primers was verified using the National Center for Biotechnology Information (NCBI) BLAST. Primers were synthesized by Shanghai Bioengineering Technology Service.
Table 1. Summary of primers for various pathogens
Primer
namePrimer sequence (5'–3') Amplicon size (bp) Target gene Ref HSV-1-F
HSV-1-RGGTCAGCTCGTGATTCTGCA
CGCATCAAGACCACCTCCTC106 UL27 − HSV-2-F
HSV-2-RCTAGTTGTCGCGGTGGGACT
TAGTACACAGTGATCGGGAT212 US5 [14] EBV-F
EBV-RCTCTAACTCCAACGAGGGCAG
GAGGGCCTCCATCATTTCC
320
LMP1
−HCMV-F
HCMV-RAATGATATCCGTACTGGGTCCCATT
TGATGATGGGGATGTTCAGCAT
350
UL83
−VZV-F
VZV-RGGGTTTTGTATGAGCGTTGG
CCCCCGAGGTTCGTAATATC447 ORF22
[15]JEV-F
JEV-RCGTTCTTCAAGTTTACAGCATTAGC
CCYRTGTTYCTGCCAAGCATCCAMCC674 E [16] WNV-F
WNV-RACCAACTACTGTGGAGTC
TTCCATCTTCACTCTACACT445 E [17] EVs-F
EVs-RTTCTGTTTCCCCGGACTGAG
TAAAAGGAAACACGGACACCCA400 VP3 − Mumps-F
Mumps-RAATATCAAGTAGTGTCGATGA
AGGTGCAAAGGTGGCATTGTC316 SH [18] Note. − is a self-designed primer; HSV-1, herpes simplex virus-1; HSV-2, herpes simplex virus-2; EBV, epstein-barr virus; HCMV, human cytomegalovirus; VZV, varicella zoster virus; JEV, Japanese encephalitis virus; WNV, west nile virus; EVs, Enteroviruses. In the initial primer pool, we included a total of 16 pairs of primers, but after optimization, the primer pool of this platform finally conducted verification and later experiments against nine pairs of primers for 12 viruses, including HSV1, HSV2, EBV, HCMV, VZV, JEV, WNV, EV71, CA6, CA10, CA16, and mumps virus, with five pairs referring to relevant literature and four pairs designed in this study. We performed specificity verification on NCBI and PCR verification using the virus strain’s nucleic acid for the primers in the references. The primary reference sequences of the pathogens were downloaded from the NCBI database for the primers designed in this study, and multiple reference sequences for the same virus were compared online by muscle (https://www.ebi.ac.uk/Tools/msa/muscle/). Primers were designed using Geneious Prime software. According to the principles of primer design, primers with excessive mismatches or low specificity should be modified, eliminated, or redesigned manually.
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The nucleic acids of HSV-1, HSV-2, HCMV, and EBV were derived from the aqueous humor of clinical patients with anterior uveitis; nucleic acids for EV71, CA6, CA10, CA16, and mumps virus were obtained from pharyngeal swabs of patients who were clinically positive for these viruses; and for VZV, nucleic acids were extracted from vaccines. The other two viruses (JEV and WNV) were obtained from strains maintained in Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention. RNA was extracted from RNA virus strains using the Qiagen Viral RNA mini kit (Qiagen, #52906), and DNA was extracted from DNA virus strains using the QIAamp DNA Mini Kit (Qiagen, #51304), according to the manufacturer’s instructions.
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Prior to cDNA synthesis, RT-PCR was performed to estimate the concentration of the target template. Real-time RT-PCR was performed using a commercial kits. The reaction system was configured, and the reaction procedure was determined according to the manufacturer’s instructions.
DNA was extracted directly from viruses. For RNA viruses, RNA was extracted, reverse-transcribed into cDNA, and the DNA was amplified. PCR was used to confirm the specificity of primers for DNA viruses, and RT-PCR was used to confirm the specificity of primers for RNA viruses. The amplification products were analyzed using gel electrophoresis to assess amplification specificity, and the fragment sizes of the target bands were compared with the electrophoresis map of the PCR products detected via gel electrophoresis to determine whether they were consistent with the predicted primer-amplified fragment sizes. The PCR products were sent to Shanghai Bioengineering Engineering Service for sequencing. The sequencing results were verified using NCBI BLAST to ensure accuracy.
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Nucleic acid samples with Ct values < 20 were subjected to 10–100-fold gradient dilutions prior to cDNA synthesis. SuperScript IV VILO Master Mix (Invitrogen, #11756500) was used for cDNA synthesis, according to the manufacturer’s instructions.
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The primers in Table 1 were diluted to 10 μmol/L and mixed into one tube in equal volumes, and Q5 Hot Start High-Fidelity DNA Polymerase (NEB, #M0493L) was selected for multiplex PCR amplification. The cDNA template has a volume of 5 μL, and amplification was performed in a 50 μL reaction with primer concentration set to 0.02 μmol/L per primer. The reaction procedure for multiplex PCR was as follows: hot start at 98 °C for 30 s, followed by 30 cycles of 98 °C for 10 s, 52 °C for 20 s, 72 °C for 40 s,and 72 °C extension for 3 min.
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Amplification products were purified using AMPure XP magnetic beads (Beckman Coulter, # A63881) at a sample-to-bead ratio of 1:1. Purified DNA samples were quantified using a Qubit 4.0 fluorometer (Invitrogen; #Q33238) with Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kits (Invitrogen; #Q32851). Depending on the quantification results, all DNA samples were diluted to 0.2 to 0.3 μg/μL prior to library preparation. After diluting each sample to 0.2 μg/μL, equal volumes were mixed into one tube (e.g., 5 μL for each sample), and Qubit quantification was then used to confirm that the mixed sample concentration was indeed 0.2 μg/μL.
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DNA libraries were constructed using the Nextera XT DNA Library Preparation Kit (#FC-131-1024; Illumina) according to manufacturer’s instructions. After enzymatic fragmentation, library amplification was performed on the fragmented samples using combined dual indices (Nextera DNA Indexes, Illumina, #FC-121-1011). Library quantification was performed using a Qubit 4.0 fluorometer (Invitrogen, #cQ33238) and Qubit dsDNA HS Assay Kit (Invitrogen, # Q32851). Absolute quantification of libraries was performed using the Library Quantification Kit For Illumina. Libraries were scaled and diluted to 1.8 pmol/L and paired-ended 150-cycle high-throughput sequencing was performed on an Illumina Miniseq sequencing platform using the MiniSeq Mid Output Kit (300-cycles) (Illumina, #FS-420-1004).
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For NGS downstream data, the CLC Genomics Workbench (Qiagen, #832021) was used for data quality control (QC), read trimming, mapping refining, and consensus extraction. NC_001806(Hsv-1), NC_001798(Hsv-2), NC_001348(VZV), NC_009942(WNV), NC_007605(EBV), NC_006273(HCMV), JE_U47032(JEV), NC_009942(WNV), KX_064282(CA6), KP_289398(CA10), JQ_354992(CA16), AY_4653561(EV71), and NC_002200(Mumps virus) were used as the reference sequences.
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The primers presented in Table 1 were used for the PCR of each pathogen to determine whether they amplified the target fragment. All PCR products were sequenced (Sanger sequencing) to obtain the sequences of the target fragments, and BLAST showed that all sequences matched the reference sequences.
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Multiple PCR amplifications for different pathogens were performed after mixing the optimized primers (Table 1), and the cDNA library of the pathogens targeted in this study was then sequenced. Target sequences of the pathogens were extracted from the sequencing data at coverage depths greater than 20×. The amplicon lengths were consistent with the predicted sizes of the amplified fragments. Preliminary verification was performed using NCBI BLAST, and all sequences matched their corresponding reference sequences. The experimental data are shown in Figure 2. Panel A shows the results of the pre-experiments (primers were not optimized, and sample concentrations were not adjusted), along with the coverage and sequencing depth for targeted sequence amplification of HSV-1, HSV-2, HCMV, EBV, VZV, JEV, WNV, EV71, CA6, CA10, CA16, and mumps virus. Notably, the primer pool contained two pairs of EBV-specific primers, but amplification using one pair of primers generated few sequences, indicating that the amplification efficiency of this primer pair was poor.
Figure 2. Coverage and sequencing depth map of pathogen target sequences. Coverage and sequencing depth of pathogen target sequences result: A shows the results of the pre-experiments, showing the coverage and sequencing depth of HSV-1, HSV-2, HCMV, EBV, VZV, JEV, WNV, EV71, CA6, CA10, CA16 and Mumps target sequences; B shows the sequencing results of samples at 10-fold dilution, showing the coverage and sequencing depth of VZV, JEV, WNV, EV71, CA6, CA10, CA16 and Mumps target sequences; C shows the sequencing results of the sample at 100-fold dilution, showing the coverage and sequencing depth of HCMV, EBV, VZV, JEV, WNV, EV71, CA6, CA10, CA16 and Mumps target sequences; D shows the sequencing results of samples at 1000-fold dilution, showing the coverage and sequencing depth of HSV-1, HSV-2, HCMV, EBV, VZV, JEV, WNV, EV71,CA6, CA10, CA16 and Mumps target sequences.
Not all nucleic acid templates were derived from clinical samples; the JEV and WNV templates were extracted from virulent strains, and the VZV template nucleic acid was extracted from the vaccine. We generated serial dilutions of these nucleic acid samples (10-, 100-, and 1000-fold), and panel B shows the sequencing results for the 10-fold diluted samples (Ct values: 25.2–29.1), with the coverage and sequencing depths of the VZV, JEV, WNV, EV71, CA6, CA10, CA16, and mumps virus target sequences. Owing to the concentration limitations of some clinical samples, there were only one concentration of HSV-1 and HSV-2 and two concentrations of HCMV and EBV. Therefore, only eight pathogens are shown in panel B. Panel C of Figure 2 shows the sequencing results of the 100-fold diluted samples (Ct values: 29.1–32.7), with the coverage and sequencing depth of the target sequences for 10 pathogens (HCMV, EBV, VZV, JEV, WNV, EV71, CA6, CA10, CA16, and mumps virus). Panel D of Figure 2 shows the sequencing results for the 1000-fold diluted samples (Ct values: 33.1–35.5), with the coverage and sequencing depth of the target sequences for 12 pathogens (HSV-1, HSV-2, HCMV, EBV, VZV, JEV, WNV, EV71, CA6, CA10, CA16, and mumps virus). Amplicons were obtained from all the pathogen samples after gradient dilution. Notably, the number of reads for HCMV was the lowest, mainly due to the amplification efficiency of the primers.
We compiled the experimental data, which included the CT values for each pathogen, number of reads, and reference sequence of the spliced target sequence (Table 2). In the pre-experiments, we found that the sequencing depth was not balanced among pathogens; CA10 had the highest total reads (2,563,545 reads), whereas HSV-2 had the fewest reads (6,308 reads), which may be related to the number of viral copies in the template or the amplification efficiency of the primers. The HSV-1, HSV-2, HCMV, and EBV samples were derived from the aqueous humor of patients with anterior uveitis; the EV71, CA6, CA10, CA16, and mumps virus samples were obtained from pharyngeal swabs of patients who were clinically positive for these viruses, and the remaining samples were nucleic acids recovered from viral strains, which may explain why the sequencing depth was not balanced among the pathogens. BLAST was used to compare the amplification sequences with the Sanger sequencing results, and the findings showed remarkable uniformity for all pathogens.
Table 2. Summary of sequencing results for all pathogens
Virus Pre-experiments 10-fold 100-fold 1,000-fold Ref Ct-value Reads Ct-value Reads Ct-value Reads Ct-value Reads HSV1 27.9 8,678 — — — — 33.9 21,932 NC_001806 HSV2 28.2 6,308 — — — — 33.6 14,562 NC_001798 HCMV 30.1 36,752 — — 30.8 546 34.0 0 NC_006273 EBV 29.6 21,307 — — 29.1 46,482 33.1 23,902 NC_007605 VZV 24.6 774,363 27.0 961,166 29.4 986,698 34.0 2,069,900 NC_001348 JEV 25.8 642,001 27.4 907,263 30.4 102,001 33.5 18,209 JE_U47032 WNV 25.3 1,073,388 27.2 790,729 31.0 430,332 34.7 884,879 NC_009942 EV71 24.1 2,225,726 26.9 2,951,553 31.6 2,178,524 33.7 3,449,881 AY_4653561 CA6 26.9 2,013,312 28.1 2,973,422 32.7 2,195,624 35.5 3,538,523 KX_064282 CA10 21.1 2,401,006 29.1 2,976,273 31.2 2,213,093 33.5 3,597,826 KP_289398 CA16 24.4 2,563,545 28.8 2,959,416 31.4 2,189,642 34.6 3,513,948 JQ_354992 Mumps 23.1 903,858 25.2 928,429 30.5 479,978 33.2 119,500 NC_002200 Note. — Indicates no data. HSV-1, herpes simplex virus-1; HSV-2, herpes simplex virus-2; EBV,epstein-barr virus; HCMV, human cytomegalovirus; VZV, varicella zoster virus; JEV, Japanese encephalitis virus;WNV, west nile virus; EVs, Enteroviruses
doi: 10.3967/bes2024.032
Development of a High-throughput Sequencing Platform for Detection of Viral Encephalitis Pathogens Based on Amplicon Sequencing
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Abstract:
Objective Viral encephalitis is an infectious disease severely affecting human health. It is caused by a wide variety of viral pathogens, including herpes viruses, flaviviruses, enteroviruses, and other viruses. The laboratory diagnosis of viral encephalitis is a worldwide challenge. Recently, high-throughput sequencing technology has provided new tools for diagnosing central nervous system infections. Thus, In this study, we established a multipathogen detection platform for viral encephalitis based on amplicon sequencing. Methods We designed nine pairs of specific polymerase chain reaction (PCR) primers for the 12 viruses by reviewing the relevant literature. The detection ability of the primers was verified by software simulation and the detection of known positive samples. Amplicon sequencing was used to validate the samples, and consistency was compared with Sanger sequencing. Results The results showed that the target sequences of various pathogens were obtained at a coverage depth level greater than 20×, and the sequence lengths were consistent with the sizes of the predicted amplicons. The sequences were verified using the National Center for Biotechnology Information BLAST, and all results were consistent with the results of Sanger sequencing. Conclusion Amplicon-based high-throughput sequencing technology is feasible as a supplementary method for the pathogenic detection of viral encephalitis. It is also a useful tool for the high-volume screening of clinical samples. -
Key words:
- Viral encephalitis /
- Amplicon sequencing /
- High-throughput sequencing /
- Multipathogen detection
The authors have no conflicts of interest to declare.
&These authors contributed equally to this work.
注释:1) AUTHOR CONTRIBUTIONS: 2) CONFLICTS OF INTEREST: -
Figure 2. Coverage and sequencing depth map of pathogen target sequences. Coverage and sequencing depth of pathogen target sequences result: A shows the results of the pre-experiments, showing the coverage and sequencing depth of HSV-1, HSV-2, HCMV, EBV, VZV, JEV, WNV, EV71, CA6, CA10, CA16 and Mumps target sequences; B shows the sequencing results of samples at 10-fold dilution, showing the coverage and sequencing depth of VZV, JEV, WNV, EV71, CA6, CA10, CA16 and Mumps target sequences; C shows the sequencing results of the sample at 100-fold dilution, showing the coverage and sequencing depth of HCMV, EBV, VZV, JEV, WNV, EV71, CA6, CA10, CA16 and Mumps target sequences; D shows the sequencing results of samples at 1000-fold dilution, showing the coverage and sequencing depth of HSV-1, HSV-2, HCMV, EBV, VZV, JEV, WNV, EV71,CA6, CA10, CA16 and Mumps target sequences.
Table 1. Summary of primers for various pathogens
Primer
namePrimer sequence (5'–3') Amplicon size (bp) Target gene Ref HSV-1-F
HSV-1-RGGTCAGCTCGTGATTCTGCA
CGCATCAAGACCACCTCCTC106 UL27 − HSV-2-F
HSV-2-RCTAGTTGTCGCGGTGGGACT
TAGTACACAGTGATCGGGAT212 US5 [14] EBV-F
EBV-RCTCTAACTCCAACGAGGGCAG
GAGGGCCTCCATCATTTCC
320
LMP1
−HCMV-F
HCMV-RAATGATATCCGTACTGGGTCCCATT
TGATGATGGGGATGTTCAGCAT
350
UL83
−VZV-F
VZV-RGGGTTTTGTATGAGCGTTGG
CCCCCGAGGTTCGTAATATC447 ORF22
[15]JEV-F
JEV-RCGTTCTTCAAGTTTACAGCATTAGC
CCYRTGTTYCTGCCAAGCATCCAMCC674 E [16] WNV-F
WNV-RACCAACTACTGTGGAGTC
TTCCATCTTCACTCTACACT445 E [17] EVs-F
EVs-RTTCTGTTTCCCCGGACTGAG
TAAAAGGAAACACGGACACCCA400 VP3 − Mumps-F
Mumps-RAATATCAAGTAGTGTCGATGA
AGGTGCAAAGGTGGCATTGTC316 SH [18] Note. − is a self-designed primer; HSV-1, herpes simplex virus-1; HSV-2, herpes simplex virus-2; EBV, epstein-barr virus; HCMV, human cytomegalovirus; VZV, varicella zoster virus; JEV, Japanese encephalitis virus; WNV, west nile virus; EVs, Enteroviruses. Table 2. Summary of sequencing results for all pathogens
Virus Pre-experiments 10-fold 100-fold 1,000-fold Ref Ct-value Reads Ct-value Reads Ct-value Reads Ct-value Reads HSV1 27.9 8,678 — — — — 33.9 21,932 NC_001806 HSV2 28.2 6,308 — — — — 33.6 14,562 NC_001798 HCMV 30.1 36,752 — — 30.8 546 34.0 0 NC_006273 EBV 29.6 21,307 — — 29.1 46,482 33.1 23,902 NC_007605 VZV 24.6 774,363 27.0 961,166 29.4 986,698 34.0 2,069,900 NC_001348 JEV 25.8 642,001 27.4 907,263 30.4 102,001 33.5 18,209 JE_U47032 WNV 25.3 1,073,388 27.2 790,729 31.0 430,332 34.7 884,879 NC_009942 EV71 24.1 2,225,726 26.9 2,951,553 31.6 2,178,524 33.7 3,449,881 AY_4653561 CA6 26.9 2,013,312 28.1 2,973,422 32.7 2,195,624 35.5 3,538,523 KX_064282 CA10 21.1 2,401,006 29.1 2,976,273 31.2 2,213,093 33.5 3,597,826 KP_289398 CA16 24.4 2,563,545 28.8 2,959,416 31.4 2,189,642 34.6 3,513,948 JQ_354992 Mumps 23.1 903,858 25.2 928,429 30.5 479,978 33.2 119,500 NC_002200 Note. — Indicates no data. HSV-1, herpes simplex virus-1; HSV-2, herpes simplex virus-2; EBV,epstein-barr virus; HCMV, human cytomegalovirus; VZV, varicella zoster virus; JEV, Japanese encephalitis virus;WNV, west nile virus; EVs, Enteroviruses -
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