Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

Summary

A pan-lyssavirus nested reverse transcription polymerase chain reaction has been developed to detect specifically all known lyssaviruses. Validation using rabies brain samples of different animal species showed that this method has a sensitivity and specificity equivalent to the gold standard fluorescent antibody test and could be used for routine rabies diagnosis.

Abstract

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription polymerase chain reaction (nested RT-PCR) was developed to detect the conserved region of the nucleoprotein (N) gene of lyssaviruses. The method applies reverse transcription (RT) using viral RNA as template and oligo (dT)₁₅ and random hexamers as primers to synthesize the viral complementary DNA (cDNA). Then, the viral cDNA is used as a template to amplify an 845 bp N gene fragment in first-round PCR using outer primers, followed by second-round nested PCR to amplify the final 371 bp fragment using inner primers. This method can detect different genetic clades of rabies viruses (RABV). The validation, using 9,624 brain specimens from eight domestic animal species in 10 years of clinical rabies diagnoses and surveillance in China, showed that the method has 100% sensitivity and 99.97% specificity in comparison with the direct fluorescent antibody test (FAT), the gold standard method recommended by the World Health Organization (WHO) and the World Organization for Animal Health (OIE). In addition, the method could also specifically amplify the targeted N gene fragment of 15 other approved and two novel lyssavirus species in the 10th Report of the International Committee on Taxonomy of Viruses (ICTV) as evaluated by a mimic detection of synthesized N gene plasmids of all lyssaviruses. The method provides a convenient alternative to FAT for rabies diagnosis and has been approved as a National Standard (GB/T36789-2018) of China.

Introduction

Rabies is a worldwide zoonotic disease caused by viruses within the genus Lyssavirus¹. Lyssaviruses (family Rhabdoviridae) are single-negative-stranded RNA viruses with an approximately 12 kb genome that encodes five proteins: N, phosphoprotein (P), matrix protein (M), glycoprotein (G), and the large protein or polymerase (L). Based on nucleotide sequences of the N gene, genetic distance, and antigenic patterns, the lyssaviruses have been divided into 16 species, comprising classical rabies virus (RABV) and the rabies-related viruses (RRV): Lagos bat virus (LBV), Duvenhage virus (DUVV), Mokola virus (MOKV), European bat lyssavirus 1 (EBLV-1), European bat lyssavirus 2 (EBLV-2), Australian bat lyssavirus (ABLV), Aravan virus (ARAV), Ikoma virus (IKOV), Bokeloh bat lyssavirus (BBLV), Gannoruwa bat lyssavirus (GBLV), Irkut virus (IRKV), Khujand virus (KHUV), West Caucasian bat virus (WCBV), Shimoni bat virus (SHIBV), and Lleida bat lyssavirus (LLEBV)². Recently, two additional lyssaviruses have been identified: Kotalahti bat lyssavirus (KBLV) isolated from a Brandt’s bat (Myotis brandtii) in Finland in 2017³ and Taiwan bat lyssavirus (TWBLV) isolated from a Japanese pipistrelle (Pipistrellus abramus) in Taiwan, China in 2016–2017⁴.

All mammals are susceptible to rabies; however, no gross pathognomonic lesions or specific clinical signs permit its identification, and diagnosis can only be made in the laboratory⁵. The most widely used method for rabies diagnosis is the FAT, which is considered as the gold standard by both the WHO and the OIE^5,6. Nevertheless, the FAT can produce unreliable results on degraded/autolyzed brain tissue samples. Additionally, it cannot be used to assay biological fluid specimens such as cerebrospinal fluid (CSF), saliva, and urine, thereby largely precluding its employment in antemortem diagnosis⁷. Alternative conventional diagnostic tests, such as the rabies tissue culture infection test (RTCIT) and the mouse inoculation test (MIT), require several days⁶, a major drawback when a rapid diagnosis is essential.

Various molecular diagnostic tests (e.g., the detection of viral RNA by RT-PCR, the PCR–enzyme-linked immunosorbent assay [PCR-ELISA], in situ hybridization, and real-time PCR) are used as rapid and sensitive techniques for rabies diagnosis⁸. RT-PCR is now recommended by OIE for routine rabies diagnosis, and a heminested (hn) PCR is described in the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals to detect all lyssaviruses⁵. Here we describe a pan-lyssavirus nested RT-PCR, which allows the specific and sensitive detection of all 18 lyssavirus species comparable to or exceeding that obtained by the FAT. The principle of the method is an RT of the target RNA (conserved region of the lyssavirus N gene) into cDNA, followed by the amplification of the cDNA by two rounds of PCR. The cDNA undergoes the first-round PCR with outer primers to amplify an 845 bp fragment; then, the second-round PCR uses the first-round PCR product as a template to amplify a 371 bp fragment with inner primers. The two rounds of PCR significantly increase the sensitivity of the assay.

Protocol

The use of mice in this protocol was approved by the Administrative Committee on Animal Welfare of the Institute of Military Veterinary Medicine, the Academy of Military Medical Sciences, China (Laboratory Animal Care and Use Committee Authorization, permit number JSY-DW-2010-02). All institutional and national guidelines for the care and use of laboratory animals were followed.

1. RNA Extraction

1. Extract RNA from rabies-suspected brain tissue, skin biopsies, saliva, or CSF or from RABV-infected cell culture, using guanidinium isothiocyanate-phenol-chloroform-based extraction methods or commercially available viral RNA extraction kits. Use the prepared RNA immediately or store it at -80 °C until required.

2. Reverse Transcription of the Viral RNA

1. Remove the RT reagents listed in Table 1 from the freezer, keep them on ice, and thaw and vortex them before use.
2. Prepare 12 µL of RT reaction mix in a 0.2 mL PCR tube with the reagents listed in Table 1. Allow for pipetting variations by preparing a volume of master mix at least one reaction size greater than required.
3. Add 8 µL sample, positive control RNA or negative control to the RT reaction mix within a PCR workstation in a template room. The RT positive control is RNA extracted from the cell culture infected with fixed RABV strain CVS-11 (challenge virus standard-11) and stored at -80 °C. The negative control contains RNase-free ddH₂O.
4. Mix the contents of the RT tubes by vortexing; then, centrifuge briefly.
5. Load the reaction tubes into a thermal cycler. Set up the cDNA synthesis program with the following conditions: 42 °C for 90 min, 95 °C for 5 min, and 4 °C on hold. Set the reaction volume to 20 µL. Start the RT run.

3. First-round PCR

1. Keep the PCR reagents listed in Table 2 on ice in a clean room until use; then, thaw and vortex them.
2. Prepare the first-round PCR mix in a 0.2 mL PCR tube with the reagents listed in Table 2.
3. Add a 2 µL sample of cDNA or plasmid into the first-round PCR mix within a PCR workstation in a template room. The PCR positive control is CVS-11 cDNA prepared as mentioned in step 2.3 for the above RT method. The PCR negative control is ddH₂O.
4. Transfer the sealed tubes to a PCR thermal cycler and cycle using the parameters listed in Table 3.

4. Second-round PCR

1. Prepare the second-round PCR mix in a 0.2 mL PCR tube using the reagents listed in Table 4.
2. Add 2 µL of first-round PCR product into the second-round PCR mix. In addition, include ddH₂O as a negative control of the second-round PCR.
3. Perform PCR thermal cycling using the same parameters as given in step 3.4.

5. Analysis of the PCR Products by Electrophoresis on Agarose Gels

1. Prepare a 1.5% agarose gel by adding 1.5 g of agarose to 100 mL of Tris-acetate-EDTA (TAE) and dissolving it thoroughly by heating it in a microwave oven.
2. Add ethidium bromide (EB) (at a final concentration of 0.01%) or another commercial EB substitution. Pour the gel into the mold and leave it to solidify at ambient temperature for at least 30 min.
3. Prepare the loading samples by mixing 5 µL of each PCR product with 1 µL of 6x loading buffer.
4. Load the samples and suitable DNA marker separately into the wells and run the gel for approximately 30-45 min at 120 V until the dye line is approximately 75%-80% down the gel.
5. Turn off the power, disconnect the electrodes from the power source, and then, carefully remove the gel from the gel box.
6. Use a UV gel documenting device to visualize and photograph the DNA fragments.

6. Characterization of Nested RT-PCR

1. 6.1. Specificity and sensitivity for the detection of 18 lyssaviruses plasmids
   1. Order 18 commercial plasmids containing the full N gene of each lyssavirus (16 ICTV species and two novel species) for the PCR.
   2. Calculate the copy number of the plasmid using Avogadro’s number (N_A) and the following formula.
      [(g/µL plasmid DNA)/(plasmid length in bp x 660)] x 6.022 x 10²³ = number of molecules/µL
   3. Prepare stock solutions (2.24 x 10⁹ molecules/µL) of all 18 plasmids in ddH₂O.
   4. Perform nine 10-fold serial dilutions of all 18 plasmids in ddH₂O. Dilute 10 µL of each plasmid stock with 90 µL of ddH₂O. Vortex and centrifuge briefly.
   5. Perform PCR amplification as described in sections 3-5.
   6. Analyze the specificity and sensitivity of the nested PCR by detecting a series of lyssavirus plasmids.
2. Determination of the detection limit
   1. Adjust the titer of the rabies virus strain CVS-11 cell culture to 10^5.5 TCID₅₀/mL (virus titer determined according to the OIE Manual)⁵.
   2. Perform five 10-fold serial dilutions of CVS-11 (stock solution is 10^5.5 TCID₅₀/mL) as described in step 6.1.4.
   3. Perform the RNA extraction and nested RT-PCR amplification procedures of all viral dilutions as described in sections 1-5.
3. Comparison of the nested RT-PCR with the "gold standard" FAT
   1. Test all clinical samples by nested RT-PCR, and then, confirm the results with the FAT⁵ and N gene sequencing⁹.
   2. Use normalized data for a statistical analysis with SAS 9.1. Use the kappa test and McNemar’s chi-squared test for a statistical comparison of the diagnostic tests (SAS command: proc freq; table/agree). Calculate confidence intervals assuming abinomial distribution.
4. Evaluation of the efficacy in testing degraded samples
   1. Expose two confirmed clinical brain tissue samples of rabid dogs at 37 °C.
   2. Assay the two samples on each day of exposure at 37 °C by nested RT-PCR, the FAT, and the MIT⁵.

Representative Results

Results of nested RT-PCR to detect 18 lyssavirus species are shown in Figure 1. All PCR positive controls showed the expected 845 bp in the first- and 371 bp in the second-round amplifications with no band in the negative control. All 18 lyssaviruses produced the expected 845 and/or 371 bp bands, indicating that the nested RT-PCR detected all 18 lyssaviruses. Sixteen lyssaviruses plasmids had efficient amplification in two rounds of PCR, but two, namely ARAV and IKOV, had amplification in either the first- or second-round PCR. The sensitivity of the method varied in the detection of different lyssavirus plasmids, with limits ranging from 2.24 x 10⁰ to 2.24 x 10⁵ molecules/µL, as shown in Table 6. These differences can be attributed to the mismatches between the primers and templates due to viral sequence diversity. Furthermore, the sensitivity of detecting rabies virus CVS-11 in cell culture was 10^2.5 TCID₅₀/mL.

A total of 9,624 brain tissues from clinical specimens were tested by nested RT-PCR in comparison with the FAT and the results are summarized in Table 7, which shows that nested RT-PCR had a 100% sensitivity (CI, 97.75% to 100%) and a 99.97% specificity (CI, 99.91% to 99.99%). The accordance between the two methods was 99.07%. Three tests that were positive by nested RT-PCR but negative by the FAT were of highly decayed clinical material. These three specimens were confirmed as RABV positive by N gene sequencing.

Comparison of the test performance in the detection of the two brain specimens incubated at 37 °C (step 6.4.1 of the protocol) indicated that the nested RT-PCR could effectively detect virus in decayed brain tissues for least 17 days postdegradation, which is for a longer period of time when compared with only 7 days by the FAT and not even 1 day by the MIT. This result shows that nested RT-PCR is more sensitive in the detection of degraded samples than the FAT and the MIT.

To further validate the nested RT-PCR, 10 rabies laboratories in China were invited to conduct tests on a set of specimens. Of these, eight laboratories were provided a set of 10 blinded animal brain tissues from our laboratory, including RABV positive and negative specimens. The other two laboratories used their own specimens. All specimens had been archived and confirmed by the FAT previously. All 10 laboratories obtained results by nested RT-PCR in 100% accordance with the FAT, with no false-negatives or false-positives (Table 8), indicating that the nested RT-PCR had a high specificity and reproducibility.

Components	Volume per reaction (µL)
dNTPs (2.5 mM )	4
Random Primer (50 μM)	1.5
Oligo(dT) 15 (50 μM)	0.5
M-MLV buffer (5x)	4
M-MLV reverse transcriptase (200 IU/µL)	1
RNasin (40 IU/µL)	1
Total volume	12

Table 1: Reagents of reverse transcription for cDNA synthesis.

Components	Volume per reaction (µL)
dNTPs (10 mM)	1
Ex-Taq (5 U/μL)	0.3
Taq Buffer (10x)	5
N127 (20 μM)	1
N829 (20 μM)	1
dd H2O	39.7
Total volume	48

Table 2: Reagents of the first-round PCR.

Temperature	Time	Cycles
94 °C	2 min	1
94 °C	30 s	35
56 °C	30 s	35
72 °C	40 s	35
72 °C	10 min	1
4 °C	∞

Table 3: Cycling parameters of the first- and second-round PCR.

Components	Volume per reaction (µL)
dNTPs (10 mM)	1
Ex-Taq (5 U/μL)	0.3
Taq Buffer (10x)	5
N371F (20 μM)	1
N371R (20 μM)	1
dd H2O	39.7
Total volume	48

Table 4: Reagents of the second-round PCR.

Primer Name	Details	Direction	Sequence (5’-3’)		Nucleotide position	Product Size
N127	The first round PCR	Forward	ATGTAACNCCTCTACAATGG		-19~0	845bp
N829	The first round PCR	Reverse	GCCCTGGTTCGAACATTCT		807~825	845bp
N371F	The second round PCR	Forward	ACAATGGAKKCTGACAARATTG		-6~15	371bp
N371R	The second round PCR	Reverse	CCTGYYWGAGCCCAGTTVCCYTC		345~367	371bp

Table 5: Primer sequences of the first- and second-round PCR. Degenerate bases: N (A/T/C/G), K (G/T), R (A/G), Y (C/T), WV (G/A/C).

Lyssavirus species	Strain	Template plasmid (molecules /µL)
RABV	GQ918139.1	2.24 x 101
LBV	EU293110.1	2.24 x 102
MOKV	KF155005.1	2.24 x 101
DUVV	EU293119.1	2.24 x 101
EBLV-1	EF157976.1	2.24 x 101
EBLV-2	KF155004.1	2.24 x 101
ABLV	GU992312.1	2.24 x 100
IRKV	NC_020809.1	2.24 x 101
WCBV	NC_025377.1	2.24 x 101
KHUV	NC_025385.1	2.24 x 103
ARAV	NC_020808.1	2.24 x 102
SHIBV	NC_025365.1	2.24 x 101
BBLV	NC_025251.1	2.24 x 101
IKOV	NC_018629.1	2.24 x 105
LLEBV	NC_031955.1	2.24 x 103
GBLV	NC_031988.1	2.24 x 105
KBLV	MF960865.1	2.24 x 101
TWBLV	MF472710.1	2.24 x 101

Table 6: Detection limit of nested RT-PCR of 18 lyssaviruses.

Standard method and result		RT-nPCR Positive	RT-nPCR Negative	Correlation (%)
FAT	Positive Negative	162 3	0 9459	99.07

Table 7: Correlation between nested RT-PCR and the FAT in the detection of RABVs in clinical specimens.

Table 8: Validation results of nested RT-PCR by 10 laboratories.

Figure 1: Detection of 18 lyssaviruses by nested RT-PCR. (A) The result of the first-round PCR. (B) The result of the second-round PCR. M = DL 2000 DNA marker. Lanes 1–18 = RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, ABLV, ARAV, IKOV, BBLV, GBLV, IRKV, KHUV, LLEBV, SHIBV, WCBV, KBLV, and TWBLV, respectively. Lane 19 = PCR positive control; lane 20 = negative control for the first-round PCR; lane 21 = negative control for the second-round PCR. A positive PCR result shows a band at 845 bp in the first round and 371 bp in the second-round PCR. The amplicon of ARAV is not visible in the first round, but visible in the second-round PCR (lane 8), while the amplicon of IKOV is visible in the first round, but not visible in the second-round PCR (lane 9). Please click here to view a larger version of this figure.

Figure 2: Comparison with primer sequences shows the differential nucleotides in primer regions of 18 lyssavirus species. N127 and N829 were outer primers, N371F and N371R were inner primers. Please click here to view a larger version of this figure.

Discussion

Currently, RABV is a major lyssavirus responsible for nearly all human and animal rabies in China, as well as in other countries. In addition, an IRKV variant was first identified from a Murina leucogaster bat in the Jilin province in Northeast China in 2012¹⁰, and it has been reported to cause a dog’s death in the Liaoning Province in 2017¹¹. Most recently, a novel lyssavirus, TWBLV, was also identified from a Japanese pipistrelle bat in Taiwan, China in 2017. These results suggest that the effective detection of other lyssaviruses is also important to prevent the spill-over of the bat-borne lyssaviruses. In this regard, the pan-lyssavirus nested RT-PCR targeting the most conserved N gene region is a very useful tool, and the results have shown that it can effectively detect all 16 ICTV-approved and two novel lyssavirus species identified so far, including genetically divergent RABVs and IRKV in China (data on IRKV strain not shown). However, it is also interesting to note that ARAV was detected only by the second-round PCR primers, while IKOV was detected only by first-round PCR primers (Figure 1). To investigate the cause of this discrepancy, sequence comparison of all 18 lyssaviruses within the four primer regions was conducted, with the results showing that the 3' end nucleotide T of outer primer N829 in the first-round PCR and the second nucleotide T at the 3' end of inner primer N371F in the second-round PCR were not identical to the corresponding positions of ARAV (G) and IKOV (A) (Figure 2). Once the T of primer N829 was changed to the G of ARAV and the T of primer N371F changed to the A of IKOV, both viruses were successfully detected in PCR (data not shown). This result demonstrates the critical role of the 3' end or near-end nucleotides of primers in the successful amplification of the target region.

To evaluate the sensitivity and specificity of the method in the detection of rabies virus for clinical diagnosis and surveillance, 9,624 animal brain tissue samples were tested in the last 10 years by the FAT and nested RT-PCR in parallel. Of the 165 samples that tested rabies positive by nested RT-PCR, 162 were detected as positive by the FAT; therefore, the two methods show 99.07% accordance. The RABVs detected in these 165 samples could be classified into different lineages in Asian, Arctic-related, and Cosmopolitan clades^12,13, indicating that nested RT-PCR can cover the various genetic clades of RABVs. Three highly decayed clinical specimens were tested positive by nested RT-PCR but negative by the FAT. This result is consistent with the evaluation of the efficacy in testing two degraded samples as shown in the representative results section, indicating that nested RT-PCR is more sensitive than the FAT in the detection of viruses in highly decayed specimens.

The performance of nested RT-PCR was further confirmed by participation in the International Laboratory Comparison Test (ILCT) organized by the EU Reference Laboratory for Rabies at the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) in 2010, using a set of 12 samples (one each of RABV, EBLV-1, EBLV-2, and ABLV as positive controls, one negative control, and seven blinded samples). In the test, nested RT-PCR successfully identified all the lyssaviruses with a 100% consistency. In 2018, the method was approved as a National Standard of Rabies Diagnoses (GB/T36789-2018) by the National Technical Standardization Committee of Animal Health of China.

Materials

Name	Company	Catalog Number	Comments
50 × TAE	Various	Various
6 × loading buffer	TakaRa	9156
Agarose	US Everbright® Inc	A-2015-100g
ddH2O	Various	Various
DL 2,000 Marker	Takara	3427A
dNTPs (10 mM)	TakaRa	4019
dNTPs (2.5 mM)	TakaRa	4030
Electrophoresis System	Tanon	EPS300
Ex-Taq (5 U/μL)	TakaRa	RR001
Gel Imaging System	UVITEC	Fire Reader
Microcentifuge tubes	Various	Various
M-MLV reverse transcriptase (200 IU/µL)	TakaRa	2641A
NanoDrop 1000 Spectrophotometer	Thermoscientific	ND1000
Oligo (dT)15	TakaRa	3805
PCR Machine	BIO-RAD	T100
PCR Tubes	Various	Various
Phusion High-Fidelity DNA Polymearase	NEW ENGLAND BioLabs	M0530S
Pipettors	Various	Various
Random Primer	TakaRa	3801
RNase Inhibitor (40 IU/µL)	TakaRa	2313A
RNase-free ddH2O	TakaRa	9102
Taq Buffer (10×)	TakaRa	9152A
Tips	Various	Various
Vortex mixer	Various	Various