Development of a Real-Time Reverse Transcription-PCR Assay To Detect and Quantify Group A Rotavirus Equine-Like G3 Strains

ABSTRACT

Since 2013, group A rotavirus strains characterized as novel DS-1-like intergenogroup reassortant “equine-like G3” strains have emerged and spread across 5 continents among human populations in at least 14 countries. Here, we report a novel one-step TaqMan quantitative real-time reverse transcription-PCR assay developed to genotype and quantify the viral load for samples containing rotavirus equine-like G3 strains. Using a universal G forward primer and a newly designed reverse primer and TaqMan probe, we developed and validated an assay with a linear dynamic range of 227 to 2.3 × 10⁹ copies per reaction and a limit of detection of 227 copies. The percent positive agreement, percent negative agreement, and precision of our assay were 100.00%, 99.63%, and 100.00%, respectively. This assay can simultaneously detect and quantify the viral load for samples containing DS-1-like intergenogroup reassortant equine-like G3 strains with high sensitivity and specificity, faster turnaround time, and decreased cost. It will be valuable for high-throughput screening of stool samples collected to monitor equine-like G3 strain prevalence and circulation among human populations throughout the world.

INTRODUCTION

Group A rotaviruses (RVAs) are members of the family Reoviridae and are the primary cause of acute gastroenteritis (AGE) in children in most countries worldwide. RVAs possess a genome composed of 11 segments of double-stranded RNA (dsRNA) that encode 6 structural viral proteins (VP1, VP2, VP3, VP4, VP6, and VP7) and 5 or 6 nonstructural proteins (NSP1, NSP2, NSP3, NSP4, and NSP5/NSP6) (1, 2). The current RVA strain classification incorporates all 11 gene segments, namely, Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx, where x represents integers that specify the corresponding genotypes of the VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes, respectively (1, 2). The RVAs that infect humans typically possess either the Wa-like genogroup-1 constellation Gx-P[x]-I1-R1-C1-M1-A1-N1-T1-E1-H1 or the DS-1-like genogroup-2 constellation Gx-P[x]-I2-R2-C2-M2-A2-N2-T2-E2-H2 (1). Historically, RVAs have been genotyped using the sequence differences observed exclusively within the VP7 (Gx) and VP4 (P[x]) genes (2, 3), and the following six RVA strains predominate and represent >90% of those in global circulation: G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8] (2).

G3 (VP7) RVA strains infect humans and a broad range of animal species and remain among the most genetically diverse RVAs in contemporary circulation with 9 described lineages (4–6). Human G3P[8] RVA strains are the most commonly reported G3 strains (6) and typically possess a VP7 gene that occupies G3 lineage I and a genogroup 1 backbone (G3-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1) (4, 6, 7). Since 2013, novel DS-1-like intergenogroup reassortant (IGR) “equine-like G3” (EQL.G3) strains that possess a VP7 gene of apparent equine RVA origin have emerged and spread rapidly across 5 continents among human populations in at least 14 countries, with reports of endemic circulation (5, 6, 8–18). IGR EQL.G3 strains predominately possess the genotype constellation G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2, although IGR EQL.G3 strains possessing genotype constellations G3-P[4]/[6]- I2-R2-C2-M2-A2-N1/2-T2-E2-H2 have also been reported (6, 10, 14, 17, 19). Dispersal of EQL.G3P[8] strains that exhibit unique genetic constellations and conserved sequences distinct from all other RVA strains (5, 6, 8–18) has resulted in a globally circulating pool of highly conserved EQL.G3P[8] strains (6, 14). EQL.G3P[8] strains can also reassort with locally circulating genogroup-1 and genogroup-2 RVA strains resulting in novel EQL.G3 reassortants (6).

Molecular techniques developed to detect and genotype RVA strains (20) include one- and two-step real-time reverse transcription-PCR (qRT-PCR) assays (20–30). However, EQL.G3 strains possess sequence mutations within the VP7 gene (6) that prevent specific detection using a previously published qRT-PCR assay to genotype RVA G3 strains (20) (M. D. Esona, R. Gautam, and E. M. Katz, unpublished data). In addition, the VP7 primer set designed by Gouvea et al. (31), which is still used worldwide as a WHO-recommended RVA genotyping primer set, has been shown to mistype EQL.G3 strains as G1 strains (32). Only qualitative molecular assays (conventional RT-PCR, Sanger sequencing, and next-generation sequencing) are available currently to genotype RVA EQL.G3 strains (6, 8, 32), but these assays typically exhibit lower throughput and increased cost compared with qRT-PCR assays (33, 34). Therefore, development of a qRT-PCR assay to detect EQL.G3 strains would provide an ideal molecular assay for high-throughput testing for these increasingly prevalent human RVA strains and, if run in a quantitative manner, could provide information on the viral load in specimens.

In this study, we developed and validated a novel one-step TaqMan qRT-PCR assay to detect the RVA EQL.G3 (VP7) genotype and quantify the viral load for samples containing EQL.G3 strains (hereafter this assay is referenced as “EQL.G3 assay”). We propose that the assay be used for rapid screening of RVA-positive samples to detect and quantify EQL.G3 strains to provide valuable data for monitoring EQL.G3 strain prevalence and circulation among human populations throughout the world.

MATERIALS AND METHODS

Ethics statement.

Clinical specimens were obtained from the US National Rotavirus Strain Surveillance System which had been determined as “public health practice- enhanced surveillance” under Centers for Disease Control and Prevention (CDC) Public Health nonresearch determination RD 2010 5941. Clinical specimens were also collected at sentinel surveillance sites participating in the rotavirus surveillance network in the Latin American and Caribbean regions in compliance with the Pan American Health Organization (PAHO) surveillance guidelines for rotavirus (https://iris.paho.org/handle/10665.2/49140). All clinical specimens tested in this study were deidentified and could not be traced back to patient or hospital case identifiers.

Oligonucleotide design.

The EQL.G3 assay developed during this study included the universal G forward primer “G-consensus-FP” that targeted the conserved 5′ end of the RVA VP7 gene (20) and a novel reverse primer and probe (“EQL.G3-RP’ and ‘EQL.G3-Probe,” respectively) (Table 1). The probe sequence was created in part by modifying previously described forward primer “EQG3FWD” (8). The reverse primer and probe sequences were designed to maximize dissimilarity with the VP7 gene sequences found within RVA strains possessing a non-equine-like G3 genotype ([G3*], hereafter strains possessing a non-equine-like G3 genotype are referenced using “G3*”). Using gene sequences obtained from GenBank, multiple alignments were prepared using the Molecular Evolutionary Genetics Analysis Version 6.0 software (MEGA6) (35) and multiple candidate probe and primer sets were designed manually from the alignment consensus sequence that included degenerate bases to account for nucleotide variation among the VP7 gene sequences existent among EQL.G3 strains. Theoretical oligonucleotide specificity for the EQL.G3 genotype was in part established through alignment of the reverse primer and probe sequences against the VP7 gene consensus sequences obtained for nontarget genotypes (G1, G2, G3*, G4, G9, and G12) and GenBank searches using the Basic Local Alignment Search Tool (blastn; https://blast.ncbi.nlm.nih.gov/Blast.cgi) (36). Internal sequence modifications were included to increase the melting temperatures (T_ms) of the primers and probe to 57°C to 58°C and 65°C, respectively (Table 1). The probe was labeled with a 5′ 6-carboxyfluorescein (FAM) reporter dye and a 3′ black hole quencher-1 (BHQ1) (Biosearch Technologies, GBR) (Table 1). All primers and probes were synthesized by the Biotechnology Core Facility at the CDC, Atlanta, GA.

TABLE 1.

Summary of the oligonucleotide characteristics for the primers and probe developed for the EQL.G3 assay

Oligonucleotide	Nucleotide sequence (5′–3′)a	Fluorophore-quencher (5′–3′)	Concn/reaction (nM)	Nucleotide position (bp)b	Tm (°C)	Amplicon length (bp)	Oligonucleotide source
G-consensus-FPc	TAG{C}TCYTTTTRATGTATGGTAT	N/ad	400	37–59	57	399	20
EQL.G3-RPe	CTGARAAAGAAG[C]RATGTCTGTATACTC	N/a	400	406–433	58		Modified from reference 8
EQL.G3-Probe	CTRCATA[C]GYTAATT{C}TACACAAGGAG	FAMf-BHQ1g	200	242–268	65		This study

^a{C}, AP-dC (G-Clamp); [C], C-5 propynyl-dC (pDC); Y, C or T; R, A or G.

^bPositions numbered according to EQL.G3 strain D388 VP7 gene sequence, GenBank accession no. KU059771.

^cForward primer.

^dN/a, not applicable.

^eReverse primer.

^fFAM, 5′ 6-(3′,6′-Dipivaloylfluoresceinyl-6-carboxamido)-hexyl-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (Glen Research, catalog no. 10-5901-95).

^gBHQ, 3′ Black Hole Quencher-1 (Biosearch Technologies, catalog no. SCG5-5041G).

Clinical specimens.

RVA-positive (n = 385) and -negative stool specimens (n = 85), which had been tested using the Premier Rotaclone enzyme immunoassay (EIA) (Meridian Diagnostics, Inc., OH, USA) and/or a qRT-PCR assay targeting the NSP3 gene (29), were used to screen, optimize, and validate the EQL.G3 assay (Table 2), including 247 EQL.G3-positive specimens from 4 countries. The RVA-positive samples were genotyped previously using a combination of conventional multiplexed one-step RT-PCR (37), Sanger sequencing (38), either or both VP7 and VP4 genotype-specific multi- or singleplexed qRT-PCR (20), and next-generation sequencing (6). Stool specimens were stored at −20°C or 4°C at the surveillance sites until they were shipped on dry ice or cold packs to the CDC, Atlanta, GA. Upon receipt to the CDC, specimens were stored at 4°C prior to nucleic acid extraction and confirmatory testing by EIA and then at −80°C until the RNA was extracted.

TABLE 2.

Summary of clinical stool specimens used to screen, optimize, and validate the EQL.G3 assay^a

RVA G-typeb	No. of samples
G1	33
G2	17
G3*	54
EQL.G3c	247
G4	3
G8	3
G9	16
G12	12
RVA negative	85

^aSelected specimens included those from which infection by RVA and/or the RVA genotype assignment were previously established by the RSMET at the CDC, Atlanta, GA; n = 470.

^bInfection by RVA and/or the RVA genotype assignment were previously established using ≥2 molecular assays as described in the “Clinical specimens” section of the text.

^cEQL.G3 samples were obtained from Colombia, the Dominican Republic, Haiti, and Paraguay.

RNA extraction.

RNA was extracted from 10% stool suspensions, prepared in phosphate-buffered saline (Applied Biosystems, CA, USA) using the MagMax-96 viral RNA isolation kit (Applied Biosystems) on the automated Kingfisher Flex purification system (ThermoFisher Scientific, MA, USA) or the MagNA pure compact (MPC) RNA isolation kit (Roche Applied Science, USA) on the MPC System (Roche Applied Science). All RNA extractions were performed according to the manufacturer’s instructions with minor modifications as described previously (6). All resulting RNA extracts were immediately stored at −80°C until qRT-PCR testing.

Reference virus strains.

Reference RVA strains possessing nontarget genotypes, RVA vaccine strains (Rotarix [GSK Biologics, Belgium] and RotaTeq [Merck, PA, USA]), and non-RVA viral pathogens causing AGE (norovirus, sapovirus, astrovirus, enteric adenovirus) (see Table S1 in the supplemental material) were used to screen, optimize, and validate the EQL.G3 assay. RNA from stool samples containing norovirus, sapovirus, astrovirus, and vaccine stocks was extracted using the MPC-based methods described for stool samples, except that in the case of the vaccines, 98.0 μl of undiluted vaccine stocks was used in place of supernatant from 10% stool suspensions. RNA was also extracted from 13 reference RVA strains propagated in MA104 cells (see Table S2 in the supplemental material) using methods identical to those for the Rotarix and RotaTeq RNA extractions. Total nucleic acid (TNA) was extracted from an enteric adenovirus type 40 isolate (a DNA virus) using the MPC nucleic acid isolation kit I according to the manufacturer’s “external lysis protocol,” aside from minor modifications as described for total RNA extraction within the “RNA extraction” section, on the MPC System (Roche Applied Science, USA) using the “Total_NA_Plasma_external_lysis” instrument protocol. Both RNA and TNA extracts were stored at −80°C until qRT-PCR testing.

dsRNA control transcripts.

The EQL.G3 dsRNA RVA transcript was synthesized and, along with nontarget dsRNA RVA transcripts, was used to screen, optimize, and validate the EQL.G3 assay. Nontarget dsRNA RVA transcripts (G1, G2, G3*, G4, G9, and G12) and the EQL.G3 dsRNA RVA transcript were prepared as described by Gautam et al. 1990 (20). The EQL.G3 transcription template was prepared by using T7 tailed primer pairs for the VP7 gene (see Table S3 in the supplemental material) and RNA extracted from EQL.G3 strain “HTI-EQL.G3” (20), a stool sample collected from Haiti that was genotyped and sequence confirmed at the CDC (M. D. Esona and E. M. Katz, unpublished data). The concentration of the EQL.G3 dsRNA RVA transcript was measured at 260 nm using a Nanodrop Lite spectrophotometer (ThermoFisher Scientific, MA, USA) and used to calculate viral copy number with the number of dsRNA molecules/reaction = [mass (ng) × 6.022 × 1023 (molecules/mol)]/[length of amplicon × 650 (g/mol) × 109 (ng/g)]. Hereafter, RVA genotype-specific dsRNA transcripts are referenced using the format “Gx transcript” (e.g., G1 transcript and EQL.G3 transcript).

Screening and optimization of the EQL.G3 assay.

Multiple probe and primer sets were designed to target the EQL.G3 genotype, and assay candidates were screened using each of the oligonucleotide sets by testing all Gx transcripts, RNA extracted from reference RVA strains, and a panel of clinical stool specimens possessing common G types (G1, G2, G3*, EQL.G3, G4, G9, and G12). The oligonucleotide sets exhibiting promising preliminary performance were selected for further evaluation, from which the set exhibiting sigmoidal amplification curves with the lowest quantification cycle (C_q) values was selected for assay optimization. Five distinct probe:primer concentrations/ratios were tested (100:200 nM, 100:300 nM, 100:400 nM, 200:400 nM, and 200:600 nM); the probe:primer concentrations/ratio exhibiting target amplification at the lowest C_q value with low background amplification was selected for in-depth assay validation (Table 1). The most promising assay then was validated using the seven dsRNA transcripts, RNA extracted from clinical stool specimens, reference RVA strains, and non-RVA AGE viruses.

qRT-PCRs were performed in MicroAmp fast optical 96-well reaction plates (Applied Biosystems, CA, USA) using the one-step RT-PCR master mix kit (MilliporeSigma, MA, USA). Each 25-μl reaction mixture contained 8.25-μl nuclease free water; 12.5-μl 2× one-step RT-PCR master mix (includes recombinant Thermus thermophilus [rTth] DNA polymerase); 1.25 μl 50 mM manganese(II) acetate [Mn(OAc)₂]; 0.25 μl of each primer (400 nM each); 0.5-μl TaqMan probe (200 nM) (Table 1); and 2.0-μl RNA template, extraction control, or water in the case of no-template controls. The thermal cycling conditions were as follows: 5 min at 95°C (dsRNA denaturation), 30 min at 50°C (RT), 1 min at 95°C (cDNA denaturation), and 45 PCR amplification cycles consisting of 15 s at 95°C and annealing for 1 min at 60°C with fluorescent signals collected during the annealing phase. TNA extracted from enteric adenovirus type 40 was tested using identical reaction conditions to those described above; however, the thermal cycling conditions were modified to remove the RT step. All testing was conducted using the 7500 fast real-time PCR system in the “Fast 7500” run mode and the 7500 fast system SDS v1.4 21 CFR part 11 module (Applied Biosystems) software. C_q values were determined by setting the analytical threshold (ΔRn value) and baseline according to the manufacturer’s instructions. TNA extracted from enteric adenovirus type 40 was tested using identical reaction conditions to those described above; however, the thermal cycling conditions were modified to remove the RT step. All samples and controls were tested in duplicate, and a test result was considered positive if both replicates from a single sample produced amplification curves that crossed the analytical threshold before 45 cycles.

Assay performance.

The EQL.G3 assay underwent linearity testing using serial 10-fold dilutions of EQL.G3 transcripts that were prepared in nuclease-free water containing 100-ng/μl yeast RNA (Invitrogen, CA, USA; prevents diluted transcripts from adhering to vial walls) and subsequently spiked with 1.0 × 10⁵ PFU/μl of MS2 bacteriophage (ZeptoMetrix, NY, USA) internal process control (IPC) at a ratio of 1:50. These “assay standards” were tested in triplicate, and if all three replicates produced a sigmoidal amplification curve (Fig. 1), they were used to generate a standard curve and to determine the linear dynamic range (33). Intra-assay variance was calculated using the standard deviation (SD) for the C_q variance among test replicates (33). Linear regression analyses were applied to the standard curve to calculate the correlation coefficient (r²) value, the slope and y intercept for the line of best fit, and the assay amplification efficiency (E) (E = 10^−1/slope – 1) (33) (Fig. 1).

FIG 1.

Amplification curves and a graph of the standard curve (insert) resulting from linearity testing using serial 10-fold dilutions of the RVA EQL.G3 dsRNA transcript (10⁻² through 10⁻¹²) spiked with MS2 bacteriophage RNA, obtained using the EQL.G3 assay (detection of the MS2 IPC not included, as described in the text). Using the analytical threshold (ΔRn, green line), amplification curves (black) exhibit 10-fold dilutions of the EQL.G3 transcript ranging from 10⁻² to 10⁻⁹ corresponding to average C_q values of 14.5 through 40.5, respectively. Linear regression analyses of the standard curve (insert) resulted in r² of 0.99818, slope of −3.66213, and y intercept of 48.43505. The fluorescent signals from the EQL.G3 transcript dilutions 10⁻¹⁰ through 10⁻¹², RVA-negative extraction controls, and no-template controls are exhibited within the blue outline.

The limit of detection (LOD) for the EQL.G3 assay was determined by testing EQL.G3 transcript dilutions within the range of the expected detection limit for the assay (see Table S4 in the supplemental material) using 20 replicates per dilution, and the average C_q value was used to determine the average viral copy number detected per reaction for each concentration (Table S4). Intra-assay variance was reported as the SD for the C_q variance among test replicates (33).

The qualitative diagnostic accuracy of the EQL.G3 assay was determined using the “per-sample agreement for detection of the EQL.G3 genotype (positive or negative),” resulting from a comparison of the EQL.G3 assay test results with previously determined genotyping results (Table 2) (39, 40). The results were used to calculate percent positive agreement and percent negative agreement with reference genotyping assays for the EQL.G3 assay (Table 3) as described previously (41). The 95% score confidence intervals (95% CI) were calculated for percent positive agreement and percent negative agreement (Table 3) as described previously (41).

TABLE 3.

Summary of the data used to determine the qualitative diagnostic accuracy for the EQL.G3 assay^a

Detection of the RVA EQL.G3 (VP7) genotypeb (new test: EQL.G3 assay)	No. of samples with RVA EQL.G3 (VP7) genotype (reference standard) result
	Positivec	Negative	Total
Positivec	248	1	249
Negative	0	262	262
Total	248	263	511
% Agreement	100.00 (95% CId 98.47–100)	99.62% (95% CId 97.88–99.93)

^aThe resulting per-sample agreement between the qualitative detection of the EQL.G3 genotype was determined using the test under evaluation (the EQL.G3 assay) and the reference values established using the reference standards. All values are no. of samples unless otherwise indicated.

^bThe following samples were included: clinical specimens, reference virus strains, RVA vaccines, and dsRNA control transcripts.

^cA test result was considered positive if both replicates from a single sample resulted in a sigmoidal amplification curve that crossed the analytical threshold before 45 cycles and all assay control reactions yielded the expected results.

^dThe 95% CI were calculated for percent positive agreement and percent negative agreement using the formula: [100% × (Q1 − Q2)/Q3, 100% × (Q1 + Q2)/Q3] (41), where the quantities of Q1, Q2, and Q3 were for computed for each 95% CI as described previously (41).

The qualitative precision for the EQL.G3 assay was determined through repeatability (intra-assay variance) and reproducibility (interassay variance) testing (33). RNA samples previously determined as positive or negative (n = 12 positive, 12 negative) for detection of the EQL.G3 genotype were tested in triplicate by three different operators on different days over a 2-week period. Positive samples were known to include the assay target at concentrations over a 3-log concentration range for the assay, namely, 10⁶ to 10⁹ copies/μl RNA extract. Repeatability of the assay was reported as the percentage of samples tested by a single operator for which all replicates (n = 3) yielded concordant assay detection results. Reproducibility of the assay was reported as the percentage of samples tested by all operators for which all replicates (n = 9) yielded concordant assay detection results.

RESULTS

The best-performing EQL.G3 assay incorporated oligonucleotides G-consensus-FP, EQL.G3-RP, and EQL.G3-Probe at concentrations of 400:400:200 nM, respectively, per reaction (Table 1).

Assay performance.

EQL.G3 assay standards with dilution factors 10⁻² through 10⁻⁹ (1.326 through 1.326 × 10⁻⁷ng/μl) yielded amplification curves that crossed the analytical threshold (ΔRn = 24,999.998) for all three replicates (Fig. 1). The linear dynamic range was 2.3 × 10⁹ through 227 copies per reaction (1.15 × 10⁹ through 113.5 copies per μl) corresponding to average C_q values of 14.5 through 40.5, respectively (Fig. 1). Intra-assay variance among test replicates was 0.014 to 0.553 cycles. Linear regression analyses resulted in r² of 0.9982 and E of 87.53% with approximately 3.3 cycles (slope, −3.6621; y intercept, 48.4351) recorded between average C_q values of consecutive 10-fold dilutions (Fig. 1). The LOD for the EQL.G3 assay was 227 (95% CI, 198 to 260) copies per reaction (113.5 copies per μl), and 95% of replicates were detected at 31.5 copies per μl (Table S4).

All target and nontarget clinical specimens (Table 2; Table S1), 27 of 28 RNA reference virus strains, the adenovirus type 40 isolate, both RVA vaccines, and all dsRNA transcripts yielded the expected positive and negative results for the detection of the EQL.G3 genotype (n = 248 and 262, respectively) (Table 3). Reference RVA G3* strain CC425, G3*-transcript, and all clinical specimens possessing a G3* genotype (n = 54) yielded the expected negative results for the detection of the EQL.G3 genotype except one reference strain, Ro1845. The percent positive agreement and percent negative agreement of the EQL.G3 assay were calculated to be 100.00% (95% CI, 98.47 to 100.00%) and 99.63% (95% CI, 97.88 to 99.93%), respectively (Table 3). All 24 samples tested by all 3 operators yielded the expected positive and negative results for detection of the EQL.G3; therefore, precision for the assay was calculated to be 100.00%.

DISCUSSION

In this study, we developed and validated a novel quantitative one-step TaqMan qRT-PCR assay that targets the RVA EQL.G3 (VP7) genotype to provide a sensitive, specific, and accurate molecular technique to monitor the continued emergence and spread of IGR EQL.G3 strains. The EQL.G3 assay was efficient and yielded a robust linear dynamic range that exhibited highly sensitive detection of the EQL.G3 genotype, exhibited high linearity across 8 orders of magnitude, and was comparable in performance to previously published multiplex qRT-PCR assays developed to detect and quantify other RVA VP7 genotypes (20). The low levels of intra-assay variance observed among test replicates indicated that the EQL.G3 assay is highly repeatable when using RNA samples possessing RVA EQL.G3 strains at concentrations across the linear dynamic range and at or above the LOD for the assay. Our assay was also highly accurate, as evident from the detection of the EQL.G3 target genotype among target and nontarget RNA samples and exhibited a near-universal lack of detection of RNA samples extracted from RVA G3* strains and nontarget genotypes. Repeatability and reproducibility of the EQL.G3 assay were confirmed empirically through the qualitative intra- and interassay variance and precision values, which indicated the assay is highly precise.

This EQL.G3 assay can simultaneously detect EQL.G3 strains and quantify the viral load in stool specimens. Previously, this would require use of a qualitative genotyping assay (6, 8) followed by viral load quantification using a qRT-PCR assay, such as the one that targets the NSP3 gene (29). In a limited number of instances when the same samples were tested by the two quantitative assays, the NSP3 and EQL.G3 copy number estimates differed by 1.4 to 2.0 logs, and this difference could be the result of the lower efficiency of the EQL.G3 assay (87.53% versus 94.69 for NSP3), but an additional assay comparison is needed. The EQL.G3 assay, however, offers high-throughput testing with fast turnaround time and decreased cost and is well suited for preliminary or confirmatory detection of EQL.G3 strains. As such, the EQL.G3 assay is ideal for rapid screening of RVA-positive samples to detect and quantify EQL.G3 strains to provide valuable data for monitoring EQL.G3 strain prevalence and contemporary circulation among human populations worldwide.

Development of the EQL.G3 assay incorporated reproducible and robust procedures and the use commercially available kits and reagents to standardize and streamline the assay for use across multiple laboratories. Selection of the one-step RT-PCR master mix kit (MilliporeSigma, MA, USA) was in part based on the kit’s successful use as part of other qRT-PCR assays developed to detect, genotype, and quantify RVA strains (29, 42). In addition, the one-step EQL.G3 assay included dsRNA denaturation, reverse transcription, and PCR amplification in a single tube using rTth DNA polymerase with uninterrupted thermal cycling conditions to reduce the manipulation of samples, decrease the probability of sample cross-contamination, and facilitate rapid generation of results (20, 30). The universal forward primer G-consensus-FP was included within the EQL.G3 assay because it effectively binds to a highly conserved VP7 gene region existent among RVA strains irrespective of their genotype, as evident from its use within previous multiplexed qRT-PCR-based RVA genotyping assays (20). The continued use of universal G-consensus-FP within multiplex qRT-PCR-based VP7 genotyping assays should reduce the number of oligonucleotides required during the development of associated multiplexed qRT-PCR-based RVA genotyping assays and simplify the assay optimization process.

Unexpectedly, the EQL.G3 assay yielded false-positive detection of RNA extracted from reference RVA G3* strain Ro1845. Interestingly, strain Ro1845 possesses a VP7 open reading frame (ORF) nucleotide sequence that occupies phylogenetic G3 lineage III and was reported as a human RVA strain possessing 11 genes of putative canine/feline origin (6, 43). These characteristics differ significantly from those reported for EQL.G3 strains (6) that possess VP7 ORF nucleotide sequence(s) that occupy phylogenetic G3 lineage IX (6). The ORF sequence for the Ro1845 VP7 gene and the “EQL.G3 ORF consensuses sequence” shared 85.4% nucleotide identity (838/981 nucleotides), but sequence identity was higher in the EQL.G3-Probe (25/27, 92.6%) and EQL.G3-RP (24/28, 85.7%) binding regions (Table S2) with nucleotide differences all located in the interior of each oligonucleotide. Thus, it is possible that the EQL.G3 assay will occasionally mistype G3 strains of feline/canine origin; however, these strains are rare in human populations (44). It is also important to note that select rare human RVA genotypes (e.g., G6 and G20) were not tested during assay validation due to an inability to acquire specimens possessing such genotypes; therefore, further evaluation of this assay through testing of additional samples possessing rare human RVA genotypes may prove beneficial.

Since 2013, the rapid emergence and spread of EQL.G3 strains has been in part attributed to a general fitness advantage and possibly Rotarix vaccine-induced selective immune pressure (6, 16) that has allowed for their increased prevalence in the postvaccine introduction era (6). As a result, RVA EQL.G3 strains capable of reassortment with locally circulating RVA (6) have emerged and spread to multiple continents and countries, including reports of endemic circulation (5, 6, 8–17). We propose that the EQL.G3 assay be used for high-throughput screening of stool samples collected as part of global RVA surveillance and to support future studies investigating RVA vaccine effectiveness (45) and monitor the increasingly apparent effects of vaccine-induced immune selection on local strain ecology (16, 46–48).