Since 2013, group A rotavirus strains characterized as novel DS-1-like inter-genogroup reassortant 'equine-like G3' strains have emerged and spread across five 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 2.3 × 10(9) - 227 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 inter-genogroup reassortant equine-like G3 strains with high sensitivity and specificity, faster turnaround time, and decreased cost and 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.

BACKGROUND: Rotavirus is a common cause of severe pediatric acute gastroenteritis (AGE). Two vaccines are licensed in the United States and have demonstrated high effectiveness against moderate to severe disease. However, fewer data are available on rotavirus vaccine effectiveness (VE) against milder disease. METHODS: We leveraged active surveillance data from Kaiser Permanente Northwest (KPNW) to calculate rotavirus VE against medically attended rotavirus illness among age-eligible children. We utilized a test-negative case-control design and applied 4 distinct case definitions based on reverse-transcription-quantitative real-time PCR (qRT-PCR) assay and enzyme immunoassay (EIA) test results. VE was calculated as 100*(1 - Odds Ratio), and models were adjusted for age group. RESULTS: The VE analysis population comprised 842 children, 799 (95%) of which had mild disease requiring at most a clinic visit, and 698 (83%) of whom were fully vaccinated against rotavirus. Age-adjusted VE was 70% (95% confidence interval [CI]: 37 - 86%) against disease defined solely by qRT-PCR results, 72% (95% CI: 31 - 89%) against disease as defined by qRT-PCR with a quantification cycle (Cq) value <27, 73% (95% CI: 32 - 90%) against disease that was qRT-PCR positive but EIA negative, and 62% (95% CI: -20 - 88%) against disease defined solely by EIA. Results were similar when restricting to disease resulting in at most an ambulatory clinic or emergency department visit. CONCLUSIONS: These results support the effectiveness of rotavirus vaccination in protecting U.S. children from mild to moderate as well as severe disease. Our findings are also useful to show the effectiveness of rotavirus vaccination against qRT-PCR-defined illness.

Inter-genogroup reassortant group A rotavirus (RVA) strains possessing a G3 VP7 gene of putative equine origin (EQL-G3) have been detected in humans since 2013. Here we report detection of EQL-G3P[8] RVA strains from the Dominican Republic collected in 2014-16. Whole-gene analysis of RVA in stool specimens revealed 16 EQL-G3P[8] strains, 3 of which appear to have acquired an N1 NSP1 gene from locally-circulating G9P[8] strains and a novel G2P[8] reassortant possessing 7 EQL-G3-associated genes and 3 genes from a locally-circulating G2P[4] strain. Phylogenetic/genetic analyses of VP7 gene sequences revealed nine G3 lineages (I-IX) with newly-assigned lineage IX encompassing all reported human EQL-G3 strains along with the ancestral equine strain. VP1 and NSP2 gene phylogenies suggest that EQL-G3P[8] strains were introduced into the Dominican Republic from Thailand. The emergence of EQL-G3P[8] strains in the Dominican Republic and their reassortment with locally-circulating RVA could have implications for current vaccination strategies.

A previously published one-step real-time quantitative reverse transcription-PCR (qRT-PCR) targeting the group A rotavirus (RVA) NSP3 gene is a sensitive technique for detection of RVA (1). This assay used the GeneAmp EZ recombinant thermostable Thermus thermophilus (rTth) RNA PCR kit (Applied Biosystems, Inc., Foster City, CA, USA), which was discontinued in July 2014. Thus, a replacement kit was needed to perform the assay. | Here, we report evaluation of a one-step RT-PCR master mix kit (EMD Millipore Corporation, Billerica, MA, USA) for detection of the RVA NSP3 gene. RNA extraction with an MS2 bacteriophage RNA (ZeptoMetrix, Buffalo, NY, USA) internal process control (IPC) (1,–3) was conducted as described previously. Each 25-μl reaction mixture contained 7.75 μl nuclease-free water, 12.5 μl 2× one-step RT-PCR master mix (rTth DNA polymerase, antibody, buffer, deoxynucleotides), 1.25 μl 50 mM manganese(II) acetate [Mn(OAc)2], the NSP3 (400 nM) and MS2 (400 nM) forward and reverse oligonucleotide primers and probes (NSP3-FAM, MS2-Texas Red, with the probes at 100 nM), and 2 μl of undenatured RNA extract. NSP3 and MS2 primer/probe sequences were published previously (1,–4). Assay modifications for use of the EMD Millipore kit were as follows: NSP3 primer and probe concentrations were reduced to 400 nM and 100 nM, respectively; thermal cycling conditions were changed to 40 cycles of 15 s at 95°C and 1 min at 60°C; and the assay was run on an Applied Biosystems 7500 Fast real-time PCR system in fast mode instead of standard mode. Establishment of the limit of detection and efficiency of the EMD Millipore-based assay was as described previously (1), except that the calculation to determine the double-stranded RNA (dsRNA) copy number per reaction was amended to use RNA template volume rather than the ratio of RNA template volume to overall reaction volume (see Fig. S1 in the supplemental material).