Development of a Quadruplex RT-qPCR for the Detection of Porcine Rotaviruses and the Phylogenetic Analysis of Porcine RVH in China

Abstract

Rotavirus A species (RVA), RVB, RVC, and RVH are four species of rotaviruses (RVs) that are prevalent in pig herds, and co-infections occur frequently. In this study, a quadruplex real-time quantitative RT-PCR (RT-qPCR) for the simultaneous detection of four porcine RVs was developed by designing specific primers and probes based on the VP6 gene of RVA, RVB, RVC, and RVH, respectively. The method showed high specificity and could only detect RVA, RVB, RVC, and RVH, without cross-reaction with other porcine viruses; showed excellent sensitivity, with a limit of detection (LOD) of 1.5 copies/µL for each virus; showed good repeatability, with intra-assay coefficients of variation (CVs) of 0.15–1.14% and inter-assay CVs of 0.07–0.96%. A total of 1447 clinical fecal samples from Guangxi province in China were tested using the developed quadruplex RT-qPCR. The results showed that RVA (42.71%, 618/1447), RVB (26.95%, 390/1447), RVC (42.92%, 621/1447), and RVH (13.68%, 198/1447) were simultaneously circulating in the pig herds, and the co-infection rate of different species of rotaviruses was found to be up to 44.01% (579/1447). The clinical samples were also detected using one previously reported method, and the coincidence rate of the detection results using two methods was more than 99.65%. The phylogenetic tree based on the VP6 gene sequences of RVH revealed that the porcine RVH strains from Guangxi province belonged to the genotype I5, which was closely related to Japanese and Vietnamese strains. In summary, an efficient, sensitive, and accurate method for the detection and differentiation of RVA, RVB, RVC, and RVH was developed and applied to investigate the prevalence of porcine RVs in Guangxi province, China. This study is the first to report the prevalence of porcine RVH in China.

Interspecies recombination in NSP3 gene in the first porcine rotavirus H in Russia identified using nanopore-based metagenomic sequencing

“…Otherwise, in co-infections, RVH was noted more rarely: RVA+RVH (2.14%), RVB+RVH (0.83%), and RVC+RVH (1.66%). It is also interesting that the RVA + RVB + RVC + RVH infection showed a positivity rate of 2.07% (30/1447) ( 21 ).…”

“…Subsequently, the RVH circulation was confirmed in pigs of different ages in the USA ( 11 , 12 ), Japan ( 13 , 14 ), Brazil ( 15 , 16 ), South Africa ( 17 ), Vietnam ( 18 ), and some European countries, such as Spain ( 19 ) and Italy ( 20 ). A recent large-scale study in China has also indicated a high prevalence of RVH in the local swine herds ( 21 ). Furthermore, the virus has also been observed in bats in west-central Africa ( 22 ) and Korea ( 23 ).…”

Identification of Putative Novel Rotavirus H VP7, VP4, VP6 and NSP4 Genotypes in Pigs

“…In pigs, RVH was first described in fecal samples of animals affected by diarrhea between 1991 and 1995 in Japan [ 12 ]. Between 2008 and 2022, the presence of porcine RVH was also found in the United States [ 13 ], Brazil [ 14 ], South Africa [ 15 ], Vietnam [ 16 ] and China [ 17 ] as well as some European countries, such as Spain [ 18 ], Italy [ 7 ] and Russia [ 19 ]. In the period 2016–2022, the rates of detection of RVH in symptomatic pigs ranged between 9 and 14% in Spain [ 18 ], Italy [ 7 ], Brazil [ 20 ] and China [ 17 ].…”

“…Between 2008 and 2022, the presence of porcine RVH was also found in the United States [ 13 ], Brazil [ 14 ], South Africa [ 15 ], Vietnam [ 16 ] and China [ 17 ] as well as some European countries, such as Spain [ 18 ], Italy [ 7 ] and Russia [ 19 ]. In the period 2016–2022, the rates of detection of RVH in symptomatic pigs ranged between 9 and 14% in Spain [ 18 ], Italy [ 7 ], Brazil [ 20 ] and China [ 17 ]. These values indicate that RVH is relatively widespread in swine populations, although the impact of RVH on porcine herds in terms of costs and animal health has not been assessed.…”