Despite the presence of a plethora of different types of diagnostic assays, nucleic acid-based assays offer an added advantage of high specificity and sensitivity with the possibility of a lower detection limit [37]. Recently, mRT-PCR is being used as one of the most important diagnostic methods for rapidly and simultaneously detecting viral pathogens. And hence, different mRT-PCR assays for simultaneously detecting nine [38], six [25, 39], five [40], four [23, 24, 26, 41], three [42] and two [43, 44] swine enteric viruses have been developed. Due to the asymptomatic nature of SaV, EMCV, RVA, and AstV infections, these viruses have received insufficient attention, and no single diagnostic assay capable of simultaneously detecting these viruses from porcine fecal samples has been developed.
A mRT-PCR assay capable of simultaneously detecting SaV, EMCV, RVA, and AstV RNA from porcine fecal samples was developed in this study. Our assay and other previously developed assays [23, 26]for other viruses suggest that sRT-PCR may be more specific than mRT-PCR, but the improvement in turnaround time and cost-effectiveness would compensate for this minor reduction in sensitivity. Yet, compared to sRT-PCR, mRT-PCR is more economical and rapid. The sensitivity of the developed mRT-PCR assay using plasmids containing the specific viral target fragments was 2.5 X 103 copies for each template. Similarly, Zhao, et al. [23] reported a similar mRT-PCR assay sensitivity of 2.17 × 103, 2.1 × 103, 1.74 × 104and 1.26 × 104 for porcine epidemic diarrhea virus, transmissible gastroenteritis virus, RVA, and porcine circovirus 2, respectively. Thus, the currently developed mRT-PCR assay has similar sensitivity with Hu, et al. [41] and Liu, et al. [26], a higher sensitivity compared to Day, et al. [45] and Cagirgan and Yazici [46], and lower sensitivity compared to Liu, et al. [40]. Further, 130 fecal samples were tested for checking the difference in sensitivity of sRT-PCR and mRT-PCR assays. Test results of mRT-PCR showed an overall concordance rate of 98.5% (128/130) (Table 2) to the test results of sRT-PCR. Besides sensitivity, test results of specificity and reproducibility of this assay indicate that similar to the sRT-PCR, the developed mRT-PCR could be employed to detect SaV, EMCV, RVA, and AstV from porcine fecal samples. Similarly, previous reports [25, 38] and others suggested that mRT-PCR could be a highly sensitive and specific assay that could be used for rapid detection of enteric swine viruses.
To further confirm the validity of the developed mRT-PCR assay, 280 porcine fecal samples were tested and the positive rates of SaV, EMCV, RVA and AstV was found to be 24.6% (69/280), 5% (14/280), 4.3% (12/280), and 17.5% (49/280) respectively. This shows that, compared to the other two viruses, SaV has the highest positive rate and RVA the lowest positive rate. Similarly, SaV, EMCV, RVA, and AstV have been detected in diarrheic and non-diarrheic swine feces [5, 11–13, 15], highlighting the importance of detecting these viruses in swine farms. In swine, coinfection of enteric viruses is common. In this study, a coinfection rate of 6.4% (18/280), 2.86% (8/280), 2.86% (8/280), 2.1% (6/280), 2.1% (6/280) and 0.35% (1/280) for SaV & AstV, SaV & EMCV, RVA& AstV, EMCV& AstV, SaV & RVA, and for EMCV & RVA respectively has also been observed. Similar to the current study, coinfections of pigs with different enteric viruses have been reported from China [25, 47], the United States[48] and Belgium [49].
A phylogenetic analysis was also made to explore further the epidemiologic characteristics of the detected viruses. The phylogenetic analysis revealed that the SaV detected in this study belongs to the GIII genogroup. Similar to this study, previous research [50, 51] indicated that from the eight SaV genogroups detected in swine [31, 32], the GIII genogroup is the most prevalent in China. Phylogenetic analysis of the partial sequence of the 3D region of EMCV, similar to previous studies from China [52, 53], revealed that the EMCV variants detected in this study clustered into two groups. Yuan, et al. [53] assigned the EMCV-30 strain (DQ288856) from the USA and the Korean strains K3 (EU780148) and K11 (EU780149) to the EMCV group one lineage while strains PV2 (X87335) and D variant (M37588) to group two lineage. Based on Yuan, et al. [53], three of the EMCV strains identified in this study may belong to group one lineage, regardless of their clustering pattern. Based on the VP7 gene sequence, Group A rotaviruses are classified into 36 G genotypes, of which 12 G-genotypes (G1–G6, G8–G12, and G26) have been identified in porcine [18, 54]. All three RVA variants detected in this study are closely grouped with strains NJ2012 (MT874988.1) and TA-3-1 (KT820771.1), both of which are G9 genotypes, implying that the RVA variants detected in this study could be G9 genotype. According to phylogenetic analysis, the AstV variants detected in this study belong to AstV4. Similarly, Previous reports from the United States [55], Thailand [56] and Slovakia[8] indicated that AstV4 is the most prevalent type of AstV. In Contrast, a higher prevalence of AstV2 from China [57] and AstV5 from China's Hunan province [58] have also been observed, highlighting the importance of further AstV epidemiology study. These findings lend credence to the notion that coinfection of these viruses is common in swine. Through recombination and mutations, the coinfection of viruses accelerates the evolution of coinfected viruses [59, 60], allowing more virulent virus strains to emerge. As a result, the developed mRT-PCR assay could play a critical role in controlling and preventing SaV, EMCV, RVA, and AstV through early and accurate detection.