In this study, a marked detection rate of RVCs in asymptomatic piglets in two parts of Maharashtra state and in different years was reported. Until, now a single study documented from India with a 12% prevalence rate of RVC in symptomatic pigs [16]. The majority of RVC prevalence studies reported from other countries have been carried out in diarrheic and / or non-diarrheic pigs with higher detection rate in diarrheic pigs [4, 5, 8, 12, 23, 24]. The studies on asymptomatic pigs were documented from the USA, Ireland, Western Kenya and Uganda, with 4.4–69.9% detection rates [4, 5, 23, 24].The variable rotavirus prevalence reported from different countries has been attributed to differences in population density, age groups, geographic locations, climate and environment [24, 25]. In addition, variable reliability of diagnostic methods employed routinely could also affect detection of Rotaviruses due to continuous accumulation of point mutations, reassortment events and cross species transmission possibly resulting into new genotypes and / or lineages in the RVC genome [26].
Similar RVC detection rates in the specimens collected in 2009 from northern and in 2013 from western parts of Maharashtra state in different seasons of the year suggest the possibility of continuous circulation of this pathogen in pigs of this region through asymptomatic infections as described earlier [8]. Among RVC positive specimens (n = 30), 20 and 7 were tested positive for RVA and RVB, respectively in earlier studies [18, 19]. It is to be noted that in spite of dual infection (RVA + RVC in 7; RVB + RVC in 2) of rotaviruses in nine pigs, diarrheal symptoms were not evident in any of these animals. Previously, lactogenic protection provided by sows was suspected to be the reason for the asymptomatic infection in RVC positive piglets [8].
Among 31 G genotypes identified to date majority belong to porcine RVC [27]. The data on the genomes of porcine RVCs mainly available from Japan and indicate circulation of G1, G3, G5, G6, G7, G9, G12 and G13 genotypes of the VP7 along with P1, P4–P9 of the VP4; I1, I4, I6 and I7 of the VP6 and M1, M3, M6 of the VP3 genes in Asia [14–16, 28]. In the present study, G1, P1, and E5 genotypes of the VP7, VP4, and NSP4 genes respectively, of porcine RVC strains were identified in combination with either of the two genotypes of VP6 gene namely I7 and I10. RVC strains from diarrheic pigs described earlier in India have been reported to carry a combination of porcine E4-E8 (NSP4) and H6 (NSP5) genotypes and human I2 (VP6) genotype, and thus suggested occurrence of reassortment events [16]. Interestingly, the phylogenetic analysis of RVC strains (Uttar Pradesh, India) with accession numbers KX374480/2015 and KX374482/2016 available in GenBank (Shanmuganathan et.al, unpublished data) showed clustering with I7 and I10 genotypes respectively, of VP6 gene, and thereby supported the conclusion that such RVC strains circulated in India for a long time.
To date, limited genotyping data available on asymptomatic pigs indicates dominance of G1 globally and occurrence of G3 (Belgium) and G6 (Ireland), G12 and G13 (Japan) genotype strains [5, 28, 29]. Whole-genome characterization of RVC strains from asymptomatic pigs has so far been reported for a very few strains [28, 29]. Here, the characterization of a study strain classified it as G1-P1-I7-R1-C1-A1-N5-T5-E5-H1 genotypes of the VP7, VP4, VP6, P1, VP2, NSP1, NSP2, NSP3, NSP4, NSP5 genes respectively of porcine RVC. The phylogenetic analysis of the VP3 gene classified study strain as M3 genotype consisting of strains isolated from both porcine and human species. Previously, RVC strains from terrestrial mammals were classified, with a single genotype noted in all genes with the exception of two genotypes in VP3 (M2 and M3) gene [15]. The possibility of reassortment events for M3 genotype of VP3 was suggested due to clustering of RVC strains originated from human and porcine species in to a single lineage with high PNI values [15, 21] (Fig. 1f). The scanty studies on human specimens from Western and Eastern India documented circulation of M2 genotype of RVCs [21, 30]. Therefore, more studies in humans and animals of same geographical regions are necessary for confirmation.
In spite of use of multiple primer pairs, reported earlier [20, 21, 31] as well as designed in the present study amplification of complete of NSP5 gene failed (Supplementary Table 2). The amplification of the segmented genome of rotaviruses using gene specific primers is the most commonly used method. However, failure in sequencing of atypical and un-typeable Rotavirus strains with variable frequency, location, season, and environment has been reported may be due to reassortment possibilities not predicted by the detection and genotyping RT-PCR assays [32, 33]. Primer independent, Next-generation sequencing approach needs to be tested for analysis of remaining genome of the NSP5 gene in future [34].
In the absence of or limited data from India on RVC strains the high nucleotide similarity of most (except for the VP6 and NSP4) genes of the study strain is noted in comparison with cogent data from Japan/ other countries (Table 2b). In addition exceptionally higher (VP6, VP1, VP3, NSP4, NSP5) or lower (NSP1) PNI values, of the study strain as compared to the recommended cut off standard [15, 27] were observed (Table 2b). These observations emphasize the need of more data from India and other countries for validation of the genotyping criteria and elucidation of the evolutionary relationship of the RVC strains.
The current study has limitations with regard to the low number of fecal specimens analyzed and use of fecal specimens, which had been stored for a long time. The effect on the RVC detection rate is likely negligible as double stranded RNA has been described to remain stable in providing accurate assessment of environmental and biological systems [35]. Further, failure to characterize complete genomes of all the study strains could be due to low viral load that caused subclinical infection of RVC.
Despite the host-specificity of many RVC genotypes, evidence of cross-species transmission and reassortment and recombination events have also been documented [11, 16, 36–39]. Additionally, high RVC antibody prevalence described in rural as compared to the urban region was suspected to be due to the proximity of humans with animals [40]. On the backdrop of these data, identification of RVC strains at a notable level in asymptomatic pigs in the present study highlights its significance in the perpetuation of the virus in the environment and emphasizes the need to conduct epidemiological and molecular studies to know the disease burden and genomic diversity existing in RVC strains circulating in different regions. The data obtained will be useful for the development of more accurate diagnostic tools.