First Detection of Rotavirus C in Asymptomatic Pigs from India with Unexpected Dominance and Characterization of the Structural and Nonstructural Genes

Epidemiological and molecular investigation was conducted on Rotavirus C (RVC), the viral agent documented with rising prevalence rate, disease severity and cross species transmission and large outbreak potential. Fecal specimens of pigs collected from two cities of Maharashtra state, India tested for RVC showed 20.1% detection rate with majority below 3 months of age. No signicant difference in detection rates was observed in the specimens collected in 2009 from Northern and in 2013 from Western parts of Maharashtra. The phylogenetic analyses showed presence of the I7 and I10 genotypes of the VP6 gene and representative strains with G1 and E5 genotypes of the VP7 and NSP4 gene respectively. Full genome characterization of a single strain showed presence of G1, P1, I7, R1, C1, M3, A1, N5, T5, E5, H1 genotypes of the VP7, VP4, VP6, VP1, VP2, VP3, NSP1, NSP2, NSP3, NSP4 and NSP5 genes respectively. This is the rst evidence of detection of porcine RVC in asymptomatic pigs in India as well as with highest detection rate reported in asymptomatic pigs till date globally. Identication of porcine RVC at two time intervals and two different parts of Maharashtra state indicates the possibility of continuous circulation of RVC in pig population through asymptomatic infections. strain ; E1 genotype strain for the NSP4; H4 genotype canine strain NSP5 The study strain classied as A1 genotype (NSP1 gene) during phylogenetic analysis showed PNI values between 82.6 and 83.2 with other A1 genotype strains. These PNI values were lower than the recommended cut off value of 84% for NSP1 gene.


Introduction
MJF3:ATHAGACCAGCDGCYATGAC and VP6-MJR1: AGCCACATAGTTCACATTTCATC). Initially, 5µl of RNA was denatured at 97°C for 5 min and was rapidly chilled on ice for 5 minutes followed by addition of master mix for RT-PCR. The cycling conditions were initial reverse transcription step at 45°C for 30 min followed by 45 cycles of ampli cation (1 min at 94°C, 1 min at 55°C and 1 min at 68°C) and a nal extension of 7 min at 68°C in a thermal cycler. The ampli ed PCR products were analyzed using agarose gel electrophoresis and visualized under UV transilluminator. All RT-PCR positive products were excised from the gel and puri ed using QIA quick gel extraction kit (QIAgen, Hilden, Germany). The nucleotide sequencing of the puri ed PCR products was carried out using the Big Dye Terminator cycle sequencing kit v 3.1 (Applied Biosystems, Foster city, CA) followed by puri cation using DyeEx 2.0 Spin Kit, (Qiagen, USA). The puri ed DNA was sequenced in ABI PRISM 3130XL Genetic Analyzer (Applied Biosystems, USA). BLAST (www.ncbi.nlm.nih.gov/blast) tool in NCBI database was used to determine the nucleotide identity of the sequences. Phylogenetic tree was constructed by using Maximum Likelihood method with 1000 bootstrap support by selecting the best suited model for each gene using molecular evolutionary genomic analyzer software MEGA 6.0 [24]. The reference strains included in the study for phylogenetic analysis were isolated from diverse host species namely porcine, human, bovine and canine representing all genotypes of the RVC and were retrieved from GenBank. The attempt to characterize full genome of RVC strains was carried out on number of available specimens.
Molecular characterization of the few RVC positive strains was possible for the VP7 and NSP4 genes and single strain was characterized for all 11 genes using corresponding gene speci c primers [  A total of nine RVC positive specimens were ampli ed using the NSP4 gene speci c primers reported earlier for the characterization human RVC strains (Table 2). Further, phylogenetic analysis of the nucleotide sequences of the nine study strains classi ed them in to E5 genotype ( Figure 3). PNI within these study strains was 97-100%. Identity of these strains with GenBank E5 genotypes strains and Indian strain was 85.3-87.3% and 96.7% -97.5% whereas identity with human, bovine and canine species was 64.5%, 64.8-65.7% and 74.5-75.1% respectively. Among these nine RVC strains two were with I10 genotype for the VP6 gene (NIV94306, NIV94340).
Successful characterization of all 11 genes was possible only for single strain (NIV094290/Nagpur /2009) using different primer pairs ( Table 2). The nucleotide sequence data for 81.8% of genome (14504 bp) was obtained with more than 70% sequence data for each gene with exception of the VP3 (53.3%) and NSP5 genes (19.2%) ( Table 3).
The PNI values and phylogenetic analysis classi ed RVC strain as G1, P1, I7, R1, C1, M3, A1, N5, T5, E5, H1 genotype of the VP7, VP4, VP6, VP1, VP2, VP3, NSP1, NSP2, NSP3, NSP4 and NSP5 gene respectively (  of it is restricted to north eastern states. Till date only a single study documented from India (north eastern, northern and southern parts) with 12% prevalence rate of RVC in symptomatic pigs [21]. In the present study fecal specimens of asymptomatic pigs (n=149) from Western India showed 20.1% detection rate. Majority of the RVC prevalence studies reported so far, were conceded in mixed population of diarrheic and non-diarrheic pigs with detection rates higher in diarrheic pigs [7,17,27]. The studies solely on asymptomatic pigs were documented from Ireland, Western Kenya and Uganda with 4.4 -8.7% detection rates [9,28]. Overall, this is the rst study from India on recognition of the RVC in asymptomatic pigs as well as with highest detection rate reported in asymptomatic pigs till date globally. The high prevalence of RVC in symptomatic pigs has been allocated to high density of pig population in Tororo sub-country of Uganada [13]. Further, the high detection rate may possibly be due to use of sensitive diagnostic method. Comparative utility of RT-PCR assays needs to be examined from time to time due to the continuous accumulation of point mutations in rotavirus genome, reassortment events and possibility of cross species transmission leading to introduction of new genotypes and /or lineages [29]. Therefore, the nucleotide alignment of GenBank strains of porcine RVC (n=59), the one which was used for designing primers of the present study was also used for the analysis of primers reported by Amimo et.al (2017)  Previously, highest RVC infection rate has been documented in post weaning pigs (22-55 days) and in older pigs (5-6 months) [11,28]. In non diarrheic pigs from USA, 6.6% prevalence rate of RVC in weaning pigs was reported [27]. It has been reported that insu cient lactogenic protection provided by gilts plays a key role in the development and increase in clinical RVC disease [30]. Maternally derived circulating RVA speci c antibodies have been reported to mediate high levels of passive protection against human RVA disease [31]. However, no such studies reported till date on passive immunity against RVC. Therefore, asymptomatic RVC infections observed in the present study point out the possibility of lactogenic protection of pigs and need of further serological studies to con rm the same.
Among the scanty data available on genetic characterization of RVC strains from Asian countries, circulation of the G1, G3, G5, G6, G7, G9, G12 and G13 genotypes of the VP7, P1, P4-P9 genotypes of the VP4, I1, I4, I6 and I7 genotypes of the VP6 and M1, M3, M6 genotypes of the VP3 gene have been reported [18 -20, 32]. A single study from India reported circulation of the I2, E4-E8, H6 genotypes of the VP6, NSP4 and NSP5 genes respectively among diarrheic pigs [21]. In the present study two genotypes of the VP6 gene namely I7 and I10 along with G1, P1 and E5 genotypes of the VP7, VP4 and NSP4 genes were identi ed respectively. Till date, studies solely on asymptomatic pigs indicated dominance of the G1, occurrence of the G6 genotype in Ireland and G3P5 genotype in a single strain from Belgium [9,33]. Genotyping data was not available for the studies on asymptomatic pigs reported from Western Kenya, eastern Uganda and China [17,28]. Whole genome characterization of the RVC strains from asymptomatic pigs has so far been reported for a single Belgium strain with G3, P5, I11, R1, C1, A7, N8, T5, E5, H1 genotype of the VP7, VP4, VP6, VP1, VP2, NSP1, NSP2, NSP3, NSP4 and NSP5 gene respectively whereas the VP3 gene remained ungrouped [33]. Full genome characterization of a present study strain classi ed it as G1, P1 , I7, R1,   C1, M3, A1, N5, T5, E5, H1 genotype of the VP7, VP4, VP6, VP1, VP2, VP3, NSP1, NSP2, NSP3, NSP4 and NSP5 gene respectively. High nucleotide similarity of the study strain with strains originated from different countries (except for the VP6 and NSP4 gene) and for majority genes with Japanese strains may be due to lack of genomic data from India ( Table 3). The cut-off values of PNI for identi cation of different genotypes have been described earlier [20].
Using the same criterion, for few genes (VP6, VP1, VP3, NSP4 and NSP5) overlapping PNI values of the study strain with other genotype strains rather than the designated ones were observed exceptionally (Table 3). During NSP1 gene phylogenetic analysis study strain grouped with A1 genotype with 94% bootstrap support however, lower PNI values than the recommended cut off value of 84% were observed as compared to A1 genotype strains. Overall, more molecular data from India and different parts of the world is required for validation of the genotyping criteria suggested by Suzuki et.al (2017) as well as to elucidate the evolutionary relationship of the RVC strains.
The main limitation of the current study is long term storage of the clinical specimens. Reduction in RVC detection rate possibly will be negligible as double stranded RNA are known to be more stable and reported to have wide applications for accurate assessment in environmental and biological systems [34]. Failure to characterize complete genome of the remaining study strains was mostly due to low viral load as the infection were subclinical as well as shortage in amount of few fecal specimens.
Apart from this the increase in disease severity as well as prevalence rate (from 10 to 46%) of RVC over the period has been reported [11,35]. The rise in RVC infections in piglets was attributed to large scale RVA vaccination carried out to control and mitigate disease especially in developed countries [7,11,14]. Even though, host speci c (bovine, canine, human, and porcine) nature of the RVC genotypes is known, (excluding the VP6 and VP3 gene in which human and porcine strains share evolutionary relationship) evidences of cross species transmission and reassortment events have been documented [15,21,36]. Identi cation of porcine RVC in bovine, bovine RVC in porcine, porcine RVC in human and human RVC in porcine species has been reported [21,[37][38][39][40]. In addition, high RVC antibody prevalence documented in rural as compared to urban region was suspected to be due to close proximity of the humans with animals [38]. Therefore, RVC the viral agent with escalating prevalence rate and disease severity over the period needs to be monitored closely keeping in mind its cross species transmission and large outbreak potential.
Identi cation of porcine RVC in large number of asymptomatic pigs in the present study may possibly have signi cance in perpetuation of the virus in the environment. Molecular characterization studies on RVC strains isolated from age matched weaning and post weaning, diarrheic and non-diarrheic piglets/pigs from the same geographical regions will be useful to de ne the role of maternal immunity and genotypes of RVC in symptomatic or asymptomatic infections. The epidemiological and molecular studies need to be conducted to know exact disease burden and genomic diversity existing in RVC strains circulating in different parts of the world. The data obtained will be useful for development of more accurate diagnostic tools and for future vaccine candidate.

Data Availability
The datasets generated during the current study are available in the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/).

Ethics approval
The study was approved by the institutional Animal Ethics committee on 3 rd November 2014.