Presence of Human G Rotavirus Genotypes Amongst Bovine Calves and its Combination with P Genotypes

Rotavirus are non-enveloped double-stranded RNA (dsRNA) viruses belonging to the family Reoviridae and it has a triple layered protein capsid of 75 nm, surrounding the viral genome consisting of 11 segments of double-stranded RNAwhich encodes six structural (VP1, VP2, VP3, VP4, VP6 and VP7) and six non–structural proteins (NS1, NS2, NS3, NS4, NS5 and NS6) (Greenberg and Estes, 2009). The proteins in the mature virus particle determine the host specificity, cell entry and enzymatic functions necessary for the production of viral transcripts and contain epitopes that generate immune responses. Rotaviruses have been classified into nine serological species or groups (A–I) based on the antigenic properties of inner capsid protein (VP6) according to the International Committee on Taxonomy of Viruses (ICTV) (Sharad et al., 2018). The RVs belonging to species A, B and C (RVA, RVB and RVC, respectively) are known to infect human and various animals, whereas the species D, E, F, G, H and I (RVD, RVE, RVF, RVG, RVH and RVI respectively) have only been recovered from animals, and mostly birds (Ball et al., 2005; Matthijnssens et al., 2010). Rotaviruses have segmented double stranded RNA (ds RNA) genome, which can be separated into discrete segments by RNA-PAGE. Viruses classified as Group A rotaviruses (RV-A), including BoRV-A, posses two independent neutralization antigens, namely VP4 and VP7, on the outer capsid, that specify the P type (protease-sensitive protein) and G type (glycoprotein), respectively. It has therefore been proposed that serotypic classification of rotaviruses should account for both VP7 (G) (Hoshino et al., 1984) and VP4 (P) (Snodgrass et al., 1992) specificities. Epidemiologically, RVA is the most important cause of human and animal diarrhea. Specifically, RVA strains have been categorized based on (i) the antigenic properties of VP6, VP7 and VP4 (subgroups, G–serotypes and P–serotypes, respectively); (ii) the migration pattern of the RNA genome segments when subjected to polyacrylamide gel electrophoresis (long, short, super–short or atypical electropherotypes) and (iii) nucleotide sequence analysis (genotypes) (Estes and Kapikian, 2007). To date, 28 G types and 39 P types of RVA have been identified in humans and animals as per the minutes of 7^th Rotavirus Classification Working Group (Matthijnssens and Theuns, 2015). Rotavirus is the most common viral pathogens causing diarrhea in young cattle. The study was undertaken to have an update on the prevalence of Group A Rotavirus genotypes amongst bovines from central India thus aiding in better understanding of interspecies transmission, evolution and strategic planning to combat rotavirus infection.

Sample collection and processing
 
A total of 200 faecal samples were collected from diarrheic cow and buffalo calves (up to the age of 3 months), from different regions of Jabalpur viz. Adhartal Dairy Farm, Military Dairy Farm, Gorakhpur Gausala, Jamtara, Pariyat Dairy Farms, Gaur Dairy Farms, Kaladehi Bargi, Padmaniya Shahdol. The samples were kept on ice and transported to laboratory for processing. 10% suspension of the faecal material was prepared with phosphate buffer saline (PBS, pH 7.2) by dissolving one gram of faeces in 10 ml PBS.
 
ELISA and RNA-PAGE
 
All the fecal samples were subjected to Bio X Bovine Rotavirus Sandwich ELISA kit – (Product ID- BIO K 343/2) (Singh et al., 2015) and RNA-PAGE analysis for initial screening of all the bovine faecal samples for RV presence by observing the 11 genome segments and their typical migration pattern. For electrophoresis, resolving gel (7.5%) and stacking gel (5%) were prepared as per the method of Laemmli (1970).
 
RNA extraction and reverse transcription for VP6, VP4 and VP7
 
Total RNA was isolated using Tri reagent LS according the manufacturers protocol. RNA from 22 positive samples both from ELISA and RNA PAGE were further subjected to RT-PCR for VP6, VP4 and VP7 gene. RNA isolated was reverse transcribed using random primers and reverse transcriptase of Revert Aid First Strand cDNA synthesis kit, Thermo Scientific, USA. The reaction mix was prepared in a 0.5 ml micro centrifuge tube, using Template RNA (0.1 ng -5 µg) 1 µl, Random Hexamer 1 µl and Nuclease Free Water 10 µl. After denaturing at 65°C for 5 min, followed by snap cooling on ice, the following reagents were added to the tube: 5X Reaction Buffer 4 µl, RiboLockRNase Inhibitor (20u/µl) 1 µl, 10 mM dNTP Mix 2 µl, RevertAid M-MuLVRT (200 u/µl) 1 µl and final volume 20 µl. The reaction mixture was kept for 5 min at 25°C followed by 42°C for 1 hr, finally terminated the reaction at 70°C for 5 min. The cDNA (3 µl) thus obtained was used for PCR and the remaining was kept at -20°C until further use.

RT-PCR for VP7 gene to amplify near full length copies of rotavirus gene 9 coding for VP7 protein of rotavirus. The reaction mixture consist of RNA 7 µl, Bov9Com5 (10 µM) 1.5 µl, Bov9Com3 (10 µM) 1.5 µl, this mixture was denatured at 97°C for 5 minutes and immediately chilled on ice. Following reagents were then added RNase inhibitor (10 U) 1.0 µl, 0.1 M Dithiothreitol (DTT) 1.0 µl, 5X RTbuffer 4.0 µl, 100 mM dNTP mix 0.6 µl, Reverse transcriptase (5 U): 0.5 µl, Nuclease Free Water 2.9 µl, with final volume 20 µl. The reaction mixture was mixed well and incubated at 42°C for 1 hour followed by denaturation of RNA-cDNA hybrid at 95°C for 2 minutes and chilled on ice after a brief spin.
 
PCR for amplification of VP6, VP4 and VP7 gene.
 
All samples positive in Bio X diagnostic ELISA and RNA-PAGE were further subjected to amplification of VP6, VP4 and VP7 gene. The reaction for VP6 and VP4 gene was carried out in a 0.2 ml tube, with 2X PCR Master Mix 12.5 µl, Forward primer (20pMole/µl) 1 µl, Reverse Primer (20pMole/µl) 1 µl (Table 1), DMSO 1 µl, cDNA 3.0 µl, Nuclease Free Water 6.5 µl, with final reaction volume of 25 µl. The PCR components were mixed and reaction was set with the amplification condition: Intial denaturation for 5 min at 95°C, followed by 30 cycles at 95ºC for 30 sec, 48°C 30 sec, extension at 72°C for 30 sec and final extension at 72°C for 10 mins.

PCR amplification of near full length VP7 gene was carried out in a reaction mixture consisting of reverse transcriptase product 5 µl, 100 mM dNTP 0.2 µl, 10X PCR buffer 2.5 µl, MgCl₂ (25 mM) 1 µl, Bov9Com5 (10 µM) Forward primer 0.5 µl, Bov9Com3 Reverse primer (10 µM) 0.5 µl (Table 1), Taq DNA polymerase (3 U/µl) 0.6 µl and Nuclease free water 14.7 µl, final volume 25 µl. Production of 1011 bp product was considered as positive result. PCR reaction was set with the amplification condition : 94°C for 2 min, 30 cycles of 94°C 1 min, 50°C 1 min, 72°C for 2 min with final extension at 72°C for 7 min as per Wani et al. (2004) with slight modifications. The amplicons so generated were resolved on agarose (2%) gelelectrophoresis.


 
Multiplex-semi-nested P and G genotyping PCR
 
The first round PCR amplicons of VP4 and VP7 genes were diluted 1:100 with NFW.  The resulting diluted PCR products were then used as a template in a semi-nested PCR for P-typing and G-typing, respectively.
 
G genotyping PCR
 
The PCR product of the VP7 gene was diluted to 1:20 with NFW and subjected to second round of amplification as described above using generic primer Bov9Com3 and a cocktail of typing primers specific for G1, G3, G5, G6,G8 and G10 (Table 2). The reaction was carried out as follows 2X PCR Master Mix 12.5 μl, Bov9Com3 (10 pMole/μl) 1.0 μl, G1 (10 pMole/μl) 0.5 μl, G3 (10 pMole/μl) 0.5 μl, G5 (10 pMole/μl) 0.5 μl, G6 (10 pMole/μl) 0.5 μl, G8 (10 pMole/μl) 0.5 μl, G10 (10 pMole/μl) 0.5 μl, Template 2.0 μl, Nuclease free water 8.0 μl with Total reaction volume 25.0 μl. PCR reaction was set with the amplification condition 95°C 8 min, followed by 30 cycles of 94°C 45 sec, 52°C 45 sec, 72°C 90 sec, with final extension of 72°C for 10 min. The amplicons so generated were resolved on agarose (2%) gel electrophoresis.


    
P genotyping PCR
 
The P genotyping PCR was carried out by using a common forward primer and a set of three different reverse primers (P1, P5 and P11) corresponding to three different P genotypes for bovine RVA. The First round PCR amplicons of VP4 were diluted 1:100 with NFW. The resulting diluted PCR products were then used as a template in a semi-nested PCR for typing. The reaction was carried out as follows: 2X PCR Master Mix 12.5 μl, Forward primer (10 pMole/μl) 1.0 μl, P1 (10 pMole/μl) 0.5 μl, P5 (10 pMole/μl) 0.5 μl, P11 (10 pMole/μl) 0.5 μl, Template 1.0 μl, Nuclease free water 8.0 μl, Total reaction volume 25.0 μl PCR reaction was set with the amplification condition as 95°C 5 min, 30 cycles of 95°C 45 sec, 48°C 30 sec, 72°C 1.5 min with final extension of 72°C 10 min. The amplicons so generated were resolved on agarose (2%) gel electrophoresis.

Rotaviruses are continuously evolving due to lack of proofreading activity in RNA polymerase and segmented nature of genome which leads to frequent reassortment. The global distribution, wide host range and presence of numerous G and P types demands frequent surveillance of rotavirus infection. RT-PCR, ELISA and hybridization assays have been used to detect the G and P types of field strains of group A rotavirus but RT- PCR assay using type specific primers is now-a-days most widely used technique for strain characterization. RT-PCR based assay has been found to be 5000 times more sensitive than hybridization based screening assay (Xu et al., 1990). It has an advantage over ELISA because the later can sometimes yield false positive results (Pedley and McCrae, 1984).

Out of 22 ELISA and RNA-PAGE positive diarrheic samples of calves, in 22 (100%), 15 (68.18%) samples we could amplify VP6 and VP4 gene segments, respectively. Gene product of VP6 is indicated by 227 bp (Fig 1a, 1b and 1c) amplicon while, 864 bp product showed the presence of partial length VP4 gene segment (Fig 2a, 2b and 2c) by RT-PCR. All the eight cow and 14 buffalo samples of total 22 samples subjected for VP6 gene showed 36.36% and 63.63% amplification. For VP4 gene total positive samples were fifteen, out of which 03 (20%) were form cow calf and 12 (80%) were from buffalo calf (Table 3).









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Fifteen samples (68.18%), out of twenty two samples screened for VP7 gene were found positive for VP7 (Fig 3a, 3b, 3c). Amongst the total 15 samples 03 (20%) were from cow and 12 (80%) were from buffalo. All the samples which showed positive result for VP4 gene were also found positive for VP7 gene as well.







The presence of VP6 gene is demonstrated by other researchers as well which are in accordance with our findings. Basera et al., (2010) detected 10 (10/13, 76.9%) samples positive for VP6 antigen. Mukhtar et al., (2016) reported the amplification of VP6 gene (33.3%) in 04 diarrheic samples out of total twelve ELISA positive samples. Singh et al., (2015) reported the amplification of VP6 gene in 12/ 100 (12%) fecal samples from Mathura region.

Multiplex semi nested PCR was performed on VP4 and VP7 amplified products of 15 samples for the P and G typing of Rotavirus. The P genotypes were determined (Fig 4a and 4b) by using 1:100 diluted VP4 amplicons in a multiplex semi-nested PCR assay using cocktail of P typing primers common to bovine RVA. Of the 15 RV positive samples P typed, P [11] was found in 11 (73.33%), P[5] in one (6.66%), mixed P[5]P[11] in one (6.66%) while the 02 samples (13.33%) were untypeable. Out of the 15 samples G typed, G3 was seen in 04 (26.66%) samples, G1G3 in 08 (53.33%), G1G5 in 01 (06.66%) samples, while one (06.66%) sample had G3G8 (Fig 5a and 5b). One sample (06.66%) was non-typeable (Table 4). The G and P genotype combinations as determined by RT-PCR are listed in Table 5. Out of the 12 samples which were G and P genotyped by RT-PCR, G3P[11] was seen in 0 2 (16.66%) samples, G3P[5]  in 01 (08.33%) samples, G1G5P [11] in 01 (08.33%) sample, G1G3P[11] in 07 (58.33%) samples, while 01 (08.33%) samples had a mixed G1G3P[5]P[11] combination.









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Due to segmented nature of the RNA genome and wide host range, vast genetic and antigenic diversity exists among different isolates of rotavirus. There are many G and P types RVA circulating amongst human and animal population. Human rotaviruses most commonly belong to G types 1-4, 9 and P types [4] and [8] (Gentsch et al., 1996) whereas bovine rotaviruses most commonly belong to G types 6, 8 and 10 and P types [1], [5], [6] or [11] (EL-Attar et al., 2002). G10 strains commonly occur in combination with P [11], P [5] and P[1] (Pisanelli et al., 2005). A G10 P [15] strain was reported for the first time in a lamb infected with rotavirus in China (Shen et al., 1993). In India, so far, bovine diarrhea associated with G10 rotavirus has been seen in combination with P[11] (Beg et al., 2010; Minakshi et al., 2005; Saravanan et al., 2006), P[6] (Gulati et al., 2007), P[14] (Ghosh et al., 2007) and P[3] (Manuja et al., 2008). Despite clear evidence of host range restriction, a number of animal gene segments, mostly those encoding the neutralizing antigens (defining G and P types), have been identified repeatedly in humans in different parts of the world during surveillance studies (Gentsch et al., 2005; Santos et al., 2005), providing evidence that animals may act as a source of virus and/or of genetic material for evolutionary diversification of human rotaviruses. For example, strains such as G3 (found commonly in species such as cats, dogs, monkeys, pigs, mice, rabbits and horses), G5 (pigs and lambs), G9 (pigs and lambs) and G10 (cattle) have been isolated from the human population throughout the world (Gentsch et al., 1996). The G9 strains reported in humans (Cubitt et al., 2000) which are found in lambs and pigs (Santos et al., 1999), may have been become established in humans during the past two decades through transfer from animals. There are a number of studies reporting rotavirus strain distribution in animals or humans in India but they do not provide any geographic or temporal comparisons of distribution among animals and humans (Kang et al., 2001; Nataraja et al., 2009). This is also similar to the lack of such reports worldwide with only a few studies that have compared the strains isolated from animals and humans simultaneously in the same region (Steyer et al., 2008; Van der Heide et al., 2005).

During the present study, 15 samples were genotyped by using multiplex semi-nested PCR assay. The G genotyping PCR assay showed G3 as the most predominant G type found in 04 (26.66%) samples (Table 4), while 10 (66.66%) samples showed mixed G types (two or more G types in same sample), amongst which G1G3 was most predominant 08(53.33%), while G1G5 and G3G8 one each (6.66%). This study was in contrast to the earlier reports which detected G10 as the most predominant G-type in India (Varshney et al., 2002; Saravanan et al., 2006). Gulati et al., (1999) detected G10 (83%) as the most predominant G type in India followed by G6 (6%). Minakshi et al., (2005) also reported a frequency of G10 and G6 genotypes to be 86.61 and 13.39%, respectively, in Indian bovine rotavirus isolates. Reidy et al. (2006) reported G6 as most prevalent, followed by G10. Our findings are in accordance with the findings of Sravani et al., (2014), who detected G3 as most predominant with 43.75% and combination of G3G8 as 50%. They also recorded that prevalence detected by RNA-PAGE is higher (19.16%) as compared to RT- PCR (13.3%) thus underlying the benefit of simultaneous use of atleast two test for improved diagnostic potential. Malik et al., (2012) also found 52.9% of G3 and 47% of mixed G types in the samples, with G3G8 and G3G10 60.6%. Among the mixed genotypes we found G1G3 the most predominant one which is supported by the findings of Das et al., (2002) who reported the presence of G1G3 genotype in human fecal sample reported the presence of 1% G1G3 genotype. Castillo et al., (2000) also reported 02 diarrheic samples of children from Mexico having G1G3 genotype. Ezung et al. (2014) also reported the presence of G1G3 genotype in human diarrheic sample from Uttarakhand region. This is probably the first report of depicting G1G3 from bovine diarrheic samples as per our knowledge. G1 and G3 genotypes are most commonly reported from human diarrheal cases, the presence of these genotypes in bovines suggest the transmission of rotavirus strains from human to bovines. This might be because of close proximity between human and bovine.

As per our findings, among the G type one sample was non typeable which is supported by the findings of Klingenberg et al., (1999) where they found 07 samples non typeable for any of the G types. Reidy et al., (2006) also found 03 (3.2%) samples non typeable. Niture et al., (2011) could not type any buffalo sample for G genotype. The high proportion of non typeable strains is a common feature of rotavirus characterization in most developing countries, where up to 30% of strains may remain un typed (Aminu et al., 2010). These strains may represent common strains with accumulated point mutation in the currently used primer binding sites, animal rotaviruses including G5, G6, G10 and G12 or new unidentified rotavirus strains. Sequence analysis and the constant monitoring of genetic drift and shift would aid researchers in the developing world in the typing of circulating rotavirus strains.

The P genotyping PCR assay showed P[11] as the most predominant P type as it was found in 11 (73.33%) samples (Table 4), while one (6.66%) sample each showed P[5]P[11] mixed P type and P[5] whereas two (13.33%) sample was un typeable. Although P[1] typing primers were incorporated in the multiplex semi-nested typing PCR assay, none of the isolates was typed as P[1]. This study is in conformity with the earlier report by Gulati et al., (1999), who reported P[11] in 94% of the samples genotyped. Falcone et al., (1999) reported 55% of P[11] and 42.3% of P[5], interestingly he also reported none sample positive for P[1], similar to our findings. Reidy et al. (2006) found P[5]P[11] combination in 11% i.e. six out of 54 samples positive for this combination.                           

As shown in (Table 5) the most predominant G and P type combination as determined by PCR assay was G1G3P[11] (58.33%) followed by G3P[11] (16.66%) and G3P[5], G1G5P[11] and G1G3P[5]P[11] one each (8.33%). This again is in contrast with the earlier report by Gulati et al., (1999) who reported G10P[11] (81%) as the most predominant G and P type combination among Indian bovine population, followed by G6P[1] (3%) and G6P[11] (3%). Saravanan et al., (2006) also reported G10P[11] from Tamil Nadu as the most predominant genotype combination. Beg et al., (2010) also reported from Kashmir G10P[11] as the most predominant (80.6%) combination followed by G8P[11] (7.7%). However, Manuja et al., (2008) reported unusual G10P[3] as the most predominant (73.3%) combination from north India followed by G6P[11] (26.7%). Ezung et al (2014) reported predominant G and P type combination in bovine samples was 23.07% G3G6G10P[11] followed by 15.38% each of G3P[11], G10P[1]P[11], G3G10P[1]P[11] and G3G6G10P[1], while 7.69% had G10P[1] and G3 P[1]P[11] combination. In HRV, most predominant G and P type combination was G1G3P[8] (28.57%) followed by G1P[4] (14.28%) from Uttarakhand and G3P[8] (14.28%) from Nagaland. Malik et al., (2012) detected G3P[11] as most prevalent genotype and mixed genotypes as G3G10P[11], G3G8P[11], G3G8P[1]P[11] and G3G10 P[1]P[11]. Mixed genotypes have been commonly reported in bovine RVA. Minakshi et al., (2005) reported two isolates with extra-genomic bands in RNA-PAGE, with mixed G and P types (one isolate with G6G10 and another with P[1]P[11]). Mixed infection has also been reported in bovines from India by Sharma et al., (2009) and Beg et al., (2010). Mixed genotype infections can pave a way for reassortment between co-infecting RV strains and emergence of novel genotypes.

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Knowledge of G and P types of group A rotavirus and their combinations in a particular geographical location is very important prerequisite as far as the prophylactic and control measures against the rotavirus infection are concerned. Genotypic strains prevalent in and around Jabalpur had been determined first time.

The presence of such large number of combinations of G and P types suggests genetic reassortment amongst the circulating strains. The presence of G1 and G3 genotypes are commonly found in humans, its presence in bovine samples indicate interspecies transmission. There should be continuous monitoring of rotavirus A strains circulating in different species.

We would like to thank, The Dean, College of Veterinary Science and Animal Husbandry, Jabalpur for providing all needed assistance and facilities for conducting the above work.