Prevalence of Group A Rotaviruses (RVA) in Pig Population of North East India

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1 Journal of Immunology and Immunopathology Vol. 16, No. 1&2, January-December, 2014: DOI: / IndianJournals.com A product of Diva Enterprises Pvt. Ltd. Research Article Prevalence of Group A Rotaviruses (RVA) in Pig Population of North East India DP Bora 1 *, M Bora 2, B Borah 3, B Bezborah 4, DK Sarma 5, YPS Malik 6 and TK Dutta 7 1 Assistant Professor, 3 Junior Research Fellow, 4 Senior Research Fellow, 5 MVSc Student, Department of Microbiology, College of Veterinary Science, Assam Agricultural University, Khanapara campus, Guwahati , Assam, India 2 PhD Scholar, Division of Virology, Indian Veterinary Research Institute, Izatnagar, Bareilly , Uttar Pradesh 6 Senior Scientist & National Fellow, Division of Biological Standardization, Indian Veterinary Research Institute, Izatnagar, Bareilly , Uttar Pradesh, India 7 Associate Professor, Department of Veterinary Microbiology, College of Veterinary Science & Animal Husbandry, CAU, Selesih, Aizwal , Mizoram, India *Corresponding author id: drdpbora@gmail.com ABSTRACT Bora DP, Bora M, Borah B, Bezborah B, Sarma DK, Malik YPS and Dutta TK. (2014). Prevalence of group A rotaviruses (RVA) in pig population of North East India. J. Immunol. Immunopathol. 16(1&2): Group A rotaviruses (RVA) have been recognised as an important cause of piglet diarrhoea which is a major threat causing serious economic losses in swine industry of north east India. Apart from RVA, there are many other agents which are directly or indirectly associated with piglet diarrhoea. Thus accurate and timely diagnosis is very much essential to establish the etiology of piglet diarrhoea. In the present study, RVA was detected in faecal samples of diarrhoeic pigs using RNA-PAGE and RT-PCR, collected from different states of Northeast India. Overall, of 601 faecal samples collected, 134(22.29%) samples were found to be positive for presence of RVA by RNA-PAGE. In RT-PCR, RVA was detected by amplification of VP4 and VP7 genes of RV, where 178 (29.61%) samples were found positive. Among the states, Assam and Tripura recorded highest prevalence of RV among pig population in comparison to other states of NER. Keywords: Diarrhoea, Piglet, GARV, VP4 gene, VP7 gene, PCR INTRODUCTION Group A Rotaviruses (RVA) are the most common etiological agents of severe diarrhoea in human infants as well as in young ones of most farm animal species worldwide (Estes and Kapikian, 2007, Malik et al., 2014). In piglets, RV-induced diarrhoea has been a major concern to pig farmers worldwide. North-East India (NER) being a potential hub for pig farming accounts for approximately two-third of total pig population of the country. Rotavirus is a member of the family Reoviridae characterised by non-enveloped triple-layered viral particles with a viral genome composed by 11 double-stranded RNA segments (dsrna). RVs are classified in seven groups (A G), according to the antigenic variability of the inner capsid protein VP6. Group A RVs are further classified into G and P types based on the genetic and antigenic variation of the two outer capsid proteins VP7 (glycoprotein) and VP4 (protease sensitive protein), respectively (Matthijnssens et al., 2008). Further, on the basis of relative migration patterns of RNA segments in PAGE, rotaviruses are classified into electropherotypes. The electrophoretic pattern of the dsrna segments by PAGE determines the group of RV (Herring et al., 1982). Earlier studies from this region have 53

2 DP Bora, M Bora, B Borah, B Bezborah, DK Sarma, YPS Malik and TK Dutta demonstrated isolation of pig rotavirus from local piglets of Assam in MA 104 cell lines (Barman et al., 1998; Bora et al., 2007; Neog et al., 2011), nevertheless; there is no report of detection of rotaviruses from other states of NER. The present study was therefore focused on the detection of rotaviruses from pigs with further characterisation by RNA-PAGE and RT-PCR. MATERIALS AND METHODS Collection of Samples A total of 601 numbers of freshly voided faecal samples form diarrhoeic piglets were collected. Samples were collected from different states of NER as detailed in Table 1. Approximately 4 g of faecal sample were collected either from freshly voided faeces or obtained by rectal evacuation from the animals. Suspension of each samples were prepared in 0.06 M phosphate buffer saline (ph-7.2) by diluting in a ratio of 1:4, followed by centrifugation at 12,000g at 4 C for 30 mins and the supernatant was collected and preserved at -20 C until further use. Detection of RV in faecal samples Detection of RV in the faecal samples were detected by using RNA-PAGE and RT-PCR as per method of Herring et al., 1982 and Gouvea et al., Ribonucleic acid poly acrylamide gel electrophoresis (RNA PAGE) Twenty percent of faecal suspension was used for viral RNA genome extraction using phenol chloroform mixture (Herring et al. 1982) and 0.1 M sodium acetate buffer (ph 5.0) containing 1% (w/v) sodium dodecyl sulphate (SDS). RNA quantity and quality was assessed using a Nanodrop Spectrophotometer (ND-1000; Thermo Scientific, USA) and was analysed by RNA-PAGE in 7.5% polyacrylamide slab gels using 3% spacer gel in a discontinuous buffer system (Laemmli, 1970) without SDS. Electrophoresis was carried out at V for 2-3 hour in room temperature using vertical slab gel electrophoresis apparatus (Biorad, USA). The gel was further stained with silver nitrate (AgNO 3 ) solution to generate electropherotype of the fragmented viral RNA (Svensson et al., 1986). RT-PCR for amplification of VP7 and VP4 genes of RV Reverse transcription of suspected faecal samples for amplification of full length VP7 gene was performed for which viral RNA was extracted using QIAamp Viral RNA Kit (Qiagen) following manufacturer s protocol and was used as templates in RT-PCR. One step RT-PCR was performed using set of generic primers as described earlier (Gouvea et al., 1990) in two reaction steps holding a total reaction volume of 50 µl. In RT-1 reaction, the mixture of template and10 pmol primer was denatured at 95 C for 5 minutes in a thermal cycler (VERITI 96 wells, Applied Biosystems) and immediately chilled on ice. RT-2 reaction for cdna synthesis and gene amplification was performed in the same tube by adding 5X RT buffer, 10mM dntp mix, 25 mm MgSO 4, 5 U of AMV RT enzyme, 5U of Taq polymerase (Invitrogen) and DMSO under following Table 1: Details of the samples collected from different states of North-East India Name of the state No. of samples Number positive screened RNA PAGE RT-PCR Assam (26.88) 71 (33.49) Meghalaya (22.47) 27 (30.33) Arunachal Pradesh (22.36) 25 (32.89) Nagaland (20.68) 17 (29.31) Manipur 32 5 (15.62) 8 (25.00) Mizoram 80 7 (8.75) 11(13.75) Tripura (29.62) 19 (35.18) Total (22.29) 178(29.61) Figures in parenthesis indicate percentage 54 Vol. 16, No. 1&2, January-December, 2014

3 Prevalence of Group A Rotaviruses (RVA) in pig population of North East India thermal cycling profile: Pre-incubation at 48 C for 45 minutes, denaturation at 94 C for 1 minute, annealing at 46 C for 2 minutes, extension at 68 C for 3 minute and repeated the above steps for 39 cycles following final extension for 10 minutes at 68 C and wind up by hold at 4 C. The PCR amplified product was confirmed by agarose gel electrophoresis using 1.5% agarose (Amresco) containing ethidium bromide (0.5 mg/ml) in 0.5X Tris borate EDTA (TBE) buffer and the gel was visualised under gel documentation system (Gel Logic 212 PRO). For amplification of VP4 gene, RNA was extracted similarly. The primers, con2 and con3 were used to reverse transcribe the VP4 gene as described by Gentsch et al. (1992). The RT PCR of VP4 gene was in two steps. The RT-1 reaction which consisted of RNA (4µl), 20 pmol of each primer and RNase In (40U, Thermo Scientific) was subjected to a denaturing temperature of 95 C for 5 minutes and then immediately chilled on ice followed by RT-2 reaction. The RT-2 reaction was carried out by adding the master mix comprising of 5X RT buffer (Thermo Scientific), 3% DMSO, 1.5mM MgCl 2 (Thermo Scientific), 10mM dntps (Thermo Scientific), RevertAid H Minus Reverse Transcriptase (100U, Thermo Scientific), Taq DNA Polymerase (2.5U, Thermo Scientific) and nuclease-free water (to make up the final volume up to 20 µl) in the RT-1 reaction tube with thermal conditions as: initially 48 C for 45 minutes; then, 30 cycles of 94 C for 1 minute, 46 C for 2 minutes, 68 C for 3 minutes followed by final extension at 68 C for 10 minutes and hold at 4 C. Analysed 5µl from each PCR product on 1.2% agarose gel (SRL) and was visualised in gel documentation system (DNR Gel Logic 212 PRO). rotavirus (4:2:3:2) in all positive samples (Figure 1). In RT-PCR, on amplification of VP4 and VP7 genes using specific primers, desired amplification of 876bp and 1062 bp could be observed corresponding to VP4 and VP7 genes respectively (Figure 2 and 3). Of 601 faecal samples subjected to RT-PCR, 178 (29.61%) samples were found positive for presence of RV. Among the different states of NER, highest RV positivity was recorded in Tripura state (35.18%) followed by the Assam state (33.49%). The lowest RV positivity was recorded in the Mizoram state (13.75%). More numbers of samples were found positive for RV by RT-PCR method in comparison to RNA-PAGE. DISCUSSION Rotavirus is a major cause of acute gastroenteritis in humans and animals worldwide (Saif et al., 1996). In pigs, it has emerged as a potential threat causing piglet diarrhoea in newborn piglets (Jhala and Raghavan, 1998; Malik et al., 2014). The north eastern states of India, being a hub for pig husbandry need extensive study on prevalence of RV-induced diarrhoea among pig population. The present study was undertaken to determine the prevalence of RV infection in pig population by detection of RV antigen 1,2,3,4 5 & 6 7, 8, 9 L1 L2 L3 L4 L5 L6 L7 RESULTS 10 & 11 During the study, a total of 601 numbers of faecal samples from different states of North-East India were collected and analysed for detection of RV antigen. Details of the samples collected along with the results of RV detection are presented in Table 1. Results of RV detection by RNA-PAGE showed that overall, 134 (22.29%) samples were found to be positive for RV. The RNA PAGE analysis revealed the typical 11 segmented RNA genome of rotavirus with characteristic band migration pattern of group A Figure 1: RNA-PAGE for RV detection showing characteristic 4:2:3:2 pattern. L1: Positive RV control, L2- L5: Field samples; L6: Negative sample, L7: Negative control Journal of Immunology and Immunopathology 55

4 DP Bora, M Bora, B Borah, B Bezborah, DK Sarma, YPS Malik and TK Dutta segmented RNA genome of rotavirus with characteristic band migration pattern of group A rotavirus (4:2:3:2) in all positive samples (Figure 1). In RT-PCR, on amplification of VP4 and VP7 genes using specific primers, desired amplification of 1062 bp and 876bp could be observed corresponding to VP7 and VP4 genes respectively (Figure 2 and 3). Of 601 faecal samples subjected to RT-PCR, 178 (29.61%) samples were found positive for presence of RV. Figure 2: VP7 amplified regions of Rotavirus from faecal samples of pigs of NER Figure 3: VP4 amplified regions of Rotavirus from pigs of NER in faecal samples collected from piglets affected with diarrhoea. For this, RV antigen was detected in faecal sam ples by RNA- PAGE followed by PCR amplification of VP4 and VP7 genes of RV. During the study, a total of 601 numbers of faecal samples from different states of North-East India were collected and analysed for detection of RV antigen. Details of the samples collected along with the results of RV detection are presented in Table 1. Results of RV detection by RNA-PAGE showed that overall, 134 (22.29%) samples were found to be positive for RV. The RNA PAGE analysis revealed the typical 11 Use of polyacrylamide gel electrophoresis for detection and characterisation of RV has been reported earlier (Herring et al., 1982; Chauhan and Singh, 1993; Wani et al., 2003). Analysis of RNA electropherotypes provides evidence for the genetic diversity and heterogeneity of the rotaviruses circulating in a population. In the present study also, the group A RV was detected successfully with characteristic band migration pattern of group A rotavirus (4:2:3:2) in all positive samples. However in RT-PCR, the number of positive samples was more signifying the more sensitivity of the test. Rotavirus associated diarrhoea has been reported earlier from different places of India (Kusumakar et al., 2008; Chouhan and Singh, 2003). Among the different states of North-East India, RV was invariably associated with cases of piglet diarrhoea. However, in pig population of Assam and Tripura state, prevalence of RV was found to be more both in RNA- PAGE as well as in RT-PCR (Table 1). Mizoram state was found to have the lowest prevalence of RV followed by Manipur state in the collected samples. The samples in these states were collected from organised farms which may the reason of low detection of RV in comparison to other states. Association of RV with piglet diarrhoea in Assam state has been reported in many occasions earlier (Barman et al., 2003; Bora et al., 2007; Sharma et al., 2013). However, no such report on RV detection in pigs of the other states of NER India has been reported so far. To the authors knowledge, this is the first report of this kind on detection RV in pig population of different states of North-East India. Further characterisation of RV from these states will be of immense help in identifying the epidemiology of RV in NER India. 56 Vol. 16, No. 1&2, January-December, 2014

5 Prevalence of Group A Rotaviruses (RVA) in pig population of North East India ACKNOWLEDGEMENTS The authors would like to thank the Department of Biotechnology, Government of India, for providing financial help to carry out the research under DBT Twinning project on Development of improved diagnostics with Monitoring and Characterisation of viral and bacterial pathogens associated with piglet diarrhoea in North-Eastern region of India to carry out the research work. REFERENCES silver-stained polyacrilamide gels. J. Clin. Microbiol., 16(3): 473. Jhala MK and Raghavan R (1998). Detection and isolation of porcine rotavirus from diarrhoeic piglets. Indian J. Virol., 14: Kapikian AZ, Hoshino Y and Chanock RM (2001). Rotaviruses, In Fields Virology (Lippincott Williams & Wilkins, London) pp Kusumakar AL, Savita, Malik YPS, Minakshi P and Prasad, G (2008). Occurrence of mammalian group A rotavirus in swine population of central India. Indian J Anim Sci., 78: Barman NN, Barman B, Sarma DK and Pansaert M B (2003). Prevalence of rotavirus, transmissible gastroenteritis virus and porcine epidemic diarrhea virus antibodies in pigs of Assam, India. Indian J Anim Sci., 73: Barman NN, Sarma DK and Pensaert M (1998). Detection of swine rotavirus and transmissible gastroenteritis virus in piglets with diarrhea by sandwich ELISA. Indian J. Anim. Sci., 68(9): Bora DP, Barman NN and Bhattacharyya DK (2007). Isolation of rotavirus in MA104 cell line from diarrhoeic piglets of Assam. Indian J. Virol., 18(1): Chauhan RS and Singh NP (1993). Rotavirus infection in calves: Diagnosis by polyacrylamide gel electrophoresis. Indian J. Anim. Sci., 63(1): 1-3. Desselberger U and van Ranst M (2008). Recommendations for the classification of Estes M and Kapikian A (2007). Rotaviruses. In: Fields Virology, Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizmzn B (eds) 5 th edn. Kluver Health/ Lippincott, Williams & Wilkins, Philadelphia, pp Gentsch JR, Glass RI, Woods P, Gouvea V, Gorziglia M, Flores J, Das BK and Bhan MK (1992). Identification of group A rotavirus gene 4 types by polymerase chain reaction. J. Clin. Microbiol., 30: Gouvea V, Glass RI, Woods K, Taniguchi K, Clarke HF, Forrester B and Fang ZY (1990). Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J. Clin. Microbiol., 28: Herring AJ, Inglis NF, Ojelt CK, Snodgrass DR and James D (1982). Rapid diagnosis of rotavirus infection by direct detection of viral nucleic acid in Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: Malik YPS, Kumar N, Sharma K, Bora DP and Dutta TK (2014). Rotavirus diarrhea in piglets: A review on epidemiology, genetic diversity and zoonotic risks. Indian J. Animal Sci., 84(10): Matthijnssens J, Ciarlet M, Rahman M, Attoui H, Banyai K, Estes MK, Gentsch JR, Iturriza-Gomara M, Kirkwood CD, Martella V, Mertens PP, Nakagomi O, Patton JT, Ruggeri FM, Saif LJ, Santos N, Steyer A and Taniguchi K (2008). Recommendations for the classification of group A rotaviruses using all 11 genomic RNA segments. Arch. Virol., 153: Neog BK, Barman NN, Bora DP, Dey SC and Chakraborty A (2011). Experimental infection of pigs with group A rotavirus and enterotoxigenic Escherichia coli in India: gross, histopathological and immunopathological study. Vet Ital., 47: Saif LJ, Ward LA, Yuan L, Rosen BI and To TL (1996). The gnotobiotic piglet as a model for studies of disease pathogenesis and immunity to human rotaviruses. Arch Virol Suppl., 12: Sharma R, Bora DP, Chakraborty P, Das S and Barman NN (2013). Circulation of group A rotaviruses among neonates of human, cow and pig: study from Assam, a north eastern state of India. Indian J. Virol., 24: Svensson L, Uhnoo I, Grandien M and Wadell G (1986). Molecular epidemiology of rotavirus infections in Upsala, Sweden in (1981): Disappearance of a predominant electropherotype. J. Med. Virol., 18: Wani SA, Bhat MA, Qureshi S, Ishaq SM, Buchh AS, Samanta I and Ashrafi MA (2003). Epidemiology of diarrhea caused by rotavirus and E.coli in calves in Kashmir Valley. Indian Vet. J., 81: Journal of Immunology and Immunopathology 57