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Infection, Genetics and Evolution 25 (2014) 20–27
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Infection, Genetics and Evolution
journal homepage: www.elsevier .com/locate /meegid
Molecular analysis of non structural rotavirus group A enterotoxin geneof bovine origin from India
http://dx.doi.org/10.1016/j.meegid.2014.04.0041567-1348/� 2014 Elsevier B.V. All rights reserved.
Abbreviations: RVA, rotavirus group A; NSP4, nonstructural protein 4; RVs,rotaviruses; aa, amino acid; nt, nucleotide; CT, cytoplasmic tail; NCBI, The NationalCenter for Biotechnology Information; NJ, Neighbor Joining; SLAC, Single LikelihoodAncestor Counting; FEL, Fixed Effect Likelihood; MEME, Mixed Effects Model ofEvolution; FUBAR, Fast Unbiased Bayesian AppRoximation; NCDV, nebraska calfdiarrhea virus.⇑ Corresponding author. Tel.: +91 581 2302777; fax: +91 581 2301757.
E-mail addresses: [email protected] (Y.S. Malik), [email protected](N. Kumar), [email protected] (K. Sharma), [email protected](S. Ghosh), [email protected] (K. Bányai), [email protected](G. Balasubramanian), [email protected] (N. Kobayashi), [email protected] (J. Matthijnssens).
Yashpal Singh Malik a,⇑, Naveen Kumar a, Kuldeep Sharma a, Souvik Ghosh b, Krisztián Bányai c,Ganesh Balasubramanian d, Nobumichi Kobayashi e, Jelle Matthijnssens f
a Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, Indiab Department of Hygiene, Sapporo Medical University School of Medicine, S 1, W 17, Chuo-Ku, Sapporo, Hokkaido 060-8556, Japanc Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Hungária krt. 21, Budapest 1143, Hungaryd National Institute of Cholera and Enteric Diseases (NICED), Kolkata, West Bengal, Indiae Sapporo Medical University School of Medicine, Chuo-Ku, Sapporo, Japanf Laboratory for Clinical and Epidemiological Virology, KU Leuven, REGA Institute, Minderbroedersstraat 10, B3000 Leuven, Belgium
a r t i c l e i n f o a b s t r a c t
Article history:Received 30 January 2014Received in revised form 1 April 2014Accepted 4 April 2014Available online 16 April 2014
Keywords:Bovine rotavirusIndiaNonstructural protein 4GenotypesEvolutionSelection analysis
The rotavirus enterotoxin NSP4 (nonstructural protein 4), plays a pivotal role in viral morphogenesis aswell as pathogenesis. In this study, the NSP4 gene of rotavirus group A (RVA) isolates of bovine origin iso-lated in several states of India from 2008 to 2011 were characterized. The complete open reading frame of23 RVA strains were sequenced and analyzed phylogenetically. Genotype E1 was detected for the firsttime in bovines from India, in addition to the more common bovine genotype E2. Sequence similarityanalysis of the E1 sequences showed a close genetic relatedness to human strains. Six of the bovine E2genotypes strains clustered near bovine and unusual human strains (possible human animal reassortant)from Thailand, while the remaining E2 sequences clustered with Indian bovine strains. Analysis pointedout one positively selected site (154aa), believe to be part of an antigenic region and 123 negativelyselected sites. Unexpectedly, a pentameric NSP4 structure of the coiled coil domain in the E1 carryingstrains and a monomeric NSP4 in RVA strain P14 (E2) was predicted based on homology modeling, poten-tially affecting the biological properties of NSP4. The close relationship between bovine and human rota-virus strains further highlights the complex interaction among rotaviruses of different species.
� 2014 Elsevier B.V. All rights reserved.
1. Introduction stranded RNA, encoding six structural and six nonstructural
Group A rotaviruses (RVA) are a very common cause of diar-rheal diseases worldwide, causing a high morbidity and mortalityamong humans and huge economic losses in animal industry(Estes and Kapikian, 2007). Rotaviruses (RVs) belong to the familyReoviridae, and possess a genome of 11 segments of double
proteins. The identification of the nonstructural protein 4 (NSP4)as the first viral enterotoxin (Ball et al., 1996) has increased scien-tific interest to better understand its structure, sequence variabilityamong RVAs infecting different species and mechanism of action ininducing secretory diarrhea in suckling mice.
NSP4 is a 175 amino acids transmembrane, endoplasmic reticu-lum (ER) specific glycoprotein with pleotropic functions andcontains an uncleaved signal sequence at the N terminus followedby three hydrophobic domains namely H1 (7–21 aa), H2 (29–47aa) and H3 (67–85 aa) and a coiled a helical domain (95–137 aa)(Estes, 2001). The amino terminal region (1–44 aa) of the gene islocated in the lumen of the endoplasmic reticulum, whereas itscarboxyl terminal region (45–175 aa) constitutes the cytoplasmictail (CT) that exhibits all the known important biological propertiesassociated with the protein which include alteration of Ca2+
homeostasis by the release of Ca2+ from the endoplasmic reticulum(Dong et al., 1997; Hyser et al., 2010; Tian et al., 1994), membranepermeabilization (Newton et al., 1997), Ca2+ and VP4 binding
Y.S. Malik et al. / Infection, Genetics and Evolution 25 (2014) 20–27 21
(Estes and Kapikian, 2007), double layered particle interaction fortransport in the lumen of the endoplasmic reticulum for matura-tion into triple layered particles (Au et al., 1993; O’Brien et al.,2000) and diarrhea induction in newborn mouse pups (Ball et al.,1996; Horie et al., 1999; Jagannath et al., 2006).
At least 15 E genotypes (E1–E15) based on an 85% identity cutoff values for the NSP4 gene have been identified so far(Matthijnssens et al., 2011; Papp et al., 2012). The RVA genotypesE1 (Wa like), E2 (DS-1 like) and E3 (AU-1), previously known asgenotypes B, A and C, respectively have been detected in humansand animals (Matthijnssens et al., 2011). Each NSP4 genotypeappears to segregate more or less according to the RVA host species(Ciarlet et al., 2000) suggestive of predominance of a particulargenotype in a particular species. The rotavirus NSP4 sequenceshave been analyzed in more detail for human (Araújo et al.,2007; Ben et al., 2012; González Ochoa et al., 2013; Tavareset al., 2008) and avian species (Mori et al., 2002), but bovine spe-cies remains limitedly studied until now. In this study, 23 NSP4sequences of bovine RVA strains from India were sequenced toidentify their genotypes and sequence variability, and furthermorethe data were used to investigate selection/evolution pressure andstructure prediction using homology modeling.
2. Materials and methods
2.1. Sample collection and preparation
A total of 23 RVA positive diarrheic faecal samples collectedduring active surveillance for the presence of enteric viruses withinthe bovine (cattle and buffalo) population of India from 2008 to2011, were analyzed in this study (Table 1). The faecal sampleswere processed by making 10% faecal suspension (w/v) in phos-phate buffered saline (0.01 M, pH 7.4; Sigma, USA) followed bycentrifugation at 2000�g for 10 min and the filtration of upperaqueous layer through 0.22 lm syringe filter (MDI, India). The fil-trates were archived and stored at �20 �C until further use.
2.2. Extraction of viral RNA and reverse transcription polymerasechain reaction
The viral RNA extraction and cDNA synthesis were carried outas per the method described earlier (Malik et al., 2012). The full
Table 1Bovine rotavirus NSP4 sequences considered in this study from different geographical reg
S. No. Rotavirus strains Place of isolation RotaC2.0 ba
1 RVA/Buffalo-wt/IND/B54/2008/G3P[8] Haryana E12 RVA/Buffalo-wt/IND/B111/2009/GXP[X] Haryana E23 RVA/Buffalo-wt/IND/BRV133/2010/G3P[11] Haryana E24 RVA/Cattle-wt/IND/CC156/2010/G3P[X] Haryana E25 RVA/Cattle-wt/IND/B108/2011/GXP[X] Haryana E26 RVA/Buffalo-wt/IND/B47/2008/GXP[X] Madhya Pradesh E27 RVA/Buffalo-wt/IND/B48/2008/G3P[3] Madhya Pradesh E28 RVA/Buffalo-wt/IND/B68/2008/G3P[X] Madhya Pradesh E29 RVA/Buffalo-wt/IND/B72/2008/G10P[1] Madhya Pradesh E210 RVA/Buffalo-wt/IND/B100/2008/G3P[3] Madhya Pradesh E211 RVA/Cattle-wt/IND/C51/2008/GXP[X] Madhya Pradesh E212 RVA/Cattle-wt/IND/C1/2010/GXP[X] Madhya Pradesh E213 RVA/Buffalo-wt/IND/B212/2010/GXP[X] Madhya Pradesh E114 RVA/Cattle-wt/IND/MF10/2010/G3P[3] Madhya Pradesh E215 RVA/Cattle-wt/IND/15E/2009/GXP[1] Uttarakhand E216 RVA/Cattle-wt/IND/P9/2009/G3P[1] Uttarakhand E117 RVA/Cattle-wt/IND/P14/2009/G3P[1] Uttarakhand E218 RVA/Cattle-wt/IND/P970/2009/G3P[1] Uttarakhand E219 RVA/Cattle-wt/IND/C5/2010/GXP[X] Uttarakhand E220 RVA/Cattle-wt/IND/Bov1/2009/G3P[1] Uttar Pradesh E221 RVA/Buffalo-wt/IND/Bov2/2009/G3P[1] Uttar Pradesh E222 RVA/Buffalo-wt/IND/BE3/2010/G6P[X] Uttar Pradesh E223 RVA/Cattle-wt/IND/H6/2011/GXP[X] West Bengal E2
length NSP4 gene (743 bp) was amplified using degenerateprimers: (NSP4(1–28)[+] GGCTTTWAAAAGTTCTGTTCCGAGAGAG;NSP4(722–743)[�] TAAGACCRTTCCYTCCATTAAC) and the reactionwas performed at initial denaturation at 95 �C for 5 min followedby 35 cycles of 30 s at 95 �C, 30 s at 55 �C, and 90 s at 72 �C, andfinal extension at 72 �C for 5 min. The PCR amplicons were gelpurified using the GeneJet Gel Extraction Kit (Fermentas, EU),cloned into the pGEMT Easy Vector cloning system (Promega)and sequencing was outsourced to M/S SciGenom Labs Pvt. Ltd.,Kerala, India.
2.3. Sequence and phylogenetic analysis
The NSP4 sequences used in this study (n = 23) along with otherNSP4 sequences from India and across the world were retrievedfrom The National Center for Biotechnology Information (NCBI)(http://www.ncbi.nlm.nih.gov/). Sequences were aligned by Clu-stalW and a dendrogram was constructed in MEGA5 (Tamuraet al., 2011) by Neighbor Joining (NJ) statistical method using theMaximum Composite Likelihood substitution model with 2500bootstrap replicates. The nucleotide and deduced amino acidsequence identities of NSP4 genes were analysed with NSP4sequences of rotaviruses from different species and geographicallocations published in GenBank database. Furthermore, the RotaCv2.0 web based tool for RVA classification (http://rotac.rega-tools.be) was used to determine the sequence E genotypes (Maeset al., 2009).
2.4. Selection pressure analysis
Site specific selection pressure was measured on gene segment10 using the HyPhy software implemented in the Datamonkey webserver (Kosakovsky Pond and Frost, 2005a). In this study, a total of119 NSP4 coding sequences of bovine rotavirus A (96 sequencesretrieved from NCBI and 23 sequences of present study) werechosen and this server accepted 80 sequences by declining the sim-ilar/duplicate sequences (Supplementary data 1). The sites underselection pressure were evaluated using classic maximum likeli-hood methods i.e. Single Likelihood Ancestor Counting (SLAC)model and Fixed Effect Likelihood (FEL) model (Kosakovsky Pondand Frost, 2005b) and other more recently developed methods;Mixed Effects Model of Evolution (MEME) capable of identifying
ions of India.
sed genotyping Amplicons size (bp) GenBank accession Nos. References
534 HQ433436 Unpublished554 JF831958 Unpublished568 HQ157345 Unpublished519 HQ157349 Unpublished500 JX442776 Unpublished724 HM591492 Unpublished724 HM591493 Unpublished739 HQ433435 Unpublished549 HQ157346 Unpublished724 HM591494 Unpublished566 HQ157350 Unpublished677 JF831956 Unpublished678 JF831954 Unpublished517 JX442768 Unpublished737 HQ171910 Unpublished744 JF689836 Unpublished534 HQ157344 Unpublished568 HQ157348 Unpublished726 JF831947 Unpublished726 JF689837 Unpublished663 JF831952 Unpublished497 JF831957 Unpublished573 JX442789 Unpublished
22 Y.S. Malik et al. / Infection, Genetics and Evolution 25 (2014) 20–27
both episodic and pervasive positive selection (Murrell et al.,2012), and the Fast Unbiased Bayesian AppRoximation (FUBAR)method that can detect positive selection under a model fasterthan the existing fixed effects likelihood models through the intro-duction of an ultrafast Markov chain Monte Carlo (MCMC) routineand that allows to visualize Bayesian inference for each site(Murrell et al., 2013). All these methods were employed usingthe best nucleotide substitution model, 010010 (HKY85 model),chosen by Akaike Information Criterion (AIC) on a defined Neigh-bor Joining (NJ) phylogenetic tree after running GARD (GeneticAlgorithm for Recombination Detection) to detect recombination(Kosakovsky Pond et al., 2006). The strength of selection pressurewas determined on the basis of the ratio of nonsynonymous (dN)to synonymous (dS) substitutions per site (x = dN/dS). To avoid ahigh false positive rates, due to the reduced number of sequences,sites with P values <0.1 for SLAC, FEL and MEME models, and aposterior probability >0.90 for FUBAR were accepted as candidatesfor selection.
2.5. Homology modeling of a helical coiled coil domain
To determine the three dimensional protein structure models,homology modeling was performed using SWISS MODEL(Schwede et al., 2003). The consensus of both genotypes E1(n = 3) and E2 (n = 20) were BLASTed to identify the most closelyrelated templates and best fitter model was selected based onstructure assessment and model quality estimation parameters.The global indicator quality model i.e. Z score QMEAN (Benkertet al., 2008) (a composite score consisting of a linear combinationof 6 model reliability parameters i.e. Cb interaction energy, Allatom pairwise energy, Solvation energy, Torsion angle energy, Sec-ondary structure agreement, Solvent accessibility agreement) and
Fig. 1. Deduced amino acid sequences comparison of rotavirus enterotoxin cytoplasmicthis study. Particular changes at amino acid positions among E1 & E2 rotavirus NSP4 genmembrane destabilizing domain; TD, tetramerization domain; IBD, integrin binding dtransducing domain; ISVD, inter-species variation domain; DLP binding domain, d(For interpretation of the references to colour in this figure legend, the reader is referre
local indicators [Anolea mean force potential (Melo andFeytmans, 1998) and Gromos empirical force field energy (vanGunsteren et al., 1996)] were carefully analyzed. The Z scoreQMEAN value ranges between 0 and 1 with higher values for bettermodels. Based on all these parameters, models were constructedfor a helical coiled coil domain using the best fitted templatesfor genotype E1 (PDB: 3miw); E2 and the unusual E2 variant strainP14 (PDB: 1g1i).
The online server for B cell epitope prediction of Iowa State Uni-versity i.e. BCPREDS Server 1.0 (http://ailab.cs.iastate.edu/bcpreds/predict.html) was used to identify putative linear epitopes withinthe NSP4 protein by fixed length epitope prediction method withepitope length of a 20-mer and specificity of 90%.
3. Results
3.1. NSP4 sequences comparison and phylogenetic analysis
On sequencing the complete ORFs of 23 RVA strains used in thisstudy, 20 belonged to genotype E2 and 3 to the E1 genotype basedon the web server tool, RotaC v2.0. Bovine RVA strains with an E1NSP4 genotype have never been reported in India before thisstudy; and only a single bovine RVA strain with an E1 was availablein NCBI database for a South Korean bovine RVA strain (GenBank:JX971578) which is believe to be an interspecies transmitted virusfrom porcine origin (Park et al., 2013). Comparing the amino acidsequences of the genotype E1, E2 and bovine reference strain NCDVrevealed point changes at nineteen positions distributed in variousbiological functional domains (marked with a red box in Fig. 1).However, based on the amino acid sequence alignment of E2strains, 3 main variants were observed, represented by Bov1, B72and P14. Sequence similarity analysis showed that the two bovine
tail with representation of biologically functional domains of 23 RVA strains used inotype isolates and reference strains are highlighted in colored boxes. PMDD, plasmaomain; Ca2+ & DIR, Ca2+ binding and diarrhea inducing region; Signal TD, signalouble layered particle binding domain; E1 & E2, Rotavirus NSP4 Genotypes.d to the web version of this article.)
Y.S. Malik et al. / Infection, Genetics and Evolution 25 (2014) 20–27 23
E1 RVA strains B54 and B212 were found most closely related tohuman strain VU08 and human reference strain Wa, both isolatedin the USA (identities of 96.4–98.3% at nucleotide and 93.8–96.8%at amino acid level) while a third E1 strain (P9) was most closelyrelated to a single bovine (B85) and several human rotavirusstrains from India, France and the USA (Fig. 2). When these threestrains when compared with porcine like RVA strains with a geno-type E1 isolated from bovine faecal samples (K5) in South Korea,
Fig. 2. Phylogenetic tree based on nucleotide sequences of bovine rotavirus NSP4 used idifferent host species. Construction of the tree was based on NJ method. The numbers atbar indicates the estimated 0.05 nt substitutions per site.
sequence similarity ranged from 92.4% to 94.9% at nucleotide and90.3–93.1% at the amino acid level.
Among the bovine E2 genotypes, six strains (B47, B48, Bov1,Bov2, B100, MF10) clustered closely with bovine and unusualhuman E2 genotype strain (partially of animal origin) fromThailand (CMH049) and India (BE1) with high identities of98.7–99.6% at the nucleotide and 97.7–98.8% at the amino acid lev-els, while the remaining E2 genotypes clustered with Indian bovine
n this study (indicated as black dots) along with other reference NSP4 sequences ofthe nodes indicate percentages of bootstrap support from 2500 replicates. The scale
24 Y.S. Malik et al. / Infection, Genetics and Evolution 25 (2014) 20–27
E2 genotype carrying RVA strain with sequence similaritiesbetween 97.9–100% at the nucleotide and 97.7–100% at the aminoacid levels (Fig. 2). The bovine strain P14 clustered with Indianbovine E2 genotype strains, such as strain 79, but with lower iden-tities of 94.2% at nucleotide and 90.0% at amino acid levels.
3.2. Selection in NSP4 genes
To increase the robustness of the selected sites, we applied fourmethods (SLAC, FEL, MEME, and FUBAR) on a total of 80 sequencesof bovine E1 and E2 genotypes. Sites with x > 1 and p values <0.1for SLAC, FEL and MEME models and a posterior probability >0.90for FUBAR were accepted as candidates for positive selection. Themean ratio of nonsynonymous (dN) to synonymous (dS) substitu-tions per site by SLAC method was 0.10. We identified one positive(position 154) and 123 negative (70% codons) selection sites basedon the site by site analysis by at least two methods (Supplementarydata 2). At position 154, the most common residue was polar posi-tive arginine, found in 52.1% of the NSP4 protein sequencesfollowed by polar positive lysine (47.9%) in the rest of thesequences.
3.3. a helical coiled coil domain analysis
We compared the heptad repeat sequences from the consensussequence of the Indian bovine E1 genotype strains with that of theconsensus of all three E2 variants and the bovine reference strain,NCDV. The a helical coiled coil domain representation throughhelical wheel model denoted as (a–g)n was constructed by Draw-Coil v1.0, where ‘a’ and ‘d’ are typically nonpolar core residuesfound at the interface of the two helices, whereas ‘e’ and ‘g’ are sol-vent exposed, polar residues that give specificity between the twohelices through electrostatic interactions (Grigoryan and Keating,2008).
Comparison of the three helical wheel projections (Fig. 3)revealed that R137 (polar positive) at position ‘d’ of helical wheelin E1 and NCDV, is replaced by Q137 (polar neutral) in E2 andP14 while in E2 and NCDV, polar positive aa (H131 at ‘e’; K133at ‘g’) are replaced by polar neutral residues in E1 (Y131 at ‘e’;
Fig. 3. Helical wheel projections of a helical coiled coil domain (95137aa) of NSP4s fromE2 and the genotype E2 variant P14. Each heptad (abcdefg)n of NSP4s starts at isoleucineand E1 genotype) or glutamine (E2 and P14) at 137aa in the d-positions. Single letter amblue (polar positive), yellow (polar neutral), and black (nonpolar neutral) and aminoreferences to colour in this figure legend, the reader is referred to the web version of th
N133 at ‘g’). The strain P14 differed from all these three strainsin having a nonpolar amino acid (P131) at ‘e’.
Further to analyze the effects of these changes on the threedimensional structure of the coil, we performed homology model-ing with the templates retrieved from the PDB (Protein Data Bank)after choosing the best fitted model based on the Z score QMEAN.Interestingly, three different oligomeric structures (pentameric inE1, tetrameric in E2 and monomeric in E2 variant P14) werepredicted by the model (Fig. 4).
4. Discussion
Studies on various gene segments of RVA are increasing; how-ever, segment 10 which encodes the viral enterotoxin, particularlyin bovines remains unexplored. In this study, the prevalence ofgenotypes, sequence variability, selection/evolution pressure andmutational substitutions within 23 NSP4 genes of bovine RVAisolates were studied. The sequence analysis grouped the bovineNSP4 genes of 23 RVA strains into two genotypes, E1 and E2. TheE1 genotype in bovines was detected for the first time from India,showing a prevalence of 13% in addition to the most frequentlydetected genotype in bovine, E2 (83%). No particular genotype seg-regation was found with respect to the host species (buffalo orcattle) or Indian states (data not shown).
All the three E1 genotype strains and six of the E2 genotypesequences of this study clustered with E1 human and E2 human/bovine genotypes (CMH049; possible reassortant with animalstrains), respectively. On looking over the genetic background ofsix of the E2 genotype sequences and CHM049 strain, which wereG3 and G2 respectively, both believed to be of bovine-human reas-sortant types and needs further investigations on origin of thesestrains. The frequent movement of people across the stategeographical boundaries and close proximity of humans and ani-mals (Mukherjee et al., 2010, 2011) also play an important rolein emergence of such viruses, providing evidence that evolutionof animal rotaviruses is tightly intermingled with that of humanrotaviruses.
The selection pressure analysis acting on gene segment 10 ofbovine RVAs identified one positively selected site (position 154
bovine reference strain (NCDV), Indian bovine genotypes E1, Indian bovine genotypeat position 95aa in all the four strains at the d-positions and ends at arginine (NCDVino acid codes are colored based on the polarity and charge, i.e. red (polar negative),acid change at particular positions are also highlighted. (For interpretation of theis article.)
Fig. 4. Protein structure homology modeling of bovine rotavirus NSP4 protein’s a helical coiled coil domain originated from this study using SWISS MODEL. Pentameric (A),Tetrameric (B) and Monomeric (C) a helical coiled coil domains of consensus Genotype E1, E2 and E2 variant P14. Inaccuracy per residue is depicted using a color gradientfrom blue (more reliable) to red (potentially unreliable). Every protein structure is sidelined with their respective QMEAN6 or Z scores. The pseudo-energies of thecontributing parameters are also given with their Z scores with respect to scores obtained for high resolution experimental structures of similar size solved by X raycrystallography. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Y.S. Malik et al. / Infection, Genetics and Evolution 25 (2014) 20–27 25
aa). Except for an immunodominant region containing conforma-tional epitope (135–175 aa) identified by single chain variablefragment (scFv) antibodies generated by phage display(Rodriguez Diaz et al., 2004), the epitopes on the enterotoxin pro-tein are not well defined so far. Epitopes are generally enrichedwith tyrosine and tryptophan with charged polar amino acidsand thereby preferred in protein protein interfaces due to theircapability to form a multitude of interactions (Bogan and Thorn,1998; Jackson, 1999; Rubinstein et al., 2008). Computational anal-ysis of epitope prediction revealed that the location of this posi-tively selected site falls within a predicted linear B cell epitope
(152–172 aa) which could explain why this site is under a positiveantibody pressure.
The mean dN/dS for bovine rotaviral NSP4 gene was 0.10 (SLACmethod) which is more compared to NSP2 gene (0.08 for N1 geno-type and 0.05 for N2 genotype) and lower than VP7 gene (0.17)(Donker and Kirkwood, 2012; Pybus et al., 2007). The NSP4 proteinis under strong negative selection pressure suggesting its impor-tant role in maintaining the long term stability of biologicalfunctional domains by removing deleterious mutations. In general,the nonstructural proteins whose functions is associated with theviral replication or pathogenesis need to remain conserved to
26 Y.S. Malik et al. / Infection, Genetics and Evolution 25 (2014) 20–27
maintain their biological functions and are thereby expected to beunder negative selection. Conversely, the proteins on the outercapsid of a virus are exposed constantly to the host immunesystem, and are therefore likely under positive selective pressureto evade the host immune response (Loewe, 2008).
Since the discovery of the first viral enterotoxin, its diarrheainducing domain has fascinated a lot of researchers to work on itexhaustively. We investigated heptad repeats spanning residues95–137 suggestive of oligomeriza tion via a helical coiled coilinteraction (Bowman et al., 2000; Taylor et al., 1996) and alsocontaining the diarrhea inducing domain within it. Interestingly,a pentameric NSP4 structure of the bovine E1 genotype and amonomeric structure of the P14 strain were predicted, rather thanthe more common tetrameric structures using homology model-ing. The crystal structure of a pentameric NSP4 complex of anasymptomatic human strain, ST3 (genotype E1) has been revealedrecently (Chacko et al., 2011). The initially described pentamericNSP4 structure was from asymptomatic human RVA strain,whereas in this study, these strains also produced diarrhea inbovines despite having the pentameric structure.
In conclusion, our study identified the RVA genotype E1 for thefirst time from the Indian bovine population and the existence ofthree variants of genotype E2 on the basis of amino acid sequencecomparison. The findings further add to the diversity of existingcirculating RVA genotypes of NSP4. Homology based pentamericNSP4 structure of the coiled coil domain in E1 carrying strains(B54, B212 and P9) isolated from diarrheic cattle calves from Indiacreated interest since this pentameric structure has been describedfrom the asymptomatic humans only. Therefore, detailed analysisof these three E1 carrying strains and P14 strain (E2) would be ofinterest to investigate if these predicted coiled coil domain struc-tures exist in vivo and could be associate with differences inbiological activity.
Acknowledgment
The authors wish to thank Director, Indian Veterinary ResearchInstitute (IVRI), Izatnagar, India for the financial and infrastructuralsupport.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.meegid.2014.04.004.
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