Molecular Characterization of VP4 and NSP4 Genes from Rotavirus Strains Infecting Neonates and Young Children in Belem,´ Brazil Joana D’Arc P

Virus Research 126 (2007) 149–158

Molecular characterization of VP4 and NSP4 genes from rotavirus strains infecting neonates and young children in Belem,´ Brazil Joana D’Arc P. Mascarenhas a,∗, Alexandre C. Linhares a, Yvone B. Gabbay a, Clarissa S. Lima a, Sylvia de Fatima´ S. Guerra a, Luana S. Soares a, Darleise S. Oliveira a, Jackson C. Lima b, Olinda Macedoˆ a, Jose´ Paulo G. Leite c a Se¸cão de Virologia, Instituto Evandro Chagas, Secretaria de Vigilância em Saúde, Ministério da Saúde, Rodovia BR 316 KM 07, S/N, Levilândia, 67.030-000 Ananindeua, Pará, Brazil b Departamento de Informática em Saúde, Universidade Federal de São Paulo, São Paulo, Brazil c Departamento de Virologia, Instituto Oswaldo Cruz, Funda¸cão Oswaldo Cruz, Rio de Janeiro, Brazil Received 10 November 2006; received in revised form 8 February 2007; accepted 10 February 2007 Available online 21 March 2007

Abstract Several reports have identified P[6] specificities in humans and in animals in different countries of the world, but few sequence data are available in public databases. In this work we have characterized the VP4 strains bearing P[6] specificity and NSP4 genotypes among diarrheic young children and diarrheic and non-diarrheic neonates from three studies previously conducted in Belem,´ Northern region of Brazil. As the to VP8* fragment, we observed a close relationship to both human prototypes of lineage P[6]-Ia (bootstrap of 99%) and porcine sublineages Ib and Ic (89.2–98.1% aa similarity and mean of 95%). With regards to the NSP4, the samples clustered into genotypes A and B. Of note, of the 27 P[6] strains analyzed in the present study and classified as genotype B, 8 (29.6%) were more similar to porcine prototypes when VP8* and NSP4 genes are compared, and were recovered, one from a neonate and seven from diarrheic children. These preliminary findings reinforce that further investigations are needed to assess the relative frequencies of P[6] strains in our region, as well as to investigate the potential for interspecies transmission involving humans and animals, particularly pigs. © 2007 Elsevier B.V. All rights reserved.

Keywords: P[6] genotype; Neonates; Diarrheic children; Interspecies transmission

1. Introduction segments of double-stranded RNA (dsRNA), which encodes six structural (VP1–VP4, VP6 and VP7) and six non-structural Worldwide, group A human rotaviruses (HRV) are important (NSP1–NSP6) proteins (Estes, 2001; Kapikian et al., 2001). Pro- cause of severe gastroenteritis among infants and young chil- teolytic cleavage of VP4 generates two smaller polypeptides dren, as well as in young animals of a wide variety of species designated VP5* and VP8*. Antigenic analyses have demon- (Kapikian et al., 2001). The Rotavirus comprises a single genus strated that the VP8* subunit possesses the major epitopes within the Reoviridae family with a genome consisting of 11 responsible for serotype specificity (Estes and Cohen, 1989). Human group A comprises at least 27 P and 15 G genotypes coding by VP4 and VP7 genes, respectively (Estes, 2001; Kapikian ∗ Corresponding author. Tel.: +55 91 32142016; fax: +55 91 32142006. et al., 2001; Khamrin et al., 2007; Martella et al., 2006b; Rahman E-mail addresses: [email protected] (J.D.P. Mascarenhas), [email protected] (A.C. Linhares), et al., 2005; Rao et al., 2000; Steyer et al., 2007). [email protected] (Y.B. Gabbay), The rotavirus non-structural protein NSP4, encoded by gene [email protected] (C.S. Lima), segment 10, has 175 amino acids (aa) in length. NSP4, specifi- [email protected] (S.d.F.S. Guerra), cally 22 aa corresponding to residues 114–135, appears to induce [email protected] (L.S. Soares), diarrhea in young mice and may be a key determinant of rotavirus [email protected] (D.S. Oliveira), NSP4 [email protected] (J.C. Lima), pathogenicity (Ball et al., 1996; Tian et al., 1995). The [email protected] (O. Macedo),ˆ gene has been classified into groups A, B and C. Group A com- jpgleite@ioc.fiocruz.br (J.P.G. Leite). prises five genotypes: A (KUN), B (Wa), C (AU-1), D (EW) and

E (avian-like) (Ciarlet et al., 2000; Ito et al., 2001; Mori et al., al., 2001, 2002; Banyai´ et al., 2004; Fang et al., 2002; Laird et al., 2002). 2003; Leite et al., 1996; Mascarenhas et al., 2002, 2006, 2007; Rotavirus strains bearing P[6] genotype represent one of the Page and Steele, 2004; Salu et al., 2003; Santos et al., 1994; three most common VP4 specificities associated with human Steele and Ivanoff, 2003; Steele and Sears, 1996; Timenetsky et infections (Santos and Hoshino, 2005). Originally, the M37 al., 1994; Volotao˜ et al., 2006). and RV3 prototypes with P[6] serotype have been associated Although several reports have documented P[6] specificity with avirulent neonatal strains recovered from asymptomatic and it has been identified in humans and animals in different neonates (Flores et al., 1986; Hoshino et al., 1985), and was countries of the world, few sequence data are available in public the basis for developing candidate vaccines (Barnes et al., 1997, databases (Banyai´ et al., 2004). These studies have been sup- 2002; Flores et al., 1990; Midthun et al., 1991; Vesikari et al., ported by phylogenetic analysis and suggest that interspecies 1991). transmission may occur (Martella et al., 2006a; Rahman et al., Worldwide, P[6] genotype strains have been recovered from 2003). diarrheic and non-diarrheic neonates (Cunliffe et al., 2002; In this study we analyzed VP4 and NSP4 genes from Linhares et al., 2002; Pager et al., 2000; Steele et al., 1992, 27 P[6] rotavirus strains obtained from diarrheic and non- 1995) and young children (Adah et al., 1997, 2001; Araujo´ et diarrheic neonates and diarrheic young children during 1990

Fig. 1. Characteristics of VP4 P[6] rotavirus isolates from three surveys conducted in Belem,´ Brazil. Study A, Surveillance for young children participating in a trial with a reassortant tetravalent vaccine against rotavirus (RRV-TV) conducted from 1990 to 1992 (Linhares et al., 1996; Mascarenhas et al., 2002); Study B, surveillance for rotavirus infection in a neonatal care unit wards at a public hospital in Belem,´ Brazil, conducted from 1996 to 1998 (Linhares et al., 2002; Mascarenhas et al., 2006, 2007); Study C, surveillance for hospitalized diarrheic young children and those attending an outpatient heath unit conducted from May 1998 to May 2000 (Gabbay et al., 2002; Mascarenhas et al., 2003). J.D.P. Mascarenhas et al. / Virus Research 126 (2007) 149–158 151 and 2000, from studies previously conducted in Belem,´ Northern Prism 3100 automatic sequencer (Applied Biosystems, Foster Brazil. City, CA, USA). The sequences obtained for the VP4 and the NSP4 genes were assembled and analyzed with the BioEdit 2. Materials and methods Sequence Alignment Editor (Version 7.0.5.2). A phylogenetic analysis was performed using MEGA package (Version 3.1). 2.1. Patients and specimens Distances between sequences were analyzed using the neighbor- joining algorithm based on the Kimura two-parameters distance All rotavirus samples used were obtained from three studies estimative method for nucleotide (Kimura, 1980). Bootstrap previously conducted in Belem,´ Brazil, where P[6] genotypes resembling (over 2000 replicates). could be determined. The Study A involved diarrheic young Prototype strains for VP4 and NSP4 genes were obtained from children with ages ranging from 9 to 24 months, who partici- GenBank at the National Center for Biotechnology Information, pated in a trial with the Rhesus-human reassortant tetravalent USA (http://www.ncbi.nlm.nih.gov), through the conducting of rotavirus vaccine (RRV-TV), from 1990 to 1992 (Linhares et Blast and nucleotide search. al., 1996; Mascarenhas et al., 2002). The Study B was a surveillance for rotavirus infection from May 1996 to May 1998 among 3. Results neonates admitted to a hospital in Belem,´ Brazil (Linhares et al., 2002; Mascarenhas et al., 2006, 2007). The Study C was con- 3.1. Characterization of rotavirus strains by PAGE, PCR ducted among hospitalized young children with diarrhea and and sequence analysis those attending an outpatient health unit in a 2-year surveillance study in Belem,´ Brazil, conducted from May 1998 to May Fig. 1 shows the characteristics of 27 P[6] VP4 strains, includ- 2000 (Gabbay et al., 2002; Mascarenhas et al., 2003). Fig. 1 ing corresponding RNA profile, VP7 and NSP4 specificities. The summarizes the main characteristics of these studies. Overall, three strains from Study A were genotyped as G4 and NSP4 B. 27 samples were available in sufficient amount and were then Sixteen out of 17 strains from Study B displayed short electro- selected from the original studies, as follows: 3 from the Study pherotype and were genotyped as G2 and NSP4 A. A more A, 17 from Study B and 7 from Study C. The neonates and wide variation in electropherotypes, G and NSP4 genotypes diarrheic children lived in the outskirts of Belem,´ Brazil, under was observed in the Study C, which showed G2, G4 and G9 poor sanitation conditions and in close contact with domestic associated with either NSP4 A or B. animals, including pigs. 3.2. Comparison of amino acid sequence of VP8* gene 2.2. RT-PCR amplification of the VP4, VP7 and NSP4 from rotaviruses P[6] genotypes genes A phylogenetic tree was constructed using the deduced aa Rotavirus dsRNA was extracted from 10% fecal suspensions sequences of the VP4 protein (270 aa), corresponding to aa by using guanidinium isothiocyanate-silica nucleic acid extrac- 13–282 of the coding region of the gene (Fig. 2). Of the 27 P[6] tion, as described previously (Boom et al., 1990), including strains analyzed to VP8*, 26 clustered with the M37-like P[6]- modifications of Araujo´ et al. (2001). Polyacrylamide gel elec- I genetic lineage (bootstrap value 91%), with 19 strains being trophoresis (PAGE) was carried out in Tris–glycine buffer and more similar to human prototypes of sublineage P[6]-Ia (boot- rotavirus genome profile was defined following electrophoresis strap of 99%), with aa similarity ranging from 90.7 to 98.8%. of extracted dsRNA through vertical 5% acrylamide bisacry- The seven remaining strains fell into a distinct group and they did lamide gels (Pereira et al., 1983). The reverse transcription not group specifically with any of the four representative sub- was carried out using SuperScriptTM (Invitrogen, Carlsbad, CA, lineages proposed by Martella et al. (2006a). These P[6] strains USA) and the cDNA was amplified to generate fragments of 876 were more similar to porcine sublineages Ib and Ic (89.2–98.1% and 738 bp corresponding to gene-encoding the VP4 and NSP4 aa similarity and mean of 95%), rather than to human sublineage proteins, respectively. Used primer sets were Con2/Con3 and Ia (84.4–93.7% and mean of 91%). The strain HST-327 did not Jrg30/Jrg31, as described by Gentsch et al. (1992) and Cunliffe cluster with any of the lineages analyzed, showing mean sim- et al. (1997), respectively. The G genotyping was carried out ilarity of 90% (lineage IV), 88% (lineage V), 88% (lineage I), as described by Das et al. (1994). All PCR products to VP4 83% (lineage II) and 85% (lineage III). and NSP4 were purified using QIAquick® PCR purification kit Analysis of the aa sequences revealed several aa residues con- (Qiagen, Hilden, Germany). served within the P[6]-I human and animal strains but differed from the lineages II, III, IV and V (Fig. 3). Synapomorphisms 2.3. DNA sequencing and phylogenetic analysis (shared-derived aa substitutions) were observed among the P[6]- I strains in residues 105-L, 134-T, 189-S and 196-V. In addition, The purified PCR products were automatically sequenced by several residues were completely conserved only within the using the Con3/Con2 (VP4 gene) and jrg30/jrg31 (NSP4 gene). P[6]-Ia human strains in residues 91-V, 98-K, 101-I, 147-V, the Sequencing was carried out by the dideoxynucleotide chain ter- triplet YNS at positions 170–172 and 202-V. HST-327 strain minator method, using the ABI Prism Big Dye Terminator Cycle showed synapomorphisms with P[6] human and animal strains Sequencing Ready Reaction Kit (Applied Biosystems) on a ABI in residues 30-N (lineages IV and V), 51-G (IV), 112-T (V), 152 J.D.P. Mascarenhas et al. / Virus Research 126 (2007) 149–158

129-S (III), 189-T (II, III and V) and 196-I (III, IV, V) (data not shown).

3.3. Comparison of amino acid sequences with NSP4 genotypes

Fig. 4 shows that genotypes A and B formed distinct groups with bootstrap values of 100 and 99%, respectively. However, genotype B was subdivided in two groups related to human and porcine prototypes and this made it difficult to analyze the divergence these samples. All genotype B samples, including seven samples recovered from diarrheic children and one from a diarrheic neonate, were more closely related to porcine prototypes. The 19 remaining samples were gathered into genotype A, showing to be closely related to human rotaviruses. Three of these were recovered from diarrheic children, four from diarrheic neonate and twelve from non-diarrheic neonates. Nev- ertheless, when a sequence of human origin from this genotype was analyzed, a slight divergence (4%) could be seen between asymptomatic prototypes (M37, RV3 and ST3) and symptomatic Wa, symptomatic children (5%), symptomatic neonates (4%) and asymptomatic neonates (4%). Among the P[6] strains classified as genotype A, it was not observed a significant difference between diarrheic and non- diarrheic strains when symptomatic prototypes (RV5, S2, KUN and DS-1), were compared with symptomatic and asymptomatic neonates (both 4%) and symptomatic children (3%). Of note, of the 27 P[6] strains analyzed in the present study and classified as genotype B, 8 (29.6%) were more similar to porcine prototypes when VP8* and NSP4 genes are compared, and were recovered, one from a neonate and seven from diarrheic children.

3.4. Lack of correlation between sequence changes and expression of symptoms

With regards to functional sites of the NSP4 gene, samples with P[6] of genotype B differed from genotype A because of alterations (D–S/N) in the second site of glycosylation at aa 19. In the domain H3, changes were observed (I/L–V) and (I–F/L) at aa 68 and 76, respectively. When the toxic peptide was analyzed, Fig. 2. Phylogenetic analyses based on the VP8* amino acid sequences changes were observed (H–Y) and (I/A/V–M/T) at aa 131 and of the P[6] strains using on the neighbor joining method within MEGA 135, respectively. Changes of (P/T–S) were also observed in aa * package. The sequences data reported in this paper to VP8 were submitted 138 of the region of linking of VP4 and of (S–A) in region of to GenBank with accession numbers (DQ525192–DQ525201). The references under comparison were ST3 (L33895), M37 (L20887), RV3 (U16299), 1076 linking of VP6 at aa 174 (Fig. 5). (P11198), US1205 (AF079356), MW23 (AJ278253), 134-04-10 (AY955299), Five aa differences were identiﬁed as shown in aa 16 (S–L); 134-04-11 (AY955300), 221-04-13 (AY955302), 221-04-7 (AY955303), 134- aa 60 (A–T); aa 124 (I–V); aa 146 (M–K); aa 162 (R–G), but 04-8 (AY955301), 51-04 (AY955306), 51-02 (AY955304), 51-03 (AY955305), any aa changes were noted when comparing P[6] strains from BP1338-00 (AJ621507), BP271-00 (AJ621502), BP720-93 (AJ621503), diarrheic and non-diarrheic neonates and those from diarrheic 134-04-7 (AY955310), BP1227-02 (AJ621505), 221-04-21 (AY955309), 221-04-19 (AY955307), 221-04-20 (AY955308), Gottfried (M33516), young children (Fig. 5). RMC321 (AF523677), DS-1 (AJ540227), Wa (L34161), NB-140 (DQ070447), NB-150 (DQ299877), NB-156 (DQ070450), NB-162 (DQ070452), NB- 4. Discussion 171 (DQ070454), NB-174 (DQ070455), NB-175 (DQ070456), NB-189 (DQ070457), NB-208 (DQ070460), NB-211 (DQ070462), NB-233 In our study, the P[6] genotype has been detected using the 3T- (DQ070464), NB-242 (DQ070465), NB-244 (DQ822476), NB-287 (DQ070467), NB-301 (DQ070468), NB-308 (DQ070469) and NB-321 1 primer described by Gentsch et al. (1992) which is capable of (DQ070470). recognizing M37-like P[6]. In this study, all samples were ampli- ﬁed and sequenced using this primer which has been described J.D.P. Mascarenhas et al. / Virus Research 126 (2007) 149–158 153

Fig. 3. Multiple sequence alignment of the human rotavirus P[6] strains with the P[6] human rotaviruses RV3, ST3, M37, US1205 and P[6] porcine rotavirus Gottfried and other P from human and animal rotaviruses. Dots indicate similarity to ST3 human prototype and (?) indicate sequence not determined. in other studies recognizing strains of porcine origin (Racz et al., ilar to human P[6]-I strains, rather than to the porcine prototype 2000; Teodoroff et al., 2005). Martella et al. (2006a) analyzing strain Gottfried, P[6]-II. In our study, the samples have clus- P[6] porcine rotavirus strains from various geographical areas in tered with the M37-like, some strains exhibiting more similarity Southern Europe, Spain and Italy, found P[6] strains more sim- to the human sublineage P[6]-Ia, that is, synapomorphisms in 154 J.D.P. Mascarenhas et al. / Virus Research 126 (2007) 149–158

six residues, while other strains had more similarity to porcine sublineages Ib and Ic (synapomorphisms in three residues). When comparing the VP8* gene of the rotavirus strains from diarrheic and non-diarrheic neonates and diarrheic young children, we did not observe significant aa differences. These results corroborate previous reports from Kirkwood et al. (1996), Pager et al. (2000) and Santos et al. (1994). Previous investigations have shown that NSP4 genotype A was largely predominant among neonates in nurseries in Belem,´ Brazil, having P[6]G2 specificity (Mascarenhas et al., 2006, 2007). In the present study, while all P[6]G4 strains are associated with NSP4 genotype B and were obtained from diarrheic young children, P[6]G2 and P[6]G9 strains were associated with genotype A. In contrast, Lee et al. (2000) comparing P[6]G1 rotaviruses from babies with and without diarrhea have shown that strains were associated with NSP4 genotype B. Pager et al. (2000) analyzing P[6]G4 rotavirus strains, found that strains isolated from diarrheic and non-diarrheic neonates differed in their NSP4 gene. In contrast, our P[6] strains did not differ with regards to their NSP4 gene when genotypes B and A were compared. Kirkwood et al. (1996) comparing P[6]G3 from non-diarrheic neonates in Melbourne, Australia, found isoleucine at aa residue 135 of NSP4 shared by rotavirus prototypes from non-diarrheic and diarrheic children. In the present study we found isoleucine at residue 135, only in two diarrheic children, shared by both non-diarrheic and diarrheic prototypes. Tyrosine 131 NSP4 protein has been postulated to be critical for the diarrheagenic activity of the toxic peptide (Ball et al., 1996). In the present study samples with genotype B (genetic group II) showed histidine in diarrheic young children and samples with genotype A (genetic group I) showed tyrosine in both diarrheic and non-diarrheic neonates and diarrheic young children. These results are in agreement with those reported by Cunliffe et al. (1997) who found tyrosine conserved only within genetic group I, whereas in genetic groups II and III histidine was more frequently found at residue 131. Although it has been suggested that NSP4 region spanning Fig. 4. Boostrapping consensus tree was constructed based on the neighbor joining method within MEGA package using the NSP4 amino acid sequences aa residues 114–135 plays a critical role in rotavirus pathogen- of the P[6] strains, human and animal prototypes. The genotypes A, B, C, D esis (Angel et al., 1998; Ball et al., 1996) and several reports and E were included in analyse. The numbers in the branches are bootstrap had analyzed this toxigenic region, the majority of them failed support valued for 2000 replicates. The sequences data reported in this to identify any specific aa substitutions indicative of either viru- paper to NSP4 gene were submitted to GenBank with accession numbers lence or avirulence related to this region (Horie et al., 1997; Lee (DQ525182–DQ525191 and EF089269). The references under comparison were ST3 (U59110), Wa (AF200224), M37 (U59109), RV3 (U42628), CH613 et al., 2000; Mascarenhas et al., 2006). As also supported by our (AF173193), TE56 (AF173200), CH52 (AF173183), CH624 (AF173199), TI25 data, it is likely that NSP4 gene may not be the sole determinant (AF173212), CH638 (AF173202), CH631 (AF173201), TA371 (AF173208), of pathogenicity. RMC321 (AF541921), OSU (D88831), A2 (AB180978), RV5 (U59103), Genetic and phylogenetic analyses of structural and DS-1 (AF174305), S2 (U59104), KUN (D88829), TF85 (AF174304), 1250 non-structural genes have indicated a consistent pattern (AJ236756), TA34 (AF174300), TA3 (AF174298), AU1 (D89873), EW (U96335), AvRV-1 (AY062937), Ch-1 (AB065287), NB-140 (DQ070395), of evolutionary relationships between certain animal and NB-150 (DQ299876), NB-156 (DQ070398), NB-162 (DQ070400), NB- human rotaviruses (Cunliffe et al., 1997; Horie et al., 1997; 171 (DQ070402), NB-174 (DQ070403), NB-175 (DQ070404), NB-189 Matthijnssens et al., 2006). (DQ070405), NB-208 (DQ070408), NB-211 (DQ070410), NB-233 The P[6] strains analyzed in the present study were from (DQ070412), NB-242 (DQ070413), NB-287 (DQ070415), NB-301 neonates and young children living in the outskirts of Belem,´ (DQ070416), NB-308 (DQ070417) and NB-321 (DQ070418). Brazil, under poor sanitation conditions.and in close proximity to animals, particularly pigs. Such findings allow us to postulate an interspecies transmission. By the way, a high rate of interspecies transmission has been noted among genotype B strains. J.D.P. Mascarenhas et al. / Virus Research 126 (2007) 149–158 155

Fig. 5. Comparation between ST3 with P[6] rotavirus strains isolates from diarrheic and non-diarrheic neonates to those from diarrheic young children showing functional sites among NSP4 genotypes A and B. Residues that differ from this consensus are shown. Dots indicate similarity to ST3 human prototype. 156 J.D.P. Mascarenhas et al. / Virus Research 126 (2007) 149–158

Importantly, a study conducted in Rio de Janeiro with samples Araujo,´ I.T., Fialho, A.M., de Assis, R.M., Rocha, M., Galvao, M., Cruz, C.M., from diarrheic children has shown that NSP4 genotypes A and B Ferreira, M.S., Leite, J.P., 2002. Rotavirus strain diversity in Rio de Janeiro, were circulating, however, all genotype B strains were of human Brazil: characterization of VP4 and VP7 genotypes in hospitalized children. J. Trop. Pediatr. 48, 214–218. origin (Araujo´ et al., in press). Araujo,´ I.T., Heinemann, M.B., Mascarenhas, J.D.P., De Assis, R.M., Fialho, In Brazil, several reports have shown strains of porcine ori- A.M., Leite, J.P. Molecular analysis of NSP4 and VP6 genes of rotavirus gin, specially G5 and G9 genotypes, causing diarrhea in children, strains recovered from hospitalized children in Rio de Janeiro, Brazil. J. suggesting a frequent exchange of genetic material among co- Clin. Virol., in press. circulating human and animal rotavirus strains (Alfieri et al., Ball, J.M., Tian, P., Zeng, C.Q., Morris, A.P., Estes, M.K., 1996. Age-dependent diarrhoea induced by a rotaviral nonstructural glycoprotein. Science 272, 1996; Leite et al., 1996; Mascarenhas et al., 2002; Santos and 101–104. Hoshino, 2005). A limitation of our study is that no fecal speci- Banyai,´ K., Martella, V., Jakab, F., Melegh, B.M., Szucs,¨ G., 2004. Sequencing mens were collected from pigs living in the same environment, so and phylogenetic analysis of human genotype P[6] rotavirus strains detected a more firm conclusion can not be drawn as to the proposed inter- in Hungary provides evidence for genetic heterogeneity within the P[6] VP4 species transmission. Simultaneous surveillance and sequencing gene. J. Clin. Microbiol. 42, 4338–4343. Barnes, G.L., Lund, J.S., Adams, L., Mora, A., Mitchell, S.V.,Caples, A., Bishop, of rotavirus genes from animals and humans are essential for a R.F., 1997. Phase 1 trial of a candidate rotavirus vaccine (RV3) derived from better understanding of the relationship between co-circulating a human neonate. J. Paediatr. Child Health 33, 300–304. strains with common and uncommon G/P combinations. Barnes, G.L., Lund, J.S., Mitchell, S.V.,De Bruyn, L., Piggford, L., Smith, A.L., In summary, with regards to P[6] genotype, we did not Furmedge, J., Masendycz, P.J., Bugg, H.C., Bogdanovic-Sakran, N., Carlin, observe significant aa differences between either non-diarrheic J.B., Bishop, R.F., 2002. Early phase II trial of human rotavirus vaccine candidate RV3. Vaccine 20, 2950–2956. and diarrheic neonates or diarrheic young children in compari- Boom, R., Sol, C.J.A., Salimans, M.M.M., Jansen, C.L., Wertheim-Van Dillen, son with non-diarrheic or diarrheic prototypes included in this P.M.E., Van Der Noordaa, J., 1990. Rapid and simple method for purifica- analysis. These preliminary findings warrant further and broader tions of nucleic acids. J. Clin. Microbiol. 28, 495–503. investigations to assess the relative frequencies of P[6] strains Ciarlet, M., Liprandi, F., Conner, M.E., Estes, M.K., 2000. Species specificity in our region, as well as to explore the potential for interspecies and interspeccies relatedness of NSP4 genetic groups by comparative NSP4 analysis of animal rotaviruses. Arch. Virol. 145, 371–383. transmission involving humans and animals, particularly pigs. Cunliffe, N.A., Rogerson, S., Dove, W., Thindwa, B.D.M., Greensill, J., Finally, genetic reassortment events in nature, even in the context Kirkwood, C.D., Broadhead, R.L., Hart, C.A., 2002. Detection and char- of possible interspecies transmission, may constitute a challenge acterization of rotaviruses in hospitalized neonates in Blantyre, Malawi. J. for future vaccination strategies. Clin. Microbiol. 40, 1534–1537. Cunliffe, N.A., Woods, P.A., Leite, J.P.G., Das, B.K., Ramachandran, M., Bhan, M.K., Hart, C.A., Glass, R.I., Gentsch, J.R., 1997. Sequence analysis of Acknowledgements NSP4 gene of human rotavirus allows classification into two main genetic groups. J. Med. Virol. 53, 41–50. We gratefully acknowledge the valuable technical support Das, B.K., Gentsch, J.R., Cicirello, H.G., Woods, P.A., Ramachandran, M., provided by Mrs. Euzeni Maria de Fatima´ Menezes and Antoniaˆ Gupta, A., Kumar, R., Bhan, M.K., Glass, R.I., 1994. Characterization of Alves. Clarissa Silva Lima, Sylvia de Fatima´ dos Guerra, rotavirus strains from newborns in New Delhi, India. J. Clin. Microbiol. 32, 1820–1822. Luana Silva Soares and Jackson Cordeiro Lima received a grant Estes, M.K., Cohen, J., 1989. Rotavirus gene structure and function. Microbiol. fellowship from the Brazilian National Council for the Devel- Rev. 53, 410–449 (Review). opment of Science and Technology (CNPq). This work was Estes, M.K., 2001. Rotaviruses and their replication. In: Knipe, D.M., How- partially supported by a grant from Para´ State Secretary of Sci- ley, P.M. (Eds.), Fields Virology, fourth ed. Lippincott Williams & Wilkins, ence and Technology (SECTAM/FUNTEC/PA), IEC/SVS/MS, Philadelphia, pp. 1747–1785. Fang, Z.Y., Yang, H., Qi, J., Zhang, J., Sun, L.W., Tang, J.Y., Ma, L., Du, Z.Q., IOC/FIOCRUZ and CNPq. He, A.H., Xie, J.P., Lu, Y.Y., Ji, Z.Z., Zhu, B.Q., Wu, H.Y., Lin, S.E., Xie, H.P., Griffin, D.D., Ivanoff, B., Glass, R.I., Gentsch, J.R., 2002. Diversity References of rotavirus strains among children with acute diarrhea in China. J. Clin. Microbiol. 40, 1875–1878. Adah, M.I., Wade, A., Taniguchi, K., 2001. Molecular epidemiology of rotavirus Flores, J., Midthun, K., Hoshino, Y., Green, K., Gorziglia, M., Kapikian, A.Z., in Nigeria: detection of unusual strains with G2P[6] and G8P[1] specificities. Chanock, R.M., 1986. Conservation of the fourth gene among rotaviruses J. Clin. Microbiol. 39, 3969–3975. recovered from asymptomatic newborn infants and its possible role in atten- Adah, M.I., Rohwedder, A., Olaleye, O.D., Durojaiye, O.A., Werchau, H., 1997. uation. J. Virol. 60, 972–979. Further characterization of field strains of rotavirus from Nigeria VP4 geno- Flores, J., Perez-Schael, I., Blanco, M., White, L., Garcia, D., Vilar, M., type P6 most frequently identified among symptomatically infected children. Cunto, W., Gonzalez, R., Urbina, C., Boher, J., Mendez, M., Kapikian, J. Trop. Pediatr. 43, 267–274. A.Z., 1990. Comparison of reactogenicity and antigenicity of M37 rotavirus Alfieri, A.A., Leite, J.P., Nakagomi, O., Kaga, E., Woods, P.A., Glass, vaccine and Rhesus-rotavirus-based quadrivalent vaccine. Lancet 336, R.I., Gentsch, J.R., 1996. Characterization of human rotavirus genotype 330–334. P[8]G5 from Brazil by probe-hybridization and sequence. Arch. Virol. 141, Gabbay, Y.B., Leite, J.P.G., Mascarenhas, J.D.P., Ferreira, L.M., Soares, L.S., 2353–2364. Ferreira, V.S., Alves, A.S., Menezes, E., Linhares, A.C., 2002. Ocorrenciaˆ Angel, J., Tang, B., Feng, N., Greenberg, H.B., Bass, D., 1998. Studies of the do genotipo G9 de rotav´ırus em Belem,´ Brasil. In: XIII Congresso Brasileiro role for NSP4 in the pathogenesis of homologous murine rotavirus diarrhea. de Infectologia Pediatrica,´ Salvador, Ba, p. 69. J. Infect. Dis. 177, 455–458. Gentsch, J.R., Glass, R.I., Woods, P., Gouvea, V., Gorziglia, M., Flores, J., Das, Araujo,´ I.T., Ferreira, M.S., Fialho, A.M., Assis, R.M., Cruz, C.M., Rocha, B.K., Bhan, M.K., 1992. Identification of group A rotavirus gene 4 types by M., Leite, J.P., 2001. Rotavirus genotypes P[4]G9, P[6]G9, and P[8]G9 in polymerase chain reaction. J. Clin. Microbiol. 30, 1365–1373. hospitalized children with acute gastroenteritis in Rio de Janeiro, Brazil. J. Horie, Y., Masamune, O., Nakagomi, O., 1997. Three major alleles of rotavirus Clin. Microbiol. 39, 1999–2001. NSP4 proteins identified by sequence analysis. J. Gen. Virol. 78, 2341–2346. J.D.P. Mascarenhas et al. / Virus Research 126 (2007) 149–158 157

Hoshino, Y., Wyatt, R.G., Flores, J., Midthun, K., Kapikian, A.Z., 1985. Matthijnssens, J., Rahman, M., Martella, V., Xuelei, Y., De Vos, S., De Leener, Serotypic characterization of rotaviruses from asymptomatic human neona- K., Ciarlet, M., Buonavoglia, C., Van Ranst, M., 2006. Full genomic analysis tal infections. J. Clin. Microbiol. 21, 425–430. of human rotavirus strain B4106 and lapine rotavirus strain 30/96 provides Ito, H., Sugiyama, M., Masubuchi, Y., Mori, Y., Minamoto, N., 2001. Com- evidence for interspecies transmission. J. Virol. 80, 3801–3810. plete nucleotide sequence of a group A avian rotavirus genome and a Midthun, K., Halsey, N.A., Jett-Goheen, M., Clements, M.L., Steinhoff, M., comparison with counterparts of mammalian rotaviruses. Virus Res. 75, King, J.C., Karron, R., Wilson, M., Burns, B., Perkis, V., Samorodin, 123–138. R., Kapikian, A.Z., 1991. Safety and immunogenicity of human rotavirus Kapikian, A.Z., Hoshino, Y., Chanock, R.M., 2001. Rotaviruses. In: Knipe, vaccine strain M37 in adults, children and infants. J. Infect. Dis. 164, D.M., Howley, P.M. (Eds.), Fields Virology, fourth ed. Lippincott Williams 792–796. & Wilkins, Philadelphia, pp. 1787–1833. Mori, Y., Borgan, M.A., Ito, N., Sugiyama, M., Minamoto, N., 2002. Diarrhoea- Khamrin, P., Maneekarn, N., Peerakome, S., Chan-it, W., Yagyu, F., Okitsu, inducing activity of avian rotavirus NSP4 glycoproteins, which differ greatly S., Ushijima, H., 2007. Novel porcine rotavirus of genotype P[27] shares from mammalian rotavirus NSP4 glycoproteins in deduced amino acid new phylogenetic lineage with G2 porcine rotavirus strain. Virology 361, sequence, in suckling mice. J. Virol. 76, 5829–5834. 243–252. Page, N.A., Steele, A.D., 2004. Antigenic and genetic characteriation of serotype Kimura, M., 1980. A simple method for estimating evolutionary rate of base G2 human rotavirus strains from the African Continent. J. Clin. Microbiol. substitution through comparative studies of nucleotide sequences. J. Mol. 42, 595–600. Evol. 16, 111–120. Pager, C.T., Alexander, J.J., Steele, A.D., 2000. South African G4P[6] asymp- Kirkwood, C.D., Coulson, B.S., Bishop, R.F., 1996. G2P2 rotaviruses causing tomatic and symptomatic neonatal rotavirus strains differ in their NSP4, diarrhoeal disease in neonates differ in VP4, VP7 and NSP4 sequence from VP8*, and VP7 genes. J. Med. Virol. 62, 208–216. G3P2 strains causing asymptomatic neonatal infection. Arch. Virol. 141, Pereira, H.G., Azeredo, R.S., Leite, J.P., Barth, O.M., Sutmoller, F., De Farias, 1661–1676. V., Vidal, M.N., 1983. Comparison of polyacrylamide gel electrophoresis Laird, A.R., Gentsch, J.R., Nakagomi, T., Nakagomi, O., Glass, R.I., 2003. (PAGE), immuno-electron microscopy (IEM) and enzyme immunoassay Characterization of serotype G9 rotavirus strains isolated in the United States (EIA) for the rapid diagnosis of rotavirus infection in children. Mem. Inst. and India from 1993 to 2001. J. Clin. Microbiol. 41, 3100–3111. Oswaldo Cruz, Rio de Janeiro 78, 483–490. Lee, C.N., Wang, Y.L., Kao, C.L., Zao, C.L., Lee, C.Y., Chen, H.N., 2000. Racz, M.L., Kroeff, S.S., Munford, V., Caruzo, T.A., Durigon, E.L., Hayashi, Nsp4 gene analysis of rotaviruses recovered from infected children with and Y., Gouvea, V., Palombo, E.A., 2000. Molecular characterization of porcine without diarrhoea. J. Clin. Microbiol. 38, 4471–4477. rotaviruses from the southern region of Brazil: characterization of an atypical Leite, J.P.G., Alfieri, A.A., Woods, P.A., Glass, R.I., Gentsch, J.R., 1996. genotype G[9] strain. J. Clin. Microbiol. 38, 2443–2446. Rotavirus G and P types circulating in Brazil: characterization by RT- Rahman, M., De Leener, K., Goegebuer, T., Wollants, E., Van der Donck, I., PCR, probe hybridization, and sequence analysis. Arch. Virol. 141, 2365– Van Hoovels, L., Van Ranst, M., 2003. Genetic characterization of a novel, 2374. naturally occurring recombinant human G6P[6] rotavirus. J. Clin. Microbiol. Linhares, A.C., Gabbay, Y.B., Mascarenhas, J.D.P., De Freitas, R.B., Oliveira, 41, 2088–2095. C.S., Bellesi, N., Monteiro, T.A.F., Lins-Lainson, Z., Ramos, F.L.P., Rahman, M., Mattthijjnssens, J., Nahar, S., Podder, G., Sack, D.A., Azim, T., Valente, S.A., 1996. Immunogenicity, safety and efficacy of Rhesus- Van Ranst, M., 2005. Characterization of a novel P[25]G11 human Group human, reassortant rotavirus vaccine in Belem,´ Brazil. Bull. W.H.O. 74, A rotavirus. J. Clin. Microbiol. 43, 3208–3212. 491–500. Rao, C.D., Gowda, K., Reddy, B.S., 2000. Sequence analysis of VP4 and VP7 Linhares, A.C., Mascarenhas, J.D.P., Gusmao,˜ R.H.P., Gabbay, Y.B., Fialho, genes of nontypeable strains identifies a new pair of outer capsid proteins A.M., Leite, J.P.G., 2002. Neonatal rotavirus infection in Belem,´ North- representing novel P and G genotypes in bovine rotaviruses. Virology 276, ern Brazil: nosocomial transmission of a P[6]G2 strain. J. Med. Virol. 67, 104–113. 418–426. Salu, O.B., Audu, R., Geyer, A., Steele, A.D., Oyefolu, A.O.B., 2003. Molecular Martella, V., Banyai,´ K., Ciarlet, M., Iturriza-Gomara, M., Lorusso, E., De epidemiology of rotaviruses in Nigeria: detection of unusual strains G2P[6] Grazia, S., Arista, S., Decaro, N., Elia, G., Cavalli, A., Corrente, M., Lavazza, and G8P[1] specificities. J. Clin. Microbiol. 41, 913–914. A., Baselga, R., Buonavoglia, C., 2006a. Relationships among porcine and Santos, N., Gouvea, V., Timenetsky, M.C., Clark, H.F., Riepenhoff-Talty, M., human P[6] rotaviruses: evidence that the different human P[6] lineages Garbarg-Chenon, A., 1994. Comparative analysis of VP8* sequences from have originated from multiple interspecies transmission events. Virology rotaviruses possessing M37-like VP4 recovered from children with and 344, 509–519. without diarrhea. J. Gen. Virol. 75, 1775–1780. Martella, V., Ciarlet, M., Banyai,´ K., Lorusso, E., Cavalli, A., Corrente, M., Elia, Santos, N., Hoshino, Y., 2005. Global distribution of rotavirus serotypes/ G., Arista, S., Camero, M., Desario, C., Decaro, N., Lavazza, A., Buon- genotypes and its implication for the development and implementation of an avoglia, C., 2006b. Identification of a novel VP4 genotype carried by a effective rotavirus vaccine. Rev. Med. Virol. 15, 29–56. serotype G5 porcine rotavirus strain. Virology 346, 301–311. Steele, A.D., Ivanoff, B., 2003. Rotavirus strains circulating in Africa dur- Mascarenhas, J.D.P., Gabbay, Y.B., Caniceiro, A.M., Soares, L.S., Menezes, E., ing 1996–1999: emergence of G9 strains and P[6] strains. Vaccine 17, Bayma, A.G., Leite, J.P.G., Linhares, A.C., 2003. Genomic characterization 361–367. of rotavirus strains among children participating in a surveillance for diar- Steele, A.D., Sears, J.F., 1996. Characterisation of rotaviruses recov- rhoea in Belem,´ Para.´ In: XIV National Meeting of Virology, Florianopolis,´ ered from neonates with symptomatic infection. S. Afr. Med. J. 86, SC, p. 233. 1546–1549. Mascarenhas, J.D.P., Leite, J.P.G., Lima, J.C., Heinemann, M.B., Oliveira, D.S., Steele, A.D., van Niekerk, M.C., Geyer, A., Bos, P., Alexander, J.J., 1992. Fur- Araujo,´ I.T., Soares, L.S., Gusmao,˜ R.H.P., Gabbay, Y.B., Linhares, A.C., ther characterisation of human rotaviruses isolated from asymptomatically 2007. Detection of a neonatal human rotavirus strain with VP4 and NSP4 infected neonates in five South Africa. J. Med. Virol. 38, 22–26. genes of porcine origin. J. Med. Microbiol. 56 (4), 524–532. Steele, A.D., van Niekerk, M.C., Mphalele, M.J., 1995. Geographic distribution Mascarenhas, J.D.P., Linhares, A.C., Bayma, A.P.G., Lima, J.C., Sousa, M.S., of human rotavirus VP4 genotypes and VP7 serotypes in five South African Araujo,´ I.T., Heinemann, M.B., Gusmao,˜ R.H.P., Gabbay, Y.B.,Leite, J.P.G., regions. J. Clin. Microbiol. 33, 1516–1519. 2006. Molecular analysis of VP4, VP7, and NSP4 genes of P[6]G2 rotavirus Steyer, A., Poljsak-Prijatelj, M., Barlic-Maganja, D., Jamnikar, U., Mijovski, genotype strains recovered from neonates admitted to hospital in Belem,´ J.Z., Marin, J., 2007. Molecular characterization of a new porcine rotavirus Brazil. J. Med. Virol. 78, 281–289. P genotype found in an asymptomatic pig in Slovenia. Virology 359, Mascarenhas, J.D.P.,Linhares, A.C., Gabbay, Y.B.,Leite, J.P.G.,2002. Detection 275–282. and characterization of rotavirus G and P types from children participating Teodoroff, T.A., Tsunemitsu, H., Okamoto, K., Katsuda, K., Kohmoto, M., in a rotavirus vaccine trial in Belem,´ Brazil. Mem. Inst. Oswaldo Cruz, Rio Kawashima, K., Nakagomi, T., Nakagomi, O., 2005. Predominance of de Janeiro 97, 113–117. porcine rotavirus G9 in Japanese piglets with diarrhea: close relationship of 158 J.D.P. Mascarenhas et al. / Virus Research 126 (2007) 149–158

their VP7 genes with those of recent human G9 strains. J. Clin. Microbiol. Vesikari, T., Ruuska, T., Koivu, H.P., Green, K.Y., Flores, J., Kapikian, A.Z., 43, 1377–1384. 1991. Evaluation of the M37 human rotavirus vaccine in 2- to 6-month old Tian, P., Estes, M.K., Hu, Y., Ball, J.M., Zeng, C.Q., Schilling, W.P., 1995. infants. Pediatr. Infect. Dis. 10, 912–917. The rotavirus nonstructural glycoprotein NSP4 mobilizes Ca2+ from the Volotao,˜ E.M., Soares, C.C., Maranhao,˜ A.G., Rocha, L.N., Hoshino, Y., San- endoplasmic reticulum. J. Virol. 69, 5763–5772. tos, N., 2006. Rotavirus surveillance in the City of Rio de Janeiro, Brazil, Timenetsky, M.C.S.T., Santos, N., Gouvea, V., 1994. Survey of rotavirus G and during 2000–2004: detection of unusual strains with G8P[4] or G10P[9] P types associated with human gastroenteritis in Sao˜ Paulo, Brazil, from speciﬁcities. J. Med. Virol. 78, 263–272. 1986 to 1992. J. Clin. Microbiol. 32, 2622–2624.