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Phylogenetic analysis of nonstructural protein 5 (NSP5) gene sequences in porcine rotavirus B strains
Infection, Genetics and Evolution 12 (2012) 1661–1668
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Phylogenetic analysis of nonstructural protein 5 (NSP5) gene sequences in porcine rotavirus B strains Tohru Suzuki a,⇑, Junichi Soma b, Ayako Miyazaki a, Hiroshi Tsunemitsu a a b
Viral Disease and Epidemiology Research Division, National Institute of Animal Health, National Agriculture and Food Research Organization, Ibaraki, Japan Research and Development Section, Institute of Animal Health, National Federation of Agricultural Co-operative Association, Chiba, Japan
a r t i c l e
i n f o
Article history: Received 24 April 2012 Received in revised form 22 June 2012 Accepted 28 June 2012 Available online 2 August 2012 Keywords: Porcine rotavirus B Nonstructural protein 5 Sequence analysis Genetic classification
a b s t r a c t Porcine rotavirus B (RVB) has frequently been detected in the diarrhea of suckling and weaned pigs. Because it is difficult to propagate RVBs serially in cell culture, little genetic information about RNA segments other than VP7, NSP1 and NSP2 is available for porcine RVBs. We conducted a phylogenetic analysis focusing on nonstructural protein 5 (NSP5) using 22 porcine RVB strains, which were identified in fecal samples collected around Japan. Sequence analysis showed that NSP5 genes of RVBs contain one ORF, in contrast to the corresponding gene of RVAs that have two ORFs. Comparison of NSP5 amino acid sequences from porcine RVBs with other RVBs revealed that eight serine and serine/threonine residues at the N- and C-terminal regions are highly conserved among RVBs. Phylogenetic analysis also indicated the presence of six clusters (H1–H6) including rat, human, porcine and bovine plus ovine clusters with cut-off values of 78% at the nucleotide level. Moreover, the NSP5 genes of porcine RVBs were divided to three clusters. The data presented here demonstrates that several porcine RVBs with distinctive genotypes are circulating among farms throughout Japan. Our findings provide important new insights into the evolutionary dynamics of RVBs. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Rotaviruses, a member of the Reoviridae family, are a major etiological agent of gastroenteritis in humans and animals worldwide. Their genome is composed of 11 double-stranded RNAs (dsRNAs) encoding six structural proteins (VP1–4, VP6–7) and five or six nonstructural proteins (NSP1–6). On the basis of their antigenic relationships and genomic characteristics, rotaviruses are classified into seven species (A–G) (Matthijnssens et al., 2011). Rotavirus B (RVB) has been primarily associated with nationwide outbreaks of diarrhea in adults in China, and then spreading among Asian countries outside China, including India, Bangladesh and Myanmar (Hung et al., 1984; Chen et al., 1985; Krishnan et al., 1999; Sanekata et al., 2003; Kelkar and Zade, 2004; Aung et al., 2009). Apart from in humans, RVBs have also been detected in rodents, cows, pigs and sheep (Eiden et al., 1992; Chang et al., 1997; Shen et al., 1999; Tsunemitsu et al., 1999; Barman et al., 2004; Ghosh et al., 2007; Kuga et al., 2009). Bovine RVBs have been detected in sporadic cases and outbreaks
⇑ Corresponding author. Address: Viral Disease and Epidemiology Research Division, National Institute of Animal Health, National Agriculture and Food Research Organization, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan. Tel.: +81 29 838 7765; fax: +81 29 838 7844. E-mail addresses: [email protected], [email protected] (T. Suzuki). 1567-1348/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2012.06.016
of diarrhea in calves and adult cows from India, Japan and USA. On the other hand, porcine RVBs have been identified in gastrointestinal diseases of suckling and weaned pigs, and shown to cause acute, transitory diarrhea in experimentally inoculated gnotobiotic pigs (Theil et al., 1985; Janke et al., 1990). Furthermore, epidemiological surveys of RVB infections with ELISA in cattle and pig herds from Japan and the UK demonstrated a high antibody prevalence in sera (Brown et al., 1987; Tsunemitsu et al., 2005). However, RVBs are shed at low levels and cannot be propagated in cell culture, preventing progress in their molecular study (Saif, 1990). The full-genome sequence has been determined for a large number of rotavirus A (RVA) strains. Moreover a full genome-based genotyping system composed of genotypes of 11 RNA segments has been recently proposed for the classification of RVAs (Matthijnssens et al., 2008a,b, 2010). In contrast, available genetic data for RVBs has been extremely limited, and genetic diversity in RNA segments other than VP7 gene has been scarcely analyzed. However, several bovine and human RVB strains that have sequence data available for most of their genome, in addition to a few human RVB and rat RVB (IDIR) strains already published, have been analyzed recently (Eiden et al., 1992; Sen et al., 2001; Ahmed et al., 2004; Yang et al., 2004; Yamamoto et al., 2010; Ghosh et al., 2010). Furthermore, genetic divergence and the classification of NSP1 and NSP2 genes from several porcine RVBs has been
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Table 1 The origin of 22 porcine RVBs in fecal samples from suckling, weaned and fattening pigs.
a b c d
RVB strain
GenBank ID
Prefecture and farmsa
Time-point of sampling
Age or stageb
Diarrheac
Genotyped
PB-Kyushu PB-S5 PB-S22-3 PB-S24-11 PB-S26-1 PB-S40-1 PB-68-C17 PB-68-D5 PB-68-G4 PB-93-H2 PB-93-I5 PB-70-H3 PB-70-H5 PB-71-H5 PB-103-1 PB-98-3 PB-104-3 PB-91-L1 PB-91-Z4 PB-72-I2 PB-87-Z2 PB-107-G16
AB713975 AB713976 AB713977 AB713978 AB713979 AB713980 AB713981 AB713982 AB713983 AB713987 AB713973 AB713984 AB713985 AB713986 AB713988 AB713992 AB713989 AB713993 AB713994 AB713990 AB713991 AB713974
Kagoshima, A Kagoshima, B Aomori, C Yamagata, D Aomori, E Miyagi Gifu, G Gifu, G Gifu, G Gifu, G Gifu, G Chiba, H Chiba, H Chiba, H Miyazaki, I Miyazaki, I Chiba, J Chiba, J Chiba, J Ibaraki, K Ibaraki, K Tochigi, L
April 2001 May 2002 August 2002 September 2002 September 2002 May 2003 August 2007 August 2007 August 2007 October 2008 October 2008 August 2007 August 2007 August 2007 March 2009 February 2009 March 2009 October 2008 October 2008 August 2007 September 2008 April 2009
5 41 8 29 15 15 Suckling 30 100 Weaned Weaned 30 30 30 9 5 3 150 Weaned 60 Weaned 6
+ + + + + + + + + + + +
H4 H3 H4 H4 H4 H4 H4 H4 H3 H4 H5 H4 H4 H4 H4 H5 H4 H5 H5 H5 H5 H4
Different letters mean that each RVB strain originates from a different farm. The number is days of age. Samples with uncertain age are shown in the breeding stage: suckling; 0–34 days old, weaned; 30–90 days old. The presence of diarrhea when the sample is collected is shown. Each genotype as shown in Fig. 3.
elucidated subsequent to the classification of RVB VP7 including porcine RVBs in human, rat, bovine and partial ovine RVBs (Kuga et al., 2009; Suzuki et al., 2011, 2012). Further genetic information on the remaining segments of porcine RVBs is required to uncover accurately the status of the molecular evolution of RVBs, since fullgenome analysis of RVBs is important for understanding of not only ecology and evolution of the pathogen, but also the mechanisms involved in genetic diversity such as gene reassortment and/or crossing of the host-species barrier. Genetic characterization in RVBs depends largely on comparative analysis with RVAs, which warrant investigation. The viroplasms are the viral factories in which the genome is replicated and packaged into virions. Of the several viral proteins located in the viroplasms, NSP2 and NSP5 are essential for viroplasms formation (Fabbretti et al., 1999; Eichwald et al., 2004a). NSP5 has a high number of serine and threonine residues and various posttranslational modifications, like O-glycosylation and polyphosphorylation (Welch et al., 1989; Gonzalez and Burrone, 1991). Although there are some reports on the phosphorylational modification mechanisms of NSP5 protein involving autokinase and cellular kinases, the role of NSP5 phosphorylation is still unclear and elusive (Afrikanova et al., 1998; Eichwald et al., 2002, 2004b; Sen et al., 2006; Bar-Magen et al., 2007). NSP5 also binds to ssRNA and dsRNA without sequence specificity (Vende et al., 2002). Moreover, RNA interference inhibition of NSP5 synthesis reduces dsRNA production (Campagna et al., 2005; Lopez et al., 2005). Taken together, these studies suggest an important role for NSP5 in the replication of the viral genome. On the other hand, while little is known of the properties of NSP5 in RVB, the deduced amino acid sequence of some RVBs has revealed serine/threonine-rich proteins, typical for NSP5 of RVAs. In the present study, we determined the nucleotide sequences of NSP5 genes in several porcine RVB strains from fecal specimens collected around Japan in order to investigate the origin and genetic relatedness of their RVBs. Moreover, phylogenetic analyses of NSP5 of porcine RVBs identified in this study along with NSP5 of other RVBs previously published, may enhance our understanding of the molecular evolution of RVBs.
2. Materials and methods 2.1. Origin of samples As a passive surveillance of diarrhea, 22 fecal samples from suckling and weaned pigs collected at twelve farms around Japan from 2001 to 2009 are summarized in Table 1. Viral RNA was extracted from 10% fecal suspensions in minimum essential medium (MEM) using LS (Invitrogen, CA, USA) according to the manufacturer’s instructions. The RNA samples were examined for VP7 genes of RVA, RVB and RVC by RT–PCR with respective specific primers (Gouvea et al., 1990; Tsunemitsu et al., 1996; Kuga et al., 2009). 2.2. Cloning and sequencing of the NSP5 gene from porcine RVB PB-93I5 strain The full-length nucleotide sequence of the NSP5 gene from porcine RVB PB-93-I5 strain was determined using the single primer amplification method as mentioned by Wakuda et al. (2005). Briefly, ligation of the single amino-group linked oligonucleotide primer to the 30 -ends of both strands of the viral dsRNA, columnbased purification and concentration of the ligated RNA and RT–PCR reactions were carried out. The PCR products were cloned into the pCR2.1 TOPO vector (Invitrogen, CA, USA), and consequently four positive clones were obtained. Thereafter, the four clones were sequenced using the BigDye Terminator v3.1 cycle sequencing kit on an automated ABI Prism 3130 Genetic Analyzer (Life technologies Corp.CA, USA). 2.3. RT–PCR amplification of NSP5 genes from several porcine RVB strains In order to determine the ORFs of NSP5 genes from other porcine RVB strains, two pairs of oligonucleotide primers, NSP5-1F [50 -GAGATAGGTAGTGGCTGGAA-30 ; nucleotide (nt) position 10– 29 of the PB-93-I5] and NSP5-1R [50 -ACTCTAGCACTAAGGGTTT-30 ;
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Fig. 1. Alignments of full-length NSP5 nucleotide sequences from representative porcine and other RVB strains. Sequence data constructed by the Clustal W method using the Lasergene software. The position of primers designed in this study is shown by underlining. The sites of initial and terminal codons of each RVB NSP5 genes are boxed. The GenBank accession numbers are as follows: RVB strains, ADRV (M34380); Bang117 (GU391311); DB180 (AY347928); IDIR (D00912); KB63 (AF079158).
nt position 603–621 of the PB-93-I5], and NSP5-2F [50 -GGAAACG TTGTACTGACTACTC-30 ; nt position 26–47 of the Bang117] and NSP5-2R [50 -TTGACCCCAGTAGATGACAG-30 ; nt position 586–605 of the Bang117] were designed by reference to the full genome of PB-93-I5 and human RVB Bang117 strains, respectively (Fig. 1). RT-PCR was conducted using the Onestep RT-PCR kit (Qiagen, CA, USA). The reaction was performed at 50 °C for 30 min and 95 °C for 15 min, followed by 35 cycles of 94 °C for 1 min, 48 °C for 1 min and 72 °C for 2 min, and then a final extension at 72 °C for 10 min. The products were cleaned with MicroSpin
columns (S-400 HR, GE Healthcare, UK), and then sequenced using the BigDye Terminator v3.1 cycle sequencing kit on an automated ABI Prism 3130 Genetic Analyzer (Life technologies Corp., CA, USA). 2.4. Sequence and phylogenetic analyses Sequence data were aligned by the Clustal W method using the MegAlign program of the Lasergene software (DNASTAR, WI, USA). In models of the nucleotide substitution, the general time reversible (GTR) is selected according to the Akaike information criterion
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(a)
(b)
Fig. 2. Alignments of NSP5 amino acid sequences from representative porcine and other RVB strains (a) including host species and genotypes from representative RVA and RVC strains (b). Sequence data constructed by the Clustal W method using the Lasergene software. Identical and similar residues are shown by asterisks (⁄) and dot (.), respectively. Serine and/or threonine residues conserved among RVBs and between RVAs and RVCs are highlighted in gray. Gaps introduced to optimize the alignment are indicated by dashes. The GenBank accession numbers are as follows: RVA strains, OSU (P19715); SA11 (AAK15267); Wa (V01191); RVC strains Bristol (Q00682); Cowden (P36358); Shintoku (P34718).
(AIC) with the jModelTest program (Guindon and Gascuel, 2003; Posada, 2008). Phylogenetic analysis was conducted by using the GTR model of maximum likelihood (ML) method with the MEGA 5 program (Tamura et al., 2011). Genetic distances were calculated by the Poisson correction parameter at the amino acid level and the Kimura 2-parameter correction at the nucleotide level. A cut-off value was calculated according to the definition recommended by the Rotavirus Classification Working Group (RCWG) (Matthijnssens et al., 2008a,b). Briefly, the percentage identities between the complete ORFs of available NSP5 genes from other RVBs including human, rat, bovine and ovine RVBs in GenBank, as well as those of NSP5 genes from porcine RVBs determined in this study, were calculated using the sequence distances program of the Lasergene software. A pairwise identity frequency graph was constructed by plotting all the calculated pairwise identities in a graph with the percentage identities in the X-axis and the frequency of each of the calculated pairwise identities in the Y-axis.
Consequently, a cut-off percentage for genetic classification of RVB NSP5 was estimated as the percentage separating the intragenotype identities, the nucleotide/amino acid identities between strains belonging to the same genotype and the intergenotype identities, the nucleotide/amino acid identities between strains belonging to different genotypes. The phylogenetic trees were constructed using the maximum likelihood method with bootstrapping with 1000 replicates (Saitou and Nei, 1987). 3. Results 3.1. Sequence analysis of the NSP5 gene of porcine RVB PB-93-I5 strain Twenty-two viral RNA samples used in this study were positive for RVB VP7 but not RVA or RVC VP7 genes by RT–PCR using either pair of specific primers (data not shown). Of these samples, a full-genome sequence analysis of the NSP5 gene of porcine RVB
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Table 2 NSP5 sequence identities (%) in nucleotide (upper right) and amino acids (lower left) among genotypes grouped by phylogenetic analysis.
a
Each genotype includes the strains shown in Fig. 3.
PB-93-I5 strain, which might be positioned at the origin of other porcine RVB strains from our previous phylogenetic analysis of RVB NSP1 (Suzuki et al., 2011), was firstly performed using the single primer amplification method. The four pCR2.1 TOPO clones obtained were identical on sequencing analysis. The full-length of PB-93-I5 NSP5 gene was 625 bp long as for bovine and ovine RVB strains (Fig. 1). In addition, neither of the first two in-frame AUGs or any significant out-of-frame ORF could be identified in its full-genome sequence, suggesting it contained only one ORF. Moreover, the NSP5 deduced amino acid sequence was distinctive from other RVB strains, while several serine residues were highly conserved among RVBs (Fig. 2). Comparison of the NSP5 gene of porcine RVB PB-93-I5 strain with those of human, bovine and rat RVB strains indicated low identities of 50.9–73.1% in nt, 44.2– 72.9% in amino acids (aa). 3.2. Sequence identification of NSP5 genes from several porcine RVB strains There were distinctive sequence differences at both terminals of the PB-93-I5 NSP5 gene between those of human RVBs but not bovine RVBs, and hence two pairs of oligonucleotide primers were designed on the basis of human RVB Bang117 NSP5 sequence, in addition to PB-93-I5 NSP5 sequence (Fig. 1). The ORFs of NSP5 genes from 21 porcine RVB strains were amplified by RT–PCR reactions using either primer and sequenced. Substantial diversity in NSP5 nucleotide sequences was found among 22 porcine RVB strains (50.9–99.8%), which was larger than that among human (88.5–100%) and bovine RVB strains (93.0–100%). Percent identities of NSP5 amino acid sequences among porcine RVBs also exhibited relatively variability (40.1–100%) as compared with those among human and bovine strains (90.6–100% and 97.0–100%). Moreover, comparison of NSP5 nucleotide and amino acid sequences among RVBs of human, bovine and rat strains indicated that porcine RVBs had a high level of diversity distinct from other RVBs (Table 2). 3.3. Phylogenetic analysis of RVB NSP5 The genetic classification of RVB NSP5 was performed on the basis of a value that was estimated from the frequency distribu-
tion of pairwise sequence identities according to the definition recommended by the RCWG (Matthijnssens et al., 2008a,b). The cut-off value for the division of genotypes was defined as 78% at the nt level. A phylogenetic dendrogram of NSP5 nucleotide sequences on RVB strains showed them to be genetically classified into six genotypes (Fig. 3). Although the functions of RVB NSP5 genes is still unknown, we would be provisionally defined the new NSP5 genotypes with the argument of having a concordant nomenclature as follows: rat cluster (H1), human cluster (H2), porcine clusters (H3–5) and bovine plus ovine cluster (H6). Notably, porcine RVB strains were widely divided into three clusters. 4. Discussion In contrast with human, rat and bovine RVBs, little information about RNA segments other than those encoding the VP7, NSP1 and NSP2 genes is available for porcine RVBs (Eiden et al., 1992; Ghosh et al., 2010; Yamamoto et al., 2010; Kuga et al., 2009 Suzuki et al., 2011, 2012). With no prior genomic knowledge, the single primer amplification method is useful (Lambden et al., 1992; Potgieter et al., 2002, 2009; Wakuda et al., 2005; Maan et al., 2007). In our analysis using this method, we firstly determined the nucleotide sequence of the NSP5 gene from porcine RVB PB-93-I5 strain, and thereafter those of NSP5 ORFs from 21 porcine RVB strains by RT-PCR with two sets of primers (Fig. 1). There were large variations in nucleotide sequences within (50.9–99.8% identities) and between (50.7–73.9%) strains (Table 2). Moreover, neither of the first two in-frame AUGs or the significant out-of-frame ORF could be detected in their full-genome sequences or in other RVBs, suggesting the presence of just one ORF encoding only NSP5, in contrast to the cognate gene of RVA SA11 strain has two ORFs encoding NSP5 and NSP6 (Mitchell and Both, 1988; Welch et al., 1989). Alignment of NSP5 amino acid sequences with porcine and other RVB strains indicated eight highly conserved serine and serine/threonine residues (Fig. 2). Although the positions and mechanisms of phosphorylational modification of the NSP5 protein are still unclear, NSP5 is known to be activated by complex hyperphospholyration of serine residues among RVAs (Poncet et al., 1997; Afrikanova et al., 1998; Eichwald et al., 2002, 2004b; Sen et al., 2006; Bar-magen et al., 2007; Sotelo et al., 2010). While this
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(a)
100
99
99
88
(b)
Frequency of different identities
0.1
KB63 (Ovine) DB176 (Bovine) 92 98 DB180 (Bovine) 93 DB101 (Bovine) RUBV106 (Bovine) 99 RUBV282 (Bovine) 99 PB-87-Z2 09/08 (Porcine) 97 PB-72-I2 08/07 (Porcine) PB-98-3 02/09 (Porcine) 100 PB-91-Z4 10/08 (Porcine) 90 PB-91-L1 10/08 (Porcine) 99 PB-93-I5 10/08 (Porcine) 100 PB-93-H2 10/08 (Porcine) PB-68-D5 08/07 (Porcine) PB-107-G16 04/09 (Porcine) PB-71-H5 08/07 (Porcine) 92 PB-103-1 03/09 (Porcine) PB-Kyushu 04/01 (Porcine) PB-S24-11 09/02 (Porcine) PB-S40-1 05/03 (Porcine) PB-104-3 03/09 (Porcine) PB-70-H3 08/07 (Porcine) 73 100 PB-70-H5 08/07 (Porcine) 87 PB-S22-3 08/02 (Porcine) PB-S26-1 09/02 (Porcine) PB-68-C17 08/07 (Porcine) PB-68-G4 08/07 (Porcine) PB-S5 05/02 (Porcine) 91 MMR-B1 (Human) 96 IDH-084 (Human) IC-008 (Human) Bang373 (Human) 93 85 Bang117 (Human) 100 CAL-1 (Human) WH-1 (Human) 94 ADRV (Human) IDIR (Rat)
H6
H5
H4
H3
H2
H1
120
78%
100 80
Intergenotype identities 60
Intragenotype identities 40 20 0 50
60
70
80
90
100
Identities Fig. 3. Phylogenetic tree (a) and distribution of pairwise identity frequencies (b) in RVB NSP5 nucleotide sequences. Phylogenetic dendrogram constructed by the GTR model of maximum likelihood method, which was statistically selected by jModelTest, with the MEGA 5 program. Percentage bootstrap values of less than 70% are not indicated. The dotted line represents the division of genotypes with a cut-off value of 78%. NSP5 genotypes (H1–H6) of RVB strains are shown on the right. Bar, 0.1 substitutions per site. Porcine RVBs are indicated the date (month/year) when they were collected after strain. The GenBank accession numbers are as follows: human RVB strains, Bang373 (AY238394); IC-008 (GU377223); IDH-084 (GU377234); MMR-B1 (GU370060); WH-1 (AY539863); bovine RVB strains, DB101 (AY347930); DB176 (AY347929); RUBV106 (DQ987863).
finding suggests that RVB NSP5 may also be a phosphoprotein as is typical for RVA NSP5, nevertheless an important serine site of phosphorylation has not been identified. Furthermore, three regions (aa 1–7, 48–77, 121 to C terminus) of the NSP5 amino acid sequences are conserved between porcine and other RVBs, which were different from the regions that were conserved between RVAs and RVCs (Sotelo et al., 2010). However, this fact suggests the possibility that the N- and C-terminal regions of RVB NSP5, despite
low level amino acid identity, are also critical for formation of viroplasm-like structures as with RVA NSP5 (Fabbretti et al., 1999; Mohan et al., 2003). Taken together, further molecular analysis of RVB NSP5 is awaited to confirm any possible functional similarities. In RVA classification, the RCWG estimated a suggestion of the sequence identity cut-off value on RVA NSP5 genes in the definition of genotype as 91% in nt (Matthijnssens et al., 2008a,b). In this
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study, the cut-off values based on the distribution of RVB NSP5 were 78% identity in nt and 77% identity in aa (data not shown), which were relatively lower than that of RVA. Thus, these data support the notion that RVB strains from different animal species might have diverged from each other over a longer period of time than RVAs (Petric et al., 1991; Eiden et al., 1992; Tsunemitsu et al., 1999). Interestingly, NSP5 from porcine RVBs were distributed among three genotypes, in contrast to those from other RVBs, which belonged to a monophyletic genotype. These patterns imply that RVB strains infecting different animal species, especially porcine RVB strains have large genetic diversity that has evolved in accordance with the inherent variation of the host. In conclusion, the present study firstly demonstrated the full-length nucleotide sequence of NSP5 from PB-93-I5 strain and ORFs of NSP5 genes from 21 porcine RVB strains. The data presented in this study also indicates that multiple porcine RVB strains with different genotypes co-circulate at several farms around Japan. Moreover, RVB NSP5 genes could be classified into six genotypes according to genetic relatedness and the species of origin. Notably, the NSP5 genes of porcine RVBs were divided into three clusters. Our findings provide new insights for genetic characterization of porcine RVBs. Further genetic data for porcine RVBs will be essential to elucidate accurately the molecular evolution of RVBs.
Acknowledgements This study was supported in part by a grant-in-aid for scientific research from Ministry of Health, Labor and Welfare of Japan and by a grant from the National Institute of Animal Health.
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Phylogenetic analysis of nonstructural protein 5 (NSP5) gene sequences in porcine rotavirus B strains Tohru Suzuki a,⇑, Junichi Soma b, Ayako Miyazaki a, Hiroshi Tsunemitsu a a b
Viral Disease and Epidemiology Research Division, National Institute of Animal Health, National Agriculture and Food Research Organization, Ibaraki, Japan Research and Development Section, Institute of Animal Health, National Federation of Agricultural Co-operative Association, Chiba, Japan
a r t i c l e
i n f o
Article history: Received 24 April 2012 Received in revised form 22 June 2012 Accepted 28 June 2012 Available online 2 August 2012 Keywords: Porcine rotavirus B Nonstructural protein 5 Sequence analysis Genetic classification
a b s t r a c t Porcine rotavirus B (RVB) has frequently been detected in the diarrhea of suckling and weaned pigs. Because it is difficult to propagate RVBs serially in cell culture, little genetic information about RNA segments other than VP7, NSP1 and NSP2 is available for porcine RVBs. We conducted a phylogenetic analysis focusing on nonstructural protein 5 (NSP5) using 22 porcine RVB strains, which were identified in fecal samples collected around Japan. Sequence analysis showed that NSP5 genes of RVBs contain one ORF, in contrast to the corresponding gene of RVAs that have two ORFs. Comparison of NSP5 amino acid sequences from porcine RVBs with other RVBs revealed that eight serine and serine/threonine residues at the N- and C-terminal regions are highly conserved among RVBs. Phylogenetic analysis also indicated the presence of six clusters (H1–H6) including rat, human, porcine and bovine plus ovine clusters with cut-off values of 78% at the nucleotide level. Moreover, the NSP5 genes of porcine RVBs were divided to three clusters. The data presented here demonstrates that several porcine RVBs with distinctive genotypes are circulating among farms throughout Japan. Our findings provide important new insights into the evolutionary dynamics of RVBs. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Rotaviruses, a member of the Reoviridae family, are a major etiological agent of gastroenteritis in humans and animals worldwide. Their genome is composed of 11 double-stranded RNAs (dsRNAs) encoding six structural proteins (VP1–4, VP6–7) and five or six nonstructural proteins (NSP1–6). On the basis of their antigenic relationships and genomic characteristics, rotaviruses are classified into seven species (A–G) (Matthijnssens et al., 2011). Rotavirus B (RVB) has been primarily associated with nationwide outbreaks of diarrhea in adults in China, and then spreading among Asian countries outside China, including India, Bangladesh and Myanmar (Hung et al., 1984; Chen et al., 1985; Krishnan et al., 1999; Sanekata et al., 2003; Kelkar and Zade, 2004; Aung et al., 2009). Apart from in humans, RVBs have also been detected in rodents, cows, pigs and sheep (Eiden et al., 1992; Chang et al., 1997; Shen et al., 1999; Tsunemitsu et al., 1999; Barman et al., 2004; Ghosh et al., 2007; Kuga et al., 2009). Bovine RVBs have been detected in sporadic cases and outbreaks
⇑ Corresponding author. Address: Viral Disease and Epidemiology Research Division, National Institute of Animal Health, National Agriculture and Food Research Organization, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan. Tel.: +81 29 838 7765; fax: +81 29 838 7844. E-mail addresses: [email protected], [email protected] (T. Suzuki). 1567-1348/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2012.06.016
of diarrhea in calves and adult cows from India, Japan and USA. On the other hand, porcine RVBs have been identified in gastrointestinal diseases of suckling and weaned pigs, and shown to cause acute, transitory diarrhea in experimentally inoculated gnotobiotic pigs (Theil et al., 1985; Janke et al., 1990). Furthermore, epidemiological surveys of RVB infections with ELISA in cattle and pig herds from Japan and the UK demonstrated a high antibody prevalence in sera (Brown et al., 1987; Tsunemitsu et al., 2005). However, RVBs are shed at low levels and cannot be propagated in cell culture, preventing progress in their molecular study (Saif, 1990). The full-genome sequence has been determined for a large number of rotavirus A (RVA) strains. Moreover a full genome-based genotyping system composed of genotypes of 11 RNA segments has been recently proposed for the classification of RVAs (Matthijnssens et al., 2008a,b, 2010). In contrast, available genetic data for RVBs has been extremely limited, and genetic diversity in RNA segments other than VP7 gene has been scarcely analyzed. However, several bovine and human RVB strains that have sequence data available for most of their genome, in addition to a few human RVB and rat RVB (IDIR) strains already published, have been analyzed recently (Eiden et al., 1992; Sen et al., 2001; Ahmed et al., 2004; Yang et al., 2004; Yamamoto et al., 2010; Ghosh et al., 2010). Furthermore, genetic divergence and the classification of NSP1 and NSP2 genes from several porcine RVBs has been
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Table 1 The origin of 22 porcine RVBs in fecal samples from suckling, weaned and fattening pigs.
a b c d
RVB strain
GenBank ID
Prefecture and farmsa
Time-point of sampling
Age or stageb
Diarrheac
Genotyped
PB-Kyushu PB-S5 PB-S22-3 PB-S24-11 PB-S26-1 PB-S40-1 PB-68-C17 PB-68-D5 PB-68-G4 PB-93-H2 PB-93-I5 PB-70-H3 PB-70-H5 PB-71-H5 PB-103-1 PB-98-3 PB-104-3 PB-91-L1 PB-91-Z4 PB-72-I2 PB-87-Z2 PB-107-G16
AB713975 AB713976 AB713977 AB713978 AB713979 AB713980 AB713981 AB713982 AB713983 AB713987 AB713973 AB713984 AB713985 AB713986 AB713988 AB713992 AB713989 AB713993 AB713994 AB713990 AB713991 AB713974
Kagoshima, A Kagoshima, B Aomori, C Yamagata, D Aomori, E Miyagi Gifu, G Gifu, G Gifu, G Gifu, G Gifu, G Chiba, H Chiba, H Chiba, H Miyazaki, I Miyazaki, I Chiba, J Chiba, J Chiba, J Ibaraki, K Ibaraki, K Tochigi, L
April 2001 May 2002 August 2002 September 2002 September 2002 May 2003 August 2007 August 2007 August 2007 October 2008 October 2008 August 2007 August 2007 August 2007 March 2009 February 2009 March 2009 October 2008 October 2008 August 2007 September 2008 April 2009
5 41 8 29 15 15 Suckling 30 100 Weaned Weaned 30 30 30 9 5 3 150 Weaned 60 Weaned 6
+ + + + + + + + + + + +
H4 H3 H4 H4 H4 H4 H4 H4 H3 H4 H5 H4 H4 H4 H4 H5 H4 H5 H5 H5 H5 H4
Different letters mean that each RVB strain originates from a different farm. The number is days of age. Samples with uncertain age are shown in the breeding stage: suckling; 0–34 days old, weaned; 30–90 days old. The presence of diarrhea when the sample is collected is shown. Each genotype as shown in Fig. 3.
elucidated subsequent to the classification of RVB VP7 including porcine RVBs in human, rat, bovine and partial ovine RVBs (Kuga et al., 2009; Suzuki et al., 2011, 2012). Further genetic information on the remaining segments of porcine RVBs is required to uncover accurately the status of the molecular evolution of RVBs, since fullgenome analysis of RVBs is important for understanding of not only ecology and evolution of the pathogen, but also the mechanisms involved in genetic diversity such as gene reassortment and/or crossing of the host-species barrier. Genetic characterization in RVBs depends largely on comparative analysis with RVAs, which warrant investigation. The viroplasms are the viral factories in which the genome is replicated and packaged into virions. Of the several viral proteins located in the viroplasms, NSP2 and NSP5 are essential for viroplasms formation (Fabbretti et al., 1999; Eichwald et al., 2004a). NSP5 has a high number of serine and threonine residues and various posttranslational modifications, like O-glycosylation and polyphosphorylation (Welch et al., 1989; Gonzalez and Burrone, 1991). Although there are some reports on the phosphorylational modification mechanisms of NSP5 protein involving autokinase and cellular kinases, the role of NSP5 phosphorylation is still unclear and elusive (Afrikanova et al., 1998; Eichwald et al., 2002, 2004b; Sen et al., 2006; Bar-Magen et al., 2007). NSP5 also binds to ssRNA and dsRNA without sequence specificity (Vende et al., 2002). Moreover, RNA interference inhibition of NSP5 synthesis reduces dsRNA production (Campagna et al., 2005; Lopez et al., 2005). Taken together, these studies suggest an important role for NSP5 in the replication of the viral genome. On the other hand, while little is known of the properties of NSP5 in RVB, the deduced amino acid sequence of some RVBs has revealed serine/threonine-rich proteins, typical for NSP5 of RVAs. In the present study, we determined the nucleotide sequences of NSP5 genes in several porcine RVB strains from fecal specimens collected around Japan in order to investigate the origin and genetic relatedness of their RVBs. Moreover, phylogenetic analyses of NSP5 of porcine RVBs identified in this study along with NSP5 of other RVBs previously published, may enhance our understanding of the molecular evolution of RVBs.
2. Materials and methods 2.1. Origin of samples As a passive surveillance of diarrhea, 22 fecal samples from suckling and weaned pigs collected at twelve farms around Japan from 2001 to 2009 are summarized in Table 1. Viral RNA was extracted from 10% fecal suspensions in minimum essential medium (MEM) using LS (Invitrogen, CA, USA) according to the manufacturer’s instructions. The RNA samples were examined for VP7 genes of RVA, RVB and RVC by RT–PCR with respective specific primers (Gouvea et al., 1990; Tsunemitsu et al., 1996; Kuga et al., 2009). 2.2. Cloning and sequencing of the NSP5 gene from porcine RVB PB-93I5 strain The full-length nucleotide sequence of the NSP5 gene from porcine RVB PB-93-I5 strain was determined using the single primer amplification method as mentioned by Wakuda et al. (2005). Briefly, ligation of the single amino-group linked oligonucleotide primer to the 30 -ends of both strands of the viral dsRNA, columnbased purification and concentration of the ligated RNA and RT–PCR reactions were carried out. The PCR products were cloned into the pCR2.1 TOPO vector (Invitrogen, CA, USA), and consequently four positive clones were obtained. Thereafter, the four clones were sequenced using the BigDye Terminator v3.1 cycle sequencing kit on an automated ABI Prism 3130 Genetic Analyzer (Life technologies Corp.CA, USA). 2.3. RT–PCR amplification of NSP5 genes from several porcine RVB strains In order to determine the ORFs of NSP5 genes from other porcine RVB strains, two pairs of oligonucleotide primers, NSP5-1F [50 -GAGATAGGTAGTGGCTGGAA-30 ; nucleotide (nt) position 10– 29 of the PB-93-I5] and NSP5-1R [50 -ACTCTAGCACTAAGGGTTT-30 ;
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Fig. 1. Alignments of full-length NSP5 nucleotide sequences from representative porcine and other RVB strains. Sequence data constructed by the Clustal W method using the Lasergene software. The position of primers designed in this study is shown by underlining. The sites of initial and terminal codons of each RVB NSP5 genes are boxed. The GenBank accession numbers are as follows: RVB strains, ADRV (M34380); Bang117 (GU391311); DB180 (AY347928); IDIR (D00912); KB63 (AF079158).
nt position 603–621 of the PB-93-I5], and NSP5-2F [50 -GGAAACG TTGTACTGACTACTC-30 ; nt position 26–47 of the Bang117] and NSP5-2R [50 -TTGACCCCAGTAGATGACAG-30 ; nt position 586–605 of the Bang117] were designed by reference to the full genome of PB-93-I5 and human RVB Bang117 strains, respectively (Fig. 1). RT-PCR was conducted using the Onestep RT-PCR kit (Qiagen, CA, USA). The reaction was performed at 50 °C for 30 min and 95 °C for 15 min, followed by 35 cycles of 94 °C for 1 min, 48 °C for 1 min and 72 °C for 2 min, and then a final extension at 72 °C for 10 min. The products were cleaned with MicroSpin
columns (S-400 HR, GE Healthcare, UK), and then sequenced using the BigDye Terminator v3.1 cycle sequencing kit on an automated ABI Prism 3130 Genetic Analyzer (Life technologies Corp., CA, USA). 2.4. Sequence and phylogenetic analyses Sequence data were aligned by the Clustal W method using the MegAlign program of the Lasergene software (DNASTAR, WI, USA). In models of the nucleotide substitution, the general time reversible (GTR) is selected according to the Akaike information criterion
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(a)
(b)
Fig. 2. Alignments of NSP5 amino acid sequences from representative porcine and other RVB strains (a) including host species and genotypes from representative RVA and RVC strains (b). Sequence data constructed by the Clustal W method using the Lasergene software. Identical and similar residues are shown by asterisks (⁄) and dot (.), respectively. Serine and/or threonine residues conserved among RVBs and between RVAs and RVCs are highlighted in gray. Gaps introduced to optimize the alignment are indicated by dashes. The GenBank accession numbers are as follows: RVA strains, OSU (P19715); SA11 (AAK15267); Wa (V01191); RVC strains Bristol (Q00682); Cowden (P36358); Shintoku (P34718).
(AIC) with the jModelTest program (Guindon and Gascuel, 2003; Posada, 2008). Phylogenetic analysis was conducted by using the GTR model of maximum likelihood (ML) method with the MEGA 5 program (Tamura et al., 2011). Genetic distances were calculated by the Poisson correction parameter at the amino acid level and the Kimura 2-parameter correction at the nucleotide level. A cut-off value was calculated according to the definition recommended by the Rotavirus Classification Working Group (RCWG) (Matthijnssens et al., 2008a,b). Briefly, the percentage identities between the complete ORFs of available NSP5 genes from other RVBs including human, rat, bovine and ovine RVBs in GenBank, as well as those of NSP5 genes from porcine RVBs determined in this study, were calculated using the sequence distances program of the Lasergene software. A pairwise identity frequency graph was constructed by plotting all the calculated pairwise identities in a graph with the percentage identities in the X-axis and the frequency of each of the calculated pairwise identities in the Y-axis.
Consequently, a cut-off percentage for genetic classification of RVB NSP5 was estimated as the percentage separating the intragenotype identities, the nucleotide/amino acid identities between strains belonging to the same genotype and the intergenotype identities, the nucleotide/amino acid identities between strains belonging to different genotypes. The phylogenetic trees were constructed using the maximum likelihood method with bootstrapping with 1000 replicates (Saitou and Nei, 1987). 3. Results 3.1. Sequence analysis of the NSP5 gene of porcine RVB PB-93-I5 strain Twenty-two viral RNA samples used in this study were positive for RVB VP7 but not RVA or RVC VP7 genes by RT–PCR using either pair of specific primers (data not shown). Of these samples, a full-genome sequence analysis of the NSP5 gene of porcine RVB
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Table 2 NSP5 sequence identities (%) in nucleotide (upper right) and amino acids (lower left) among genotypes grouped by phylogenetic analysis.
a
Each genotype includes the strains shown in Fig. 3.
PB-93-I5 strain, which might be positioned at the origin of other porcine RVB strains from our previous phylogenetic analysis of RVB NSP1 (Suzuki et al., 2011), was firstly performed using the single primer amplification method. The four pCR2.1 TOPO clones obtained were identical on sequencing analysis. The full-length of PB-93-I5 NSP5 gene was 625 bp long as for bovine and ovine RVB strains (Fig. 1). In addition, neither of the first two in-frame AUGs or any significant out-of-frame ORF could be identified in its full-genome sequence, suggesting it contained only one ORF. Moreover, the NSP5 deduced amino acid sequence was distinctive from other RVB strains, while several serine residues were highly conserved among RVBs (Fig. 2). Comparison of the NSP5 gene of porcine RVB PB-93-I5 strain with those of human, bovine and rat RVB strains indicated low identities of 50.9–73.1% in nt, 44.2– 72.9% in amino acids (aa). 3.2. Sequence identification of NSP5 genes from several porcine RVB strains There were distinctive sequence differences at both terminals of the PB-93-I5 NSP5 gene between those of human RVBs but not bovine RVBs, and hence two pairs of oligonucleotide primers were designed on the basis of human RVB Bang117 NSP5 sequence, in addition to PB-93-I5 NSP5 sequence (Fig. 1). The ORFs of NSP5 genes from 21 porcine RVB strains were amplified by RT–PCR reactions using either primer and sequenced. Substantial diversity in NSP5 nucleotide sequences was found among 22 porcine RVB strains (50.9–99.8%), which was larger than that among human (88.5–100%) and bovine RVB strains (93.0–100%). Percent identities of NSP5 amino acid sequences among porcine RVBs also exhibited relatively variability (40.1–100%) as compared with those among human and bovine strains (90.6–100% and 97.0–100%). Moreover, comparison of NSP5 nucleotide and amino acid sequences among RVBs of human, bovine and rat strains indicated that porcine RVBs had a high level of diversity distinct from other RVBs (Table 2). 3.3. Phylogenetic analysis of RVB NSP5 The genetic classification of RVB NSP5 was performed on the basis of a value that was estimated from the frequency distribu-
tion of pairwise sequence identities according to the definition recommended by the RCWG (Matthijnssens et al., 2008a,b). The cut-off value for the division of genotypes was defined as 78% at the nt level. A phylogenetic dendrogram of NSP5 nucleotide sequences on RVB strains showed them to be genetically classified into six genotypes (Fig. 3). Although the functions of RVB NSP5 genes is still unknown, we would be provisionally defined the new NSP5 genotypes with the argument of having a concordant nomenclature as follows: rat cluster (H1), human cluster (H2), porcine clusters (H3–5) and bovine plus ovine cluster (H6). Notably, porcine RVB strains were widely divided into three clusters. 4. Discussion In contrast with human, rat and bovine RVBs, little information about RNA segments other than those encoding the VP7, NSP1 and NSP2 genes is available for porcine RVBs (Eiden et al., 1992; Ghosh et al., 2010; Yamamoto et al., 2010; Kuga et al., 2009 Suzuki et al., 2011, 2012). With no prior genomic knowledge, the single primer amplification method is useful (Lambden et al., 1992; Potgieter et al., 2002, 2009; Wakuda et al., 2005; Maan et al., 2007). In our analysis using this method, we firstly determined the nucleotide sequence of the NSP5 gene from porcine RVB PB-93-I5 strain, and thereafter those of NSP5 ORFs from 21 porcine RVB strains by RT-PCR with two sets of primers (Fig. 1). There were large variations in nucleotide sequences within (50.9–99.8% identities) and between (50.7–73.9%) strains (Table 2). Moreover, neither of the first two in-frame AUGs or the significant out-of-frame ORF could be detected in their full-genome sequences or in other RVBs, suggesting the presence of just one ORF encoding only NSP5, in contrast to the cognate gene of RVA SA11 strain has two ORFs encoding NSP5 and NSP6 (Mitchell and Both, 1988; Welch et al., 1989). Alignment of NSP5 amino acid sequences with porcine and other RVB strains indicated eight highly conserved serine and serine/threonine residues (Fig. 2). Although the positions and mechanisms of phosphorylational modification of the NSP5 protein are still unclear, NSP5 is known to be activated by complex hyperphospholyration of serine residues among RVAs (Poncet et al., 1997; Afrikanova et al., 1998; Eichwald et al., 2002, 2004b; Sen et al., 2006; Bar-magen et al., 2007; Sotelo et al., 2010). While this
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(a)
100
99
99
88
(b)
Frequency of different identities
0.1
KB63 (Ovine) DB176 (Bovine) 92 98 DB180 (Bovine) 93 DB101 (Bovine) RUBV106 (Bovine) 99 RUBV282 (Bovine) 99 PB-87-Z2 09/08 (Porcine) 97 PB-72-I2 08/07 (Porcine) PB-98-3 02/09 (Porcine) 100 PB-91-Z4 10/08 (Porcine) 90 PB-91-L1 10/08 (Porcine) 99 PB-93-I5 10/08 (Porcine) 100 PB-93-H2 10/08 (Porcine) PB-68-D5 08/07 (Porcine) PB-107-G16 04/09 (Porcine) PB-71-H5 08/07 (Porcine) 92 PB-103-1 03/09 (Porcine) PB-Kyushu 04/01 (Porcine) PB-S24-11 09/02 (Porcine) PB-S40-1 05/03 (Porcine) PB-104-3 03/09 (Porcine) PB-70-H3 08/07 (Porcine) 73 100 PB-70-H5 08/07 (Porcine) 87 PB-S22-3 08/02 (Porcine) PB-S26-1 09/02 (Porcine) PB-68-C17 08/07 (Porcine) PB-68-G4 08/07 (Porcine) PB-S5 05/02 (Porcine) 91 MMR-B1 (Human) 96 IDH-084 (Human) IC-008 (Human) Bang373 (Human) 93 85 Bang117 (Human) 100 CAL-1 (Human) WH-1 (Human) 94 ADRV (Human) IDIR (Rat)
H6
H5
H4
H3
H2
H1
120
78%
100 80
Intergenotype identities 60
Intragenotype identities 40 20 0 50
60
70
80
90
100
Identities Fig. 3. Phylogenetic tree (a) and distribution of pairwise identity frequencies (b) in RVB NSP5 nucleotide sequences. Phylogenetic dendrogram constructed by the GTR model of maximum likelihood method, which was statistically selected by jModelTest, with the MEGA 5 program. Percentage bootstrap values of less than 70% are not indicated. The dotted line represents the division of genotypes with a cut-off value of 78%. NSP5 genotypes (H1–H6) of RVB strains are shown on the right. Bar, 0.1 substitutions per site. Porcine RVBs are indicated the date (month/year) when they were collected after strain. The GenBank accession numbers are as follows: human RVB strains, Bang373 (AY238394); IC-008 (GU377223); IDH-084 (GU377234); MMR-B1 (GU370060); WH-1 (AY539863); bovine RVB strains, DB101 (AY347930); DB176 (AY347929); RUBV106 (DQ987863).
finding suggests that RVB NSP5 may also be a phosphoprotein as is typical for RVA NSP5, nevertheless an important serine site of phosphorylation has not been identified. Furthermore, three regions (aa 1–7, 48–77, 121 to C terminus) of the NSP5 amino acid sequences are conserved between porcine and other RVBs, which were different from the regions that were conserved between RVAs and RVCs (Sotelo et al., 2010). However, this fact suggests the possibility that the N- and C-terminal regions of RVB NSP5, despite
low level amino acid identity, are also critical for formation of viroplasm-like structures as with RVA NSP5 (Fabbretti et al., 1999; Mohan et al., 2003). Taken together, further molecular analysis of RVB NSP5 is awaited to confirm any possible functional similarities. In RVA classification, the RCWG estimated a suggestion of the sequence identity cut-off value on RVA NSP5 genes in the definition of genotype as 91% in nt (Matthijnssens et al., 2008a,b). In this
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study, the cut-off values based on the distribution of RVB NSP5 were 78% identity in nt and 77% identity in aa (data not shown), which were relatively lower than that of RVA. Thus, these data support the notion that RVB strains from different animal species might have diverged from each other over a longer period of time than RVAs (Petric et al., 1991; Eiden et al., 1992; Tsunemitsu et al., 1999). Interestingly, NSP5 from porcine RVBs were distributed among three genotypes, in contrast to those from other RVBs, which belonged to a monophyletic genotype. These patterns imply that RVB strains infecting different animal species, especially porcine RVB strains have large genetic diversity that has evolved in accordance with the inherent variation of the host. In conclusion, the present study firstly demonstrated the full-length nucleotide sequence of NSP5 from PB-93-I5 strain and ORFs of NSP5 genes from 21 porcine RVB strains. The data presented in this study also indicates that multiple porcine RVB strains with different genotypes co-circulate at several farms around Japan. Moreover, RVB NSP5 genes could be classified into six genotypes according to genetic relatedness and the species of origin. Notably, the NSP5 genes of porcine RVBs were divided into three clusters. Our findings provide new insights for genetic characterization of porcine RVBs. Further genetic data for porcine RVBs will be essential to elucidate accurately the molecular evolution of RVBs.
Acknowledgements This study was supported in part by a grant-in-aid for scientific research from Ministry of Health, Labor and Welfare of Japan and by a grant from the National Institute of Animal Health.
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