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. 2015 Apr 8;32(8):2060–2071. doi: 10.1093/molbev/msv088

Emerging OP354-Like P[8] Rotaviruses Have Rapidly Dispersed from Asia to Other Continents

Mark Zeller 1,*, Elisabeth Heylen 1, Susan Damanka 2, Corinna Pietsch 3, Celeste Donato 4, Tsutomu Tamura 5, Ruta Kulkarni 6, Ritu Arora 6, Nigel Cunliffe 7, Leena Maunula 8, Christiaan Potgieter 9,10, Sana Tamim 11, Sarah De Coster 1, Elena Zhirakovskaya 12, Salwa Bdour 13, Helen O’Shea 14, Carl D Kirkwood 4, Mapaseka Seheri 15, Martin Monene Nyaga 15, Jeffrey Mphahlele 15, Shobha D Chitambar 6, Ron Dagan 16, George Armah 2, Nina Tikunova 12, Marc Van Ranst 1, Jelle Matthijnssens 1,*
PMCID: PMC4833074  PMID: 25858434

Abstract

The majority of human group A rotaviruses possess the P[8] VP4 genotype. Recently, a genetically distinct subtype of the P[8] genotype, also known as OP354-like P[8] or lineage P[8]-4, emerged in several countries. However, it is unclear for how long the OP354-like P[8] gene has been circulating in humans and how it has spread. In a global collaborative effort 98 (near-)complete OP354-like P[8] VP4 sequences were obtained and used for phylogeographic analysis to determine the viral migration patterns. During the sampling period, 1988–2012, we found that South and East Asia acted as a source from which strains with the OP354-like P[8] gene were seeded to Africa, Europe, and North America. The time to the most recent common ancestor (TMRCA) of all OP354-like P[8] genes was estimated at 1987. However, most OP354-like P[8] strains were found in three main clusters with TMRCAs estimated between 1996 and 2001. The VP7 gene segment of OP354-like P[8] strains showed evidence of frequent reassortment, even in localized epidemics, suggesting that OP354-like P[8] genes behave in a similar manner on the evolutionary level as other P[8] subtypes. The results of this study suggest that OP354-like P[8] strains have been able to disperse globally in a relatively short time period. This, in combination with a relatively large genetic distance to other P[8] subtypes, might result in a lower vaccine effectiveness, underscoring the need for a continued surveillance of OP354-like P[8] strains, especially in countries where rotavirus vaccination programs are in place.

Keywords: OP354-like P[8], rotaviruses, emerging viruses, reassortment

Introduction

Group A rotaviruses (RVAs) are an important cause of diarrhea in children under 5 years old, with approximately 450,000 deaths worldwide due to rotaviruses gastroenteritis annually (Tate et al. 2012). Rotaviruses are a member of the Reoviridae family and possess a segmented genome consisting of 11 gene segments of which only the shortest segment is polycistronic (Estes and Kapikian 2007). The outer capsid of rotavirus consists of VP7 and VP4 proteins, and together they determine the G-genotype and the P-genotype, respectively. Currently, 27 G-genotypes and 37 P-genotypes have been described (Matthijnssens et al. 2011; Trojnar et al. 2013). However, only a few genotypes regularly infect humans worldwide: G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8] (Santos and Hoshino 2005; Bányai et al. 2012; Matthijnssens and Van Ranst 2012). In addition, rotaviruses with other genotype combinations are known to circulate locally, for example G9P[6] and G12P[6] in South Asia (Uchida et al. 2006; Mukherjee et al. 2010; Miles et al. 2012), and G2P[6] and G8P[6] in Sub-Saharan Africa (Todd et al. 2010; Heylen et al. 2014; Seheri et al. 2014).

Zoonotic rotavirus infections are not uncommon in humans, and occasionally rotavirus gene segments of animal origin establish themselves permanently in the human population through a mechanism by which animal rotaviruses become increasingly “humanized” by one or multiple reassortment with human rotavirus gene segments (Matthijnssens, Rahman, et al. 2010). Most humanized rotavirus strains possess an animal derived VP7 segment in combination with a P[8] VP4 gene and a human Wa-like backbone (e.g. G9P[8], G11P[8] and G12P[8] strains) (Matthijnssens and Van Ranst 2012). The reason for this is still largely unclear, but it is likely that the very high prevalence of Wa-like rotaviruses, antigenic pressure against established G-genotypes and possibly host specificity play an important role. As a result, the genetic diversity of the VP7 protein observed in human rotaviruses is relatively large when compared with other rotavirus proteins, including VP4. Human P-genotypes are mostly, but not entirely, restricted to the P[4], P[6], and P[8] genotypes (Santos and Hoshino 2005; Todd et al. 2010; Iturriza-Gómara et al. 2011; Matthijnssens and Van Ranst 2012). As VP4 is involved in several structural and functional interactions such as viral particle maturation in the endoplasmic reticulum, cell attachment, and cell membrane penetration (Estes and Kapikian 2007; Trask et al. 2012), genetic variability of this protein in humans is more restricted than that found in VP7. This feature is exploited by the currently available RVA vaccines; Rotarix (GlaxoSmithKline Inc., Belgium) is a live oral attenuated human G1P[8] rotavirus vaccine, and RotaTeq (Merck & Co. Inc.) is a human-bovine reassortant pentavalent vaccine comprising the human G1-G4 genotypes and the human P[8] genotype (Matthijnssens, Joelsson, et al. 2010). The detrimental effect of the introduction of unusual G-genotypes in the human RVA population on vaccine effectiveness has thus far been mitigated due to the Wa-like background that accompanies these unusual G-genotypes. Therefore, RotaTeq and Rotarix are still able to provide sufficient protection against disease through their VP4 P[8] genotype, cross protection between different G-genotypes and possibly also through other gene segments.

In some countries where Rotarix has been used as the main or only vaccine, a relatively high prevalence of G2P[4] strains has been reported compared with rotavirus seasons prior to vaccine introduction (Gurgel et al. 2007; Zeller et al. 2010; Paulke-Korinek et al. 2013; da Silva Soares et al. 2014). In addition, the effectiveness of Rotarix against G2P[4] RVAs has been shown to be slightly less than against Wa-like genotypes (Braeckman et al. 2012; Ichihara et al. 2014; Matthijnssens et al. 2014). Although all 11 gene segments in G2P[4] RVAs are heterotypic from the Rotarix vaccine strain, generation of cross-protective antibodies against the two outer capsid proteins probably play an important role in protection. Because the genetic backbone of the RotaTeq vaccine originates from a bovine RVA (Matthijnssens, Joelsson, et al. 2010), the main mode of protection is through the two outer capsid proteins as they elicit protective responses. Therefore, an increased genetic diversity in VP4 in combination with a G-genotype not present in the vaccines could pose a threat to RVA vaccine effectiveness.

Relatively recently, a new lineage of P[8] VP4 genes has been detected in different parts of Europe, Africa, and Asia (Maunula and Bonsdorff 1995; Cunliffe et al. 2001; Zade et al. 2009; Tamura et al. 2010; Zhirakovskaia et al. 2012). Although limited sequence information is available to date, these so-called OP354-like P[8] VP4 genes appear to be genetically divergent to the other P[8] lineages, including the most common lineage globally: P[8]-3 (Zeller et al. 2012). This large genetic diversity compared with other P[8] genes raises questions about the evolutionary origins of OP354-like P[8] genes. Possibly, rotaviruses with OP354-like P[8] genes have been circulating unnoticed in the human population for a prolonged period of time. This hypothesis is supported by the fact that strains with a OP354-like P[8] gene have been detected on four different continents to date. In this study, we infer the evolutionary history of OP354-like P[8] rotaviruses sampled from 1988 to 2012 across Europe, Africa, America, and Asia, in order to elucidate their global spread and putative origins.

Results

Viral Data Set

This study is a global effort to better understand the emergence of OP354-like P[8] strains and for this we composed a unique data set comprising the complete VP4 genes of 98 OP354-like P[8] viruses detected on four continents. The majority of the samples were collected in Asia (Asian part of Russia (24), Bangladesh (14), Israel (8), India (4), Pakistan (3), Vietnam (3), Jordan (2), China (2), Japan (1), and South-Korea (1)), followed by Europe (Belgium (12), Germany (3), Finland (2), and Ireland (1)), Africa (South-Africa (7), Ghana (4), Malawi (3), Ethiopia (2), Togo (1)), and North America (Canada (1)). The oldest strain was detected in Finland in 1988, whereas the most recent isolates were detected in Japan and Ethiopia in 2012.

OP354-Like P[8] VP4 Genes Are Distantly Related to VP4 Genes Belonging to Other P[8] Lineages

A maximum likelihood tree was constructed to infer the genetic relationships between OP354-like P[8] VP4 genes and the most common P-genotypes in humans, P[4] and P[8]. P[8] strains can be subdivided into four lineages (P[8]-1–P[8]-4), of which the latter is also referred to as the OP354-like P[8] lineage. Lineage P[8]-2 and P[8]-3 are the most closely related lineages (median genetic difference: 6.4% [3.3–10.3%]) (fig. 1). Lineage P[8]-1 is more distantly related to both P[8]-2 and P[8]-3 (median: 9.4% [5.3–12.3%]). The OP354-like P[8] lineage possessed the highest genetic distance to all other P[8] lineages (median: 11.3% [8.9–13.6%]), including lineage P[8]-1 and P[8]-2, which contain the Rotarix and RotaTeq P[8] VP4 gene, respectively. P[8]-1, P[8]-2, and P[8]-3 strains were even more distantly related to P[4] strains (median: 12.8% [10.9–15.9%]) than to strains belonging to the OP354-like P[8] lineage. However, of all P[8] and P[4] strains, the largest genetic distance was observed between P[4] and OP354-like P[8] strains (median: 14.5% [13.0–16.4%]). On the amino acid level genetic distances among the various P[8] lineages and P[4] were smaller than on the nucleotide level (fig. 1). However, the median distance of OP354-like P[8] strains to other P[8] lineages was 6.3% (4.4–11.8%), whereas the median distance of P[4] strains to P[8]-1, P[8]-2, and P[8]-3 strains was 8.4% (6.9–17.2%). Notably, P[4] strains were also most divergent to OP354-like P[8] strains (median: 10.6% [9.0–14.3%]) on the amino acid level. In comparison, the genetic diversity within the OP354-like P[8] lineage was small on both the nucleotide (median: 2.1% [0.0–4.0%]) and amino acid level (median: 1.2% [0.0–3.3%]). Thus, OP354-like P[8] strains can be genetically characterized as divergent from other P[8] lineages on both the nucleotide and amino acid level.

Fig. 1.

Fig. 1.

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Maximum likelihood tree of the VP4 gene of 1,137 P[4] and P[8] isolates. Only bootstrap values for the lineage-defining nodes are shown. Different lineages within P[8] and P[4] are indicated in different colors (panel A). Nucleotide identity frequencies between P[4] and the four lineages of P[8] are shown based on 1,137 sequences (panel B). Panel C shows identity frequencies on the amino acid level.

OP354-Like P[8] VP4 Genes Are Found in Three Distinct Clusters

To investigate the evolutionary dynamics of OP354-like P[8] genes, we constructed a time-measured Bayesian phylogenetic tree using the BEAST package (Drummond and Rambaut 2007; Drummond et al. 2012). We estimated the evolutionary rate of the OP354-like P[8] gene at 1.30 × 10−3 (95% HPD: 0.97–1.69 × 10−3) nucleotide substitutions per site per year, resulting in a time to the most recent common ancestor (TMRCA) of all OP354-like P[8] genes of 1987 (1985–1988) (table 1). Over time, three main clusters have been formed as shown in the maximum clade credibility (MCC) tree (fig. 2): Cluster I included OP354-like P[8] VP4 genes detected in Europe, the Middle East, and Sub-Saharan Africa, whereas cluster II contained only isolates from South Asia and Sub-Saharan Africa. The third cluster contained OP354-like P[8] VP4 genes isolated from all global regions, including East Asia and the Americas (fig. 2). The TMRCAs for these three clusters were relatively recent, ranging from 1996 to 2001 (table 1). In addition, four strains were found outside these three clusters: two strains isolated in India, RVA/Human-wt/IND/NIV935070/1993/G2P[8] and RVA/Human-wt/IND/NIV951454/1995/G1P[8], which clustered separately but close to the trunk and two strains from Finland, RVA/Human-wt/FIN/Fin-301/1988/G1P[8] and RVA/Human-wt/Fin/Fin-302/1988/G1P[8], which cluster close to the root. Rotaviruses with a OP354-like P[8] gene from a single sampling location were sometimes present in two or three clusters, most notably isolates from Belgium, Israel, South Africa, and Russia. However, the different clusters often contained strains isolated in different rotavirus seasons suggesting multiple independent introductions of OP354-like P[8] strains in these countries. In other cases, OP354-like P[8] rotaviruses detected in one location tended to cluster together (e.g., Bangladesh and Russia), suggesting that after an initial seeding event, localized epidemics of OP354-like P[8] rotaviruses have occurred. OP354-like P[8] strains isolated from neighboring countries were also frequently found in different clusters. For example, OP354-like P[8] strains from Bangladesh were genetically more closely related to OP354-like P[8] strains from Sub-Saharan Africa and more distantly related to rotaviruses isolated from the geographically closer countries India and Pakistan (fig. 2).

Table 1.

Evolutionary Rate and TMRCA of OP354-Like P[8] Strains.

N 98
Sampling time interval 1988–2012
Evolutionary rate 1.30 × 10−3 (95% HPD: 0.97–1.69 × 10−3)
TMRCA All OP354-like P[8] 1987 (1985–1988)
TMRCA cluster I 2000 (1997–2004)
TMRCA cluster II 1996 (1994–1998)
TMRCA cluster III 2001 (1998–2004)
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Fig. 2.

Fig. 2.

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MCC tree of 98 OP354-like P[8] strains. Branches are color-coded according to their most probable location state. The three different clusters are indicated by the green arrows and the timescale is indicated below the tree. Location probabilities at the tree root and the three cluster roots are indicated in the pie charts.

OP354-Like P[8] VP4 Genes Are Associated with Multiple VP7 G-Genotypes

Although we were able to obtain the G-genotype for every strain, sequence information was often missing or limited to a small part of the VP7 gene. Therefore, we investigated to what extent strains with a OP354-like P[8] gene reassort by incorporating the G-genotype of a virus as a discrete state in the Markov chain Monte Carlo (MCMC) framework. Strains with a OP354-like P[8] VP4 were found in combination with five different G-genotypes, G1, G2, G3, G4, and G9. Most strains possessed either a G9 genotype (n = 44) or a G1 genotype (n = 32), whereas G4 (n = 15) and G3 (n = 5) genotypes were less frequently found. Two OP354-like P[8] strains were associated with a G2 genotype, which is relatively rarely found in combination with a P[8] VP4 gene. In every cluster at least three different G-genotypes were found, suggesting frequent reassortment of OP354-like P[8] strains with other human rotaviruses (fig. 3). Although strains in localized epidemics most of the time possessed a similar G-genotype, there was also evidence that reassortment events occurred during local outbreaks as occasionally multiple genotypes were present. This was most clearly observed in Bangladesh (cluster II) and Russia (cluster III). The trunk of the phylogenetic tree was mainly dominated by the G1-genotype, suggesting that the combination of a OP354-like P[8] in combination with a G1 genotype probably yields the fittest constellation as is seen in other P[8] subtypes. Also the root state probability was highest for G1 (60.3%, vs. 23.6% for G2), which indicates that early OP354-like P[8] strains probably possessed the G1 genotype.

Fig. 3.

Fig. 3.

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MCC tree of 98 OP354-like P[8] strains. Branches are color-coded according to their most probable G-genotype. The three different clusters are indicated by the green arrows and the timescale is indicated below the tree. Genotype probabilities at the tree root and the three cluster roots are indicated in the pie charts.

We used the Bayes factor (BF) test to calculate significant nonzero rates of reassortment between G-genotypes and identified the strongest rate between G4 and G2 (BF: 100.7), which could be completely attributed to a localized epidemic in Russia in cluster III (figs. 3 and 4). Also reassortment from G1 to G4 was observed in the same epidemic (BF: 20.9). G1 and G9 were the most abundant genotypes present in our sample set, and strong rates of reassortment were found between these two genotypes (BF: 31.4 and 12.2, for G9 to G1 and vice versa, respectively).

Fig. 4.

Fig. 4.

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Network of reassortment rates between different G-genotypes. Circles are drawn proportional to the number of OP354-like P[8] strains with that particular genotype. The connections are colored according to their origin and indicate the direction of genotype change. For example, the green connection between G1 and G9 indicates the reassortment rate from G1 to G9, whereas the blue connection indicates the reassortment rate from G9 to G1. Connections between circles are proportional to the strength of the reassortment as measured by BFs. Only connections with a BF >3 are shown in bright colors.

Strains with OP354-Like P[8] VP4 Genes Are Globally Dispersed and Migrate Large Distances in a Short Time Period

To investigate viral migration across different regions, we applied a phylogeographic analysis, considering each geographic region as a discrete state and using a BF test to identify significant migration rates. For this, the world was divided into six regions: East Asia and Oceania, Europe, Middle East and North Africa, South Asia, Sub-Saharan Africa, and The Americas (fig. 5).

Fig. 5.

Fig. 5.

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Global migration pattern of OP354-like P[8] strains. Viral migration is indicated by the connections between the different regions and the width of the connection is proportional to the strength of the transmission rate. The color of the connections indicates the origin and hence the direction of migration. For example, the purple connection between East Asia and Oceania and Europe indicates the transmission rate from East Asia and Oceania to Europe, whereas the green connection indicates the transmission rate from Europe to East Asia and Oceania. Only connections with a BF >3 are shown in bright colors. The size of the circles in each region is proportional to the number of samples. Approximate isolation locations are indicated in small circles.

Transmission links were strongest between South Asia and Sub-Saharan Africa (BF: 148.3) and can be primarily attributed to several migration events within cluster II in the early 90’s and in 2007. Despite the strong transmission link with Sub-Saharan Africa, South Asian migration rates to other regions were weak. Only seven OP354-like P[8] strains were sampled from the East Asia and Oceania region, but they possessed the most statistically significant transmission rates. In particular, the transmission rate to Europe was strong (BF: 37.2), which was caused by several independent migration events including from Vietnam to Ireland, Germany and Russia, and from China to Belgium (figs. 2 and 5). OP354-like P[8] strains from Europe were seeded to the Middle East and Sub-Saharan Africa and are mainly the result of migration events occurring in cluster I. Only one OP354-like P[8] strain was sampled in the Americas, which clustered close to Belgian, Japanese, and Chinese isolates, hence the significant transmission rates with the European and East Asian regions (BF: 3.02 and 17.0, respectively).

Dynamics of the OP354-Like P[8] VP4 Gene Over Time

To analyze location and genotype dynamics over time, a time-slice analysis was made each year and the proportion of branches belonging to a particular region or genotype was determined. The OP354-like P[8] gene was present in South Asia throughout the sampling period and until 1994 the majority of branches showed the highest probability for South Asia (fig. 6A). From 1995 until 2003, a significant proportion of branches was observed in Sub-Saharan Africa. Although almost throughout the entire sampling period branches were present with the highest probability for Europe, only after 2003 a sharp increase was seen together with the Middle East and East Asia. The longitudinal analysis of G-genotypes showed that for most of the sample period branches were approximately equally divided between the G1 and G9 genotype (fig. 6B). Before 1995 however, G1 was the most prevalent genotype. The high proportion of branches with the G2 genotype between 1987 and 1992 can be completely attributed to a relatively old isolate from India, RVA/Human-wt/IND/NIV935070/1993/G2P[8].

Fig. 6.

Fig. 6.

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Analyses of viral dynamics during the sampling period. The MCC trees were sliced every year and the proportion of branches with the most probable location or genotype state is indicated in panel A and B, respectively. Regions included are Sub-Saharan Africa (AF), The Americas (AM), East Asia and Oceania (EAO), Europe (EU), Middle East and North Africa (MENA), and South Asia (SA). The relative population size throughout the sampling period is shown in panel C.

We implemented a GMRF skyride to investigate viral population dynamics across the sampling period. From 1988 until 2007/2008, a steady increase in the viral population size was observed, resulting in a 25 times increase in population size in 2007/2008 when compared with 1988. After 2007/2008 the viral population size decreases again, probably as an artifact of the limited number of OP354-like P[8] genes that have been sequenced in more recent years (fig. 6C).

Discussion

In this study, we have traced the spatial and temporal dynamics of OP354-like P[8] VP4 in rotaviruses to elucidate its evolution in the human population. Compared with other VP4 P[8] lineages, strains with the OP354-like P[8] gene have emerged relatively recently and this allowed us to track their evolutionary pathway in more detail than is generally possible for already established, omnipresent rotavirus genotypes or subgenotypic lineages. We have composed a unique set of 98 strains with a OP354-like P[8] gene and showed that they have spread to at least four continents in less than 20 years. South and East Asia played a key role in the migration of the OP354-like P[8] strains as they act as a source for subsequent seeding events to Europe, Sub-Saharan Africa, and North America. Previously, it has been hypothesized that reassortment events leading to omnipresent G12P[8] rotaviruses took place in Asia and the data presented in this study also suggest that Asia is also the most important staging ground for rotaviruses bearing an OP354-like P[8] VP4 (Rahman et al. 2007). It is unclear if the OP354-like P[8] gene is continuously circulating in strains in Sub-Saharan Africa, Western Europe, and the Americas as little evidence was found for localized epidemics in subsequent rotavirus seasons in these regions. Clearly, this is in sharp contrast with Bangladesh and Russia, where OP354-like P[8] rotaviruses could now be considered as endemic.

We used 98 OP354-like P[8] VP4 sequences for our analyses but this is likely only a small fraction of currently circulating OP354-like P[8] rotaviruses worldwide. We cannot rule out that this will affect our modeling of viral migration. For instance, in addition to this data set a number of shorter OP354-like P[8] sequences from Myanmar and Thailand are known (Ghosh et al. 2010; Khananurak et al. 2010). It has been shown that commonly used RT-polymerase chain reaction (PCR) multiplex genotyping assays are unable to distinguish OP354-like P[8] strains from other P[8] strains or even fail to detect OP354-like P[8] strains at all due to mismatches in the primer-binding site (Nagashima et al. 2010). Fortunately, recent modifications of the rotavirus RT-PCR multiplex assay specifically included an additional primer to detect OP354-like P[8] VP4s, which could lead to an increased detection rate of OP354-like P[8] strains (Nagashima et al. 2010). More widespread use of this assay will likely result in much higher detection rates and could possibly also increase the detection rate of OP354-like P[8] strains in the Americas, where thus far only one OP354-like P[8] strain has been detected. However, the main conclusion that OP354-like P[8] rotaviruses are mainly seeded from Asia to other continents is probably only mildly affected by these biases. For example, it was previously reported that a German child infected with OP354-like P[8] strain RVA/Human-wt/GER/GER15-08/2008/G1P[8] had been traveling to Vietnam weeks before the onset of symptoms (Pietsch et al. 2011). Our phylogenetic analysis showed that GER15-08 was indeed closely linked to three Vietnamese OP354-like P[8] rotaviruses, which also possessed a similar G-genotype as GER15-08 and were only 0.7% different on the nucleotide level (figs. 2 and 3, cluster III). Possibly, this represents an important way for the introduction of novel (variants of) genotypes from developing countries to Europe and North America.

The vaccine effectiveness of Rotarix and RotaTeq against OP354-like P[8] strains is currently unknown, yet this information could be important for the reduction of rotavirus gastroenteritis in South Asia, as OP354-like P[8] rotaviruses are relatively prevalent in this region and vaccine usage still limited. Because the VP4 genes present in the vaccines belong to P[8] lineage I and P[8] lineage II for Rotarix and RotaTeq, respectively (Matthijnssens, Joelsson, et al. 2010; Zeller et al. 2012), it is possible that protection afforded by the vaccines against OP354-like P[8] rotaviruses might be less than against other more prevalent P[8] lineages (Zeller et al. 2012). Particularly, since the relatively large genetic distance between OP354-like P[8] strains and both vaccine strains is also translated to multiple differences in antigenic epitopes. These amino acid differences were predominantly found in the 8-1 epitope of VP8*, but also extended to VP5* (Zeller et al. 2012) and could result in a decreased vaccine effectiveness. Currently, there are a few indications that infections with OP354-like P[8] rotaviruses could also have a more severe disease outcome than infections with other P[8] strains belonging to other lineages (Nguyen et al. 2008; Dong et al. 2011). The more severe disease outcome together with a potential reduced vaccine effectiveness warrants a close monitoring of OP354-like P[8] strains, in particular in countries where national immunization programs are in place.

We have found rotaviruses with an OP354-like P[8] VP4 can contain a wide range of G-genotypes, those commonly associated with a Wa-like genotype constellation (G1, G3, G4, and G9) and also two G2 genotypes. This suggests that the VP4 OP354-like P[8] gene frequently reassorts in a similar way as other P[8] VP4 lineages. Complete genome sequences of OP354-like P[8] strains are relatively rare, and currently 13 are available. Among these, all but one showed a typical human Wa-like genetic background. RVA/Human-wt/ZAF/MRC-DPRU2144/2003/G9P[8] was the only strain that possessed a DS-1-like genotype constellation. In general, P[8] genotypes are rarely found in combination with a DS-1-like genetic background and whether the DS-1-like background of MRC-DPRU2144 is a rarity among OP354-like P[8] strains or not, can only be determined by collecting more complete genome data of OP354-like P[8] RVAs.

In three of the earliest OP354-like P[8] strains identified, MRC-DPRU2144 (2003), Fin-301 and Fin-302 (both 1988), a two-nucleotide insertion in the 5′-end noncoding region of the VP4 segment was observed (data not shown). Although we have determined the 5′-end of 41 OP354-like P[8] strains, including strains belonging to cluster II (data not shown), only MRC-DPRU2144 and both strains from Finland possessed this insertion. To our knowledge, this insertion is also absent in any other human P[4], P[6], or P[8] rotavirus. The fact that rotaviruses detected in South Africa and Finland contain a similar insertion is surprising and could indicate a wide, but seemingly unnoticed, spread in humans in the past of this particular variant. Alternatively, this particular variant could have been circulating in animals for a prolonged period of time and MRC-DPRU2144, Fin-301, and Fin-302 could be the result of two independent interspecies transmission events. The only other animal in which P[8] rotaviruses have been detected to date are swine, making them also the prime suspect as a reservoir for OP354-like P[8] rotaviruses (Halaihel et al. 2010; Midgley et al. 2012; Amimo et al. 2014). However, the few porcine P[8] strains that have been characterized did not belong to the OP354-like lineage. A third possibility could be that these insertions were generated de novo in both locations. Thus, sequencing of archival human and animal strains will be helpful to answer this question. Whether OP354-like P[8] will be an important genotype in the human rotavirus population in the future remains to be seen: genotypes such as P[6] are of local importance, but are still not globally prevalent (Santos and Hoshino 2005). Other previously emerging genotypes such as G9 and G12 now circulate worldwide (Matthijnssens, Heylen, et al. 2010). Rotavirus surveillance remains one of the most powerful tools to address this question in the future.

Materials and Methods

Sample Collection

For some OP354-like P[8] genes only short nucleotide sequences are available in GenBank. For this study an attempt was made to retrieve fecal samples of all rotavirus strains possessing an OP354-like P[8] gene available in GenBank in order to determine the nucleotide sequence of the complete open reading frame (ORF). We have determined the VP4 ORF of 54 OP354-like P[8] strains that were detected during rotavirus surveillance in Bangladesh, Belgium, Germany, Ghana, Israel, Jordan, Pakistan, and Russia. In addition, we completed the VP4 ORF of 16 OP354-like P[8] strains isolated from Finland, India, Germany, Malawi, Pakistan, Vietnam, and Ireland of which a short fragment of VP4 was already deposited in GenBank. These were supplemented with 28 completely sequenced OP354-like P[8] genes that were already deposited in GenBank, resulting in a total data set of 98 OP354-like P[8] genes. A detailed overview of the viral strains and accession numbers used in this study can be found in supplementary table S1, Supplementary Material online.

RNA Extraction, RT-PCR, and Sequencing

For each stool sample, viral RNA was extracted using the QIAamp Viral RNA mini-kit (Qiagen/Westburg, The Netherlands) according to the manufacturer’s instructions. Ten microliters RNA were denatured at 95°C for 2 min, and RT-PCR was carried out using the Qiagen One Step RT-PCR kit (Qiagen/Westburg, The Netherlands) using forward primer VP4_1-17F (5′-GGC TAT AAA ATG GCT TCG C-3′), in combination with the reverse primer VP4_P[8]_2328R (5′-CAT TGT AGA ATT ARY TGT TCA ATT CTA TTC C-3). The RT-PCR was carried out with an initial RT step at 50°C for 30 min; Taq polymerase activation was at 95°C for 15 min, followed by 35 cycles of amplification (30 s at 94°C, 30 s at 45°C, and 3 min at 72°C), with a final extension of 10 min at 72°C. When sufficient sample material was available, the 5′- and 3′-ends of the VP4 ORF were covered using a single-primer amplification method as described previously using the internal primers 5′-ATG ACC CCA ATT GAC TGG-3′ and primer 5′-CGG ATT CTC CAG TTA TAT CAG C-3′, respectively (Matthijnssens et al. 2006).

The PCR amplicons were purified with EXO-SAP-it (Affymetrix) and sequenced using the dideoxynucleotide chain termination method with the ABI PRISM BigDye Terminator Cycle Sequencing Reaction kit (Applied Biosystems) on an automated sequencer (ABI PRISM 3130). Sequencing was performed with the forward and reverse primers used for the RT-PCR. In addition, primer-walking was performed to cover the complete ORF of the VP4 segment. The chromatogram sequence files were analyzed using Chromas 2.3 (Technelysium, Queensland, Australia), and consensus sequences were prepared using SeqMan II (DNAstar, Madison, WI).

Phylogenetic Analysis

The 70 newly sequenced VP4 genes were analyzed together with 28 OP354-like P[8] sequences deposited in GenBank. In total, 98 sequences were used for Bayesian phylogenetic tree reconstruction applying an MCMC analysis as implemented in the BEAST package (v1.8.1) (Drummond and Rambaut 2007). The VP4 phylogeny was estimated using the SRD06 model (Hasegawa–Kishino–Yano with four category gamma distributed rate variation among sites allowing for invariant sites) and an uncorrelated lognormal relaxed molecular clock. Changes in relative population size were reconstructed with a GMRF skyride plot. In total, five independent MCMC runs of 108 generations each were conducted sampling every 10,000 generations after discarding 10% as burn-in. The MCC tree of the OP354-like P[8] gene was summarized with TreeAnnotator and visualized using FigTree. To determine the viral transmission network a phylogeographic model was used dividing the world in six different regions (East Asia and Oceania, Europe, Middle East and North Africa, South Asia, Sub-Saharan Africa, and The Americas), which were used as discrete states (Lemey et al. 2009). We used SPREAD for calculation of BFs to determine strong links of transmission (Bielejec et al. 2011). In addition, we also used a discrete model to estimate the probability of the G-genotype along every branch of the MCC tree.

The VP4 maximum likelihood tree was constructed in Mega 5 with 1,000 bootstrap replicates using the OP354-like P[8] data set supplemented with all available (near-)complete P[4] and P[8] VP4 genes in GenBank (n = 1,137; Tamura et al. 2011). A general time reversible model allowing for invariant sites was used to model nucleotide substitution rates. Mega 5 was also used to calculate pairwise genetic distances (P-distance option) between P[8] and P[4] VP4 genes.

Supplementary Material

Supplementary table S1 is available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).

Supplementary Data
supp_32_8_2060__index.html (918B, html)

Acknowledgments

M.Z. was supported by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT Vlaanderen).

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