By nucleotide sequence identities and phylogenetic analyses of the nearly full-length nucleotide sequences, the VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4- NSP5 genes of strains E1911, R588 and Y128 were assigned to the G1-P-I1-R1-C1-M1-A1-N1-T1-E1-H1 genotypes, respectively (A–K). Therefore, all the three Chinese G1P RVA strains exhibited a typical Wa-like genotype constellation. With the exception of the NSP3 gene of strain E1911, strains E1911, R588 and Y128, detected in an infant, a child and an adult, respectively, were found to be closely related (nucleotide sequence identities of 97.5–99.8%) to each other (A–K).
The VP7 genes of strains E1911, R588 and Y128 exhibited high nucleotide sequence identities (99%) to those of several other recent G1 strains. Phylogenetically, the VP7 genes of the Chinese G1P strains clustered into a subcluster (shown as G1-L6-S1) that primarily consisted of G1 strains from China and Japan within G1-Lineage 6, different from those of the G1 strains contained in the RVA vaccines RotarixTM
(Lineage 5) and RotaTeqTM
(Lineage 1) (A). By multiple alignment, the deduced amino acid sequences of the VP7 of the Chinese G1P strains exhibited 17–18, 14–15 and 21–22 mismatches with those of the prototype G1P strain Wa, VP7 of RotarixTM
and G1 component of RotaTeqTM
, respectively. To date, the RVA P strains have been classified into two genetically distinct subtypes, Pa and Pb (also referred to as OP354-like RVAs) [19
]. The VP4 genes of strains E1911, R588 and Y128 were closely related (nucleotide sequence identities of 99%) to those of the recent Pa strains from different countries, and phylogenetically, appeared to cluster within the same subcluster (shown as Pa-S1) as the P component of RotaTeqTM
, whilst the VP4 gene of RotarixTM
was found to cluster into the other subcluster (designated as Pa-S2) within the Pa lineage (B).
Deduced amino acid residues defining the RVA VP4 and VP7 epitopes have been identified by neutralization escape mutants and identifying surface exposed amino acid residues that show intergenotypic variability among prevalent human G- and P- genotypes [20
]. With the exception of a single residue in VP4 of E1911, these amino acid residues were conserved among the three Chinese G1P strains (, ). Recently, the nucleotide sequences of the VP4 and VP7 genes of the G1 and P strains contained in the currently licensed rotavirus vaccines RotarixTM
have been reported [24
], allowing us to compare for the first time these genes with those of the Chinese G1P strains. With the amino acids defining the VP7 epitopes of G1 strains in RotarixTM
, the Chinese G1P strains differed in 5 and 7 residues, respectively (). Alignment of the amino acid residues defining the VP4 neutralization domains revealed 8 mismatches between the Chinese G1P strains and the P strain in RotarixTM
(). On the other hand, strains Y128 and R588 differed in 5 residues, whilst strain E1911 exhibited 4 mismatches with those in the VP4 of the P strain in RotaTeqTM
Figure 1 (A-K) Phylogenetic trees constructed from the nucleotide sequences of VP7, VP4, VP6, VP1-3 and NSP1-5 genes of rotavirus strains RVA/Human-wt/CHN/E1911/2009/G1P, RVA/Human-tc/CHN/R588/2005/G1P,RVA/Human-tc/CHN/Y128/2004/G1P, with those of the (more ...)
Figure 2 Alignment of the amino acid residues defining the neutralization domains (designated as 7-1a, 7-1b and 7-2 ) of VP7 between the G1 strains in Rotarix™ and RotaTeq™ and Chinese RVA strains Y128, R588 and E1911. Green indicates the residues (more ...)
Figure 3 Alignment of the amino acid residues corresponding to those defining the VP4 neutralization domains (designated as 8-1, 8-2, 8-3 and 8-4 in the VP8* subunit (A) and 5-1, 5-2, 5-3, 5-4 and 5-5 in the VP5* subunit (B) of VP4) ) between the P strains (more ...)
Among the other genes, the VP1-3, VP6, NSP2 and NSP4-5 genes of strains E1911, R588 and Y128 were closely related (nucleotide sequence identities of 97–99%) to those of several Wa-like common human RVA strains, such as G1, G3, G9, and/or G12, detected in the 2000s from different countries (C-F, H and J-K). Phylogenetically, the NSP1 genes of the recent Wa-like common human RVAs appeared to be grouped into two distinct subclusters, designated as A1-S1 and A1-S2, within genotype A1 (G). The NSP1 genes of strains E1911, R588 and Y128 were found to be closely related (nucleotide sequence identities of 97-99%) to those of the recent G1P and G3P strains within subcluster A1-S2 (G). The NSP3 genes of strains Y128 and R588 shared high nucleotide sequence identities (97-99%), and phylogenetically, clustered with several recent Wa-like common human G1,G3, G9 and G12 strains to form a subcluster (shown as T1-S1) (I). On the other hand, the NSP3 gene of strain E1911 shared low nucleotide sequence identities of 89.2% with those of Y128 and R588, and exhibited a maximum nucleotide sequence identity of 99.5% to that of G9P strain RVA/Human-tc/KOR/CAU09-376/2009/G9P from South Korea. Phylogenetically, the NSP3 genes of strains E1911 and CAU09-376 clustered together (shown as subcluster T1-S2), near the subcluster of porcine-like human G9 strains from India (strains RVA/Human-wt/IND/RMC321/ 1990/G9P, RVA/Human-wt/IND/mani-97/2006/G9P and RVA/Human-wt/IND/mcs/13-07/ 2007/G9P) [26
], and were genetically distinct from those of strains Y128, R588, other recent Wa-like common human strains, and the prototype G1P strain, Wa (I). Strain E1911 was found to share nucleotide sequence identities of 93.3%, 92.4% and 91.3% with the NSP3 genes of strains mani-97, mcs/13-07 and RMC321, respectively.
Taken together, with the exception of the NSP3 gene of E1911, the genomes of the Chinese RVA G1P strains E1911, R588 and Y128 were found to be more closely related to those of the recent Wa-like common human strains, such as G1, G3, G9, G12, from different countries than those of the prototype G1P strain Wa, or other old Wa-like strains (A-K). On the other hand, phylogenetically, the NSP3 gene of strain E1911 clustered near those of the porcine-like human G9 strains from India, and taken together, these strains appeared to share a more common origin with those of the porcine RVAs than those of the Wa-like common human strains (I). Since the remaining genes of E1911 were closely related to those of Y128 and R588, it is likely that strain E1911 acquired its NSP3 gene through inter-genotype reassortment events. The infant infected with strain E1911 lived in the central city of Wuhan, and therefore, it is unlikely that he came in direct contact with a pig. It may be possible that the infant was infected from food or water contaminated with strain E1911, or from an adult who came in close contact with pigs. However, analysis of the NSP3 genes of locally circulating and other Chinese porcine RVA strains might be required to pinpoint the exact source of the NSP3 gene of E1911. To date, the NSP3 gene sequences of only a single porcine and a few human RVA strains are available from China, as evident from the GenBank database. Recently, the whole genomes of four Chinese human G3P RVA strains have been sequenced in our laboratory (unpublished data). None of these human RVA strains from China were found to possess a porcine-like NSP3 gene [28,29, unpublished data]. Nevertheless, genetic analyses of several human and porcine strains might be required to determine as to whether the porcine-like NSP3 genes are common in RVAs prevailing in Chinese children, or strain E1911 is rare in nature.
In conclusion, whole genomic analyses of the recent Chinese G1P strains revealed a stable Wa-like genetic backbone that might be circulating in majority of the recent Wa-like common human RVAs, such as the G1P, G3P, G4P and G9P strains, worldwide. It has been hypothesized that RVAs with this genetic backbone have the ability to propagate extremely well in the human host, as evidenced from the detection of large numbers of Wa-like human RVA strains across the globe [11
]. Comparison of the whole genomes of the Chinese G1P strains from different age groups revealed a close genetic relationship among these RVAs, suggesting that genetically identical G1P strains might be circulating among children and adults in Wuhan city, China. Although the present study provided important insights into the origin and overall genetic makeup of the widely circulating human G1P RVA strains in China, it was limited to only three recent strains. Whole genomic analyses of additional RVA strains from different geographical regions might be required to gain a proper understanding of the evolutionary dynamics of the primary RVA strains in China.
The currently licensed RVA vaccines, RotarixTM
, have been found to be effective against the common human RVA strains, resulting in substantial declines in rotavirus and/or diarrhea-related hospitalization in many countries [30
]. Although there were concerns on the efficacy of the monovalent G1P vaccine, RotarixTM
, against completely heterotypic strains, RotarixTM
was shown to be effective against the common human G2P RVAs in Brazil [30
]. However, these vaccines are yet to be introduced in China. Since the Chinese G1P RVAs share close genetic similarity with those of recent G1P RVAs from other countries where routine RVA vaccination has yielded good results, it might be possible that these vaccines will cause a sharp reduction in the number of cases of RVA associated diarrhea in China. However, comparisons of the amino acid residues defining the VP7 and VP4 antigenic domains revealed several mismatches between the recent Chinese G1P strains and the G1 and P strains contained in RotarixTM
. Implications of these changes on the efficacy of these vaccines, if any, need to be monitored after their introduction in China. There is evidence that genes other than VP7 and VP4 might influence the immune response in the host following RVA vaccination [3
]. Continuous vaccine-induced immunological pressure may cause changes in these genes that are detrimental to the efficacy of the current RVA vaccines [3
]. Therefore, large scale whole genome-based studies on common human RVA strains from different countries are required to identify these vaccine-induced changes in the RVA genome. To our knowledge, this is the first report on the whole genomic analysis of G1P RVA strains from China.