Molecular Genotyping of Irish Rotavirus Strains Fiona OHalloran and Seamus Fanning

1. Introduction

Rotavirus is the primary etiological agent of gastroenteritis in infants and young children worldwide (1). In developing countries, it is estimated that rotavirus is responsible for one-third of all diarrhea-associated hospitalizations and 873,000 deaths annually (2,3). In industrialized countries, where mortality from rotavirus is low, infection is widespread, and nearly all children experience an episode of rotavirus diarrhea in the first 3-5 yr of life (4,5).

Rotaviruses have important antigenic specificities including serogroup and serotype, and all viruses are classified accordingly. They are divided into seven morphologically indistinguishable but antigenically defined serogroups, delineated A through G (6,7). The human infecting rotaviruses include groups A, B, and C, and it is well documented that group A rotaviruses are the major causative agents of diarrheal diseases in children (8,9). They are responsible for 125 million cases of diarrhea annually (10,11).

Within each serogroup, distinct serotypes exist. In group A rotavirus, serotype is specified by two viral proteins, VP4 and VP7. The neutralizing antibody response that is evoked by the antigenic determinants on VP4 and VP7 play an important role in protective immunity (12). The rotavirus genome consists of 11 double-stranded (ds) RNA segments, and each genomic segment encodes a different protein. A dual system of reporting rotavirus serotype exists because the VP4 and VP7 proteins are encoded by different genes and thus can segregate independently (13). The serotypes derived from VP7 are defined as G-serotypes. Currently 14 G-serotypes have been identified, and only 10 of these have been recovered from humans (12). The predominating G-types worldwide are G1, G2, G3, and G4, with G1 being the most prevalent type (14-16). Serotypes G5, G6, G8-G10, and G12 are rarely identified in humans and are usually recovered from animals. However, some of these unconventional types are now being frequently reported in humans, including G5, G8, and G9 (17-20).

The serotypes derived from VP4 are designated P-types (P from protease-sensitive protein) (21). Currently 13 P-serotypes have been reported, and 9 of these have been identified in humans (22). The two main P-types that predominate include P[8] and P[4] (23), and an association between G- and P-type has been observed (12). Extensive epidemiological studies in several health care systems characterizing rotavirus strains have identified the prevalent serotypes circulating in children within different populations. The predominating types recognized were G1P[8], G2P[4], G3P[8], and G4P[8] (10,24,25).

Several laboratory strategies are available to identify and characterize rotavirus isolates. Owing to the segmented nature of the rotavirus genome, gel electrophoresis is often useful and can identify genetic heterogeneity within isolates as well as monitor virus transmission (26). Migration of the dsRNA segments in polyacrylamide gels is the most frequently used gel electrophoresis method, producing distinct electropherotype patterns. A classification scheme was devised for group A rotavirus strains, based on their migration patterns (27). All 11 gene segments are divided into four groups based on their migration to one of four characteristic regions in a nondenaturing polyacrylamide gel. Group I includes gene segments 1-4; group II contains segments 5 and 6; group III contains segments 7-9; and group IV contains the remaining segments, 10 and 11 (Fig. 1). Within each group distinct dsRNA banding patterns are observed resulting from differences in the rates of migration of cognate gene segments. These variations (or heterogeneity, as it is often referred to) are attributed to base sequence polymorphisms, together with, distinctive secondary and tertiary structure of each dsRNA fragment (28,29). Sequence and corresponding structural differences can be detected based on the altered migration pattern(s) of the dsRNA segments under native conditions. The migration rate of segments 10 and 11 (group IV) broadly divides the migration patterns into two groups. Fast migration of these two gene segments generates a long electropherotype, and slow migration is referred to as a short electropherotype (Fig. 2A). Some authors have reported an association between specific G-types and a given electropherotype. For example, G1 and G4 strains predominantly demonstrate a long electropherotype, whereas G2 strains usually have short electropherotypes (30).

This classification scheme is specific to the genome profiles characteristic of group A rotavirus, which are the most prevalent rotavirus strains found in both humans and animals. The migration patterns demonstrated by nongroup A rotavirus are markedly different, as the gene segments do not migrate to the same four distinct regions of the gel. Electropherotyping may therefore be used to differentiate between different rotavirus serogroups. The presence of more than 11 segments in the electropherotype indicates that more than one strain has infected the cells, and this is indicative of a mixed infection (Fig. 2B). Mixed infections can potentially lead to the formation of novel reassortant strains. These strains often demonstrate atypical migration patterns and thus can be identified by their particular array of genome segments in polyacryla-mide gels (31).

Rotavirus serotypes can be defined by antigen-based methods (e.g., enzyme-linked immunoassays) or molecular protocols, including reverse transcriptase (RT)-mediated

Group I Group II Group III Group IV

iii n

Region 1 Region 2 Region 3 Region 4

Fig. 1. A typical migration pattern displayed by group A rotavirus on a nondenaturing Polyacrylamide gel. The 11 dsRNA segments migrate to one of four distinct regions on the gel and are grouped accordingly.

Fig. 2. Electropherotype patterns of rotavirus RNA generated by polyacrylamide gel electrophoresis. (A) Lanes 1-3 contain the typical "long" electropherotypes associated with group A and lanes 4-5 contain "short" electropherotypes. (B) A mixed electropherotype pattern isolated from a single rotavirus isolate.

Fig. 2. Electropherotype patterns of rotavirus RNA generated by polyacrylamide gel electrophoresis. (A) Lanes 1-3 contain the typical "long" electropherotypes associated with group A and lanes 4-5 contain "short" electropherotypes. (B) A mixed electropherotype pattern isolated from a single rotavirus isolate.

polymerase chain reaction (PCR). Standard serological methods for virus identification often lack the required sensitivity to distinguish usefully between alternate isolates of a virus serotype. In addition, these strategies cannot detect newly evolved viral variants or identify the corresponding nucleotide mutations and amino acid substitutions that alter antigenic specificities. Molecular techniques including probe hybridization, restriction enzyme analysis, and in particular PCR-based assays have provided additional biochemical and serological information. These strategies have recognized technical advantages over conventional methods.

Nucleotide sequence analysis of each of the 11 dsRNA genomic segments from different rotavirus strains identified unique features relevant to the structure of rotavirus genes. These were conserved in all 11 dsRNA segments (32). Significantly, the 5'- and 3'- ends of each genomic segment were found to be highly conserved among all strains analyzed. Each RNA segment had a single 5'-guanylate followed by a conserved sequence motif forming part of a 5'-noncoding region. Immediately downstream was located an open reading frame (ORF) coding for the specific protein product. The latter ORF was followed by another noncoding consensus sequence at the 3'-end. Using these sequence data, oligonucleotide primers were designed to be complementary to the negative ends of the VP4- and VP7-encoding RNA strands. These forward (VP7F; VP4F) and reverse (VP7R; VP4R) primer pairs (Table 1) were used in RT-PCR assays under defined reaction conditions to amplify the full-length (1062-bp) VP7 gene segment and a partial (867 bp) VP4 gene segment (33,34). Comparative sequencing of the corresponding gene segments encoding VP4 and VP7 provided sufficient unique data to facilitate the design of molecular serotyping strategies.

Sequence analysis of VP7 gene segments from several different strains identified six discrete regions, designated A-F, with significant amino acid sequence divergence (Fig. 3). These regions were shown to be unique among different serotypes but highly conserved within any given serotype. Using each of these six variable regions as a blueprint for a distinct serotype, specific primers, whose sequences were complementary to the negative RNA strand of the VP7 gene, were designed (34). These serotype-specific primers, along with a common (reverse) primer complementary to the 3'-end of the opposite strand, were included in a PCR reaction cocktail. Under defined conditions, PCR products of discrete segment lengths were generated. The variable regions under investigation are located at discrete distances from the distal end of the VP7 gene; thus the characteristic size of each amplicon is itself an indication of the viral serotype. Figure 3 shows a schematic representation of this PCR-serotyping strategy. Corresponding sizes of each amplicon are dependent on the primer design (35). Furthermore, the major human G-types (G1-G4) and other G-types can now be identified using this strategy. All major animal serotypes including G5, G6, G10, and G11 (36) can also be identified. This molecular strategy is sufficiently sensitive and has been extensively applied to several epidemiological studies to examine the geographical distribution of human rotavirus G-types (14,17,22,37-40). A useful feature of this PCR typing protocol is its ability to detect mixed G-type infections. In this event, when more than one VP7 G-serotype is present, additional amplicons are detected after conventional agarose gel electrophoresis.

Table 1

Oligonucleotide Primers Used in PCR Amplification Methods

Table 1

Oligonucleotide Primers Used in PCR Amplification Methods

Primer

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Responses

  • annalise young
    What is the genome size of group A rotavirus?
    3 years ago

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