Molecular Genotyping of Irish Rotavirus Strains

Abstract

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).

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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).

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.

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 polyacrylamide gels (31).

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

Table 1 Oligonucleotide Primers Used in PCR Amplification Methods

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.

Fig. 3.

Schematic representation of the seminested PCR assay that identifies the G-type of a rotavirus isolate. The locations of the variable regions on gene segment 9 are indicated. PCR primers identify distinct serotypes by amplifying PCR products of defined length (see also Table 1 for primer sequences).

For complete serotype characterization of a viral strain, both the P-serotype (VP4) and G-seroytype (VP7) must be determined. Gentsch et al. (33), using a similar strategy, devised a PCR protocol that would facilitate the direct identification of rotaviral P-types. Comparative sequencing studies of VP4 genes from different strains revealed genetically distinct regions that were again used to design P-typing primers. These primers generated PCR products that were characteristic for the different P-types.

The potential genomic dynamics of the rotavirus necessitates continuous surveillance to monitor rotavirus infection and facilitate detection of novel strains. The combined use of electropherotyping and PCR serotyping protocols is useful in defining the epidemiology of rotavirus and identifying any novel strains in a population.

2 Materials

2.1 RNA Isolation Procedures for Rotavirus (see Note 1)

2.1.1 Inactivation of Endogenous Ribonucleases (see Note 2 )

• 1. Diethyl-pyrocarbonate (DEPC).

• 2. RNase AWAY (Molecular Bioproducts, San Diego, CA).

2.1.2 RNA Extraction: SDS/Proteinase K Digestion-Phenol Chloroform Extraction

• 1. 10 mg/mL Proteinase K, prepared using DEPC water.

• 2. 10% (w/v) Sodium-dodecyl sulfate (SDS); prepared using DEPC water.

• 3. Chloroform.

• 4. Absolute ethanol.

• 5. 1∶1 Phenol/chloroform (see Note 3 ).

• 6. Sterile 1.5-mL Eppendorf tubes.

2.1.3 Removal of Contaminating Genomic DNA (see Note 4 )

• 1. 70% (v/v) Ethanol.

• 2. 3∶1 Phenol/chloroform.

• 3. 3 M Sodium acetate.

• 4. DNase 1 (RNase free; Roche Diagnostics, East Sussex, UK).

• 5. 10X Tris buffer: 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 5 mM MgCl₂.

• 6. Nuclease free water (Promega, Madison, WI).

2.2 Electropherotying of Rotavirus RNA Genomes

2.2.1 Polyacrylamide Gel Electrophoresis (PAGE)

• 1. 38% (w/v) Acrylamide/bisacrylamide mix: prepared by dissolving 74 g of acrylamide (molecular biology grade) and 2 g bis-acrylamide (molecular biology grade) in 200 mL DEPC-water. Store in a dark bottle at room temperature.

• 2. 1 M Tris-HCl, pH 6.8.

• 3. 1.5 M Tris-HCl, pH 8.8.

• 4. 10% (w/v) Ammonium persulfate (Sigma, Poole, UK).

• 5. 10% (w/v) SDS.

• 6. TEMED (N,N,N′,N′-Tetramethylethylenediamine; Sigma).

• 7. Isobutanol.

• 8. 5X Tris-glycine buffer: 25 mM Tris-HCl, 250 mM glycine, pH 8.3, 0.1% (w/v) SDS.

• 9. Loading buffer: 62 mM Tris, 2% (w/v) SDS, 0.001% (w/v) bromophenol blue, 10% (v/v) glycerol.

2.2.2 Staining and Detection of Polyacrylamide Gels

• 1. Buffer 1: 10% (v/v) ethanol, 0.5% (v/v) glacial acetic acid.

• 2. 0.011 M Silver stain.

• 3. Buffer 2: 0.75 M NaOH containing 0.1 M formaldehyde and 0.0023 M sodium borohydride.

• 4. Buffer 3: 0.07 M sodium carbonate.

2.3 PCR Amplification Methods

• 1. 100 mM Stocks of each deoxyribonucleoside triphosphate (dNTP) dATP, dCTP, dGTP and dTTP (Promega).

• 2. dNTP working stock solutions containing 1.25 mM of each dNTP, prepared by diluting 2.5 µL of each dNTP in 190 µL of sterile water.

• 3. 10X PCR buffer: 100 mM Tris-HCl, pH 9.0, 500 mM KCl (Promega).

• 4. 25 mM MgCl₂ (Promega).

• 5. 5 U/mL Taq DNA Polymerase (Promega).

• 6. AMV (avian myeloblastosis virus)-reverse transcriptase (AMV-RT; Promega).

• 7. 5X AMV buffer: 250 mM Tris-HCl, pH 8.3, 250 mM KCl, 50 mM MgCl₂, 2.5 mM spermidine, 50 mM dithiotreitol (DTT).

• 8. 1 mM DTT (Sigma).

• 9. Dimethyl sulphoxide (DMSO).

• 10. Rnasin® ribonuclease inhibitor (Promega).

• 11. Sterile mineral oil (Sigma).

• 12. Sterile water.

• 13. Oligonucleotide primers ( Table 1 ) were adjusted to the required concentrations. (All primers were obtained from Oswel Scientific and purified by high-performance liquid chromatography prior to use.)

2.4 Purification of PCR Products

• 1. PCR purification kit (QIAGEN, West Sussex, UK).

2.5 Conventional Agarose Gel Electrophoresis

• 1. Agarose (Promega).

• 2. 10X Tris-acetate EDTA (TAE): 40 mM Tris-HCl, pH 7.8, 40 mM glacial acetic acid, 2 mM EDTA.

• 3. 10X Gel loading buffer: 6.6 mL glycerol, 3.3 mL 10X TAE, 100 µL of 10% (w/v) bromophenol blue.

• 4. Ethidium bromide (10 mg/mL; Sigma).

• 5. DNA molecular weight markers: a range of DNA markers are commercially available (Roche Diagnostics) that have various fragment molecular weight ranges. In this study three DNA markers were used, including DNA marker XIV, which has 16 fragments that range in size from 100 to 1500 bp with an additional band at 2642 bp; DNA marker III providing 13 fragments with sizes ranging from 0.12 to 21.2 kbp; and DNA marker V, which contains 22 fragments ranging in sizes from 8 to 587 bp.

3 Methods

3.1 RNA Isolation Procedures for Rotavirus

3.1.1 Inactivation of Endogenous Ribonucleases

• 1. Prepare ribonuclease-free water by adjusting the volume of water required to contain 0.2% (w/v) DEPC.

• 2. Incubate the treated water at 37°C for 12 h and then autoclave at 121°C for 30 min.

• 3. Immerse glassware and nonsterile plasticware in a solution of 0.2% (v/v) DEPC and then autoclave at 121°C for 30 min.

• 4. Prepare buffers used in RNA procedures using DEPC-treated water. Buffers that cannot be autoclaved should be sterilized by filtration through a 0.2-µm filter.

3.1.2 RNA Extraction

• 1. Into a sterile 1.5-mL Eppendorf tube add the required volume of fecal filtrate (typically 400 µL). Adjust the volume to contain 1% (w/v) SDS and 100 µg/mL of proteinase K. Mix by pipeting and incubate at 37°C for 1 h.

• 2. To each reaction mixture add an equal volume of phenol/chloroform mixture (1∶1) and vortex the sample for 30 s.

• 3. Centrifuge at 11,000g for 10 min, which separates the sample into two phases.

• 4. Collect the aqueous phase into a new sterile 1.5-mL Eppendorf tube and add an equal volume of chloroform.

• 5. Mix the sample by vortexing for 30 s followed by centrifugation at 11,000g for 10 min. Again collect the aqueous phase.

• 6. To precipitate the template 11 dsRNA segments, add 2 vol of cold absolute ethanol to the aqueous phase, and incubate the sample at −80°C overnight (12–18 h).

• 7. Recover the purified dsRNA by centrifugation (11,000g for 10 min) and then resuspend the pellet of RNA obtained in 100 µL nuclease-free water.

3.1.3 Removal of Contaminating Genomic DNA

• 1. Incubate the total RNA recovered (approx 100 µL) with 2 µL DNase 1 and 10.2 µL 10X Tris buffer (to give a final working solution of 1X) at 37°C for 30 min. This step allows sufficient time for any copurifying chromosomal DNA to be degraded.

• 2. To this solution add 80 µL of phenol/chloroform (3∶1) reagent and vortex the sample for 1 min.

• 3. Centrifuge at 11,000g for 5 min and collect the aqueous phase as before into a sterile 1.5-mL Eppendorf tube.

• 4. Finally, precipitate the DNA-free purified dsRNA with 10 µL of 3 M sodium acetate and 300 µL of cold absolute ethanol.

• 5. Incubate the above overnight at −20°C and recover the dsRNA by centrifugation for 10 min at 10,000 rpm.

• 6. Wash the dsRNA pellet with 1 mL of cold, 70% (v/v) ethanol and resuspend the pellet in 100 µL DEPC-water.

• 7. Purified RNA can be analyzed by conventional agarose gel (1.5%) electrophoresis and rotaviral genomic segments visualized after staining with ethidium bromide (0.1 mg/mL). Store the dsRNA at −80°C.

3.2 Examination of the Electropherotype Patterns of Rotavirus Strains

3.2.1 Polyacrylamide Gel Electrophoresis

• 1. Vertical gel electrophoresis apparatus (Life Technologies, Paisley, UK).

• 2. Initially wash the glass plates in 1% (w/v) SDS, rinse in deionized water, and dry the plates completely.

• 3. Prepare 70 mL of a 10% (w/v) polyacrylamide gel by initially preparing the resolving gel (see Note 5 ).

• 4. Add (in order) 23.28 mL distilled water, 13.2 mL 38% (w/v) acrylamide mix, 12.5 mL 1.5M Tris-HCl, pH 8.8, 1.5 mL 10% (w/v) SDS, 0.5 mL 10% (w/v) ammonium persulfate, and 0.02 mL TEMED.

• 5. Pour this mixture immediately into the gel cast leaving sufficient space for the stacking gel (approx 2 cm).

• 6. Overlay the acrylamide solution with isobutanol (see Note 6 ) and allow sufficient time for polymerization to occur (approx 30 min).

• 7. Pour off the overlay and wash the gel surface several times with deionized water.

• 8. Prepare 20 mL of the stacking gel (see Note 5 ) by adding (in order) 11.78 mL distilled water, 5.3 mL 38% (w/v) acrylamide mix, 2.5 mL 1 M Tris-HCl, pH 6.8, 0.2 mL 10% (w/v) SDS, 0.2 mL 10% (w/v) ammonium persulfate, and 0.02 mL TEMED. Mix the components and pour directly onto the resolving gel.

• 9. Insert a Teflon comb into the stacking gel and continue polymerization for a further 30 min at room temperature.

• 10. Remove the comb and wash the preformed wells with deionized water to remove any unpolymerized acrylamide.

• 11. Place the gel into the electrophoresis apparatus and add electrophoresis buffer to the top and bottom buffer reservoirs.

• 12. Prepare samples for electrophoresis by mixing 20 µL of sample RNA and 20 µl of gel loading dye (see Note 7 ).

• 13. Load all of this mixture directly onto the gel and perform electrophoresis for 16 h at 25 mA using 1X Tris-glycine, pH 8.3 buffer.

3.2.2 Staining and Detection of dsRNA Segments in Polyacrylamide Gels

• 1. Following electrophoresis, fix the gel for 30 min in a 10% (v/v) ethanol-acetic acid solution.

• 2. Silver stain with 0.011 M silver nitrate for 2 h then briefly (1 min) rinse in deionized water.

• 3. Transfer the gel to the developing solution (buffer 2) for 20 min, or until the RNA segments are clearly visible (not longer than 45 min).

• 4. Increase the intensity/visibility of the segments by immersing the gel in a solution of freshly prepared 0.07 M sodium carbonate for 10 min.

• 5. Visualize the electrophoretic patterns (electropherotypes) over a white light box.

3.3 PCR Methods

3.3.1 Reverse Transcriptase (RT)-Mediated PCR

• 1. Denature 1–5 µL of the dsRNA template (see Note 8 ) by heating to 97°C in the presence of 3.5 µL DMSO for 5 min (see Note 9 ). Place the reaction tubes immediately on ice.

• 2. To amplify full-length gene segment 9 (1062 bp) encoding VP7, assemble a PCR reaction cocktail containing the following: 5 µL 10X PCR buffer, 8 µL of the working dNTP mix (final concentration of each dATP, dCTP, dGTP, dTTP was 200 µM), 1.5 mM MgCl₂, 10 pmol of each primer ( Table 1 ), 2.5 U Taq DNA polymerase, 4 U AMV-RT, 0.5 µL RNasin, and 1 mM DTT (which is added to the denatured template; see Note 10 ). The final reaction volume is 50 µL. Include an appropriate negative control containing all of the above except template dsRNA.

• 3. Centrifuge the tubes for 10 s and overlay the mixture with 100 µL of sterile mineral oil to prevent evaporation during thermal cycling.

• 4. Perform the RT step at 45°C for 30 min.

• 5. Then heat all reaction tubes to 70°C to inactivate the AMV-RT enzyme and cool on ice for 1 min. This step results in the production of a single-stranded complementary DNA (cDNA) copy of one of the double-stranded RNA molecules.

• 6. Amplify the cDNA products by PCR using the following cycling parameters: 94°C for 1 min, 48°C for 2 min, 72°C for 1 min (30 cycles) and finally 72°C for 7 min (1 cycle) to complete extension (see Note 11 ).

• 7. To amplify partial length gene segment 4 (867 bp), the same reaction conditions are required, with the substitution of 40 pmol of each VP4 forward and VP4 reverse primer ( Table 1 ) to target gene segment 4 specifically.

• 8. Perform reverse transcription of the dsRNA template as previously described ( step 4 above). The reaction parameters for the VP4 primer pair are as follows; 94°C for 1 min, 53°C for 2 min, 72°C for 1 min (30 cycles), with a final extension at 72°C for 7 min (1 cycle).

• 9. Resolve RT-PCR products in a 1.5% (w/v) agarose gel, containing ethidium bromide (0.1 mg/mL) and visualize by UV illumination.

3.3.2 Purification of PCR Products

• 1. Purify RT-PCR products using a PCR purification kit according to the manufacturer’s instructions (QIAGEN).

The RT-PCR products are subsequently used in seminested PCR assays to identify the G- and P-type of each isolate. In addition, the VP7 serotype could also be identified by direct DNA sequencing of the RT-PCR fragments followed by nucleotide comparison with sequenced strains in the databases using BLAST search tools (http://www.ncbi.nih.nlm.gov/BLAST).

3.3.3 Seminested PCR

• 1. Analyze PCR products from the RT-PCR reactions by seminested PCR to identify G- and/or P-types.

• 2. Identify the G-type of each isolate by adding sufficient template (see Note 12 ) to 5 µL of 10X PCR buffer (100 mM Tris-HCl, pH 9.0, 500 mM KCl), 8 µL of stock dNTP mix, 1 mM MgCl₂, 10 pmol of each G-serotype primer, and 10 pmol of the common reverse primer, RVG9 ( Tables 1 and 2 and Fig. 3 ).

• 3. Add 1 U Taq DNA polymerase to a final volume of 50 mL.

• 4. Overlay all reaction mixtures with 100 mL of sterile mineral oil.

• 5. As thermal cycling conditions for amplification, use the following parameters: 97°C for 1 min, 46°C for 2 min, 72°C for 1 min (15 cycles), final extension at 72°C for 7 min (1 cycle).

• 6. Identification of the P-type isolates requires adding sufficient VP4 template to 5 µL 10X PCR buffer (100 mM Tris-HCl, pH 9.0, 500 mM KCl), 8 µL of stock dNTP mix, 1.5 mM MgCl₂, 2.5 U Taq DNA polymerase, 20 pmol of each P-typing primer, and 20 pmol of the VP4 common primer, Con2 ( Tables 1 and 3 ).

• 7. The cycling parameters for the P-typing reaction are as follows; 97°C for 1 min, 50°C for 2 min, 72°C for 1 min (20 cycles), with a final extension at 72°C for 5 min (1 cycle).

• 8. Visualize all PCR products by agarose gel (1.5%) electrophoresis as previously described in Subheading 3.3.1 .

Table 2 Characteristics of G-Typing Primers

Table 3 Characteristics of P-Typing Primers

4 Notes

• 1. The RNA isolation procedure described was used to isolate RNA directly from fecal matter. The method can also be applied to tissue culture samples, but the quality and quantity of RNA may not be as good. Alternative methods are also available.

• 2. Contamination with exogenous ribonucleases can result in the degradation of RNA; therefore proper pretreatment of reagents and experimental apparatus is a prerequisite. To reduce ribonuclease contamination of laboratory equipment, all surfaces and apparatus can be thoroughly wiped with RNase AWAY (Molecular Bioproducts, San Diego, CA). Glassware and nonsterile plasticware can be immersed in a solution of 0.2% (v/v) DEPC and then autoclaved at 121°C for 30 min. Gloves should be worn at all times during procedures.

• 3. Caution: care should be taken when handling phenol/chloroform. As phenol is an acid, it can cause skin burns. This reagent should only be handled in a safety cabinet with extraction ventilation.

• 4. The presence of DNA in RNA samples could potentially interfere with subsequent analysis of the rotaviral genome and therefore it should be removed. DNA is degraded by treatment with a specific endonuclease enzyme, DNase 1. This enzyme digests DNA but does not affect the integrity of RNA.

• 5. Prepare sufficient gel volumes according to the PAGE apparatus used.

• 6. Isobutanol is added to prevent oxygen from diffusing into the gel, as this would inhibit polymerization.

• 7. The loading buffer described in Subheading 2.2.1. can be substituted for another appropriate loading buffer if preferred. The volume of sample that is loaded into the gel will depend on the size of the gel wells and thus can be adjusted accordingly.

• 8. Using excess RNA template can be detrimental to the RT-PCR assay, as it appears to inhibit the reaction. In general, small volumes of dsRNA template (1–5 µL) often produce better results.

• 9. Stronger binding forces exist between RNA-RNA compared with DNA-DNA and DNA-RNA; thus more stringent denaturing conditions are required to prevent strand reannealing. Prolonged denaturation (5 min) and the addition of nucleic acid destabilizers, such as DMSO, in the RT step are often recommended (34).

• 10. It is not essential but recommended to include RNasin in the assay to inactivate any contaminating ribonucleases that may be present in the reaction.

• 11. The thermocycler used in this study was a Pharmacia ATAQ controller (Pharmacia, Uppsala, Sweden).

• 12. The amount of cDNA template used in the serotyping PCR assays can be identified by performing initial template titrations. In general, 1–2 µL of cDNA template is sufficient for amplification. However, if the RT-PCR products are very weak, use more template.