Globally, rotavirus infection is the leading cause of severe diarrhoea in children. On the basis of 2 outer layer viral proteins, VP7 and VP4, rotaviruses are classified into 16 G and 27 P types, respectively. In humans, the genotype G1P is predominant, followed by G2P, G3P, and G4P (1).
The high genetic variability of rotavirus has been documented through molecular epidemiological studies (1). Emerging genotypes (including G9, G12, G5, and G8), and increasing numbers of G-P combinations have been reported over time (1). Mechanisms responsible for fast rotavirus evolution include point mutations, accumulating at a high rate, and genetic reassortment events (2).
Viraemia has been proved to occur commonly in children with acute rotavirus diarrhoea (3–6). However, information on genetic characterisation of strains associated with systemic infection is poor. The aim of our study is to evaluate prospectively children hospitalised for acute rotavirus diarrhoea and genotype strains obtained from blood and stool samples. Moreover, nucleotide sequences within the VP4 and VP7 genes of strains obtained from blood and stool specimens of the same patients were compared.
PATIENTS AND METHODS
The study subjects were 21 children with acute diarrhoea, who were admitted to the Division of Paediatrics, Department of Paediatrics, University of Florence, from January 1 to April 31, 2006. The childrens' ages ranged from 15 days to 10.4 years (median 1.2 years). Blood and stool samples for rotavirus detection were taken from each patient on the first day of hospitalisation before administration of any treatment. Samples were stored in aliquots at −70°C, until tested.
Detection of VP6 Rotavirus Antigen in Stool Specimens
Stool specimens were tested for VP6 rotavirus antigen by immunochromatographic assay (Orion Diagnostica Inc, Espoo, Finland), according to the manufacturer's instructions. Quality control of the laboratory performance was assessed by the United Kingdom National External Quality Assessment Service, Central Public Health Laboratory, London.
Detection of Double-stranded RNA of Rotavirus in Faecal and Blood Specimens
Extraction of Rotavirus dsRNA
RNA was extracted from whole blood and stool samples using the nucleic acid binding system QIAamp Viral RNA kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. The extracted viral RNA was stored at −70°C.
RT-PCR of VP6 Gene Segment
Rotavirus RNA was detected by nested reverse transcriptase-polymerase chain reaction (RT-PCR) with primers designed on the basis of the nucleotide sequence of genome segment 6 of group A rotavirus as previously described (7).
VP7 and VP4 Genotyping of Rotavirus Strains
The full-length VP7 gene (1062-nt) was reverse transcribed and amplified using the SuperScript RT-PCR system (Invitrogen, Carlsbad, CA) with the primer pair Beg9/End9 (8). Blood specimens were subjected to 2 other steps of amplification using the internal primers 9Con1/sEnd9 (9) and VP7-F/VP7-R primers (10). Determination of the G genotypes was performed on amplicons obtained using a pool of internal primers specific for the G1, G2, G3, G4, and G9 genotypes in combination with the appropriate reverse consensus primer (11). The VP4 gene was reverse transcribed and amplified using the generic primers Con3 (11) and 3';com VP4 (12). A second amplification, using Con3 and Con2 primers, was performed, to increase sensitivity for blood specimens. The P genotyping was carried out on second step amplicons using internal primers specific for the P, P, P, P, P, and P genotypes (11).
Sequence and Phylogenetic Analysis
Amplicons were directly sequenced using primers Beg9/End9 or VP7F/VP7R for the VP7 gene and Con2/Con3 for VP4. Amplification products were purified using the Montage PCR centrifugal filter devices (Millipore Corporation, Bedford, MA) before sequencing using the CEQ2000 Dye Terminator Cycle Sequencing Quick Start Kit (Beckman Coulter Inc, Fullerton, CA). Sequences were resolved using an automated sequencer (CEQ, Beckman Coulter Inc).
Rotaviruses were detected in the stool samples taken from 11 of 21 (52.4%) children. Among the latter 11 children, 7 (63.6%) displayed positive rotavirus RNA in blood samples.
Faecal Rotavirus Strains Typing
All faecal rotavirus strains detected in the specimens from these children were VP7 and VP4 genotyped, there being 5 G1P, 1 G9P, and 1 mixed G1-G4P cases of infection.
Blood Rotavirus Strains Typing
Only 2 of 7 rotavirus strains recovered from blood samples were typeable both for VP7 and VP4. The first was G1P, the second was G4P, and a third blood strain was typeable only for VP7, being a G1 genotype, but not for VP4 (Table 1). All of the children recovered completely within a few days.
Sequence and Phylogenetic Analysis
Considering patient number 2, comparing G1 sequences of strains obtained from blood and stool samples 100% and 98.8% of identities in VP7 and VP4, respectively, were identified. The VP4 nucleotide differences were responsible for 2 substitutions (Ile→Val and Asn→Ser at positions 106 and 113) at the amino acid level.
Considering patient number 3, 99.7% of identity between sequences of G1 strains obtained from faecal and blood samples of the same patients. One amino acidic substitution (Ser→Asp) at position 94 in VP7 was evidenced.
G1 nucleotide sequences of patients number 2 and number 3 differed by 2.9% between each other and by 2.1% and 2.7%, respectively, with the phylogenetically closest G1 rotavirus sequence (strain PA73/04) recovered in Italy (13).
Considering patient number 4, a mixed G1-G4P (8) infection was documented by nested RT-PCR, by G1 strains could not be sequenced. Thus comparison of VP7 and VP4 sequences of G4 strains isolated from blood and faecal samples was also performed for this patient, and revealed a 100% identity.
An identity of 99.35% and 98.3% respectively, was observed with the closest G4 and P sequences of Italian rotavirus strains (PA36/00 and PA67/04) (14).
In the present pilot prospective study, molecular characterisation of VP7 and VP4 genotypes and phylogenetic analyses were performed on rotavirus amplicons obtained from blood and stool samples from 7 Italian children with rotavirus viraemia.
All faecal rotavirus strains were G and P typed by nested-RT-PCR, and all but 1 were identified as G1P, the most common genotype in Italy (14). Alternatively, G typing of rotavirus in blood samples was successfully performed only in 3 of 7 strains, and P typing was possible only in 2 of 7 strains. The presence of lower RNA level in the blood, as compared with stool specimens, and the occurrence of PCR inhibitors in serum may explain the difficulty in typing blood strains (8). Similarly, in a recent study, typing was only possible in 5 of 16 strains in blood samples from children with acute rotavirus gastroenteritis and positive serum VP6 RT-PCR (15).
Our data suggest that a variety of rotavirus G types can be found in blood because both G1 and G4 types were identified. Moreover, we previously documented 1 G9P type in blood sample from a child hospitalised for acute rotavirus diarrhoea in 2005 (unpublished data). Similarly, in a recent study on systemic rotavirus infection, Blutt and Conner (6) speculated, on the basis of the detection of different G and P specificities in faecal strains from viraemic patients, that rotavirus viraemia was not limited to a specific genotype. Also, Arista et al (13) identified both the G1P and the G9P types in blood samples.
In our study, comparing VP7 and VP4 nucleotide sequences from blood and stool samples obtained from the same patient, complete correspondence was not observed in the 2 cases. Differences of 0.3% and of 1.2% in the VP7 and VP4 sequences were found considering patients number 3 and number 2, respectively. These limited nucleotide divergences were responsible for amino acid substitutions in both cases. The amino acidic substitution Ser→Asp at position 94 of VP7 gene is particularly interesting because the residue 94 is crucial in identifying the G1 type (14). A bigger difference in the VP4 sequences, with 2 amino acid substitutions (Ile→Val and Asn→Ser at position 106 and 113) was demonstrated in strain from patient number 2. These amino acids are included in the most prominent area of divergence within the VP4 sequence (residues 92 to 192). Substitutions in this area may also be involved in rotavirus neutralisation. In a detailed review on neutralisation sites for VP8 (15), neutralising monoclonal antibody (MAb) 5D9 was demonstrated to bind to amino acid 114 of rhesus rotavirus strain, 1 residue aside the amino acid 113, where 1 substitution occurred. Moreover, ntMAb 1A9 binds amino acid residue 100, which is in the same β-sheet as the amino acid 106. Because this region of VP8 is spatially close to the sugar-binding pocket of human rotaviruses (15), it may be speculated that residue 106 may be involved in rotavirus binding of the cell receptor. These differences may indicate a faecal co-infection by multiple strains.
In a recent study on Indian children, 2 of 5 typeable serum strains showed different genotypes from stool strains (G2[P4] vs G12[P8] and G2[P4] vs G8[P4]) (16). In our study, such a great genetic discordance between stool and blood strains was not observed, because when typeable, only limited nucleotide divergences were documented. In 1 child we observed a co-infection (G1-G4P) in the stool, whereas only 1 genotype was identified in the serum (G4P). This result may indicate that the extraintestinal spreading was limited to 1 genotype in this patient, even if the difficulties encountered in typing serum strains should also be considered. On the whole, these findings support the hypothesis that, during co-infection by multiple strains, a preferential extraintestinal dissemination of some strains may occur, possibly because of differential growth characteristics and/or tissue tropism (16).
An alternative explanation for our findings is that, given the natural great genetic variability of rotaviruses, it is also possible that new variants emerged after some cycles of replication. This occurs in the context of other viral infections, including hepatitis C, human immunodeficiency virus, cytomegalovirus, and varicella-zoster virus, and may be associated with variable tropism characteristics (17). Similarly, antigenic variations of rotaviruses, within a genotype may be a mechanism by which viral variants emerge to escape host immunity, causing extraintestinal disease (18). On the contrary, it must be considered that the gut, where massive viral replication occurs, is more likely to be the site for generation of viral mutants. Possibly, viraemic strains sequenced represent the virus population, which was prevalent in the gut at the time of passage into the blood. However, because of the limited number of positive samples tested, any conclusion in this regard would be speculative.
Our study has several limitations. The presence of infectious viruses in the blood through isolation in cell culture or animals was not demonstrated. Nevertheless, RNA detection in the blood strongly suggests the presence of viraemia. In this respect, Blutt et al (19) demonstrated the presence of infectious virus through cell culture in the blood of children with rotavirus infection and positive antigen and/or RNA in blood. Moreover, this is a pilot study on a limited number of children, and the results should be interpreted with caution. However, our data may be of interest and may stimulate further research. Larger investigations may elucidate the role of VP4 and VP7 amino acid differences observed between faecal and blood strains. Such studies may be of help in better defining rotavirus evolution, in the light of future vaccination strategies.
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