Original Articles: Gastroenterology
Intestinal viruses are known to produce viral gastroenteritis, human rotaviruses and human noroviruses being the 2 major threats to the health of infants (1). Most of the studies to date have focused on elucidating whether the gut microbiota or supplementation of gut microbiota with probiotics can help fight pathogenic intestinal viruses, mostly rotavirus (2). Two independent reports have shown that the gut microbiota may not protect against pathogen intestinal viruses. Kane et al (3) used the mouse mammary tumour virus to show that the gut microbiota plays an essential role in the evasion of mouse mammary tumour virus to the immune system. In the second study, Kuss et al (4), using a poliovirus model, showed that intestinal microbiota promote enteric virus replication and systemic pathogenesis in mice. A few studies have, however, examined the total human viral populations in healthy infants (5,6). In the present study, we assayed the presence of intestinal viruses in the faeces of 6-month-old breast-fed infants from Malawi, to shed some light on the viral microbiota.
The study population comprised 44 healthy 6-month-old infants, who were enrolled in an epidemiological clinical trial assessing the effect of selected dietary interventions on early childhood growth (registration no.: NCT00524446), which has been described elsewhere (7). The trial adhered to the principles of the Declaration of Helsinki and regulatory guidelines in Malawi. Written informed consent was obtained from all of the participant mothers and the trial protocol was reviewed and approved by the College of Medicine's research and ethics committee (University of Malawi) and the ethical committee of the Pirkanmaa Hospital District, Finland.
The stool samples were collected at enrollment into the clinical trial, which started when infants were 6 months old and before the participants had started receiving any nutritional interventions. The infants were recruited in 2 close locations: Lungwena Health Centre (n = 38) and the Malindi Hospital catchment area (n = 6). The clinical characteristics and patterns of dietary intakes of the infants at enrollment are shown in Table 1.
Viral RNA and DNA were extracted from stools using the NucleoSpin RNA-DNA virus KIT (Macherey-Nagel, Bethlehem, PA). Stool suspensions (10%) were centrifuged, and the supernatants were processed as indicated by the manufacturer. RNA and DNA were finally eluted in 50 μL of RNAse-DNAse–free H2O and stored at −80°C until use. RNA was retrotranscribed using the Maxima First-Strand cDNA Synthesis Kit (Fermentas GmbH, St Leon-Rot, Germany) as per the manufacturer's instruction.
The detection and characterization of viruses were performed using a combination of several molecular techniques. Polymerase chain reaction (PCR) was used for adenovirus, whereas reverse transcription-PCR (RT-PCR) was used for detection of enterovirus, rotavirus, norovirus, and astrovirus (8,9).
Positive PCR amplifications were sequenced to confirm the results, sequence analyses were carried out with DNAMAN (version 4.0) for Windows (Lynnon BioSoft, Pointe-Claire, Canada), and sequence similarities were analysed with the BLAST program (blast.ncbi.nlm.nih.gov/Blast.cgi) (10).
Complementary DNA was subjected to quantitative PCR (qPCR), with the primer pair EV1 and EV2. qPCR was performed using a LightCycler (version 2.0) system (Roche, Indianapolis, IN) and LC Fast Start DNA Master SYBR green I (Roche). The reaction mixture (10 μL) contained 5 μL of 2 times master mix, 0.5 μL of each primer (10 μmol/L), and 1 μL of cDNA. Reaction mixtures without a template were run as controls. The cycling conditions were as follows: 95°C for 10 minutes, followed by 45 cycles of 3 steps consisting of denaturation at 95°C for 10 seconds, primer annealing at 60°C for 20 seconds, and primer extension at 72°C for 20 seconds. Absolute quantification was performed after comparing the crossing points of each sample with a regression curve constructed with known amounts of control DNA. For statistical analysis, SPSS 17.0 software (SPSS Inc, Chicago, IL) was used. The χ2 test was used to establish differences in the virus prevalence between the studied groups. Comparisons among data of >2 groups of infants were done by applying the Kruskal-Wallis test. A P < 0.05 was considered statistically significant. The possible correlation between variables was studied by applying the Spearman rank correlation coefficient, and significance was established at 0.5%.
Half of the samples were positive for human enteroviruses (HuEV, 22/44, 50%) as demonstrated by nested PCR (Table 2). Gastroenteritis and diarrhoea episodes were not found at sampling time points in any of the infants harbouring enteroviruses, suggesting the “asymptomatic” presence of the enterovirus. This finding confirms the higher presence of enteroviruses in healthy infants as described in Western countries previously, but with a lower number of infants studied (5,6,11). All of the positive samples were successfully sequenced, and the sequences possessed >90% identities with known HuEV strains after BLAST analysis of the sequences (Table 2). Three of the samples (sample IDs 7, 113, and 117) were positive for strains that are included in the oral poliovirus vaccine. These positives are probably the result of recent vaccinations (12,13). Most of the positive samples could be quantified by qPCR, giving a result of 105 to 106 viral copies per gram of stool. Some positive samples gave a signal that was below the quantification limit (Table 2); this can be explained by the fact that nested PCR is usually more sensitive than direct PCR used for the quantification purposes. The statistical analyses (Table 3) showed that there were no seasonal differences in the prevalence, amounts, and genogroups of the detected enteroviruses. Infants who consumed animal milk (cows or goats) had higher tendency to harbour enteroviruses (P = 0.061).
One sample was found to be positive for rotavirus (accession no. AB807827). The sequencing of the VP7 gene amplification product showed that this sample belongs to the G1 genotype. The infant had diarrhoea at the time of sampling, and diarrhoea and fever lasted 7 and 12 days, respectively. These would be because of the rotavirus infection.
We have used a classical molecular approach to look for the most common and widespread human intestinal viruses that include adenovirus, rotavirus, norovirus, astrovirus, and entorovirus. We chose this option because metagenomic approaches were not suitable for detecting human enteric viruses: the vast majority of viruses found with these approaches in the human gut are bacteriophages and pathogenic plant viruses with a poor representation of human viruses (14,15). Hence, we can state that asymptomatic enteroviruses can be seen in healthy infants in rural Malawi.
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Keywords:© 2014 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,
enteric viruses; enterovirus; healthy infants