Among the 429 samples collected from children with allcause AGE on the day of hospitalization, 287 samples (67%) were positive for one or more enteric viruses, whereas 142 samples (33%) were negative for viral nucleic acids (Table 1). RVA was the most frequently detected pathogen, with a prevalence of 40.1% (172 cases), followed by norovirus and adenovirus infections, 11.4% (49 cases) and 4.7% (20 cases), respectively. Astroviruses, sapoviruses, enteroviruses, and orthoreoviruses were detected in 1.9%, 1.4%, 1.2%, and 0.2% of cases, respectively. Among the 287 positive samples, 261 were positive for one virus, while 26 were co-infected with two or more different enteric viruses, with a predominance of RVA, including co-infections with RVA and AdV or RVA and NoV, which were detected in 7 cases (1.6%), and RVA and AstV, which was identified in 4 cases (0.9%). In addition, single cases of co-infection with AdV/AstV, RVA/RVC, RVA/NoV/EnV/AstV, SaV/AstV, EnV/NoV, and RVA/EnV/NoV were detected.
Table 1. Distribution of enteric viruses detected in samples from children with AGE, according to their age (n = 429)
Next, the distribution of enteric viruses implicated in AGE etiology in different age groups (0–6, 7–12, 13–24, and 24–60 months) was estimated (Table 1). The lower levels of viral etiology in the age group 0–6 months is likely due to the presence of transplacentally transmitted maternal specific antibodies.
Furthermore, a substantial increase in RVA frequency (up to 66.7%) in children with AGE was observed during March and April, compared with other months (27.4%–42.9%) (Fig. 2).
Figure 2. Distribution of rotavirus A and noroviruses detected in clinical samples according to month. The graph shows the change in the proportion of individuals positive for rotaviruses (RVA; blue line) and noroviruses (NoV; red line) during the year, determined by the percentage of samples with positive PCR results (n = 429).
No viral nucleic acids were detected in 38 of 42 specimens (90.5%) collected from healthy children (without AGE manifestation) during the summer of 2010. In four samples, adenoviral DNA (two cases with Ct values of 25.0 and 30.0) and sapoviral RNA (two cases with Ct values of 23.3 and 27.3) were detected. In contrast, among samples collected from children with AGE in summer 2010 (n = 42), enteric viruses were identified in 67.7% of cases with a predominance of RVAs (35.7%), followed by noroviruses (9.5%), adenoviruses, enteroviruses, and mixed infection (each 7.1%). Notably, a high proportion of RVA infection was detected in hospitalized children with AGE registered in summer 2010 (35.7%), which is not typical for the summer period in regions with a temperate continental climate.
In 42 of 165 secondary stool specimens collected twice, nucleic acid of a virus other than that detected in primary samples was identified, indicating that these were nosocomial infections (25.5%). Nosocomial infections were identified with rotaviruses, noroviruses, astroviruses, and adenovirus in 22 cases (52.4%), 12 cases (28.6%), 3 cases (7.1%), and 1 case (2.4%), respectively. In four cases (9.5%), nosocomial infection with two or three viruses was detected. Furthermore, a high linear correlation (R2 = 0.94) was determined between the viral population structure of nosocomial infections and that in children with AGE hospital admissions.
Nosocomial infections were significantly more prevalent in younger infants: median age, 5 (CI 3–9) months versus 13 (CI 6–23) months for children without nosocomial infections (P = 0.0007, M–W). Seventy-eight per cent of patients with nosocomial enteric viral infections (32 of 41) had clinical manifestations, such as reduced general condition, vomiting, and diarrhea. In 31 cases, severe clinical manifestations were associated with RVA (20 cases), NoV (8 cases), or both viruses (3 cases). AstV, AdV, RVC, and ReV were detected more frequently (4 among 9 cases) in children without clinical manifestations of nosocomial infection. Furthermore, among children with clinical manifestations of nosocomial viral infections, the viral load, determined by real-time PCR (according cycle threshold (Ct) values), was higher than in children without clinical manifestations of nosocomial infection [Ct = 19.5 (CI 16.1-22.5) vs. 26.9 (CI 22.0-30.6), P = 0.010, M–W]. Thus, nosocomial infection with enteric viruses was observed in 24.85% of hospitalized children, while accompanying clinical manifestations were present in only 19.4% of cases. The presence of clinical manifestations correlated with younger age and, probably, higher levels of virus replication activity.
RVA positive samples (n = 212) were analyzed by multiplex type-specific real-time PCR for G/[P]-genotyping. For the 170 RVA strains detected in clinical specimens, the G/[P]-genotype, or a variant of the VP7 or VP4 genes, was determined (Fig. 3). The predominant genotype was G4P, detected in 38.7% of cases, whereas genotypes G1P, G9P, G3P, G2P varied in frequency from 11.8% to 3.3%. In almost 20% of cases, the RVA genotype was not determined, predominantly due to the low viral load in collected samples, and probably also because of insufficient sensitivity of the genotyping system. The 2012–2013 period was characterized by an unusually high proportion of the G9P genotype (30%).
Figure 3. Distribution of RVA G/[P]-genotypes detected in clinical samples between 2009 and 2014 in Moscow. Each segment represents the relative distribution of an RVA G/[P] genotype based on data from type-specific multiplex real-time PCR analysis of fecal extracts collected from children with rotaviral enteritis (n = 212). The total figures for the whole period are presented. Mix mixed infection, P/T partially typed.
The reliability of the data obtained by type-specific PCR was confirmed by selective Sanger sequencing of the VP4 and VP7 genes fragments. Phylogenetic analysis was performed based on partial sequences of the VP4 (segment 4) and VP7 (segment 9) genes derived from 664 and 885 bp PCR amplicons, respectively. In total, 44 gene fragment sequences (11 VP4 and 33 VP7, GenBank accession numbers: KT000090–KT000133) were sequenced. To determine the taxonomy of the RVA strains, phylogenetic trees were generated using the Maximum Likelihood Method and the Kimura two-parameter evolution model, based on the partial VP4 and VP7 RVA gene nucleotide sequences (Fig. 4). The bootstrap values of tree nodes uniting particular variants of the genes ranged from 88% to 100%, indicating strong support for these nodes. Overall, in 100% of cases the studied strains could be grouped according to the genotype of the reference strains and VP4 and VP7 genes variants, previously determined by typespecific real-time RT-PCR, indicating the high accuracy of RVA genotyping by PCR and the reliability of the data obtained. Among RVA strains with genotype undetermined by type-specific real-time RT-PCR, two strains with rare gene variants were identified by sequencing: G6 and P (GenBank accessions KT000122 and KT000133).
Figure 4. A Phylogenetic tree based on partial nucleotide sequences of the RVA VP7 (A) and VP4 (B) gene from sequenced clinical samples and reference strains. Phylogenetic trees were generated using MEGA6 (Tamura et al. 2013) with the maximum likelihood method and the Kimura two-parameter model, based on the partial nucleotide sequences of the RVA VP7 and VP4 genes. The names of clinical samples include the GenBank accession number, the sample isolation location (Moscow), a laboratory identification number, the year of isolation, and the gene variant determined by PCR. Reference strain names are indicated by GenBank accession number, strain (isolate) name, country and year of isolation, and genotype. Three-letter country codes were used according to ISO 3166-1. Numbers at the nodes are bootstrap values based on 1000 replications. Reference strains are labeled as filled square. Bootstrap cutoff values are 95% (A) and 85% (B).
It should be noted that the lengths of the sequenced PCR products determined in this study were less than 50% of the VP4 gene open reading frame sequence while, according to the recommendations for the RVA classification by Rotavirus Classification Working Group, to define the genotype of an RVA strain, at least 50% of the open reading frame sequence should be determined. Therefore, an additional reference method for G/P-genotyping of all sequenced specimens by multiplex RT-PCR and agarose gel electrophoresis was used, as recommended by the WHO "Manual of rotavirus detection and characterization methods". This analysis revealed a 100% agreement of the results obtained by the two methods.
The Prevalence of Enteric Viruses in AGE Etiology
The Frequency of Asymptomatic Carriage of Enteric Viruses During Summer
Nosocomial Enteric Viral Infections
Distribution of RVA Genotypes G and P
Table Supplementary Table S1. Primers and probes for multiplex real-time RT-PCR-detection of AdV, EnV, RVA, NoV, AstV, SaV, ReV, RVC, genotyping of rotaviruses and sequencing of VP7 and VP4 genes fragments.
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