From a case-control survey to a diagnostic viral gastroenteritis panel for testing of general practitioners’ patients

Objective To evaluate the pathogenicity of a broad range of 11 possible gastroenteritis viruses, by means of statistical relationships with cases vs. controls, or Ct-values, in order to establish the most appropriate diagnostic panel for our general practitioner (GP) patients in the Netherlands (2010–2012). Methods Archived stool samples from 1340 cases and 1100 controls were retested using internally controlled multiplex real-time PCRs for putative pathogenic gastroenteritis viruses: adenovirus, astrovirus, bocavirus, enterovirus, norovirus GI and GII, human parechovirus, rotavirus, salivirus, sapovirus, and torovirus. Results The prevalence of any virus in symptomatic cases and asymptomatic controls was 16.6% (223/1340) and 10.2% (112/1100), respectively. Prevalence of astrovirus (adjusted odds ratio (aOR) 10.37; 95% confidence interval (CI) 1.34–80.06) and norovirus GII (aOR 3.10; CI 1.62–5.92) was significantly higher in cases versus controls. Rotavirus was encountered only in cases. We did not find torovirus and there was no statistically significant relationship with cases for salivirus (aOR 1,67; (CI) 0.43–6.54)), adenovirus non-group F (aOR 1.20; CI 0.75–1.91), bocavirus (aOR 0.85; CI 0.05–13.64), enterovirus (aOR 0.83; CI 0.50–1.37), human parechovirus (aOR 1.61; CI 0.54–4.77) and sapovirus (aOR 1.15; CI 0.67–1.98). Though adenovirus group F (aOR 6.37; CI 0.80–50.92) and norovirus GI (aOR 2.22, CI: 0.79–6.23) are known enteropathogenic viruses and were more prevalent in cases than in controls, this did not reach significance in this study. The Ct value did not discriminate between carriage and disease in PCR-positive subjects. Conclusions In our population, diagnostic gastroenteritis tests should screen for adenovirus group F, astrovirus, noroviruses GI and GII, and rotavirus. Case-control studies as ours are lacking and should also be carried out in populations from other epidemiological backgrounds.

Parechovirus A Infection of the Intestinal Epithelium: Differences Between Genotypes A1 and A3

Human parechovirus (PeV-A), one of the species within the Picornaviridae family, is known to cause disease in humans. The most commonly detected genotypes are PeV-A1, associated with mild gastrointestinal disease in young children, and PeV-A3, linked to severe disease with neurological symptoms in neonates. As PeV-A are detectable in stool and nasopharyngeal samples, entry is speculated to occur via the respiratory and gastro-intestinal routes. In this study, we characterized PeV-A1 and PeV-A3 replication and tropism in the intestinal epithelium using a primary 2D model based on human fetal enteroids. This model was permissive to infection with lab-adapted strains and clinical isolates of PeV-A1, but for PeV-A3, infection could only be established with clinical isolates. Replication was highest with infection established from the basolateral side with apical shedding for both genotypes. Compared to PeV-A1, replication kinetics of PeV-A3 were slower. Interestingly, there was a difference in cell tropism with PeV-A1 infecting both Paneth cells and enterocytes, while PeV-A3 infected mainly goblet cells. This difference in cell tropism may explain the difference in replication kinetics and associated disease in humans.

Virome of respiratory secretion from children with unknown etiological acute respiratory disease revealed recombinant human parechovirus and other significant viruses

Using viral metagenomics, viral nucleic acid in 30 respiratory secretion samples collected from children with unknown etiological acute respiratory disease were investigated. Sequences showing similarity to human parainfluenza virus 1, anellovirus, bocavirus, coxsackievirus A4, human parechovirus (HPeV), and alphaflexivirus were recovered from these samples. Complete genomes of one anellovirus, one coxsackievirus A4, three parechoviruses were determined from these libraries. The anellovirus (MW267851) phylogenetically clustered with an unpublished anellovirus (MK212032) from respiratory sample of a Vietnamese patient, forming a separate branch neighboring to strains within the genus Betatorquevirus. The genome of coxsackievirus A4 (MW267852) shares the highest sequence identity of 96.4% to a coxsackievirus A4 (MN964079) which was identified in clinical samples from children with Hand, Foot, and Mouth Disease (HFMD). Two (MW267853 and MW267854) of the three parechoviruses belong to HPeV-1 and the other one (MW267855) belongs to HPeV-6. Recombination analysis indicated that an HPeV-1 (MW267854) identified in this study is a putative recombinant occurred between HPeV-1 and HPeV-3. Whether these viruses have association with specific respiratory disease calls for further investigation.

Phylogenetic molecular evolution and recombination analysis of complete genome of human parechovirus in Thailand

Human parechovirus (HPeV), which is a member of the Picornavirus group of viruses, is a pathogen that is reported to be associated with manifestations that include respiratory tract involvement, gastroenteritis, sepsis-like symptom, and central nervous system complication. Until now, nineteen genotypes have been identified. The lack of proofreading property of viral RNA-dependent RNA polymerase (RdRp) together with recombination among the intra- and inter-genotypes of the virus results in high diversity. However, data specific to the molecular evolutionary perspective of the complete genome of HPeV remains limited. This study aimed to analyze the phylogenetic, molecular evolution, and recombination characteristics of the complete genome of HPeV strains isolated in Thailand during 2009–2012. Fifty-eight samples that were previously confirmed to be HPeV positive and then evaluated for genotyping were subjected to complete genome amplification to generate ten overlapping PCR fragments using a set of in-house designed primers. The same position of the viral genome was read in triplicate using direct Sanger sequencing. All samples were classified into the same previously defined genotypes in both whole-genome and VP1 phylogenic tree. However, sample B1091/HPeV14/2011 exhibited discordant grouping between whole-genome and VP1 on the phylogenetic tree. Bootscan analysis revealed that B1091/HPeV14/2011 inherited from two genotypic viruses, including VP1 from HPeV14, and the rest of the genome from HPeV1B. The results of this study provide important insights into the molecular evolution of and recombination in the viral genome of HPeV that will improve and accelerate our ability to develop treatment and prophylactic strategies in the future.