First whole genome sequencing and analysis of human parechovirus type 3 causing a healthcare-associated outbreak among neonates in Hungary

Abstract

Purpose: In November 2023, the National Reference Laboratory for Enteroviruses (Budapest, Hungary) received stool, pharyngeal swab and cerebrospinal fluid samples from five newborns suspected of having human parechovirus (PEV-A) infection. The neonates were born in the same hospital and presented with fever and sepsis-like symptoms at 8-9 days of age, and three of them showed symptoms consistent with central nervous system involvement. PEV-A positivity was confirmed by quantitative reverse transcription polymerase chain reaction.

Methods: To determine the PEV-A genotype responsible for the infections, fecal samples of four neonates were subjected to metagenomic sequencing. For further analyses, amplicon-based whole genome sequencing was performed directly from the clinical samples.

Results: On the basis of whole genome analysis, sequences were allocated to PEV-A genotype 3 (PEV-A3) and consensus sequences were identical. Two ambiguities were identified in the viral protein 1 (VP1) region of all sequences at a frequency of 17.7-53.7%, indicating the simultaneous presence of at least two quasispecies in the clinical samples. The phylogenetic analysis and similarity plotting showed that all sequences clustered without any topological inconsistencies between the P1 capsid and P2, P3 non-capsid regions, suggesting that recombination events during evolution were unlikely.

Conclusion: Our findings suggest that the apparent cluster of cases were microbiologically related, and the results may also inform future investigations on the evolution and pathogenicity of PEV-A3 infections.

Keywords: Genotyping; Human parechovirus 3; Infection of neonates; Phylogenetic analysis; Whole genome sequencing.

Fig. 1

PEV-A routine diagnostic procedure followed by genotyping and genome analysis using metagenomic sequencing and amplicon-based whole genome sequencing (National Reference Laboratory for Enteroviruses, National Center for Public Health and Pharmacy, Budapest, Hungary)

Fig. 2

VP1 region, positions 71–140. Ambiguities in sequence PP176216 in positions 119 (G > U, 48.0%) and 123 (U > A, 53.7%) indicating the simultaneous presence of at least two quasispecies. Ambiguity (W) in position 123 is also present at a frequency over 40% in sequences PP176217 and PP176213, and at ≤ 40% frequency in the other sequences (software: Geneious Prime 2021.2.2.)

Fig. 3

Phylogenetic tree based on the complete coding region using the GTR + G + I model. The analysis included the eight whole genome sequences of the present study and 33 sequences and reference sequences representing global PEV-A strains derived from the Genbank database. Study sequences clustered together and were allocated to PEV-A genotype 3. Scale bars indicate nt substitutions per site and bootstrap values on the branches were calculated using 1000 replicates