Abstract
BackgroundHuman parechoviruses (HPeVs) are among the most frequently detected picornaviruses in humans. HPeVs are usually associated with mild gastrointestinal and respiratory symptoms with the exception of HPeV3 which causes neonatal sepsis and CNS infection. Previous studies showed various results in culturing different HPeV genotypes, inducing only a low cytopathic effect (CPE).MethodsIn vitro growth characteristics of the different HPeV genotypes in a range of 10 different cell lines are scored with CPE and measured in the supernatant by real time PCR. In the optimal cell line for each genotype a standard neutralization assay with the available HPeV antibodies (Abs) was performed and scored by CPE and measured by real time PCR.ResultsAll six HPeV types were able to replicate on the RD99, A549, and Vero cell lines. HPeV1 was the only genotype able to replicate on all cell lines. Most efficient growth of HPeV1, 2, 4, 5, and 6 was shown on the HT29 cell line, while HPeV3 was unable to replicate on HT29. In all cases viral replication could be measured by real time PCR before CPE appeared. The polyclonal Abs available against HPeV1, 2, 4 and 5 all showed neutralization of their respective genotype after 7 days with inhibition of >60% in real time PCR and full inhibition of CPE, although cross-neutralization is shown. Replication of HPeV3 could only be inhibited by 12% by the anti-HPeV3 (aHPeV3) Ab and no inhibition of CPE was shown after 7 days.ConclusionWhen replication is monitored by PCR, growth of HPeV genotypes 1 to 6 is supported by most of the cell lines tested, where viral replication is measured before appearance of CPE. A combination of HT29 and Vero cells would therefore support replication of all culturable HPeV types, so viral replication could be detected by PCR within 3 days for all genotypes.In addition, we showed efficient neutralization for HPeV1, 2, 4, 5, while cross- neutralization was shown between these types, indicating possible common neutralizing epitopes. For HPeV3 no efficient (cross-) neutralization was shown, indicating different neutralizing epitopes for HPeV3 compared to the other HPeV genotypes.
Highlights
Human Parechoviruses are single stranded RNA viruses belonging to the Picornaviridae family
All six Human parechoviruses (HPeVs) types were able to replicate on the RD99, A549, and Vero cell lines (Figure 1, left panel)
Rapid replication kinetics with lower titers were found on HT29, Caco-2 and LLCMK2, while replication kinetics were slow on SH-SY-5Y and SK-N-SH cells
Summary
Human Parechoviruses are single stranded RNA viruses belonging to the Picornaviridae family. 16 genotypes are known [5,6,7,8,9,10], HPeV1 and HPeV2 were first isolated in the 1950’s, whereas the third type was only described in 2004 This late discovery of HPeV could be due to difficulties of HPeVs detection in cell culture, because of only a low induction of CPE, most pronounced for HPeV3 and HPeV6. In addition it was shown that only 42% of clinical samples positive for HPeV by PCR could be cultured, using 6 different cell lines [12]. Previous studies showed various results in culturing different HPeV genotypes, inducing only a low cytopathic effect (CPE)
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