Nonpolio enteroviruses can also be recovered from non-reproducible cytopathology in L20B cell line

Abstract

Samples showing cytopathology (CPE) on initial inoculation into L20B cell line but with no observed or reproducible CPE on passage in L20B or RD are considered negative for both poliovirus and nonpolio enteroviruses (NPEVs). The phenomenon is termed ‘non-reproducible CPE’. Its occurrence is usually ascribed to the likely presence of reoviruses, adenoviruses and other non-enteroviruses. This study aimed to investigate the likelihood that NPEVs are also present in cases with non-reproducible CPE.Twenty-six (26) cell culture suspensions were analyzed in this study. The suspensions were collected from the WHO National Polio Laboratory, Department of Virology, College of Medicine, University of Ibadan. The suspensions emanated from 13 L20B cell culture tubes that showed cytopathology within 5 days of inoculation with fecal suspension from AFP cases. However, on passage into one each of RD and L20B cell lines, the CPE was not reproducible. All samples were subjected to RNA extraction, cDNA synthesis, the WHO recommended VP1 RT-seminestedPCR assay, species resolution PCR assay, sequencing and phylogenetic analysis.Six (6) samples were positive for the VP1 RT-seminested PCR assay. Only four of which were positive by the species resolution PCR assay. The four amplicons were sequenced, however, only three (3) were successfully identified as Coxsackievirus A20 (2 isolates) and Echovirus 29 (1 isolate).The results of this study unambiguously showed the presence of NPEVs (particularly CVA20 and E29) in cell culture supernatants of samples with CPE on initial inoculation into L20B cell line but with no observed or reproducible CPE on passage in RD cell line. Therefore, like reoviruses, adenoviruses and other non-enteroviruses, NPEVs can also be recovered in cases with non-reproducible CPE.

Isolation and Identification of Enterovirus D111 in a Child with Acute Flaccid Paralysis in Nigeria

“…This phenomenon of NR-CPE has been suggested to be caused by nonspecific cytotoxicity, Adenovirus or Reoviruses (to which the Rotaviruses belong) (WHO, 2007). However, we (Adeniji et al, 2018) recently showed that EVs could also contribute to NR-CPE. In this study, we go a step further to find out how often the three (Adenoviruses, group A Rotaviruses and EVs) virus types are present in cell culture supernatants (CCS) recovered from L20b cell culture tubes with NR-CPE.…”

“…to 30%(Adeniji et al, 2018) of CCSs from NR-CPE on R-L cell lines might contain EVs. D94, the EGT could not assign it to any EV type using the VP2 and VP4 data.…”

“…Adeniji et al, 2018) had previously shown that EVs could be recovered from CCSs of NR-CPE by direct RT-snPCR(Nix et al, 2006, WHO, 2015. We(Adeniji et al, 2018) also showedthat EVs were detectable by direct RT-snPCR in 30.8% (4/13; 2 each of EV-Cs and EV-Bs) of the CCSs. We have taken a step further to recover the EV-B and EV-C isolates on RD (Adewumi et al, 2017) and Hep-2 (Faleye et al, 2018b) cell lines, respectively.…”