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

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.

Echovirus 29 (E29) is a member of Species Enterovirus B (EV-B) in the genus Enterovirus, family Picornaviridae, order Picornavirinae. In Nigeria, molecular characterization of E29 was first described in 2002. In 2015, we found that a new clade of E29 had replaced that described in Nigeria in 2002-2003. To date, E29 isolates described from Nigeria were isolated in cultures of RD cell line. In 2016, we characterised an E29 strain that did not show cytopathology on RD cell line within the recommended 10 days of culture.Here we show that the E29 in question grows with evident CPE in RD cell culture like other members of the clade when allowed to stay in culture for 13 to 14 days. The findings of this study therefore suggest that some of the samples declared negative for enteroviruses by the current WHO cell culture based detection algorithm might be false negatives. It is therefore encouraged that those particularly interested in non-polio enteroviruses endeavour to maintain at least 14 days incubation in cell culture in a bid to accommodate NPEVs like E29 that might need longer time to develop CPE especially when present at low titre.

Echovirus 29 (E29) is a member of Species Enterovirus B (EV-B) in the genus Enterovirus,family Picornaviridae, order Picornavirinae. In Nigeria, molecular characterization of E29 was first described in 2002. In 2015, we found that a new clade of E29 had replaced that described in Nigeria in [2002][2003]. To date, E29 isolates described from Nigeria were isolated in cultures of RD cell line. In 2016, we characterised an E29 strain that did not show cytopathology on RD cell line within the recommended 10 days of culture.Here we show that the E29 in question grows with evident CPE in RD cell culture like other members of the clade when allowed to stay in culture for 13 to 14 days. The findings of this study therefore suggest that some of the samples declared negative for enteroviruses by the current WHO cell culture based detection algorithm might be false negatives. It is therefore encouraged that those particularly interested in non-polio enteroviruses endeavour to maintain at least 14 days incubation in cell culture in a bid to accommodate NPEVs like E29 that might need longer time to develop CPE especially when present at low titre.