RESUMO
PURPOSE: We recently showed that enteroviruses (EVs) andenterovirus species C (EV-C) in particular were abundant in faecal samples from children who had been diagnosed with acute flaccid paralysis (AFP) in Nigeria but declared to be EV-free by the RD-L20B cell culture-based algorithm. In this study, we investigated whether this observed preponderance of EVs (and EV-Cs) in such samples varies by geographical region. METHODOLOGY: One hundred and eight samples (i.e. 54 paired stool suspensions from 54 AFP cases) that had previously been confirmed to be negative for EVs by the WHO-recommended RD-L20B cell culture-based algorithm were analysed. The 108 samples were made into 54 pools (27 each from North-West and South-South Nigeria). All were subjected to RNA extraction, cDNA synthesis and the WHO-recommended semi-nested PCR assay and its modifications. All of the amplicons were sequenced, and the enteroviruses identified, using the enterovirus genotyping tool and phylogenetic analysis. RESULTS: EVs were detected in 16 (29.63â%) of the 54 samples that were screened and successfully identified in 14 (25.93â%). Of these, 10 were from North-West and 4 were from South-South Nigeria. One (7.14â%), 2 (14.29â%) and 11 (78.57â%) of the strains detected were EV-A, EV-B and EV-C, respectively. The 10 strains from North-West Nigeria included 7 EV types, namely CV-A10, E29, CV-A13, CV-A17, CV-A19, CV-A24 and EV-C99. The four EV types recovered from South-South Nigeria were E31, CV-A1, EV-C99 and EV-C116. CONCLUSION: The results of this study showed that the presence of EVs and consequently EV-Cs in AFP samples declared to be EV-free by the RD-L20B cell culture-based algorithm varies by geographical region in Nigeria.
Assuntos
Enterovirus Humano C/genética , Enterovirus Humano C/isolamento & purificação , Infecções por Enterovirus/epidemiologia , Fezes/virologia , Paraplegia/virologia , Doença Aguda , Adolescente , Técnicas Bacteriológicas , Linhagem Celular , Criança , Pré-Escolar , Enterovirus Humano C/classificação , Enterovirus Humano C/crescimento & desenvolvimento , Infecções por Enterovirus/virologia , Feminino , Humanos , Masculino , Nigéria/epidemiologia , Filogenia , Reação em Cadeia da PolimeraseRESUMO
Between 2005 and 2011, 23 lineages of circulating vaccine-derived polioviruses (cVDPVs) were detected in Nigeria with nonstructural region (NSR) of non-polio enterovirus C (NPEV-C) origin. However, no information exists on NPEV-C strains recombining with oral poliovirus type 2 vaccine strains (OPV2) to make type 2 cVDPVs (cVDPV2s) in Nigeria. This study was therefore designed to investigate the probable contribution of NPEV-Cs recently isolated in the region to the emergence of cVDPV2s. Eleven enterovirus C (EV-C) strains (8 NPEV-Cs and 3 PV2s) previously isolated by the authors were analysed in this study. All 11 isolates were assayed for cell-line-dependent growth restriction in four cell lines (LLC-MK2, MCF-7, RD and L20B). Subsequently, the isolates were subjected to RT-PCR specific for VP1 and 3Dpol/3'-UTR of EV-C. All PCR products were sequenced, and phylogenetic analysis was performed. All eight NPEV-Cs replicated exclusively in the MCF-7 cell line, while the three PV2s replicated in all four cell lines. The eight NPEV-Cs were identified as CVA13 (7 isolates) and CVA20 (1 isolate) by VP1 analysis, while all 11 isolates were confirmed to be EV-Cs by 3Dpol/3'-UTR analysis. In addition, phylogeny violations suggested that some cVDPVs might have recombined with common ancestors of the NPEV-Cs described in this study. This was confirmed by the scatter plot of divergence in VP1 against that of 3Dpol/3'-UTR sequences for pairs of isolates. The results of this study showed that the NSR of unknown origin found in cVDPVs from the region might have come from NPEV-Cs (e.g., CVA13 and CVA20) circulating in Nigeria.
Assuntos
Enterovirus Humano C/crescimento & desenvolvimento , Infecções por Enterovirus/epidemiologia , Infecções por Enterovirus/virologia , Vacina Antipólio Oral , Poliovirus/crescimento & desenvolvimento , Recombinação Genética , Linhagem Celular , Enterovirus Humano C/genética , Humanos , Dados de Sequência Molecular , Nigéria/epidemiologia , Filogenia , Poliovirus/genética , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
BACKGROUND: The purpose of this study was to establish an ex vivo model of coxsackievirus infection since there seems to be no suitable disease model currently. METHODS: Human conjunctival epithelial cells (HCECs) were cultured for 2 weeks in a serum-free air-liquid interface system to produce a multilayered structure. The cells were infected with coxsackievirus A24 (CVA24). Histological changes were investigated by staining the cells with H&E and DAPI, and apoptosis was evaluated using the TUNEL technique. Virus replication was measured in HeLa cells infected with viral progeny from multilayered HCECs, after 1 and 3 days, using the TCID(50) method. RESULTS: Cultured HCECs formed multiple layers. The cells showed characteristics of conjunctival epithelial cells and goblet cells, being immunohistochemically positive for CK19 and MUC5AC, respectively. CVA24 replicated readily in cultured multilayered HCECs. A mild cytopathic effect was noted 1 day after viral inoculation. Cell damage was extensive at 3 days. TUNEL imaging confirmed that the cytopathology was attributable to apoptosis. The TCID(50) data from HeLa cells indicated that the virus was actively replicating at both 1 and 3 days after inoculation. CONCLUSIONS: This novel infection model may be useful in investigating the pathogenesis of acute hemorrhagic conjunctivitis and the effectiveness of antiviral treatments.
