Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) likely emerged from a zoonotic spill-over event and has led to a global pandemic. The public health response has been predominantly informed by surveillance of symptomatic individuals and contact tracing, with quarantine, and other preventive measures have then been applied to mitigate further spread. Non-traditional methods of surveillance such as genomic epidemiology and wastewater-based epidemiology (WBE) have also been leveraged during this pandemic. Genomic epidemiology uses high-throughput sequencing of SARS-CoV-2 genomes to inform local and international transmission events, as well as the diversity of circulating variants. WBE uses wastewater to analyse community spread, as it is known that SARS-CoV-2 is shed through bodily excretions. Since both symptomatic and asymptomatic individuals contribute to wastewater inputs, we hypothesized that the resultant pooled sample of population-wide excreta can provide a more comprehensive picture of SARS-CoV-2 genomic diversity circulating in a community than clinical testing and sequencing alone. In this study, we analysed 91 wastewater samples from 11 states in the USA, where the majority of samples represent Maricopa County, Arizona (USA). With the objective of assessing the viral diversity at a population scale, we undertook a single-nucleotide variant (SNV) analysis on data from 52 samples with >90% SARS-CoV-2 genome coverage of sequence reads, and compared these SNVs with those detected in genomes sequenced from clinical patients. We identified 7973 SNVs, of which 548 were “novel” SNVs that had not yet been identified in the global clinical-derived data as of 17^th June 2020 (the day after our last wastewater sampling date). However, between 17^th of June 2020 and 20^th November 2020, almost half of the novel SNVs have since been detected in clinical-derived data. Using the combination of SNVs present in each sample, we identified the more probable lineages present in that sample and compared them to lineages observed in North America prior to our sampling dates. The wastewater-derived SARS-CoV-2 sequence data indicates there were more lineages circulating across the sampled communities than represented in the clinical-derived data. Principal coordinate analyses identified patterns in population structure based on genetic variation within the sequenced samples, with clear trends associated with increased diversity likely due to a higher number of infected individuals relative to the sampling dates. We demonstrate that genetic correlation analysis combined with SNVs analysis using wastewater sampling can provide a comprehensive snapshot of the SARS-CoV-2 genetic population structure circulating within a community, which might not be observed if relying solely on clinical cases.

Echoviruses, a serotype of enteroviruses, infect millions of people globally and there is no specific drug treatment or vaccine available for its management. The screening of medicinal plants used locally for the treatment of infectious diseases, can provide a reliable option in the discovery of potent therapeutic compounds. This study was carried out to investigate the antiviral activities of 27 medicinal plant extracts, belonging to 26 different plant species, selected from Nigerian ethnobotany, against echovirus 7, 13 and 19 serotypes (E7, E13 and E19, respectively). The plants were macerated in methanol and the cytotoxicities of the crude extracts were evaluated on the rhabdomyosarcoma cell line using the MTT assay. The antiviral activity of the plant extracts and fractions against echoviruses (E7, E13, and E19) was determined using the neutralisation assay, an assay that measures the inhibition of cytopathic effect on cell culture. The crude extract of Macaranga barteri leaves had the highest cytotoxicity with CC50 value of 0.27 μg/mL. This was followed by Crinum jagus (9.88 μg/mL) and Terminalia ivorensis (12.14 μg/mL). The antiviral screening showed that ten out of the 27 crude plant extracts tested were active on E7 and E19, inhibiting the cytopathic effect of the virus in tissue culture. None of the extracts inhibited the cytopathic effect caused by E13 serotype. Amongst the active plant extracts, the methanol extract of M. barteri leaves had the highest antiviral activity on both E7 and E9 with IC50 values of 0.028 and 0.0017 ng/mL, respectively, followed by the Ageratum conyzoides extract (0.208 μg/mL, E7; 0.006 μg/mL, E19) and Mondia whitei extract (0.038 μg/mL, E7; 0.005 μg/mL, E19). Amongst the fractions of M. barteri, the DCM fraction was most the active and selective on E7 (IC50 = 0.0075 ng/mL; SI = 19,896.54) and E19 (IC50 = 0.0175 ng/mL; SI = 8581.24). Our research has demonstrated that Macaranga barteri extracts has potent antiviral activity against echoviruses E7 and E19, and our findings suggest that this extract may have potential as a therapeutic agent in the treatment of enteroviral infections.

The need to investigate the contribution of non-polio enteroviruses to acute flaccid paralysis (AFP) cannot be over emphasized as we move towards a poliovirus free world. Hence, we aim to identify non-polio enteroviruses recovered from the faeces of children diagnosed with AFP in Nigeria. Ninety-six isolates, (95 unidentified and one previously confirmed Sabin poliovirus 3) recovered on RD cell culture from the stool of children <15 years old diagnosed with AFP in 2014 were analyzed. All isolates were subjected to RNA extraction, cDNA synthesis and three different PCR reactions (one panenterovirus 5′-UTR and two different VP1 amplification assays). VP1 amplicons were then sequenced and isolates identified. 92.71% (89/96) of the isolates were detected by at least one of the three assays as an enterovirus. Precisely, 79.17% (76/96), 6.25% (6/96), 7.30% (7/96) and 7.30% (7/96) of the isolates were positive for both, positive and negative, negative and positive, as well as negative for both the 5′-UTR and VP1 assays, respectively. In this study, sixty-nine (69) of the 83 VP1 amplicons sequenced were identified as 27 different enterovirus types. The most commonly detected were CV-B3 (10 isolates) and EV-B75 (5 isolates). Specifically, one, twenty-four and two of the enterovirus types identified in this study belong to EV-A, EV-B and EV-C respectively. This study reports the circulating strains of 27 non-polio enterovirus types in Nigerian children with AFP in 2014 and Nigerian strains of CV-B2, CV-B4, E17, EV-B80, EV-B73, EV-B97, EV-B93, EV-C99 and EV-A120 were reported for the first time. Furthermore, it shows that being positive for the 5′-UTR assay should not be the basis for subjecting isolates to the VP1 assays.

Human enteroviruses (EVs) are highly prevalent in sewage and have been associated with human diseases with complications leading to severe neurological syndromes. We have used a recently developed molecular method to investigate the presence of EVs in eight samples collected in 2017–2018 from water streams contaminated by drainage channels in three different locations in Nigeria. A total of 93 human EV strains belonging to 45 different serotypes were identified, far exceeding the number of strains and serotypes found in similar samples in previous studies. Next generation sequencing analysis retrieved whole-capsid genomic nucleotide sequences of EV strains belonging to all four A, B, C, and D species. Our results further demonstrate the value of environmental surveillance for the detection of EV transmission of both serotypes commonly associated with clinical syndromes, such as EV-A71, and those that appear to circulate silently but could eventually cause outbreaks and disease. Several uncommon serotypes, rarely reported elsewhere, were detected such as EV-A119, EV-B87, EV-C116, and EV-D111. Ten EV serotypes were detected in Nigeria for the first time and two of them, CV-A12 and EV-B86, firstly described in Africa. This method can be expanded to generate whole-genome EV sequences as we show here for one EV-D111 strain. Our data revealed phylogenetic relationships of Nigerian sewage strains with EV strains reported elsewhere, mostly from African origin, and provided new insights into the whole-genome structure of emerging serotype EV-D111 and recombination events among EV-D serotypes.

Recently, a cell-culture independent protocol for detection of enteroviruses from clinical specimen was recommended by the WHO for surveillance alongside the previously established protocols. Here, we investigated whether this new protocol will show the same enterovirus diversity landscape as the established cell-culture dependent protocols. Faecal samples were collected from sixty apparently healthy children in Ibadan, Nigeria. Samples were resuspended in phosphate buffered saline, RNA was extracted, and the VP1 gene was amplified using WHO recommended RT-snPCR protocol. Amplicons were sequenced and sequences subjected to phylogenetic analysis. Fifteen (25%) of the 60 samples yielded the expected band size. Of the 15 amplicons sequenced, 12 were exploitable. The remaining 3 had electropherograms with multiple peaks and were unexploitable. Eleven of the 12 exploitable sequences were identified as Coxsackievirus A1 (CVA1), CVA3, CVA4, CVA8, CVA20, echovirus 32 (E32), enterovirus 71 (EV71), EVB80, and EVC99. Subsequently, the last exploitable sequence was identified as enterobacteriophage baseplate gene by nucleotide BLAST. The results of this study document the first description of molecular sequence data on CVA1, CVA8, and E32 strains present in Nigeria. The result further showed that species A enteroviruses were more commonly detected in the region when cell-culture bias is bypassed.