Molecular characterization of non‐polio enteroviruses isolated from acute flaccid paralysis patients in Uganda

Abstract Enteroviruses (EVs) are RNA viruses that can cause many clinical syndromes including acute flaccid paralysis (AFP). Within the global polio laboratory network, EVs are categorized either as polioviruses or non‐polio enteroviruses (NPEVs). Specific NPEVs have been described in polio‐like residual paralytic events in AFP patients. Retrospective analysis of 112 NPEV isolates from AFP patients was performed and thirty one NPEV types were identified of which 91% were Enterovirus B and 9% were Enterovirus A species. The NPEVs were distributed across the country with most patients in the eastern region (41/89; 46.1%). The highest proportion of patients were children less than 5 years (77/89; 86.5%) and male patients were more common (54/89; 60.7%). Echovirus 11 (11/89; 12.4%) was frequently observed and phylogenetic analysis of these sequences revealed high diversity. Coxsackievirus B5 (CV‐B5), CV‐B6, E21, and EV‐B69 were only seen in patients with residual paralysis. Analyses of the EV‐A71 sequence indicated a unique genogroup.

America and Europe have observed clustered cases of neurological illnesses (now termed acute flaccid myelitis) following outbreaks of respiratory illness due to EV D68 (EV-D68). [7][8][9] These observations suggest a causal relationship between NPEVs and paralytic disease.
EVs can undergo both inter and intra species recombination resulting into genetic divergence. 10 Different EV types are classified based on sequence divergences in the capsid viral protein 1 (VP1) coding region. 11 Conventionally, EVs are classified by the global polio laboratory network into two broad categories that is the polioviruses (e.g. wild polioviruses and vaccine derived polioviruses) and the NPEVs. NPEVs are prevalent globally, and in Africa and Uganda, 12

| Sequencing of part of the VP1 region
A sensitive semi-nested RT-PCR (RT-snPCR) was performed as described by Nix et al. 16 For quality control, RNA from a sample whose genotype had already been identified as well as the RNA extraction controls were included as positive and negative controls respectively. Amplification products were separated and visualized on 1% ethidium bromide (10 mg/ml) stained agarose gels and sized against a 100 bp molecular weight marker (Lonza). The DNA was then purified using the QIAquick PCR purification kit (QIAGEN) following the manufacturer's guidelines.
The purified DNA was diluted to a final concentration of 7.5 µg/µl and sequenced using a BigDye Terminator V3.1 ready reaction cycle sequencing kit (Thermo Fisher Scientific). In brief, for each 10 µl reaction mixture, 5 µl of the PCR product, 2 µl of Big Dye 5 × buffer, 2 µl of BigDye, and 0.08 µl of each of primers AN88 and AN89 at a final concentration of 0.08 µmol/L were mixed. Amplification was done at 96°C for 1 min and 25 PCR cycles of 96°C/10 s, 50°C/5 s, 60°C/4 m.
When sequencing failed with primers AN88/AN89, it was repeated with primers AN232/AN233 under similar PCR conditions. The sequencing reaction products were cleaned using the BigDye XTerminator purification kit (Life technologies) following the manufacturer's guidelines and finally loaded onto a 3500xL genetic analyzer (Applied Biosystems).

| Enterovirus strain identification and phylogenetic analysis
The partial VP1 sequences obtained were analyzed using sequencher GenBank. 17 Classification was done based on the algorithm proposed by Oberste et al. where a partial VP1 nucleotide sequence identity score of at least 75% to any enterovirus prototype strain indicated type identity. 18 The types obtained with BLAST were corroborated using the Enterovirus Genotyping Tool (v0.1, RIVM) developed by National Institute for Public Health and the Environment, Netherlands (RIVM). 19 Determination of EV types was also demonstrated by construction of phylogenetic trees using the partial VP1 sequences obtained and the respective reference sequences. All study and reference sequences were aligned using the ClustalW alignment program within the Molecular Evolutionary Genetics Analysis V7 (MEGA7) software. 20 Trees were inferred with the Maximum Likelihood method and the robustness of the nodes was tested with 1000 bootstrap replications and bootstrap support values > 80 are shown at the nodes. All partial VP1 sequences from this study were deposited in GenBank with the accession numbers MT661794-MT661882.   paralysis. CV-B6 has previously been reported in two patients with residual paralysis in India. 26 Male patients (54/89; 60.7%) were predominantly infected with NPEVs as was observed in Zambia (54.9%), Nigeria (63%), and Mongolia (56%). 23,39,40 Enteroviruses were also predominant in children below 5 years, similar to reports from India. 26 However, this may be due to higher prevalence or pathogenicity of enteroviruses in younger children or a bias towards inclusion of this age-group in AFP surveillance programs.

| Data management and analysis
Enterovirus A71 (EV-A71) and CV-A16 are common causes of hand, foot and mouth disease (HFMD), and have caused recurrent genogroup that has not been reported before (Figure 3). Only seven (7) EV-A71 genogroups (genogroups A-G) were previously recognized. 41

| Limitations and strengths of the study
The sample size was small but can contribute to future meta-analyzes in which significance testing might establish a statistical correlation be-

| CONCLUSION
This study revealed a high NPEV type diversity in patients clinically diagnosed with AFP. Some NPEV types were only observed in patients with residual paralysis. This facet of the study adds to the understanding of the natural history of such infections in Uganda and globally. The detection of EV-A71, which is most commonly associated with HFMD but also associated with severe neurological disease (including permanent paralysis) and fatal outcomes (neurogenic pulmonary edema), calls for formulation of effective long term strategies to monitor NPEV circulation in Uganda. The views expressed in this publication are those of the author(s) and

ACKNOWLEDGMENTS
not necessarily those of AAS, NEPAD Agency, Wellcome Trust or the UK government.

CONFLICT OF INTERESTS
The authors declare that they have no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.