Coxsackievirus A6 U.K. Genetic and Clinical Epidemiology Pre- and Post-SARS-CoV-2 Emergence

Abstract

Coxsackievirus A6 (CVA6) has become increasingly clinically relevant as a cause of Hand, Foot and Mouth Disease (HFMD) globally since 2008. However, most laboratories do not routinely determine the enteroviral type of positive samples. The non-pharmaceutical measures introduced to curb transmission during the COVID-19 pandemic may also have perturbed CVA6 epidemiology. We thus aimed to determine the prevalence, clinical presentation and genetic relationship of CVA6 across three complete epidemic seasons: one pre-SARS-CoV-2 emergence and two post-SARS-CoV-2 emergence in our regional healthcare setting. Surplus diagnostic nucleic acid from diagnosed enteroviral positives diagnosed between September and December of 2018 and between May 2021 and April of 2023 was subject to VP1 gene sequencing to determine the CVA6 cases and interrogate their phylogenetic relationship. The confirmed CVA6 cases were also retrospectively clinically audited. CVA6 infections were identified in 33 and 69 individuals pre- and post-pandemic, respectively, with cases peaking in November of 2018 and 2022, but in October of 2021. HFMD was the primary diagnosis in 85.5% of the post-pandemic cases, but only 69.7% of the pre-pandemic cases, where respiratory and neurological symptoms (45.5% and 12.1%, respectively) were significantly elevated. A complete VP1 sequence was retrieved for 94% of the CVA6 cases, revealing that studied infections were genetically diverse and suggestive of multiple local and international transmission chains. CVA6 presented a significant clinical burden in our regional U.K. hospital setting both pre- and post-pandemic and was subject to dynamic clinical and genetic epidemiology.

Keywords: CVA6; HFMD; coxsackievirus A6; enterovirus; genetic epidemiology; hand, foot and mouth disease.

Figure 1

Sequence-confirmed CVA6 prevalence by month. The 2018 samples (red line) were only assessed between September and December, then enteroviral typing recommenced in May of 2021 (blue) and was continued through the entirety of 2022 (yellow) to the end of April in 2023 (grey).

Figure 2

Molecular phylogenetic analysis of complete CVA6 VP1 gene of study and available reference sequences. The phylogenetic tree includes 27 novel sequences from 2018 (red), 28 novel sequences from 2021 (green), 31 novel sequences from 2022 (blue), 5 novel sequences from 2023 (purple) and 4358 publicly available lineage D sequences from GenBank. The tree was constructed using the maximum likelihood method with the SYM+I+R6 model in IQ-TREE2. Bootstrapping based on 1000 replications was performed, bootstrap values were omitted for clarity. Tree is midpoint rooted, with a scale of 0.002. A simplified phylogeny of the study sequences and down-sampled reference set is presented in Figure S4.

Figure 3

Phylogenetic analysis of CVA6 complete VP1 subgroups 1–3. Molecular phylogenetic analysis of full VP1 CVA6 gene using the maximum likelihood method with the SYM+I+R6 model in IQ-TREE2. Analysis was completed to include the subgroup 1, 2 and 3 sequences highlighted in Figure 2, including 21 novel sequences from 2018 (red), 2 novel sequences from 2021 (green), a novel sequence from 2022 (blue) and 133 publicly available sequences from GenBank. Where branches have been collapsed for clarity (represented by triangles), the country of origin and year for all sequences has been annotated. Bootstrapping based on 1000 replications is shown next to each branch at a scale of 0.002, with bootstrap values below 70 omitted. Tree is midpoint rooted.

Figure 4

Phylogenetic analysis of CVA6 complete VP1 subgroups 6 and 7. Molecular phylogenetic analysis of full VP1 CVA6 gene using the maximum likelihood method with the SYM+I+R6 model in IQ-TREE2. Analysis was completed to include the subgroup 6 and 7 sequences highlighted in Figure 2, including 26 novel sequences from 2021 (green), 22 novel sequences from 2022 (blue), 4 novel sequences from 2023 (purple) and 6 publicly available sequences from GenBank. Bootstrapping based on 1000 replications is shown next to each branch at a scale of 0.002, with bootstrap values below 70 omitted. Tree is midpoint rooted.