Epidemiology and etiology of HFMD in Nanchang from 2019 to 2022
From 2019 to 2022, CVA6 and CVA16 were two major pathogens, accounting for 34.0%-59.6% and 14.9%-31.4% EV-positive cases, respectively. However, other un-serotyped enteroviruses (UEV) accounted for 13.7-%-33.2% EV-positive cases during 2019-2022. Overall, the UEV accounted for a larger proportion in 2019 (33.2%) and 2021 (31.3%) compared with that in 2020 (20.8%) and 2022 (13.7%). On the contrast, proportion of CVA6-associated cases increased in 2020 (59.6%) and 2022 (57.0%) compared with that in 2019 (43.7%) and 2021 (34.0%) (Supplementary Figure 2). This result indicated that the COVID-19 outbreaks and the associated measures had different levels of impact on EVs. In geographical level, we analyzed and compared the positive rate (Figure 3A-D) and serotype proportion (Figure 4) in different districts and counties, in which their population densities were displayed in Figure 3E. Overall, the EV-positive rate of the HFMD cases was high in urban and suburb areas. In northwest areas AY and XJ, the positive rate had a downtrend, while it was an uptrend in the suburb area NC from 2019 to 2022. During this period, Proportion of CVA6-infected cases maintained in a low volatility in different areas apart from a very low proportion in QSH but high proportion in DH and JX in 2022. This data supports the predominance of CVA6 in Nanchang regions in the past few years. As for CVA16, a relatively high volatility was observed in outskirt areas like JX and AY. The 0% proportion of CVA16 in some areas might be due to the COVID-19 intervention. In addition, UEV proportion in QSH district was the highest reaching up to 60%.
Characterization of UEV in Nanchang
As the dominance of CVA6 becomes more pronounced in HFMD cases, the proportion UEV tends to decline. However, there is an average 24% UEV cases with unspecified serotypes during 2019-2022. The serotypes and their proportion in UEV cases were determined using a nested RT-PCR, and an expected PCR product (~389-bp) was obtained as shown in 2% agarose gel electrophoresis (Figure 5A). A total of 46 UEV cases were randomly selected during 2019-2022 for serotyping described above, among them 6 serotypes were determined by sequencing and then online blast in NCBI. Among them CVA4, CVA2, CVA5 and CVB3 were the major agents accounting for 34.8%, 23.9%, 23.9% and 10.9%, respectively (Figure 5B). And a partial 5’ UTR-based phylogenetic tree was constructed for the representative strains of 6 serotypes CVA4, CVA2, CVA5, CVB3, CVA9 and Echovirus 9 (ECHO9) (Figure 5C). After pairwise sequence identity analysis, we found that the partial 5’UTR region was conservative in CVB3 but hypervariable in CVA2, CVA4 and CVA5 (Figure 5D). According to the proportion of these serotypes in UEV, the estimated proportions for CVA4, CVA2, CVA5, CVB3, CVA9 and ECHO9 in EV-positive cases were 8.6%, 5.9%, 5.9%, 2.7%, 1.1% and 0.5%, respectively. This result suggests that enhanced etiological surveillance is essential, especially when the proportion of UEV rises.