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Environmental surveillance is an effective approach in investigating circulating enteroviruses and had been conducted in the cities of Jinan and Linyi since February 2008 and April 2010, respectively. This study analyzed 46 sewage samples collected in the two cities in 2011 and found that echovirus 6 (E6) was the predominant serotype, with 134 isolates (65 in Jinan and 69 in Linyi) from 23 (50%) samples. This differs from the 2010 data that found 29 E6 isolates in Jinan and only 3 in Linyi. Phylogenetic analysis of the VP1 coding region showed that all environmental E6 samples from 2008 to 2011 (n = 167) segregated into two lineages and revealed an increase in VP1 gene diversity in 2011, suggesting that the increased number of E6 detections reflects a real epidemic in the two cities. Most Linyi isolates (n = 61, or 88%) in 2011 segregated into sublineage 1a, together with 18 Jinan isolates in 2011. Interestingly, the ancestral VP1 sequence of sublineage 1a inferred using the maximum-likelihood method had 100% identity with the sequence of one environmental isolate from Jinan in August 2010, suggesting an intercity spread from Jinan to Linyi. By Bayesian phylodynamic methods, the most recent common ancestor of Linyi isolates in sublineage 1a dated back to 24 December 2010, revealing that this sublineage was likely imported into Linyi from August to December in 2010. This study demonstrates that environmental surveillance is a sensitive method in tracing transmission pathways of circulating enteroviruses among different regions and reveals that E6-associated aseptic meningitis is an emerging concern in China.

Intercity Spread of Echovirus 6 in Shandong Province, China: Application of Environmental Surveillance in Tracing Circulating Enteroviruses