Journal of Medical Internet Research - International Scientific Journal for Medical Research, Information and Communication on the Internet #Preprint #PeerReviewMe: Warning: This is a unreviewed preprint. Readers are warned that the document has not been peer-reviewed by expert/patient reviewers or an academic editor, may contain misleading claims, and is likely to undergo changes before final publication, if accepted, or may have been rejected/withdrawn. Readers with interest and expertise are encouraged to sign up as peer-reviewer, if the paper is within an open peer-review period. Please cite this preprint only for review purposes or for grant applications and CVs (if you are the author). Background: Social media have been increasingly adopted by health agencies and professionals to disseminate information, interact with public, and understand public opinion. Among them, the Centers for Disease Control and Prevention (CDC) is arguably one of the first government health agencies to adopt social media during health emergencies and crisis. It had been active on Twitter during the 2016 Zika epidemic that caused 5,168 domestic cases in the United States. Objective: This study aims to quantify the temporal variabilities in CDC’s tweeting activities over the course of Zika epidemic, public engagement in these CDC-initiated tweets (i.e., retweets and replies), and Zika case counts. It then compares the patterns of these three sets of data to identify the discrepancy and consistency among actual domestic Zika case counts, CDC response on Twitter, and public engagement in this topic. Methods: All of the CDC-initiated tweets published in 2016 with corresponding retweets and replies were collected from 67 CDC-associated Twitter accounts. Both univariate (ARIMA model) and multivariate time series analyses (CCF, mutual Shannon information entropy, ARIMAX model, Granger test) were performed in each quarter of 2016 for domestic Zika case counts, CDC tweeting activities, and public engagement in the CDC-initiated tweets. Results: CDC sent out more than 84% of its Zika tweets in the first quarter of 2016 when Zika case counts were low in the 50 U.S. states and territories. While Zika case counts increased dramatically in the second and the third quarters, CDC efforts on Twitter plunged. Time series of public engagement in the CDC-initiated tweets generally differed among quarters and from that of original CDC tweets based on ARIMA model parameters. Original CDC tweets and public engagement both had highest mutual information with Zika case counts in the 2nd quarter. Public engagement in the original CDC tweets was also substantially influenced by and preceded actual Zika epidemic. Conclusions: There were substantial discrepancies among CDC’s original tweets regarding Zika, public engagement in these tweets, and actual Zika epidemic. The patterns of these discrepancies also varied between different quarters in 2016. We discovered that although CDC was very effective in early warning of Zika in the first quarter of 2016, it failed to respond in a timely manner on Twitter during the rest of the Zika epidemic. Public engagement in CDC’s original tweets served as a more prominent predictor of actual Zika epidemic than number of CDC’s original tweets later in the year.