Proceedings of the Estonian Academy of Sciences, 2012, 61, 4, 320–329 doi: 10.3176/proc.2012.4.07 Available online at www.eap.ee/proceedings The Estonian H1N1 influenza 2009 outbreak was highly underestimated Regina Saar a , Diivi Põdersoo b , Mari Järvelaid c , Linda Tuubel d , Jaanus Suurväli a , Anu Nutt a , Merle Saaremäe a , Tiiu Saar b , and Sirje Rüütel Boudinot a* a Department of Gene Technology, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia b Department of Infectious Diseases, Estonian University of Life Sciences, Kreutzwaldi 62, 51014 Tartu, Estonia c Northern Service of Health Board of Estonia, Paldiski mnt. 81, 10617 Tallinn, Estonia d Faculty of Medicine, University of Tartu, Ravila 19, 50411 Tartu, Estonia Received 13 July 2011, revised 12 December 2011, accepted 3 January 2012, available online 20 November 2012 Abstract. The H1N1 influenza strain Mexico 2009 (H1N1pandemic09) led to mild symptoms (with no or low fever) in Estonia during the 2009–2010 outbreak. Due to the lack of clinical signs, it was difficult to estimate the real spreading of this influenza virus in Estonia and no cases of H1N1 influenza were officially registered in animals either. We used an ELISA method to screen blood sample collections for the presence of anti-H1N1 and anti-H3N2 antibodies. All sera were also tested with the hemagglutination inhibition (HI) assay. Out of the 123 samples from human patients, 23 (i.e. 18.7%) were seropositive for the H1N1pandemic09 virus. In addition, blood samples from six persons were positive for both H1N1 and H3N2 viruses, while according to the data from the Estonian Health Board, people aged 15–65 had a general disease rate of around 3.9%. Almost all of the tested animals from two herds (out of four studied) were seropositive for H1N1pandemic09. The seven HA protein sequences isolated from Estonia were aligned with a consensus sequence of the pandemic H1N1 HA sequences from Mexico using ClustalW, and 12 amino acids substitutions were found. Key words: influenza H1N1, human, porcine, ELISA, hemagglutination inhibition. INTRODUCTION * In Europe, the H1N1 influenza strain Mexico 2009 (H1N1pandemic09) [1], also known as ‘swine flu’, appeared to be a mild to moderate disease affecting preferentially school-age children. Elderly adults were underrepresented in severe cases [2–6]. However, the true proportion of infected persons could not be well assessed due to the lack of serological evidence of asymptomatic cases. Asymptomatic and mild cases are missed by current reporting techniques of influenza and only a few studies provide assessment of seroprevalence during an epidemic [3,7]. Also the H1N1pandemic09 generally led to only mild symptoms in Estonia during the 2009–2010 out- break. Hence, due to the lack of clinical signs, it was difficult to estimate the real spreading of this influenza virus in Estonia. No cases of H1N1 influenza were * Corresponding author, Sirje.boudinot@gmail.com officially registered in animals either during this period. However, even a mild H1N1 influenza infection would afford a good level of protection [8,9]. The H1N1 vaccine became available only at the end of the out- break. Consequently, only 13.4% of the persons belong- ing to high-risk groups and about 2.7% of the whole Estonian population were vaccinated [10]. The aim of this study was to assess how well the spread of H1N1pandemic09 during the 2009–2010 out- break in Estonia was estimated. Our hypothesis was that the outbreak was highly underestimated, which would deserve a definitive epidemiologic demonstration. The potential consequences of underestimation are hereby discussed. MATERIALS AND METHODS To provide an easy way to test the H1N1pandemic09 influenza virus (IV) seropositivity, we designed an