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Beijing, China from 2015 to 2017. Nasopharyngeal swabs and sputum were collected from 30 sentinel hospitals in Beijing and subjected to EV and EV-D68 detection by real-time PCR. The VP1 gene region and complete genome sequences of EV-D68 positive cases were analyzed. Of 21816 ARTI cases, 619 (2.84%) were EV positive and 42 cases were EV-D68 positive. The detection rates of EV-D68 were 0 (0/6644) in 2015, 0.53% (40/7522) in 2016 and 0.03% (2/7650) in 2017, respectively. Two peaks of EV-D68 infections occurred in late summer and early-winter. Ten cases (23.81%) with upper respiratory tract infection and 32 cases (76.19%) presented with pneumonia, including 3 cases with severe pneumonia. The phylogenetic analysis suggested 15 subclade D3 strains and 27 subclade B3 strains of EV-D68 were circulated in China from 2016 to 2017. A total of 52 amino acid polymorphisms were identified between subclades D1 and D3. These data suggest an upsurge of EV-D68 occurred in Beijing in 2016, the new subclade D3 emerged in 2016 and co-circulated with subclade B3 between 2016 and 2017. Enteroviruses (EV) are small, non-enveloped viruses with a single-stranded, positive-sense RNA in the family Picornaviridae 1,2 . The length of enteroviruses is approximately 7.5 kilobases, and divided into 15 species (EV-A to L and rhinovirus A to C) 3-6 . Seven species, including EV-A to D and rhinovirus (RV) A to C, caused a wide spectrum of diseases in human 7,8 . Enterovirus D68 (EV-D68), a member of the species EV-D, was first identified from bronchiolitis and pneumonia in California in 1962 9 . EV-D68 infections have been rarely reported until the worldwide outbreaks 10-18 . A nationwide outbreak of EV-D68 associated with severe respiratory tract infection was reported in the USA from mid-August 2014 to January 15, 2015 and 1,153 cases were confirmed with clinical characteristics of patients mainly resembling pneumonia with severe dyspnea syndrome during this epidemic 19 . In China, sporadic cases of EV-D68 were reported between 2006 and 2014 13,20-23 . The similar results were found that few patients confirmed with EV-D68 infections during the surveillance among US 24 , China 21 in 2015. However, an upsurge of EV-D68 infections occurred in 2016, ranging from Europe to North America, including Netherlands, France, Sweden and United states 24-27 . In addition, sporadic cases have been reported in Hong Kong, Italy, Germany, Portugal and UK in 2016 28, 29 . Earlier phylogenetic analysis revealed that EV-D68 strains circulating worldwide during 1961-2013 were divided into three clades (clades A to C) or lineages (lineages 1 to 3) 10,11,29-31 . With the deepening of the study, the clades of EV-D68 were further refined. After the outbreak of the United States in 2014, EV-D68 was grouped into four clades and five subclades, including clade A, clade B (subclades B1 to B3), clade C and clade D (subclades D1 to D2) 32,33 . The subclade B1 and B3, as the major subclade, circulated around the world in 2014 and in 2016, respectively. To raise awareness of this upsurge of EV-D68 infection, we performed an EV-D68 surveillance study on ARTI cases from the Respiratory Virus Surveillance System (RVSS) in Beijing and analyzed the epidemiological and clinical characteristics of EV-D68 infections from 2015 to 2017 in Beijing. Results A total of 21, 816 ARTI cases were collected from RVSS from January 2015 to December 2017. The median age of them was 36 years old (range: 1 day to 101 years). Among 21, 816 cases, 619 (2.84%) cases were enterovirus positive and 42 (0.19%) cases were EV-D68 positive. 359 cases were successfully amplified among 619 enterovirus positive cases, including 326 cases belonging to EV-A to D, 26 cases and 7 cases belonging to RV-A and RV-C respectively, and no case belonging to RV-B. Of 326 cases belonging to EV-A to D, they were genotyped to 30 serotypes including 9 of EV-A, 16 of EV-B, 4 of EV-C and 1 of EV-D and the dominant serotypes were CVA6, EV-D68, CVA4, CVA2, CVA10, CVA5 and CVA12, accounting for 16.27%, 12.88%, 11.83%, 11.54%, 8.28%, 5.62% and 5.03%, respectively (Fig. 1) . No samples of EV-D68 co-infected with other enteroviruses were found. The annual detection rates of EV-D68 were 0 (0/6, 644) in 2015, 0.53% (40/7, 522) in 2016 and 0.03% (2/7, 650) in 2017, respectively (Table 1) . Twenty cases were aged younger than 18 years, 15 were aged 18 to 60 years and 7 were aged over 60 years. No statistically significant difference of EV-D68 positive rates was identified in the different age groups in 2016 (P = 0.062). Statistical analysis. The differences of EV-D68 positive cases among different age groups were performed using chi-square test. The comparison of EV-D68 different subclades proportions in different age groups were performed using fisher's exact test. The comparison of respiratory diseases by different subclades of EV-D68 were performed using fisher's exact test. A two-sided P value of less than 0.05 was considered to be statistically significant. All analyses were performed using SPSS software, version 17.0.
A:
Upsurge of Enterovirus D68 and Circulation of the New Subclade D3 and Subclade B3 in Beijing OPEN