Upsurge of Enterovirus D68 and Circulation of the New Subclade D3 and Subclade B3 in Beijing, China, 2016

We conducted a surveillance among acute respiratory tract infection (ARTI) cases to define the epidemiology, clinical characteristics and genetic variations of enterovirus D68 (EV-D68) in 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.

There were 10 cases (23.81%) with URTI and 32 (76.19%) cases with pneumonia. Three cases, including one baby and two old men, were diagnosed with severe pneumonia and two of them were admitted to the ICU ward. No acute flaccid paralysis was found among 42 EV-D68 cases. They had been physically healthy without underlying diseases and immunocompromised before causing ARTI by EV-D68, and the prognosis was satisfactory after treatment The common clinical characteristics included cough (95.24%), fever (71.43%), expectoration (66.67%) and pharyngalgia (28.57%). About 35.7% of EV-D68 infections had hyperpyrexia symptoms (39 °C-40.5 °C). The CXR findings showed 26 cases with the pulmonary consolidation, four cases with interstitial lesion and two cases with pleural effusion. Four cases who were diagnosed with pneumonia were accompanied with other pathogen infections (one with H3N2, one with respiratory syncytial virus and two with mycoplasma). Similar to the tree based on VP1 gene region, the phylogenetic tree based on the complete genome that 9 of EV-D68 strains from this study and one strain detected in 2017 in USA (GenBank Accession No. MG757146) belonged to the new subclade D3, which were grouped into a new cluster separated from the subclades D1 and D2 strains of clade D over the world before 2017 with 90.5-97.0% identity in nucleotide. Homology comparison showed fifteen strains of subclade D3 in this study sharing 98.9-99.9% identity in nucleotide and 99.5-99.7% identity in amino acid with each other, and 98.2-98.5% identity in nucleotide and 99.1%-99.2% identity in amino acid with the American strain (GenBank Accession No. MG757146).

Phylogenetic analysis of EV-D68 strains.
On basis of the complete polypeptide sequences of 2190 amino acids, a total of 52 amino acid polymorphisms were identified between subclade D1 and D3, which may further support the new cluster of D3. There were 17 amino acid polymorphisms identified from subclade B3 in Beijing between 2014 and 2016 and 12 amino acid polymorphisms identified from subclade B3 between American and Chinese strains in 2016 (Fig. 5).

Molecular epidemiology of EV-D68 circulating in Beijing.
The analysis of our sequences in this study and our prophase study 23 collected from RVSS showed subclades B1, B3, D1 and D3 appeared to dominate in different years in Beijing between 2011 and 2017. Both subclade D1 and B1 were co-circulating in 2011, only subclade D1 was sequentially detected in 2012 and co-circulated with subclade B1 in 2013 once again, but not detected in the subsequent years. However, subclade B3 was detected for the first time as single prevalent lineage in 2014. The new subclade D3 was first detected in 2016 and co-circulated with subclade B3 during 2016 to 2017.
Between 2011 and 2014, EV-D68 infections appeared mainly in the people younger than 18 years of age (11/12) in our previously study 18 . However, more of EV-D68 infections appeared in the adult group from 18 to 60 years of age (15/42) and elderly group over 60 years of age (7/42) in Beijing, that 11 cases were caused by subclade B3 and 11 by subclade D3. No statistical difference of age was identified among EV-D68 positive patients caused by subclades B3 or D3 (P = 0.130). www.nature.com/scientificreports www.nature.com/scientificreports/ Of 27 cases caused by subclade B3, 6 cases were diagnosed with URTI and 21 cases with pneumonia, including 1 cases with severe pneumonia. Of 15 cases caused by the new subclade D3 in 2016, 4 cases were diagnosed with URTI and 11 cases with pneumonia, including 2 cases with severe pneumonia. There was no statistical differences of the proportions on pneumonia or severe pneumonia caused by subclade B3 or D3 (both P > 0.05) ( Table 2).

Discussion
The outbreaks of ARTI caused by EV-D68 had been reported in various countries in 2014, such as the United States, Canada, Brazil, Norway, Netherlands and other countries 19 . Recent study in USA showed a significant decrease of EV-D68 detection in 2015, and subsequently another outbreak occurred in 2016 24 . The study in the Netherlands showed very limited activity of EV-D68 was observed in 2015 and an upsurge in 2016 25 . The      35 . In this study, the upsurge of EV-D68 with 40 cases confirmed was in 2016, and significant decreased in 2017 with only 2 cases confirmed. In recent years, there was a trend that the peak of EV-D68 appeared every two years.
However, the circulation of diverse EV-D68 subclades in China were slightly different from that in the world. Notable, it showed a new subclade of EV-D68 strains in Beijing from 2016 to 2017. One study by Cyril et al. showed that 2.6-7.1% diversity in nucleotide identity of EV-D68 VP1 gene region could be divided into different subclades 31 . The diversities of EV-D68 VP1 gene region in nucleotide were 2.6-3.8% between subclade D1 and D3, and 5.7-6.4% between subclade D2 and D3. Therefore, we defined the new subclade of EV-D68, subclade D3. In this study, the phylogenetic analysis showed subclade B3 strains with highly homologous were grouped into the same evolutionary branch with strains in other countries in 2016, but in separated evolutionary branches with the strains in Beijing, Hong Kong, Shenzhen and Taiwan during 2014 to 2015.
In the United States, two subclades, a major subclade B1 and a minor subclade B2, had co-circulated during the outbreak in 2014 14 and subclade B3 had emerged and caused outbreak in 2016 24 . In the Netherlands, both a major subclade B1 and a minor subclade D were detected in 2014, and only subclade B3 upsurged in 2016 25 . In addition, the subclade B3 was detected in Sweden in 2016 27  Homology analysis showed multiple mutations in the 2C protein region which inhibits NF-κB mediating the adaptive immunity and innate immunity of the body to enterovirus 36 . Therefore, the phenomenon of the high mutation in 2C protein region needed to be further study.
Several clinical characteristics should be noticed in our patients who had EV-D68 subclades B3 and D3 infections in this study with the comparison of previously studies. First of all, there were the major URTI and a minor Beijing and other countries. The GenBank accession numbers of complete genomes in this study were from MH341711 to MH341734. The phylogenetic relationships were estimated by the maximum-likelihood method with 1,000 the bootstrap replicates in MEGA6. GenBank accession numbers, the countries, the years and the clades were shown for each EV-D68 strains. Clade A was indicated in yellow, subclade B1 in purple, B2 in gray, B3 in blue, clade C in green, subclade D1 in pink, D2 in brown, and new subclade D3 in red. (a) The phylogenetic tree of EV-D68 strains; (b), the phylogenetic tree of strains in subclade B3; (c), the phylogenetic tree of strains in clade D.
www.nature.com/scientificreports www.nature.com/scientificreports/ pneumonia causing by EV-D68 in China [20][21][22][34][35][36] . But 32 cases in this study were diagnosed with pneumonia including 3 cases with severe pneumonia. In addition, studies showed the majority of EV-D68 positive cases were children, elderly patients and a few adult around world 12,19,21,28 . In Hong Kong, their study firstly demonstrated EV-D68 could caused severe respiratory diseases in elderly people 28 . Several reports from China, US, Sweden, Netherlands, Denmark also identified a few adults or elderly people with ARTI, but there was no statistical analysis to show the characteristics of EV-D68 distribution among different age groups of people 21,24-28 . Our demonstrated EV-D68 could cause ARTI among all age groups of people equally. Last, Subclades B3 and D3 could lead lobes to pulmonary consolidation and interstitial lesion, even pleural effusion.
In conclusion, our study revealed an upsurge of EV-D68 in Beijing in 2016, and the new subclade D3 emerged in 2016 co-circulated with subclade B3 during 2016 to 2017 period and the patients of EV-D68 infection among all age groups. The prevalence and genetic variations of EV-D68 in China was of great significance to EV-D68 prevention in the world. Whether the emergence of new subclade D3 would cause outbreaks in the future should arouse our attention.

Patients. RVSS established by the Beijing Center for Disease Prevention and Control (BJCDC) in 2011 and
now covers 30 sentinel hospitals distributing in all 16 districts of Beijing. This surveillance has obtained ethics approval from the Ethics Committee at BJCDC and experiments were performed in accordance with relevant guidelines and regulations. At recruitment, written informed consent had been obtained from the patient or the guardian. For research involving human participants under the age of 18 years (including donors of tissue samples), informed consent had been obtained from a parent and/or legal guardian. All clinical specimens were collected from RVSS. Clinical specimens, including nasopharyngeal swabs and sputum, were collected from acute respiratory tract infection (ARTI) and sent to BJCDC for laboratory diagnostic testing. ARTI cases in this study included upper respiratory tract infection (URTI) and pneumonia (including severe pneumonia). Diagnosis according to the criterions established by Pediatrics Society and Respiratory Society, Chinese Medical Association 37-39 .

Real-time PCR for detection of EV-D68 and other enterovirus. Total nucleic acid (RNA and DNA)
was extracted from the clinical specimens by Thermo Scientific ™ KingFisher ™ Flex Magnetic Particle Processors (Thermo Fisher). EV-D68 and other enteroviruses were simultaneously detected in one tube with a commercial real-time RT-PCR kit (cat. no. CN08-4G-100, Jiangsu uninovo biological technology company, China). A panel of respiratory pathogens, including influenza virus A (pandemic influenza virus H1N1, seasonal influenza A virus H3N2) and B, respiratory syncytial virus, parainfluenza virus 1 to 4, human adenovirus, human rhinovirus, human metapneumovirus, human coronavirus (NL63, OC43, 229E and HKU1), human bocavirus, mycoplasma and chlamydia, were also detected in these specimens with commercial real-time RT-PCR kits (cat. no. CN12-33-100 and cat. no. CN09-4-100, Jiangsu Uninovo Biological Technology Co. Ltd., China). The serotypes of enteroviruses were identified with the the seminested and conventional RT-PCR method for amplification of VP1 sequences (cat. no. C81401180, Invitrogen GoldScript, United States) 40 .
Phylogenetic analysis of VP1 gene regions and complete genomes. The fragments of EV-D68 positive specimens were amplified and sequenced with EV-D68 specific primers 23 . The consensus sequences were assembled using BioEdit software, version 7.0.9. The phylogenetic tree of VP1 gene region was estimated using the GTR + G model in maximum-likelihood method and the complete genome was estimated using GTR + G + I model in maximum-likelihood method, with bootstrap analysis of 1,000 replicates, in MEGA software, version 6.0. The amino acid polymorphisms and substitutions identified by sequence analysis were plotted on graphs using the Weblogo, version 3.0.