The present study was approved by the Istituto di ricovero e Cura a Carattere Scientifico (IRCCS) Policlinico San Matteo Ethical Committee, and informed consent was obtained from parents or guardians in compliance with the Helsinki Declaration and for the consent to publish individual clinical data (Table 1).
Pediatric samples. A total of 974 archived nasopharyngeal aspirates (NPA) from children <24 months old and hospitalized for ARDs (mainly bronchiolitis, pneumonia, bronchitis, bronchospasm or wheezing) were examined in the present study. Samples were collected from October 2004 to November 2010, at the microbiology laboratory of the Azienda Ospedaliera in Melegnano. Samples collected during the 2004–2006 period were part of previous studies to characterize the infections of human respiratory syncytial virus (HRSV) and other respiratory viruses identified recently in infants with ARDs [11, 12]. In all cases NPAs were collected as part of the standard diagnostic practice to assess the presence of HRSV. Clinical data including comorbidities or subsequent bacterial infections, the duration of hospitalization, the presence of hypoxia, fever >38°C or gastrointestinal symptoms at the time of diagnosis were also available for most infants.
Transplant recipients’ samples. A total of 175 archived NPAs were obtained from 50 allogeneic and 4 autologous haematopoietic stem cell transplant recipient patients recruited at the Division of Hematology, IRCCS Policlinico San Matteo (Pavia, Italy) regardless of respiratory symptoms, as described [13, 14]. Respiratory samples were consecutively obtained after informed consent from October 1st, 2004 to April 2007. One to nine samples from each patient were collected every 30 days up to 180 days after transplantation. At each time point clinical data were recorded.
All samples were extracted by using Qiagen RNA mini kit (Qiagen, Germany), in accordance with the manufacturer’s protocol.
To investigate the prevalence of HEV109 in children and haematopoietic stem cell transplant recipient patients, specific primers targeting the UTR region were used (EV109 VP1 123 F, 5′-GGA GAC TGG AGC AAC TAG TAA AG-3′; EV109 VP1 363R, 5′-GGT GAA CAT TTC CAA TTT CCT ACG-3′). To better characterize the novel HEV 109 strains the VP4/VP2 region was also amplified using the following primers: P2-4-Rw GCA TCI GGY ARY TTC CAC CAC CAN CC; VP2-4-Fw GGG ACC AAC TAC TTT GGG TGT CCG TGT (Figure 2). The sensitivity of amplification protocols was determined by cloning each amplified target regions into pCR2.1 plasmid vector (TA Cloning Kit; Invitrogen), serially diluted from 106 copies to 1 copy as previously described [11, 13, 16] Amplification protocols ensured the detection of 5 and 15 DNA copies/reaction of HEV109 target regions respectively (data not shown).
After agarose gel electrophoresis visualization, all the amplification products were sequenced bidirectionally to confirm amplification specificity. Molecular identification and typing of HEV109 positive samples, were performed with MEGA 3.1 software after ClastalW alignment and manual sequence editing with BioEdit. Phylogenetic relationships were estimated using MEGA V3.1, (neighbor-joining method by using Tajima-Nei model as estimated by using Modeltest; the α value used in MEGA was previously estimated directly from the data by using PAUP).
Since multiple infections are frequently detected in respiratory samples of patients with respiratory symptoms and to better clarify the pathogenetic role of the novel enterovirus in co-infections, all specimens positive for HEV109 were also assayed for the presence of other respiratory viruses, including parainfluenza viruses (PIV 1–3), influenza A and B viruses, human metapneumovirus (HMPV), human respiratory syncytial virus (HRSV), adenoviruses, human coronaviruses (hCoV), human bocavirus (hBoV) and for WU (WUPyV) and KI (KIPyV) polyomaviruses using a multiplex PCR strategy (Seeplex RV12 ACE Detection, Seegene, Rockville) and in house protocols [11–13]. Presence of human rhinoviruses (HRVs) in HEV109 positive samples from infants and in all samples from immunosuppressed patients were also investigated by molecular analysis and phylogenetic reconstruction. Samples were subjected to first round one step reverse transcription and amplification (Super-ScriptIII One Step RT-PCR with Platinum Taq (Invitrogen) after RNA extraction using Primers P1-1 (CAA GCA CTT CTG TYW CCC C) and P3-1 (ACG GAC ACC CAA AGT AG) and the following thermal profile: 20 min 50°C, 10 min 95°C, followed by 15 'touchdown' cycles (The steps were 94°C for 20 s, 68°C down to 52°C (2°C intervals) for 30 s), and 25 cycles at 94°C 30 s, 52°C 30 s and a final extention step at 68°C for 40 s.; 5 μl of first RT-PCR reaction underwent a semi-nested amplification using the Platinum TaqDNA Polimerase (Invitrogen) and the forward primer P1-1 and three reversed primers P2-1 (TTA GCC ACA TTC AGG GGC), P2-2 (TTA GCC ACA TTC AGG AGC C) and P2-3 (TTA GCC GCA TTC AGG GG) as described previously with minor modifications . In all amplification protocols primers were used at a final concentration of 200 nM. Amplification specificity was confirmed by sequencing and phylogenetic internal control used to exclude contaminations [18, 19].
Since this amplification protocol amplified also HEV109 homologous target sequence, in co-infected samples, amplifications products after gel electrophoresis (data not shown) were individually gel extracted (using Qiagen mini elute gel extraction kit) and sequenced to confirm amplification specificity. In all HRV samples when visualized in the gel only the nested amplification product of about 300 bp was observed. In the co-infected samples instead, after both amplification reaction, an additional 390 bp band was indeed always present that when sequenced independently corresponded to the HEV109 genome amplified with the outer primers. We cannot exclude that a deeper clonal analyses of each band would have demonstrated the presence of both viruses in both amplification products, however in the mono-infected transplant recipient patient the inner amplification product corresponded to the HEV only when sequenced.