1. Human rhinovirus species occurrence among adults with respiratory tract infection and asymptomatic individuals during two consecutive winter seasons Zlateva KT 1, van Nieuwkoop S 1, Coenjaerts F 2, Farzad N 1, Verheij T 3, Little P 4, Ieven M 5 and Claas EC 1 on behalf of the GRACE Study Group 1 Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands 2 Deparment of Virology, UMC Utrecht, Utrecht, The Netherlands 3 Julius Center for Health Sciences and Primary Care, UMC Utrecht 4 School of Medicine, University of Southampton, Southampton, UK 5 Department of Microbiology, University Hospital Antwerp, Antwerp, Belgium Leiden University, Leiden, The Netherlands GRACE project: https://www.grace-lrti.org/portal/en-GB/Homepage.htm 4. CONCLUSIONS HRV-A was the most commonly detected species among adult outpatients in Europe during the winter seasons 2007/2008 and 2008/2009. Multiple HRV genotypes were present in each community and closely related VP1 sequences were obtained from several distant locations, indicating broad distribution of certain HRV strains. The observed tendency towards geographic clustering, suggests for predominant circulation of particular HRV genotypes in each community. A higher level of genetic variability was identified among HRV-C isolates. HRV-A HRV9 V1-08/09 HRV32 V1-08/09 HRV67 V2-07/08 HRV38 HRV60 V0-08/09 V1-08/09 HRV74 V1-08/09 HRV15 V1-07/08 HRV19 HRV94 HRV64 HRV82 V1-07/08 V1-08/09 V2-08/09 V1-08/09 HRV96 HRV61 HRV73 HRV13 HRV41 V1-08/09 HRV23 V1-08/09 HRV30 V1-08/09 HRV49 HRV2 V1-08/09 V1-07/08 V1-08/09 V1-07/08 V1-08/09 HRV43 HRV75 V1-08/09 HRV34 HRV50 HRV18 V1-08/09 V1-07/08 V1-08/09 HRV21 V1-07/08 V1-08/09 HRV57 V2-08/09 V1-08/09 HRV33 HRV11 HRV76 V1-08/09 HRV90 V1-07/08 HRV24 V1-07/08 V1-08/09 V2-07/08 HRV81 V2-07/08 HRV16 V1-08/09 HRV1 HRV1B V1-08/09 V1-07/08 HRV54 HRV98 V1-08/09 HRV56 HRV85 V2-08/09 HRV40 V2-07/08 HRV59 V1-08/09 HRV63 HRV39 V2-08/09 V1-08/09 HRV10 HRV100 HRV77 V1-08/09 V2-08/09 HRV44 HRV29 V1-08/09 V1-07/08 V1-08/09 HRV31 HRV47 V1-08/09 V1-07/08 V1-08/09 V1-07/08 HRV25 HRV62 V1-08/09 V1-07/08 V0-07/08 HRV36 HRV89 HRV58 HRV88 V0-07/08 HRV7 HRV78 HRV46 HRV80 V1-07/08 HRV65 HRV51 HRV71 A101 V1-08/09 HRV68 HRV20 HRV28 HRV53 V2-08/09 V2-07/08 HRV45 HRV8 HRV95 V1-08/09 V1(2)*-07/08 V1-08/09 0.05 99 100 95 99 100 81 100 92 97 96 92 100 99 100 96 100 99 100 98 96 100 82 100 99 100 99 100 98 100 99 100 76 90 100 99 83 100 99 100 99 100 87 99 96 100 79 100 99 100 99 100 92 100 99 100 HRV-B V1-08/09 HRV70 HRV17 HRV91 HRV69 V1-08/09 V2-07/08 HRV52 V1-08/09 HRV48 V1-08/09 HRV86 V2-07/08 HRV6 HRV37 HRV72 HRV14 HRV3 HRV35 HRV79 HRV92 V1-08/09 HRV83 HRV5 HRV42 HRV99 HRV26 V2-07/08 HRV27 HRV93 HRV97 HRV84 V1-V2*-07/08 V1-08/09 0.05 100 99 100 82 100 97 100 97 100 94 77 98 83 100 94 100 97 100 99 HRV-C NY-074/2004-2005 V1-08/09 V1-07/08 AU-QPM/2003 V0-07/08 HK-026/2005 V1-07/08 CA-NAT001/2001 V1-08/09 V1-07/08 V1-08/09 V0 -07/08 V1-08/09 HK-025/2005 V1-08/09 HK-024/2005 V1-08/09 CA-NAT045/2003 V1-08/09 Shanghai-N10/2007 V1-08/09 Shanghai-N4/2006 V1-07/08 V0-07/08 V1-08/09 0.05 100 87 100 96 100 92 100 99 100 99 87 BARCELONA (SP) MATARO (SP) LODZ (PL) SZCZECIN (PL) BIALYSTOCK (PL) GENBANK UTRECHT(NL) ATWERP (BE) GENT (BE) SOUTHAMPTON (GB) ROTENBURG (DE) CARDIFF (GB) 3.2. Genetic diversity and circulation patterns of HRV isolates (Figure 3) Multiple VP1 genotypes were present among the GRACE HRV-A, B and C strains with a tendency for close clustering of strains isolated from the same geographic area. For example HRV-A sequences from Antwerp, Szczecin, Gent, Rotenburg, and Lodz obtained during the 2008/2009 season, grouped closely together. Furthermore the majority of HRV-A isolates from Spain (Mataro and Barcelona) grouped closely together in 3 main clades. Geographic clustering was also observed for HRV-B strains from Spain (Mataro and Barcelona), and Antwerp and between HRV-C isolates from Poland (Lodz and Bialystock), and Antwerp. However, we also detected close clustering of VP1 sequences obtained from communities in different countries, suggesting for global distribution of rhinovirus strains. Two pairs of identical sequences were identified among the HRV-B and HRV-A clinical isolates. A higher degree of sequence variation was detected among HRV-C clinical strains, for which the estimated paiwise amino acid identity was as low as 58% in comparison to 63% and 72%, respectively, for HRV-A and HRV-B GRACE viruses. 3. RESULTS 3.1. HRV species prevalence and distribution of HRV infections in Europe (Figure 2) From the total of 357 (74% of 482) real-time HRV positive samples, 196 (55%) were successfully amplified and analysed by nucleotide sequencing. The genotyped HRV strains were obtained from 179 patients and 16 asymptomatic adults. HRV-A accounted for 67% (131/196) of the positive GRACE samples, HRV-C for 17% (33/354) and HRV-B 13% (26/357). In addition 6 samples (3% of 357) were identified to be positive for HEV, including 1 HEV 68 (HEV-D), 1 Human coxsackievirus A13 (HEV-C), 2 HEV 104 (HEV-C), 2 HEV 109 (HEV-C). Figure 2 shows the predominance of mainly HRV-A in the different geographic origins. Infections caused by HRV-C were more frequently observed in Antwerp and Gent during 2007/2008 season. HRV-C viruses were not detected in Cardiff and Szczecin during 2008/2009 season as well as in Southampton and Mataro during both seasons. HRV-B infections were observed during both epidemic seasons in 6 communities, but were not detected in Gent, in Southampton during 2007/2008 and also in the three communities in Poland during the 2008/2009 season. HRV-A: 131 (67 %)HRV-B: 26 (13%)HRV-C: 33 (17%)HEV: 6 (3%) 0 2 4 6 8 10 12 14 16 18 20 08/09 ANTWERP BE SOUTHAMPTON UK ROTENBURG DE MATARO SP SZCZECIN PL UTRECHT NL CARDIFF UK BARCELONA SP BIALYSTOCK PL LODZ PL GENT BE 07/08 Epidemic seasons07/08 29% 08/09 68% 07/08 25% 08/09 52% 07/08 44% 08/09 17% 07/08 45% 08/09 44% 07/08 36% 08/09 4% 07/08 38% 08/09 71% 07/08 24% 08/09 70% 07/08 44% 08/09 48% 07/08 75% 08/09 44%21% 08/09 63%Genotyped HRVs (%) Number of genotyped HRVs N.S.* N.S.*: no samples available for this season 2.2. Primer design and semi nested PCR assays for VP1 amplification (Figure1) Degenerate primers were used to amplify the VP1 capsid gene of HRVs. Primers were developed based on an alignment of 139 HRV sequences (98 HRV-A, 32 HRV-B and 9 HRV-C) and an additional internal VP1-B primer was designed from an alignment of 84 HRV-B sequences. 3’-UTR HRV genome organization and primers locations CapsidNon-structural proteins P1P2P3 V4V2V3V12A2B2C3A 3C pro 3D pol 5’-UTR 3B 3. ABI sequencing with VP3 and VP1 primers HRV-VP3 senseHRV-2A antisense ~1.5 kb 1. One-step RT-PCR (Qiagen) ~ 1 kb 2. Semi-nested PCR (Biorad IQ supermix) HRV-VP1 antisenseHRV-VP3 sense HRV-VP1-B antisense HRV-VP3 sense sn-PCR1 sn-PCR2 2. METHODS 2.1. Clinical specimens and study cohort A total of 482 nasopharyngeal swabs, real-time PCR positive for HRV, were collected at 11 primary care centers located in 6 European countries, during two consecutive seasons (2007-2009) as part of the GRACE study (Genomics to combat Resistance against Antibiotics in Community-acquired LRTI in Europe, EU framework 6, funded contract PL518226). The studied specimens were from 481 adults (age 18-89 years; mean 48 years) with a respiratory disease at their first (V1 samples) and/or second (V2 samples) visit to the general practitioner. In addition, samples from asymptomatic control subjects (V0 samples) were available. 2007/2008 2008/2009 V1 V2 V0 17 (9%) 27 (9%) 30 (15%) 43 (15%) 148 (76%)217 (76%) 1. INTRODUCTION Human rhinoviruses (HRVs) are classified into three species: HRV-A, HRV-B and HRV-C and are the leading cause of respiratory tract infections in humans. The disease outcomes associated with different HRV species and genotypes are poorly established. Several studies have documented high incidence and more severe illness caused by HRV-A and HRV-C viruses, however most of them have focused mainly on hospitalized pediatric patients during one season. Objective: To investigate HRV species prevalence, genetic diversity and circulation patterns among adults outpatients and asymptomatic individuals during the 2007/2008 and 2008/2009 winter seasons. Table 1. Seasonal distribution of HRV positive GRACE samples 2.3. Sequence analysis Phylogenetic analysis was performed with ~800 nt fragments of the VP3-VP1 genome region of 90 HRV-A, 17 HRV-B and 19 HRV-C GRACE isolates compared to respectively 71 HRV-A, 24 HRV-B and 9 HRV-C GenBank reference sequences. Neighbor-joining (NJ) trees were constructed with the MEGA4 software (Figure 3). The genetic similarity among HRV GRACE strains was determined with GeneDoc program.