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Journal of Clinical Virology 132 (2020) 104590 Contents lists available at ScienceDirect Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv Insights into immune evasion of human metapneumovirus: novel 180- and 111-nucleotide duplications within viral G gene throughout 2014-2017 seasons in Barcelona, Spain Maria Piñana a, Jorgina Vila b, Carolina Maldonado c, Juan José Galano-Frutos d, e, Maria Valls b, Javier Sancho d, e, f, Francesc Xavier Nuvials c, Cristina Andrés a, María Teresa Martín-Gómez a, Juliana Esperalba a, Maria Gema Codina a, Tomàs Pumarola a, *, Andrés Antón a a Respiratory Viruses Unit, Microbiology Department, Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus, Universitat Autònoma de Barcelona, Passeig Vall d’Hebron 119-129, 08035 Barcelona, Spain b Paediatric Hospitalization Unit, Paediatrics Department, Hospital Universitari Maternoinfantil Vall d’Hebron, Vall d’Hebron Barcelona Hospital Campus, Universitat Autònoma de Barcelona, Passeig Vall d’Hebron 119-129, 08035 Barcelona, Spain c Intensive Care Department, Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus, Universitat Autònoma de Barcelona, Passeig Vall d’Hebron 119-129, 08035 Barcelona, Spain d Biochemistry and Molecular and Cell Biology Department, Sciences Faculty, Universidad de Zaragoza, Zaragoza, Spain e Biocomputation and Complex Systems Physics Institute (BIFI). Joint Units BIFI-IQFR (CSIC) and GBs-CSIC, Universidad de Zaragoza, Zaragoza, Spain f Aragon Health Research Institute (IIS Aragón), Zaragoza, Spain A R T I C L E I N F O A B S T R A C T Keywords: Background: Human metapneumovirus (HMPV) is an important aetiologic agent of respiratory tract infection Human metapneumovirus (RTI). This study aimed to describe its genetic diversity and clinical impact in patients attended at a tertiary duplication university hospital in Barcelona from the 2014-2015 to the 2016-2017 seasons, focusing on the emerging du­ steric shielding plications in G gene and their structural properties. epidemiology genetic diversity Methods: Laboratory-confirmed HMPV were characterised based on partial-coding F and G gene sequences with clinical impact MEGA.v6.0. Computational analysis of disorder propensity, aggregation propensity and glycosylation sites in viral G predicted protein sequence were carried out. Clinical data was retrospectively reviewed and further associated to virological features. Results: HMPV prevalence was 3%. The 180- and 111-nucleotide duplications occurred in A2c lineage G protein increased in prevalence throughout the study, in addition to short genetic changes observed in other HMPV lineages. The A2c G protein without duplications was calculated to protrude over F protein in 23% of cases and increased to a 39% and a 46% with the 111- and 180-nucleotide duplications, respectively. Children did not seem to be more affected by these mutant viruses, but there was a strong association of these variants to LRTI in adults. Discussion: HMPV presents a high genetic diversity in all lineages. Novel variants carrying duplications might present an evolutionary advantage due to an improved steric shielding, which would have been responsible for the reported increasing prevalence and the association to LRTI in adults. 1. INTRODUCTION respiratory syncytial virus (HRSV) [2], causing an indistinguishable symptomatology [1]. HMPV is an enveloped, lineal, negative-sense, Human metapneumovirus (HMPV) is an important aetiologic agent single-stranded RNA virus, classified into HMPV-A and HMPV-B geno­ of upper and lower respiratory tract infections (URTI and LRTI) in types and subdivided into subgenotypes A1, A2 (A2a, A2b and A2c children and adults [1]. lineages) [1,3,4], B1 and B2 (B2a and B2b lineages) [5]. HMPV belongs to the Pneumoviridae family together with human The fusion (F) and the attachment (G) proteins are the major * Corresponding author: Respiratory Viruses Unit, Microbiology Department, Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus, Universitat Autònoma de Barcelona, Passeig Vall d’Hebron 119-129, 08035 Barcelona, Spain. E-mail address: virusrespiratoris@vhebron.net (T. Pumarola). https://doi.org/10.1016/j.jcv.2020.104590 Received 4 May 2020; Received in revised form 24 July 2020; Accepted 10 August 2020 Available online 11 August 2020 1386-6532/© 2020 Elsevier B.V. All rights reserved. M. Piñana et al. Journal of Clinical Virology 132 (2020) 104590 envelope glycoproteins. F protein is the major cross-protective antigenic substitution model was determined by MEGA v6.0, and the lowest determinant and is highly conserved between genotypes (88%) [4]. Bayesian information criterion score model was used and evaluated with Hence, it is the main target for most vaccine strategies under develop­ 1,000 bootstrap resamplings. ment [6]. Differently, G protein is weakly immunogenic [7], with 28% genetic divergence between genotypes and 74-82% intra-genotype [4]. 2.4. Computational analysis, generation of unfolded ensembles and In addition, 180- and 111-nucleotide duplications have been recently geometric analyses of G predicted protein sequence described into G protein’s ectodomain [8–11]. The aims of this study were to describe circulation pattern, genetic The propensity to adopt disordered conformations of three G se­ diversity and clinical impact of HMPV in paediatric and adult population quences with and without nucleotide duplications was analysed using attended at a tertiary university hospital in Barcelona from the 2014- the MetaDisorder server [14], their propensity to aggregate using the 2015 to the 2016-2017 seasons, focusing on the emergence and spread Pasta 2.0 server [15], and the prediction of potential N- and O-glyco­ of variants carrying these two nucleotide duplications. sylation sites using NetNGlyc 1.0 [16] and NetOglyc 4.0 [17] servers, respectively. 2. MATERIALS AND METHODS Ensembles consisting of 2,000 unfolded conformations were gener­ ated for each of the three G sequences using the ProtSA server [18]. The 2.1. Sample collection PDB file of each conformation was analysed to compute the distance between the N atom of the first extracellular residue (Asn52) and the From October/2014 to May/2017, respiratory specimens (nasopha­ more distant atom, as well as the radius of gyration of the particular ryngeal aspirates, nasal and pharyngeal swabs, bronchoaspirates, conformation. bronchoalveolar, bronchoselective and tracheal washes and sputums) were received for the laboratory-confirmation of respiratory viruses 2.5. Clinical features from children and adults attended at the Hospital Universitari Vall d’Hebron with suspicion of respiratory tract infection (RTI). Institu­ Demographic (age and sex) and clinical features (URTI/LRTI, co- tional Review Board approval (PR(AG)161/2016) was obtained from morbidities, co-infections, antibiotic use, need, type and length of res­ the hospital’s Clinical Research Ethics Committee. piratory support, length of hospital stay, ICU admission or exitus) of HMPV-laboratory confirmed cases were retrospectively reviewed from 2.2. Respiratory viruses’ detection medical records and related to viral features. Patients included in the demographic study were those with clinical presentation of URTI or During the HRSV and influenza epidemics, rapid tests were per­ LRTI, whilst patients with other symptoms rather than respiratory were formed for a fast diagnosis, which were based on immunocromatog­ excluded from the study. For the severity study, only patients hospital­ raphy (Alere BinaxNOW® Influenza A&B/RSV, Alere, USA), ised due to LRTI were included, and exclusion criteria were those cases immunofluorescence (Sofia RSV FIA, Quidel, USA) or real-time RT-PCR with other symptoms rather than LRTI and hospitalisation due to other (GenXpert Flu/RSV XC, Cepheid, USA). Samples received out of HRSV/ clinical reasons even though the patient manifested RTI. FLUV epidemics or negative for rapid tests were analysed by immuno­ fluorescence (D3 Ultra 8™ DFA Respiratory Virus, Diagnostic HYBRIDS, 2.6. Statistical analysis USA) or mainly by real-time RT-PCR (Anyplex II RV16, Seegene, Korea, until 2015; Allplex Respiratory Panels 1-3, Seegene, Korea, since 2015). Data were analysed with R software v3.5.1. For categorical data, Chi- Total nucleic acids were extracted using NucliSense easyMAG (Bio­ squared or Fisher’s exact test were performed. For numerical variables, t Mérieux, Marcy l’Etoile) and kept at -80 ◦ C. student, Mann-Withney, ANOVA or Kruskall-Wallis tests were per­ formed according to the need. Statistical significance was taken at the p- 2.3. HMPV phylogenetic analysis value <0.05. Both partial F and G genes were retrospectively sequenced from all 3. RESULTS HMPV laboratory-confirmed samples. The amplification was performed using the One-Step RT-PCR kit (Qiagen, Hilden, Germany), conditions in 3.1. HMPV epidemiology Table 1. PCR products were purified using Exo-SAP-IT (USB, Affymetrix Inc., Cleveland, USA) and sequenced by the ABI Prism BigDye Termi­ A total of 20,132 samples of 14,769 patients were tested, of which nator v3.1 (Applied Biosystems, Carlsbad, USA) on the ABI PRISM 9,370 (47%) were laboratory-confirmed for at least one respiratory 3130xl Genetic Analyzer (Thermo Fisher Scientific, Waltham, USA). virus. Though being in a similar period, HRSV and influenza epidemics Nucleotide sequences were edited and assembled using MEGA v6.0 [12]. varied between seasons (Fig. 1A), being stablished during weeks 45/ A collapse to haplotypes was done with ALTER server [13]. The best fit 2014-13/2015 in the first season, 47/2015-16/2016 in the second Table 1 Primers and PCR conditions. CAN97-83 Fragment length Gene Primers Initial Final PCR conditions Reference (bp) position position GF: 6,247 6,268 GAGAACATTCGRRCRATAGAYA 50ºC×30’ – 95 ◦ C×15’ – 45x (95 ◦ C×30” – 59 ◦ C×30” – Ludewick HP et al., G 924 GR: 72 ◦ C×1’) – 72ºc×10’ 2005 7,149 7,170 AGATAGACATTRACAGTGGATT FF: 3,693 3,713 CAATGCWGGRATAACACCAGC 50ºC×30’ – 95 ◦ C×15’ – 45x (95 ◦ C×30” – 55 ◦ C×30” – Designed for this F 745 FR: 72 ◦ C×1’) – 72 ◦ C×10’ study 4,416 4,437 ATTGAAYTGATCYTCAGGAAAC 2 M. Piñana et al. Journal of Clinical Virology 132 (2020) 104590 season and 43/2016-11/2017 in the third season. Overall these 14,769 3.2. Genetic characterisation of viral strains patients, 11,185 (76%) samples were collected during all 3 consecutive HRSV/influenza epidemics and 3,584 (24%) out of these epidemics Phylogenetic analyses of HMPV F and G sequences from 387 strains (Supplementary Table 1). Regarding the methods for detection, from all revealed that HMPV-A (201/387, 52%) and HMPV-B (185/387, 48%) these 14,769 patients, 3,316 samples (22%) were tested with the co-circulated, one HMPV-A/B was observed. The remaining 20/407 immunofluorescence assay, while 8,483 (57%) were tested with the (5%) HMPV could not be characterised due to low RNA quality or low real-time RT-PCR multiplex assays. Importantly, 656 (4%) samples viral load. HMPV-B (61%) predominated during the 2014-2015 season which tested negative for the immunofluorescence assay were re-tested and HMPV-A (62%) during 2015-2016. No difference in circulation was with the real-time RT-PCR multiplex assays. The remaining samples had observed during the 2016-2017 season (Table 3). been tested with a rapid test. The use of immunofluorescence and PCR- Phylogenetic analyses of F (382, 94%) and G (365, 90%) sequences based assays were changing throughout the period of study, decreasing (Fig. 2) showed congruent results. Overall, 11 (3%) samples belonged to for immunofluorescence assays and increasing for molecular methods A2a, 37 (10%) to A2b, 153 (40%) to A2c, 106 (27%) to B1 and 79 (20%) (59% vs 27% in 2014-2015; 15% vs 57% in 2015-2016 and <1% vs 74% to B2 (Table 3). in 2016-2017, respectively). Genetic characterisation of A2 G revealed that A2a and A2c se­ HMPV was laboratory-confirmed in 423 (2%) respiratory samples quences generally had a length of 220 aa, and A2b of 218 due to pre­ (nasopharyngeal swabs or aspirates, bronchoaspirates, bronchoalveolar mature stop codons. Genetic characterisation of 153 A2c strains washes or tracheal aspirates) from 407 (3%) patients, 221 (3%) of them revealed the presence of the novel 180- (A2c180dup; 46; 30%) and 111- occurring in the paediatric population and 186 (3%) in adults, showing nucleotide (A2c111dup; 13; 9%) duplications into G ectodomain with an important prevalence in the adult population, higher than in children increasing prevalence (Table 3). While all A2c180dup clustered together, in the 2015-2016 season (Table 2). This prevalence varied according to two subgroups could be observed in the F phylogenetic tree (Fig. 2). whether it was during HRSV/influenza epidemics or not (Supplemen­ Differently, A2c111dup G clustered into 2 groups but their F genes clus­ tary Table 1). During the HRSV/influenza epidemics, 82-95% of HMPV tered together (except NSVH2017-09-82477). laboratory-confirmed samples previously had a negative result in the B1 G clustered into two phylogenetic groups (I and II), differing in rapid tests for HRSV and influenza. Over the 3 seasons, the proportion of the acquisition of a premature stop codon in the 232 aa (relative to HMPV laboratory-confirmed samples tested by immunofluorescence KU375606) in all strains belonging to group II (Fig. 2), but one decreased while those tested by PCR-based assays increased (61%-11%- (NSVH2015-12-87728). In addition, two sequences presented deletions 0% vs 47%-91%-100%, respectively and by season). of one (NSVH2017-04-59510) or two aa (NSVH2016-03-50135). Ge­ 75/407 (18%) HMPV cases presented co-infections with 88 respira­ netic characterisation of 10 B2a and 69 B2b G sequences revealed the tory viruses: rhinovirus (26, 30%), adenovirus (15, 17%), human acquisition of short duplications. Whereas B2a group, represented by bocavirus (15, 17%), enterovirus (11, 13%), HRSV (6, 7%), human CAN98-75 strain (AY297748), presented the aa KE in the 160-161 po­ coronavirus (HCoV) 229E (5, 6%), HCoV OC43 (5, 6%), HCoV NL63 (2, sitions, B2b group presented 1 or 2 duplications (KEKE and KEKEKE). 2%), human parainfluenza 3 (1, 1%), influenza A (1, 1%) and B (1, 1%). Also, a group differed from the rest of B2b group presenting an R after Weekly distribution of HMPV showed a higher circulation from only one KE, which could probably be a mutation of the duplication of K February to April in the first two seasons, but started at mid December in (Fig. 2). 2016-2017 season (Fig. 1B). The peaks of incidence of the first two The sequences of the present study were submitted to GenBank seasons were in March, but the last season presented a pattern with two (MN617398-MN617753). peaks. Fig. 1. Seasonality. The X-axis represent the timeline of the study, the left Y-axis represents the number of total samples received and the right Y-axis represent the number of samples of each specific virus. HRSV/influenza epidemics period are framed in a black dotted box. A) shows the seasonality of HRSV, Influenza A virus, Influenza B virus and HMPV. B) shows the seasonality of HMPV. 3 M. Piñana et al. Journal of Clinical Virology 132 (2020) 104590 Table 2 Demographic data of total patients and patients with HMPV. Total patients Patients with HMPV Season Pediatric Adult Total Pediatric Adult Total 2014 - 2015 1,939 1,744 3,683 54 3% 36 2% 90 2% 2015 194 547 741 3 2% 2 <1% 5 1% 2015 - 2016 2,591 2,209 4,800 64 2% 87 4% 151 3% 2016 222 564 786 10 5% 0 0% 10 1% 2016 - 2017 2,832 1,927 4,759 90 3% 61 3% 151 3% Overall 7,778 6,991 14,769 221 3% 186 3% 407 3% Table 3 Distribution of genotypes and lineages throughout the study period. Genetic Season Total group 2014-2015 2015 2015-2016 2016 2016-2017 A1 0 0% 0 0% 0 0% 0 0% 0 0% 0 0% A2a 9 10% 1 20% 0 0% 0 0% 1 1% 11 3% A2b 12 14% 0 0% 22 16% 1 10% 2 1% 37 10% A2c 13 15% 0 0% 65 46% 8 80% 67 47% 153 40% A2cwt 11 85% 0 0% 46 71% 6 75% 31 46% 94 61% A2c180dup 2 15% 0 0% 18 28% 1 12.5% 25 37% 46 30% A2c111dup 0 0% 0 0% 1 1% 1 12.5% 11 17% 13 9% B1 37 42% 4 80% 22 16% 1 10% 42 29% 106 27% B2 17 19% 0 0% 31 22% 0 0% 31 22% 79 20% B2a 5 29% 0 0% 2 6% 0 0% 3 10% 10 13% B2b 12 71% 0 0% 29 94% 0 0% 28 90% 69 87% Total 88 100% 5 100% 140 100% 10 100% 143 100% 386 100% A2cwt: A2c strains without any duplication A2c180dup: A2c strains carrying the 180-nucleotide duplication A2c111dup: A2c strains carrying the 111-nucleotide duplication Fig. 2. Phylogenetic trees. Phylogenetic trees of F and G sequences from (A) HMPV-A and (B) HMPV-B, respectively. The analyses were performed on G sequences from nucleotide position 6340–6891 in reference to CAN97-83 strain (accession number AY297749) for HMPV-A and 6319-6921 CAN98-75 (accession number AY297748) for HMPV-B. On F sequences, nucleotide positions were 3846-4287 in reference to CAN97-83 strain and 3843-4284 in reference to CAN98-75. All 4 phylogenetic trees were inferred by using the maximum likelihood method, based on the Kimura 2-parameter for the F gene and General Time Reversible for the G gene. Numbers at the tree branch nodes represent the measure of support calculated by the bootstrap method (1000 replicates); only those exceeding 70% are shown. Sequences are marked with solid circles or triangles depending on whether they were collected during the seasonal or the interseasonal period (2014-2015 and 2015 in light grey, 2015-2016 and 2016 in dark grey and 2016-2017 in black). A2c sequences with duplications in the G protein have their name in bold turquoise for the 180-nucleotide duplication and in bold pink for the 111-nucleotide duplications. 4 M. Piñana et al. Journal of Clinical Virology 132 (2020) 104590 3.3. Structural biology of G protein The pre-fusion conformation of the F trimer is calculated to protrude 13 nm [21]. According to the distance distributions of the three en­ The ectodomain ensemble of the non-glycosylated form of G protein sembles, the actual fraction of G protein’s ectodomain protruding more of NSVH2015-06-62150 (A2cwt) was simulated [18–20], resulting in a than 13 nm from the membrane amounts to 23% in the A2cwt, and it composition of conformations with maximal length (Dmax) of 4.5-22.2 increases to 39% in A2c111dup and 46% and A2c180dup. nm and radii of gyration (Rg) of 1.9-7.4 nm (Fig. 3). The MetaDisorder server predicted that both duplications were as fully disordered as A2cwt, with no self-aggregation segments. Also, the 3.4. Clinical impact of human metapneumovirus glycosylation pattern showed a similar distribution of the numerous O- glycosylation sites (Fig. 4), with 23-26 additional O-glycosylation sites Due to the absence of clinical information (2), non-amplification (20) in A2c180dup and 12-13 in A2c111dup. or manifestation of other syndromes rather than URTI or LRTI (20), Once verified that duplications did not differ from A2cwt, 2000- clinical features of 203 paediatric and 162 adult cases were finally conformation unfolded ensembles were generated for NSVH2017-09- studied (Table 4). 78834 (A2c111dup) and NSVH2015-19-63118 (A2c180dup). A2c111dup The median age of paediatric patients presenting LRTI (1.17; IQR increased size to Dmax 5.1-27.3 nm and Rg 2.4-9.0 nm, while A2c180dup 0.49-2.61) was significantly lower (p 0.011) than those with URTI (2.18; increased Dmax 5.2-29.4 nm and Rg 2.4-9.9 nm (Fig. 3). IQR 0.88-6.41). A2c lineage was more associated to LRTI (p 0.032) than other lineages, but A2c with duplications were not associated with a Fig. 3. Maximum distance and radius of gy­ ration analyses of the disordered ensembles of G protein. A) Representation of the maximum distance measured between the N atom of the first extracellular residue (Asn52) and the more distant atom to this. B) Depiction of the radius of gyration calculated for an in­ dividual conformation. C, E, G) Histograms of the maximum distances measured in the disor­ dered ensemble for the wild type, 111- and 180- nucleotide duplications variants of the G pro­ tein, respectively. Insets at the right-hand part of each panel depict scatter-plots of maximum distances versus structure. D, F, H) Histograms of the radius of gyration of structures in the disordered ensemble for the wild type, 111- and 180-nucleotide duplications variants of the G protein, respectively. Similarly, insets at the right-hand part of each panel show scatter-plots of radius of gyration versus structure of each ensemble. 5 M. Piñana et al. Journal of Clinical Virology 132 (2020) 104590 Fig. 4. Glycosylation pattern of HMPV-A G protein. Multiple alignment of deduced G amino acid sequences of HMPV-A. Only positions reflecting an amino acid change or a putative N- or O-glycosylated site are shown, amino acids were numerated following the reference sequence CAN99-81 (accession number AY574224). Amino acid positions 40 and 41 correspond to the transmembrane domain, while the remaining positions correspond to the ectodomain. The 180-nucleotide du­ plications in the ectodomain are framed in a turquoise box, the 111-nucleotide duplications are in a pink box. N- and O-glycosylated sites are marked in purple. higher risk of LRTI compared to other strains (p 0.743 and 0.202). genetic recombination has been described for HMPV. All subgenotypes The median age of adult patients presenting LRTI (74.7; IQR 61.3- were detected except A1, suggesting it has extinguished and been 82.8) was significantly higher (p 0.001) than those with URTI (66.8; replaced by A2, according to previous studies [3]. According to the data IQR 53.7-77.1), each year of age increasing 1.03 times the odds of of the present study, A2c lineage appears to be replacing A2a and A2b. having LRTI. No lineages or subgenotypes were more associated to LRTI Moreover, A2c strains with duplications might be replacing A2cwt in the (p 0.052 and 0.246). Cardiopathy was associated to LRTI (OR 4.2, IC near future, as they might present an improved mechanism of immune 95% 2-9.43, p < 0.001). A2c strains with duplications were significantly evasion. In fact, a group in Japan observed that A2c111dup had totally more associated to LRTI, compared to all other strains (OR 2.83, IC 95% replaced the rest of A2 strains [25], including A2c180dup. Interestingly, 1.17-6.84, p 0.018) or to A2cwt (OR 3.45, IC 95% 1.22-9.77, p 0.034). our group has observed how both A2c111dup and A2c180dup have replaced For the severity study, only patients hospitalised due to LRTI (176) together the rest of HMPV-A viruses, being the latter more prevalent were considered, being 116 (66%) paediatric (Table 5) and 60 (34%) [26]. adult patients (Table 6). Children infected with A2 were more likely to Different lengths of G protein have been observed due to premature be admitted in the ICU (OR 5.14, IC 95% 1.06-24.95, p 0.031). No other stop codons, as previously described [3]. A2b and A2c lineages included variables were found to be significant. viruses with G proteins of 218 and 220 aa respectively; and two different genetic groups (I and II) could be distinguished within B1 subgenotype, 4. DISCUSSION with a difference of 10 aa in length, which might evolve into novel lineages. Also, nucleotide duplications can lengthen the G aa sequence, This study reports recent data on prevalence, genetic diversity, such as long duplications in A2c, and short duplications in B2. For B2 structural biology of G protein and clinical features of HMPV in Barce­ viruses, KE duplications or KER variants should be monitored next lona, Spain. seasons to reveal whether they confer an evolutionary advantage. The The positivity rate of HMPV was similar to recent reports [4,22,23]. deletions observed seem not to have been fixed in the viral population. Interestingly, the prevalence in adults was similar or even higher than in Once these A2c111dup and A2c180dup were described, one of the aims children, which emphasizes the importance of HMPV in adults. HMPV was to study their structural properties. G has a heavily glycosylated prevalence increased throughout the three seasons, probably due to the pattern [21], enhanced by the emergence of duplications that increase higher implementation of molecular methods, though there might be an the number of potential glycosylation sites. Although it is a very disor­ underestimation, as a large number of positive samples for HRSV and dered protein and seems to have numerous random conformations, a influenza by rapid assay were not tested for other respiratory viruses. composition of these conformations could be done. This prediction Most co-infections were with rhinoviruses, adenoviruses and bocavi­ suggests that both A2c180dup and A2c111dup proteins protrude more than ruses, as previously reported [23,24]. A2cwt. This finding supports the hypothesis of Leyrat [21], who sug­ HMPV presented a clear seasonality, as previously described [2,6, gested that G protein had a shielding function towards F protein, 24]. Interestingly, the last season presented a different pattern, showing masking its antigenic epitopes, and at the same time validates the hy­ two different peaks in one epidemic season without changes among pothesis that these novel long duplications would enhance this immune circulating genotypes. Interestingly, the prevalence of HMPV was higher evasion mechanism, as it would hide more efficiently F epitopes [8]. out of the HRSV/influenza epidemics in the first season but did not vary Sequences of the newly described A2c lineage [3,4] were compared in the second and third seasons. This could be due to the higher to sequences of the previously described A2b1 and A2b2 sublineages implementation of PCR-based assays in detriment to the use of immu­ [27,28] and clustered together; that is to say, A2b and A2c lineages are nofluorescence assays. Moreover, the great majority of HMPV exactly the same as A2b1 and A2b2, respectively. This misunderstanding laboratory-confirmed samples during these epidemics were previously between the genetic classification used in several articles highlights the tested by rapid assays and had a negative result, which would suggest urgent need of an official classification, as well as universal criteria to that there might be many more samples that would be positive for HMPV define new genotypes or lineages. but are not tested due to a HRSV or influenza positive result when HMPV Furthermore, clinical impact was also assessed. As in literature [2], circulation is coincidental with influenza epidemics. Thus, HMPV LRTI is more common in children under 2 and adults over 65. Moreover, prevalence could be underestimated due to the lack of search of this adults have an increase of 1.03 times the odds of suffering LRTI every virus when samples are HRSV or influenza laboratory-confirmed during passing year. The presence of chronic medical conditions as cardiopa­ the epidemics. thy, more frequent in the elderly, may be responsible for this, so HMPV Genetic characterisation revealed that both genotypes co-circulated should be tightly surveilled in these cases. Comorbidities are also asso­ with a shift in predominance, as expected [2]. However, there was an ciated with LRTI in children, especially respiratory comorbidities and unpredicted co-predominance in the third season, which could be due to immunodepression. In this study, prematurity and cardiopathies were either an intermediate alternation of genotypes or the emergence of not associated with a major risk of developing LRTI in children, in HMPV-A viruses with new antigenic features that would evade the im­ opposite to previous studies [29–32]. munity created on the previous season. Paediatric and adult patients underwent more antibiotic treatment Congruent classification of both F and G genes was expected, as no when manifesting LRTI than URTI. However, only 8% of children and 6 M. Piñana et al. Journal of Clinical Virology 132 (2020) 104590 Table 4 Clinical features of patients infected with HMPV. Pediatric cases Adult cases Factor URTI (n = 42) LRTI (n = 161) p URTI (n = 77) LRTI (n = 85) p Season* 0.908 0.020 2014-2015 10 (25.6 %) 42 (26.1 %) 14 (18.4 %) 19 (22.6 %) 2015-2016 10 (25.6 %) 44 (27.3 %) 46 (60.5 %) 33 (39.3 %) 2016 1 (2.56 %) 9 (5.59 %) 0 (0.00 %) 0 (0.00 %) 2016-2017 18 (46.2 %) 66 (41.0 %) 16 (21.1 %) 32 (38.1 %) Age 2.18 [0.88;6.41] 1.17 [0.49;2.61] 0.011 66.8 [53.7;77.1] 74.7 [61.3;82.8] 0.001 Age group 0.001 0.031 0-2 years old 19 (45.2 %) 95 (59.0 %) 0 (0.00 %) 0 (0.00 %) 2-5 years old 10 (23.8 %) 52 (32.3 %) 0 (0.00 %) 0 (0.00 %) 5-15 years old 11 (26.2 %) 14 (8.70 %) 0 (0.00 %) 0 (0.00 %) 15-64 years old 2 (4.76 %) 0 (0.00 %) 34 (43.6%) 23 (27.4%) >64 years old 0 (0.00 %) 0 (0.00 %) 44 (56.4%) 61 (72.6%) Sex 1.000 0.197 Female 18 (42.9 %) 67 (41.6 %) 45 (58.4 %) 40 (47.1 %) Male 24 (57.1 %) 94 (58.4%) 32 (41.6%) 45 (52.9 %) Subgenotype 0.763 0.052 A/B 0 (0.00 %) 0 (0.00 %) 0 (0.00 %) 1 (1.18 %) A2 24 (57.1 %) 83 (51.6 %) 37 (48.1 %) 46 (54.1 %) B1 11 (26.2 %) 44 (27.3 %) 17 (22.1 %) 26 (30.6 %) B2 7 (16.7 %) 34 (21.1 %) 23 (29.9 %) 12 (14.1 %) Sublineage 0.032 0.246 A2a 5 (11.9%) 2 (1.2%) 1 (1.3 %) 2 (2.4 %) A2b 6 (14.3%) 14 (8.7%) 7 (9.1 %) 7 (8.3 %) A2c 13 (31.0%) 67 (41.6 %) 29 (37.7 %) 37 (44.0 %) B1 11 (26.2) 44 (27.3%) 17 (22.1%) 26 (31.0%) B2a 0 (0.00 %) 5 (3.1%) 3 (3.9 %) 2 (2.4 %) B2b 7 (16.7%) 29 (18.0%) 20 (26.0 %) 10 (11.9%) Duplication 0.768 0.048 111 1 (2.4 %) 5 (3.1%) 1 (1.3 %) 6 (7.1 %) 180 6 (14.3%) 16 (9.9 %) 7 (9.1 %) 15 (17.6 %) no 35 (83.3%) 140 (87.0%) 69 (89.6%) 64 (75.3%) Comorbidities 0.046 0.937 Yes 13 (31.0 %) 80 (49.7 %) 63 (81.8 %) 71 (83.5 %) Non 29 (69.0 %) 81 (50.3 %) 14 (18.2 %) 14 (16.5 %) Respiratory comorbidities <0.001 0.799 Asthma 1 (2.38 %) 32 (19.9 %) 0 (0.00 %) 0 (0.00 %) Pneumopathy 0 (0.00 %) 20 (12.42%) 0 (0.00 %) 0 (0.00 %) EPOC 0 (0.00 %) 0 (0.00 %) 15 (19.5 %) 19 (22.4 %) Non 41 (97.6 %) 109 (67.7 %) 62 (80.5 %) 66 (77.6 %) Cardiopathy 0.532 <0.001 Yes 2 (4.76 %) 14 (8.70 %) 11 (14.3 %) 35 (41.2 %) Non 40 (95.2 %) 147 (91.3 %) 66 (85.7 %) 50 (58.8 %) Oncohematology 0.276 0.022 Yes 4 (9.52 %) 8 (4.97 %) 31 (40.3 %) 19 (22.4 %) Non 38 (90.5 %) 153 (95.0 %) 46 (59.7 %) 66 (77.6 %) Immunodepression 0.008 0.203 Immunodeficiency 1 (2.38 %) 2 (1.24 %) 23 (29.9 %) 17 (20.0 %) TPH 3 (7.14 %) 0 (0.00 %) 0 (0.00 %) 0 (0.00 %) Non 38 (90.5 %) 159 (98.8 %) 54 (70.1 %) 68 (80.0 %) Diabetes mellitus . 0.114 Yes 0 (0.00 %) 0 (0.00 %) 12 (15.6 %) 23 (27.1 %) Non 42 (100 %) 161 (100 %) 65 (84.4 %) 62 (72.9 %) Prematurity 0.422 . Yes 3 (7.14 %) 21 (13.0 %) 0 (0.00 %) 0 (0.00 %) Non 39 (92.9 %) 140 (87.0 %) 77 (100 %) 85 (100 %) Chronic kidney disease . 0.221 Yes 0 (0.00 %) 0 (0.00 %) 9 (11.7 %) 17 (20.0 %) Non 42 (100 %) 161 (100 %) 68 (88.3 %) 68 (80.0 %) Bacteria co-infection 0.244 0.005 Yes 4 (9.52 %) 7 (4.35 %) 10 (13.0 %) 28 (32.9 %) Non 38 (90.5 %) 154 (95.7 %) 67 (87.0 %) 57 (67.1 %) Viral co-infection 0.384 1000 Yes 11 (26.2 %) 30 (18.6 %) 5 (6.49 %) 5 (5.88 %) Non 31 (73.8 %) 131 (81.4 %) 72 (93.5 %) 80 (94.1 %) Antibiotic <0.001 <0.001 Yes 7 (16.7 %) 85 (52.8 %) 42 (54.5 %) 79 (92.9 %) Non 35 (83.3 %) 76 (47.2 %) 35 (45.5 %) 6 (7.06 %) Duplication vs the rest 0.743 0.018 A2c w/ duplication 7 (22.6 %) 21 (17.9 %) 8 (13.3 %) 21 (36.2 %) Other types 24 (77.4 %) 96 (82.1 %) 69 (86.7 %) 64 (63.8 %) A2c sublineage 0.202 0.034 A2c w/ duplication 7 (53.8 %) 21 (31.3 %) 8 (10.4 %) 21 (24.7 %) A2c w/o duplication 6 (46.2 %) 46 (68.7 %) 21 (89.6 %) 16 (75.3 %) 7 M. Piñana et al. Journal of Clinical Virology 132 (2020) 104590 Table 5 Severity of HMPV disease in paediatric patients. HFNC = high flux nasal cannula; CO = conventional oxygenotherapy (low flow nasal cannula or oxygen mask); MV = mechanical ventilation. Pediatric patients Subgenotypes Duplications vs other strains A2c sublineage Factor A2c w/ A2c w/ A2c w/o A2 B1 B2 p Other strains p p duplication duplication duplication Length hospital 4.50 5.00 5.50 5.00 5.00 5.00 4.00 0.880 0.199 0.166 stay [3.00;7.75] [4.00;7.00] [3.00;7.25] [4.00;11.00] [3.00;7.00] [4.00;11.0] [3.00;6.00] Respiratory support 1.000 0.297 0.406 Yes 47 (81.0 %) 27 (79.4 %) 19 (79.2 %) 14 (93.3%) 79 (78.2%) 14 (93.3 %) 25 (78.1 %) Non 11 (19.0 %) 7 (20.6 %) 5 (20.8 %) 1 (6.67%) 22 (21.8%) 1 (6.67 %) 7 (21.9 %) Type of respiratory 0.520 0.140 0.391 support HFNC 13 (22.4 %) 7 (20.6 %) 7 (29.2 %) 3 (20.0%) 24 (23.8%) 3 (20.0 %) 7 (21.9 %) CO 28 (48.3 %) 20 (58.8 %) 10 (41.7 %) 8 (53.3%) 50 (49.5%) 8 (53.3 %) 16 (50.0 %) MV 6 (10.3 %) 0 (0.00 %) 2 (8.33 %) 3 (20.0%) 5 (5.00%) 3 (20.0 %) 2 (6.25 %) Non 11 (19.0 %) 7 (20.6 %) 5 (20.8 %) 1 (6.67%) 22 (21.8%) 1 (6.67 %) 7 (21.9 %) Length respiratory 3.00 4.00 3.00 3.00 4.00 3.00 3.00 0.874 0.208 0.122 support [2.00;5.75] [2.00;5.75] [2.00;6.00] [3.00;9.00] [2.00;5.00] [3.00;9.00] [2.00;4.25] ICU admission 0.031 0.633 1.000 Yes 9 (15.5 %) 0 (0.00 %) 2 (8.33 %) 2 (13.3%) 9 (8.9%) 2 (13.3 %) 5 (15.6 %) Non 49 (84.5 %) 34 (100 %) 22 (91.7 %) 13 (86.7%) 92 (91.1%) 13 (86.7 %) 27 (84.4 %) 6.00 13.0 9.00 5.00 9.00 4.00 Length ICU stay . [.;.] 0.340 0.340 0.155 [4.00;7.00] [9.00;17.0] [7.50;10.50] [4.00;7.00] [7.50;10.5] [4.00;6.00] Exitus . . . Yes 0 (0 %) 0 (0 %) 0 (0 %) 0 (0 %) 0 (0 %) 0 (0%) 0 (0%) Non 58 (100 %) 34 (100 %) 24 (100 %) 15 (100%) 101 (100%) 15 (100 %) 32 (100 %) Table 6 Severity of HMPV disease in adult patients. HFNC = high flux nasal cannula; CO = conventional oxygenotherapy (low flow nasal cannula or oxygen mask); MV = mechanical ventilation Adult patients Subgenotypes Duplications vs other strains A2c sublineage Factor A2c w/ A2c w/ A2c w/o A2 B1 B2 p Other strains p p duplication duplication duplication Length hospital 4.00 9.00 4.00 4.00 6.00 4.00 2.50 0.327 0.442 0.787 stay [2.00;9.00] [2.00;12.0] [2.00;7.25] [2.00;9.00] [2.00;10.50] [2.00;9.00] [2.00;7.50] Respiratory 0.655 0.639 0.596 support Yes 30 (90.9 %) 15 (88.2 %) 8 (80.0 %) 11 (84.6%) 42 (89.4%) 11 (84.6 %) 13 (92.9 %) Non 3 (9.09 %) 2 (11.8 %) 2 (20.0 %) 2 (15.4%) 5 (10.6%) 2 (15.4 %) 1 (7.14 %) Type of respiratory 0.145 0.780 0.450 support HFNC 4 (12.1 %) 1 (5.88 %) 1 (10.0 %) 2 (15.4%) 4 (8.5%) 2 (15.4 %) 1 (7.14 %) CO 25 (75.8 %) 9 (52.9 %) 6 (60.0 %) 8 (61.5%) 32 (68.1%) 8 (61.5 %) 12 (85.7 %) MV 1 (3.03 %) 5 (29.4 %) 1 (10.0 %) 1 (7.69%) 6 (12.8%) 1 (7.69 %) 0 (0.00 %) Non 3 (9.09 %) 2 (11.8 %) 2 (20.0 %) 2 (15.4%) 5 (10.6%) 2 (15.4 %) 1 (7.14 %) Length respiratory 2.00 3.00 1.00 1.00 2.00 1.00 1.00 0.304 0.449 0.938 support [1.00;5.00] [1.00;10.0] [1.00;5.50] [1.00;4.00] [1.00;6.00] [1.00;4.00] [1.00;4.25] ICU admission 0.151 0.182 1.000 Yes 2 (6.06 %) 4 (23.5 %) 2 (20.0 %) 0 (0%) 8 (17%) 0 (0.00 %) 1 (7.14 %) Non 31 (93.9 %) 13 (76.5%) 8 (80.0 %) 13 (100%) 39 (83%) 13 (100 %) 13 (92.9 %) 9.50 11.0 10.5 10.5 15.0 Length ICU stay 0.939 . [.;.] . . [.;.] . [6.75;12.2] [5.00;21.0] [8.75;12.2] [5.5;15.25] [15.0;15.0] Exitus 0.096 1.000 1.000 Yes 2 (6.06 %) 1 (5.88 %) 3 (30.0 %) 1 (7.69%) 5 (10.6%) 1 (7.69 %) 1 (7.14 %) Non 31 (93.9 %) 16 (94.1 %) 7 (70.0 %) 12 (92.3%) 42 (89.4%) 12 (92.3 %) 13 (92.9 %) 30% of adults treated with antibiotics had a positive bacterial culture. strains with duplication cause more severe disease could be demon­ Hence, over-antibiotic prescription is still reported. strated neither in children nor in adults. Regarding infections by A2c, children seemed to be as affected by The increasing prevalence of viral variants carrying a duplication A2c with duplications as by A2cwt or other lineages, as it is probably a into the ectodomain of the G protein throughout the study period, the primary infection. Instead, A2c with duplications were more associated association of A2c111dup and A2c180dup with more severe disease in with LRTI in adults than A2cwt or other lineages. Although adults should adults, and the prediction of an enhancing steric shielding of the G have an efficient immune response [6], they have 3.45 times more odds protein masking antigenic epitopes of the F protein suggest that these of manifesting LRTI when infected by A2c with duplication than by duplications might confer an evolutionary advantage contributing to the A2cwt. This suggests that it might be acting as a primary infection, which immune evasion during the infection. This mechanism would be similar supports the hypothesis of G protein’s steric shielding over F protein. to that described for other viruses which have been reported to evade the HMPV is known for the many immune evasion strategies it has, so this immune response due to the glycosylation they present in their enve­ could be a new mechanism developed in recent years [33,34]. Whether lopes [35]. Given that F protein is the main target for most vaccine 8 M. Piñana et al. Journal of Clinical Virology 132 (2020) 104590 strategies currently under development, the fact that it could be masked [14] L.P. Kozlowski, J.M. Bujnicki, MetaDisorder: a meta-server for the prediction of intrinsic disorder in proteins, BMC Bioinformatics. 13 (2012) 1, https://doi.org/ by G should be taken into account. 10.1186/1471-2105-13-111. [15] I. Walsh, F. Seno, S.C.E. Tosatto, A. Trovato, PASTA 2.0: An improved server for Declaration of Competing Interest protein aggregation prediction, Nucleic Acids Res. 42 (2014) 301–307, https://doi. org/10.1093/nar/gku399. [16] R. Gupta, E. Jung, S. Brunak, Prediction of N-glycosylation sites in human proteins, The authors declare no conflicts of interest. Prep, 2004. [17] C. Steentoft, S.Y. Vakhrushev, H.J. Joshi, Y. Kong, M.B. Vester-Christensen, K.T.-B. ACKNOWLEDGEMENTS G. Schjoldager, K. Lavrsen, S. Dabelsteen, N.B. Pedersen, L. Marcos-Silva, R. Gupta, E. Paul Bennett, U. Mandel, S. Brunak, H.H. Wandall, S.B. Levery, H. 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