Conceived and designed the experiments: FB AZ. Performed the experiments: A.Piralla EP FR GC. Analyzed the data: A.Piralla EP FR GC AM FB. Contributed reagents/materials/analysis tools: VE GI A.Pesenti PGC. Wrote the paper: A.Piralla FB.
The authors have declared that no competing interests exist.
Since its appearance in 2009, the pandemic influenza A(H1N1) virus circulated worldwide causing several severe infections.
Respiratory samples from patients with 2009 influenza A(H1N1) and acute respiratory distress attending 24 intensive care units (ICUs) as well as from patients with lower respiratory tract infections not requiring ICU admission and community upper respiratory tract infections in the Lombardy region (10 million inhabitants) of Italy during the 2010–2011 winter-spring season, were analyzed.
In patients with severe ILI, the viral load was higher in bronchoalveolar lavage (BAL) with respect to nasal swab (NS), (p<0.001) suggesting a higher virus replication in the lower respiratory tract. Four distinct virus clusters (referred to as cluster A to D) circulated simultaneously. Most (72.7%, n = 48) of the 66 patients infected with viruses belonging to cluster A had a severe (n = 26) or moderate ILI (n = 22). Amino acid mutations (V26I, I116M, A186T, D187Y, D222G/N, M257I, S263F, I286L/M, and N473D) were observed only in patients with severe ILI. D222G/N variants were detected exclusively in BAL samples.
Multiple virus clusters co-circulated during the 2010–2011 winter-spring season. Severe or moderate ILI were associated with specific 2009 influenza A(H1N1) variants, which replicated preferentially in the lower respiratory tract.
The emergence of a new pandemic influenza A strain in 2009 has raised global concerns for health organizations. Although moderate in general, severe cases of influenza-like illness (ILI) during the 2009 pandemic have been associated with the emergence of specific virus variants. In particular, the D222G or N mutations have been observed in cases of severe disease
The aim of this study was to investigate the prevalence of 2009 influenza A(H1N1) variants with mutated hemagglutinin (HA) in upper and lower respiratory tract of patients with severe infections.
In the framework of the severe influenza A surveillance program in Lombardy (Northern Italy, nearly 10 million inhabitants out of a total of about 60 million), our reference laboratories collected respiratory samples from patients admitted to intensive care units (ICUs) as well as from inpatients hospitalized at units other than ICUs, and from patients with community acquired infections referred by sentinel practitioners. Patients were stratified according to strict clinical criteria, and 2009 A(H1N1) HA sequences were analyzed in severe cases and in a comparable number of moderate and mild cases.
Particular consideration was paid both to the three structural elements (190-helix, 220-loop and 130-loop) in the HA involved in receptor binding site (RBS) domains and to the four antigenic sites (Sa, Sb, Ca and Cb), where point mutations could alter the ability of the virus to infect cells and the antibody reactivity
From November 1, 2010 to March 6, 2011, nasal swab (NS) and/or bronchoalveolar lavage (BAL) specimens collected from 45 patients with severe ILI admitted to 24 ICUs in the Lombardy region were analyzed. A comparable number of patients with moderate ILI (N = 40) or mild ILI (N = 54) were analyzed in parallel. Analyses were carried out at S.S. Virologia Molecolare, Fondazione IRCCS Policlinico San Matteo, Pavia or Dipartimento di Sanità Pubblica-Microbiologia-Virologia, Università degli Studi di Milano.
Patients were stratified using the criteria proposed by Zarychanski et al.
The local Ethics Committee consent was not required since according to a Regional Surveillance and Preparedness Plan (DGR IX/1046, 22 Dec. 2010 and DGR 5988, 30 Jun 2011), diagnostic and clinical management of patients admitted at hospitals in the Lombardy Region with severe and moderate ILI included prospective influenza A detection, subtyping and sequencing which was centralized at the two Regional Reference Laboratories (Molecular Virology Unit, IRCCS Policlinico San Matteo, Pavia and Dipartimento di Sanità Pubblica-Microbiologia-Virologia, Università degli Studi di Milano, Milan). Mild respiratory infections were collected by sentinel pratictioners and anonymously analyzed at the reference laboratory in Milan, in the frame of the National Surveillance Plan (Influnet). Informed consent was not necessary since patients with severe and mild ILI were included in a Regional diagnostic and clinical management protocol. Data were anonymously analyzed according to a Regional Surveillance and Preparedness Plan. Mild ILI were collected and analyzed within the National Surveillance Plan (Influnet).
Detection and quantification of influenza A viruses was routinely performed on NS specimens by a real-time RT-PCR developed at the Centers for Disease Control, Atlanta, USA and available on the WHO website: (
The 2009 influenza A(H1N1) HA gene was amplified directly from clinical specimens using the Superscript III One-step RT-PCR amplification kit (Invitrogen, Carlsbad, USA). Primer sequences were as follows: forward primer, HA-F0 (nt 1–28)
Sequences were assembled using the Sequencher software, version 4.6 (Gene Codes Corporation, Ann Arbor, USA). Nucleotide alignments were constructed using the ClustalW method with MEGA version 4.1 software
NCBI GenBank accession numbers for our nucleotide sequences are JF801855-JF801909 and JN017095-JN017181.
Comparison of viral loads from paired respiratory samples was performed with the Wilcoxon rank sum test for continuous paired variables. The Fisher's exact test for categorical variables was used for analysis of mutation frequencies between groups of patients.
This study included 139 patients with a 2009 influenza A(H1N1) virus infection: 45 (32.4%) critically ill patients with severe ILI, 40 (28.8%) patients with moderate ILI and 54 (38.8%) patients with mild ILI.
As shown in
Group of patients (no.) | P value |
||||||
Data | All patients (N = 139) | Severe (45) | Moderate (40) | Mild (54) | Severe |
Severe |
Moderate |
Gender Number (%) | |||||||
Male | 86 (61.9%) | 29 (64.4%) | 26 (65.0%) | 31 (57.4%) | |||
Female | 53 (38.1%) | 16 (35.6%) | 14 (35.0%) | 23 (42.6%) | |||
Age |
|||||||
Median (years) | 47 | 52 | 49 | 34 |
|
ns | ns |
Range | 1 mo-85 | 25–83 | 1 mo-85 | 1–83 |
mo, month.
for one patient this information was not available.
P value>0.10 is considered not significant (ns); significant P value under 0.05 is in bold.
In the 13 patients with paired NS and BAL samples (12 severe ILI and 1 moderate ILI), virus load was significantly higher in the lower respiratory tract secretions (median 2.2×107 RNA copies/ml, range 7×104–3.8×108 RNA copies/ml) than in upper respiratory tract samples (median 4.2×104 RNA copies/ml, range 1–1.4×107; p = 0.003) (
A: Comparison of viral load in paired nasal swabs (NS) and bronchoalveolar lavage (BAL) samples. B: Distribution of 222 polymorphisms in paired NS and BAL specimens. NA, not available; ND, not done due to low viral load.
The overall nucleotide identity of analyzed HA sequences with the reference vaccine strain A/California/07/2009(H1N1) ranged between 98.2% and 99.6%, with a maximum of 13 amino acid changes.
As shown in
A: Geographic distribution of patients categorized by clinical outcome. B: Phylogenetic tree based on influenza 2009 A(H1N1) viruses HA nucleotide sequences. Sequences of strains circulating in 2009–2011 are represented in italics (NCBI accession numbers or GISAID numbers are provided). NCBI GenBank accession numbers for our nucleotide sequences are JF801855-JF801909 and JN017095-JN017181. †, deceased patient.
Cluster C, containing 37 (26.6%) sequences, was characterized by S185T and S451N substitutions, and corresponded to a cluster observed in the UK
Cluster D, which included 12 (8.6%) sequences, was closely related to one of the two clusters circulating in the UK during the 2010–2011 seasonal epidemic
Most amino acid substitutions were located in the HA1 region containing the major antigenic epitopes. All sequences analyzed belonged to clade 7 and were characterized by the fixed amino acid mutation S203T (
The amino acid changes in the receptor binding sites (RBS) are highlighted in black. The amino acid changes in the antigenic sites are highlighted in grey. †, deceased patient; URTI, upper respiratory tract infection; LRTI, lower respiratory tract infection; ARDS, acute respiratory disease syndrome.
Several HA mutations were observed throughout the three patient groups. On the whole, 59 HA residue positions were found with at least one change compared to vaccine reference strain A/California/07/2009(H1N1) (
The frequency of mutations in RBSs, antigenic sites and amino acids fixed in each cluster among patient groups was investigated (
Patient categories (no.) | P value |
||||||||
Location of mutations | Mutations |
Severe (45) | Moderate (40) | Mild (54) | Severe |
Severe/Moderate |
Severe |
Moderate |
|
Receptor Binding Sites | Loop 220 | D222G/N/Y | 8/45 (17.8%) | 1/40 (2.5%) | 1/54 (1.8%) |
|
0.08 |
|
nns |
D222N | 3/45 (6.7%) | 0/40 (0%) | 0/54 (0%) | 0.09 | ns | ns | ns | ||
D222G | 5/45 (11.1%) | 0/40 (0%) | 0/54 (0%) |
|
ns | 0.06 | ns | ||
D222Y | 0/45 (0%) | 1/40 (2.5%) | 1/54 (1.8%) | ns | ns | ns | ns | ||
I216V | 26/45 (57.8%) | 22/40 (55.0%) | 18/54 (33.3%) |
|
|
ns | 0.06 | ||
I216T | 1/45 (2.2) | 1/40 (2.5%) | 0/54 (0%) | ns | ns | ns | ns | ||
Helix 190 | S183P | 0/45 (0%) | 3/40 (7.5%) | 9/54 (16.7%) |
|
|
0.10 | ns | |
A186T | 1/45 (2.2%) | 0/40 (0%) | 0/54 (0%) | ns | ns | ns | ns | ||
D187Y | 2/45 (4.4%) | 0/40 (0%) | 0/54 (0%) | ns | ns | ns | ns | ||
Loop 130 | A134T | 0/45 (0%) | 3/40 (7.5%) | 9/54 (16.7%) |
|
|
0.10 | ns | |
Antigenic sites | Sa | N125D | 7/45 (15.5%) | 1/40 (2.5%) | 16/54 (29.6%) |
|
|
0.06 |
|
S162N | 0/45 (0%) | 0/40 (0%) | 1/54 (1.8%) | ns | ns | ns | ns | ||
K163R | 0/45 (0%) | 1/40 (2.5%) | 0/54 (0%) | ns | ns | ns | ns | ||
Sb | S185T | 12/45 (26.7%) | 13/40 (32.5) | 12/54 (22.2%) | ns | ns | ns | ns | |
D187Y | 2/45 (4.4%) | 0/40 (0%) | 0/54 (0%) | ns | ns | ns | ns | ||
Ca1 | R205K | 27/45 (60.0%) | 22/40 (55.0%) | 18/54 (33.3%) |
|
|
ns | 0.06 | |
Ca2 | H138Q | 21/45 (46.7%) | 21/40 (52.5%) | 17/54 (31.5%) | ns |
|
ns | 0.08 | |
A141S/T | 0/45 (0%) | 5/40 (12.5%) | 10/54 (18.5%) |
|
|
|
ns | ||
Cb | L70H | 0/45 (0%) | 0/40 (0%) | 1/54 (1.8%) | ns | ns | ns | ns | |
S74N | 0/45 (0%) | 0/40 (0%) | 1/54 (1.8%) | ns | ns | ns | ns | ||
Cluster A | V249L | 26/45 (57.8) | 22/40 (55.0%) | 18/54 (33.3%) |
|
|
ns | 0.06 | |
E356A/G |
20/38 (52.6%) | 17/31 (54.8%) | 17/45 (37.8%) | ns | ns | ns | ns | ||
Cluster B | D94E | 4/45 (8.9%) | 0/40 (0%) | 4/54 (7.4%) | ns | ns | ns | ns | |
Cluster C | S451N |
11/38 (28.9%) | 9/32 (28.1%) | 9/45 (20.0%) | ns | ns | ns | ns | |
Cluster D | I295V |
0/43 (0%) | 2/38 (5.3%) | 8/52 (15.4%) |
|
|
ns | ns |
Amino acids numbering start after signal peptide DTLC.
P value>0.10 is considered not significant (ns); P value between 0.05 and 0.10 was considered a trend of significance; significant P value under 0.05 are in bold.
Data not available for all sequences.
D222G/N amino acid changes were detected more frequently in sequences from patients with severe ILI (8/45; 17.7%) than in those from patients with mild (0/54, 0%; p = 0.01) or moderate ILI (0/40, 0%; p = 0.03). The D222G/N changes were observed only in BAL samples from five patients with severe ILI, while the HA sequences in the paired NS samples showed the wild-type 222D (
Besides D222G/N changes, an additional mutation in the RBS domain (D187Y) was observed. It was detected only in viruses from two patients with severe ILI, one requiring mechanical ventilation and the other requiring an ECMO procedure.
To address the difference in antigenic properties, the mutations in antigenic epitope regions (Sa, Sb, Ca1, Ca2 and Cb) were investigated (
Six potential N-glycosylation sites were found in all HA sequences analyzed and corresponded with positions 11, 23, 87, 277, 287, and 482. The sequence named A/Pavia/09/2011 possessed an additional N-glycosylation site at residue 163 due to an amino acid change from serine (S) to asparagine (N), with a glycosylation score of 0.727. This patient presented with mild symptoms of ILI.
This study was aimed at investigate the prevalence of 2009 influenza A(H1N1) variants with mutated hemagglutinin (HA) in upper and lower respiratory tract of patients with severe infections in the 2010–2011 seasonal epidemic to extend observations reported during the 2009 pandemic
In keeping with preliminary data published in the ECDC report released in February
In the molecular analysis of HA sequences, particular attention was paid to the amino acid variations occurring in the HA structural elements involved in both the receptor-binding sites and the antigenic sites. Some of the substitutions observed were prominently exposed on the surface of the HA trimer (at residue 125, 134, 183, 185, 216) but others were buried. With respect to the reference vaccine strain A/California/07/2009(H1N1), a higher number of amino acid changes in the antigenic sites and, to a lower extent, in RBSs was observed in virus strains from patients with mild ILI than in those with severe or moderate ILI. This suggests that HA divergence in virus strains sustaining mild upper respiratory tract infections thus expressing a great spreading capacity might be the result of multiple rounds of selection by herd immunity. In contrast, the HA sequences of virus strains from patients with a severe infection were genetically more conserved, probably due to the evolutionary bottleneck represented by the receptor specificity. In fact, virus variants associated with severe infection are sequestered in the lower respiratory tract and, hence, the switch of receptor specificity of these viruses from α2-6 to α2-3-linked sialosides seems to decrease the capacity of the virus to infect the upper respiratory tract, thus impairing the spreading ability of those aggressive variants.
Consistent with data reported during the 2009 pandemic
In our series, D222Y variants, previously reported in severe cases
Further studies on the receptor binding specificity and affinity will clarify both the role of such HA mutants on viral tropism and their contribution to exacerbation of disease.
In this study, patients with severe influenza infections showed a significantly higher viral load in lower respiratory tract secretions compared to paired upper respiratory tract ones. These findings confirm and extend the data found in the 2009 influenza pandemic
A limitation of this study is the relatively small set of data analyzed and the pathogenetic role of the virus mutants observed remains to be confirmed in a larger cohort. On the other hand, the infection by the 2009 influenza A(H1N1) strain was moderate in general and the prospective enrollment, according to strict clinical criteria, of 45 critically ill patients required the combined effort of 24 ICUs. Thus, this data set, although limited, appears unique to shed more light on the mechanisms of virulence of 2009 influenza A(H1N1). Although the comparison of different respiratory specimens might be questionable, our data are in agreement with results by Giannella et al
In conclusion, our results highlight that HA D222G/N mutation was detected exclusively in lower respiratory tract secretions. The findings of this study on the circulation of new variants of 2009 influenza A(H1N1) stress the importance of continuous monitoring of influenza virus evolution. Prompt analysis of severe infections by adopting appropriate sampling guidelines will promote better understanding of the epidemiology of mutated viruses with respect to clinical outcome.
We thank all the technical staff for handling the specimens and performing the assays. We thank Mrs Daniela Sartori for manuscript editing and Mrs Laurene Kelly for English revision. Members of the Severe Influenza A Task Force are as follows: