Arch Virol DOI 10.1007/s00705-013-1934-x

BRIEF REPORT

Hemagglutinin variants of influenza A(H1N1)pdm09 virus with reduced affinity for sialic acid receptors G. Ayora-Talavera • L. Cetina-Montejo A. Matos-Patro´n • L. Romero-Beltra´n



Received: 19 August 2013 / Accepted: 21 November 2013 Ó Springer-Verlag Wien 2013

Abstract In influenza A H1 virus, amino acids at position 190 and 225 of HA affect replication and transmission. In this study, we show that the mutation D190Y in the HA of influenza AH1N1pdm09 virus reduces the affinity of the virus for sialic acid receptors expressed at the surface of red blood cells from different species without affecting virus replication in MDCK cells.

Introduction Receptor binding of influenza virus hemagglutinin (HA) is a key determinant of host range and transmission of viruses to human and animal hosts. Avian influenza viruses preferentially recognise cell receptors containing sialic acid linked in an a-2,3 (SAa2,3) conformation to the terminal sugar of the chain, whereas human influenza viruses preferentially recognise receptors linked in an a-2,6 conformation (SAa2,6). Changes in key amino acids in the receptor-binding site (RBS) of the influenza virus HA are known to alter receptor recognition [7]. Genetic analysis of the H1N1 virus that caused the human pandemic in 1918 showed that residues D190 and D225 were key determinants for effective binding to the human type of receptor SAa2,6. In fact, a single G?D substitution at residue 225 in the RBS altered the pattern of SA recognition, and also the transmission efficiency of the virus [5, 10, 11].

G. Ayora-Talavera (&)  L. Cetina-Montejo  A. Matos-Patro´n  L. Romero-Beltra´n Centro de Investigaciones Regionales Dr. Hideyo Noguchi, Universidad Autonoma de Yucatan, Av. Itzaes #490 x 59, Centro, C. P. 97000 Merida, Yucatan, Mexico e-mail: [email protected]

The HA gene of the influenza H1N1pdm09 virus has its origin in the classical swine lineage, which itself was derived from the 1918 human pandemic strain. The novel H1N1 virus contains the amino acids D190 and D225 in the RBS of its HA. Human isolates of influenza H1N1pdm09 virus with the amino acid 225G, which is found in avianlike H1 viruses, have been detected worldwide, and the biological significance of this mutation for the severity of disease has been recognised [2, 9, 13]. To our knowledge, no changes at residue 190 in H1N1pdm09 viruses affecting receptor-biding recognition have been reported previously. Here, we described the occurrence of an H1N1pdm09 virus with a single amino acid change, D190Y, in the hemagglutinin receptor-binding site with reduced affinity to sialic acid receptors.

Results and discussion The influenza virus strain used in this study was identified as A/Yucatan/5783/2009 (H1N1pdm09) (Yuc/5783 hereafter), isolated from the bronchial lavage of a severe case of infection. The virus was grown in MDCK cells following standard procedures. Viruses were harvested after cytopathic effect was observed, and viral titre was assayed by a conventional hemagglutination (HA) assay with 1 % turkey red blood cells (RBCs). This particular type of RBC was chosen due to the previously reported preference of the H1N1pdm09 virus to agglutinate this type of cells. Initial growth of the virus in MDCK cells directly from the clinical sample (P0) produced no HA titre with turkey RBCs. Sequence analysis of the hemagglutinin gene of the Yuc/ 5783 isolate from P0, along with other isolates from severe and fatal clinical outcomes of infection, revealed a point mutation at residue D190Y in the Yuc/5783 virus. It was

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G. Ayora-Talavera et al.

a

b Influenza A

Influenza AH1

Fig. 1 Infectivity of A/Yuc/5783/09 in MDCK cells and detection by real-time RT-PCR. a A plaque assay was performed by serial dilution of the virus stock in serum-free Dulbecco’s modified Eagle’s medium (DMEM), and cells were incubated for 1 hour at room temperature with each virus dilution and then overlaid with 3 % low-melting agarose in DMEM supplemented with TPCK-trypsin at a final concentration of 2 lg/ml. Once the agar/medium was solid, plates

were incubated for 72 hours and stained with 0.4 % crystal violet in methanol. b Virus detection was performed for AH1N1pdm09 virus according to the CDC protocol. The plot indicates amplification of the M gene (influenza A, left panel) and the pH1 HA gene (influenza AH1, right panel). Yuc/5783, green curve; Yuc/2370, red curve; Yuc/ 5761, blue curve; negative control, black

further passaged in MDCK cells to increase viral titre (P1 and 2), but no HA reaction with turkey RBCs was observed in either passage. Sequence analysis of the HA gene from passages P1 and P2 revealed a conserved D190Y mutation. To rule out that the negative result in the HA assay was due to a low viral titre, a plaque assay in MDCK cells with viruses from P2 was performed using standard protocols. Results from the plaque assay showed limited viral growth with 2.6 9 104 PFU/mL at the 10-2 dilution (Fig. 1a). The plaque phenotype showed a predominant population of viruses with large plaques, although small plaques were also present. In addition, viral growth was assessed by rRT-PCR using the CDC protocol to detect pH1N1 virus [12]. From this, the M and the HA genes of Yuc/5783 were detected at Ct values of 18 and 21, respectively, as indicators of viral replication (Fig. 1b). The amplification curves for the mutant virus were similar to the control HA-positive viruses Yuc/2370 and Yuc/5761, which were isolated from mild and severe cases of infection, respectively. Therefore, the

lack of receptor binding by Yuc/5783 was not due to a low virus titre. Thus, we hypothesized a possible effect of the mutation D190Y on the receptor-recognition ability of the Yuc/5783 isolate. To test this, the virus was subjected to HA assay using different sources of RBCs, including turkey, chicken, swine and guinea pig. Viruses from P2, which produced plaques and positive amplification by rRT-PCR, were used for the assay. Interestingly, no HA titre was observed with any of the RBCs tested (Fig. 2a). In contrast, Yuc/5761 was able to bind to both chicken and turkey RBCs and had the the highest titre with the guinea pig cells but no affinity for swine erythrocytes. Yuc/2370 bound to turkey and guinea pig RBCs, but not chicken or swine RBCs (Fig. 2a). Sequence analysis of the HA gene from all three viruses showed two differences between the three isolates, including the D190Y mutation, which was detected only in Yuc/5783, and G158E, which was detected in the Yuc/ 5761 virus and is associated with adaptation to cell culture

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Influenza A(H1N1)pdm09 virus HA mutants

a

CC

Guinea Pig

Swine

Chicken

Turkey

Guinea Pig

Swine

Chicken

Turkey

Guinea Pig

Swine

Chicken

Turkey

1:2 1:4 1:8 1:16 1:32 1:64

1:128 1:256 Yuc/5783 - D190Y

Yuc/2370

Yuc/5761 - G155E

b

Fig. 2 Receptor-binding affinity of Yuc/5783 determined by HA assay and solid-phase binding assay. a Hemagglutination properties of viruses were determined using RBCs from different animal species. b Binding curves of Yuc/5783, Yuc/2370 and Yuc/5761 to sialylglycopolymers 6’SLN (green line) and 3’SLN (red line). Polycarbonate 96-well plates were coated with 100 ll of purified viruses in carbonate buffer, pH 9.6, at the equivalent of 4 HA units and incubated overnight at 4 °C. The virus suspension was removed, and polymers were serially diluted in reaction buffer (PBS/0.5 % Tween

containing 5 lM zanamivir) and incubated at 4 °C for four hours. Plates were washed four times and incubated for 1 hour at room temperature with a 1:500 dilution of streptavidin/HRP antibody in reaction buffer. After washing, plates were incubated with TMB until a blue color developed, and the reaction was stopped by addition of 0.5 M of HCl. The optical density at 450 nm was recorded, and data were analysed using GraphPad software. The data represent the average of triplicate experiments

[6]. These results suggested that the mutation D?Y in the 190 helix had a dramatic effect in the Yuc/5783 virus by decreasing to undetectable levels its affinity for SA receptors expressed at the surface of red blood cells. Since the isolate Yuc/5783 still replicated in MDCK cells, this suggests that different SA isoforms are used by the HA of the virus to initiate recognition and binding, followed by replication in MDCK cells. Comparative sequence analysis of the HA gene of Yuc/5783 and the vaccine strain A/Cal/ 4/09 indicated no additional changes in the 130 loop (residues 135 to 138) or in the 220 loop (residues 221 to 228), which form the sides of the receptor-binding site. To confirm these results, the Yuc/5783 virus was also tested using a solid-phase binding assay with synthetic 3’SLN and 6’SLN glycopolymers [1]. The binding curves obtained in this assay showed reduced affinity for 6’SLN and no binding to 3’SLN for the Yuc/5783. Affinity for both glycopolymers, with a preference for 6’SLN, was detected for the Yuc/5761 virus, in agreement with the

results of the HA assay (Fig. 2b). Yuc/2370 showed a preference for 6’SLN and lower affinity for 3’SLN. To rule out that all of the previous results were due to insufficient concentrations of virus, an HA assay was performed on different virus preparations from both mutant and controls. The results for the mutant virus were consistent; no HA activity was observed with virus from any of the different passages or with the purified virus, which had a viral titre that was one log higher than that of the unpurified virus. In contrast, the purified control Yuc/2370 had a viral titre similar to that of the mutant and did agglutinate RBCs. The control virus Yuc/5761 showed the highest HA titres and the highest viral titre in PFU/ml. Furthermore, we performed a third passage from the mutant virus (P3); surprisingly, this virus stock did agglutinate turkey RBCs at low titre (Table 1). Sequence analysis of the HA gene of the P3 virus revealed a reversion of the mutation to the wild-type Y190D. To evaluate if the D190Y mutation had an effect on the virus replication rate, the kinetics of virus growth in

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G. Ayora-Talavera et al. Table 1 Viral titres of mutant and control viruses from different MDCK culture passages HA titrea

Virus

PFU/ ml

Swine

Guinea pig

Turkey

Chicken

Yuc/5783 purifiedb

ND

ND

\1:1

\1:1

1.1 9 105

Yuc/5783 (P2) (13.09.12)

\1:1

\1:1

\1:1

\1:1

ND

Yuc/5783 (P2) (2.03.13)

\1:1

\1:1

\1:1

\1:1

2.6 9 104

Yuc/5783 (P3)

ND

ND

1:4

\1:1

ND

Yuc/5761 purified

1:16

1:128

1:128

1:64

2.5 9 108

Yuc/5761

1:4

1:32

1:32

1:4

1.3 9 107

Yuc/2370 purified

\1:1

1:32

1:64

\1:1

1.6 9 105

Yuc/2370

\1:1

1:32

1:32

\1:1

4.2 9 104

ND, not determined a

Viruses were serially diluted (1:1 to 1:128) in PBS and incubated with a 1 % RBCs suspension from different species at room temperature for 45 minutes b

Viruses were purified by ultracentrifugation at 25,000 g for 2 hours in a 30 % sucrose cushion. The virus pellet was resuspended in PBS, aliquoted, and stored until use

Virus Titre Log10 PFU/ml

10

2370

5783

5761

8

****

6

**** 4

2

0 0

12

24

36

48

Time (Hrs)

Fig. 3 Growth kinetic curve of mutant and control viruses. MDCK cells were infected at an MOI of 0.001 PFU/ml. After one hour of viral adsorption, cells were washed three times with PBS and incubated with serum free DMEM supplemented with TPCK-trypsin at a final concentration of 2 lg/ml. Supernatant from infected cells was harvested every 12 hours postinfection. Virus titres were determined by plaque assay. Data represent the average of triplicate experiments

MDCK cells were examined. At a multiplicity of infection (MOI) of 0.001 PFU/cell, all three viruses showed a similar rate of replication at 12, 36 and 48 hours postinfection.

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However, at 24 hours there was a significant difference in viral titre between the control Yuc/5761 and the mutant and Yuc/2370 (Fig. 3). This suggested that although affinity for short-chain sugars was almost lost, as shown in the binding assay, the mutant D190Y virus was still able to replicate in MDCK cells. These data suggested that the amino acid change D190Y did not restrict virus binding to SA receptors expressed in MDCK cells. In this study, we report for the first time the occurrence of a mutation at residue D190Y of an A(H1N1)pdm09 virus with altered receptor-binding properties. This mutation caused a dramatic reduction in the affinity for sialic acid receptors, either expressed at the surface of RBCs from different species or from a synthetic source. A recent report describes a mutation at the same residue, E190K, in a plaque-purified H1HA virus that, in contrast to our findings, enhances the receptor-binding affinity of the virus. These results support the notion that amino acid changes at the 190 residue have an effect on the receptorbinding phenotype of H1HA viruses [4]. The D190Y mutation has been detected in human A(H1N1)pdm09 influenza viruses isolated from Italy, although it is a highly conserved residue in pandemic H1 viruses (http://flusurver.bii.a-star.edu.sg). A search in the NCBI public database revealed only one 2009 H1HA sequence with a D190N mutation and five 2010 H1HA sequences with the D190G mutation. Both of these mutations have been associated with host adaptation in embryonated hen’s eggs [3]. The lack of an original clinical sample was an impediment to determining if the Y190 mutation arose from host adaptation, considering that the patient suffered from a severe infection. Recent work by Gen et al. demonstrated attenuation of the mutant virus E190K in mice [4]. It would be of interest to analyse the biological role of our mutant virus D190Y in an animal model to investigate if is related to the severe infection in the patient, considering the origin of the clinical sample (bronchial lavage). The mutant Yuc/5783 and the virus control Yuc/2370 had weak binding to synthetic glycans when analysed in the solid-phase binding assay. However, the weak avidity of the pH1N1 virus for SAa2,6 glycans has been shown using arrays or in the ELISA-plate assay using either whole virus preparations or recombinant HAs [14, 15]. Likewise, compared to the new H7N9 virus, pH1N1 shows binding to 30 SLN and 60 SLN glycopolymers just above the detection limit of the assay, similar to our results [8]. Avian influenza viruses of the H1 subtype are well known to require host-adapted mutations at residues 190 and 225 to be transmitted efficiently between humans. Previous work has shown that the egg-adapted mutation D190N not only increased binding affinity to SAa2,3 receptors but also impaired the ability to recognise SAa2,6

Influenza A(H1N1)pdm09 virus HA mutants

receptors. In contrast, the amino acid change E190K resulted in recognition of both SAa2,6 and SAa2,3 receptors [3, 4]. The role of residue 190 in receptor shifting has also been demonstrated for the reconstructed 1918 influenza virus [5]. As part of influenza surveillance, virus isolation in cell culture or embryonated chicken eggs followed by hemagglutination or hemagglutination inhibition assays is a crucial methodology for virus characterization. Special attention has been placed on viruses isolated from severe or fatal cases of infection with the influenza A(H1N1)pdm09 virus in order to detect any indicators of viral pathogenicity. Viral variants with mutations in the Sb antigenic site and altered receptor-binding properties, as we have described here, should be considered during influenza surveillance. Acknowledgments cyt-2009-131965.

This work was supported by grant SEP/Cona-

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Hemagglutinin variants of influenza A(H1N1)pdm09 virus with reduced affinity for sialic acid receptors.

In influenza A H1 virus, amino acids at position 190 and 225 of HA affect replication and transmission. In this study, we show that the mutation D190Y...
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