A(H1N1)pdm09 detection and isolation in immunocompromised patients
Influenza A virus was detected in oropharyngeal swab samples collected from patients with lymphoblastic leukemia (Patient A: female, 37 years old) and relapsed lymphoma (Patient B: female, 38 years old), with real-time RT-PCR Ct values of 28 and 29, respectively. A(H1N1)pdm09 viruses were identified by additional H1 subtype detection at respective Ct values of 28 and 29 (Table 3). Plaques were observed within 48 h following specimen inoculation in MDCK cells, with the harvested supernatant exhibiting an HA titer of 256. The isolates were named A/Korea/S0002/2019 and A/Korea/S0003/2019.
Table 3: A(H1N1)pdm09 virus detection in immunocompromised patients by real-time RT-PCR.
Patient
|
Genetic detection using real-time RT-PCR
|
IFV A
|
IFV B
|
A/H1
|
A/H3
|
IPC
|
A
|
26.1
|
UD*
|
25.4
|
UD*
|
26.3/26.2
|
B
|
27.0
|
UD*
|
25.8
|
UD*
|
32.1/34.0
|
UD*: undetected
Genetic characterization
NA genetic analysis for screening drug resistance, phylogeny, and variation
The H275Y NA substitution (N1 numbering), associated with strong drug resistance, was observed in (GISAID: EPI1602908 and EPI1602906) of A(H1N1)pdm09 isolates from both immunocompromised patients. Other NA drug-resistance substitutions (V116A, I117V, Q136K, D151A, Y155H, R156K, D198V, I222V, R224K, Q226H, E227D, E277Q, R293K, N294S, E425G, and I436N) were not detected. The NA sequences of A/Korea/S0002/2019 and A/Korea/S0003/2019 most closely matched with those of A/Brisbane/02/2018 (98.5% and 98.3% amino-acid identity, respectively). The viruses could be distinguished in the phylogenetic tree (Fig. 1). A/Korea/S0002/2019 had six amino acid differences (T13I, Q51K, F74S, H275Y, D416N, and T452I) relative to A/Brisbane/02/2018 (Table 4), while A/Korea/S0003/2019 had eight (T13I, I29M, P93S, I99V, H275Y, G298A, V321I, and V394I). Table 4 shows the amino acid substitutions in NA compared with A/Brisbane/02/2018 and their reported effects.
Table 4: NA genetic mutations compared with A/Brisbane/02/2018 (N1 numbering).
Virus
|
Amino acid identity with A/Brisbane/02/2018 (%)
|
T13I
|
I29M
|
Q51K
|
F74S
|
P93S1,2
|
I99V2
|
H275Y3
|
G298A1
|
V321I1
|
V394I2
|
D416N1,2
|
T452I1,2,4
|
A/Korea/S0003/2019
|
98.3
|
○
|
○
|
|
|
○
|
○
|
○
|
○
|
○
|
○
|
|
|
A/Korea/S0002/2019
|
98.5
|
○
|
|
○
|
○
|
|
|
○
|
|
|
|
○
|
○
|
Frequency (%)
|
99.79
|
0.65
|
18.14
|
16.78
|
0.34
|
0.89
|
0.58
|
0.75
|
1.13
|
1.37
|
36.25
|
18.76
|
1Binding small ligand; 2viral oligomerization interfaces; 3strong drug resistance; 4binding host protein
○ substitution of the amino acid indicated
HA phylogeny and variation
In the HA phylogenetic tree, two viruses belonged to clade 6B.1A, including A/Brisbane/02/2018 and a virus similar to a 2018–2019 season-isolated A(H1N1)pdm09 in the Republic of Korea. A/Korea/S0002/2019 and A/Korea/S0003/2019 HA sequences best matched with those of A/Brisbane/02/2018 (98.6 and 98.9% amino-acid identity, respectively). The two viruses fell into different subclades according to discrepancies at residues 158, 202, 277, and 521. A/Korea/S0002/2019 belonged to subclade 6B.1A5 and A/Korea/S0003/2019 to subclade 6B.1A4 (Fig. 2). Table 5 shows the amino-acid substitutions in the HA gene compared with A/Brisbane/02/2018 and their reported effects.
Table 5: HA genetic variation compared with A/Brisbane/02/2018 (H1 numbering).
Virus
|
Amino acid identity with A/Brisbane/02/2018 (%)
|
G62R1,2,3
|
N146D1,2,3
|
A158E 1,2,3
|
T202I 1,2,3
|
R240Q 1,3,4
|
N277D2
|
A299P1,2
|
V315I1,2
|
K5212
|
A/Korea/S0003/2019
|
98.94
|
○
|
○
|
○
|
|
○
|
|
○
|
○
|
|
A/Korea/S0002/2019
|
98.59
|
○
|
○
|
|
○
|
○
|
○
|
○
|
○
|
○
|
Frequency (%)
|
84.19
|
17.76
|
0.97
|
19.77
|
99.87
|
29.46
|
84.85
|
85.38
|
3.08
|
1Binding small ligand; 2viral oligomerization interfaces; 3antibody recognition sites; 4host cell receptor binding
○ substitution of the amino acid indicated
NA inhibition assay
A/Korea/S0003/2019 and A/Korea/S0002/2019 viruses exhibited significantly reduced inhibition by oseltamivir and peramivir with > 100 IC50 fold-changes relative to drug-sensitive virus, but were normally susceptible to zanamivir (Table 6). However, every isolate collected from the KINRESS in 2018–2019 was susceptible to all three drugs.
Table 6: Neuraminidase inhibition assay for A(H1N1)pdm09 viruses isolated in immunocompromised patients
Virus
|
Type(Subtype)
|
Genotype
|
NAI resistance
(IC50 fold change)
|
Oseltamivir
|
Zanamivir
|
Peramivir
|
A/Korea/S0003/2019
|
A(H1N1)pdm09
|
Variant (H275Y)
|
HRI
179.33
|
NI
2.56
|
HRI
117.74
|
A/Korea/S0002/2019
|
Variant (H275Y)
|
HRI
221.58
|
NI
0.44
|
HRI
168.48
|
2018–2019 isolates
|
A(H1N1)pdm09
|
Wild
|
0.1–1.2
|
0.3–0.7
|
0.1
|
A(H3N2)
|
Wild
|
0.1–0.2
|
0.4–0.8
|
0.2–0.3
|
B
|
Wild
|
9–16
|
0.7–1.6
|
0.3–0.4
|
Antigenic characterization
The two viruses were well inhibited by antisera from ferrets immunized with A/Michigan/45/2015 and A/Brisbane/02/2018, which are northern-hemisphere influenza A(H1N1)pdm09 isolates collected in 2018–2019 and 2019–2020, respectively, with less than two-fold reduced HI titer relative to homologous virus (Table 7).
Table 7: Hemagglutinin inhibition assay.
Virus
|
Hemagglutination inhibition titer
|
Post-infection ferret antisera
|
A/Michigan/45/2015 Egg
|
A/Brisbane/02/2018 Egg
|
A/Korea/S0003/2019 MDCK
|
640
|
640
|
A/Korea/S0002/2019 MDCK
|
320
|
640
|
A/Michigan/45/2015 MDCK
|
1280
|
640
|
A/Brisbane/02/2018 MDCK
|
640
|
1280
|