Background
Methods
Clinical specimen collection
Viral RNA extraction
One-step multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR)
Type/Subtype | Primer/probe | Sequence (5′–3′) | Target (bp) |
---|---|---|---|
A | Forward | AATCCTGTCACCTCTGACTAAGG | Matrix (98) |
Reverse | CATTYTGGACAAAKCGTCTACG | ||
Probe | FAM-TGCAGTCCTCGCTCAC-MGBNFQ | ||
B | Forward | GAATGCTGTCAATGAATATTGAGGG | Nucleoprotein (77) |
Reverse | CATTGAGTCATTCATCATCTTGAGTAGAT | ||
Probe | VIC-TCCTTTGACATCTGCAT-MGBNFQ | ||
H1 | Forward | AACAATTCAACAGACACTGTAGACACAGT | Hemagglutinin (144) |
Reverse | GGGCTACCCCTCTTAGTTTRCATAGTTT | ||
Probe | FAM- ATGTAACAGTAACACACTCTGT-MGBNFQ | ||
H3 | Forward | GGAATGGTTTGTCATTGGGAAT | Hemagglutinin (95) |
Reverse | AAGCTCAATAATGAGRTCAGATGCA | ||
Probe | VIC-CTTCCATTTGGAGTRATRCA-MGBNFQ | ||
IPC | Forward | AGATTTGGACCTGCGAGCG | GAPDH (65) |
Reverse | GAGCGGCTGTCTCCACAAGT | ||
Probe | Cy5-TTCTGACCTGAAGGCTCTGCGCG-BHQ |
Virus isolation in MDCK cell lines
Hemagglutinin (HA) and neuraminidase (NA) sequencing
Primers (F/R) | Gene (subtype) | Locus | Sequence (5′–3′) |
---|---|---|---|
A-uni-HA-1F | HA | 1–28 | TATTCGTCTCAGGGAGCAAAAGCAGGGG |
HA-H1-345F | HA (H1) | 345–365 | GGAACGTGTTACCCAGGAGAT |
HA-H1-680F | HA (H1) | 680–701 | TCAAGCCGGAAATAGCAATAAG |
HA-H1-1137F | HA (H1) | 1137–1156 | AGCCGACCTGAAGAGCACAC |
HA-H1-714R | HA (H1) | 691–714 | CCTCACTTTGGGTCTTATTGCTAT |
HA-H1-1093R | HA (H1) | 1072–1093 | CATCCATCTACCATCCCTGTCC |
HA-H1-1330R | HA (H1) | 1309–1330 | GCATTGTAAGTCCAAATGTCCA |
A-uni-HA-1807R | HA | 1773–1807 | ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTTT |
A-uni-NA-1F | NA | 1–29 | TATTGGTCTCAGGGAGCAAAAGCAGGAGT |
H1-NA-324F | NA (N1) | 324–345 | GGTTCCAAGGGGGATGTGTTTG |
H1-NA-744F | NA (N1) | 744–766 | TGGACAGGCCTCATACAAGATCT |
H1-NA-645R | NA (N1) | 622–645 | AGTGTCTGTTATTATTCCGTTGTA |
H1-NA-1010F | NA (N1) | 985–1010 | CAGTATCGTCTAATGGAGCAAATGGA |
H1-NA-1057R | NA (N1) | 1036–1057 | CTATCCAAACACCATTGCCGTA |
A-uni-NA-1442R | NA | 1407–1442 | ATATGGTCTCGTATTAGTAGAAACAAGGAGTTTTTT |
Genetic characterization
NA inhibition assay
Hemagglutination inhibition (HI) assay
Results
A(H1N1)pdm09 detection and isolation in immunocompromised patients
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 |
Genetic characterization
NA genetic analysis for screening drug resistance, phylogeny, and variation
Virus | Amino acid identity with A/Brisbane/02/2018 (%) | T13I | I29M | Q51K | F74S | P93Sa,b | I99Vb | H275Yc | G298Aa | V321Ia | V394Ib | D416Na,b | T452Ia,b,d |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
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 |
HA phylogeny and variation
Virus | Amino acid identity with A/Brisbane/02/2018 (%) | G62Ra,b,c | N146Da,b,c | A158E a,b,c | T202I a,b,c | R240Q a,c,d | N277Db | A299Pa,b | V315Ia,b | K521b |
---|---|---|---|---|---|---|---|---|---|---|
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 |
NA inhibition assay
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
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 |