RIEMS Influenza A Typing assay-

On the way to version

RITA2.0

Kareem E. Hassan, Timm Harder

20th June, 2019

Riems influenza A typing (RITA)

RITA version 2

32 RT-qPCRs, 14 HA, 9 NA subtypes

inclusion of new variants and new emerging IAVs,

inclusion of new subtypes (H14, H15),

improved sensitivity and specificity avoiding current

cross reactivity of H1/H6/H8, H2/H5, H7/H10/H15,

new array format (24 wells) for more cost-effective

performance,

new chemistry (lyophilized ingredients, ready-to-use

plates).

En route to RITA 2.0

Modification and development of new oligos for

Past problems with specificity

* - Cross reactivity in RITA 1.0; about 6-12 higher Cq values compared

to specific target

Influenza A variability

HA similarities

NA similarities

Identifying problems in RITA

1, finding salvage options

Sequence repositories,

AIV, also other hosts,

geographic range?

Recently emerging

mutants

Inclusivity versus

specificity

Stages of improving RITA

Designing new primers

and probes for all HA

including new H14 and

H15 and NA

Evaluation of new RT-

qPCR chemistry

In silico planning

Evaluation of each set

of primers and probe

Cross reactivity?

Wet lab work

Full evaluation of as many as possible different

viruses with respect to temporal and geographical

origin, clade and pathogenicity;

Multicenter study

SubtypeSample no.

total Avian Human Porcine EquineHost

unknown

H1 63 10 17 32 4

H2 20 19 1

H3 52 25 11 15 1

H4 20 20

H5 32 32

H6 67 67

H7 42 40 1 1

H8 5 5

H9 43 29 14

H10 32 30

H11 14 12 1

H12 1 1

H13 10 10

H14 1 1

H15 1 1

H16 4 4

N1 140 91 16 30 4

N2 120 73 11 22 14

N3 24 24

N4 10 10

N5 6

N6 28 23 4 1

N7 38 34 3 1 1

N8 27 26 1

N9 11 9 1 1

RITA 2.0 full evaluation (Riems)

Subtype RITA 1, problems RITA 2, tested RITA 2, cross reactivity

H1 H11-H3-H10 H11-H3-H10-H5 NOT found

H2 H5 H5 including egyptian H5N8 NOT found

H3 H1 H1 NOT found

H4 H14 NOT found

H5

H6 H10 NOT found

H7 H10 H10-H15 NOT found

H8 H11-12-13 NOT found

H9 NOT found

H10 H6 H7-H15-H6 NOT found

H11 H1 H1 NOT found

H12

H13

H14 H4 NOT found

H15 H7-H10 NOT found

H16 H13 NOT found

N1 N2-N3-N6-N7 N2-N3-N4-N6-N7 NOT found

N2 N1-N3 N1-N3 NOT found

N3 N1-N7 N1-N7 NOT found

N4

N5 N8 NOT found

N6 N9-N3 N9-N3 NOT found

N7 N2-N5-N8 N2-N5-N8 NOT found

N8 N5-N6 N5-N6 NOT found

N9 N3-N6 N3-N6 NOT found

RITA 2, cross reactivity evaluation

1 2 3 4

RITA 2.0 new plate layout

Version 1,

FAM, HEX

Version 2,

FAM, HEX

Example, H9 viruses

RITA 1; H9 primers and probe: flat curves versus PTC

RITA 2; restored sigmoidal

kinetics after adaptation of

reverse primer and probe

PTC

PTC

Larger scale

Adaptation, H9 viruses

Changes in oligonucleotide sequences (Hassan et al., 2019, accepted in TBED)

Designation Oligonucleotides Conc1 Reference

AIV-H9 RITA assay2

RITA F1 Fw ATGGGGTTTGCTGCC 20 μM

(Hoffmann et al., 2016)RITA R Rv TTATATACAAATGTTGCAYCTG 20 μM

RITA FAM Probe FAM-TTCTGGGCCATGTCCAATGG-BHQ1 5 μM

AIV-H9 modified

RITA2-H9-F1 New Fw CAATGGGGTTYGCTGCCT 10 μM This study

RITA2-H9-F2 New Fw CAATGGGRKTTGCTGCCT 10 μM This study

RITA2 –H9-R Rv TTATATACARATGTTGCAYCTG 20 μM This study

RITA2 –H9-FAM Probe FAM-TTYTGGGCCATGTCIAATGGRTC-BHQ1 5 μM This study

AIV-N2 RITA assay2

RITA F1 Fw AGTCTGGTGGACYTCAAAYAG 20 μM

(Hoffmann et al., 2016)RITA R Rv ATGBCTATATAAGCTTTCGCAATT 20 μM

RITA FAM Probe FAM-AGGCTCATGGCCTGATGG-BHQ1 5 μM

AIV-N2 modified

RITA2 –N2-F1 Fw AGTCTGGTGGACYTCAAAYAG 10 μM (Hoffmann et al., 2016)

RITA2-N2- F2 –New Fw CAGAGTRTGGTGGACITC 10 μM This study

RITA2-N2-R –New Rv TTG CGA AAG CTT AYA TNG VCA T 20 μM This study

RITA2-N2-Fam-New Probe FAM-CAT CAG GCC ATG AGC CTG TYC CAT-BHQ1 5 μM This study

Increased sensitivity, H9 viruses

Direct comparison of an Egyptian H9N2 G1-B lineage field sample

RITA 1

RITA 2

18.8 23.0

Sample code

Cq value

RITA 1 RITA 2

H9 N2 H9 N2

AR517/18 25.544 31.78 24.87 25.02

AR537/18 17.234 25.02 14.10 15.23

AR538/18 17.014 26.10 17.72 19.80

AR544/18 20.024 27.53 18.89 21.02

AR540/18 Neg 18.01 15.52 15.78

AR542/18 Neg 33.05 Neg 31.56

AR551/18 Neg 29.89 27.04 28.01

AR563/18 Neg 27.56 23.90 25.01

AR591/18 Neg 32.12 30.05 29.90

Example, H3N1/BE virus

Direct comparison of two H3N1 isolates of Belgian origin (source: private

diagnostic laboratory in Germany), RITA 1 vs RITA2

17.3

20.5

21.2

24.2

Towards the final goal

RITA 2 new plate layout: 3 columns/sample (-25%)

Lyophilized primers, probes and chemistry ready in

each well (

Only sample RNA to be added (2.5 µL/well diluted to

20 µL final)

Select labs for multicenter study and distribute plates:

Co-work with Seibersdorf lab of IAEA/FAO