“Future of microarray techniques for study of viral diseases” Guy Vernet Importance of viruses genetic variability • Viruses target different host species, organs or cells, • Polymorphisms in non-structural genes impact viral replication and clinical outcome of infection, • Polymorphisms can also affect response to drug or immune treatment and vaccination efficacy.
Detection of viral polymorphisms has several interests • Pathogens identification: – sequences specific for genus, family, species, types, subtypes or even individual viral strains can be used for identification purposes in the diagnosis of patients disease or in epidemiological surveys, – for known and unknown pathogens • Typing and detection of variants of clinical interest: – the detection of single variants at key genome positions in genotyping assays can be used as a signature for virulence, transmissibility, resistance to treatment and for molecular epidemiology surveys • Gene expression: – the analysis of host genes expression changes during infection- is another aspect where genome information can be the basis of laboratory tools especially useful for studies of the mechanisms of viral diseases and their treatment. Microarrays as laboratory tools for research and clinic • Numerous examples of the use of microarrays have been described in the last 15 years, • Most of them are based on oligonucleotides hybridization but microarrays using antigens or antibodies have been described, • Both DNA and protein-microarrays are useful tools in the three domains: – pathogens identification, – typing and variants detection, – gen expression analysis.
Principle Multiplex Hybridization Reading, DNA/RNA amplification data mining extraction /labelling Chips versus beads microarrays Probes and proteins can be coated on: • glass slides (= chips) • on « beads » Only chips can currently provide high-density (thousands of probes).
Spotting versus in-situ synthesis of probes on chips Advantages Drawbacks •Custom Chips •Low-density •Low-cost manufacturing •Complexity of Spotting and reading equipment manufacturing (ccd) •Lack of robustness •Long probes •High-density •« Supplier mode » In-situ •Result robustness •Shorter probes (redundancy) •Cost of platform (laser) synthesis •Robust manufacturing process Pathogens identification The use of microarrays allows the simultaneous detection of numerous pathogens in a « syndrome-based » approach
•Affymetrix 2L-tilling •40588 probes
•The assay can efficiently detect enteroviruses, flaviviruses and herpesviruses •It can differentiate virus species among flaviviruses and herpesviruses (including closely sequence-related HSV1 and 2) •Enteroviruses can be typed in VP1 Recent examples of syndrome-based chips • Ayodeji M et al., 2009. W ater-borne diseases. • 13,000 elements, • hepatitis A virus, human coxsackieviruses A and B, genogroups I and II of Norovirus, and hum an rotavirus, • tested on strains only, « The results obtained with HAV and CV indicated that the hybridization profile thus generated can be used to identify closely related viral strains » • Shi J et al., 2009. Encephalitis. • herpes simplex virus type 1 and type 2, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, and human herpes virus 6, • probes in highly conserved regions of the DNA polymerase gene, • tested on 290 CSF specimens, « Compared with the results of TaqMan PCR, the sensitivity of PCR- microarray technology was 91.7% , the specificity was 100% »
Respiratory Viruses Panel (Luminex/Abbott) Luminex's xMAP technology
Microarrays are promising technologies but multiplex RT-PCR may be sufficient Multiplex real-time PCR with multi-channel machines FTD, Luxembourg 1 tube 5-6 tubes real-time Concordance: 94.1 % Currently, because of its automation on the Infiniti analyzer, the microarray assay described herein is the most adaptable system for clinical laboratories.
Virus discovery: GreeneChip Pr. Ian Lipkin, Columbia University, NYC • Sequence database containing 228,638 viral sequences. • 3 distinct genomic target regions for every family or genus of vertebrate virus (1 conserved, 2 more variable). • 29,495 probes (60-mer oligonucleotides): – 9,477 probes for vertebrate viruses (1,710 species), – 11,479 16S rRNA bacterial probes, – 1,120 18S rRNA fungal probes – 848 18S rRNA parasite probes • Random amplification, specific second step PCR + labeling: – Reverse transcription with a random octamer linked to a specific primer sequence, – After digestion with RNase H, amplification using the above primer and a primer targeting the specific primer sequence, – Labeling in a subsequent PCR with the specific primer sequence linked to a capture sequence for 3 DNA dendrimers containing >300 fluorescent reporter molecules Future of microarrays for pathogens identification • Microarrays are well adapted for large panels of highly variable pathogens like viruses, • Multiplex amplification is a bottleneck: – sensitivity is lower than single amplification, – sequence variabilty of viruses is well addressed at the detection level but multiple primers may be needed for a single virus. • Automation, integration, cost reduction, robustness: – beads-based assays are more easily automated and integrated, • Competition to microarrays by multiplex real-time PCR for known viruses and by next-generation sequencing for virus discovery
Typing and variants detection Keys for treatment tailoring and monitoring and for epidemiology Hepatitis B microarray: amplification strategy X sp rt tp sp PreS2 S C PreS1 S 1 AS 1 1.7 kb 1.5 kb S 2 AS 2 S 3 0.7 kb Nested-PCR rt / S S 1’ AS 1’ for low viral loads
Hepatitis B: Affymetrix chip • HBV sequences alignment for design : > 1400 sequences • Spots size 11µm • 520 000 probes per chip • Probes length : 20 mers • Re-sequencing using a set of 4 probes for each nucleotide Hepatitis B microarray: mutations detection and genotyping Positions (a.a.) All mutants (Nt) to Role to be detected be detected tp 0 0 Resistance sp 2 10 rt 66 197 preS1 3 6 preS2 6 16 Vaccine escape S 98 339 BCP 33 112 preC 14 55 Clinical outcome C 21 115 X 55 144 Cancer 298 994 Total •Genotyping (8A to H): based on 812 informative positions along the genome
Quasi-species analysis Genotyping performances
Mutations detection SEQUENCING SEQUENCING rt 169 WT MUT rt 204 WT MUT Ind Ind n=164 (98.8 %) I T L n=164 (82.3%) M I V S WT I 161 1 WT M 104 2 1 3 T 0 I 1 10 MUT MUT L 0 1 V 2 12 21 Ind 1 S 0 Ind 3 1 3 0 rt 173 WT MUT rt 236 WT MUT Ind Ind n=164 (96.3%) V L G n=123 (48.0%) N T Y WT V 150 1 1 WT N 57 L 3 8 1 MUT T 2 MUT G 0 Y 0 Ind 0 Ind 58 6 0 rt 180 WT MUT rt 250 WT MUT Ind Ind n=164 (81,7%) L M P n=114 (94.7%) M V WT L 104 1 WT M 107 M 2 28 MUT V 5 0 MUT C 0 Ind 1 1 Ind 14 13 2 rt 181 WT MUT Y Ind Y A n=164 (81.7%) A T G V A R R WT A 124 1 R R A A T 2 O O R MUT G 0 R IC IC M V 2 12 M Ind 20 2 1 0 rt 184 WT MUT Ind n=164 (93.9%) T S A G N I WT T 152 1 S 1 1 2 A 0 MUT G 1 0 N 0 I 1 Ind 2 3 0 rt 194 WT MUT Ind n=164 (100%) A T WT A 164 MUT T 0 Ind 0 rt 202 WT MUT Ind n=163 (95.1%) S I G WT S 155 I 0 MUT G 5 0 Ind 3 0 Influenza typing: a few recent examples • Li X et al., 2009,Taizhou Affynigen Biotechnologies, Inc.,Shanghai, China • a microarray with 46 short virus-specific oligonucleotides for detecting influenza A virus of 5 subtypes: H1N1, H1N2, H3N2, H5N1, and H9N2. « When tested with 225 clinical samples, 20 were detected to be positive using our microarray-based assay, whereas only 10 were positive by the conventional culture method » • Huang Y et al. , 2009, Nanogen, Inc., San Diego, California • an electronic microarray to simultaneously type influenza A and B viruses and to distinguish influenza A virus subtypes H1N1 and H3N2 from the potentially pandemic avian virus subtype H5N1. « In a study of 146 human clinical specimens that had previously been shown to be positive for influenza virus or another respiratory virus, the assay showed a clinical sensitivity of 96% and a clinical specificity of 100% » • Gall A et al. , 2009, Friedrich-Loeffler-Institut, Riems, Germany • a low-density microarray for the detection and typing of avian influenza virus (AIV). One probe for the conserved matrix gene and 97 probes targeting the HA0 cleavage-site region. « For validation, a panel of 92 influenza A viruses which ncluded 43 reference strains representing all 16 HA subtypes was used. All reference strains were correctly typed with respect to their HA subtypes and pathotypes, including HPAIV H5N1/ Asia »
Recommend
More recommend
