Viral quantification and filogenetic analysis Chikungunya virus strains imported to Italy Antonino Di Caro
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1 Viral quantification a filogenetic analysis Chikungunya virus strains imported to Italy Antonino Di Caro National Institute for Infectious Diseases L. Spallanzani, Rome
2 BACKGROUND 1 Since 2005, more than 1000 imported CHIKV cases detected among European a American travellers CHIKV diagnosis is essentially based on virus isolation, Ab detection (IF, ELISA, HAI) a RT-PCR assays. The RT-PCR is the method of choice for early detection a confirmation of virus in clinical samples. Necessity to improve laboratory tools for rapid diagnosis a patients monitoring Most available diagnostic methods targeted E1 envelope protein. Other targets were capsid (C), E2 a nsp1 viral genomic regions.
3 AIM To develop a real-time RT-PCR method (qpcr) able to detect a quantify a CHIKV gene product more conserved than E1 or other structural proteins
4 Primers/Probe name CHK142f CHK142r CHK probe1 CHK probe2 Sequence 5 - CCC GAG AGA CTC GCC AAT TA 5 - TGT GTA AGC AGA ATG TTG GCG T 5 - CCT GGA CAG AAA CAT CTC TGG AAA GAT CGG- Fluorescein Red 610- ACT TAC AAG CAG TAA TGG CCG TGC CAG AC -Phosphate Amplicon size bp 142 Primers a FRET probes targeting nsp1 region
5 Dynamic range:7 Log10 Sensitivity: 20 cp/reaction Specificity: 100% No cross-reactivity with other viruses
6 qpcr validation Vero E6 cells treated 24h with IFNa (1-10,000 IU/ml), infected with CHIKV (MOI 0.01) Log copies/ml viralrna Viral Infectivity Log TCID50/ml INF α U/ml IFNα U/ml 1 IFNα exerted a dose-depeent inhibition of virus replication, assessed as both viral infectivity a viral RNA production.
7 Log copies/ml viralrna 7 Viral Infectivity INF α U/ml IFNα In these experiments, virus infectivity a RNA titres showed very good correlation Log TCID50/ml r = P< D I C T g o L Log cp/ml
8 Application of CHIKV qpcr to clinical samples viremia was detected during the acute phase; viral load ranged from 1.3x10 5 to 5.9x10 8 cp/ml Patient ID, geer, age Travel history, beginning of symptoms Sampling date RT-PCR (nsp1 /E1) qpcr (cp/ml) Viral Isolation (strain designation) Ab IFA IgG (titers) Ab IFA IgM CG L, M, 48 Mauritius 27/03/06 31/03/06 28/04/06 Pos * 1.3 x 10 5 ** *** 1:320 Pos Pos TAM, M, 45 Mauritius 03/04/06 05/04/06 27/04/06 Pos 1.3 x 10 8 Pos (CHKV ITA1 TAM) 1:320 *** Pos RS, M, 56 Seychelles 30/05/06 06/06/06 10/10/06 Pos 2.2 x :320 MR, F, 35 Iia 07/09/06 09/09/06 29/09/06 Pos 5.9 x 10 8 Pos (CHKV ITA3 MR) 1:320 Pos *: not done; ** neg:<4x10 3 cp/m; *** neg:<1:20; pos:>1:20 (titre if available)
9 BACKGROUND 2 Mutations of E1 CHIKV as molecular signatures of the Iian Ocean outbreak: M269V, D284E a A226V. A226V mutation, initially absent, appeared in >90% of the isolates collected after Dicember 2005 in Réunion. A226V increase viral fitness in Aedes albopictus (widespread vector). Concerns about possible spreading in Italy (vectors A. albopictus widespread)
10 AIM To characterize 7 CHIKV isolates, 5 imported to Italy (Mauritius a Iia) a 2 obtained during the recent 2007 Italian outbreak, with respect to molecular signatures of the Iian Ocean outbreak in E1
11 Phylogenetic tree of partial E1 gene (895 bp, of CHIKV S27) Iian Ocean Central Africa Central/east African genotype Asia West Africa
12 Sequences of 1013 bp fragment of E1 ( respect to the S27) Chikingunya virus strain Provenance Time of isolation A226V E1 mutations M269V D284E S27 (prototype strain) Tanzania 1953 No No No LR2006 OPY1 Reunion 2006 ITA1_TAM_06 Mauritius February 06 ITA2_BMI_06 Mauritius March 06 ITA3_CGO_06 Mauritius April 06 IND-06-AP3 Iia 2006 No ITA4_MRA_06 Iia September 06 No MIV-69 Iia 2007 ITA5_JEM_0 Iia July 07 ITA7_BIO_07 Italy September 07 ITA8_VEN_07 Italy September 07 ITA07-RA1 Italy, mosquito September 07 SG EHIss622 Singapore 2008 No
13 Phylogenetic tree of partial E1 gene (895 bp, of CHIKV S27) Iian Ocean Central Africa Central/east African genotype Asia West Africa
14 CONCLUSIONS The A226V mutation, was absent in the strains circulating in Iia a imported from Iia to Italy in 2006 No outbreak in Italy was originated by Iian strains without the mutation The mutation appeared in Iia in 2007, a was present in sporadic cases imported to Italy a in epidemic cases from the outbreak Outbreak triggered in 2008 by Iian strains without the mutation, was easily controlled (Singapore) The presence of A226V in the isolate from Iia changed over time, acquiring after 2006 the E1 mutation associated with enhanced fitness in Aedes albopictus Acquisition a fixation of the A226V mutation may be a common pathway of Chikungunya outbreak explosion, in a parallel interplay with the mosquito vector dynamics.
15 Fabrizio Carletti Concetta Castilletti Roberta Chiappini Licia Bordi Laboratory of Virology, National Institute for Infectious Diseases L. Spallanzani, Rome, Italy Giuseppe Ippolito, Maria R. Capobianchi Vittorio Sambri Francesca Cavrini Laboratory of Microbiology, Alma Mater Studiorum Bologna University, Italy Next, please!
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Received 1 April 2008/Returned for modification 5 June 2008/Accepted 16 July 2008
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