Il ruolo del laboratorio nella diagnosi delle zoonosi e delle malattie trasmesse da vettori IRCSS Lazzaro Spallanzani –Roma 1-2 ottobre 2014 Maria Grazia Cusi Toscana Virus was isolated from Phlebotomus perniciosus on Monte Argentario (Grosseto) in 1971 (Verani, 1984). In 1983, it was isolated for the first time from a young woman with lymphocytic meningitis (Leoncini, 1983). Genus Human disease Orthobunyavirus La Crosse encephalitis virus, others Vector mosquito Toscana virus, Sandfly Fever Sicilian and Naples viruses, Rift Valley Fever virus, etc. Sandfly P. Perniciosus P. Perfiliewi Nairovirus Crimean-Congo hemorrhagic fever virus Tick Tospovirus Plant virus. No known human disease Thrips Hantavirus Hemorrhagic fever with renal syndrome Hantavirus pulmonary syndrome Tick Incubazione: da pochi giorni a due settimane Infezioni inapparenti o pauci-sintomatiche con sintomatologia simil-influenzale e self-limitante (Febbre, eritema, linfoadenopatia) Meningite con o senza sintomatologia encefalica (Rigidità nucale, perdita di coscienza, tremori, paresi; Manifestazioni inusuali (Ischemia, sordità, idrocefalo, cambio di personalità, fascite e miosite) NON ESISTE TERAPIA SPECIFICA! NS gene N gene M gene L gene Phylogenetic analysis of TOSV strains: 2 circulating genotypes (A, B) Collao X. et al. Emerg. Infect. Dis. 15: 2009 Toscana Virus meningitis in Siena area during the 1993-2013 years 40 36 35 37 31 29 27 30 28 26 25 23 25 25 24 22 20 19 20 15 18 13 17 14 16 16 11 10 5 0 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 COURSE OF TOSV INFECTION Viremia IgG IgM Incubation 2d 14d ≥ 1 month 7d 1 year 15d Diagnosi di infezione da Toscana Virus RT-PCR+ RT-PCR (CSF) or virus isolation on cell culture PCR-, IgM+, IgG- Neuroinvasive infection with TOSV Test a 2° serum sample after 1-2 weeks PCR-, IgM+, IgG+ Serology IgMIgG (serum/plasma) IgM-, IgG+ Past infection with TOSV IgM+/-, IgG+ Neuroinvasive infection with TOSV The virus grows on Vero cells and causes a lytic cytophatic effect. The viral load is sometimes too low that virus isolation from CSF is not reliable. CSF and not blood is the elective sample for Toscana virus isolation, since it is very difficult to identify the short period of viremia. Molecular detection of Phleboviruses The relatively low viral load in biological samples makes nucleic acid detection necessary Some aspects should be kept in mind for reliable results: • Time of sampling • Appropriate storage of sample (preferably -80°C) Toscana virus detection Small (S) genomic segment is the most abundant molecule during viral infection. The nucleoprotein (N) coding gene on the S segment is the most conserved gene among circulating TOSV strains. It is the best target for virus detection by amplification! •RT nested-PCR: Virus specific primers •Pan-Phlebovirus degenerated primers •Real time PCR Use of pan-phlebovirus oligos • Degenerated primers matching on the L segment; • Rapid and simultaneous detection of several phleboviruses in a sample including: TOSV; SFSV/SFNV; RVFV; PTV and UUKV • Need to sequence amplification product in order to identify viral species • TOSV specific detection by using a degenerated reverse primer (ATos2-) in the second-round PCR Primer Target gene Position (nt) Sequence Assay NPhlebo1+ L 2047-2069 5'-ATGGARGGITTTGTIWSICIICC-3' RT-PCR NPhlebo1- L 2600-2575 5'-AARTTRCTIGWIGCYTTIARIGTIGC-3' RT-PCR NPhlebo2+ L 2074-2094 5'-WTICCIAAICCIYMSAARATG-3' Nested PCR NPhlebo2- L 2318-2296 5'-TCYTCYTTRTTYTTRARRTARCC-3' Nested PCR ATos2- L 2209-2190 5'-RTGRAGCTGGAAKGGIGWIG-3' Nested PCR Amplicon size Reference 554 244 126 Sanchez-Seco et al., 2003 Pan-Phlebovirus assay MW K- 1 2 3 4 5 6 7 8 K- K+ MW 1 2 3 4 K+ Pools of sandflies 244 bp 244 bp Sanflies pools tested by RT-nested-PCR with degenerated primers. Pools 2-4 were then tested with TOSV specific ATos2- primer. Whole blood MW K- K- K- K- WB1 WB2 CSF K+ MW K- K- K- CSF1 CSF2 K+ Human samples 244 bp 244 bp Phlebovirus detection by degenerated primers on whole blood samples from healthy donors. Phlebovirus detection by degenerated primers on TOSV positive (#1) and negative (#2) CSFs. Serological investigations • Enzyme-linked immunosorbent assay (ELISA) • Indirect immunofluorescence (IFA) • Immunoblotting (IB) • Immuno-chromatographic assay (ICA) • Plaque-reduction neutralization test (PRNT) Toscana virus ELISA FAST, SENSITIVE AND SPECIFIC ASSAY Recombinant nucleoprotein (rN) (Schwarz et al., 1995; Soldateschi et al., 1999; Valassina et al., 1998; Magurano et al., 1999; Ciufolini et al., 1999). – – – – – The N protein is the most immunogenic TOSV antigen. Highly expressed and purified in prokaryotic cells. Safer use compared to live virus as antigen source. Good correlation of specificity and sensitivity in comparison to the gold standard (IFA) and plaque-reduction neutralization test (PRNT). Possible cross-reactivity with anti-SFNV due to high similarity sequence of the N protein. Only one commercial ELISA test for serological diagnosis of TOSV infection is available on the market (DIESSE, Diagnostica Senese, Italy). Immunofluorescence (IFA) Useful alternative to PRNT!! • Simple and fast; • Aspecific reaction due to rheumatoid factor during IgM detection. • Cross-reactivity with TOSV and SFNV • Background reaction due to sample properties (haemolysed and/or lipemic sera) NEED OF SAMPLE PRETREATMENT NEED OF CONFIRMATORY TEST (PRNT) Commercial assay is available from Euroimmun Mosaic Sandfly fever virus 1: Toscana, Sicilian, Naples and Cyprus viruses. Mosaic Phlebovirus 1: Toscana, Sicilian and Rift Valley Fever viruses. Both tests permit IgG and/or IgM detection by using specific secondary conjugated antibody. The manufacturer provides and efficient RF adsorbent for IgM detection. Cross-reaction was reported between Toscana/Naples and Sicilian/Cyprus serotypes Anti-TOSV IgG/IgM detection by Immunoblotting (IB) Antigen Immunoglobulin class Reference TOSV infected cell lysate IgG/IgM Magurano et al., 1999 Purified virus IgG/IgM Schwarz et al. , 1996 rN protein IgG/IgM Ciufolini et al., 1999 Valassina et al., 1998 Schwarz et al., 1998 Mikrogen diagnostik, Neuried, Germany In this system, human sera show a high reactivity to the N protein, while the response to glycoproteins is highly variable due to the denaturing treatment of the proteins before gel loading, which destroys their conformational epitopes, no longer recognised by specific antibodies. Currently, an immunoblot kit is available by Mikrogen which permits the simultaneous detection of IgG and IgM antibodies against the Toscana virus N-antigen. Immunochromatographic assay (ICA) An immunochromatographic assay (ICA) was developed for human anti-TOSV IgG or IgM detection by InBios International (Seattle, WA, USA) (Houghton R. et al., J Virol Methods, 2013). The sensitivity of the new assay compared to commercial ELISA test was 98.5% for IgM and 90.1% for IgG, while specificity was 100% in both cases. Time to test: 15min Control line Test line 1: TOSV IgM/IgG negative sera. 2: TOSV IgG positive sera. 3: TOSV IgM positive sera. This is a new diagnostic approach that is easier and faster than conventional tests 1 2 3 • TOSV can be considered an emergent pathogen circulating in the Mediterranean area and in other countries; • Diagnosis should be performed on CSF and/or serum by molecular detection or serological assays; • RT-PCR and real time PCR are the gold standard assays in direct diagnosis; • ELISA and IFA are serological tests available on market for TOSV specific IgM/IgG research.