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Development and evaluation of a real-time RT-PCR assay for Sindbis virus detection
Journal of Virological Methods 179 (2012) 185–188
Contents lists available at SciVerse ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Development and evaluation of a real-time RT-PCR assay for Sindbis virus detection Jussi Sane a,∗ , Satu Kurkela a,b , Lev Levanov a , Simo Nikkari d , Antti Vaheri a,b , Olli Vapalahti a,b,c a
Infection Biology Research Program, Research Programs Unit, Department of Virology, Haartman Institute, Faculty of Medicine, University of Helsinki, Helsinki, Finland Department of Virology and Immunology, HUSLAB, Helsinki, Finland Division of Microbiology and Epidemiology, Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland d Centres for Military Medicine and for Biological Threat Preparedness, Helsinki, Finland b c
a b s t r a c t Article history: Received 21 September 2011 Received in revised form 11 October 2011 Accepted 27 October 2011 Available online 3 November 2011 Keywords: Sindbis virus Pogosta disease Detection Real-time RT-PCR Viral load
Sindbis virus (SINV) is an arthropod-borne alphavirus found widely in Eurasia, Africa and Oceania. Clinical SINV infection, characterized by rash and arthritis, is reported primarily in Northern Europe. The laboratory diagnosis of SINV infection is based currently on serology. A one-step TaqMan® real-time RT-PCR assay was developed for the detection of SINV and evaluated its clinical performance with acute-phase serum samples. The specificity and sensitivity of the real-time PCR assay were assessed using cell cultured Finnish SINV strains. The applicability of the assay for diagnostic use was evaluated using 58 serum samples from patients infected with SINV. The real-time RT-PCR assay was specific and sensitive for the detection of SINV in cell culture supernatants with a 95% detection limit of 9 genome copies/reaction determined by probit analysis. However, in the assay only 7/58 (12%) of serum samples were positive of which two were also positive by conventional nested PCR assay and none by virus isolation. This novel assay is specific and sensitive for detection of SINV and can be used for example for screening SINV in wildlife. However, molecular diagnostic techniques using serum samples seem to be of limited value for the diagnosis of human SINV infection due to the short and low viraemia of infection with SINV. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Arthropod-borne Sindbis virus (SINV) is a mosquito-borne enveloped single-stranded RNA virus of the genus Alphavirus, family Togaviridae (Weaver et al., 2005). SINV is found in Eurasia, Africa and Oceania (Hubalek, 2008) but clinical infections are reported almost exclusively from Northern Europe, particularly from Finland (Kurkela et al., 2004; Skogh and Espmark, 1982; L’vov et al., 1985). Acute SINV infection, named Pogosta disease in Finland, is characterized by fever, rash and arthritis (Kurkela et al., 2005; Turunen et al., 1998; Sane et al., 2011). Long-term joint manifestations can persist even for years (Kurkela et al., 2008). SINV epidemics have a peculiar cyclic appearance in Finland and during large outbreaks several hundreds of cases are serologically verified (Brummer-Korvenkontio et al., 2002; Sane et al., 2010). Population density fluctuations of grouse, which are considered probable natural hosts for the virus, and alterations in climatic and weather
Abbreviations: SINV, Sindbis virus; MGB, minor groove binder; Ct, threshold cycle; nsP, non-structural protein. ∗ Corresponding author at: University of Helsinki, Haartmaninkatu 3, P.O. Box 21, 00014 Helsinki, Finland. Tel.: +358 9 191 26608, fax: +358 9 191 26491. E-mail address: jussi.sane@helsinki.fi (J. Sane). 0166-0934/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2011.10.021
conditions, may be involved in the observed epidemiological pattern (Brummer-Korvenkontio et al., 2002). More recently, there is an increased awareness of SINV also in other parts of Europe, such as in Germany where SINV was for the first time isolated from mosquitoes in 2009 (Jost et al., 2010). SINV is also used as a vector in cancer therapy trials (Quetglas et al., 2010). The laboratory diagnosis of SINV is based currently on serology. Only approximately 40% of patients show IgM antibodies within the first week of illness (Kurkela et al., 2005). As patients usually seek medical care within the first days of illness (Sane et al., 2011), a negative antibody test result during the first week is common. Therefore, a second serum sample is frequently required to establish the diagnosis of acute SINV infection. Diagnostic methods based on real-time nucleic acid detection could enable a more prompt diagnosis. This would be especially useful during epidemics when a large number of samples are processed within a short time period. Thus far, a few conventional PCR (Kurkela et al., 2005; Hörling et al., 1993) and real-time RT-PCR (Jost et al., 2010; He et al., 2005) assays have been described for SINV detection but assays for the diagnosis of human SINV infection have not been described previously or their clinical performance and validity evaluated. The aim of this study was to develop a one step real-time RTPCR assay for the detection of SINV using TaqMan® chemistry and to evaluate its clinical performance with acute-phase serum samples.
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2. Material and methods 2.1. Patient samples Fifty-eight first serum samples from Pogosta disease patients from whom sera was taken during the acute phase of SINV infection were available for this study. The samples were obtained from the Department of Virology and Immunology of the Helsinki University Hospital Laboratory. A seroconversion between paired sera and/or a positive IgM test result in a single serum, using a highly specific (95.2% and 97.6% for IgM and IgG, respectively) and sensitive (97.6% and 100% for IgM and IgG, respectively) enzyme inmmunoassay (EIA) (Manni et al., 2008) was required for the diagnosis of acute SINV infection. All the 58 patients fulfilled the criteria for serological diagnosis of SINV infection. The sera were collected during the epidemic years 2002 (n = 24) and 2009 (n = 34). Fifty samples were IgM and IgG negative; 8 samples were IgM positive or had a borderline IgM test result. The sera were aliquoted and stored at −70 ◦ C prior to RNA extraction. 2.2. Viral RNA extraction RNA was extracted from 140 l of serum or viral culture supernatant using QiAmp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. 2.3. Primer and probe design A one-step real-time RT-PCR based on TaqMan® chemistry was developed for the detection of SINV RNA. Conserved regions of SINV were identified using an alignment of complete protein-coding area of Finnish SINV strains isolated previously in our laboratory and further sequenced for this study (part of the genome published earlier (Kurkela et al., 2004), as well as the complete SINV genomes accessible in GenBank (Fig. 1). Sequence alignment was performed with Bioedit Sequence Alignment editor (Hall, 1999). The primers and the probe were designed with Primer Express Software version 3.0 (Applied Biosystems, Foster City, USA) and selected within the non-structural protein nsP1 region (Fig. 1). 2.4. Generation of SINV RNA transcript The target region (74 bp) was amplified from Ilomantsi-2002C strain and cloned into pGEM® -T cloning vector (Promega, Madison,
USA). The presence of the insert was verified by sequencing and restriction enzyme analysis. After linearization of the plasmid by digestion with BsaI, RNA was generated using RiboMAXTM Large Scale RNA production system with SP6 polymerase (Promega, Madison, USA) according to the manufacturer’s instructions. The transcribed RNA was then treated with DNAse and purified with RNeasy Mini Kit (QIAGEN, Hilden, Germany). Finally, RNA was quantified by spectrophotometry and copy number of RNA was calculated based on its concentration, length, and the molecular weight. 2.5. One-step real-time RT-PCR The real-time RT-PCR was performed with Quantitect One Step Probe RT-PCR Kit (QIAGEN, Hilden, Germany) in a final volume of 25 l containing 5 l of template RNA, 400 nM of primers and 250 nM of probe. The TaqMan® probe (Applied Biosystems, Foster City, USA) was labeled at the 5 -end with reporter 6carboxyfluorescein (FAM) and at the 3 -end with minor groove binder (MGB)-non-fluorescent quencher (NFQ). The assay was carried out using ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, USA) with the following cycling parameters: 50 ◦ C for 30 min, 95 ◦ C for 15 min, 45 cycles of 95 ◦ C for 15 s and 60 ◦ C for 1 min. Alternative primers and a probe labeled with quencher tetramethyl-6-carboxyrhodamine (TAMRA) were also designed within the E1-protein region (nucleotide position according to SINV Ockelbo strain; forward primer 5 -CACCCCGCACAAAAATGAC3 at position 11258-11276, probe at position 11284-11309 and reverse primer 5 -AAAAGGGCAAACAGCCAACTC-3 at position 11312-11332) and a subset of samples was analysed with this test format using the same assay parameters as described above. 2.6. Conventional nested RT-PCR and virus isolation Serum samples with a positive test result in the SINV real-time RT-PCR were additionally analysed with a previously described conventional nested RT-PCR assay that has been able to detect SINV in whole blood samples and skin biopsies taken from patients with SINV infection (Kurkela et al., 2005). Furthermore, virus isolation was attempted from these samples on Vero cells using a modification of a previously described protocol that has yielded SINV isolates from whole blood and skin biopsies (Kurkela et al., 2005).
Fig. 1. Alignment of the primer/probe binding regions in nsP1-protein (5 to 3 direction) of different SINV strains and Whataroa virus, SFV and CHIKV. Conserved nucleotides are shown with dots.
J. Sane et al. / Journal of Virological Methods 179 (2012) 185–188
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In brief, 50 l of serum was added to cells in 25-cm2 flasks. After a 1-h incubation, fresh culture medium was added, and the cells were observed for cytopathic effect. 2.7. Evaluation of the one-step real-time PCR assay The assay specificity was evaluated with viral RNA derived from supernatants of five cell cultured Finnish SINV strains previously isolated from humans (Kurkela et al., 2004). In addition, RNA from cell cultured Semliki Forest (SFV, expression vector VA7) (Vähä-Koskela et al., 2003) and Chikungunya (CHIKV, Ross strain) alphaviruses, tick-borne encephalitis flavivirus (TBEV, strain Kumlinge A52), and Puumala (PUUV) hantavirus-positive spleen and liver tissue from a PUUV-infected patient were tested. First serum samples (n = 24) from patients suspected with SINV infection but confirmed SINV-antibody negative in paired samples were also analysed to further evaluate the specificity. The precise analytical sensitivity was assessed by 10-fold dilutions of in vitro transcribed SINV RNA (ranging from 1.42 to 1.42 × 107 copies per reaction) in 8 replicates at each concentration. The limit of detection was calculated with probit regression model (Finney, 1971) with 95% probability endpoint. The probit analysis was performed with SPSS software (version 18, USA). Sensitivity was also assessed in plaque forming units (PFU) by determining the lowest concentration at which positive signal is obtained. Plaque titration assay for Ilomantsi-2002A virus strain was performed. In brief, serial dilutions of virus were added to confluent Vero E6 cells in cell culture plate. After an incubation of 1 hour at 37 ◦ C, agarose (0.5%) overlay medium was added to cells and the plates were incubated for 4 days. Cells were then stained with crystal violet and the plaques were counted. Virus titer was 9.0 × 106 PFU/ml. RNA was extracted from 10-fold dilutions of this virus stock and analysed by the real-time RT-PCR assay.
Fig. 2. Probit regression analysis of SINV real-time RT-PCR. The analysis was based on serial dilutions of in vitro transcribed SINV RNA in 8 replicates at each concentration. The probability of positive result is plotted against the concentration of SINV RNA. The 95% detection limit is indicated with the dashed lines.
positive) and this format was not pursued further for additional samples. Conventional nested PCR was able to detect SINV RNA in 2/7 of those positive with the real-time RT-PCR. These samples originated from patients whose skin biopsies were previously found positive with conventional nested PCR and from whom SINV strains (Ilomantsi-2002A and Johannes) were isolated (Kurkela et al., 2004). No infectious virus could be recovered from any of the seven serum samples by virus isolation in Vero cells.
3. Results
4. Discussion
The real-time RT-PCR assay detected viral RNA of all five Finnish SINV strains in cell culture supernatants with similar threshold cycle (Ct) values (approximately 16 in dilution of 10−2 ). The limit of detection determined by probit analysis (concentration giving a positive real-time RT-PCR result in 95% of samples) was 9 copies/reaction (Fig. 2). End-point dilution showed that the lowest template concentration that gave positive signal was 1.4 copies/reaction (120 copies/ml) and using plaque titrated virus stock, 0.001 PFU/reaction (0.09 PFU/ml). Cell culture supernatants of SFV, CHIKV and TBEV, as well as PUUV-infected tissue samples were subjected to the assay in order to evaluate the specificity, and none gave false-positive signals. In addition, all the SINVseronegative (n = 24) sera were non-reactive in the test. Of the acute-phase serum samples from patients infected with SINV, 7/58 (12%) were positive in the real-time RT-PCR assay. All the SINV RNA positive samples were negative for SINV IgM and IgG, although, according to the inclusion criteria of the study, these patients had seroconverted to SINV by the time of the second samples. In 4/7 (57%) of the samples only one of the duplicates was positive in repeated runs. Ct values for positive samples were high ranging from 35 to 37.3 and the viral load of the samples approximated from the in vitro RNA standard dilutions was in the range of 130–660 viral copies/ml of serum. The use of higher primer concentration (up to 900 nM) or longer reverse transcription time (45 min) did not improve the detection sensitivity. The positive samples were also tested with primers and TAMRAlabeled probe designed within the E1 protein. However, poor sensitivity was observed with this test format (3/7 samples were
A one-step real-time RT-PCR assay was developed for the detection of SINV and its applicability as a diagnostic tool for human SINV infection was evaluated. This would be particularly valuable as the first samples of patients infected with SINV are usually IgM and IgG negative. Earlier studies suggest a narrow viraemic window and low level of viraemia in acute SINV infection (Kurkela et al., 2004; Manni et al., 2008), unlike for the related, but more severe CHIKV infection where high viral loads, up to 109 copies/mL of plasma, have been reported [Panning et al., 2008]. The current data support this finding, and as only 12% of the serum samples were positive with high Ct values in the assay corresponding to levels of less than 103 SINV copies/ml of sample, we suggest that real-time RT-PCR from serum samples is not a practical approach for the laboratory diagnosis of SINV. Furthermore, for several positive samples only one of the duplicates was positive. This indicates a very low viral load and uneven Poisson distribution of template virus in the replicates. The possibility for contamination was minimized by routine measures, and negative controls were not amplified in any of the runs. We acknowledge that by increasing the extraction volume and concentrating the sample, the sensitivity of the assay may have improved resulting in increased proportion of positive samples. However, the RNA extraction kit (QIAGEN, Hilden, Germany) is optimized for the volume of 140 l and this has been sufficient for real-time RT-PCR assays used in routine diagnosis of viral diseases such as CHIK fever, where viral RNA is regularly found during the first week of illness (Edwards et al., 2007; Panning et al., 2008). A protocol requiring substantially larger volumes would probably diminish the applicability of the assay for clinical purposes as
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additional time consuming steps would be required. Also, only a restricted sample volume is often available for a single diagnostic test and only this amount was available to us properly stored (−70 ◦ C) to ensure preservation of RNA. The assay was able to detect all available Finnish SINV strains from cell culture supernatants without any cross-reactivity to other viruses tested. No unspecific amplification in sera negative for SINV was observed. Due to high sequence similarity in the primer binding region of the nsP1 protein, the assay can potentially detect all SINV strains of which sequence is available in the Genbank. The observation on the low viral load in serum during acute infection may have implications as to the pathogenesis of SINV infection. Evidence suggests that SINV can readily infect human macrophages, and macrophage-derived pro-inflammatory response contributes to the pathogenesis of arthritis (AssuncaoMiranda et al., 2010). It is possible that after inoculation, SINV targets cells circulating in blood such as monocytes that further disseminate virus to different tissues (e.g. skin, joints, muscle), while the presence of SINV in serum remains short-lasting. In a previous study, SINV RNA was detected in 5 of 73 acute-phase whole blood samples with nested RT-PCR (Kurkela et al., 2005). Our preliminary data show that the real-time RT-PCR assay can detect SINV RNA in whole blood but due to lack of proper sample panel, comprehensive comparative analysis of the whole blood and serum samples cannot be undertaken at this time. It remains to be determined what are the mechanisms behind the million-fold difference in the viral load between CHIKV and SINV as both replicate readily to high titers in cell cultures and what is the role of the viral load for pathogenesis No infectious virus could be isolated from the real-time RTPCR positive sera in this study further demonstrating a very low viral load in serum. CHIKV has been successfully isolated only from serum samples that contained >107 RNA copies/ml (Panning et al., 2008), which is considerably more than levels of SINV RNA (<103 RNA copies/ml) approximated in this study. SINV has been isolated from a whole blood sample of an acutely ill patient once (Kurkela et al., 2004). Furthermore, despite frequent attempts made in our laboratory (unpublished data) and elsewhere (Hörling et al., 1993; Jupp et al., 1986), only one isolate has been recovered from human serum in China (Zhou et al., 1999). However, some doubts have been raised about the genuineness of this isolate due to its unexpected position in phylogenetic analyses and high sequence similarity with the widely used laboratory strain AR86 (Lundstrom and Pfeffer, 2010). 5. Conclusions The real-time RT-PCR assay described in this study can detect specifically and with high sensitivity SINV in cell culture supernatants. This assay is a sensitive molecular diagnostic tool for detecting SINV and may be used for purposes such as screening the virus in mosquitoes or from potential viral reservoirs. However, using molecular diagnostic techniques for serum samples does not seem to be useful for the diagnosis of human SINV infection due to short and low viraemia of SINV, resulting in low detection rates in clinical serum samples. Conflicts of interest None declared. Acknowledgements This investigation was funded by the Emil Aaltonen Foundation, the Helsinki Biomedical Graduate School, the Centre for
Military Medicine and the Academy of Finland. The skilful technical assistance of Irina Suomalainen and Kirsi Aaltonen is greatly appreciated. Ethical approval was obtained from the coordinating Ethics Committee of the Hospital District of Helsinki and Uusimaa (permission nr. 127/13/03/00/2009). References Assuncao-Miranda, I., Bozza, M.T., Da Poian, A.T., 2010. Pro-inflammatory response resulting from Sindbis virus infection of human macrophages: implications for the pathogenesis of viral arthritis. J. Med. Virol. 82, 164–174. Brummer-Korvenkontio, M., Vapalahti, O., Kuusisto, P., Saikku, P., Manni, T., Koskela, P., Nygren, T., Brummer-Korvenkontio, H., Vaheri, A., 2002. Epidemiology of Sindbis virus infections in Finland 1981–96: possible factors explaining a peculiar disease pattern. Epidemiol. Infect. 129, 335–345. Edwards, C.J., Welch, S.R., Chamberlain, J., Hewson, R., Tolley, H., Cane, P.A., Lloyd, G., 2007. Molecular diagnosis and analysis of Chikungunya virus. J. Clin. Virol. 39, 271–275. Finney, D.J., 1971. Probit Analysis, 3rd ed. Cambridge University Press, Cambridge. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids Symp. Ser. 41, 95–98. He, L.F., Xu, L.H., Cao, Y.X., Wang, L.H., Liu, W.B., Fu, S.H., Liang, G.D., 2005. Detection of Sindbis virus-specific nucleic acid with SYBR GREEN I real time PCR assay. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 19, 347–352. Hörling, J., Vene, S., Franzen, C., Niklasson, B., 1993. Detection of Ockelbo virus RNA in skin biopsies by polymerase chain reaction. J. Clin. Microbiol. 31, 2004–2009. Hubalek, Z., 2008. Mosquito-borne viruses in Europe. Parasitol. Res. 103 (Suppl. 1), S29–S43. Jost, H., Bialonski, A., Storch, V., Gunther, S., Becker, N., Schmidt-Chanasit, J., 2010. Isolation and phylogenetic analysis of Sindbis viruses from mosquitoes in Germany. J. Clin. Microbiol. 48, 1900–1903. Jupp, P.G., Blackburn, N.K., Thompson, D.L., Meenehan, G.M., 1986. Sindbis and West Nile virus infections in the Witwatersrand-Pretoria region. S. Afr. Med. J. 70, 218–220. Kurkela, S., Helve, T., Vaheri, A., Vapalahti, O., 2008. Arthritis and arthralgia three years after Sindbis virus infection: clinical follow-up of a cohort of 49 patients. Scand. J. Infect. Dis. 40, 167–173. Kurkela, S., Manni, T., Myllynen, J., Vaheri, A., Vapalahti, O., 2005. Clinical and laboratory manifestations of Sindbis virus infection: prospective study, Finland, 2002–2003. J. Infect. Dis. 191, 1820–1829. Kurkela, S., Manni, T., Vaheri, A., Vapalahti, O., 2004. Causative agent of Pogosta disease isolated from blood and skin lesions. Emerg. Infect. Dis. 10, 889–894. Lundstrom, J.O., Pfeffer, M., 2010. Phylogeographic structure and evolutionary history of Sindbis virus. Vector Borne Zoonotic Dis. 10, 889–907. L’vov, D.K., Skvortsova, T.M., Gromashevskii, V.L., Berezina, L.K., Iakovlev, V.I., 1985. Isolation of the causative agent of Karelian fever from Aedes sp. mosquitoes. Vopr. Virusol. 30, 311–313. Manni, T., Kurkela, S., Vaheri, A., Vapalahti, O., 2008. Diagnostics of Pogosta disease: antigenic properties and evaluation of Sindbis virus IgM and IgG enzyme immunoassays. Vector Borne Zoonotic Dis. 8, 303–311. Panning, M., Grywna, K., van Esbroeck, M., Emmerich, P., Drosten, C., 2008. Chikungunya fever in travelers returning to Europe from the Indian Ocean region, 2006. Emerg. Infect. Dis. 14, 416–422. Quetglas, J.I., Ruiz-Guillen, M., Aranda, A., Casales, E., Bezunartea, J., Smerdou, C., 2010. Alphavirus vectors for cancer therapy. Virus Res. 153, 179–196. Sane, J., Guedes, S., Kurkela, S., Lyytikainen, O., Vapalahti, O., 2010. Epidemiological analysis of mosquito-borne Pogosta disease in Finland, 2009. Euro Surveill. 15, 19462–19470. Sane, J., Guedes, S., Ollgren, J., Kurkela, S., Klemets, P., Vapalahti, O., Kela, E., Lyytikainen, O., Pekka Nuorti, J., 2011. Epidemic Sindbis virus infection in Finland: a population-based case–control study of risk factors. J. Infect. Dis. 204, 459–466. Skogh, M., Espmark, A., 1982. Ockelbo disease: epidemic arthritis-exanthema syndrome in Sweden caused by Sindbis-virus like agent. Lancet 1, 795–796. Turunen, M., Kuusisto, P., Uggeldahl, P.E., Toivanen, A., 1998. Pogosta disease: clinical observations during an outbreak in the province of North Karelia, Finland. Br. J. Rheumatol. 37, 1177–1180. Vähä-Koskela, M.J., Tuittila, M.T., Nygardas, P.T., Nyman, J.K., Ehrengruber, M.U., Renggli, M., Hinkkanen, A.E., 2003. A novel neurotropic expression vector based on the avirulent A7(74) strain of Semliki Forest virus. J. Neurovirol. 9, 1–15. Weaver, S.C., Frey, T.K., Huang, H.V., Kinney, R.M., et al., 2005. Togaviridae. In: Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L.A. (Eds.), Virus Taxonomy, Classification and Nomenclature of Viruses, 8th ICTV Report of the International Committee on Taxonomy of Viruses. Elsevier/Academic Press, New York. Zhou, G., Liang, G., Li, L., 1999. Complete nucleotide sequence of the nonstructural gene of alphavirus YN87448 strain isolated in China and its relationship to other Sindbis viruses. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 13, 314–320.
Contents lists available at SciVerse ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Development and evaluation of a real-time RT-PCR assay for Sindbis virus detection Jussi Sane a,∗ , Satu Kurkela a,b , Lev Levanov a , Simo Nikkari d , Antti Vaheri a,b , Olli Vapalahti a,b,c a
Infection Biology Research Program, Research Programs Unit, Department of Virology, Haartman Institute, Faculty of Medicine, University of Helsinki, Helsinki, Finland Department of Virology and Immunology, HUSLAB, Helsinki, Finland Division of Microbiology and Epidemiology, Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland d Centres for Military Medicine and for Biological Threat Preparedness, Helsinki, Finland b c
a b s t r a c t Article history: Received 21 September 2011 Received in revised form 11 October 2011 Accepted 27 October 2011 Available online 3 November 2011 Keywords: Sindbis virus Pogosta disease Detection Real-time RT-PCR Viral load
Sindbis virus (SINV) is an arthropod-borne alphavirus found widely in Eurasia, Africa and Oceania. Clinical SINV infection, characterized by rash and arthritis, is reported primarily in Northern Europe. The laboratory diagnosis of SINV infection is based currently on serology. A one-step TaqMan® real-time RT-PCR assay was developed for the detection of SINV and evaluated its clinical performance with acute-phase serum samples. The specificity and sensitivity of the real-time PCR assay were assessed using cell cultured Finnish SINV strains. The applicability of the assay for diagnostic use was evaluated using 58 serum samples from patients infected with SINV. The real-time RT-PCR assay was specific and sensitive for the detection of SINV in cell culture supernatants with a 95% detection limit of 9 genome copies/reaction determined by probit analysis. However, in the assay only 7/58 (12%) of serum samples were positive of which two were also positive by conventional nested PCR assay and none by virus isolation. This novel assay is specific and sensitive for detection of SINV and can be used for example for screening SINV in wildlife. However, molecular diagnostic techniques using serum samples seem to be of limited value for the diagnosis of human SINV infection due to the short and low viraemia of infection with SINV. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Arthropod-borne Sindbis virus (SINV) is a mosquito-borne enveloped single-stranded RNA virus of the genus Alphavirus, family Togaviridae (Weaver et al., 2005). SINV is found in Eurasia, Africa and Oceania (Hubalek, 2008) but clinical infections are reported almost exclusively from Northern Europe, particularly from Finland (Kurkela et al., 2004; Skogh and Espmark, 1982; L’vov et al., 1985). Acute SINV infection, named Pogosta disease in Finland, is characterized by fever, rash and arthritis (Kurkela et al., 2005; Turunen et al., 1998; Sane et al., 2011). Long-term joint manifestations can persist even for years (Kurkela et al., 2008). SINV epidemics have a peculiar cyclic appearance in Finland and during large outbreaks several hundreds of cases are serologically verified (Brummer-Korvenkontio et al., 2002; Sane et al., 2010). Population density fluctuations of grouse, which are considered probable natural hosts for the virus, and alterations in climatic and weather
Abbreviations: SINV, Sindbis virus; MGB, minor groove binder; Ct, threshold cycle; nsP, non-structural protein. ∗ Corresponding author at: University of Helsinki, Haartmaninkatu 3, P.O. Box 21, 00014 Helsinki, Finland. Tel.: +358 9 191 26608, fax: +358 9 191 26491. E-mail address: jussi.sane@helsinki.fi (J. Sane). 0166-0934/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2011.10.021
conditions, may be involved in the observed epidemiological pattern (Brummer-Korvenkontio et al., 2002). More recently, there is an increased awareness of SINV also in other parts of Europe, such as in Germany where SINV was for the first time isolated from mosquitoes in 2009 (Jost et al., 2010). SINV is also used as a vector in cancer therapy trials (Quetglas et al., 2010). The laboratory diagnosis of SINV is based currently on serology. Only approximately 40% of patients show IgM antibodies within the first week of illness (Kurkela et al., 2005). As patients usually seek medical care within the first days of illness (Sane et al., 2011), a negative antibody test result during the first week is common. Therefore, a second serum sample is frequently required to establish the diagnosis of acute SINV infection. Diagnostic methods based on real-time nucleic acid detection could enable a more prompt diagnosis. This would be especially useful during epidemics when a large number of samples are processed within a short time period. Thus far, a few conventional PCR (Kurkela et al., 2005; Hörling et al., 1993) and real-time RT-PCR (Jost et al., 2010; He et al., 2005) assays have been described for SINV detection but assays for the diagnosis of human SINV infection have not been described previously or their clinical performance and validity evaluated. The aim of this study was to develop a one step real-time RTPCR assay for the detection of SINV using TaqMan® chemistry and to evaluate its clinical performance with acute-phase serum samples.
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2. Material and methods 2.1. Patient samples Fifty-eight first serum samples from Pogosta disease patients from whom sera was taken during the acute phase of SINV infection were available for this study. The samples were obtained from the Department of Virology and Immunology of the Helsinki University Hospital Laboratory. A seroconversion between paired sera and/or a positive IgM test result in a single serum, using a highly specific (95.2% and 97.6% for IgM and IgG, respectively) and sensitive (97.6% and 100% for IgM and IgG, respectively) enzyme inmmunoassay (EIA) (Manni et al., 2008) was required for the diagnosis of acute SINV infection. All the 58 patients fulfilled the criteria for serological diagnosis of SINV infection. The sera were collected during the epidemic years 2002 (n = 24) and 2009 (n = 34). Fifty samples were IgM and IgG negative; 8 samples were IgM positive or had a borderline IgM test result. The sera were aliquoted and stored at −70 ◦ C prior to RNA extraction. 2.2. Viral RNA extraction RNA was extracted from 140 l of serum or viral culture supernatant using QiAmp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. 2.3. Primer and probe design A one-step real-time RT-PCR based on TaqMan® chemistry was developed for the detection of SINV RNA. Conserved regions of SINV were identified using an alignment of complete protein-coding area of Finnish SINV strains isolated previously in our laboratory and further sequenced for this study (part of the genome published earlier (Kurkela et al., 2004), as well as the complete SINV genomes accessible in GenBank (Fig. 1). Sequence alignment was performed with Bioedit Sequence Alignment editor (Hall, 1999). The primers and the probe were designed with Primer Express Software version 3.0 (Applied Biosystems, Foster City, USA) and selected within the non-structural protein nsP1 region (Fig. 1). 2.4. Generation of SINV RNA transcript The target region (74 bp) was amplified from Ilomantsi-2002C strain and cloned into pGEM® -T cloning vector (Promega, Madison,
USA). The presence of the insert was verified by sequencing and restriction enzyme analysis. After linearization of the plasmid by digestion with BsaI, RNA was generated using RiboMAXTM Large Scale RNA production system with SP6 polymerase (Promega, Madison, USA) according to the manufacturer’s instructions. The transcribed RNA was then treated with DNAse and purified with RNeasy Mini Kit (QIAGEN, Hilden, Germany). Finally, RNA was quantified by spectrophotometry and copy number of RNA was calculated based on its concentration, length, and the molecular weight. 2.5. One-step real-time RT-PCR The real-time RT-PCR was performed with Quantitect One Step Probe RT-PCR Kit (QIAGEN, Hilden, Germany) in a final volume of 25 l containing 5 l of template RNA, 400 nM of primers and 250 nM of probe. The TaqMan® probe (Applied Biosystems, Foster City, USA) was labeled at the 5 -end with reporter 6carboxyfluorescein (FAM) and at the 3 -end with minor groove binder (MGB)-non-fluorescent quencher (NFQ). The assay was carried out using ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, USA) with the following cycling parameters: 50 ◦ C for 30 min, 95 ◦ C for 15 min, 45 cycles of 95 ◦ C for 15 s and 60 ◦ C for 1 min. Alternative primers and a probe labeled with quencher tetramethyl-6-carboxyrhodamine (TAMRA) were also designed within the E1-protein region (nucleotide position according to SINV Ockelbo strain; forward primer 5 -CACCCCGCACAAAAATGAC3 at position 11258-11276, probe at position 11284-11309 and reverse primer 5 -AAAAGGGCAAACAGCCAACTC-3 at position 11312-11332) and a subset of samples was analysed with this test format using the same assay parameters as described above. 2.6. Conventional nested RT-PCR and virus isolation Serum samples with a positive test result in the SINV real-time RT-PCR were additionally analysed with a previously described conventional nested RT-PCR assay that has been able to detect SINV in whole blood samples and skin biopsies taken from patients with SINV infection (Kurkela et al., 2005). Furthermore, virus isolation was attempted from these samples on Vero cells using a modification of a previously described protocol that has yielded SINV isolates from whole blood and skin biopsies (Kurkela et al., 2005).
Fig. 1. Alignment of the primer/probe binding regions in nsP1-protein (5 to 3 direction) of different SINV strains and Whataroa virus, SFV and CHIKV. Conserved nucleotides are shown with dots.
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In brief, 50 l of serum was added to cells in 25-cm2 flasks. After a 1-h incubation, fresh culture medium was added, and the cells were observed for cytopathic effect. 2.7. Evaluation of the one-step real-time PCR assay The assay specificity was evaluated with viral RNA derived from supernatants of five cell cultured Finnish SINV strains previously isolated from humans (Kurkela et al., 2004). In addition, RNA from cell cultured Semliki Forest (SFV, expression vector VA7) (Vähä-Koskela et al., 2003) and Chikungunya (CHIKV, Ross strain) alphaviruses, tick-borne encephalitis flavivirus (TBEV, strain Kumlinge A52), and Puumala (PUUV) hantavirus-positive spleen and liver tissue from a PUUV-infected patient were tested. First serum samples (n = 24) from patients suspected with SINV infection but confirmed SINV-antibody negative in paired samples were also analysed to further evaluate the specificity. The precise analytical sensitivity was assessed by 10-fold dilutions of in vitro transcribed SINV RNA (ranging from 1.42 to 1.42 × 107 copies per reaction) in 8 replicates at each concentration. The limit of detection was calculated with probit regression model (Finney, 1971) with 95% probability endpoint. The probit analysis was performed with SPSS software (version 18, USA). Sensitivity was also assessed in plaque forming units (PFU) by determining the lowest concentration at which positive signal is obtained. Plaque titration assay for Ilomantsi-2002A virus strain was performed. In brief, serial dilutions of virus were added to confluent Vero E6 cells in cell culture plate. After an incubation of 1 hour at 37 ◦ C, agarose (0.5%) overlay medium was added to cells and the plates were incubated for 4 days. Cells were then stained with crystal violet and the plaques were counted. Virus titer was 9.0 × 106 PFU/ml. RNA was extracted from 10-fold dilutions of this virus stock and analysed by the real-time RT-PCR assay.
Fig. 2. Probit regression analysis of SINV real-time RT-PCR. The analysis was based on serial dilutions of in vitro transcribed SINV RNA in 8 replicates at each concentration. The probability of positive result is plotted against the concentration of SINV RNA. The 95% detection limit is indicated with the dashed lines.
positive) and this format was not pursued further for additional samples. Conventional nested PCR was able to detect SINV RNA in 2/7 of those positive with the real-time RT-PCR. These samples originated from patients whose skin biopsies were previously found positive with conventional nested PCR and from whom SINV strains (Ilomantsi-2002A and Johannes) were isolated (Kurkela et al., 2004). No infectious virus could be recovered from any of the seven serum samples by virus isolation in Vero cells.
3. Results
4. Discussion
The real-time RT-PCR assay detected viral RNA of all five Finnish SINV strains in cell culture supernatants with similar threshold cycle (Ct) values (approximately 16 in dilution of 10−2 ). The limit of detection determined by probit analysis (concentration giving a positive real-time RT-PCR result in 95% of samples) was 9 copies/reaction (Fig. 2). End-point dilution showed that the lowest template concentration that gave positive signal was 1.4 copies/reaction (120 copies/ml) and using plaque titrated virus stock, 0.001 PFU/reaction (0.09 PFU/ml). Cell culture supernatants of SFV, CHIKV and TBEV, as well as PUUV-infected tissue samples were subjected to the assay in order to evaluate the specificity, and none gave false-positive signals. In addition, all the SINVseronegative (n = 24) sera were non-reactive in the test. Of the acute-phase serum samples from patients infected with SINV, 7/58 (12%) were positive in the real-time RT-PCR assay. All the SINV RNA positive samples were negative for SINV IgM and IgG, although, according to the inclusion criteria of the study, these patients had seroconverted to SINV by the time of the second samples. In 4/7 (57%) of the samples only one of the duplicates was positive in repeated runs. Ct values for positive samples were high ranging from 35 to 37.3 and the viral load of the samples approximated from the in vitro RNA standard dilutions was in the range of 130–660 viral copies/ml of serum. The use of higher primer concentration (up to 900 nM) or longer reverse transcription time (45 min) did not improve the detection sensitivity. The positive samples were also tested with primers and TAMRAlabeled probe designed within the E1 protein. However, poor sensitivity was observed with this test format (3/7 samples were
A one-step real-time RT-PCR assay was developed for the detection of SINV and its applicability as a diagnostic tool for human SINV infection was evaluated. This would be particularly valuable as the first samples of patients infected with SINV are usually IgM and IgG negative. Earlier studies suggest a narrow viraemic window and low level of viraemia in acute SINV infection (Kurkela et al., 2004; Manni et al., 2008), unlike for the related, but more severe CHIKV infection where high viral loads, up to 109 copies/mL of plasma, have been reported [Panning et al., 2008]. The current data support this finding, and as only 12% of the serum samples were positive with high Ct values in the assay corresponding to levels of less than 103 SINV copies/ml of sample, we suggest that real-time RT-PCR from serum samples is not a practical approach for the laboratory diagnosis of SINV. Furthermore, for several positive samples only one of the duplicates was positive. This indicates a very low viral load and uneven Poisson distribution of template virus in the replicates. The possibility for contamination was minimized by routine measures, and negative controls were not amplified in any of the runs. We acknowledge that by increasing the extraction volume and concentrating the sample, the sensitivity of the assay may have improved resulting in increased proportion of positive samples. However, the RNA extraction kit (QIAGEN, Hilden, Germany) is optimized for the volume of 140 l and this has been sufficient for real-time RT-PCR assays used in routine diagnosis of viral diseases such as CHIK fever, where viral RNA is regularly found during the first week of illness (Edwards et al., 2007; Panning et al., 2008). A protocol requiring substantially larger volumes would probably diminish the applicability of the assay for clinical purposes as
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additional time consuming steps would be required. Also, only a restricted sample volume is often available for a single diagnostic test and only this amount was available to us properly stored (−70 ◦ C) to ensure preservation of RNA. The assay was able to detect all available Finnish SINV strains from cell culture supernatants without any cross-reactivity to other viruses tested. No unspecific amplification in sera negative for SINV was observed. Due to high sequence similarity in the primer binding region of the nsP1 protein, the assay can potentially detect all SINV strains of which sequence is available in the Genbank. The observation on the low viral load in serum during acute infection may have implications as to the pathogenesis of SINV infection. Evidence suggests that SINV can readily infect human macrophages, and macrophage-derived pro-inflammatory response contributes to the pathogenesis of arthritis (AssuncaoMiranda et al., 2010). It is possible that after inoculation, SINV targets cells circulating in blood such as monocytes that further disseminate virus to different tissues (e.g. skin, joints, muscle), while the presence of SINV in serum remains short-lasting. In a previous study, SINV RNA was detected in 5 of 73 acute-phase whole blood samples with nested RT-PCR (Kurkela et al., 2005). Our preliminary data show that the real-time RT-PCR assay can detect SINV RNA in whole blood but due to lack of proper sample panel, comprehensive comparative analysis of the whole blood and serum samples cannot be undertaken at this time. It remains to be determined what are the mechanisms behind the million-fold difference in the viral load between CHIKV and SINV as both replicate readily to high titers in cell cultures and what is the role of the viral load for pathogenesis No infectious virus could be isolated from the real-time RTPCR positive sera in this study further demonstrating a very low viral load in serum. CHIKV has been successfully isolated only from serum samples that contained >107 RNA copies/ml (Panning et al., 2008), which is considerably more than levels of SINV RNA (<103 RNA copies/ml) approximated in this study. SINV has been isolated from a whole blood sample of an acutely ill patient once (Kurkela et al., 2004). Furthermore, despite frequent attempts made in our laboratory (unpublished data) and elsewhere (Hörling et al., 1993; Jupp et al., 1986), only one isolate has been recovered from human serum in China (Zhou et al., 1999). However, some doubts have been raised about the genuineness of this isolate due to its unexpected position in phylogenetic analyses and high sequence similarity with the widely used laboratory strain AR86 (Lundstrom and Pfeffer, 2010). 5. Conclusions The real-time RT-PCR assay described in this study can detect specifically and with high sensitivity SINV in cell culture supernatants. This assay is a sensitive molecular diagnostic tool for detecting SINV and may be used for purposes such as screening the virus in mosquitoes or from potential viral reservoirs. However, using molecular diagnostic techniques for serum samples does not seem to be useful for the diagnosis of human SINV infection due to short and low viraemia of SINV, resulting in low detection rates in clinical serum samples. Conflicts of interest None declared. Acknowledgements This investigation was funded by the Emil Aaltonen Foundation, the Helsinki Biomedical Graduate School, the Centre for
Military Medicine and the Academy of Finland. The skilful technical assistance of Irina Suomalainen and Kirsi Aaltonen is greatly appreciated. Ethical approval was obtained from the coordinating Ethics Committee of the Hospital District of Helsinki and Uusimaa (permission nr. 127/13/03/00/2009). References Assuncao-Miranda, I., Bozza, M.T., Da Poian, A.T., 2010. Pro-inflammatory response resulting from Sindbis virus infection of human macrophages: implications for the pathogenesis of viral arthritis. J. Med. Virol. 82, 164–174. Brummer-Korvenkontio, M., Vapalahti, O., Kuusisto, P., Saikku, P., Manni, T., Koskela, P., Nygren, T., Brummer-Korvenkontio, H., Vaheri, A., 2002. Epidemiology of Sindbis virus infections in Finland 1981–96: possible factors explaining a peculiar disease pattern. Epidemiol. Infect. 129, 335–345. Edwards, C.J., Welch, S.R., Chamberlain, J., Hewson, R., Tolley, H., Cane, P.A., Lloyd, G., 2007. Molecular diagnosis and analysis of Chikungunya virus. J. Clin. 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