Development and evaluation of a real-time RT-PCR assay for Sindbis virus detection. J Virol Methods

Infection Biology Research Program, Research Programs Unit, Department of Virology, Haartman Institute, Faculty of Medicine, University of Helsinki, Helsinki, Finland.
Journal of virological methods (Impact Factor: 1.78). 11/2011; 179(1):185-8. DOI: 10.1016/j.jviromet.2011.10.021
Source: PubMed


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.

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    • "The major symptoms are joint inflammation, rash, fever, fatigue , and headache (Kurkela et al. 2005, Sane et al. 2012a). The joint symptoms may persist for months or even years (Skogh and Espmark 1982, Niklasson and Espmark 1986, Niklasson et al. 1988, Kurkela et al. 2008). "
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    ABSTRACT: Mosquito-borne alphaviruses have the potential to cause large outbreaks throughout the world. Here we investigated the causative agent of an unexpected Sindbis virus (SINV) outbreak during August-September, 2013, in a previously nonendemic region of Sweden. Mosquitoes were collected using carbon dioxide-baited CDC traps at locations close to human cases. The mosquitoes were initially screened as large pools by SINV-specific quantitative RT-PCR, and the SINV-positive mosquitoes were species determined by single-nucleotide polymorphism (SNP) analysis, followed by sequencing the barcoding region of the cytochrome oxidase I gene. The proportion of the collected mosquitoes was determined by a metabarcoding strategy. By using novel strategies for PCR screening and genetic typing, a new SINV strain, Lövånger, was isolated from a pool of 1600 mosquitoes composed of Culex, Culiseta, and Aedes mosquitoes as determined by metabarcoding. The SINV-positive mosquito Culiseta morsitans was identified by SNP analysis and sequencing. After whole-genome sequencing and phylogenetic analysis, the SINV Lövånger isolate was shown to be most closely similar to recent Finnish SINV isolates. In conclusion, within a few weeks, we were able to detect and isolate a novel SINV strain and identify the mosquito vector during a sudden SINV outbreak.
    Full-text · Article · Feb 2015 · Vector borne and zoonotic diseases (Larchmont, N.Y.)
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    ABSTRACT: Traditional tick taxonomy based on morphological characters is recently challenged by data generated from DNA analysis and several revisions in tick families have been proposed accordingly. Thus, names of some tick genera and their taxonomic positions have changed, species moved from one rank to another, while other names were invalidated. In this chapter, we update the genus Hyalomma species names as compiled from recent re-descriptions of species and tick reviews up to year 2011. Hyalomma species are known vectors of large numbers of parasites and pathogens transmitted to humans and livestock in different parts of the world making these ticks the economically most important ixodids.
    No preview · Chapter · Jan 2012
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    ABSTRACT: Sindbis virus (SINV) is a mosquito-borne alphavirus found in Eurasia, Africa, and Oceania. Clinical SINV infection is characterized by febrile rash and arthritis and sometimes prolonged arthralgia and myalgia. The pathophysiological mechanisms of musculoskeletal and rheumatic disease caused by SINV are inadequately understood. We studied the muscle pathology of SINV infection ex vivo by examining a unique muscle biopsy obtained from a patient with chronic myalgia and arthralgia 6 months after acute SINV infection and assessed potential genetic predisposing factors by determining the human leukocyte antigen (HLA) and complement factor C4 genes and proteins. In addition, we performed in vitro SINV infections of primary human myoblasts and myotubes. In the muscle biopsy we found evidence of muscle regeneration due to previous necrotic lesions likely caused by earlier SINV infection. We showed that human myoblasts and myotubes were susceptible in vitro for SINV infection as the cells became immunoreactive for viral antigens and cytopathic effect was observed. The patient was homozygous for HLA-B*35 alleles and heterozygous for HLA-DRB1*01 and HLA-DRB1*03 alleles and had total deficiency of C4B protein. This study provides new insights concerning pathological processes leading to chronic symptoms in SINV infection and demonstrates for the first time the susceptibility of human myogenic cells to SINV infection.
    Full-text · Article · May 2012 · The Journal of Infectious Diseases
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