sybr-green-i and Hemorrhagic-Fever-with-Renal-Syndrome

Hemorrhagic fever with renal syndrome (HFRS) is a severe, viral zoonotic disease which occurs worldwide, particularly in Asia and Europe. In China, the Hantaan virus (HTNV) and the Seoul virus (SEOV) are known to be the most prevalent causative agents of HFRS. Since no protective vaccines or effective treatments are available for human use, accurate and reliable diagnostic methods are essential for disease surveillance. In the present study, the viral loads in cell culture supernatant, infected mice blood and clinical serum samples were quantified using the SYBR‑Green I-based reverse transcription-quantitiative polymerase chain reaction (RT-qPCR) assay, which targeted the S gene sequence of the HTNV and SEOV genomes. The cRNA of these two viruses were synthesized as a positive control and 10-fold serially diluted from 1x105 to 1x100 copies/µl. Standard curves were generated by plotting the mean cycle threshold (Ct) values versus copy numbers. The standard curve of HTNV had a correlation coefficient (R2) of 0.994, efficiency of amplification (E) of 101.9%, and the slope of -3.278, whereas that of SEOV had an R2 of 0.993, E of 104.8%, and the slope of -3.212. The minimum detection limit of the RT-qPCR assay for HTNV and SEOV was 101 copies/µl. Two qPCR assays were successfully established for the detection of HTNV and SEOV, respectively. Taken together, these findings demonstrate that using the SYBR‑Green I-based RT-qPCR assay, the HTNV and SEOV may be genotyped precisely without cross-reactivity. Furthermore, viral RNA may be detected and quantified in cells, mice and infected individuals, which may be useful in epidemiological studies as well as for early monitoring and further preventative treatment against SEOV and HTNV-induced diseases.

Topics: Animals; Benzothiazoles; Diamines; Gene Dosage; Genome, Viral; Genotype; Hantaan virus; Hemorrhagic Fever with Renal Syndrome; Host-Pathogen Interactions; Humans; Mice; Organic Chemicals; Quinolines; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; RNA, Complementary; RNA, Viral; Sensitivity and Specificity; Seoul virus; Species Specificity

Hantaan virus (HTNV), which belongs to the genus Hantavirus, causes hemorrhagic fever with renal syndrome (HFRS) mainly in China. The diagnosis of HFRS depends on clinical manifestations and serological tests. A SYBR Green I based one-step real-time PCR assay was established in this study to detect HTNV. The HTNV standard curves were generated by plotting mean cycle threshold (Ct) values versus 10-fold serial dilutions of a previous titrated HTNV stock over a wide range of concentrations (1×10(7) to 1PFU/ml). The minimum detection limit of the assay was 1PFU/ml, and it was 100-fold more sensitive than conventional RT-PCR. Melting curve analysis indicated that there were no primer-dimers and non-specific products in the assay. No cross-reaction was observed with Seoul virus (SEOV). The specificity of the asssay was also verified by nuleotide sequencing of the PCR products. Intra- and inter-assay variability data were analyzed to examine the reproducibility of the assay. HTNV viral loads in HFRS patients were also investigated with the assay. These results indicated that the one-step real-time PCR assay is useful for detecting HTNV and for monitoring the viral loads.

Topics: Benzothiazoles; China; Diamines; Hantaan virus; Hemorrhagic Fever with Renal Syndrome; Humans; Molecular Diagnostic Techniques; Organic Chemicals; Quinolines; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Sensitivity and Specificity; Staining and Labeling; Virology

Dobrava hantavirus (DOBV) infection was diagnosed in a previously healthy 46-y-old hunter suffering from severe haemorrhagic fever with renal syndrome (HFRS). Specific IgM antibodies against DOBV were identified by an immunofluorescence assay, while viral nucleic acid was detected by the molecular method, confirming the diagnosis. Our results reveal an existing risk of DOBV transmission to humans in Hungary.

Topics: Antibodies, Viral; Benzothiazoles; Diamines; Fluorescent Dyes; Hantavirus Infections; Hemorrhagic Fever with Renal Syndrome; Humans; Immunoglobulin M; Male; Middle Aged; Organic Chemicals; Orthohantavirus; Phylogeny; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral