sybr-green-i and Hantavirus-Infections

Hantaviruses are members of the family Bunyaviridae and are an emerging cause of disease worldwide with high lethality in the Americas. In Brazil, the diagnosis for hantaviruses is based on immunologic techniques associated with conventional RT-PCR. A novel one-step SYBR Green real-time RT-PCR was developed for the detection and quantitation of Araraquara (ARAV) and Rio Mamore hantavirus (RIOMV). The detection limit of assay was 10 copies/μL of RNA in vitro transcribed of segment S. The specificity of assay was evaluated by melting curve analysis, which showed that the Araraquara virus amplified product generated a melt peak at 80.83 ± 0.89 °C without generating primer-dimers or non-specific products. The assay was more sensitive than conventional RT-PCR and we detected two samples undetected by conventional RT-PCR. The one-step SYBR Green real-time quantitative RT-PCR is specific, sensible and reproducible, which makes it a powerful tool in both diagnostic applications and general research of ARAV and RIOMV and possibly other Brazilian hantaviruses.

Topics: Benzothiazoles; Brazil; Diamines; DNA Primers; DNA, Viral; Fluorescent Dyes; Hantavirus Infections; Humans; Organic Chemicals; Orthohantavirus; Quinolines; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity

Dobrava hantavirus (DOBV) belongs to the genus Hantavirus of the family Bunyaviridae, and is carried by yellow necked and striped field mice (Apodemus flavicollis and Apodemus agrarius), respectively. The aim of this study was to detect and genetically characterize new DOBV strains in rodents captured in the Transdanubian region of Hungary. Rodent corpses were dissected and lung tissues were used for hantavirus detection by SYBR Green-based real-time RT-PCR using specific primers located in the S-segment of the virus genome. A total of 22 captured animals of the Apodemus species were tested for the presence of DOBV. Three out of the 22 mice were positive. Phylogenetic and molecular sequence analyses showed that Hungarian DOBVs were most closely related to those viruses detected from A. agrarius mice in Slovenia. Based on our new data from the region we concluded that extended reservoir studies would be necessary in the future.

Topics: Animals; Benzothiazoles; Diamines; DNA Primers; Hantavirus Infections; Hungary; Molecular Sequence Data; Murinae; Organic Chemicals; Orthohantavirus; Phylogeny; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; Rodent Diseases; Sequence Analysis, DNA

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