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Detection and differentiation of Schmallenberg, Akabane and Aino viruses by one-step multiplex reverse-transcriptase quantitative PCR assay.

ABSTRACT: Schmallenberg virus (SBV), Akabane virus (AKAV) and Aino virus (AINV) are members of the Simbu serogroup within the genus Orthobunyavirus, family Bunyaviridae, which can cause reproductive disorders including abortion, stillbirth and congenital malformation in ruminants. Because, the clinical signs are similar, confirmatory diagnosis requires viral detection to differentiate infection between these three viruses.In this study, a one-step multiplex reverse-transcriptase quantitative PCR (one-step mRT-qPCR) was developed for the simultaneous detection and differentiation of SBV, AKAV and AINV.The detection limit of the one-step mRT-qPCR for SBV, AKAV and AINV were 2.4 copies (10 (0.6) TCID 50/ml), 96.2 copies (10 (1.5) TCID 50/ml) and 52.3 copies (10 (1.2) TCID 50/ml), respectively. Various field samples such as bovine serum, bovine whole blood, bovine brain, goat serum and Culicoides were analyzed using the one-step mRT-qPCR and compared with previously published RT-qPCRs. The test results of the field samples were identical for the one-step mRT-qPCR and RT-qPCRs, which showed all samples to be negative for SBV, AKAV and AINV, except for one bovine brain sample (1/123) that was positive for AKAV.The one-step mRT-qPCR allows for the simultaneous detection of three viral pathogens (SBV, AKAV and AINV) that cause reproductive failure.

SUBMITTER: Lee JH

PROVIDER: S-EPMC4619357 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Detection and differentiation of Schmallenberg, Akabane and Aino viruses by one-step multiplex reverse-transcriptase quantitative PCR assay.

Lee Ji-Hye JH Seo Hyun-Ji HJ Park Jee-Yong JY Kim Sung-Hee SH Cho Yun Sang YS Kim Yong-Joo YJ Cho In-Soo IS Jeoung Hye-Young HY

BMC veterinary research 20151024

<h4>Background</h4>Schmallenberg virus (SBV), Akabane virus (AKAV) and Aino virus (AINV) are members of the Simbu serogroup within the genus Orthobunyavirus, family Bunyaviridae, which can cause reproductive disorders including abortion, stillbirth and congenital malformation in ruminants. Because, the clinical signs are similar, confirmatory diagnosis requires viral detection to differentiate infection between these three viruses.<h4>Methods</h4>In this study, a one-step multiplex reverse-trans ...[more]

PMID: 26497392

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Project description:BackgroundChandipura virus (CHPV), a member of family Rhabdoviridae was attributed to an explosive outbreak of acute encephalitis in children in Andhra Pradesh, India in 2003 and a small outbreak among tribal children from Gujarat, Western India in 2004. The case-fatality rate ranged from 55-75%. Considering the rapid progression of the disease and high mortality, a highly sensitive method for quantifying CHPV RNA by real-time one step reverse transcriptase PCR (real-time one step RT-PCR) using TaqMan technology was developed for rapid diagnosis.MethodsPrimers and probe for P gene were designed and used to standardize real-time one step RT-PCR assay for CHPV RNA quantitation. Standard RNA was prepared by PCR amplification, TA cloning and run off transcription. The optimized real-time one step RT-PCR assay was compared with the diagnostic nested RT-PCR and different virus isolation systems [in vivo (mice) in ovo (eggs), in vitro (Vero E6, PS, RD and Sand fly cell line)] for the detection of CHPV. Sensitivity and specificity of real-time one step RT-PCR assay was evaluated with diagnostic nested RT-PCR, which is considered as a gold standard.ResultsReal-time one step RT-PCR was optimized using in vitro transcribed (IVT) RNA. Standard curve showed linear relationship for wide range of 102-1010 (r2 = 0.99) with maximum Coefficient of variation (CV = 5.91%) for IVT RNA. The newly developed real-time RT-PCR was at par with nested RT-PCR in sensitivity and superior to cell lines and other living systems (embryonated eggs and infant mice) used for the isolation of the virus. Detection limit of real-time one step RT-PCR and nested RT-PCR was found to be 1.2 x 100 PFU/ml. RD cells, sand fly cells, infant mice, and embryonated eggs showed almost equal sensitivity (1.2 x 102 PFU/ml). Vero and PS cell-lines (1.2 x 103 PFU/ml) were least sensitive to CHPV infection. Specificity of the assay was found to be 100% when RNA from other viruses or healthy individual was used.ConclusionOn account of the high sensitivity, reproducibility and specificity, the assay can be used for the rapid detection and quantitation of CHPV RNA from clinical samples during epidemics and from endemic areas. The assay may also find application in screening of antiviral compounds, understanding of pathogenesis as well as evaluation of vaccine.

Dataset Information

Detection and differentiation of Schmallenberg, Akabane and Aino viruses by one-step multiplex reverse-transcriptase quantitative PCR assay.

Publications

Detection and differentiation of Schmallenberg, Akabane and Aino viruses by one-step multiplex reverse-transcriptase quantitative PCR assay.

Similar Datasets

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