Diagnostics-in-a-Suitcase: Development of a portable and rapid assay for the detection of the emerging avian influenza A (H7N9) virus

Background: In developing countries, the necessary equipment for the diagnosis is only available in few central laboratories, which are less accessible and of limited capacity to test large numbers of incoming samples. Moreover, transport conditions of samples are inadequate and therefore lead to unreliable results. Objectives: The development of rapid, inexpensive, and simple test would allow mobile detection of viruses. Study Design: A suitcase laboratory “Diagnostics-in-a-Suitcase” (56 × 45.5 × 26.5 cm) containing all necessary reagents and devices to perform reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed. As an example, Two RT-RPA assays for the detection of hemagglutinin (H) and neuraminidase (N) genes of the novel avian influenza (H7N9) virus were established. Results: Sensitivities were 10 and 100 \{RNA\} molecules for the \{H7\} and the \{N9\} RT-RPA assays, respectively. Assays were performed at a single temperature (42 °C). Results were obtained within 2-7 minutes. The \{H7N9\} RT-RPA assays showed neither a cross-detection of any other respiratory viruses affecting humans and/or birds nor of the human or chicken genomes. All reagents were used, stored, and transported at ambient temperature, i.e. cold chain independent. In addition, the Diagnostics-in-a-Suitcase was operated by a solar power battery. Conclusions: The developed assay protocol and mobile setup performed well. Moreover, it can be easily implemented to perform diagnosis at airport, quarantine stations, or farms for rapid on-site viral nucleic acid detection

Reverse transcription recombinase polymerase amplification assay for the detection of middle East respiratory syndrome coronavirus

The emergence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in the eastern Mediterranean and imported cases to Europe has alerted public health authorities. Currently, detection of MERS-CoV in patient samples is done by real-time RT-PCR. Samples collected from suspected cases are sent to highly-equipped centralized laboratories for screening. A rapid point-of-care test is needed to allow more widespread mobile detection of the virus directly from patient material. In this study, we describe the development of a reverse transcription isothermal Recombinase Polymerase Amplification (RT-RPA) assay for the identification of MERS-CoV. A partial nucleocapsid gene RNA molecular standard of MERS-coronavirus was used to determine the assay sensitivity. The isothermal (42°C) MERS-CoV RT-RPA was as sensitive as real-time RT-PCR (10 RNA molecules), rapid (3-7 minutes) and mobile (using tubescanner weighing 1kg). The MERS-CoV RT-RPA showed cross-detection neither of any of the RNAs of several coronaviruses and respiratory viruses affecting humans nor of the human genome. The developed isothermal real-time RT-RPA is ideal for rapid mobile molecular MERS-CoV monitoring in acute patients and may also facilitate the search for the animal reservoir of MERS-CoV

Recombinase polymerase amplification assay for rapid detection of lumpy skin disease virus

Background  Lumpy skin disease virus (LSDV) is aCapripoxvirusinfecting cattle and Buffalos. Lumpy skin disease (LSD) leads to significant economic losses due to hide damage, reduction of milk production, mastitis, infertility and mortalities (10 %). Early detection of the virus is crucial to start appropriate outbreak control measures. Veterinarians rely on the presence of the characteristic clinical signs of LSD. Laboratory diagnostics including virus isolation, sequencing and real-time polymerase chain reaction (PCR) are performed at well-equipped laboratories. In this study, a portable, simple, and rapid recombinase polymerase amplification (RPA) assay for the detection of LSDV-genome for the use on farms was developed.  Results  The LSDV RPA assay was performed at 42 °C and detected down to 179 DNA copies/reaction in a maximum of 15 min. Unspecific amplification was observed with neither LSDV-negative samples (n= 12) nor nucleic acid preparations from orf virus, bovine papular stomatitis virus, cowpoxvirus, Peste des petits ruminants and Blue tongue virus (serotypes 1, 6 and 8). The clinical sensitivity of the LSDV RPA assay matched 100 % (n= 22) to real-time PCR results. In addition, the LSDV RPA assay detected sheep and goat poxviruses.  Conclusion  The LSDV RPA assay is a rapid and sensitive test that could be implemented in field or at quarantine stations for the identification of LSDV infected case

Development of mobile laboratory for viral hemorrhagic fever detection in Africa

Background In order to enable local response to viral haemorrhagic fever outbreaks a mobile laboratory transportable on commercial flights was developed. Methodology The development progressed from use of mobile real time RT-PCR to mobile Recombinase Polymerase Amplification (RT-RPA). The various stages of the mobile laboratory development are described. Results A brief overview of its deployments, which culminated in the first on site detection of Ebola virus disease (EVD) in March 2014 and a successful use in a campaign to roll back EVD cases in Conakry in the West-Africa Ebola virus outbreak are described. Conclusion The developed mobile laboratory successfully enabled local teams to perform rapid viral haemorrhagic fever disgnostics

Full-length genome sequence of Ntaya virus

Presentation of pyrosequencing data and phylogenetic analysis for the full genome of Ntaya virus, type virus of the Ntaya virus group of the Flaviviridae isolated in Cameroon in 1966

A field-deployable reverse transcription recombinase polymerase amplification assay for rapid detection of the Chikungunya virus

Background  Chikungunya virus (CHIKV) is a mosquito-borne virus currently transmitted in about 60 countries. CHIKV causes acute flu-like symptoms and in many cases prolonged musculoskeletal and joint pain. Detection of the infection is mostly done using RT-RCR or ELISA, which are not suitable for point-of-care diagnosis.  Methodology/Principal Findings  In this study, a reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of the CHIKV was developed. The assay sensitivity, specificity, and cross-reactivity were tested. CHIKV RT-RPA assay detected down to 80 genome copies/reaction in a maximum of 15 minutes. It successfully identified 18 isolates representing the three CHIKV genotypes. No cross-reactivity was detected to other alphaviruses and arboviruses except O'nyong'nyong virus, which could be differentiated by a modified RPA primer pair. Seventy-eight samples were screened both by RT-RPA and real-time RT-PCR. The diagnostic sensitivity and specificity of the CHIKV RT-RPA assay were determined at 100%.  Conclusions/Significance   The developed RT-RPA assay represents a promising method for the molecular detection of CHIKV at point of need

Recombinase polymerase amplification assay for rapid detection of Monkeypox virus

In this study, a rapid method for the detection of Central and West Africa clades of Monkeypox virus (MPXV) using recombinase polymerase amplification (RPA) assay targeting the G2R gene was developed. MPXV, an Orthopoxvirus, is a zoonotic dsDNA virus, which is listed as a biothreat agent. RPA was operated at a single constant temperature of 42°C and produced results within 3 to 10 minutes. The MPXV-RPA-assay was highly sensitive with a limit of detection of 16 DNA molecules/μl. The clinical performance of the MPXV-RPA-assay was tested using 47 sera and whole blood samples from humans collected during the recent MPXV outbreak in Nigeria as well as 48 plasma samples from monkeys some of which were experimentally infected with MPXV. The specificity of the MPXV-RPA-assay was 100% (50/50), while the sensitivity was 95% (43/45). This new MPXV-RPA-assay is fast and can be easily utilised at low resource settings using a solar powered mobile suitcase laboratory

Biosafety standards for working with Crimean-Congo haemorrhagic fever virus

In countries from which Crimean-Congo haemorrhagic fever (CCHF) is absent, the causative virus CCHF virus (CCHFV) is classified as a hazard group 4 agent and handled in containment level 4. In contrast, most endemic countries out of necessity have had to perform diagnostic tests under biosafety level (BSL) -2 or -3 conditions. In particular, Turkey and several of the Balkan countries have safely processed more than 100000 samples over many years in BSL-2 laboratories. It is therefore advocated that biosafety requirements for CCHF diagnostic procedures should be revised, to allow the required tests to be performed under enhanced BSL-2 conditions with appropriate biosafety laboratory equipment and personal protective equipment used according to standardized protocols in the affected countries. Downgrading of CCHFV research work from Cl-4, BSL-4 to Cl-3, BSL-3 should also be considered.Additional co-authors: Gülay Korukluoglu, Pieter Lyssen, Ali Mirazimi, Johan Neyts, Matthias Niedrig, Aykut Ozkul, Anna Papa, Janusz Paweska, Amadou A Sall, Connie S Schmaljohn, Robert Swanepoel, Yavuz Uyar, Friedemann Weber, Herve Zelle

Genetic characterization of Yug Bogdanovac virus

We present pyrosequencing data and phylogenetic analysis for the full genome of Yug Bogdanovac virus (YBV), a member of the Vesicular stomatitis virus serogroup of the Rhabdoviridae isolated from a pool of Phlebotomus perfiliewi sandflies collected in Serbia in 1976. YBV shows very low nucleotide identities to other members of the Vesicular stomatitis virus serogroup and does not contain a reading frame for C′/C proteins

Genetic characterization of Bhanja virus and Palma virus, two tick-borne phleboviruses

The genomes of Bhanja virus (BHAV) and Palma virus (PALV) two tick-borne viruses hitherto grouped into the Bhanja virus antigenic complex of the Bunyaviridae were determined by pyrosequencing. Phylogenetic analysis groups all three segments of BHAV and PALV into a distinct clade of tick-borne phleboviruses together with the newly described severe fever with thrombocytopenia syndrome virus and Uukuniemi virus. The terminal signature sequences which are signatures for taxonomic grouping and important for virus replication and RNA transcription show marked differences in the L- and S-segments