Two real-time TaqMan PCRs for the detection of Alongshan Virus (ALSV), a new member of the tick-borne Flaviviridae family

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Abstract

Introduction: Alongshan Virus (ALSV) is a tick-borne Flaviviridae. Ticks carrying ALSV have been identified in various regions, including China, Russia, Finland, Switzerland, and Germany, mainly within Ixodes ricinus and Ixodes persulcatus species. Hypothesis/Gap Statement: However, the clinical relevance and the pathogenicity of ALSV in humans is still unknown. Aim: We aimed to validate and integrate a real-time polymerase chain reaction (PCR) assay on our in-house molecular diagnostic platform to accurately detect ALSV and then further investigate its clinical relevance and its potential role as agent of meningoencephalitis in humans. Methodology: We customized two ALSV-specific Taqman RT-PCR assays targeting the NS3 and NS5 genes to align with the genetic diversity of ALSV sequences and the technical features of TaqMan probes-related technologies. Positive controls included ALSV RNA from infected Swiss ticks and synthetic plasmids including the target region of the RT-PCR assays. Results: To determine the limit of detection of the ALSV PCR assay, we used serial dilutions of the synthetic plasmid. Amplification rates of 100%, 92%, and 20% were respectively achieved for 1000, 100, and 10 copies per reaction for the NS3 PCR and 100%, 100%, 20% for the NS5 PCR. Intra- and inter-run reproducibility, evaluated over five independent runs with plasmid dilutions representing 100 and 10 DNA copies per reaction, met diagnostic standards. The assay exhibited 100% specificity (71/71) when tested against (i) a panel of cerebrospinal fluid pathogens (n=41), including bacteria, viruses, and fungi, and (ii) CSF specimens from our biobank (n=30), collected during winter when local tick exposure is unlikely due to low temperatures. In contrast specific amplification was obtained in ALSV-infected tick samples. Conclusions: Two ALSV real-time PCRs were validated for rapid, sensitive, and specific detection of ALSV RNA. Integration of ALSV PCR into open molecular diagnostic platform such as our, allows for syndromic testing alongside other encephalitis-associated viruses, such as Tick-Borne encephalitis virus or West Nile virus. This PCR will facilitate high-throughput screening of ticks for surveillance purposes. Moreover, this assay could be integrated into other in-house molecular diagnostic platform, in order to accurately detect ALSV to further explore ALSV pathogenic role.

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  1. Comments to Author

    All information seems to be there but some parts require a more in depth explanation, and the methods section slightly restructured so that the findings of the paper are clear. L1 - Use the phrase Taqman PCR assays instead of Taqman PCRs, as that is what has been created here L45 - Here it says amplification rates of 100%, 92% and 20% for 1000, 100 and 10 copies respectively for NS3, but in table 2 it says 100%, 100% and 92% for those copy numbers. L54 - Again use the phrase Taqman PCR assays throughout the whole manuscript L69 - Some more background on named viruses such as single or double stranded, enveloped etc L108-117 - This paragraph could be better structured - i.e. starting with where samples were obtained from and how they were handled and/or treated, with machinery used for each step …

  2. Thank you for submitting your manuscript for publication in Access Microbiology. It has been examined by expert reviewers who have concluded that the work is of potential interest to the readership of Access Microbiology. However, based on the comments received, it is clear that a major revision of this manuscript will be required before a decision can be made on its publication. I will be pleased to consider a revised manuscript along with a document including a point by point response to each of the reviewers comments. Your revised manuscript may be returned to one or more of the original reviewers, along with your itemised response to the reviewers’ comments.

  3. Comments to Author

    The manuscript describes the development of two new RT-qPCR assays for the detection of Alongshan Virus (ALSV). Overall, the methods and results appear to be scientifically sound. However, a lot of information and details are missing. The manuscript is difficult to follow and would benefit from a clearer structure. There are also inconsistencies in the used terminology. Please see below for specific comments. Abstract L33: Why is it important to develop and validate new RT-qPCR assays? L36-37: The aim of the manuscript is to develop new RT-qPCR assays for Alongshan Virus (ALSV). While the role of ALSV in meningoencephalitis in humans is not investigated in this manuscript, rapid and precise diagnostics of ALSV will help to further understand the role the virus plays in meningoencephalitis. …