RNA reference materials with defined viral RNA loads of SARS-CoV-2-A useful tool towards a better PCR assay harmonization.

SARS-CoV-2, the cause of COVID-19, requires reliable diagnostic methods to track the circulation of this virus. Following the development of RT-qPCR methods to meet this diagnostic need in January 2020, it became clear from interlaboratory studies that the reported Ct values obtained for the different laboratories showed high variability. Despite this the Ct values were explored as a quantitative cut off to aid clinical decisions based on viral load. Consequently, there was a need to introduce standards to support estimation of SARS-CoV-2 viral load in diagnostic specimens. In a collaborative study, INSTAND established two reference materials (RMs) containing heat-inactivated SARS-CoV-2 with SARS-CoV-2 RNA loads of ~107 copies/mL (RM 1) and ~106 copies/mL (RM 2), respectively. Quantification was performed by RT-qPCR using synthetic SARS-CoV-2 RNA standards and digital PCR. Between November 2020 and February 2021, German laboratories were invited to use the two RMs to anchor their Ct values measured in routine diagnostic specimens, with the Ct values of the two RMs. A total of 305 laboratories in Germany were supplied with RM 1 and RM 2. The laboratories were requested to report their measured Ct values together with details on the PCR method they used to INSTAND. This resultant 1,109 data sets were differentiated by test system and targeted gene region. Our findings demonstrate that an indispensable prerequisite for linking Ct values to SARS-CoV-2 viral loads is that they are treated as being unique to an individual laboratory. For this reason, clinical guidance based on viral loads should not cite Ct values. The RMs described were a suitable tool to determine the specific laboratory Ct for a given viral load. Furthermore, as Ct values can also vary between runs when using the same instrument, such RMs could be used as run controls to ensure reproducibility of the quantitative measurements.

Results of cytomegalovirus DNA viral loads expressed in copies per millilitre and international units per millilitre are equivalent.

Quantification of Cytomegalovirus (CMV) DNA is required for the initiation and monitoring of anti-viral treatment and the detection of viral resistance. However, due to the lack of standardisation of CMV DNA nucleic acid tests, it is difficult to set universal thresholds. In 2010, the 1st WHO International Standard for Human Cytomegalovirus for Nucleic Acid Amplification Techniques was released. Since then CMV DNA viral load assays have been calibrated using this standard. Three external quality assessment (EQA) providers sent the same five samples to their participants and analysed the results to determine the equivalence of reporting CMV DNA results in international units per millilitre (IU/mL), and compared the difference in results reported in IU/mL with those reported in copies per millilitre (c/mL), and to determine the rate of adoption of IU/mL. About 78% of participants continue to report results in c/mL even though six of the 12 commercial assays are calibrated against the standard. The range of the results reported in IU/mL was less than those reported in c/mL indicating that the adoption of the WHO standard successfully improved the reporting of the CMV viral load. The variation in individual sample results reported by different assays, irrespective of whether in IU/mL or c/mL, is still great and therefore more standardisation of the assays is needed to allow the setting of treatment and monitoring thresholds. This study can act as a bench mark to determine rate of future adoption if reporting CMV DNA viral load results in IU/mL.

Targeting Highly Structured RNA by Cooperative Action of siRNAs and Helper Antisense Oligomers in Living Cells.

RNA target accessibility is one of the most important factors limiting the efficiency of RNA interference-mediated RNA degradation. However, targeting RNA viruses in their poorly accessible, highly structured regions can be advantageous because these regions are often conserved in sequence and thus less prone to viral escape. We developed an experimental strategy to attack highly structured RNA by means of pairs of specifically designed small interfering RNAs and helper antisense oligonucleotides using the 5' untranslated region (5'UTR) of coxsackievirus B3 as a model target. In the first step, sites accessible to hybridization of complementary oligonucleotides were identified using two mapping methods with random libraries of short DNA oligomers. Subsequently, the accessibility of the mapped regions for hybridization of longer DNA 16-mers was confirmed by an RNase H assay. Using criteria for the design of efficient small interfering RNAs (siRNA) and a secondary structure model of the viral 5'UTR, several DNA 19-mers were designed against partly double-stranded RNA regions. Target sites for DNA 19-mers were located opposite the sites which had been confirmed as accessible for hybridization. Three pairs of DNA 19-mers and the helper 2'-O-methyl-16-mers were able to effectively induce RNase H cleavage in vitro. For cellular assays, the DNA 19-mers were replaced by siRNAs, and the corresponding three pairs of siRNA-helper oligomer tools were found to target 5'UTR efficiently in a reporter construct in HeLa cells. Addition of the helper oligomer improved silencing capacity of the respective siRNA. We assume that the described procedure will generally be useful for designing of nucleic acid-based tools to silence highly structured RNA targets.

Virus-host coevolution in a persistently coxsackievirus B3-infected cardiomyocyte cell line.

Coevolution of virus and host is a process that emerges in persistent virus infections. Here we studied the coevolutionary development of coxsackievirus B3 (CVB3) and cardiac myocytes representing the major target cells of CVB3 in the heart in a newly established persistently CVB3-infected murine cardiac myocyte cell line, HL-1(CVB3). CVB3 persistence in HL-1(CVB3) cells represented a typical carrier-state infection with high levels (10(6) to 10(8) PFU/ml) of infectious virus produced from only a small proportion (approximately 10%) of infected cells. CVB3 persistence was characterized by the evolution of a CVB3 variant (CVB3-HL1) that displayed strongly increased cytotoxicity in the naive HL-1 cell line and showed increased replication rates in cultured primary cardiac myocytes of mouse, rat, and naive HL-1 cells in vitro, whereas it was unable to establish murine cardiac infection in vivo. Resistance of HL-1(CVB3) cells to CVB3-HL1 was associated with reduction of coxsackievirus and adenovirus receptor (CAR) expression. Decreasing host cell CAR expression was partially overcome by the CVB3-HL1 variant through CAR-independent entry into resistant cells. Moreover, CVB3-HL1 conserved the ability to infect cells via CAR. The employment of a soluble CAR variant resulted in the complete cure of HL-1(CVB3) cells with respect to the adapted virus. In conclusion, this is the first report of a CVB3 carrier-state infection in a cardiomyocyte cell line, revealing natural coevolution of CAR downregulation with CAR-independent viral entry in resistant host cells as an important mechanism of induction of CVB3 persistence.

Developing an effective RNA interference strategy against a plus-strand RNA virus: silencing of coxsackievirus B3 and its cognate coxsackievirus-adenovirus receptor.

Coxsackievirus B3 (CVB-3) is a plus-strand RNA virus that is believed to be the most common causal agent of viral myocarditis. Since no specific treatment for CVB-3 infections is available to date, we and others have recently started to develop RNA interference (RNAi) approaches to prevent virus propagation. Here we describe our strategy for the development of efficient small interfering RNAs (siRNAs) against viral genomes. Initially, fusion constructs of a reporter (green fluorescent protein) and viral subgenomic fragments were employed to select active siRNAs against the virus. Moreover, in an attempt to achieve sustained virus silencing and reduce the risk of generating escape mutants, only highly efficient siRNAs directed against regions of the viral genome that are unlikely to tolerate mutations were considered for virus inhibition. Two siRNAs directed against the 3D RNA-dependent RNA polymerase were found to inhibit virus propagation by 80-90%. The protective effect of the efficient siRNAs lasted for several days. Furthermore, we have first evidence that inhibition of the cellular coxsackievirus-adenovirus receptor (CAR) by RNAi also reduces the virus titre. Our strategy is likely to be applicable to other (RNA) viruses as well.