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Review
. 2021 Oct 1;13(10):1975.
doi: 10.3390/v13101975.

Best Molecular Tools to Investigate Coronavirus Diversity in Mammals: A Comparison

Petra Drzewnioková  1 , Francesca Festa  1 , Valentina Panzarin  2 , Davide Lelli  3 , Ana Moreno  3 , Barbara Zecchin  1 , Paola De Benedictis  1 , Stefania Leopardi  1
Affiliations
Review

Best Molecular Tools to Investigate Coronavirus Diversity in Mammals: A Comparison

Petra Drzewnioková et al. Viruses. .

Abstract

Coronaviruses (CoVs) are widespread and highly diversified in wildlife and domestic mammals and can emerge as zoonotic or epizootic pathogens and consequently host shift from these reservoirs, highlighting the importance of veterinary surveillance. All genera can be found in mammals, with α and β showing the highest frequency and diversification. The aims of this study were to review the literature for features of CoV surveillance in animals, to test widely used molecular protocols, and to identify the most effective one in terms of spectrum and sensitivity. We combined a literature review with analyses in silico and in vitro using viral strains and archive field samples. We found that most protocols defined as pan-coronavirus are strongly biased towards α- and β-CoVs and show medium-low sensitivity. The best results were observed using our new protocol, showing LoD 100 PFU/mL for SARS-CoV-2, 50 TCID50/mL for CaCoV, 0.39 TCID50/mL for BoCoV, and 9 ± 1 log2 ×10-5 HA for IBV. The protocol successfully confirmed the positivity for a broad range of CoVs in 30/30 field samples. Our study points out that pan-CoV surveillance in mammals could be strongly improved in sensitivity and spectrum and propose the application of a new RT-PCR assay, which is able to detect CoVs from all four genera, with an optimal sensitivity for α-, β-, and γ-.

Keywords: BoCoV; CaCoV; RT-PCR; SARS-CoV-2; coronavirus; pan-CoV; surveillance.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Flow chart for selecting the most suitable molecular protocol for pan-CoV surveillance.
Figure 2
Figure 2
Similarity between primers and their position on the consensus sequence from nucleotide alignment of 69 CoV sequences. Figures were created using Geneious Prime® 2020.1.2 (Biomatters, Auckland, New Zealand).
Figure 3
Figure 3
In vitro evaluation of primer specificity and comparison of protocols’ sensitivity. Red blocks represent serial dilution that provided positive results in all repetitions. The higher dilution at which all the replicas yielded positive results identified the LOD of the test. Red/white patterns show cases in which even lower dilution resulted in weak positivity or inconsistent results among the different repetitions. † Antigen suspension with initial titer 9 ± 1 log2 HA.
Figure 4
Figure 4
ML phylogenetic tree of coronaviruses. Sequences were aligned using the G-INS1 and default parameters implemented in Mafft using online tool: https://mafft.cbrc.jp/alignment/server [50]. ML phylogenetic trees were inferred using PhyML (version 3.0) implemented in Seaview (Lyon, France), employing the GTR+G4 substitution model, a heuristic SPR branch-swapping algorithm, and SH-like branch supports [51]; obtained trees were edited online for graphical display using iTOL on server: https://itol.embl.de/ [52]. CoV species investigated in the study are indicated in red, with branches of tested strains shown as red boxes.
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