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

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-.

Molecular Detection and Characterization of Coronaviruses in Migratory Ducks from Portugal Show the Circulation of Gammacoronavirus and Deltacoronavirus

“…Extracted nucleic acids were tested for CoVs using a broad-spectrum pan-CoV nested RT-PCR assay targeting the RNA-dependent RNA polymerase (RdRp)-conserved region with a final product size of 440 bp [ 23 ]. The sensitivity of the nested pan-CoV primers has been recently compared with different protocols by combining existing primers from different studies showing high performance and combining the chances of detecting known and unknown CoVs from all matrices [ 23 ].…”

One health surveillance strategy for coronaviruses in Italian wildlife

“…Monitoring should include the screening of other mammals, not only as a reaction to specific epidemics but also to gain a better knowledge of the diversity and distribution of CoVs. This approach would help to unravel evolutionary pathways and dynamics that might lead to the emergence of novel threats for humans [5][6][7].…”

Comparative Performance in the Detection of Four Coronavirus Genera from Human, Animal, and Environmental Specimens

“…Pan-CoV PCR or family wide PCR assays and consensus PCR has successfully detected novel CoVs in humans, whereas the detection of unidentified or novel CoVs using only one primer set might lead to false negative reporting, due to the mismatching of primers and viral targets. Pan-CoV PCR primer design using degenerate primers to overcome this problem has largely reduced the rate of false negative reporting [ 19 , 20 , 21 , 22 ].…”

“…Most of the pan-CoV PCR protocols target the amplification of CoVs belonging to the genera α-CoV and β-CoV, while fewer protocols amplify the genera γ-CoV and δ-CoV [ 20 ]. The circulation of γ-CoV and δ-CoV in mammals might be underestimated, due to the use of primers that cannot amplify γ-CoV and δ-CoV.…”