Rapid isolation and resolution immune evasion and viral fitness across contemporary SARS-CoV-2 variants

From late 2020 the world observed the rapid emergence of many distinct SARS-CoV-2 variants. At the same time, pandemic responses coalesced into significant global vaccine roll-out that have now significantly lowered Covid-19 hospital and mortality rates in the developed world. Over this period, we developed a rapid platform (R-20) for viral isolation and characterisation using primary remnant diagnostic swabs. This combined with quarantine testing and genomics surveillance, enabled the rapid isolation and characterisation of all major SARS-CoV-2 variants (all variants of concern and 6 variants of interest) globally with a 4-month period. This platform facilitated viral variant isolation and enabled rapid resolution of variant phenotype by allowing determining end point viral titers from primary nasopharyngeal swabs and through ranking of evasion of neutralising antibodies. In late 2021, when the Delta variant was dominating, Omicron rapidly emerged. Using this platform, we isolated and tested the first cases of this variant within Australia. In this setting we observed Omicron to diverge from other variants at two levels: Firstly, it ranks at the mots evasive to neutralisation antibodies compared to all VOCs and major VUIs. Secondly, it no longer engages TMPRSS2 during the late stages of fusion.

A standardized method to purify cardiomyocytes from individual mouse hearts of any age

Rationale: Primary cardiomyocytes are invaluable for understanding postnatal heart development and elucidating disease mechanisms in genetic and pharmacological models, however, a method to obtain freshly purified cardiomyocytes at any postnatal age, without using different age-dependent isolation procedures and cell culture, is lacking. Objective: To develop a standardized method that allows rapid isolation and purification of cardiomyocytes in high yield and viability from individual neonatal, infant, and adult mice. Methods and Results: Hearts of C57BL/6J mice were cannulated using a novel in situ aortic cannulation procedure optimized to allow cannulation of even the very small vessel of neonates (postnatal day 0-2, P0-2). Hearts were then subjected to Langendorff retrograde perfusion and enzymatic digestion. Cardiomyocytes were isolated after subsequent tissue disaggregation and filtration, in high yield (1.56-2.2x106 cardiomyocytes/heart) and viability (~70-100%). The larger size of infant (P10 and P13) and adult (P70), but not neonatal, cardiomyocytes relative to non-myocytes, allowed enrichment by differential centrifugation. Cardiomyocytes from all ages were further purified by immunomagnetic bead-based depletion of non-myocytes. Together, these procedures resulted in the isolation of highly purified cardiomyocytes (~94%) within 1 hour, enabling experiments using individual replicates. For example, RNA-sequencing of cardiomyocytes purified from one P2 male and female heart per litter (n=4 litters) showed distinct clustering by litters and sex differences for nine differentially expressed genes (FDR<0.005). In situ fixation via coronary perfusion, performed immediately after tissue digestion, preserved the cytoarchitecture of isolated cardiomyocytes (yielding ~94% rod-shaped cardiomyocytes at all ages), allowing capture of spindle-shaped neonatal cells undergoing mitosis, as well as enabling accurate quantitation of cardiomyocyte area and nucleation state. Conclusion: The procedures developed here provide a universal protocol for the rapid isolation and purification of high-quality cardiomyocytes from hearts of any postnatal age, even those of neonates, thereby enabling direct comparisons between individual hearts.