Unexpected thermal stability of two enveloped megaviruses, Emiliania huxleyi virus and African swine fever virus, as measured by viability PCR

Background The particle structure of Emiliania huxleyi virus (EhV), an algal infecting member of nucleocytoplasmic large DNA viruses (NCLDVs), contains an outer lipid membrane envelope similar to that found in animal viruses such as African swine fever virus (ASFV). Despite both being enveloped NCLDVs, EhV and ASFV are known for their stability outside their host environment. Method Here we report for the first time, the application of a viability qPCR (V-qPCR) method to describe the unprecedented and similar virion thermal stability of both EhV and ASFV. This result contradicts the cell culture-based assay method that suggests that virus “infectivity” is lost in a matter of seconds (for EhV) and minutes (for ASFV) at temperature greater than 50 °C. Confocal microscopy and analytical flow cytometry methods was used to validate the V-qPCR data for EhV. Results We observed that both EhV and ASFV particles has unprecedented thermal tolerances. These two NCLDVs are exceptions to the rule that having an enveloped virion anatomy is a predicted weakness, as is often observed in enveloped RNA viruses (i.e., the viruses causing Porcine Reproductive and Respiratory Syndrome (PRRS), COVID-19, Ebola, or seasonal influenza). Using the V-qPCR method, we confirm that no PRRSV particles were detectable after 20 min of exposure to temperatures up to 100 °C. We also show that the EhV particles that remain after 50 °C 20 min exposure was in fact still infectious only after the three blind passages in bioassay experiments. Conclusions This study raises the possibility that ASFV is not always eliminated or contained after applying time and temperature inactivation treatments in current decontamination or biosecurity protocols. This observation has practical implications for industries involved in animal health and food security. Finally, we propose that EhV could be used as a surrogate for ASFV under certain circumstances. Supplementary Information The online version contains supplementary material available at 10.1186/s12985-023-02272-z.


Supplementary Information Fig S1.
Quantifying EhV-86 using flow cytometry.Flow cytometry plots of 0.2 m filtrate of (A) an uninfected Emiliania huxleyi culture and (B) E. huxleyi dead culture 4 days after being infected with EhV-86.EhV-86 was maintained at 4°C before adding to E. huxleyi culture.Filtrates were fixed with 0.5% glutaraldehyde and subsequently diluted 1:100.Filtrates were then stained using SYBR gold as described in [1].Flow cytometry enumeration based on green fluorescence (FITC-A) and side scatter (SSC-A) allowed us to identify and gate EhV particles (red box) as previously described by [2].We obtained a virus count of 37,335 in 50 µL, which after taking into account the previous 100x dilution, correspond to 0.8 x 10 8 EhV mL -1 .Two regions of high (blue arrow) and low (black arrow) SYBR nucleic acid florescence were observed within the EhV-86 population of particles.3D are also shown.Note that the efficiency of the RT-PCR was only at 78% and that the 4 °C controls (triangles and squares) plotted on the standard curve gave higher TCID50 mL -1 values than the corresponding standard dilution (arrow).It is clear from the plot that the RT-PCR is inhibited at the higher PRRSV concentrations.Consequently, our calculated TCID50 mL -1 values for the S and V RT-PCR will be higher (just under 1 log) than the known starting TCID50 mL -1 value (Figure 3D).

Fig S2 .
Fig S2.Example of a standard curve used in EhV qPCR assays.Dilution of the MCP PCR amplicon (no of copies) plotted against the Ct values as obtained by real time PCR (each dot represents three technical replicates).The Ct (cycle threshold) is defined as the number of cycles required for the fluorescent signal to cross the threshold (i.e., exceeds background level).The Ct was 7.16 for our 10 8 copies of target MCP stock, and it gradually increased to a final value of 37.16 in the presence of only 10 MCP copies.The R 2 value is shown.

Fig S3 .
Fig S3.The standard curve (green line) created using a PRRSV TCID50 mL -1[3] 10-fold dilution series (green plus sign symbols, x-axis) and the Cq values (y-axis) obtained from RT-PCR.The Cq is defined as the number of cycles required for the fluorescent signal to cross the threshold (i.e., exceeds background level).The standard curve equation metrics are shown.The data obtained for Figure3Dare also shown.Note that the efficiency of the RT-PCR was only at 78% and that the 4 °C controls (triangles and squares) plotted on the standard curve gave higher TCID50 mL -1 values than the corresponding standard dilution (arrow).It is clear from the plot that the RT-PCR is inhibited at the higher PRRSV concentrations.Consequently, our calculated TCID50 mL -1 values for the S and V RT-PCR will be higher (just under 1 log) than the known starting TCID50 mL -1 value (Figure3D).

Fig
Fig S4 Infectivity of EhV-86 after temperature treatment.A log10 E. huxleyi cell density (cells mL -1 ) plot indicating the capacity of EhV to induce E. huxleyi lysis, with subsequent change in cell density in infected cultures eight days post-virus inoculation in bioassays.EhV-86 was treated for short time-intervals: 5 min up to 25 min at several temperatures from 4 °C to 60 °C.The dashed line represents the lowest accurate limit of detection for cell number enumeration.

Fig S5 .
Fig S5.Efficiency of staining EhV-86 at different temperatures using a nucleic acid (DAPI) and lipid (FM 143) stain.Box plot with minimum & maximum (vertical whiskers), sample median (horizontal line) and first (25 th percentile) and last (75 th percentile) calculated for the nucleic acid and lipid membrane ratios (mean DAPI:FM 143 ratio) for EhV-86 treated for 20 minutes at 4 °C, 60 °C, 80 °C and 100 °C.Line plots of the individual DAPI (green line) and FM 143 (red line) mean fluorescence measurements for these aforementioned treatments are shown.