While mass vaccination schemes helped end the worldwide restrictions on travel and reduced deaths due to coronavirus disease 2019 (COVID-19) significantly, new variants continue to emerge that can challenge and sometimes overcome both natural and vaccine-induced immunity. These new variants of concern can cause different pathologies to the original wild-type strain. Researchers have investigated the pathology and inflammatory response to the Delta variant in mice.
Study: SARS-CoV-2 Delta variant induces enhanced pathology and inflammatory responses in K18-hACE2 mice. Image Credit: Carl DMaster/Shutterstock
A preprint version of the study is available on the bioRxiv* server while the article undergoes peer review.
The study
The researchers evaluated the doses appropriate to challenge mice expressing human angiotensin-converting enzyme 2 (ACE2) with wild-type, Alpha, Beta, and Delta variants. They observed that at a dose of 10^3 PFU, wild-type would kill 50% of the mice, while Alpha and Beta would cause 80/100% mortality by day seven, respectively. Delta showed similar mortality levels to wild type at this dose but significantly outperformed the ancestral strain once the dose was increased to 10^4. While Alpha and Beta did cause more mortality in mice than Delta, the Delta strain caused longer-lasting symptoms in surviving mice. For further experimentation, the scientists settled on a dose of 10^3 PFU.
To better understand the manifestation of different variants, the researchers monitored rectal temperature and weight loss in infected mice. While the Alpha-infected mice showed the expected symptoms of loss of temperature and weight, the Delta-challenged mice retained body temperature and did not lose weight. Most Alpha-infected mice required euthanasia by day six, unlike those infected with Delta.
Following this, the scientists investigated the RNA burden in the mice on days two and six. They collected lung, brain, and nasal wash fluid and quantified the viral RNA burden using real-time polymerase chain reaction reverse transcription (qRT-PCR). They found very little difference in the lungs or nares, with RNA burden decreasing below detectable levels at day six. However, mice infected with the Delta strain showed no detectable RNA in the brain.
The researchers then performed histopathological analysis on the lungs of infected mice on days two and six to better understand pneumonia and encephalitis caused by the disease. They found that inflammation and the recruitment of inflammatory cells were significantly advanced in mice infected with the Delta strain compared to the Alpha strain, predominantly lymphocytes, and histiocytes. This trend continued to day six, with 36.86 marginating lymphocytes per mm length of endothelium compared to 12.19.
To further investigate this increased inflammation, the scientists examined the levels of pro-inflammatory cytokines in the lungs. The Delta-infected mice showed massively increased IL-1Beta, TNFalpha and CXCL10 compared to the mice infected with the Alpha variant. RNAseq was then performed to characterize the transcriptional profile better but revealed no significant difference in viral sgRNA.
This was followed by lung RNA sequencing reads, which revealed sharp changes in the transcriptome. On day two, both Alpha- and Delta-infected mice showed thousands of different statistically significant genes were differentially expressed compared to mice that had not been infected, and this rose sharply by day six. Both variants activate a similar set of host genes.
To investigate this further, gene ontology (GO) term analysis was performed. This showed that Delta-infected mice had a higher number of unique GO terms enriched at day six, mostly pertaining to immune responses, including antiviral, lymphocyte recruitment, and pathways associated with T cells and IFN-Beta, as well as increased expression of interferon response genes.
The scientists then looked further into the increase in the expression of interferon response genes. They found TGTP1 and IFI211 are the highest expressed genes in mice infected with Delta at day six, showing a fold change increase of ~50,000 and ~10,000, respectively. These genes are also elevated in Alpha-infected mice, although not to the same extent.
They measured type I and II interferons and lung supernatants better to understand the interferon response in Delta-infected mice and discovered modest IFNalpha and beta but significantly increased IFNgamma.
Conclusion
The authors highlight that they have helped to show that a sublethal challenge with the delta variant of SARS-CoV-2 induces a more histopathological inflammation than the alpha variant and increases interferon responses. They have also helped to clarify doses of SARS-CoV-2 variants for future studies and observed the changes in the gene expression caused by Delta. This information could be valuable for future researchers and help inform healthcare workers of patients infected by the Delta strain.
*Important notice
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
