Hypoxia-inducible factors and SARS-CoV-2 manifestations

By Bhavana KunkalikarMar 18 2022Reviewed by Aimee Molineux

A recent study posted to the bioRxiv* preprint server assessed the role of hypoxia-inducible factors (HIF) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection,  respiratory symptoms, and epithelial damage.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources

Various reports have highlighted the ability of respiratory viruses like influenza, rhinovirus, and respiratory syncytial virus in inducing anaerobic glycolysis via HIF-1α-activation. This suggests a potential role of SARS-CoV-2 in manipulating this pathway.

About the study

The present study investigated the effect of the HIF signaling pathway on SARS-CoV-2 replication, epithelial damage, and the manifestation of coronavirus disease 2019 (COVID-19) respiratory symptoms.

The study involved golden Syrian hamsters aged between six and eight weeks who were randomly grouped and individually housed within a containment level 3 facility. Each group had an equal number of female and male animals. For the study, three groups with six hamsters each were formed. The oral hypoxia-inducible factor prolyl hydroxylase inhibitor roxadustat (FG-4592) was administered twice daily.

The pre-infection treatment began 24 hours before drug administration while the post-infection treatment began 24 hours post-administration. The two treatment groups were monitored for four days after infection. The control group was treated as per the pre-infection group and treated with the vehicle only. SARS-CoV-2 was injected in all the groups via intranasal instillation.

Temperatures and weights of the animals were monitored daily while the visual inspection was performed twice daily, with symptoms like wasp-waist, ruffled hair, hunched back, or laborious breathing recorded. Nasal washes and throat swabs were collected on the first, second, and fourth days post-infection. The animals were euthanized on the fourth day and tissue samples were collected for virology and pathology assays. Direct intranasal challenge studies were also performed on groups of six hamsters.

Viral infectivity was assessed by a plaque assay using throat swabs, nasal washes, or lung homogenates. Furthermore, viral ribonucleic acid (RNA) was extracted from throat swabs or nasal washes. Quantitative reverse transcription PCR (RT-qPCR) was used to quantify the extracted viral RNA. RNA was also extracted from the homogenized lung to perform polyadenylated RNA (polyA)-enriched transcriptome sequencing. The samples also underwent immunoblotting while the SARS-CoV-2 RNAs were visualized by northern blotting.  

Results

The results showed a significant decrease in animal body temperature and weight in all the treatment groups while no marked differences were noted in the two values between the groups. The onset of laborious breathing was the first sign of disease as observed on day 2 after infection which increased in occurrences by day 4 in the control cohort. Also, a significant increase in the clinical score was found in FG-4592-treated animals, especially in the post-infection animal group.

Furthermore, labored breathing was observed in all the control group animals, in two out of the six pre-infection treatment group animals, and in none of the post-infection treatment group animals.

Terminal blood samples had a higher reticulocyte count as compared to the control group samples, indicating efficient drug treatment. Also, a reduction in messenger RNA (mRNA) levels of angiotensin-converting enzyme 2 (ACE2) was observed in the lungs of the hamsters treated with the drug. The team also noted similar HIF activation in both the treated groups, indicating comparable responses from the animals. Overall, the study showed that the drug was well tolerated while also activating HIF responses and reducing SARS-CoV-2 symptoms.

The study detected high viral RNA levels and infectious SARS-CoV-2 in the throat swabs and nasal washes after day 1 in the post-infection treatment group, which reduced over time. Animals pre-treated with the drug had reduced infectious viral burden on the second day post-infection. Also, a reduced viral burden was observed in post-infection group animals after day 4. However, the viral RNA levels remained unchanged in the nasal or throat samples.

The levels of sub-genomic (sg) RNAs and SARS-CoV-2 nucleocapsid (N) transcripts were reduced in the treatment groups. Furthermore, northern blotting showed that FG-4592 treatment reduced the levels of viral transcripts and inhibited expression of the N protein which provided evidence for the HIF antiviral activity. Also, viral sequences were retained in the genome in both treated and control tissues, indicating a lack of effect of the drug on SARS-CoV-2 sequences.

Sequencing the lung tissue obtained from treated and control animals showed that the drug caused significant changes in the form of up-regulated inflammation pathways and down-regulated genes involved in the assembly and organization of epithelial cilium. Also, the down-regulation of ciliated gene expression was less pronounced in the post-infection treatment group.

Conclusion

The study findings showed that treatment using FG-4592 before or after SARS-CoV-2 infection sufficiently reduced the SARS-CoV-2 viral burden and recovered the loss of epithelial cilia. This contributed to better clinical outcomes for COVID-19 infections. The researchers believed that this study highlighted HIF antiviral properties that may provide new therapeutic opportunities against SARS-CoV-2 infections.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources