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Principal Investigator: Elodie Ghedin
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2024
Award: $812,851
Funding agency: National Institute of Allergy and Infectious Diseases
The major aspects of this project involve characterizing SARS-CoV-2 genetic diversity and microbial communities in the respiratory tract during infection. This involves analyzing samples from COVID-19 positive subjects, and from model animal infections. As part of this project we tackle different types of studies that include:
(a) Molecular epidemiology
(b) Virus evolution and genetic diversity within infected hosts
(c) Respiratory tract microbiome profiles and host response
(a) Molecular epidemiology
Whole genome sequencing of SARS-CoV-2 has become crucial for epidemiology studies and to determine how the virus sweeps through various populations, with the transmission potential of new emerging variants. In FY24, we finalized 2 genomic epidemiology studies in collaboration with clinical and public health teams in the Dominican Republic and Haiti. We combined phylogenetics with travel history data, and used the viral diversity information to determine the number of independent introductions into each of these countries that shutdown their borders and airports early in the pandemic. From a public health perspective, this enables an evaluation of how efficient border closings are in limiting the introduction of new variants. The Caribbean is a populous region (>40 million people in the island states) at the crossroads of many trade routes and visited by tourists from around the world, yet is drastically undersampled relative to Europe and North America. We analyzed samples from Haiti in Spring 2020, and although the number of specimens sequenced in this study was small, it was sufficient to demonstrate that COVID-19 was spreading among Caribbean islands in early 2020 and to show that at least one lineage was possibly introduced from the Caribbean into North America, where it sustained community submission over a period of several months. The Haiti study is currently under review in PLoS Global Public Health.
(b) Virus evolution and genetic diversity within infected hosts
While identification of new clades and lineages, and the associated viral consensus changes help in tracking spread of the virus, few studies have been done on the minority variants present in infected individuals. These minority variants could be seeding the emergence of new clades, thus identifying them early is of relevance for preparedness and to track transmission events. We have 3 ongoing studies that focus on intra-host diversity.
The first is a collaboration with Dr. James Musser (Houston Methodist), where we use deep sequences of thousands of SARS-CoV-2 clinical samples to explore within-host diversity of the virus from a high-throughput viral surveillance program associated with a large hospital system. This project has required a complete revision of how we tackle opportunistic datasets for deep sequencing analyses and the identification of all the artifacts that can be introduced. In FY24, we developed a novel method that uses raw sequencing metrics, particularly sequencing coverage unevenness, to train a simple and fast random forest regressor for predicting viral load from raw amplicon-based sequences. Viral load, measured by clinical diagnostic RT-qPCR cycle-threshold (Ct) values, is crucial in pandemics like COVID-19 for assessing patient disease severity and stage of SARS-CoV-2 infection. Obtained from clinical nasopharyngeal swabs, it also helps guide thresholds to discard spurious mutational artifacts due to insufficient starting genomic material. Despite extensive sequencing efforts, many SARS-CoV-2 genome sequences lack viral load information. Analyzing nearly 40,000 clinical samples, half of which lacked Ct values, our model achieved an adjusted R² of 85% for linear prediction across the Ct spectrum and demonstrated generalizability across different library preparation methods when retrained on other datasets. We applied the model to logistic regressions on higher “infectious” viral loads (Ct < 30) within specific subpopulations with more severe COVID-19 and showed that predicted Ct values can also replicate a 15-day dynamic of infectious period inference. This approach, which factors sequencing coverage with nucleotide diversity, enhances the utility of millions of viral genomic samples, unlocking further study into the evolutionary dynamics of SARS-CoV-2. Two publications are currently in preparation.
In a collaborative study with Dr. Mirella Salvatore (Weill Cornell), we are currently analyzing the within-host evolution of SARS-CoV-2 in immunocompromised patients with mostly B cell defects. Persistent viral replication occurs in immunosuppressed patients with SARS-CoV-2 and viral persistence in this setting has raised concern for viral evolution and the emergence of variants. In FY24, we identified patients who had infections with SARS-CoV-2 virus carrying mutations leading to resistance against multiple antivirals. Sequence analysis showed that 9 of 15 patients analyzed carried viruses with mutations in the nsp12 (RNA dependent RNA polymerase), while four had viruses with nsp5 (3C protease) mutations. Infectious SARS-CoV-2 with a double mutation in nsp5 (T169I) and nsp12 (V792I) was recovered from respiratory secretions 77 days after initial COVID-19 diagnosis from a patient treated with remdesivir and nirmatrelvir-ritonavir. Dr. Diego Diel (Cornell U) isolated the virus and confirmed In vitro decreased sensitivity to remdesivir and nirmatrelvir, which was overcome by combined antiviral treatment. Studies in golden Syrian hamsters in the Diel Lab demonstrated efficient transmission to contact animals. We have a manuscript currently under review at Nature Communications. https://www.medrxiv.org/content/10.1101/2024.06.14.24308523v1
In another collaborative study, with Dr. Daniel Chertow (VRC/NIAID), we are determining the diversity of SARS-CoV-2 across tissues from an autopsy case from a patient with an inborn genetic disease that leads to immunocompromise. In FY24, we finalized the genetic analyses across 32 tissues and identified multiple genotypes replicating with mutations specifically in the receptor-binding domain of the Spike protein. A number of these mutations were predicted to impact the stability and binding energy to the ACE2 receptor. Dr. Mehul Suthar (Emory) isolated viruses for which he performed functional assays to test for binding and entry efficiency for 5 spike haplotypes. A manuscript is currently in preparation.
(c) Respiratory tract microbiome profiles and host response
In our first study of the airway microbiome of COVID19 patients, we focused on the metagenomic and metatranscriptomic analysis of cross-sectional BAL samples collected from mechanically ventilated patients during the first wave of the pandemic in NYC. However, while these patients all had severe disease requiring intubation, the mortality rate was much lower in this cohort because only stable patients could undergo bronchoscopy. In a follow-up study that we continued in FY24, we analyzed longitudinal samples from 70 patients for which 1-5 samples were collected up to 6 weeks follow-up. From the same cohort, we also did a focused study on patients who are immunocompromised. This cohort has rich metadata on therapeutic regimens and symptoms. We are currently drafting 2 publications. In FY24 we also published a study for the same cohort where we looked at immune responses in the airways and their association with poor outcome in critically ill COVID-19 patients. These data highlight the critical role of local adaptive immunity in the airways as a key defense mechanism against primary SARS-CoV-2 infection.
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