Human endogenous retrovirus K in the respiratory tract is associated with COVID-19 physiopathology (preprint)

Critically ill COVID-19 patients under invasive mechanical ventilation (IMV) are at greatly increased risk of death compared to the general population. While some drivers of COVID-19 disease progression, such as inflammation and hypercoagulability, have been identified, they do not completely explain the mortality of critically ill COVID-19 patients, making a search for overlooked factors necessary. A recent study examined the virome of tracheal aspirates from 25 COVID-19 patients under IMV. These samples were compared to tracheal aspirates from non-COVID patients and nasopharyngeal swabs from individuals with mild COVID-19. Critically ill COVID-19 patients had elevated expression of human endogenous retrovirus K (HERV-K), and elevated HERV-K expression in tracheal aspirate and plasma was associated with early mortality in those same patients. Among deceased patients, HERV-K expression was associated with IL-17-related inflammation, monocyte activation, and increased consumption of clotting factors. A subsequent in vitro experiment found that exposure to SARS-CoV-2 increased HERV-K expression in human primary monocytes from healthy donors. This preliminary study only included 25 individuals but implicates HERV-K in the physiopathology of COVID-19 and suggests that HERV-K could be used as a biomarker of disease severity in COVID-19 patients. 

Human endogenous retrovirus K activation in the lower respiratory tract of severe COVID-19 patients associates with early mortality (preprint)

Critically ill 2019 coronavirus disease patients (COVID-19) under invasive mechanical ventilation (IMV) are 10- to 40-times more likely to die than the general population. Although progression from mild to severe COVID-19 has been associated with hypoxia, uncontrolled inflammation and coagulopathy, the mechanisms involved in progression to severity are poorly understood. By analyzing the virome from tracheal aspirates (TA) of 25 COVID-19 patients under IMV, we found higher levels and differential expression of human endogenous retrovirus K (HERV-K) genes compared to nasopharyngeal swabs from mild cases and TA from non-COVID patients. Proteomic analysis and RT-PCR confirmed the presence of HERV-K in these patients. Moreover, increased HERV-K expression was triggered in human primary monocytes from healthy donors after experimental SARS-CoV-2 infection in vitro. In critically ill patients, higher HERV-K levels were associated with early mortality (within 14 days) in the intensive care unit. Increased HERV-K expression in deceased patients associated with IL-17-related inflammation, monocyte activation and higher consumption of clotting/fibrinolysis factors. Our data implicate the levels of HERV-K transcripts in the outcome of critical COVID-19 patients under invasive mechanical ventilation.

Viral Genetic Evidence and Host Immune Response of a Small Cluster of Individuals with Two Episodes of SARS-CoV-2 Infection (preprint)

Background: The dynamics underlying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection remains poorly understood. We added to the registered case reports of reinfection in USA, Belgium/Netherlands, Ecuador and Hong Kong, a small cluster of individuals with two episodes of 2019 coronavirus disease (COVID-19). Virus genomic analysis and the host immune response were used to characterize this group. Methods: Four individuals from Rio de Janeiro, Brazil, with clinical manifestations of COVID-19 on March and again in late May of 2020 were studied. Nasopharyngeal swabs were collected for RT-PCR and viral genome sequencing (BGI-MGI-2000). Plasma samples from the acute and convalescent phases of both infection episodes were accessed to document innate and humoral responses.Findings: After approximately 60 days of the first diagnostic episode of SARS-CoV-2 infection, the four individuals presented new clinical and molecular evidence of COVID-19. Complete SARS-CoV-2 genome sequence provided genetic evidence of reinfection. The individuals presented an enhanced innate response compared to healthy SARS-CoV-2 negative controls. Patients did not develop a neutralizing humoral immunity, possibly remaining susceptible to another episode of COVID-19. The second episode, associated with higher viral loads and clinical symptoms, likely boosted their anti-SARS-CoV-2 humoral response. Interpretation: SARS-CoV-2 reinfection was fully documented by identification of genetically distinct virus sequences in the first and second episodes for two individuals. The quantity of SARS-CoV-2-associated genetic reads and coverage of virus genome ruled out that the initial RT-PCR results were false positive. The identification that some individuals with mild COVID-19 may have controlled SARS-CoV-2 replication without developing detectable humoral immunity, opens the possibility that reinfection may be more frequent than supposed – but weakly documented.

High sensitivity detection of SARS-CoV-2 using multiplex PCR and a multiplex-PCR-based metagenomic method (preprint)

Many detection methods have been used or reported for the diagnosis and/or surveillance of COVID-19. Among them, reverse transcription polymerase chain reaction (RT-PCR) is the most commonly used because of its high sensitivity, typically claiming detection of about 5 copies of viruses. However, it has been reported that only 47-59% of the positive cases were identified by some RT-PCR methods, probably due to low viral load, timing of sampling, degradation of virus RNA in the sampling process, or possible mutations spanning the primer binding sites. Therefore, alternative and highly sensitive methods are imperative. With the goal of improving sensitivity and accommodating various application settings, we developed a multiplex-PCR-based method comprised of 343 pairs of specific primers, and demonstrated its efficiency to detect SARS-CoV-2 at low copy numbers. The assay produced clean characteristic target peaks of defined sizes, which allowed for direct identification of positives by electrophoresis. We further amplified the entire SARS-CoV-2 genome from 8 to half a million viral copies purified from 13 COVID-19 positive specimens, and detected mutations through next generation sequencing. Finally, we developed a multiplex-PCR-based metagenomic method in parallel, that required modest sequencing depth for uncovering SARS-CoV-2 mutational diversity and potentially novel or emerging isolates.