Delayed engagement of host defenses enables SARS-CoV-2 viremia and productive infection of distal organs in the hamster model of COVID-19.

Clinical presentations that develop in response to infection result from interactions between the pathogen and host defenses. SARS-CoV-2, the etiologic agent of COVID-19, directly antagonizes these defenses, leading to delayed immune engagement in the lungs that materializes only as cells succumb to infection and are phagocytosed. Leveraging the golden hamster model of COVID-19, we sought to understand the dynamics between SARS-CoV-2 infection in the airways and the systemic host response that ensues. We found that early SARS-CoV-2 replication was largely confined to the respiratory tract and olfactory system and, to a lesser extent, the heart and gastrointestinal tract but generated a host antiviral response in every organ as a result of circulating type I and III interferons. Moreover, we showed that diminishing the response in the airways by immunosuppression or administration of SARS-CoV-2 intravenously resulted in decreased immune priming, viremia, and increased viral tropism, including productive infection of the liver, kidney, spleen, and brain. Last, we showed that productive infection of the airways was required for mounting an effective and system-wide antiviral response. Together, these data illustrate how COVID-19 can result in diverse clinical presentations in which disease outcomes can be a by-product of the speed and strength of immune engagement. These studies provide additional evidence for the mechanistic basis of the diverse clinical presentations of COVID-19 and highlight the ability of the respiratory tract to generate a systemic immune defense after pathogen recognition.

One-year monitoring SARS-CoV-2 RNA surface contamination in hospitals reveals no correlation with organic material and negative pressure as a limiting factor for contamination.

Understanding transmission routes of SARS-CoV-2 is crucial to establish effective interventions in healthcare institutions. Although the role of surface contamination in SARS-CoV-2 transmission has been controversial, fomites have been proposed as a contributing factor. Longitudinal studies about SARS-CoV-2 surface contamination in hospitals with different infrastructure (presence or absence of negative pressure systems) are needed to improve our understanding of their effectiveness on patient healthcare and to advance our knowledge about the viral spread. We performed a one-year longitudinal study to evaluate surface contamination with SARS-CoV-2 RNA in reference hospitals. These hospitals have to admit all COVID-19 patients from public health services that require hospitalization. Surfaces samples were molecular tested for SARS-CoV-2 RNA presence considering three factors: the dirtiness by measuring organic material, the circulation of a high transmissibility variant, and the presence or absence of negative pressure systems in hospitalized patients' rooms. Our results show that: (i) There is no correlation between the amount of organic material dirtiness and SARS-CoV-2 RNA detected on surfaces; (ii) SARS-CoV-2 high transmissible Gamma variant introduction significantly increased surface contamination; (iii) the hospital with negative pressure systems was associated with lower levels of SARS-CoV-2 surface contamination and, iv) most environmental samples recovered from contaminated surfaces were assigned as non-infectious. This study provides data gathered for one year about the surface contamination with SARS-CoV-2 RNA sampling hospital settings. Our results suggest that spatial dynamics of SARS-CoV-2 RNA contamination varies according with the type of SARS-CoV-2 genetic variant and the presence of negative pressure systems. In addition, we showed that there is no correlation between the amount of organic material dirtiness and the quantity of viral RNA detected in hospital settings. Our findings suggest that SARS CoV-2 RNA surface contamination monitoring might be useful for the understanding of SARS-CoV-2 dissemination with impact on hospital management and public health policies. This is of special relevance for the Latin-American region where ICU rooms with negative pressure are insufficient.

Dual-Reporter System for Real-Time Monitoring of SARS-CoV-2 Main Protease Activity in Live Cells Enables Identification of an Allosteric Inhibition Path.

The SARS-CoV-2 pandemic is an ongoing threat to global health, and the continuing emergence of contagious variants highlights the urgent need for additional antiviral therapy to attenuate COVID-19 disease. The SARS-CoV-2 main protease (3CLpro) presents an attractive target for such therapy due to its high sequence conservation and key role in the viral life cycle. In this study, we designed a fluorescent-luminescent cell-based reporter for the detection and quantification of 3CLpro intracellular activity. Employing this platform, we examined the efficiency of known protease inhibitors against 3CLpro and further identified potent inhibitors through high-throughput chemical screening. Computational analysis confirmed a direct interaction of the lead compounds with the protease catalytic site and identified a prototype for efficient allosteric inhibition. These developments address a pressing need for a convenient sensor and specific targets for both virus detection and rapid discovery of potential inhibitors.

Dual-Reporter System for Real-Time Monitoring of SARS-CoV-2 Main Protease Activity in Live Cells Enables Identification of an Allosteric Inhibition Path

The SARS-CoV-2 pandemic is an ongoing threat to global health, and the continuing emergence of contagious variants highlights the urgent need for additional antiviral therapy to attenuate COVID-19 disease. The SARS-CoV-2 main protease (3CLpro) presents an attractive target for such therapy due to its high sequence conservation and key role in the viral life cycle. In this study, we designed a fluorescent–luminescent cell-based reporter for the detection and quantification of 3CLpro intracellular activity. Employing this platform, we examined the efficiency of known protease inhibitors against 3CLpro and further identified potent inhibitors through high-throughput chemical screening. Computational analysis confirmed a direct interaction of the lead compounds with the protease catalytic site and identified a prototype for efficient allosteric inhibition. These developments address a pressing need for a convenient sensor and specific targets for both virus detection and rapid discovery of potential inhibitors.

Molecular accuracy vs antigenic speed: SARS-CoV-2 testing strategies.

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has hit every corner of the world faster than any infectious disease ever known. In this context, rapid and accurate testing of positive cases are essential to follow the test-trace-isolate strategy (TETRIS), which has proven to be a key approach to constrain viral spread. Here, we discuss how to interpret and combine molecular or/and antigen-based detection methods for SARS-CoV-2 as well as when they should be used. Their application can be cleverly designed as an algorithm to prevent viral dissemination according to distinct epidemiological contexts within surveillance programs.

Emergence and Spread of a B.1.1.28-Derived P.6 Lineage with Q675H and Q677H Spike Mutations in Uruguay.

Uruguay controlled the viral dissemination during the first nine months of the SARS-CoV-2 pandemic. Unfortunately, towards the end of 2020, the number of daily new cases exponentially increased. Herein, we analyzed the country-wide genetic diversity of SARS-CoV-2 between November 2020 and April 2021. We identified that the most prevalent viral variant during the first epidemic wave in Uruguay (December 2020-February 2021) was a B.1.1.28 sublineage carrying Spike mutations Q675H + Q677H, now designated as P.6, followed by lineages P.2 and P.7. P.6 probably arose around November 2020, in Montevideo, Uruguay's capital department, and rapidly spread to other departments, with evidence of further local transmission clusters; it also spread sporadically to the USA and Spain. The more efficient dissemination of lineage P.6 with respect to P.2 and P.7 and the presence of mutations (Q675H and Q677H) in the proximity of the key cleavage site at the S1/S2 boundary suggest that P.6 may be more transmissible than other lineages co-circulating in Uruguay. Although P.6 was replaced by the variant of concern (VOC) P.1 as the predominant lineage in Uruguay since April 2021, the monitoring of the concurrent emergence of Q675H + Q677H in VOCs should be of worldwide interest.

Real-Time Genomic Surveillance for SARS-CoV-2 Variants of Concern, Uruguay.

We developed a genomic surveillance program for real-time monitoring of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) in Uruguay. We report on a PCR method for SARS-CoV-2 VOCs, the surveillance workflow, and multiple independent introductions and community transmission of the SARS-CoV-2 P.1 VOC in Uruguay.

Evaluation of SYBR Green real time PCR for detecting SARS-CoV-2 from clinical samples.

The pandemic caused by SARS-CoV-2 has triggered an extraordinary collapse of healthcare systems and hundred thousand of deaths worldwide. Following the declaration of the outbreak as a Public Health Emergency of International Concern by the World Health Organization (WHO) on January 30th, 2020, it has become imperative to develop diagnostic tools to reliably detect the virus in infected patients. Several methods based on real time reverse transcription polymerase chain reaction (RT-qPCR) for the detection of SARS-CoV-2 genomic RNA have been developed. In addition, these methods have been recommended by the WHO for laboratory diagnosis. Since most of these protocols are based on the use of fluorogenic probes and one-step reagents (cDNA synthesis followed by PCR amplification in the same tube), these techniques can be difficult to perform given the limited supply of reagents in low- and middle-income countries. In order to develop an inexpensive SARS-CoV-2 detection protocol using available resources we evaluated the SYBR Green based detection of SARS-CoV-2 to establish a suitable assay. To do so, we adapted one of the WHO recommended TaqMan-based one-step real time PCR protocols (from the University of Hong Kong) to SYBR Green. Our results indicate that SYBR-Green detection of ORF1b-nsp14 target represents a reliable cost-effective alternative to increase the testing capacity.