SARS-CoV-2 egress from Vero cells: a morphological approach.

Despite being extensively studied because of the current coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interactions with mammalian cells are still poorly understood. Furthermore, little is known about this coronavirus cycle within the host cells, particularly the steps that lead to viral egress. This study aimed to shed light on the morphological features of SARS-CoV-2 egress by utilizing transmission and high-resolution scanning electron microscopy, along with serial electron tomography, to describe the route of nascent virions towards the extracellular medium. Electron microscopy revealed that the clusters of viruses in the paracellular space did not seem to result from collective virus release. Instead, virus accumulation was observed on incurved areas of the cell surface, with egress primarily occurring through individual vesicles. Additionally, our findings showed that the emission of long membrane projections, which could facilitate virus surfing in Vero cells infected with SARS-CoV-2, was also observed in non-infected cultures, suggesting that these are constitutive events in this cell lineage.

Cost-effective 3D lung tissue spheroid as a model for SARS-CoV-2 infection and drug screening.

BACKGROUND: The SARS-CoV-2 pandemic has spurred an unparalleled scientific endeavor to elucidate the virus' structure, infection mechanisms, and pathogenesis. Two-dimensional culture systems have been instrumental in shedding light on numerous aspects of COVID-19. However, these in vitro systems lack the physiological complexity to comprehend the infection process and explore treatment options. Three-dimensional (3D) models have been proposed to fill the gap between 2D cultures and in vivo studies. Specifically, spheroids, composed of lung cell types, have been suggested for studying SARS-CoV-2 infection and serving as a drug screening platform. METHODS: 3D lung spheroids were prepared by coculturing human alveolar or bronchial epithelial cells with human lung stromal cells. The morphology, size, and ultrastructure of spheroids before and after SARS-CoV-2 infection were analyzed using optical and electron microscopy. Immunohistochemistry was used to detect spike protein and, thus, the virus presence in the spheroids. Multiplex analysis elucidated the cytokine release after virus infection. RESULTS: The spheroids were stable and kept their size and morphology after SARS-CoV-2 infection despite the presence of multivesicular bodies, endoplasmic reticulum rearrangement, tubular compartment-enclosed vesicles, and the accumulation of viral particles. The spheroid responded to the infection releasing IL-6 and IL-8 cytokines. CONCLUSION: This study demonstrates that coculture spheroids of epithelial and stromal cells can serve as a cost-effective infection model for the SARS-CoV-2 virus. We suggest using this 3D spheroid as a drug screening platform to explore new treatments related to the cytokines released during virus infection, especially for long COVID treatment.

Evaluation of SARS-CoV-2 ORF7a Deletions from COVID-19-Positive Individuals and Its Impact on Virus Spread in Cell Culture.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the COVID-19 outbreak, posed a primary concern of public health worldwide. The most common changes in SARS-CoV-2 are single nucleotide substitutions, also reported insertions and deletions. This work investigates the presence of SARS-CoV-2 ORF7a deletions identified in COVID-19-positive individuals. Sequencing of SARS-CoV-2 complete genomes showed three different ORF7a size deletions (190-nt, 339-nt and 365-nt). Deletions were confirmed through Sanger sequencing. The ORF7a∆190 was detected in a group of five relatives with mild symptoms of COVID-19, and the ORF7a∆339 and ORF7a∆365 in a couple of co-workers. These deletions did not affect subgenomic RNAs (sgRNA) production downstream of ORF7a. Still, fragments associated with sgRNA of genes upstream of ORF7a showed a decrease in size when corresponding to samples with deletions. In silico analysis suggests that the deletions impair protein proper function; however, isolated viruses with partial deletion of ORF7a can replicate in culture cells similarly to wild-type viruses at 24 hpi, but with less infectious particles after 48 hpi. These findings on deleted ORF7a accessory protein gene, contribute to understanding SARS-CoV-2 phenotypes such as replication, immune evasion and evolutionary fitness as well insights into the role of SARS-CoV-2_ORF7a in the mechanism of virus-host interactions.

Boosting SARS-CoV-2 detection combining pooling and multiplex strategies.

RT-qPCR is the gold standard technique available for SARS-CoV-2 detection. However, the long test run time and costs associated with this type of molecular testing are a challenge in a pandemic scenario. Due to high testing demand, especially for monitoring highly vaccinated populations facing the emergence of new SARS-CoV-2 variants, strategies that allow the increase in testing capacity and cost savings are needed. We evaluated a RT-qPCR pooling strategy either as a simplex and multiplex assay, as well as performed in-silico statistical modeling analysis validated with specimen samples obtained from a mass testing program of Industry Federation of the State of Rio de Janeiro (Brazil). Although the sensitivity reduction in samples pooled with 32 individuals in a simplex assay was observed, the high-test sensitivity was maintained even when 16 and 8 samples were pooled. This data was validated with the results obtained in our mass testing program with a cost saving of 51.5% already considering the expenditures with pool sampling that were analyzed individually. We also demonstrated that the pooling approach using 4 or 8 samples tested with a triplex combination in RT-qPCR is feasible to be applied without sensitivity loss, mainly combining Nucleocapsid (N) and Envelope (E) gene targets. Our data shows that the combination of pooling in a RT-qPCR multiplex assay could strongly contribute to mass testing programs with high-cost savings and low-reagent consumption while maintaining test sensitivity. In addition, the test capacity is predicted to be considerably increased which is fundamental for the control of the virus spread in the actual pandemic scenario.

Hydroxypropyl-beta-cyclodextrin (HP-BCD) inhibits SARS-CoV-2 replication and virus-induced inflammatory cytokines.

COVID-19 is marked by extensive damage to the respiratory system, often accompanied by systemic manifestations, due to both viral cytopathic effects and hyperinflammatory syndrome. Therefore, the development of new therapeutic strategies or drug repurposing aiming to control virus replication and inflammation are required to mitigate the impact of the disease. Hydroxypropyl-beta-cyclodextrin (HP-BCD) is a cholesterol-sequestering agent with antiviral activity that has been demonstrated against enveloped viruses in in vitro and in vivo experimental models. We also demonstrated that HP-BCD has an immunomodulatory effect, inhibiting the production of selected proinflammatory cytokines induced by microbial products. Importantly, this drug has been used in humans for decades as an excipient in drug delivery systems and as a therapeutic agent in the treatment of Niemann pick C disease. The safety profile for this compound is well established. Here, we investigated whether HP-BCD would affect SARS-CoV-2 replication and virus-induced inflammatory response, using established cell lines and primary human cells. Treating virus or cells with HP-BCD significantly inhibited SARS-CoV-2 replication with a high selective index. A broad activity against distinct SARS-CoV-2 variants was evidenced by a remarkable reduction in the release of infectious particles. The drug did not alter ACE2 surface expression, but affected cholesterol accumulation into intracellular replication complexes, lowering virus RNA and protein levels, and reducing virus-induced cytopathic effects. Virus replication was also impaired by HP-BCD in Calu-3 pulmonary cell line and human primary monocytes, in which not only the virus, but also the production of proinflammatory cytokines were significantly inhibited. Given the pathophysiology of COVID-19 disease, these data indicate that the use HP-BCD, which inhibits both SARS-CoV2 replication and production of proinflammatory cytokines, as a potential COVID-19 therapeutic warrants further investigation.

A SARS-CoV-2 Negative Antigen Rapid Diagnostic in RT-qPCR Positive Samples Correlates With a Low Likelihood of Infectious Viruses in the Nasopharynx.

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) transmission occurs even among fully vaccinated individuals; thus, prompt identification of infected patients is central to control viral circulation. Antigen rapid diagnostic tests (Ag-RDTs) are highly specific, but sensitivity is variable. Discordant RT-qPCR vs. Ag-RDT results are reported, raising the question of whether negative Ag-RDT in positive RT-qPCR samples could imply the absence of infectious viruses. To study the relationship between negative Ag-RDT results with virological, molecular, and serological parameters, we selected a cross-sectional and a follow-up dataset and analyzed virus culture, subgenomic RNA quantification, and sequencing to determine infectious viruses and mutations. We demonstrated that RT-qPCR positive while SARS-CoV-2 Ag-RDT negative discordant results correlate with the absence of infectious virus in nasopharyngeal samples. A decrease in sgRNA detection together with an expected increase in detectable anti-S and anti-N IgGs was also verified in these samples. The data clearly demonstrate that a negative Ag-RDT sample is less likely to harbor infectious SARS-CoV-2 and, consequently, has a lower transmissible potential.

SARS-CoV-2 Variant Determination Through SNP Assays in Samples From Industry Workers From Rio de Janeiro, Brazil.

Since the first reported case in December 2019, SARS-CoV-2 infections have become a major public health worldwide. Even with the increasing vaccination in several countries and relaxing of social distancing measures, the pandemic remains a threat especially due to the emergence of new SARS-CoV-2 variants. Despite the presence of an enzyme capable of proofreading its genome, high rates of replication provide a source of accumulation of mutations within the viral genome. In this retrospective study, samples from a cohort of industry workers tested by the SESI's COVID-19 mass testing program from September 2020 to May 2021 were analyzed using a mutation panel in order to describe the circulation of currently identified SARS-CoV-2 variants within the samples obtained in Rio de Janeiro State. Our results demonstrated that the variant of interest (VOI) Zeta has been in circulation since October 2020 and reached 87% of prevalence in February 2021 followed by a decrease due to the emergence of Gamma variant of concern (VOC). Gamma was detected in January 2021 in our studied population, and its prevalence increased during the following months, reaching absolute prevalence within positive samples in May. The Alpha variant was detected only in 4-7% of samples during March and April while Beta VOC was not detected in our study. Our data agree with sequencing genomic surveillance databases and highlight the importance of continuous mass testing programs and variant detection in order to control viral spread and guide public health measures.

Prolonged SARS-CoV-2 Positivity in Immunocompetent Patients: Virus Isolation, Genomic Integrity, and Transmission Risk.

Current guidelines for patient isolation in COVID-19 cases recommend a symptom-based approach, averting the use of control real-time reverse transcription PCR (rRT-PCR) testing. However, we hypothesized that patients with persistently positive results by RT-PCR for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could be potentially infectious for a prolonged time, even if immunocompetent and asymptomatic, which would demand a longer social isolation period than presently recommended. To test this hypothesis, 72 samples from 51 mildly symptomatic immunocompetent patients with long-lasting positive rRT-PCR results for SARS-CoV-2 were tested for their infectiousness in cell culture. The serological response of samples from those patients and virus genomic integrity were also analyzed. Infectious viruses were successfully isolated from 34.38% (22/64) of nasopharynx samples obtained 14 days or longer after symptom onset. Indeed, we observed successful virus isolation up to 128 days. Complete SARS-COV-2 genome integrity was demonstrated, suggesting the presence of replication-competent viruses. No correlation was found between the isolation of infectious viruses and rRT-PCR cycle threshold values or the humoral immune response. These findings call attention to the need to review current isolation guidelines, particularly in scenarios involving high-risk individuals. IMPORTANCE In this study, we evaluated mildly symptomatic immunocompetent patients with long-lasting positive rRT-PCR results for SARS-CoV-2. Infectious viruses were successfully isolated in cell cultures from nasopharynx samples obtained 14 days or longer after symptom onset. Indeed, we observed successful virus isolation for up to 128 days. Moreover, SARS-CoV-2 genome integrity was demonstrated by sequencing, suggesting the presence of replication-competent viruses. These data point out the risk of continuous SARS-CoV-2 transmission from patients with prolonged detection of SARS-CoV-2 in the upper respiratory tract, which has important implications for current precaution guidelines, particularly in settings where vulnerable individuals may be exposed (e.g., nursing homes and hospitals).

Chikungunya Virus: An Emergent Arbovirus to the South American Continent and a Continuous Threat to the World.

Chikungunya virus (CHIKV) is an arthropod-borne virus (arbovirus) of epidemic concern, transmitted by Aedes ssp. mosquitoes, and is the etiologic agent of a febrile and incapacitating arthritogenic illness responsible for millions of human cases worldwide. After major outbreaks starting in 2004, CHIKV spread to subtropical areas and western hemisphere coming from sub-Saharan Africa, South East Asia, and the Indian subcontinent. Even though CHIKV disease is self-limiting and non-lethal, more than 30% of the infected individuals will develop chronic disease with persistent severe joint pain, tenosynovitis, and incapacitating polyarthralgia that can last for months to years, negatively impacting an individual's quality of life and socioeconomic productivity. The lack of specific drugs or licensed vaccines to treat or prevent CHIKV disease associated with the global presence of the mosquito vector in tropical and temperate areas, representing a possibility for CHIKV to continually spread to different territories, make this virus an agent of public health burden. In South America, where Dengue virus is endemic and Zika virus was recently introduced, the impact of the expansion of CHIKV infections, and co-infection with other arboviruses, still needs to be estimated. In Brazil, the recent spread of the East/Central/South Africa (ECSA) and Asian genotypes of CHIKV was accompanied by a high morbidity rate and acute cases of abnormal disease presentation and severe neuropathies, which is an atypical outcome for this infection. In this review, we will discuss what is currently known about CHIKV epidemics, clinical manifestations of the human disease, the basic concepts and recent findings in the mechanisms underlying virus-host interaction, and CHIKV-induced chronic disease for both in vitro and in vivo models of infection. We aim to stimulate scientific debate on how the characterization of replication, host-cell interactions, and the pathogenic potential of the new epidemic viral strains can contribute as potential developments in the virology field and shed light on strategies for disease control.

HIV-1 genetic diversity and resistance to antiretroviral drugs among pregnant women in Ribeirão Preto (SP), Brazil. Cross-sectional study.

BACKGROUND: Increasing genetic diversity of HIV-1 and emergence of drug-resistant mutations may reduce the efficacy of antiretroviral therapy and prophylaxis that are used to prevent mother-to-child transmission. The aim of this study was to assess the genetic diversity and prevalence of drug-resistant mutations among HIV-infected pregnant women. DESIGN AND SETTING: Cross-sectional study at an outpatient clinic for infectious diseases within gynecology and obstetrics. METHODS: This study evaluated the dynamics of HIV-1 subtypes and the prevalence of transmitted and acquired drug-resistant mutations among 38 HIV-infected pregnant women (20 previously exposed to antiretroviral therapy and 18 naive), in Ribeirão Preto (SP), Brazil, between 2010 and 2011. Genotyping was performed by means of molecular sequencing of the protease and reverse transcriptase regions of the HIV-1 pol gene. RESULTS: Subtype B was identified in 84.2% of the samples, recombinant forms between B and F in 7.9%, subtype F1 in 5.3% and the recombinant form K/F in 2.6%. No mutation associated with transmitted drug resistance was detected in the samples from the naive pregnant women, whereas mutations associated with acquired drug resistance were found in 35.0% of the pregnant women previously exposed to antiretroviral therapy. CONCLUSION: The results showed that subtype B predominated, while there was low prevalence of sequences with transmitted drug resistance.

HIV-1 genetic diversity and resistance to antiretroviral drugs among pregnant women in Ribeirão Preto (SP), Brazil. Cross-sectional study

ABSTRACT BACKGROUND: Increasing genetic diversity of HIV-1 and emergence of drug-resistant mutations may reduce the efficacy of antiretroviral therapy and prophylaxis that are used to prevent mother-to-child transmission. The aim of this study was to assess the genetic diversity and prevalence of drug-resistant mutations among HIV-infected pregnant women. DESIGN AND SETTING: Cross-sectional study at an outpatient clinic for infectious diseases within gynecology and obstetrics. METHODS: This study evaluated the dynamics of HIV-1 subtypes and the prevalence of transmitted and acquired drug-resistant mutations among 38 HIV-infected pregnant women (20 previously exposed to antiretroviral therapy and 18 naive), in Ribeirão Preto (SP), Brazil, between 2010 and 2011. Genotyping was performed by means of molecular sequencing of the protease and reverse transcriptase regions of the HIV-1 pol gene. RESULTS: Subtype B was identified in 84.2% of the samples, recombinant forms between B and F in 7.9%, subtype F1 in 5.3% and the recombinant form K/F in 2.6%. No mutation associated with transmitted drug resistance was detected in the samples from the naive pregnant women, whereas mutations associated with acquired drug resistance were found in 35.0% of the pregnant women previously exposed to antiretroviral therapy. CONCLUSION: The results showed that subtype B predominated, while there was low prevalence of sequences with transmitted drug resistance.