Development and Optimization of a Multiplex Real-Time RT-PCR to Detect SARS-CoV-2 in Human Samples.

PCR and its variants (RT-PCR and qRT-PCR) are valuable and innovative molecular techniques for studying nucleic acids. qPCR has proven to be highly sensitive, efficient, and reproducible, generating reliable results that are easy to analyze. During the COVID-19 pandemic, qPCR became the gold standard technique for detecting the SARS-CoV-2 virus that allowed to confirm the infection event, and those asymptomatic ones, and thus save millions of lives. In-house multiplex qPCR tests were developed worldwide to detect different viral targets and ensure results, follow the infections, and favor the containment of a pandemic. Here, we present the detailed fundamentals of the qPCR technique based on fluorogenic probes and processes to develop and optimize a successful multiplex RT-qPCR test for detecting SARS-CoV-2 that could be used to diagnose COVID-19 accurately.

A Comparison of Lymphoid and Myeloid Cells Derived from Human Hematopoietic Stem Cells Xenografted into NOD-Derived Mouse Strains.

Humanized mice are an invaluable tool for investigating human diseases such as cancer, infectious diseases, and graft-versus-host disease (GvHD). However, it is crucial to understand the strengths and limitations of humanized mice and select the most appropriate model. In this study, we describe the development of the human lymphoid and myeloid lineages using a flow cytometric analysis in four humanized mouse models derived from NOD mice xenotransplanted with CD34+ fetal cord blood from a single donor. Our results showed that all murine strains sustained human immune cells within a proinflammatory environment induced by GvHD. However, the Hu-SGM3 model consistently generated higher numbers of human T cells, monocytes, dendritic cells, mast cells, and megakaryocytes, and a low number of circulating platelets showing an activated profile when compared with the other murine strains. The hu-NOG-EXL model had a similar cell development profile but a higher number of circulating platelets with an inactivated state, and the hu-NSG and hu-NCG developed low frequencies of immune cells compared with the other models. Interestingly, only the hu-SGM3 and hu-EXL models developed mast cells. In conclusion, our findings highlight the importance of selecting the appropriate humanized mouse model for specific research questions, considering the strengths and limitations of each model and the immune cell populations of interest.

Humanized Mice for the Study of Dengue Disease Pathogenesis: Biological Assays.

Dengue is one of the most prevalent infectious diseases around the world, present in all continents and mainly affecting developing countries. With few tools to fight and study this disease, it is imperative to have reliable animal models that not only recapitulate human disease but also contain human components to understand the pathogenic mechanism and immune responses, allowing the development of new treatments and vaccines against dengue. Humanized mice are a significant advance in the development of in vivo models to understanding the relation of the human immune system and target organs such as the liver during the infection by dengue virus, allowing basic and preclinical research. In this chapter, we describe the use of humanized NSG mice (huNSG) for the study of dengue disease. The first model describes reconstitution of the human immune system by transplanting human CD34+ stem cells in newborn or adult NSG mice. The second model combines the reconstitution with CD34+ stem cells with the transplant of human primary hepatocytes. This dual reconstituted animal will have two of the major players involved in the development of dengue infection. However, there are still more biological components missing in this model for dengue, but researchers continue working to improve the huNSG model to reconstitute other human components.

Current Trends and Limitations in Dengue Antiviral Research.

Dengue is the most prevalent arthropod-borne viral disease worldwide and affects approximately 2.5 billion people living in over 100 countries. Increasing geographic expansion of Aedes aegypti mosquitoes (which transmit the virus) has made dengue a global health concern. There are currently no approved antivirals available to treat dengue, and the only approved vaccine used in some countries is limited to seropositive patients. Treatment of dengue, therefore, remains largely supportive to date; hence, research efforts are being intensified for the development of antivirals. The nonstructural proteins, 3 and 5 (NS3 and NS5), have been the major targets for dengue antiviral development due to their indispensable enzymatic and biological functions in the viral replication process. NS5 is the largest and most conserved nonstructural protein encoded by flaviviruses. Its multifunctionality makes it an attractive target for antiviral development, but research efforts have, this far, not resulted in the successful development of an antiviral targeting NS5. Increase in structural insights into the dengue NS5 protein will accelerate drug discovery efforts focused on NS5 as an antiviral target. In this review, we will give an overview of the current state of therapeutic development, with a focus on NS5 as a therapeutic target against dengue.

Dengue Infections in Colombia: Epidemiological Trends of a Hyperendemic Country.

Dengue is a major public health problem in hyperendemic countries like Colombia, the understanding of the epidemiological trends is important for the development of efficient public health policies. We conducted a systematic review of the epidemiologic data on dengue in Colombia from 1971 to 2020. A total of 375 relevant citations were identified, 36 of which fulfilled the inclusion criteria. The data of dengue and severe dengue cases, infection fatality rate, and serotype distribution were used to understand and identify gaps in the epidemiological knowledge in Colombia. The epidemiology of dengue in this country was characterized by five main outbreaks in 1998, 2002, 2010, 2013, and 2019 with high fatality rates in comparison with the average values reported in the Americas. The case fatality rate of severe dengue exceeded 2% and all four serotypes co-circulate throughout the country with some regional variations. Overall, the behavior of dengue in Colombia is influenced by multiple factors including seasonal temperature variation and socioeconomic conditions. Additionally, the most important barriers in the epidemiological surveillance of dengue may be due to the insufficient notification rate in some regions and the low active search for the circulation of different serotypes.

Differential replicative fitness of the four dengue virus serotypes circulating in Colombia in human liver Huh7 cells

ABSTRACT Dengue has been a significant public health problem in Colombia since the simultaneous circulation of the four dengue virus serotypes. The replicative fitness of dengue is a biological feature important for virus evolution and contributes to elucidating the behavior of virus populations and viral pathogenesis. However, it has not yet been studied in Colombian isolates. This study aimed to compare the replicative fitness of the four dengue virus serotypes and understand the association between the serotypes, their in vitro infection ability, and their replication in target cells. We used three isolates of each DENV serotype to infect Huh-7 cells at an MOI of 0.5. The percentage of infected cells was evaluated by flow cytometry, cell viability was evaluated by MTT assay, and the pathogenicity index was calculated as a ratio of both parameters. The replicative fitness was measured by the number of viral genome copies produced using quantitative PCR and the production of infectious viral progeny was measured by plaque assay. We showed that Huh-7 cells were susceptible to infection with all the different strain isolates. Nevertheless, the biological characteristics, such as infectious ability and cell viability, were strain-dependent. We also found different degrees of pathogenicity between strains of the four serotypes, representative of the heterogeneity displayed in the circulating population. When we analyzed the replicative fitness using the mean values obtained from RT-qPCR and plaque assay for the different strains, we found serotype-dependent behavior. The highest mean values of replicative fitness were obtained for DENV-1 (log 4.9 PFU/ml) and DENV-4 (log 5.28 PFU/ml), followed by DENV-2 (log 3.9 PFU/ml) and DENV-3 (log 4.31 PFU/ml). The internal heterogeneity of the replicative fitness within each serotype could explain the simultaneous circulation of the four DENV serotypes in Colombia.

Humanized Mice in Dengue Research: A Comparison with Other Mouse Models.

Dengue virus (DENV) is an arbovirus of the Flaviviridae family and is an enveloped virion containing a positive sense single-stranded RNA genome. DENV causes dengue fever (DF) which is characterized by an undifferentiated syndrome accompanied by fever, fatigue, dizziness, muscle aches, and in severe cases, patients can deteriorate and develop life-threatening vascular leakage, bleeding, and multi-organ failure. DF is the most prevalent mosquito-borne disease affecting more than 390 million people per year with a mortality rate close to 1% in the general population but especially high among children. There is no specific treatment and there is only one licensed vaccine with restricted application. Clinical and experimental evidence advocate the role of the humoral and T-cell responses in protection against DF, as well as a role in the disease pathogenesis. A lot of pro-inflammatory factors induced during the infectious process are involved in increased severity in dengue disease. The advances in DF research have been hampered by the lack of an animal model that recreates all the characteristics of this disease. Experiments in nonhuman primates (NHP) had failed to reproduce all clinical signs of DF disease and during the past decade, humanized mouse models have demonstrated several benefits in the study of viral diseases affecting humans. In DENV studies, some of these models recapitulate specific signs of disease that are useful to test drugs or vaccine candidates. However, there is still a need for a more complete model mimicking the full spectrum of DENV. This review focuses on describing the advances in this area of research.

Differential replicative fitness of the four dengue virus serotypes circulating in Colombia in human liver Huh7 cells.

Dengue has been a significant public health problem in Colombia since the simultaneous circulation of the four dengue virus serotypes. The replicative fitness of dengue is a biological feature important for virus evolution and contributes to elucidating the behavior of virus populations and viral pathogenesis. However, it has not yet been studied in Colombian isolates. This study aimed to compare the replicative fitness of the four dengue virus serotypes and understand the association between the serotypes, their in vitro infection ability, and their replication in target cells. We used three isolates of each DENV serotype to infect Huh-7 cells at an MOI of 0.5. The percentage of infected cells was evaluated by flow cytometry, cell viability was evaluated by MTT assay, and the pathogenicity index was calculated as a ratio of both parameters. The replicative fitness was measured by the number of viral genome copies produced using quantitative PCR and the production of infectious viral progeny was measured by plaque assay. We showed that Huh-7 cells were susceptible to infection with all the different strain isolates. Nevertheless, the biological characteristics, such as infectious ability and cell viability, were strain-dependent. We also found different degrees of pathogenicity between strains of the four serotypes, representative of the heterogeneity displayed in the circulating population. When we analyzed the replicative fitness using the mean values obtained from RT-qPCR and plaque assay for the different strains, we found serotype-dependent behavior. The highest mean values of replicative fitness were obtained for DENV-1 (log 4.9 PFU/ml) and DENV-4 (log 5.28 PFU/ml), followed by DENV-2 (log 3.9 PFU/ml) and DENV-3 (log 4.31 PFU/ml). The internal heterogeneity of the replicative fitness within each serotype could explain the simultaneous circulation of the four DENV serotypes in Colombia.