How structural biology has changed our understanding of icosahedral viruses.

Cryo-electron microscopy and tomography have allowed us to unveil the remarkable structure of icosahedral viruses. However, in the past few years, the idea that these viruses must have perfectly symmetric virions, but in some cases, it might not be true. This has opened the door to challenging paradigms in structural virology and raised new questions about the biological implications of "unusual" or "defective" symmetries and structures. Also, the continual improvement of these technologies, coupled with more rigorous sample purification protocols, improvements in data processing, and the use of artificial intelligence, has allowed solving the structure of sub-viral particles in highly heterogeneous samples and finding novel symmetries or structural defects. In this review, I initially analyzed the case of the symmetry and composition of hepatitis B virus-produced spherical sub-viral particles. Then, I focused on Alphaviruses as an example of "imperfect" icosahedrons and analyzed how structural biology has changed our understanding of the Alphavirus assembly and some biological implications arising from these discoveries.

Patterned PVA Hydrogels with 3D Petri Dish® Micro-Molds of Varying Topography for Spheroid Formation of HeLa Cancer Cells: In Vitro Assessment.

Cell spheroids are an important three-dimensional (3D) model for in vitro testing and are gaining interest for their use in clinical applications. More natural 3D cell culture environments that support cell-cell interactions have been created for cancer drug discovery and therapy applications, such as the scaffold-free 3D Petri Dish® technology. This technology uses reusable and autoclavable silicone micro-molds with different topographies, and it conventionally uses gelled agarose for hydrogel formation to preserve the topography of the selected micro-mold. The present study investigated the feasibility of using a patterned Poly(vinyl alcohol) hydrogel using the circular topography 12-81 (9 × 9 wells) micro-mold to form HeLa cancer cell spheroids and compare them with the formed spheroids using agarose hydrogels. PVA hydrogels showed a slightly softer, springier, and stickier texture than agarose hydrogels. After preparation, Fourier transform infrared (FTIR) spectra showed chemical interactions through hydrogen bonding in the PVA and agarose hydrogels. Both types of hydrogels favor the formation of large HeLa spheroids with an average diameter of around 700-800 µm after 72 h. However, the PVA spheroids are more compact than those from agarose, suggesting a potential influence of micro-mold surface chemistry on cell behavior and spheroid formation. This was additionally confirmed by evaluating the spheroid size, morphology, integrity, as well as E-cadherin and Ki67 expression. The results suggest that PVA promotes stronger cell-to-cell interactions in the spheroids. Even the integrity of PVA spheroids was maintained after exposure to the drug cisplatin. In conclusion, the patterned PVA hydrogels were successfully prepared using the 3D Petri Dish® micro-molds, and they could be used as suitable platforms for studying cell-cell interactions in cancer drug therapy.

Streptococcus dentisani inhibits the growth of Candida albicans and Candida glabrata: in vitro assay.

BACKGROUND: Probiotic bacteria inhibit aggregation, biofilm formation, and dimorphism of Candida spp. However, the effects of a new probiotic, Streptococcus dentisani, on the growth of Candida albicans and Candida glabrata biofilms are unknown. OBJECTIVE: To determine the effect of S. dentisani on the different phases of C. albicans and C. glabrata biofilm development. METHODS: Growth quantification and ultrastructural analyses were performed on biofilms of C. albicans ATCC 90028, C. glabrata ATCC 2001, and clinical isolates of C. albicans from oral candidiasis (CA-C1), caries (CA-CR1), and periodontal pocket (CA-P1) treated with cell suspensions of S. dentisani CECT 7746. Cell viability was determined by quantifying colony-forming units (CFU/mL). The ultrastructural analyses were done with atomic force microscopy. RESULTS: S. dentisani induced a significant reduction (p < 0.05) of CFU/mL of immature and mature biofilm in all strains of C. albicans and C. glabrata. Microscopic analysis revealed that S. dentisani reduced C. albicans density in mixed biofilm. The fungus-bacteria interaction affected cell membrane integrity in yeast. CONCLUSION: For the first time, our data elucidate the antifungal effect of S. dentisani on the development of C. albicans and C. glabrata biofilms, supporting its usefulness as a niche-specific probiotic to prevent and treat oral dysbiosis.

Rational design and production of a chimeric antigen targeting Zika virus that induces neutralizing antibodies in mice.

The Zika virus (ZIKV) is considered a public health problem worldwide due to its association with the development of microcephaly and the Guillain-Barré syndrome. Currently, there is no specific treatment or vaccine approved to combat this disease, and thus, developing safe and effective vaccines is a relevant goal. In this study, a multi-epitope protein called rpZDIII was designed based on a series of ZIKV antigenic sequences, a bacterial carrier, and linkers. The analysis of the predicted 3D structure of the rpZDIII chimeric antigen was performed on the AlphaFold 2 server, and it was produced in E. coli and purified from inclusion bodies, followed by solubilization and refolding processes. The yield achieved for rpZDIII was 11 mg/L in terms of pure soluble recombinant protein per liter of fermentation. rpZDIII was deemed immunogenic since it induced serum IgG and IgM responses in mice upon subcutaneous immunization in a three-dose scheme. Moreover, sera from mice immunized with rpZDIII showed neutralizing activity against ZIKV. Therefore, this study reveals rpZDIII as a promising immunogen for the development of a rationally designed multi-epitope vaccine against ZIKV, and completion of its preclinical evaluation is guaranteed.

Metformin Inhibits Zika Virus Infection in Trophoblast Cell Line.

Zika virus (ZIKV) infections have been associated with severe clinical outcomes, which may include neurological manifestations, especially in newborns with intrauterine infection. However, licensed vaccines and specific antiviral agents are not yet available. Therefore, a safe and low-cost therapy is required, especially for pregnant women. In this regard, metformin, an FDA-approved drug used to treat gestational diabetes, has previously exhibited an anti-ZIKA effect in vitro in HUVEC cells by activating AMPK. In this study, we evaluated metformin treatment during ZIKV infection in vitro in a JEG3-permissive trophoblast cell line. Our results demonstrate that metformin affects viral replication and protein synthesis and reverses cytoskeletal changes promoted by ZIKV infection. In addition, it reduces lipid droplet formation, which is associated with lipogenic activation of infection. Taken together, our results indicate that metformin has potential as an antiviral agent against ZIKV infection in vitro in trophoblast cells.

Production and Immunogenicity Assessment of LTp50: An Escherichia coli-Made Chimeric Antigen Targeting S1- and S2-Epitopes from the SARS-CoV-2/BA.5 Spike Protein.

Subunit vaccines stand as a leading approach to expanding the current portfolio of vaccines to fight against COVID-19, seeking not only to lower costs but to achieve long-term immunity against variants of concern and have the main attributes that could overcome the limitations of the current vaccines. Herein a chimeric protein targeting S1 and S2 epitopes, called LTp50, was designed as a convenient approach to induce humoral responses against SARS-CoV-2. LTp50 was produced in recombinant Escherichia coli using a conventional pET vector, recovering the expected antigen in the insoluble fraction. LTp50 was purified by chromatography (purity > 90%). The solubilization and refolding stages helped to obtain a stable protein amenable for vaccine formulation. LTp50 was adsorbed onto alum, resulting in a stable formulation whose immunogenic properties were assessed in BALB/c mice. Significant humoral responses against the S protein (BA.5 variant) were detected in mice subjected to three subcutaneous doses (10 µg) of the LTp50/alum formulation. This study opens the path for the vaccine formulation optimization using additional adjuvants to advance in the development of a highly effective anti-COVID-19 vaccine directed against the antigenic regions of the S protein, which are less prone to mutations.

Modulation of the Altered Intestinal Microbiota by Use of Antibiotics with a Novel Synbiotic on Wistar Rats.

The use of antibiotics unbalances the intestinal microbiota. Probiotics, prebiotics, and synbiotics are alternatives for these unbalances. The effects of a new synbiotic composed of probiotic Saccharomyces boulardii CNCM I-745 and fructans from Agave salmiana (fAs) as prebiotics were assessed to modulate the intestinal microbiota. Two probiotic presentations, the commercial probiotic (CP) and the microencapsulated probiotic (MP) to improve those effects, were used to prepare the synbiotics and feed Wistar rats subjected to antibiotics (AB). Eight groups were studied, including five controls and three groups to modulate the microbiota after the use of antibiotics: G5: AB + MP-synbiotic, G6: AB + CP-synbiotic, and G8: AB + fAs. All treatments were administered daily for 7 days. On days 7 and 21, euthanasia was performed, cecum tissue was recovered and used to evaluate histological analysis and to study microphotograph by TEM, and finally, bacterial DNA was extracted and 16S rRNA gene metabarcode sequencing was performed. Histological analysis showed less epithelial damage and more abundance of the intestinal microbiota in the groups G5, G6, and G8 in comparison with the AB control group after 7 days. Microphotograph of the cecum at 2 weeks post treatment showed that G5 and G6 presented beneficial effects in epithelial reconstruction. Interestingly, in the groups that used the synbiotic without AB (G3 and G4) in addition to contributing to the recovery of the autochthonous microbiota, it promotes the development of beneficial microorganisms; those results were also achieved in the groups that used the synbiotic with AB enhancing the bacterial diversity and regulating the impact of AB.

Ultrastructural and Functional Characterization of Mitochondrial Dynamics Induced by Human Respiratory Syncytial Virus Infection in HEp-2 Cells.

Human respiratory syncytial virus (hRSV) is the leading cause of acute lower respiratory tract infections in children under five years of age and older adults worldwide. During hRSV infection, host cells undergo changes in endomembrane organelles, including mitochondria. This organelle is responsible for energy production in the cell and plays an important role in the antiviral response. The present study focuses on characterizing the ultrastructural and functional changes during hRSV infection using thin-section transmission electron microscopy and RT-qPCR. Here we report that hRSV infection alters mitochondrial morphodynamics by regulating the expression of key genes in the antiviral response process, such as Mfn1, VDAC2, and PINK1. Our results suggest that hRSV alters mitochondrial morphology during infection, producing a mitochondrial phenotype with shortened cristae, swollen matrix, and damaged membrane. We also observed that hRSV infection modulates the expression of the aforementioned genes, possibly as an evasion mechanism in the face of cellular antiviral response. Taken together, these results advance our knowledge of the ultrastructural alterations associated with hRSV infection and might guide future therapeutic efforts to develop effective antiviral drugs for hRSV treatment.

Description and quantification of micronucleus and nuclear abnormalities in erythrocytes of the sentinel green turtle (Chelonia mydas) with fluorescence microscopy.

Contamination in marine ecosystems is of the most critical threats to marine turtles. The identification of useful biomarkers to detect and monitor the physiological and clinical effects of pollutants on these populations will allow early detection of alterations (e.g., mutagenic damages) that could risk their viability or favor the development of diseases, thus threatening the biodiversity of these ecosystems and human population. This study is aimed at describing and quantifying nuclear anomalies in peripheral blood erythrocytes of green turtles (Chelonia mydas) from three distinct foraging areas in Mexico (Akumal, Xcalak, and Punta Herrero). We developed a novel morphological index that could be used as a biomarker to identify abnormal nuclei in peripheral blood erythrocytes. Here we describe for the first time in C. mydas, with a species-specific staining protocol, distinct nuclear abnormalities such as blebbed, lobed, notched, eight shape nuclei, and binucleated cells. These nuclear abnormalities were present in > 90 % of the subjects (n = 30). Moreover, 50 % of the organisms presented erythrocytes with micronuclei. The number of nuclear abnormalities did not correlate with size of the green turtles or differ between sites, or health status. We found a higher frequency of green turtles with nuclear abnormalities in the southern region (Punta Herrero and Xcalak) with the highest frequency of micronucleus and buds. The former could be associated to the constant exposure to chemical pollutants of oceanographic origin in the southern coast of Quintana Roo. Furthermore, the increasing anthropogenic pollution in Akumal could also explain the highest variability in the number of nuclear abnormalities presented in resident individuals. We propose that a long-term monitoring programs of green turtle populations in the Mexican Caribbean that include a micronucleus test could be a useful to determine possible mutagenic damage in these animals.

Astrocytes derived from neural progenitor cells are susceptible to Zika virus infection.

Zika virus (ZIKV) was first isolated in 1947. From its isolation until 2007, symptoms of ZIKV-caused disease were limited (e.g., fever, hives, and headache); however, during the epidemic in Brazil in 2014, ZIKV infection caused Guillain-Barré syndrome in adults and microcephaly in fetuses and infants of women infected during pregnancy. The neurovirulence of ZIKV has been studied using neural progenitor cells (NPCs), brain organoids, neurons, and astrocytes. NPCs and astrocytes appear to be the most susceptible cells of the Central Nervous System to ZIKV infection. In this work, we aimed to develop a culture of astrocytes derived from a human NPC cell line. We analyze how ZIKV affects human astrocytes and demonstrate that 1) ZIKV infection reduces cell viability, increases the production of Reactive Oxygen Species (ROS), and results in high viral titers; 2) there are changes in the expression of genes that facilitate the entry of the virus into the cells; 3) there are changes in the expression of genes involved in the homeostasis of the glutamatergic system; and 4) there are ultrastructural changes in mitochondria and lipid droplets associated with production of virions. Our findings reveal new evidence of how ZIKV compromises astrocytic functionality, which may help understand the pathophysiology of ZIKV-associated congenital disease.

Production and Purification of LTB-RBD: A Potential Antigen for Mucosal Vaccine Development against SARS-CoV-2.

Most of the current SARS-CoV-2 vaccines are based on parenteral immunization targeting the S protein. Although protective, such vaccines could be optimized by inducing effective immune responses (neutralizing IgA responses) at the mucosal surfaces, allowing them to block the virus at the earliest stage of the infectious cycle. Herein a recombinant chimeric antigen called LTB-RBD is described, which comprises the B subunit of the heat-labile enterotoxin from E. coli and a segment of the RBD from SARS-CoV-2 (aa 439-504, carrying B and T cell epitopes) from the Wuhan sequence and the variant of concern (VOC)-delta. Since LTB is a mucosal adjuvant, targeting the GM1 receptor at the surface and facilitating antigen translocation to the submucosa, this candidate will help in designing mucosal vaccines (i.e., oral or intranasal formulations). LTB-RBD was produced in E. coli and purified to homogeneity by IMAC and IMAC-anionic exchange chromatography. The yields in terms of pure LTB-RBD were 1.2 mg per liter of culture for the Wuhan sequence and 3.5 mg per liter for the delta variant. The E. coli-made LTB-RBD induced seric IgG responses and IgA responses in the mouth and feces of mice when subcutaneously administered and intestinal and mouth IgA responses when administered nasally. The expression and purification protocols developed for LTB-RBD constitute a robust system to produce vaccine candidates against SARS-CoV-2 and its variants, offering a low-cost production system with no tags and with ease of adaptation to new variants. The E. coli-made LTB-RBD will be the basis for developing mucosal vaccine candidates capable of inducing sterilizing immunity against SARS-CoV-2.

Production and characterization of chimeric SARS-CoV-2 antigens based on the capsid protein of cowpea chlorotic mottle virus.

The COVID-19 pandemic has highlighted the need for new vaccine platforms to rapidly develop solutions against emerging pathogens. In particular, some plant viruses offer several advantages for developing subunit vaccines, such as high expression rates in E. coli, high immunogenicity and safety, and absence of pre-immunity that could interfere with the vaccine's efficacy. Cowpea chlorotic mottle virus (CCMV) is a model system that has been extensively characterized, with key advantages for its use as an epitope carrier. In the present study, three relevant epitopes from the SARS-CoV-2 Spike protein were genetically inserted into the CCMV CP and expressed in E. coli cultures, resulting in the CCMV1, CCMV2, and CCMV3 chimeras. The recombinant CP mutants were purified from the formed inclusion bodies and refolded, and their immunogenicity as a subunit vaccine was assessed in BALB/c mice. The three mutants are immunogenic as they induce high IgG antibody titers that recognize the recombinant full-length S protein. This study supports the application of CCMV CP as an attractive carrier for the clinical evaluation of vaccine candidates against SARS-CoV-2. Furthermore, it suggests that VLPs assembled from these chimeric proteins could result in antigens with better immunogenicity.

Construction of a Chikungunya Virus, Replicon, and Helper Plasmids for Transfection of Mammalian Cells.

The genome of Alphaviruses can be modified to produce self-replicating RNAs and virus-like particles, which are useful virological tools. In this work, we generated three plasmids for the transfection of mammalian cells: an infectious clone of Chikungunya virus (CHIKV), one that codes for the structural proteins (helper plasmid), and another one that codes nonstructural proteins (replicon plasmid). All of these plasmids contain a reporter gene (mKate2). The reporter gene in the replicon RNA and the infectious clone are synthesized from subgenomic RNA. Co-transfection with the helper and replicon plasmids has biotechnological/biomedical applications because they allow for the delivery of self-replicating RNA for the transient expression of one or more genes to the target cells.

Synthesis and immunogenicity assessment of a gold nanoparticle conjugate for the delivery of a peptide from SARS-CoV-2.

The development of vaccines is a crucial response against the COVID-19 pandemic and innovative nanovaccines could increase the potential to address this remarkable challenge. In the present study a B cell epitope (S461-493) from the spike protein of SARS-CoV-2 was selected and its immunogenicity validated in sheep. This synthetic peptide was coupled to gold nanoparticles (AuNP) functionalized with SH-PEG-NH2 via glutaraldehyde-mediated coupling to obtain the AuNP-S461-493 candidate, which showed in s.c.-immunized mice a superior immunogenicity (IgG responses) when compared to soluble S461-493; and led to increased expression of relevant cytokines in splenocyte cultures. Interestingly, the response triggered by AuNP-S461-493 was similar in magnitude to that induced using a conventional strong adjuvant (Freund's adjuvant). This study provides a platform for the development of AuNP-based nanovaccines targeting specific SARS-CoV-2 epitopes.

The Role of Virus-Like Particles in Medical Biotechnology.

Virus-like particles (VLPs) are protein-based, nanoscale, self-assembling, cage architectures, which have relevant applications in biomedicine. They can be used for the development of vaccines, imaging approaches, drug and gene therapy delivery systems, and in vitro diagnostic methods. Today, three relevant viruses are targeted using VLP-based recombinant vaccines. VLP-based drug delivery, nanoreactors for therapy, and imaging systems are approaches under development with promising outcomes. Several VLP-based vaccines are under clinical evaluation. Herein, an updated view on the VLP-based biomedical applications is provided; advanced methods for the production, functionalization, and drug loading of VLPs are described, and perspectives for the field are identified.

Challenges and Opportunities for the Biotechnology Research Community during the Coronavirus Pandemic.

The coronavirus disease 2019 (COVID-19) pandemic has presented some significant challenges to the scientific community. However, this has also offered opportunities for the pursuit of new scientific activities, and in particular for the field of biotechnology.

Packaging of Genomic RNA in Positive-Sense Single-Stranded RNA Viruses: A Complex Story.

The packaging of genomic RNA in positive-sense single-stranded RNA viruses is a key part of the viral infectious cycle, yet this step is not fully understood. Unlike double-stranded DNA and RNA viruses, this process is coupled with nucleocapsid assembly. The specificity of RNA packaging depends on multiple factors: (i) one or more packaging signals, (ii) RNA replication, (iii) translation, (iv) viral factories, and (v) the physical properties of the RNA. The relative contribution of each of these factors to packaging specificity is different for every virus. In vitro and in vivo data show that there are different packaging mechanisms that control selective packaging of the genomic RNA during nucleocapsid assembly. The goals of this article are to explain some of the key experiments that support the contribution of these factors to packaging selectivity and to draw a general scenario that could help us move towards a better understanding of this step of the viral infectious cycle.