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The COVID-19 pandemic has resulted in millions of deaths globally, and while several diagnostic systems were proposed, real-time reverse transcription polymerase chain reaction (RT-PCR) remains the gold standard. However, diagnostic reagents, including enzymes used in RT-PCR, are subject to centralized production models and intellectual property restrictions, which present a challenge for less developed countries. With the aim of generating a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples, we purified and tested recombinant enzymes and a non-proprietary buffer. The protocol utilized M-MLV RT and Taq DNA pol enzymes to perform a Taqman probe-based assay. Synthetic RNA samples were used to validate the One-Step RT-qPCR components, demonstrating sensitivity comparable to a commercial kit routinely employed in clinical settings for patient diagnosis. Further evaluation on 40 clinical samples (20 positive and 20 negative) confirmed its comparable diagnostic accuracy. This study represents a proof of concept for an open approach to developing diagnostic kits for viral infections and diseases, which could provide a cost-effective and accessible solution for less developed countries.

Red macroalgae (Phylum Rhodophyta) are considered to be an important source of biologically active metabolites; their use as functional feed ingredients in fish diets can increase disease resistance and animal growth. Furthermore, red macroalgae can be cultured in a sustainable and environmentally friendly manner. We evaluate the potential antiviral activity against the infectious salmon anemia (ISA) virus of Chilean red macroalgae as a sustainable feed additive in the Atlantic salmon (Salmo salar) diet. Lyophilized Chilean red macroalgae concentrates were elaborated and added to a commercial diet. Diets were prepared with 0.1, 1.0, and 10.0 % of Gracilaria chilensis and Pyropia columbina separately, and 0.1 and 1.0 % of a mix of both species in a 1:1 ratio. The diets were fed to S. salar over a period of 2 months. We collected data on production parameters and blood samples. The serum and its constituents were challenged with the ISA virus in the presence of Atlantic salmon kidney cells and tested ex vivo for antiviral capacity using a plaque reduction neutralization assay. Fish fed a diet containing 10 % G. chilensis showed a significantly higher specific growth rate compared with fish fed the control diet. The feed conversion ratio was not significantly affected by treatments. Sera from fish fed the algae diets showed a significant increase in antiviral activity against the ISA virus compared with sera of fish fed the control diet that did not include red macroalgae. Diets including G. chilensis (1.0 and 10 %) exhibited the largest increase in antiviral activity.

genotype 1 (PRV-1) is an emerging viral pathogen in salmon aquaculture that causes Heart and Skeletal Muscle Inflammation (HSMI), with high prevalence in salmon-producing countries such as Chile. A significant obstacle in PRV-1 vaccine development is the inability to culture the virus in vitro, which limits the scalability and production of traditional inactivated or DNA-based vaccine strategies. This study describes the development of a novel virus-like particle (VLP)-based vaccine against PRV-1. Recombinant VLP were produced by co-expressing the six structural proteins of PRV-1 (λ1, λ2, μ1, σ1, σ2, σ3) using a baculovirus-based expression system in insect cells. In addition, to enable differentiating infected from vaccinated animals (DIVA) strategies, the σ1 protein was modified by adding of a cmyc epitope tag. The results demonstrated that the native VLP vaccine (VLP6n) significantly reduced viral loads in Atlantic salmon challenged with PRV-1. Moreover, in rainbow trout, the cmyc-tagged VLP-like vaccine (VLP6c) elicited a specific antibody response against the cmyc epitope, allowing differentiation between vaccinated and naturally infected fish. Overall, this VLP-based vaccine platform represents a promising strategy for controlling PRV-1 prevalence in salmon-producing counties, supporting the implementation of serological surveillance programs.

Prenylated flavonoids are an important class of naturally occurring flavonoids with important biological activity, but their low abundance in nature limits their application in medicines. Here, we showed the hemisynthesis and the determination of various biological activities of seven prenylated flavonoids, named 7–13, with an emphasis on antimicrobial ones. Compounds 9, 11, and 12 showed inhibitory activity against human pathogenic fungi. Compounds 11, 12 (flavanones) and 13 (isoflavone) were the most active against clinical isolated Staphylococcus aureus MRSA, showing that structural requirements as prenylation at position C-6 or C-8 and OH at positions C-5, 7, and 4′ are key to the antibacterial activity. The combination of 11 or 12 with commercial antibiotics synergistically enhanced the antibacterial activity of vancomycin, ciprofloxacin, and methicillin in a factor of 10 to 100 times against drug-resistant bacteria. Compound 11 combined with ciprofloxacin was able to decrease the levels of ROS generated by ciprofloxacin. According to docking results of S enantiomer of 11 with ATP-binding cassette transporter showed the most favorable binding energy; however, more studies are needed to support this result.

Bacteria in natural environments often encounter high concentrations of metal ions, leading to the development of defense mechanisms such as chemical reduction. This process can result in the formation of nanostructures (NS) ranging from 1–100 nm, which have valuable properties for various applications, including as virucidal agents. Currently, metallic NS with virucidal activity are used in disinfectants and surface protection products. However, their production mainly relies on physical and chemical methods, which are often complex, toxic, and energy-intensive. A sustainable alternative is the biosynthesis of nanostructures. Our research focuses on the biosynthesis of gold nanostructures (AuNS) using environmental bacteria and their proteins, with the aim of exploring their potential as agents to destroy the influenza A virus. We screened bacteria under conditions with HAuCl4, identifying eight microorganisms capable of growing in high gold concentrations. Staphylococcus haemolyticus BNF01 showed the highest resistance and Au(III) reduction, growing up to 0.25 mM in HAuCl4. Bioinformatic analysis revealed five proteins with potential Au(III)-reductase activity, which were cloned and expressed in Escherichia coli. These proteins reduced gold to form AuNPs, which were purified, characterized for size, shape, and surface charge, and tested against influenza A, showing significant virucidal effects, likely due to interactions with viral proteins.

Recombinant vaccines have low-cost manufacturing, regulatory requirements, and reduced side effects compared to attenuated or inactivated vaccines. In the porcine industry, post-weaning multisystemic disease syndrome generates economic losses, characterized by progressive weight loss and weakness in piglets, and it is caused by porcine circovirus type 2 (PCV2). We designed a chimeric antigen (Qm1) to assemble the main exposed epitopes of the Cap-PCV2 protein on the capsid protein of the tobacco necrosis virus (TNV). This design was based on the Cap-N-terminal of an isolated PCV2 virus obtained in Chile. The virus was characterized, and the sequence was clustered within the PCV2 genotype b clade. This chimeric protein was expressed as inclusion bodies in both monomeric and multimeric forms, suggesting a high-molecular-weight aggregate formation. Pigs immunized with Qm1 elicited a strong and specific antibody response, which reduced the viral loads after the PCV2 challenge. In conclusion, the implemented design allowed for the generation of an effective vaccine candidate. Our proposal could be used to express the domains or fragments of antigenic proteins, whose structural complexity does not allow for low-cost production in Escherichia coli. Hence, other antigen domains could be integrated into the TNV backbone for suitable antigenicity and immunogenicity. This work represents new biotechnological strategies, with a reduction in the costs associated with vaccine development.

Antimicrobial resistance is a major global health problem, and, among Gram-positive bacteria, methicillin-resistant Staphylococcus aureus (MRSA) represents a serious threat. MRSA causes a wide range of infections, including bacteremia, which, due to the limited use of β-lactams, is difficult to treat. This study aimed to analyze 51 MRSA isolates collected in 2018 from samples of patients with bacteremia from two hospitals of the Metropolitan Health Service of Santiago, Chile, both in their resistance profile and in the identification of virulence factors. In addition, genomic characterization was carried out by the WGS of an isolate that was shown to be the one of greatest concern (N°. 42) due to its intermediate resistance to vancomycin, multiple virulence factors and being classified as ST8 PVL-positive. In our study, most of the isolates turned out to be multidrug-resistant, but there are still therapeutic options, such as tetracycline, rifampicin, chloramphenicol and vancomycin, which are currently used for MRSA infections; however, 18% were PVL positive, which suggests greater virulence of these isolates. It was determined that isolate N°42 is grouped within the USA300-LV strains (ST8, PVL+, COMER+); however, it has been suggested that, in Chile, a complete displacement of the PVL-negative ST5 clone has not occurred.

Pharmacotherapy has long been used to control viral diseases. However, its success is questionable because its use can negatively impact environmental and human health. An alternative solution is the use of functional foods and diets containing natural products, which tend to be more biodegradable than synthetic molecules and are less likely to generate resistance. Seaweed contains biologically active macronutrients and minerals that offer a natural alternative to synthetic molecules. Red seaweeds, in particular, are a rich source of anti-viral compounds. This study aimed to evaluate the effect of two edible red seaweeds, Pyropia columbina and Gracilaria chilensis cryo concentrates (RSCC), on the gene transcription levels in leukocyte proteins involved in antiviral response (INFγ, Mx, interleukin-6, cathelicidin, and lysozyme). The RSCCs were fed to fish (Salmo salar L.) at concentrations of 0.1, 1, or 10 g kg−1 for 56 days, and blood samples were collected at 8 weeks. The transcription levels of key genes associated with the antiviral response were analyzed by qRT-PCR using leukocyte mRNA as template. The Mx transcript level was significantly decreased (p < 0.05) with the RSCC diets, and lysozyme transcript levels were significantly increased (1 g kg−1P. columbina cryo concentrate). Cathelicidin, interleukin-6, and INFɣ had stable transcription levels. Importantly, RSCC modulated the immune-relevant gene that encodes the Mx antiviral protein in white blood cells.

Antigen cross-presentation is a crucial step in the assembly of an antitumor immune response leading to activation of naïve CD8 T cells. This process has been extensively used in clinical trials, in which dendritic cells generated are loaded with tumor antigens and then autotransplanted to the patients. Recently, the use of autologous transplant of dendritic cells fused with dying tumor cells has demonstrated good results in clinical studies. In this work, we generated a similar process by treating mice with dead tumor cells [cell bodies (CBs)] expressing the fusogenic protein of the infectious salmon anemia virus (ISAV). ISAV fusion protein retains its fusogenic capability when is expressed on mammalian cells and the CBs expressing it facilitates DCs maturation, antigen transfer by antigen-presenting cells, and increase cross-presentation by DCs . Additionally, we observed in the melanoma model that CBs with or without ISAV fusion protein reduce tumor growth in prophylactic treatment; however, only ISAV expressing CBs showed an increase CD4 and CD8 cells in spleen. Overall, our results suggest that CBs could be used as a complement with other type of strategies to amplify antitumor immune response.

Background ISAV is a member of the Orthomyxoviridae family that affects salmonids with disastrous results. It was first detected in 1984 in Norway and from then on it has been reported in Canada, United States, Scotland and the Faroe Islands. Recently, an outbreak was recorded in Chile with negative consequences for the local fishing industry. However, few studies have examined available data to test hypotheses associated with the phylogeographic partitioning of the infecting viral population, the population dynamics, or the evolutionary rates and demographic history of ISAV. To explore these issues, we collected relevant sequences of genes coding for both surface proteins from Chile, Canada, and Norway. We addressed questions regarding their phylogenetic relationships, evolutionary rates, and demographic history using modern phylogenetic methods. Results A recombination breakpoint was consistently detected in the Hemagglutinin-Esterase ( he ) gene at either side of the Highly Polymorphic Region (HPR), whereas no recombination breakpoints were detected in Fusion protein ( f ) gene. Evolutionary relationships of ISAV revealed the 2007 Chilean outbreak group as a monophyletic clade for f that has a sister relationship to the Norwegian isolates. Their tMRCA is consistent with epidemiological data and demographic history was successfully recovered showing a profound bottleneck with further population expansion. Finally, selection analyses detected ongoing diversifying selection in f and he codons associated with protease processing and the HPR region, respectively. Conclusions Our results are consistent with the Norwegian origin hypothesis for the Chilean outbreak clade. In particular, ISAV HPR0 genotype is not the ancestor of all ISAV strains, although SK779/06 (HPR0) shares a common ancestor with the Chilean outbreak clade. Our analyses suggest that ISAV shows hallmarks typical of RNA viruses that can be exploited in epidemiological and surveillance settings. In addition, we hypothesized that genetic diversity of the HPR region is governed by recombination, probably due to template switching and that novel fusion gene proteolytic sites confer a selective advantage for the isolates that carry them. Additionally, protein modeling allowed us to relate the results of phylogenetic studies with the predicted structures. This study demonstrates that phylogenetic methods are important tools to predict future outbreaks of ISAV and other salmon pathogens.