Unveiling the Antiviral Capabilities of Targeting Human Dihydroorotate Dehydrogenase against SARS-CoV-2.

The urgent need for effective treatments against emerging viral diseases, driven by drug-resistant strains and new viral variants, remains critical. We focus on inhibiting the human dihydroorotate dehydrogenase (HsDHODH), one of the main enzymes responsible for pyrimidine nucleotide synthesis. This strategy could impede viral replication without provoking resistance. We evaluated naphthoquinone fragments, discovering potent HsDHODH inhibition with IC50 ranging from 48 to 684 nM, and promising in vitro anti-SARS-CoV-2 activity with EC50 ranging from 1.2 to 2.3 µM. These compounds exhibited low toxicity, indicating potential for further development. Additionally, we employed computational tools such as molecular docking and quantitative structure-activity relationship (QSAR) models to analyze protein-ligand interactions, revealing that these naphthoquinones exhibit a protein binding pattern similar to brequinar, a potent HsDHODH inhibitor. These findings represent a significant step forward in the search for effective antiviral treatments and have great potential to impact the development of new broad-spectrum antiviral drugs.

Dispersion patterns of SARS-CoV-2 variants Gamma, Lambda and Mu in Latin America and the Caribbean.

Latin America and Caribbean (LAC) regions were an important epicenter of the COVID-19 pandemic and SARS-CoV-2 evolution. Through the COVID-19 Genomic Surveillance Regional Network (COVIGEN), LAC countries produced an important number of genomic sequencing data that made possible an enhanced SARS-CoV-2 genomic surveillance capacity in the Americas, paving the way for characterization of emerging variants and helping to guide the public health response. In this study we analyzed approximately 300,000 SARS-CoV-2 sequences generated between February 2020 and March 2022 by multiple genomic surveillance efforts in LAC and reconstructed the diffusion patterns of the main variants of concern (VOCs) and of interest (VOIs) possibly originated in the Region. Our phylogenetic analysis revealed that the spread of variants Gamma, Lambda and Mu reflects human mobility patterns due to variations of international air passenger transportation and gradual lifting of social distance measures previously implemented in countries. Our results highlight the potential of genetic data to reconstruct viral spread and unveil preferential routes of viral migrations that are shaped by human mobility patterns.

Potential of Cricket (Acheta domesticus) Flour as a Lean Meat Replacer in the Development of Beef Patties.

This study examined the incorporation of cricket (Acheta domesticus) flour (CF) (0, control; 5.0%, CF5.0; 7.5%, CF7.5; and 10.0%, CF10.0) as a lean meat replacer in beef patties and its impact on composition, microbiological, sensory, and technological properties, as well as its influence on the cooking process. The inclusion of CF led to beef patties with significantly higher protein levels than the control group. Additionally, an elevation in total viable (TVC) and lactic acid bacteria (LAB) counts was observed. However, Enterobacteriaceae counts remained at safe levels. CF5.0 demonstrated similar sensory scores and purchase intention to the control treatment. CF7.5 and CF10.0 showed comparable sensory scores to the control except for texture attributes. The inclusion of CF significantly reduced cooking loss and diameter reduction values. Beef patties with CF were notably firmer and had a browner color than the control. In general, the cooking process impacted the technological properties similarly in both the control and beef patties with CF. In all cooked samples, no significant differences in pH, redness (a*), or texture were observed. This study demonstrated that incorporating up to 5.0% CF into beef patties is optimal in terms of composition, technological, sensorial, and cooking properties.

The relationships among Leishmania infantum and phyllostomid bats assessed by histopathological and molecular assays.

Bats have been reported as reservoir host of Leishmania spp. worldwide, mostly by molecular detection. However, it is still unclear whether bats act as reservoirs of Leishmania infantum to sandflies vectors. In this sense, the investigation of amastigotes forms in the target organs, and the characterization of their associated inflammation, may help to clarify the epidemiological importance of bats in endemic areas for leishmaniasis. The aim of this work was to investigate the host-parasite relationships under microscopic evaluation and predict the epidemiological role of two phyllostomid bats species naturally infected by L. infantum in an endemic area for human leishmaniasis. Fragments of skin, liver and spleen of L. infantum positive and negative bats (Artibeus planirostris and Carollia perspicillata) by qPCR, were studied by histological and immunohistochemical techniques. Both groups, positive and negative, did not show differences in the histopathological study, presenting only discrete tissue changes. Liver and skin showed mild inflammatory reactions. Findings on spleen consisted of reactivity of the lymphoid follicles, expressive presence of apoptotic cells and macrophages containing abundant phagocytic cells debris. We did not find amastigote forms in tissues by histological and IHC techniques in positive qPCR bats. Our results allow us to hypothesize that phyllostomid bats seem to have an important role in reducing the risk of transmission, possibly acting as dead-end host.

Effectiveness of COVID-19 vaccines against hospitalisation in Latin America during three pandemic waves, 2021-2022: a test-negative case-control design.

Background: Vaccine effectiveness (VE) is essential to monitor the performance of vaccines and generate strategic information to guide decision making. We pooled data from six Latin American countries to estimate the effectiveness of COVID-19 vaccines in preventing laboratory-confirmed SARS-CoV-2 hospitalisation during three different pandemic waves from February 2021 to September 2022. Methods: We used a test-negative case-control design in hospitalised adults in Chile, Costa Rica, Ecuador, Guatemala, Paraguay, and Uruguay. We estimated adjusted VE by age group (18-64 and ≥65 years), vaccine type and product for primary series vaccination and booster vaccination and by time since last dose during the Omicron variant dominant period. We used mixed effects logistic regression models adjusting for sex, age, week of onset of symptom onset and pre-existing conditions with country fit as a random effect term. Findings: We included 15,241 severe acute respiratory infection (SARI) patients in the analysis. Among adults 18-64 years, VE estimates for primary series vaccination during pre-Delta and Delta periods ranged by product from 66.5% to 95.1% and from 33.5% to 88.2% for older adults. During the Omicron period, VE estimates for primary series were lower and decreased by time since last vaccination, but VE increased to between 26.4% and 57.4% when a booster was administered. Interpretation: mRNA and viral vector vaccines presented higher VE for both primary series and booster. While VE decreased over time, protection against severe COVID-19-associated hospitalisation increased when booster doses were administered. Vaccination with additional doses should be recommended, particularly for persons at increased risk of developing severe COVID-19. Funding: This work was supported by a grant from the U.S. Centers for Disease Control and Prevention (CDC) through cooperative agreements with the Pan American Health Organization/World Health Organization.

Effect of Heat Treatment on Protein Self-Digestion in Ruminants' Milk.

This study investigated whether heat treatments (raw, 63 °C for 30 min, and 85 °C for 5 min) affect protein hydrolysis by endogenous enzymes in the milk of ruminants (bovine, ovine, and caprine) using a self-digestion model. Self-digestion consisted of the incubation for six hours at 37 °C of the ruminants' milk. Free amino group concentration was measured by the o-phthaldialdehyde method, and peptide sequences were identified by chromatography-mass spectrometry. Results showed that heat treatments prior to self-digestion decreased the free NH2 by 59% in bovine milk heated at 85 °C/5 min, and by 44 and 53% in caprine milk heated at 63 °C/30 min and 85 °C/5 min, respectively. However, after self-digestion, only new free amino groups were observed for the raw and heated at 63 °C/30 min milk. ß-Casein was the most cleaved protein in the raw and heated at 63 °C/30 min bovine milk. A similar trend was observed in raw ovine and caprine milk. Self-digestion increased 6.8-fold the potential antithrombin peptides in the bovine milk heated at 63 °C/30 min. Enhancing bioactive peptide abundance through self-digestion has potential applications in the industry for functional products. Overall, heat treatments affected the free amino groups according to the species and heat treatment applied, which was reflected in the varying degrees of cleaved peptide bonds and peptides released during self-digestion.

Histopathological, immunohistochemical and molecular analyses of foetuses from a female dog naturally infected by Leishmania (Leishmania) infantum - Case report.

This report presents anatomo-histopathological, immunohistochemical and molecular analyses of an asymptomatic female dog naturally infected with Leishmania (Leishmania) infantum and her four pups. In addition to routine serological tests, the mother's positivity was confirmed by real-time PCR of a bone marrow sample; the placenta and ovaries, however, were negative with the same analysis. The pups presented no positivity in any of the techniques used. It is suggested that the gestational period and factors related to the female's immunological condition cause greater variability in the occurrence of vertical infection.

Establishment of a new equation to estimate gestational age in female dogs.

The objectives of this study were to propose a statistical model to predict the behaviour of the thickness of the uteroplacental junction as a function of the gestation period in female dogs and to determine the relationship between the thickness of the placenta and gestational age in healthy female dogs whose pregnancies had elapsed without maternal-fetal alterations. Eight Border Collie female dogs were selected, aged 3-6 (4.48 ± 0.89) and weighing 16-22 kg (19.06 ± 1.9 kg). Female dogs with gestational ages from 20 to 62 days were examined weekly using B-mode ultrasonography. Ultrasound measurements of the uteroplacental junction were organized into four distinct groups: GT1 (27-36 days of gestation), GT2 (37-46 days of gestation), GT3 (47-56 days of gestation) and GT4 (57-62 days of gestation). Based on multiple linear regression, a statistical model was proposed to predict the behaviour of the thickness of the uteroplacental junction (y) as a function of the length of gestation (x) in female dogs, where b0 is the intercept (linear coefficient) and bp is the slope of the predictors. The analysis relating GT, age and weight could predict placental thickness and resulted in a statistically significant model [F(1,28) = 153,736; p < .001; R2 = .846], but only that relating the length of pregnancy (ß = .92; t = 12.399; p < .001) predicted the thickness of the placenta according to the equation y = b0 + bp.x1 [(thickness in cm) = ß -0.3 + 0.019 × (gestation time in days)]. Only in GT4 was there no correlation between placentas within the same pregnancy (p > .05). Based on the close relationship between the development of the uteroplacental junction thickness during pregnancy and gestational age, it is possible to develop a new tool to complement gestational ultrasound evaluation in female dogs. This is important because it allows better placental evaluation in the search for significant alterations that could compromise maternal-fetal health.

In vitro bleaching efficacy of violet LED associated with 10% hydrogen peroxide and 10% carbamide peroxide.

BACKGROUND: This in vitro study evaluated the efficacy and the effect over the dental enamel surface of violet LED dental bleaching associated to different concentrations of carbamide and hydrogen peroxide. METHODS: Human dental blocks (n = 100) were randomly distributed into 5 groups: 10% hydrogen peroxide (HP10), 10% carbamide peroxide (CP10), 10% hydrogen peroxide with violet LED (VHP10), 10% carbamide peroxide with violet LED (VCP10) and 35% hydrogen peroxide (HP35). The specimens were analyzed by Vickers microhardness test (n = 50) initially, immediately after and seven days after ending the bleaching protocol. For color analysis (n = 50), the specimens were evaluated for bleaching effectiveness (ΔE2000, ΔE1976) and whiteness index (ΔWID) with EasyShade spectrophotometer, before bleaching protocol and seven days after ending the bleaching protocol. The microhardness and color data were analyzed using one-way ANOVA with post-hoc Tukey test (α = 0.05). RESULTS: The microhardness values showed difference among the investigated groups only immediately after the end of the dental bleaching (p < 0.05), with reduction for the groups HP35 (p < 0.01) and HP10 (p < 0.05), however the microhardness values were reestablished after seven days. Regarding the color changes, a difference between VHP10 and the others groups evaluated for ΔE2000 and ΔE1976 index was observed (p < 0.05). For ΔWID, there was no difference between the studied groups. CONCLUSIONS: Violet LED associated with low concentration bleaching agents did not show a negative effect on dental enamel regarding the surface microhardness. All bleaching protocols were effective, therefore, perceptible to human eyes.

The Influence of the Omicron Variant on RNA Extraction and RT-qPCR Detection of SARS-CoV-2 in a Laboratory in Brazil.

The emergence of SARS-CoV-2 variants can affect their detection via RT-qPCR. The Omicron variant has a greater affinity for the upper respiratory system and causes clinical conditions similar to bronchitis, as opposed to the pneumonitis-like conditions caused by other SARS-CoV-2 variants. This characteristic increases the viscosity of clinical samples collected for diagnosis. Coinciding with the arrival of the Omicron variant, we observed a failure in control gene expression in our laboratory. In this report, we have optimized a rapid nucleic acid extraction step to restore gene expression and detect the presence of the SARS-CoV-2 virus. We reevaluated 3000 samples, compared variant types occurring in different time periods, and confirmed that the presence of the Omicron variant was responsible for changes observed in the characteristics of these clinical samples. For samples with large amounts of mucus, such as those containing the Omicron variant, a modification to the nucleic acid extraction step was sufficient to restore the quality of RT-qPCR results.

The impact of heating and drying on protease activities of ruminant milk before and after in vitro infant digestion.

This study investigated the effect of heating (63°C/30 min or 75°C/15 s) and drying (spray-drying or freeze-drying) on plasmin, cathepsin D, and elastase activities in bovine, ovine, and caprine milk, compared to non-dried raw milk counterparts. Protease activities and protein hydrolysis were assessed before and after in vitro infant digestion with or without gastric and pancreatic enzymes. At 75°C/15 s, plasmin activity in caprine and ovine milk decreased (69-75%, p<0.05), while cathepsin D activity in spray-dried bovine milk heated increased (2.8-fold, p<0.05). Plasmin and cathepsin D activities increased (<1.2-fold, p<0.05) after in vitro digestion with pancreatin, regardless of milk species. Endogenous milk enzymes hydrolyzed more proteins than gastric enzymes during gastric digestion and contributed to small intestinal digestion. In summary, milk proteases remained active after processing with effects dependent on the species of milk, and they contributed to in vitro protein hydrolysis in the stomach and small intestine.

Rapid Epidemic Expansion of Chikungunya Virus East/Central/South African Lineage, Paraguay.

The spread of Chikungunya virus is a major public health concern in the Americas. There were >120,000 cases and 51 deaths in 2023, of which 46 occurred in Paraguay. Using a suite of genomic, phylodynamic, and epidemiologic techniques, we characterized the ongoing large chikungunya epidemic in Paraguay.

Strengthening pathogen genomic surveillance for health emergencies: insights from the World Health Organization's regional initiatives.

The onset of the COVID-19 pandemic triggered a rapid scale-up in the use of genomic surveillance as a pandemic preparedness and response tool. As a result, the number of countries with in-country SARS-CoV-2 genomic sequencing capability increased by 40% from February 2021 to July 2022. The Global Genomic Surveillance Strategy for Pathogens with Pandemic and Epidemic Potential 2022-2032 was launched by the World Health Organization (WHO) in March 2022 to bring greater coherence to ongoing work to strengthen genomic surveillance. This paper describes how WHO's tailored regional approaches contribute to expanding and further institutionalizing the use of genomic surveillance to guide pandemic preparedness and response measures as part of a harmonized global undertaking. Challenges to achieving this vision include difficulties obtaining sequencing equipment and supplies, shortages of skilled staff, and obstacles to maximizing the utility of genomic data to inform risk assessment and public health action. WHO is helping to overcome these challenges in collaboration with partners. Through its global headquarters, six regional offices, and 153 country offices, WHO is providing support for country-driven efforts to strengthen genomic surveillance in its 194 Member States, with activities reflecting regional specificities. WHO's regional offices serve as platforms for those countries in their respective regions to share resources and knowledge, engage stakeholders in ways that reflect national and regional priorities, and develop regionally aligned approaches to implementing and sustaining genomic surveillance within public health systems.

Retrospective Spatio-Temporal Dynamics of Dengue Virus 1, 2 and 4 in Paraguay.

Dengue virus (DENV) has been a major public health concern in Paraguay, with frequent outbreaks occurring since early 1988. Although control measures have been implemented, dengue remains a significant health threat in the country, and continued efforts are required for prevention and control. In response to that, in collaboration with the Central Public Health Laboratory in Asunción, we conducted a portable whole-genome sequencing and phylodynamic analysis to investigate DENV viral strains circulating in Paraguay over the past epidemics. Our genomic surveillance activities revealed the co-circulation of multiple DENV serotypes: DENV-1 genotype V, the emerging DENV-2 genotype III, BR4-L2 clade, and DENV-4 genotype II. Results additionally highlight the possible role of Brazil as a source for the international dispersion of different viral strains to other countries in the Americas emphasizing the need for increased surveillance across the borders, for the early detection and response to outbreaks. This, in turn, emphasizes the critical role of genomic surveillance in monitoring and understanding arbovirus transmission and persistence locally and over long distances.

Rapid epidemic expansion of chikungunya virus-ECSA lineage in Paraguay.

The spread of vector-borne viruses, such as CHIKV, is a significant public health concern in the Americas, with over 120,000 cases and 51 deaths in 2023, of which 46 occurred in Paraguay. Using a suite of genomic, phylodynamic, and epidemiological techniques, we characterized the ongoing large CHIKV epidemic in Paraguay. Article Summary Line: Genomic and epidemiological characterization of the ongoing Chikungunya virus epidemic in Paraguay.

A rare case of primary hepatic poorly differentiated leiomyosarcoma in a goat.

A 12-year-old Saanen goat presented with a history of hyporexia and sudden recumbency. Euthanasia was indicated due to suspicion of hepatic neoplasia associated with senility. Necropsy revealed generalized oedema and increased liver size and weight (33 × 38 × 17 cm and 10.6 kg, respectively), with a firm, multilobular mass. Fusiform to polygonal neoplastic cells, with marked pleomorphism, anisocytosis and anisokaryosis, were seen on histopathological examination of the hepatic mass. The neoplastic cells were immunohistochemically positive for alpha-smooth muscle actin and vimentin but immunonegative for pancytokeratin. The Ki-67 index was 18.8%. A poorly differentiated leiomyosarcoma was diagnosed on the basis of the gross, histopathological and immunohistochemical findings and should be included in the differential diagnosis of liver disease in goats.

Evaluation of biometry and blood concentration of heavy metals in free-living Chelonia mydas with and without fibropapillomatosis in southern Brazil.

The present study determined the presence of heavy metals in green sea turtles with and without fibropapillomatosis in Itapirubá and Cassino beaches, southern Brazil. The weight, curved length of the carapace and body index were determined and blood was collected to quantify the concentrations of cadmium, lead and mercury. A total of 51 blood samples were analyzed, being 46 positives for at least one of the metals. There was a greater number of juvenile turtles in Itapirubá, larger in size and weight than those in Cassino. No statistical differences were found between metal concentrations between regions or between turtles with and without fibropapillomatosis. There was no significant correlation between metal concentrations, biometric variables and the presence of fibropapilomatose. Metal concentrations were low in both regions, with the highest concentrations being Cd and the lowest Hg.

Results from the second WHO external quality assessment for the molecular detection of respiratory syncytial virus, 2019-2020.

Background: External quality assessments (EQAs) for the molecular detection of human respiratory syncytial virus (RSV) are necessary to ensure the standardisation of reliable results. The Phase II, 2019-2020 World Health Organization (WHO) RSV EQA included 28 laboratories in 26 countries. The EQA panel evaluated performance in the molecular detection and subtyping of RSV-A and RSV-B. This manuscript describes the preparation, distribution, and analysis of the 2019-2020 WHO RSV EQA. Methods: Panel isolates underwent whole genome sequencing and in silico primer matching. The final panel included nine contemporary, one historical virus and two negative controls. The EQA panel was manufactured and distributed by the UK National External Quality Assessment Service (UK NEQAS). National laboratories used WHO reference assays developed by the United States Centers for Disease Control and Prevention, an RSV subtyping assay developed by the Victorian Infectious Diseases Reference Laboratory (Australia), or other in-house or commercial assays already in use at their laboratories. Results: An in silico analysis of isolates showed a good match to assay primer/probes. The panel was distributed to 28 laboratories. Isolates were correctly identified in 98% of samples for detection and 99.6% for subtyping. Conclusions: The WHO RSV EQA 2019-2020 showed that laboratories performed at high standards. Updating the composition of RSV molecular EQAs with contemporary strains to ensure representation of circulating strains, and ensuring primer matching with EQA panel viruses, is advantageous in assessing diagnostic competencies of laboratories. Ongoing EQAs are recommended because of continued evolution of mismatches between current circulating strains and existing primer sets.

Ezrin immunoexpression in gastric cells of domestic cats infected with Helicobacter spp.

The aim of this study was to evaluate de immunoexpression of ezrin in gastric cells of domestic cats infected with Helicobacter spp. and with chronic gastritis. Twenty paraffin-embedded gastric samples were selected based on previous positive results for Helicobacter spp. in the Rapid Urease Test, Warthin-Starry staining and cytology. Haematoxylin-eosin stained sections was done to evaluate inflammatory cell infiltrates. Immunohistochemical analysis was done using anti-Helicobacter pylori and anti-Ezrin antibodies. The analysis of inflammatory infiltrates revealed 8/20 (40%) in score 0, 11/20 (55%) in score 1 and 1/20 (5%) in score 2. The labelling observed in the immunohistochemical analysis using anti-Helicobacter spp. antibody showed no samples with score 0; 4/20 (20%) with score 1; 7/20 35% with score 2 and 9/20 (45%) with score 3. Ezrin overexpression on the cytoplasm of parietal cells was revealed in 18 out of 20 samples (90%). Of these, 10 cases (45%) achieved the score 1; 6 cases (30%) the score 2 and 2 cases (10%) the score 3. On the surface and pit cells there was an increase in Ezrin immnoexpression in 12 out of the 20 samples (60%), of which 8 samples (40%) achieved the score 1 and 4 samples (20%) the score 2. No sample were classified in score 3. Statistically significant differences (p = 0.026) were observed between the inflammatory infiltrate in the gastric mucosa and the immunoexpression of Ezrin in the cytoplasm of parietal cells. It was concluded that ezrin had an increased immunoexpression in the gastric mucosa of cats with chronic gastritis.