Viral infections trigger cellular stress responses, and host stress proteins play key roles in antiviral defense. Here we identify stress-responsive protein arachidonate lipoxygenase-15 (ALOX15) is a critical component of mitochondrial antiviral innate immunity. Loss of Alox15 impairs mitochondrial antiviral signaling (MAVS)-mediated type I interferon production, resulting in increased susceptibility to influenza virus, an effect reversed by adeno-associated virus-mediated lung delivery of Alox15. ALOX15 translocates to mitochondria in response to H1N1 and other RNA viruses (H3N2 and human coronavirus-229E), independent of its enzymatic activity. This mitochondrial localization is also strikingly observed in peripheral blood mononuclear cells from influenza-infected individuals. Mechanistically, ALOX15 is recruited to mitochondria by polymerized MAVS, displacing the deubiquitinase USP19 and sustaining MAVS K63-linked ubiquitination and aggregation. Leveraging these insights, we developed a synergistic ALOX15 regulation-based therapeutic strategy for influenza infection by combining the ALOX15 transcriptional activator songorine with its enzymatic inhibitor PD146176. Together, our findings establish ALOX15 as an essential component of mitochondrial antiviral immunity and a promising host-directed target for antiviral therapy.
Rebound, arising from an increase in viral load after initial treatment to levels sufficient for renewed infectiousness, has been observed in COVID-19 patients, prompting concerns about patient outcomes and potential implications for transmission and disease control. To assess the impact of viral rebound on post-treatment transmission dynamics, we developed a delay differential model parameterised with characteristics of SARS-CoV-2 infection and the rebound risk observed during treatment with Paxlovid. The model explicitly accounts for the timing of treatment initiation relative to symptom onset and the duration of treatment. By varying the relative transmissibility of individuals experiencing rebound, our simulations show that rebound events comprise only a small proportion of treated cases when treatment initiation is delayed after symptom onset or when treatment duration is extended beyond the standard five-day course. However, as the proportion of symptomatic individuals receiving treatment increases, the transmissibility of rebound infections becomes a dominant factor in disease spread. A short, five-day treatment course can amplify rebound-associated transmission and substantially increase total infections when rebound transmissibility is high. In contrast, extending treatment duration from five to eight days markedly reduces the contribution of rebound cases to overall spread. Both delayed initiation and extended treatment duration decrease the risk of viral rebound, underscoring a trade-off between reducing acute transmission and mitigating post-treatment resurgence. These findings highlight the importance of integrating rebound risk into antiviral deployment strategies to improve patient outcomes and enhance population-level disease control.
Antimicrobial peptides (AMPs) represent promising scaffolds for the development of novel broad-spectrum antiviral therapeutics. Based on structural modeling data, we applied a systematic sequence downsizing approach to the 20-amino-acid AMP Hs-1 from Hypsiboas semilineatus to identify its minimal active region. A series of N- and C-terminally truncated derivatives was synthesized to preserve the predicted amphipathic α-helical core. Circular dichroism showed that the peptides adopt a random-coil conformation in aqueous solution and transition to an α-helical structure in hydrophobic environments, which is essential for membrane disruption. Antiviral screening against enveloped viruses revealed that the peptides interfere with the early stages of infection while maintaining a favorable safety profile. Notably, variant Hs-1[7-20] showed a two-to-four-fold potency enhancement over the parent peptide, indicating the successful removal of a detrimental N-terminal segment. Further optimization via N-terminal acetylation and C-terminal amidation yielded Hs-1[7-20]mod, which exhibited increased antiviral efficacy and serum stability, achieving low-micromolar IC50 values (0.5-0.7 µM) under virus pretreatment conditions while maintaining low toxicity (CC50 > 100 µM). In conclusion, Hs-1 was successfully optimized into a potent, stable, and safe antiviral candidate Hs-1[7-20]mod.
Intrinsic cellular factors that inhibit herpesvirus infection remain incompletely defined. Here, we identify TRIM5α as a restriction factor for herpes simplex virus type 1 (HSV-1). TRIM5α-mediated restriction requires its ubiquitin ligase activity, PRY-SPRY domain, and the ability to oligomerize. Mechanistically, we show that TRIM5α directly engages capsid protein VP19C and promotes the stability of the VP19C-VP23 complex and its nuclear accumulation. VP19C also activates NF-κB synergistically with TRIM5α and independently. HSV-1 counteracts this host defense by triggering proteasome-dependent TRIM5α degradation. In addition, we show that Cyclophilin A (CypA), which is incorporated into HSV-1 virions, also binds to VP19C, but enhances infection. As with HIV-1 and orthopoxviruses, the proviral activity of CypA is disrupted by cyclosporin A (CsA), but unlike the situation with these other viruses, the proviral activity of CypA is independent of TRIM5α. Notably, CsA and its non-immunosuppressive derivatives also exhibit anti-HSV-1 activity in neuronal cell lines, suggesting a potential therapy for HSV-1 encephalitis. TRIM5α and CypA also interact with orthologs of VP19C in other alpha, beta and gamma human herpesviruses. These findings reveal two distinct host pathways acting on the herpesvirus capsid and provide a foundation for comparing how TRIM5α and CypA modulate infection of unrelated virus families, offering new directions to identify shared principles of host recognition and viral evasion.
Non-viral vectors have garnered considerable attention due to their biosafety and low immunotoxicity. However, non-viral gene transfection has been limited in applicability due to low effectiveness of transfection. Among them, a naturally occurring material, chitosan (Chi), was widely studied, but the gene transfection efficiency was very low under physiological pH. Collagen (Col), the most abundant extracellular matrix (ECM), has demonstrated high cellular uptake efficiency, while Chi was recognized for its ability to promote endosomal escape. Here, we hypothesize that conjugating Col with Chi can enhance cellular uptake and hence the transfection efficiency. Specifically, we conjugated Col to Chi through amination, resulting in aminated Col-Chi (aCol-Chi), and compared the cellular uptake and gene transfection efficiency. The conjugation significantly increased the cellular uptake of aCol-Chi, which was 2.8 times higher than that of Chi, while the gene transfection efficiency of aCol-Chi was found 2.5 times higher than that of Chi. Moreover, aCol-Chi showed a higher 3D gene transfection efficiency than that of the aCol and Chi. Additionally, aCol-Chi exhibited potential for BMP2 secretion and its application in promoting osteogenic differentiation. This research highlights the development of an ECM-enhanced gene transfection reagent, aCol-Chi, with improved cellular uptake and gene transfection efficiency, holding potential for applications in gene therapy.
Community-based psychiatric interventions for people who inject drugs (PWID) have been proven to be feasible and efficient in low-middle income countries (LMIC) where psychiatric resources are scarce and stigma important. We aimed to show that, on top of mental health improvement, PWID initially diagnosed with psychiatric symptoms and who received a sustained community-based psychiatric intervention were comparable to a control population of PWID in terms of HIV/HCV exposure. In Hai Phong, Vietnam, PWID currently or previously diagnosed with psychiatric symptoms were invited to be recruited in a 12-month follow-up cohort and proposed a community-based psychiatric and harm reduction intervention supported by peers and were compared after intervention to control PWID free from any psychiatric diagnosis who benefited from harm reduction interventions. HIV/HCV exposure was assessed using a composite score taking into account sex and drug-related risk behaviors, weighted according to the viral exposure risk. Psychiatric symptoms, severity of drug use and quality of life were also assessed at each visit. After a 12-month follow-up between March 2022 and April 2023, viral exposure among PWID diagnosed with psychiatric symptoms was considered significantly non-inferior to that of the control group. Their mental health status was significantly improved and severity of drug use or quality of life did not differ significantly from controls. In LMIC, community-based psychiatric intervention supported by trained peers is feasible and efficient for most dimensions. It may represent a valuable alternative to the classical mental health system.
Hepatitis B virus (HBV), hepatitis C virus (HCV), and metabolic dysfunction-associated steatotic liver disease (MASLD) are common liver diseases. Clinical characteristics and patient-reported outcomes (PROs) of HBV and HCV patients with/without superimposed MASLD were evaluated. Cross-sectional study of clinical and PRO (FACIT-Fatigue [FACIT-F], Chronic Liver Disease Questionnaire [CLDQ], Work Productivity and Activity Impairment [WPAI] questionnaire) data from Global Liver Registry, HBV and HCV patients. MASLD was defined as Hepatic Steatosis Index (HSI) ≥ 36 with ≥ 1 cardiometabolic risk factor (overweight, type 2 diabetes, hypertension, and hyperlipidemia). Among 4649 subjects, 2063 had HBV (48 ± 13 years; 57% male; 11% advanced fibrosis; 49% MASLD); 2586 had HCV (56 ± 14 years; 47% male; 18% advanced fibrosis; 47% MASLD). HBV with/without MASLD were similar in age, sex, and noncardiometabolic comorbidities (all p > 0.05) but those with MASLD had more advanced fibrosis (p < 0.01) and significantly lower PRO scores: FACIT-F Physical Well-Being and Fatigue domains and all CLDQ (all p < 0.01) especially Fatigue domain (6% reduction in score range). HCV patients with MASLD were younger, more frequently female (p < 0.01) with similar rates of advanced fibrosis, biopsy-proven cirrhosis, and noncardiometabolic comorbidities (all p > 0.05). They had significantly lower scores in FACIT-F Physical Well-Being and all CLDQ domains (all p < 0.01); the greatest impairment was in Systemic Symptoms and Worry domains (6% reduction in score range). MASLD was independently associated with lower CLDQ scores in multivariate analysis (p < 0.05). Approximately half of HBV or HCV patients had MASLD and reported significantly worse PROs, highlighting the importance of proactive metabolic assessment and management in patients with chronic viral hepatitis.
Dried blood spot (DBS) testing enables hepatitis C virus (HCV) testing in hard‑to‑reach patient populations but typically lacks full genotype and drug resistance information. This study evaluates a commercially available hybrid‑capture whole‑genome sequencing (WGS) workflow for HCV from DBS to determine suitability for clinical diagnostic and surveillance purposes. We analysed 147 DBS and 24 plasma samples known to be HCV‑RNA positive using the QIAGEN QIAseq xHYB HepC panel. Whole genome sequences were generated using an in-house custom designed iterative bioinformatic pipeline. We assessed coverage characteristics, genotyping accuracy, resistance‑associated mutation (RAM) detection, and analytical sensitivity to determine panel performance. WGS success rates were high for both DBS (95%) and plasma (96%). Genome enrichment was uniform across major HCV genotypes, with no systematic dropouts. Genotyping showed 100% concordance with reference laboratory results including for novel genotype 1 subtypes. RAMs were detected in 27% of samples assessed, with full concordance with reference laboratory results in those with known resistance profiles. Analytical sensitivity was calculated at < 7301 IU/ml for WGS with slightly poorer performance when assessing the NS5a gene in isolation. This protocol reliably generates high‑quality HCV genomes from DBS, enabling accurate genotype, subtype, and resistance profiling. This approach overcomes limitations of venous sampling in hard-to-reach populations and could support decentralised diagnostics and enhancing genomic surveillance in populations underserved by traditional healthcare pathways.
Uterus transplantation (UTx) is an emerging therapeutic option for women with absolute uterine factor infertility, requiring intensive immunosuppression that increases susceptibility to opportunistic infections such as cytomegalovirus (CMV). Evidence guiding CMV management in UTx remains limited. The authors report a case of CMV reactivation in a 34-year-old woman with Mayer-Rokitansky-Küster-Hauser syndrome who underwent successful living-donor UTx. The patient received induction with methylprednisolone and basiliximab, followed by maintenance immunosuppression with tacrolimus, azathioprine, and prednisone. Antiviral prophylaxis with ganciclovir/valganciclovir was administered for three months. After prophylaxis discontinuation, routine surveillance detected CMV DNAemia approximately five months post-transplant, leading to treatment with valganciclovir and temporary postponement of embryo transfer. Following viral clearance, embryo transfer was successfully performed. During early pregnancy, low-level CMV DNAemia recurred. Given concerns about antiviral teratogenicity, management focused on the reduction of immunosuppression, including tacrolimus dose adjustment and discontinuation of azathioprine, with close monitoring via cervical biopsies. CMV DNAemia resolved spontaneously without antiviral therapy, and no rejection episodes or CMV disease occurred. This case highlights the complex balance between infection control and graft preservation in UTx recipients, particularly during pregnancy. It suggests that individualized reduction of immunosuppression may be a safe and effective strategy for managing CMV reactivation when antiviral therapy is contraindicated. Additionally, it supports the concept that the uterine graft may exhibit relatively low immunogenicity. Further studies are needed to define optimal immunosuppressive thresholds and standardized CMV management protocols in this unique transplant population.
Highly pathogenic avian influenza A(H5N1) viruses of clade 2.3.4.4b, genotype D1.1, are responsible for widespread outbreaks in poultry and continue to cause sporadic, sometimes severe, human infections. Herein, we characterized a wild-type (WT) influenza A(H5N1) D1.1 isolate (BC-H5N1-WT) and its H275Y neuraminidase (NA) variant (BC-H5N1-H275Y), both of which emerged on farms in British Columbia, Canada, during the fall 2024 outbreak. In vitro analysis assessed replication kinetics in MDCK cells, with supernatants collected at different days post-infection (p.i.) and titrated by TCID50 and qRT-PCR. Neuraminidase inhibitor (NAI) susceptibility was determined by NA inhibition assays, whereas susceptibility to baloxavir acid (BXA) was evaluated by plaque reduction assay. In vivo virulence was evaluated in BALB/c mice infected with serial 10-fold dilutions of each virus to monitor weight loss and mortality. Viral titers in lungs, brain, nose, kidney, spleen, and heart were quantified at day 4 p.i. The BC-H5N1-WT virus was susceptible to the four antivirals tested, whereas BC-H5N1-H275Y displayed resistance to oseltamivir and peramivir but remained susceptible to zanamivir and BXA. The BC-H5N1-WT exhibited significantly higher viral replication titers than BC-H5N1-H275Y at all tested time points and showed larger plaque sizes. In mice, BC-H5N1-WT was more virulent with LD50 values of 1.78 × 103 PFUs compared to 8.71 × 104 PFUs for BC-H5N1-H275Y, and produced higher viral titers in lungs and other organs. Despite the reduced fitness of the resistant H5N1 D1.1 variant, its emergence in the absence of viral selection pressure underscores the need for continued surveillance.
Food insecurity is highly prevalent among people living with HIV (PLHIV) in Senegal and is associated with poor HIV outcomes. Multisectoral nutrition-sensitive agricultural (MNSA) programs may address interconnected drivers of poor HIV outcomes, but evidence is limited, particularly in West Africa. This pilot study evaluated the perceptions and impact of a MNSA program implemented in Senegal. We conducted a convergent mixed methods study among adults ≥18 years of age, living with HIV and receiving ART in Ziguinchor, Senegal. For the quantitative strand, data from MNSA program participants and historical controls were compared. Quantitative measures included the Household Food Insecurity Access Scale (HFIAS), Body Mass Index (BMI), CD4 cell count, and HIV viral load. Group differences were assessed using Chi-square and Mann-Whitney U tests, and logistic regression was used to examine associations with viral suppression. For the qualitative strand, semi-structured interviews were conducted with MNSA program participants. Qualitative data were analyzed thematically. Data from 56 program participants and 59 controls were included. Viral suppression (<1000 copies/mL) was significantly higher among MNSA participants than controls (94.2% versus 77.8%, p = 0.02). Participants had higher median BMI (24.8 versus 21.7, p < 0.01) and lower prevalence of underweight. Moderate-to-severe food insecurity was lower among program participants, though differences were not statistically significant. Logistic regression showed a trend towards an association between MNSA program participation and viral suppression (OR 3.84; 95% CI 0.97-15.13). Qualitative interviews revealed five major themes: greater autonomy and empowerment, improved mental health, strengthened social cohesion, improved household nutrition and financial stability, and enhanced ART adherence. Findings from this pilot study suggest that the MNSA program is acceptable and may be associated with improvements in viral suppression, nutritional status, food security, and psychosocial well-being among PLHIV. Future studies using a randomized controlled approach are needed to further evaluate impacts, explore mechanisms, and assess potential for scalability.
Orthoflavivirus infection is intricately linked to host cell lipid metabolism, yet the function of bioactive lipids as regulators of infection remains to be elucidated. Here, we investigated the role of lipid mediator pathways, namely ALOX/COX enzymes and upstream lipases, in orthoflavivirus replication by comparing dengue virus (DENV), Zika virus (ZIKV), wild-type yellow fever virus (YFV-Asibi), and the live-attenuated vaccine strain YFV-17D. DENV, ZIKV, and YFV-Asibi, but not the vaccine strain, induced COX2 expression in Huh7 hepatoma cells, correlating with prostaglandin E2 (PGE2) levels in culture supernatants. All four viruses replicated more efficiently in COX2-, ALOX15-, and MGLL-deficient cells, indicating a broadly antiviral role for these enzymes. In contrast, DENV and ZIKV specifically induced ALOX12 expression and depended on ALOX12 for efficient viral RNA replication, as demonstrated by reduced genome copy numbers, altered dsRNA replication compartment morphology, and decreased infectious titers in ALOX12-depleted cells. Direct measurement of lipid peroxidation revealed that ZIKV infection markedly elevated lipid peroxide levels through both ALOX12-dependent and -independent mechanisms, whereas DENV infection did not cause detectable lipid peroxide accumulation. Consistent with this, the ferroptosis inhibitor ferrostatin impaired DENV replication, while the ferroptosis inducer erastin enhanced it; this proviral effect of erastin was fully abolished by ALOX12 knockdown, indicating that DENV depends entirely on ALOX12-driven lipid peroxidation. Iron chelation reduced both DENV and ZIKV infection, confirming a requirement for iron-dependent oxidative processes. The proviral role of lipid peroxidation extended beyond hepatoma cells, as ferrostatin treatment significantly reduced DENV and ZIKV infection in human microglia cells. Our results reveal virus-specific exploitation of lipid peroxidation pathways by orthoflaviviruses and identify ALOX12-dependent lipid peroxidation as a novel proviral mechanism that may represent a target for antiviral intervention.
Early sepsis recognition is challenging due to its heterogeneous presentations and limited rapid diagnostic and prognostic tools. The TriVerityTM Test, a high-multiplex host-response mRNA point-of-care test, supports clinical decision-making by estimating infection likelihood and illness severity. This study assesses TriVerity's accuracy in Dutch emergency department (ED) patients. Adult ED patients with suspected infection and Systemic Inflammatory Response Syndrome score (SIRS) ≥2 or quick Sequential Organ Failure Assessment (qSOFA) ≥2 were prospectively included by the Acutelines data/biobank. The TriVerity test provides three scores: likelihood of bacterial infection, viral infection, and illness severity. Infection status was determined post hoc by independent physicians through clinical consensus and forced adjudication. Illness severity was defined as ICU admission or death within 7 days after ED admission. Among the 467 patients included, 234 (50%) had a bacterial infection, 80 (17%) had a viral infection, 86 (18%) had a bacterial-viral co-infection, and 67 (14%) had no infection. For bacterial infections, the TriVerity assay demonstrated a rule-in specificity of 89-94% and a rule-out sensitivity of 87-92%, depending on the adjudication method (forced vs. consensus). For viral infections, the rule-in specificity was 92-95%, and the rule-out sensitivity ranged from 76 to 92%. The illness severity score showed a rule-in specificity of 83-86% and a rule-out sensitivity of 85-87%. The TriVerity test demonstrated high specificity and sensitivity for both bacterial and viral infections, with high sensitivity and modest positive predictive value in identifying patients at risk of developing severe illness.
Mosquito-borne orthoflavivirus, such as Japanese encephalitis virus (JEV), Dengue virus (DENV), and Zika virus (ZIKV), pose a serious global health threat. As obligate intracellular parasites, they often hijack the host ubiquitin system to modify their own proteins, thereby regulating the viral life cycle, host adaptation, transmission, and pathogenesis. Despite its critical roles, the precise molecular mechanisms and functional significance of viral protein ubiquitination in orthoflavivirus infection remain incompletely understood. Here, we identify JEV prM as a novel target for host-specific ubiquitination, which occurs exclusively in vertebrate hosts but not in mosquitoes. Ubiquitin conjugation at the evolutionarily conserved lysine residues (K107/108/116) in multiple mosquito-borne orthoflaviviruses (USUV, MVEV, and WNV) confers differential adaptation between vertebrate hosts and mosquito vectors. Mechanistically, prM ubiquitination serves as a recruitment signal for the ESCRT-I subunit TSG101, an early-acting component of the ESCRT machinery, which in turn serves as an adaptor to recruit downstream ESCRT components (VPS28, CHMP2A, and CHMP4B), thereby driving viral particle budding. These findings elucidate a novel mechanism by which viral protein ubiquitination regulates JEV infection and host adaptation, and provide important insights into the adaptive evolution of orthoflaviviruses across different hosts and vectors.
The circulation of arboviruses in sub-Saharan African countries remains poorly documented. The associated health burden may be underestimated and masked by the significance of malaria. Here, we have investigated acute undifferentiated fevers for arboviral infections in Mali (2016-2024). To estimate the proportion of patients with arboviral infection, and in particular dengue. A retrospective (2016-2022) and a prospective (2023-2024) studies were conducted in patients from health centers and hospitals of Mali (mainly in the Bamako region) selected by health professionals. Studies included patients with acute fever lasting less than 7 days; the prospective sub-study excluding pyogenic, urinary, tuberculosis, viral hepatitis, typhoid fever and post-traumatic infections. Blood samples were tested for arboviruses using molecular detection (including serotyping) and genomic sequencing. We collected demographic data and results of malaria testing for all patients and, in the prospective study, a set of clinical data. A total of 2,022 patients were included. Dengue virus (DENV) was the most frequently detected pathogen (retrospective study: 7.6%, 16/210 patients; prospective study: 29.5%, 535/1812 patients). We also detected chikungunya virus (n = 7), West Nile virus (n = 2) and Rift Valley fever virus (n = 1). Three serotypes of dengue were identified: DENV-2 (n = 185), DENV-1 (n = 113) and DENV-3 (n = 105); 148 DENV cases could not be typed. For each serotype, phylogenetic analyses identified a major lineage recently originating from the subregion (DENV-1-III; DENV-2-II; DENV-3-III). In contrast to malaria, the dengue detection rate was higher among patients over 18 years of age. The most frequently observed symptoms were headache, asthenia, arthralgia, myalgia and back pain. The mean number of those symptoms per patient was significantly higher in dengue patients. We recorded 6 cases of hemorrhagic dengue, but no deaths and no case requiring transfer to intensive care. Our findings confirm the threat posed by arbovirus infections in Mali, and more specifically the growing burden of dengue fever on public health. Monitoring dengue fever has become a major challenge in sub-Saharan countries in order to determine the conditions necessary for the future implementation of a dengue vaccination policy tailored to the public health objectives of these countries.
Reducing the number of drugs in combined antiretroviral therapy (cART) likely reduces toxicity. We hypothesized that dual therapy (DT) with nevirapine (NVP) and lamivudine (3TC) would be non-inferior to a virtual control without treatment failure. This multicenter study enrolled patients on cART with HIV plasma viral load (pVL) <50 cp/ml for ≥2 years and on NVP for ≥6 months. Patients were compared to a simulated virtual control group with an assumed failure rate of zero. Those with prior Non-Nucleoside Reverse Transcriptase Inhibitor failure or 3TC resistance were excluded. Treatment was simplified to DT with NVP/3TC for 48 weeks, with quarterly pVL-measurements. The primary endpoint was confirmed virologic failure (pVL ≥ 200 cp/mL). A 4% non-inferiority margin and sample size of 201 were set, with a stopping rule if three virologic failures occurred. From April 2019 to January 2023, 201 patients from five centers in Switzerland and Germany started DT, which 194 participants completed. Two patients (1.03%, 95% CI: -0.92% to 3.68%) reached the primary endpoint for failure due to adherence issues. No additional failures were observed during a 12-month post-study follow-up of 184 participants. Simplification to NVP and 3TC was as effective as the ideal virtual control. However, the results of NVP and 3TC maintenance therapy are only applicable to people living with HIV who meet the study's inclusion and exclusion criteria. Virtual controls could improve research efficiency and warrant further evaluation.
Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), imposes a significant metabolic and nutritional burden on the human host. Viral replication requires synthesis of structural proteins (S, E, M, and N), which in turn depends on an adequate supply of amino acids. While the human body can synthesize all nucleotides, it cannot synthesize eight essential amino acids (EAAs), which must be derived from dietary intake or through catabolism of endogenous protein stores such as skeletal muscle and albumin. Despite numerous dietary advisories for COVID-19 patients, including those from international agencies, none are grounded in a biochemical analysis of viral amino acid requirements. This study aimed to determine the amino acid distribution of SARS-CoV-2 structural proteins and to identify nutritionally optimal, economically accessible staple and widely consumed foods capable of counteracting the essential amino acid burden during COVID-19. In this computational study, the amino acid profiling of SARS-CoV-2 structural proteins was performed using bioinformatics analysis of genomic sequences obtained from the GenBank database. The amino acid composition of commonly consumed foods was derived from the Indian Food Composition Tables (IFCT) 2017. Viral amino acid requirements were quantitatively compared with the amino acid profiles of cereals, millets, legumes, nuts, milk, and eggs to assess their suitability in meeting essential amino acid demands. Synthesis of SARS-CoV-2 structural proteins required all 20 amino acids. Essential amino acids constituted 42.8% of the total amino acid requirement, with branched-chain amino acids (valine, leucine, and isoleucine) accounting for approximately 23%. The nutritional load per virion ranged from 1.6% for methionine to 11.3% for leucine. Rice, wheat, maize, legumes, and even expensive nuts like almond, cashew nut, pistachio nuts and walnut were insufficient in meeting the most abundantly required EAAs. In contrast, millet-based diets, particularly combinations of pearl millet with sorghum or foxtail millet, showed superior amino acid complementarity. Cow milk closely matched overall EAA requirements, while foxtail millet supplemented with eggs provided an optimal mixed-diet option. This study provides a biochemical rationale for dietary management of COVID-19 and identifies Indian traditional millets as a nutritionally effective and integrative dietary strategy to mitigate essential amino acid burden during SARS-CoV-2 infection.
Dengue fever is a mosquito-borne viral disease posing a major health challenge in tropical and subtropical regions, including Malaysia, where the climate supports year-round transmission. It affects millions annually, leading to severe illness and hospitalization, while the lack of specific antiviral treatment and the expanding range of Aedes mosquitoes make dengue a persistent global threat. To overcome this issue, this study develops a time delay SEITR-SEI model incorporating two biologically motivated delays representing the human intrinsic and vector extrinsic incubation periods. Firstly, time delay of intrinsic incubation period (IIP) in system captures the dynamics of dengue with time delay in human. The model's dynamics are examined by identifying dengue free and existing equilibria, evaluating the basic reproduction number, and analyzing local and global stability. The dengue free state is stable when the reproduction number is below one; otherwise, a stable endemic state arises. The second delay in the vector compartment represents a delay in the extrinsic incubation period (EIP), which suppresses dengue transmission while allowing the mosquito population to remain sustainable. The model was fitted using real hospital data from Penang General Hospital. These records correspond to patients admitted to Hospital between 2022 and 2023. The model parameters related to the dengue outbreak in this specific area were estimated using the least squares method. Furthermore, using these estimated parameter values, the equilibrium points of the model were also examined through the nonstandard finite difference (NSFD) scheme to ensure numerical stability and dynamic consistency of the system. In addition, a sensitivity analysis was performed to identify the most influential parameters affecting the transmission dynamics of dengue in Penang. Unlike previous studies on simple dengue fever models, this work applies optimal control to a more realistic double-delay model, where graphical comparison showed NSFD outperforming Euler. Simulation results emphasize the importance of biological control strategies, particularly eco-friendly and sustainable approach to limit dengue transmission without eliminating mosquito populations. All over Malaysia, the spread of dengue is nearly same, so the findings provide theoretical insights and practical guidance for dengue control policies in Malaysia.
In recent years, H5 subtype highly pathogenic avian influenza viruses (HPAIVs), especially clade 2.3.4.4b, have posed a global threat to poultry, cattle, and public health. Bioinformatics analysis of H5 subtype AIVs from 2000 to 2023 revealed a progressive increase in the PA-X N193S mutation, which became predominant in both avian and mammalian isolates of clade 2.3.4.4b. Using reverse genetics, we generated viruses with PA-X 193N (rWT) and 193S (rWT-N193S). The PA-X N193S mutation significantly inhibited viral polymerase activity while enhancing host shutoff. In vitro and vivo, rWT-N193S showed attenuated replication in avian and mammalian cells, reduced pathogenicity in mice, and suppressed cytokine storms. However, it enhanced uptake by dendritic cells (DCs), impairing DC maturation, activation, cytokine secretion, and CD4+ T cell proliferation. In murine nasal mucosal experiments, the PA-X N193S mutation reduced CCL5 expression, altered DC recruitment, and suppressed IL-17/MAPK signalling. These findings reveal a viral trade-off: the mutation attenuates epithelial replication and immunopathology but enhances DC uptake and disables mucosal immune functions, a strategy that likely contributed to the global predominance of PA-X N193S mutation in epidemic H5 subtype viruses.
Pathogen diagnosis in rodents through effective health monitoring programs is essential for maintaining high welfare and standards in laboratory animal facilities. Although many animal facilities are increasingly adopting real-time polymerase chain reaction testing of environmental or filter samples for pathogen detection, thereby reducing the need for sentinel animals, classical methods such as microbiological assays and microscopic examination for parasites, remain essential and relevant tools in laboratory animal diagnostics. This article describes protocols for the identification of bacterial, viral, and parasitic agents in mice and rats in accordance with the recommendations of the Federation of European Laboratory Animal Science Associations. The methodologies presented detailed procedures for sample collection from mice and rats and for screening these samples using bacterial, parasitological, and viral panels. In addition, we describe bacterial culture techniques and the use of selective and differential media for pathogen isolation, as well as step-by-step protocols for the detection of pinworms, mites, and parasite eggs. These protocols provide a practical foundation for establishing a basic diagnostic laboratory to support standardized animal husbandry and health monitoring quality. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Gathering rodent samples for pathogen screening Basic Protocol 2: Inoculation and screening of bacterial cultures from rodent samples Support Protocol 1: Identification of bacteria using differential media Basic Protocol 3: Identification of Helicobacter by PCR targeting the 16S rRNA gene Basic Protocol 4: Pinworm screening by microscopy Support Protocol 2: Confirmation and specification of parasites (pinworms and mites) by PCR Basic Protocol 5: Mite screening by microscopy Basic Protocol 6: Virus identification by serology.