Effect of Proteolytic Processing at Two Distinct Sites on Shape and Aggregation of an Anchorless Fusion Protein of Human Respiratory Syncytial Virus and Fate of the Intervening Segment

AbstractWe have examined the consequences of cleaving the fusion glycoprotein (F) of human respiratory syncytial virus (HRSV) at two distinct furin-recognition sites. Purified anchorless F is a mixture of unaggregated cone-shaped molecules and rosettes of lollipop-shaped spikes. The unaggregated molecules contain a proportion of uncleaved F0 and an intermediate, FΔ1–109, cleaved only at site I, residues 106–109. Inhibition of cleavage at site I, by two amino acid changes (R108N/R109N), reduces the proportion of aggregated molecules with a concomitant increase in the amount of unprocessed F0. Inhibition of cleavage at site II, residues 131–136, by deletion of four amino acids (Δ131–134), abrogates aggregation of anchorless F and all molecules are seen as individual cone-shaped rods. In vitro cleavage of anchorless F, or mutant Δ131–134, with trypsin at 4, 20, or 37°C, under conditions in which cleavage at site II is complete in all molecules, leads to their aggregation in rosettes of lollipop-shaped spikes. Thus, cleavage at site II is required for the structural changes in anchorless F that lead to changes in shape and to aggregation. The segment between sites I and II, residues 110–136, is not associated with anchorless F in the supernatant of infected cell cultures, indicating that it is released from the processed protein when cleavage at sites I and II is completed

Identification of Unique Blood and Urine Biomarkers in Influenza Virus and Staphylococcus aureus Co-infection: A Preliminary Study

Each year, there are estimated to be approximately 200,000 hospitalizations and 36,000 deaths due to influenza in the United States. Reports have indicated that most deaths are not directly due to influenza virus, but to secondary bacterial pneumonia, predominantly staphylococcal in origin. Here we identify the presence of candidate blood and urine biomarkers in mice with Staphyococcus aureus and influenza virus co-infection. In this pilot study, mice were grouped into four treatments: co-infected with influenza virus and S. aureus, singly infected with influenza virus or S. aureus, and a control group of uninfected mice (PBS treated). Gene expression changes were identified by DNA-microarrays from blood samples taken at day five post infection. Proteomic changes were obtained from urine samples collected at three and five days post infection using 2-D DIGE followed by protein ID by mass spectrometry. Differentially expressed genes and/or proteins were identified as candidate biomarkers for future validation in larger studies

Field-validated multiplex RT-qPCR for simultaneous detection of bovine respiratory syncytial virus and bovine parainfluenza virus-3 in bovine respiratory samples

Bovine respiratory syncytial virus (BRSV) and bovine parainfluenza virus Type 3 (BPIV3) are ubiquitous respiratory pathogens of cattle, contributing significantly to the bovine respiratory disease (BRD) complex. Rapid and reliable detection methods are essential to mitigate economic losses and improve animal welfare. This study aimed to develop and validate sensitive and specific reverse transcriptase quantitative real-time PCR (RT-qPCR) assays for the simultaneous detection of BRSV and BPIV3 in bovine respiratory samples. Primers and dual-labeled probes were designed from GenBank sequences targeting conserved regions of the BRSV N gene and BPIV3 NP gene and optimized for sensitivity and specificity. The assays were evaluated using reference strains, field isolates, and clinical samples. Analytical sensitivity was established through serial dilutions of in vitro transcribed RNA and confirmed by probit regression analysis, yielding LOD95 values of 164 genome copies for BRSV and 359 genome copies for BPIV3. The assays demonstrated high specificity (no cross-reactivity with non-target bovine respiratory viruses), and reproducibility (CV < 5%). Standard curves demonstrated strong linearity (R2 > 0.99) with amplification efficiencies of 104.2% for BRSV and 81.6% for BPIV3. Diagnostic performance was evaluated on 100 clinical samples, with monoplex RT-qPCR detecting BRSV in 19% and BPIV3 in 11% of cases, outperforming virus isolation. The multiplex assay detected 17% BRSV and 7% BPIV3 positives of cases in a single reaction. Compared to traditional virus isolation, the RT-qPCR assays detected 2.4 × more BRSV and reliably identified BPIV3-positive cases that were otherwise missed. These assays offer a robust diagnostic solution for high-throughput screening in clinical and surveillance settings

Interaction of the human respiratory Syncytial virus matrix protein with cellular adaptor protein complex 3 plays a critical role in trafficking

Human Respiratory Syncytial Virus (HRSV) is a leading cause of bronchopneumonia in infants and the elderly. To date, knowledge of viral and host protein interactions within HRSV is limited and are critical areas of research. Here, we show that HRSV Matrix (M) protein interacts with the cellular adaptor protein complex 3 specifically via its medium subunit (AP-3Mu3A). This novel protein-protein interaction was first detected via yeast-two hybrid screen and was further confirmed in a mammalian system by immunofluorescence colocalization and co-immunoprecipitation. This novel interaction is further substantiated by the presence of a known tyrosine-based adaptor protein MU subunit sorting signal sequence, YXXФ: where Ф is a bulky hydrophobic residue, which is conserved across the related RSV M proteins. Analysis of point-mutated HRSV M derivatives indicated that AP-3Mu3A- mediated trafficking is contingent on the presence of the tyrosine residue within the YXXL sorting sequence at amino acids 197-200 of the M protein. AP-3Mu3A is up regulated at 24 hours post-infection in infected cells versus mock-infected HEp2 cells. Together, our data suggests that the AP-3 complex plays a critical role in the trafficking of HRSV proteins specifically matrix in epithelial cells. The results of this study add new insights and targets that may lead to the development of potential antivirals and attenuating mutations suitable for candidate vaccines in the future

Increased Survivorship and Altered Cytokine Profile from Treatment of Influenza A H1N1-Infected Mice with Ekybion: A Drug Complex of Natural Extracts and Inorganic Compounds

Ekybion is a drug complex of 16 natural extracts and inorganic compounds designed to treat a variety of respiratory pathogens of bacterial and viral origin. It is licensed throughout Europe for the treatment of respiratory tract infections from equine parainfluenza type 3 and equine herpes virus type 1 in equine stables. The purpose of this paper was to test the efficacy of Ekybion on a well-developed animal model of influenza A infection and determine a mode of action. Experiments were performed with Balb/c mice infected with a lethal dose of influenza A/PR/8/34 H1N1 virus and treated with nebulized Ekybion every 8 h in a time-dependant or dose-dependant fashion. These experiments showed that mice treated prior to infection with Ekybion had a higher survival rates (~46%) compared with untreated animals (~0%). Paradoxically, these mice showed no significant difference in lung virus titer or weight loss. There was, however, a decrease in the level of GM-CSF, IL-6, and G-CSF cytokines in the lungs of Ekybion-treated, infected mice. It is possible that decreases in proinflammatory cytokines may have contributed to increased survivorship in Ekybion-treated influenza-infected mice

Confirmation of interaction of a novel HIV NS2 Protein with host proteins in living mammalian cells

NS2 is a novel HIV protein confirmed to be expressed from an open reading frame alongside the Pol open reading frame. Furthermore, NS2 has been shown to be critical to viral replication and to localize in the nucleolus of the nucleus. The mechanism of NS2 function is currently unknown. A yeast-2-hybrid assay (Y2H) yielded a number of possible mammalian protein interactions with NS2. Bimolecular fluorescence complementation (BiFC) is utilized to confirm these positive interactions. The first step to performing a BiFC experiment is inserting the coding sequence for the protein of interest into a vector plasmid. From the list of Y2H positive interactions, we chose to analyze programmed cell death 5 (PDCD5), heat shock protein-40 (DNAJB1), and lantibiotic synthetase C-like protein-2 (LANCL2). Our results presented here are the successful plasmid construction of pBiFC-PDCD5-YN155 and pBiFC-DNAJB1-YN155. Both of these proteins are now ready for BiFC experiments, however, LANCL2 will require a site-directed mutagenesis to remove a restriction site within the coding sequence. The next step in this project is constructing the NS2 plasmid, pBiFC-NS2-YC155. Afterwards, BiFC experiments can be conducted and finally, the mechanism of NS2 determined, which may potentially lead to HIV-1 drug therapy.Keywords: PDCD5, DNAJB1, HIV, LANCL

Myeloid Cell Leukemia-1 knockout leads to increased viral propagation of Respiratory Syncytial Virus and influenza virus in mouse embryonic fibroblast cells and A549 cells: implications in cancer therapy

IntroductionRespiratory Syncytial Virus (RSV) remains a significant global health burden, particularly affecting young children, elderly individuals, and immunocompromised patients. The antiapoptotic protein Myeloid Cell Leukemia-1 (Mcl-1) is rapidly upregulated following RSV infection; however, its functional significance in viral pathogenesis remains poorly defined.MethodsWe investigated the role of Mcl-1 during RSV infection using Mcl-1 knockout mouse embryonic fibroblasts (ΔMcl-1 MEFs) and human alveolar epithelial (A549) cells subjected to small interfering RNA (siRNA)-mediated Mcl-1 knockdown. Viral replication was quantified by plaque assays, and phenotypic effects were assessed through syncytia formation and apoptosis assays. To assess broader implications, influenza A virus replication was also evaluated in ΔMcl-1 MEFs and Mcl-1–silenced A549 cells.ResultsRSV replication was significantly enhanced in ΔMcl-1 MEFs compared to wild-type (WT) controls, with increased viral titers, larger syncytia formation, and elevated apoptosis during the late stages of infection. Consistent results were observed in A549 cells following Mcl-1 knockdown, where RSV titers increased by more than 3 log₁₀. Influenza A virus replication was also markedly elevated in ΔMcl-1 MEFs and siRNA-treated A549 cells, suggesting that Mcl-1 exerts a broad antiviral effect across multiple respiratory viruses.DiscussionThese findings indicate that Mcl-1 upregulation during RSV and influenza virus infection functions as a critical host antiviral defense mechanism, rather than a viral evasion strategy. Clinically, our results raise concerns regarding therapies that target Mcl-1, such as certain anticancer treatments, which may inadvertently increase susceptibility to severe viral infections. Careful monitoring and potential prophylactic antiviral interventions may be warranted in patients receiving Mcl-1 inhibitor therapies

Development of a rapid point-of-care dengue virus type 2 infection diagnostic assay using recombinase polymerase amplification and lateral flow device

IntroductionDengue virus (DENV) is the most rapidly spreading arbovirus globally, with over half of the world’s population at risk of infection. Early and rapid detection is crucial to ensure timely patient care, reduce healthcare burden, and prevent severe disease progression. However, conventional nucleic acid amplification techniques are often unsuitable for low-resource settings due to their equipment and procedural demands.MethodsWe evaluated a real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for the sensitive and specific detection of DENV serotype 2 (DENV2). The assay was tested using both Twista fluorometer and lateral flow detection (LFD) formats. Analytical sensitivity was determined by probit regression, while specificity was assessed against unrelated viruses and other flaviviruses. Clinical validation was performed using serum, cell culture, and FTA® card samples. Assay robustness was evaluated under varying temperatures and after freeze-thaw cycles.ResultsThe RT-RPA assay reliably amplified DENV2 at concentrations as low as 50 copies per reaction, with LOD₉₅ estimated at 38.48 copies (Twista) and 50.37 copies (LFD). No cross-reactivity was observed with respiratory syncytial virus, influenza, rabbit herpes virus, West Nile virus, or other DENV serotypes (DENV1, DENV3, DENV4). The assay successfully detected multiple DENV2 strains and maintained performance across 33°C–40°C and after repeated freeze-thaw cycles. RNA extracted from FTA® cards was successfully amplified. Clinical validation confirmed accurate detection in serum and cell culture samples, while DENV3-positive blood samples tested negative, reinforcing specificity.DiscussionThe RT-RPA/LFD assay offers a rapid, sensitive, and specific tool for DENV2 detection, compatible with low-resource and field-based settings. Its simplicity, robustness, and portability make it a promising approach for point-of-care diagnostics and outbreak surveillance in endemic regions

A Novel Lactococcal Vaccine Expressing a Peptide from the M2 Antigen of H5N2 Highly Pathogenic Avian Influenza A Virus Prolongs Survival of Vaccinated Chickens

A cost-effective and efficacious influenza vaccine for use in commercial poultry farms would help protect against avian influenza outbreaks. Current influenza vaccines for poultry are expensive and subtype specific, and therefore there is an urgent need to develop a universal avian influenza vaccine. We have constructed a live bacterial vaccine against avian influenza by expressing a conserved peptide from the ectodomain of M2 antigen (M2e) on the surface ofLactococcus lactis(LL). Chickens were vaccinated intranasally with the lactococcal vaccine (LL-M2e) or subcutaneously with keyhole-limpet-hemocyanin conjugated M2e (KLH-M2e). Vaccinated and nonvaccinated birds were challenged with high pathogenic avian influenza virus A subtype H5N2. Birds vaccinated with LL-M2e or KLH-M2e had median survival times of 5.5 and 6.0 days, respectively, which were significantly longer than non-vaccinated birds (3.5 days). Birds vaccinated subcutaneously with KLH-M2e had a lower mean viral burden than either of the other two groups. However, there was a significant correlation between the time of survival and M2e-specific serum IgG. The results of these trials show that birds in both vaccinated groups had significantly (P&lt;0.05) higher median survival times than non-vaccinated birds and that this protection could be due to M2e-specific serum IgG.</jats:p

A Novel Lactococcal Vaccine Expressing a Peptide from the M2 Antigen of H5N2 Highly Pathogenic Avian Influenza A Virus Prolongs Survival of Vaccinated Chickens

A cost-effective and efficacious influenza vaccine for use in commercial poultry farms would help protect against avian influenza outbreaks. Current influenza vaccines for poultry are expensive and subtype specific, and therefore there is an urgent need to develop a universal avian influenza vaccine. We have constructed a live bacterial vaccine against avian influenza by expressing a conserved peptide from the ectodomain of M2 antigen (M2e) on the surface of Lactococcus lactis (LL). Chickens were vaccinated intranasally with the lactococcal vaccine (LL-M2e) or subcutaneously with keyhole-limpet-hemocyanin conjugated M2e (KLHM2e). Vaccinated and nonvaccinated birds were challenged with high pathogenic avian influenza virus A subtype H5N2. Birds vaccinated with LL-M2e or KLH-M2e had median survival times of 5.5 and 6.0 days, respectively, which were significantly longer than non-vaccinated birds (3.5 days). Birds vaccinated subcutaneously with KLH-M2e had a lower mean viral burden than either of the other two groups. However, there was a significant correlation between the time of survival and M2e-specific serum IgG. The results of these trials show that birds in both vaccinated groups had significantly ( &lt; 0.05) higher median survival times than non-vaccinated birds and that this protection could be due to M2e-specific serum IgG