SARS-CoV-2 utilizes the cell surface receptor angiotensin-converting enzyme 2 (ACE2) for entry and infection in the host cell. Thus, the molecular interface of the receptor binding domain (RBD) and ACE2 is a potential clinical target for SARS-CoV-2 infection. A small molecule inhibitor of ACE2 could block the entry of SARS-CoV‐2 and its emerging variants. This study characterizes the RBD-ACE2 interaction inhibition activity and antiviral activity of GR 127935. The binding affinity of GR 127935 to ACE2 was confirmed using Surface Plasmon Resonance (SPR). The compound inhibited RBD-ACE2 interaction in the ELISA assay (IC50 = 17 µM) and effectively blocked the entry of SARS-CoV-2 pseudovirus into HEK293T-ACE2-TMPRSS2 (IC50 = 1.2 µM). Further, the anti-SARS-CoV-2 activity of GR 127935 was evaluated in vitro using the Vero cell line and a SARS-CoV-2 clinical isolate. The most prominent inhibition (EC50 = 1.6 µM) was observed when the compound was added during the virus entry step. Finally, the GR 127935 treatment of BALB/c mice infected with the mouse-adapted strain of SARS-CoV-2 resulted in decreased viral load in the lungs along with a lower histopathology score. In summary, the GR 127935 molecule binds to ACE2, inhibits the molecular interaction between RBD and ACE2, and is effective in inhibiting virus replication. Thus, it is a promising potential therapeutic compound for treating human SARS-CoV-2 infections.

Porcine circovirus type 3 (PCV3) is an emerging pathogen linked with reproductive failure, respiratory disease, dermatitis, nephropathy, and multisystemic inflammation, posing growing threats to the swine industry worldwide. Although molecular evidence of PCV3 circulation has been reported in India, there is no published information on its seroprevalence, leaving the extent of exposure and population immunity largely unknown. Serological tools are critical for epidemiological surveillance and monitoring vaccine responses; however, validated assays for PCV3 remain scarce. In the present study, we developed and evaluated an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of PCV3-specific antibodies, based on a recombinant truncated Cap protein expressed in Escherichia coli. The antigenic fragment of the Cap gene, excluding the N-terminal nuclear localization signal, was used as coating antigen and assay parameters like optimal antigen concentration, serum dilution, and conjugate conditions, were standardized. The developed ELISA exhibited high specificity, with no cross-reactivity to antisera against other common porcine viruses, and the relative diagnostic sensitivity and specificity were estimated to be 97.0 % and 94.5 %, respectively. The assay also demonstrated strong repeatability and reproducibility and was validated at four different laboratories with κ- values indicating perfect agreement Application of the assay to field sera revealed widespread seropositivity (64.25 %), underscoring its utility for epidemiological surveillance and sero-monitoring of PCV3. This study provides the first indigenous ELISA with potential application in India, and is the first report indicating widespread seropositivity of PCV3 in India.

The rising prevalence of antibiotic resistance in microbial pathogens has ignited the quest for novel antimicrobial solutions. AgNPs were synthesized by a green approach using various plant extracts, characterized, and evaluated for their antibacterial activity against Pseudomonas aeruginosa (42 strains) and Acinetobacter baumannii (15 strains). AgNPs synthesized using cinnamon leaf extract (AgNP-CLE) were found to be most effective in terms of the diameter of the zone of inhibition produced. The minimal inhibitory concentrations of AgNP-CLE against P. aeruginosa and A. baumannii were calculated to be 0.0625 mg/mL and 0.0312 mg/mL, respectively, while the minimal bactericidal concentrations were 0.125 mg/mL and 0.0312 mg/mL, respectively. AgNP-CLE was found to be nontoxic in vitro, and storage of AgNP-CLE for up to six months did not result in aggregation. The formulation of an ointment containing AgNP-CLE produced an equivalent zone of inhibition. Green-synthesized AgNPs could serve as a potential alternative to conventional antimicrobials against drug-resistant pathogens.

SARS-CoV-2 papain-like protease (PLpro) is a key antiviral target as it plays a dual role in viral replication and modulation of innate immune responses by deubiquitinating or deISGylating host proteins. Therefore, therapeutically targeting PLpro may serve as a two-pronged approach to mitigate SARS-CoV-2 infection. Interestingly, PLpro shares structural and functional similarities with cellular deubiquitinating enzymes (DUBs), and this fact was leveraged in our study to identify DUB inhibitors that target the ubiquitin/ISG15-binding site and the known substrate-binding pocket of PLpro. Among the identified compounds, flupenthixol, lithocholic acid, teneligliptin, and linagliptin markedly inhibited the proteolytic activity of purified PLpro and demonstrated potent antiviral effects against SARS-CoV-2 infection in a dose-dependent manner. Treatment with lithocholic acid and linagliptin suppressed the expression of inflammatory mediators, thereby restoring immune responses. Here, crystal structures of SARS-CoV-2 PLpro in complex with linagliptin and with lithocholic acid were determined, revealing insights into the mechanism of inhibition and unique interactions within the ubiquitin/ISG15-binding site (S2 site; Phe69, His73, Asn128, and His175) and the substrate-binding cleft. Additionally, oral and intraperitoneal treatments with linagliptin increased survival, reduced lung viral load, and ameliorated histopathological damage in a mouse-adapted model of SARS-CoV-2 infection. This study demonstrates for the first time that using DUB inhibitors that target the proteolytic activity of PLpro can simultaneously reinstate the host's immune response against SARS-CoV-2, highlighting the potential of this two-pronged therapeutic approach.

Staphylococcus aureus, a common cause of bovine mastitis, relies on several virulence factors, with biofilm formation being a key contributor to its pathogenicity. The present study investigated the occurrence of S. aureus as etiological agents in bovine mastitis with a focus on the existence of various virulence factors and antibiotic resistance status. Among 120 milk samples collected from West Bengal and Uttar Pradesh, 36 (30%) S. aureus strains were confirmed by conventional methods and PCR. Phenotypic analysis revealed hemolysin (55.55%) and coagulase production (36.11%), while molecular analysis revealed the presence of leukotoxin (luksF, 19.44%), hemolysin (hlb, 58.33%), coagulase (coa, 63.88%), and toxic shock syndrome toxin (tsst-1, 30.55%) genes. Biofilm production ability was detected in 97.22% (crystal violet assay) and 86.11% (Congo red agar assay) strains. Biofilm-associated genes, namely, icaA (80.55%, 29/36), icaB (75%, 27/36), icaC (69.44%, 25/36), icaD (86.11%, 31/36), and MSCRAMMs genes, namely, clfA (58.33%, 21/36), clfB (75%, 27/36), fnbA (75%, 27/36), fnbB (55.55%, 20/36), bap (38.88%, 14/36), bbp (83.33%, 30/36), ebps (69.44%, 25/36), eno (66.66%, 24/36), fib (41.66%, 15/36), and cna (8.33%, 3/36), were also detected. Antimicrobial resistance was observed in 88.88% isolates, with 72.22% exhibiting multidrug resistance (MDR). Among the isolates, 83.33% were methicillin-resistant S. aureus (MRSA), and mecA, femA, and femB genes were present either singly or in combination in 76.66% of the isolates. Efflux pump protein genes, namely, norA, norB, norC, mdeA, mepA, and sepA, were detected either singly or in combination in S. aureus isolates. 61.53% of MDR-MRSA isolates harbored all six efflux pump genes. According to this study, S. aureus of mastitis origin harbors various virulence, antibiotic resistance, biofilm-forming, and efflux pump genes. Bovine mastitis-derived MDR S. aureus isolates can pose a significant public health risk and need urgent attention to formulate strategies for their control and preventing transfer to the human food chain.

The inherent piezoelectric property of bone and consequent electrical stimuli direct tissue growth and repair. Bismuth ferrite (BF) is a multiferroic material and is believed to facilitate this in-situ electrical stimuli generation. Bioactive glass (BAG) reinforced with BF (BAG-BF) biocomposite has shown promising in vitro osteogenic effects. This study evaluates the efficacy of BAG-BF electroactive biocomposite with and without a static MF and rat adipose-derived mesenchymal stem cells (r-ADMSCs) in a rat craniectomy model. A 7 mm calvarial defect was created and assigned to eight groups (1-8): sham surgery, BAG, 0.5% BAG-BF without or with a static MF, rADMSCs seeded 0.5% BAG-BF, 1.5% BAG-BF without or with a static MF, and r-ADMSCs seeded 1.5% BAG-BF. In the designated groups, a 200 mT static MF was applied for 30 min on alternative days. The r-ADMSCs seeded 1.5% BAG-BF group showed the highest tissue integration, vascularization, and bone defect coverage, followed by the r-ADMSCs seeded 0.5% BAG-BF and 1.5% BAG-BF with MF groups. Micro-CT, SEM, and histopathology confirmed superior bone healing in r-ADMSCs seeded 1.5% BAG-BF group. Furthermore, RUNX2, collagen-1, MMP2, and BMP-6 expression were highest in r-ADMSCs seeded 1.5% BAG-BF group, followed by the rADMSCs seeded 0.5% BAG-BF and 1.5% BAG-BF with MF groups. Overall, the highest bone regeneration was observed in the r-ADMSCs seeded 1.5% BAG-BF group, while the groups exposed to MF also showed enhanced regeneration compared to the scaffold alone. The study highlights that combining r-ADMSCs and MF with BAGBF scaffolds has great potential to enhance bone regeneration.

The SARS-CoV-2 pandemic supercharged global efforts towards cutting-edge vaccination strategies for current threats and emerging viruses. It is imperative to develop and investigate next-generation vaccines such as chimeric Virus-Like Particles (chi-VLPs) vaccines for increased immunogenicity, ease of production, and scalability to supplement the worldwide vaccine supply. This study reports a novel bivalent vaccine design of Chimeric Alphavirus-Coronavirus Virus-Like Particles (ChAC-VLPs), displaying fusion glycoproteins of Chikungunya virus (CHIKV) and a Receptor Binding Domain (RBD) of SARS-CoV-2 on its surface. The uniqueness and versatility of ChAC-VLPs have been demonstrated via various techniques, including Western blot, Immunofluorescence, cryo-EM, and Dynamic Light Scattering (DLS). The multimeric epitope display of immunogenic antigens was validated by cell-based assays. ChAC-VLP immunized mice sera have shown substantial neutralization titers for CHIKV (PRNT50 of 1:25). Similarly, serum antibodies were detected for SARS-CoV-2 RBD as observed by antigen-specific ELISA and validated using Surface Plasmon Resonance (SPR). ChAC-VLP-immunized mice sera at a 1:10 dilution exhibited 80-95 % SARS-CoV-2 pseudovirus neutralization relative to the untreated virus control. In conclusion, this study proposes ChAC-VLPs as a potential hybrid vaccine candidate for CHIKV and SARS-CoV-2 infections and contributes valuable insights into the chi-VLPs domain and its design.

Bovine mastitis is one of the diseases that affect cattle and has a considerable detrimental impact on both the economics and animal welfare internationally. S. aureus is among the most relevant causative agents of this disease. Due to developing bacterial resistance and public health regulations, the number of antibiotics to treat mastitis is restricted. Endolysins, cell-wall-degrading enzymes of bacteriophage origin, are emerging as a promising alternative to traditional antibiotics. Moreover, combination therapy is frequently utilized to combat drug-resistant bacterial pathogens. In this study, we evaluated the therapeutic efficacy of the recombinant endolysin S.AgEndo1332 in combination with the antibiotic gentamicin. The synergistic activity of this combination was assessed in vitro using checkerboard and time-kill assays, and in vivo using a mouse mastitis model induced by mastitogenic S. aureus. S.AgEndo1332, a homolog of GBS phage B30 lysin gene, was custom synthesized, expressed by prokaryotic expression system, and purified under native purification protocol. S.AgEndo1332 showed strong antimicrobial activity against Streptococcus agalactiae (MIC 25 µg/mL) and S. aureus (MIC 50 µg/mL) strains in Tris-HCl buffer system at pH 7.5. The lytic activity of S.AgEndo1332 wasretained at -80 °C for 12 weeks. The combination of S.AgEndo1332 (12.5 µg/mL) with gentamicin (0.0594 µg/mL) was showing synergistic activity by checkerboard assay with FIC index 0.375. The dosage for the combination treatment (10X concentration, S.AgEndo1332: 125 µg/mL and gentamicin: 0.594 µg/mL) used for the in vivo trial was determined based on cell viability by the MTT assay (> 93

SARS-CoV-2 variant recurrence has emphasized the imperative prerequisite for effective antivirals. The main protease (Mpro) of SARS-CoV-2 is crucial for viral replication, making it one of the prime and promising antiviral targets. Mpro features several druggable sites, including active sites and allosteric sites near the dimerization interface, that regulate its catalytic activity. This study identified six highly efficacious antiviral SARS-CoV-2 compounds (WIN-62577, KT185, bexarotene, ledipasvir, diacerein, and simepervir) using structure-based virtual screening of compound libraries against Mpro. Using SPR and ITC, the binding of selected inhibitory compounds to the target Mpro was validated. The FRET-based protease assay demonstrated that the identified molecules effectively inhibit Mpro with IC50 values in the range from 0.64 to 11.98 μM. Additionally, in vitro cell-based antiviral assays showed high efficacy with EC50 values in the range of 1.51 to 18.92 μM. The crystal structure of the Mpro-minocycline complex detailed the possible inhibition mechanism of minocycline, an FDA-approved antibiotic. Minocycline binds to an allosteric site, revealing residues critical for the loss of protease activity due to destabilization of molecular interactions at the dimeric interface, which are crucial for the proteolytic activity of Mpro. The study suggests that the binding of minocycline to the allosteric site may play a role in Mpro dimer destabilization and direct the rational design of minocycline derivatives as antiviral drugs.

Elephant Endotheliotropic Herpesvirus (EEHV) poses a critical threat to young Asian (Elephas maximus) and African elephants (Loxodonta africana), with high mortality rates due to acute haemorrhagic symptoms from vascular endothelial damage. EEHV remains dormant in adult elephants, reactivating under stress or immune suppression. There are multiple genotypes of EEHV have been reported based on genetic heterogeneity. The diversity among EEHV subtypes, such as EEHV1A and EEHV1B, influences disease severity and host susceptibility. EEHV genomes are large and encode over 115 open reading frames, contributing to viral replication and immune evasion. Hence, generation of complete genome is essential to determine the genotypes of EEHV. However, the inability to culture EEHV in-vitro limits the study of its life cycle and pathogenesis, necessitating molecular techniques for direct analysis. Additionally, variable viral load in the infected tissues limits the success of DNA sequencing using direct sequencing. Hence, in this study several methods of viral or DNA enrichment were considered, and ultracentrifugation method was adopted to concentrate viral copies for improved genomic analysis. Ultracentrifugation demonstrates effectiveness in isolating EEHV, significantly enriching viral DNA for detection and characterization. This technique facilitates advancements in diagnostics, therapeutic development, and vaccine research, addressing the challenges of low viral loads and host DNA contamination in clinical samples.

Bluetongue (BT) is a vector-borne viral disease of multiple domestic and wild ruminants across the globe. The VP7 protein of bluetongue virus (BTV) is the major immune-dominant structural protein that is conserved across the BTV serotypes and therefore, targeted for the development of immuno-diagnostics for BT. In this study, full-length recombinant VP7 protein (rVP7) of BTV-1 was expressed in Trochoplusia ni derived insect cells (Tn5) using codon-optimized synthetic gene construct through baculovirus expression system. The seed stock of recombinant baculovirus was amplified to a high titre (>1 × 108 pfu/ml) at P2 and P3 in sf9 cells. The rVP7 was successfully produced and purified from infected Tn5 culture lysate for evaluation of the immuno-reactivity and its diagnostic potential. The purified protein showed strong reactivity in western blot analysis with the polyclonal immune serum produced against BTV core antigen in guinea pigs. An indirect ELISA (iELISA) was optimized by using the purified rVP7 for the detection of the group-specific antibodies to BTV in sheep and goats. The iELISA was found to be highly sensitive (98.9 %), specific (98.1 %), and reproducible (CV < 10 %) for detection of the antibodies to BTV in sheep and goat serum. The iELISA could detect the specific antibodies in naturally infected goat serum containing type-specific neutralizing antibodies to different BTV serotypes indicating the potential of the rVP7 for the development of the group-specific sero-diagnostics for BT.

Elephant Endotheliotropic Herpesvirus (EEHV) is a major cause of fatal hemorrhagic disease in juvenile elephants, highlighting the urgent need for reliable serological diagnostic tools for early detection and epidemiological surveillance. Effective EEHV immunodiagnostics require a conserved, stable, and immune reactive antigen capable of consistent antibody detection across viral strains. As EEHV is a non-cultivable virus encoding approximately 115 structural and non-structural proteins, careful selection of suitable diagnostic targets is essential. In the present study, an in silico approach was employed to evaluate the diagnostic potential of selected EEHV proteins. The study moves beyond descriptive in-silico characterization by integrating antigenicity, physicochemical stability, glycosylation profiling, and rational truncation to identify recombinant antigens with direct applicability in EEHV immunodiagnostic platforms. The full-length DNA polymerase (1047 aa), glycoprotein B (gB; 847 aa), and glycoprotein L (gL; 304 aa) were analyzed for sequence conservation, physicochemical properties, antigenicity, and glycosylation patterns. Multiple sequence alignment revealed a high degree of conservation among EEHV1A strains, with approximately 99% sequence identity across all three genes, underscoring their evolutionary stability and functional importance. Glycosylation prediction identified minimal post-translational modification potential within the N-terminal regions, enabling rational truncation. Based on these analyses, truncated fragments of DNA polymerase (95 aa), gB (387 aa), and gL (164 aa) were selected for recombinant antigen development. The selected fragments exhibited favorable physicochemical properties, including stability, hydrophilicity, positive net charge, and high antigenicity scores (0.5209, 0.5271, and 0.2572, respectively). These findings support the suitability of truncated EEHV proteins, particularly DNA polymerase, as recombinant antigens for competitive ELISA development and provide a rational framework for EEHV serological assay design.