Search Results (12)

Porcine extraintestinal pathogenic Escherichia coli (ExPEC) is a significant zoonotic pathogen with escalating antimicrobial resistance, underscoring the urgent need for novel therapeutics. This study aimed to investigate the therapeutic potential and mechanism of action of the antimicrobial peptide Protegrin-1 (PG-1) against a multidrug-resistant porcine ExPEC strain, PCN033. The minimal inhibitory concentration (MIC) was determined, and resistance stability was assessed through serial induction and withdrawal passages. Hemolytic activity was evaluated to gauge selectivity. A murine infection model was utilized to assess in vivo efficacy, bacterial load reduction, cytokine modulation, and histopathology. Comparative spleen transcriptomic analysis was performed to elucidate global host responses. PG-1 exhibited potent bactericidal activity (MIC = 32 μg/mL) and maintained its efficacy over multiple passages, demonstrating no induced resistance. It showed acceptable hemolytic activity and significantly improved survival, reduced bacterial loads in multiple organs, and mitigated tissue damage in mice. Transcriptomics revealed PG-1 treatment broadly tempered infection-induced hyperinflammatory responses, including NF-κB, MAPK, and TNF signaling pathways, and counteracted metabolic reprogramming. The findings conclude that PG-1 effectively integrates direct, resistance-resistant bactericidal activity with multimodal immunomodulation, representing a superior therapeutic strategy that simultaneously eliminates pathogens and restores immune homeostasis, offering a promising alternative to conventional antibiotics against MDR ExPEC infections. Full article

Pseudomonas aeruginosa is a major opportunistic pathogen with high levels of antibiotic resistance. Phage therapy represents a promising alternative for the treatment of difficult infections both alone and in combination with antibiotics. Here, we isolated and characterized three novel lytic myoviruses, Cisa, Nello, and Moonstruck. Genomic analysis revealed that Cisa and Nello belong to the Pbunavirus genus, while Moonstruck is a novel Pakpunavirus species. All lacked lysogeny, virulence, or resistance-associated genes, supporting their therapeutic suitability. Phage Nello and Moonstruck were active against P. aeruginosa Pa3GrPv, isolated from a patient with lung infection candidate for phage therapy. Moonstruck exhibited superior lytic activity with ciprofloxacin sub-MIC value (0.125 µg/mL), achieving bacterial suppression for 48 h. However, to improve the lytic efficacy of the phages on the clinical isolate, phage adaptation via serial passage was investigated. The killing efficacy of Nello was enhanced, whereas Moonstruck showed a less consistent improvement, suggesting phage-specific differences in evolutionary dynamics. Sequencing of the evolved phages revealed point mutations in tail-associated genes, potentially linked to a better phage–host interaction. These results support the use of phage–antibiotic combinations and directed evolution as strategies to enhance phage efficacy against drug-resistant infections. Overall, these findings support the therapeutic potential of the newly isolated phages in treating P. aeruginosa lung infections. Full article

A precision genome edit in the bovine CD46 gene (A₈₂LPTFS₈₇) dramatically reduced bovine viral diarrhea virus (BVDV) susceptibility in a cloned heifer. However, pathogen evolution threatens the long-term efficacy of such interventions. Here, our aim is two-fold: first, to determine whether BVDV can adapt in vitro to use the edited CD46 receptor to infect Madin–Darby bovine kidney (MDBK) cells, and second, to evaluate the ex vivo infectivity of culture-adapted viruses in cells from the CD46-edited heifer. Serial passage of BVDV on CD46-edited MDBK cells selected for virus variants capable of CD46-independent infection. Virus genome sequencing revealed mutations in the viral E^RNS gene predicted to enhance HS-mediated entry. HS adaptation was confirmed by inhibiting virus infection with heparin or Heparinase I/III treatment. A naturally occurring HS-adapted field isolate from a persistently infected calf showed similar results. However, when tested on primary cells from the CD46-edited heifer, HS-adapted viruses showed reduced infectivity in skin fibroblasts, monocytes, and lymphocytes in a manner that correlated with HS expression. Thus, although BVDV can adapt to use HS as an alternative entry receptor, HS adaptation does not overcome the protection conferred by the CD46 edit in all relevant cell types. Full article

Background/Objectives: Vaccination is important for controlling foot-and-mouth disease (FMD) in endemic regions and to lessen the effects of outbreaks in FMD-free countries. The adaptation of FMD virus to BHK cells is a necessary but time-consuming and costly step in vaccine production and can prove problematic for some isolates. Adaptation is, in part, driven by receptor availability and selects variants with altered receptor specificity that result from amino acid substitutions in the capsid proteins. Methods: To bypass the need for cell culture adaptation, we generated chimeric viruses with field-strain capsids and introduced amino acid substitutions associated with cell culture adaptation. We targeted two sites on the capsid: the canonical heparan sulphate binding site and the icosahedral 5-fold symmetry axes. Results: Our results show that some viruses with unmodified wild-type (wt) capsids grew well in BHK cells (suspension and adherent), whereas others showed poor growth. For viruses that showed good growth, the introduction of amino acid changes associated with cell culture adaptation improved the rate of growth but not virus titres or yields of 146S particles, whereas growth and 146S yields for viruses that grew poorly in BHK cells were greatly enhanced by some of the amino acid changes. For the latter viruses, the introduced changes did not appear to adversely affect virion stability or antigenicity. Conclusions: For FMD viruses that grow poorly in BHK cells, this approach could be a viable alternative to protracted adaptation by serial passage and could expedite the production of a new vaccine strain from a field virus. Full article

Antimicrobial peptides (AMPs) hold promise as alternatives to combat bacterial infections, addressing the urgent global threat of antibiotic resistance. COG1410, a synthetic peptide derived from apolipoprotein E, has exhibited potent antimicrobial properties against various bacterial strains, including Mycobacterium smegmatis. However, our study reveals a previously unknown resistance mechanism developed by M. smegmatis against COG1410 involving ClpC. Upon subjecting M. smegmatis to serial passages in the presence of sub-MIC COG1410, resistance emerged. The comparative genomic analysis identified a point mutation in ClpC (S437P), situated within its middle domain, which led to high resistance to COG1410 without compromising bacterial fitness. Complementation of ClpC in mutant restored bacterial sensitivity. In-depth analyses, including transcriptomic profiling and in vitro assays, uncovered that COG1410 interferes with ClpC at both transcriptional and functional levels. COG1410 not only stimulated the ATPase activity of ClpC but also enhanced the proteolytic activity of Clp protease. SPR analysis confirmed that COG1410 directly binds with ClpC. Surprisingly, the identified S437P mutation did not impact their binding affinity. This study sheds light on a unique resistance mechanism against AMPs in mycobacteria, highlighting the pivotal role of ClpC in this process. Unraveling the interplay between COG1410 and ClpC enriches our understanding of AMP-bacterial interactions, offering potential insights for developing innovative strategies to combat antibiotic resistance. Full article

Kemerovo virus (KEMV) is a tick-borne orbivirus transmitted by ticks of the genus Ixodes. Previous animal experimentation studies with orbiviruses, in particular the interferon receptor double knock-out (IFNAR^(−/−)) mouse model, did not indicate bias that is related to age or sex. We endeavoured to assess the effect of serial and alternated passages of KEMV in mammalian or Ixodes cells on virus replication and potential virulence in male or female IFNAR^(−/−) mice, with important age differences: younger males (4–5 months old), older males (14–15 months old), and old females (14–15 months old). After 30 serial passages in mammalian or tick cells, or alternated passages in the two cell types, older female mice which were inoculated with the resulting virus strains were the first to show clinical signs and die. Younger males behaved differently from older males whether they were inoculated with the parental strain of KEMV or with any of the cell culture-passaged strains. The groups of male and female mice inoculated with the mammalian cell culture-adapted KEMV showed the lowest viraemia. While older female and younger male mice died by day 6 post-inoculation, surprisingly, the older males survived until the end of the experiment, which lasted 10 days. RNA extracted from blood and organs of the various mice was tested by probe-based KEMV real-time RT-PCR. Ct values of the RNA extracts were comparable between older females and younger males, while the values for older males were >5 Ct units higher for the various organs, indicating lower levels of replication. It is noteworthy that the hearts of the old males were the only organs that were negative for KEMV RNA. These results suggest, for the first time, an intriguing age- and sex-related bias for an orbivirus in this animal model. Changes in the amino acid sequence of the RNA-dependent RNA polymerase of Kemerovo virus, derived from the first serial passage in Ixodes cells (KEMV Ps.IRE1), were identified in the vicinity of the active polymerase site. This finding suggests that selection of a subpopulation of KEMV with better replication fitness in tick cells occurred. Full article

SARS-CoV-2 pathogenesis, vaccine, and therapeutic studies rely on the use of animals challenged with highly pathogenic virus stocks produced in cell cultures. Ideally, these virus stocks should be genetically and functionally similar to the original clinical isolate, retaining wild-type properties to be reliably used in animal model studies. It is well-established that SARS-CoV-2 isolates serially passaged on Vero cell lines accumulate mutations and deletions in the furin cleavage site; however, these can be eliminated when passaged on Calu-3 lung epithelial cell lines, as presented in this study. As numerous stocks of SARS-CoV-2 variants of concern are being grown in cell cultures with the intent for use in animal models, it is essential that propagation methods generate virus stocks that are pathogenic in vivo. Here, we found that the propagation of a B.1.351 SARS-CoV-2 stock on Calu-3 cells eliminated viruses that previously accumulated mutations in the furin cleavage site. Notably, there were alternative variants that accumulated at the same nucleotide positions in virus populations grown on Calu-3 cells at multiple independent facilities. When a Calu-3-derived B.1.351 virus stock was used to infect hamsters, the virus remained pathogenic and the Calu-3-specific variants persisted in the population. These results suggest that Calu-3-derived virus stocks are pathogenic but care should still be taken to evaluate virus stocks for newly arising mutations during propagation. Full article

Bluetongue virus (BTV) is an arbovirus that has been associated with dramatic epizootics in both wild and domestic ruminants in recent decades. As a segmented, double-stranded RNA virus, BTV can evolve via several mechanisms due to its genomic structure. However, the effect of BTV’s alternating-host transmission cycle on the virus’s genetic diversification remains poorly understood. Whole genome sequencing approaches offer a platform for investigating the effect of host-alternation across all ten segments of BTV’s genome. To understand the role of alternating hosts in BTV’s genetic diversification, a field isolate was passaged under three different conditions: (i) serial passages in Culicoides sonorensis cells, (ii) serial passages in bovine pulmonary artery endothelial cells, or (iii) alternating passages between insect and bovine cells. Aliquots of virus were sequenced, and single nucleotide variants were identified. Measures of viral population genetics were used to quantify the genetic diversification that occurred. Two consensus variants in segments 5 and 10 occurred in virus from all three conditions. While variants arose across all passages, measures of genetic diversity remained largely similar across cell culture conditions. Despite passage in a relaxed in vitro system, we found that this BTV isolate exhibited genetic stability across passages and conditions. Our findings underscore the valuable role that whole genome sequencing may play in improving understanding of viral evolution and highlight the genetic stability of BTV. Full article

Foot-and-mouth disease (FMD) is an economically devastating animal disease. Adapting the field virus to cells is critical to the vaccine production of FMD viruses (FMDV), and heparan sulfate (HS) and Jumonji C-domain-containing protein 6 (JMJD6) are alternative receptors of cell-adapted FMDV. We performed serial passages of FMDV O/SKR/Andong/2010, classified as the O/Mya-98 topotype/lineage and known as a highly virulent strain, to develop a vaccine seed virus. We traced changes in the amino acid sequences of the P1 region, plaque phenotypes, and the receptor usage of the viruses, and then structurally analyzed the mutations. VP3 H56R and D60G mutations were observed in viruses using the HS receptor and led to changes in the hydrogen bonding between VP3 56 and 60. A VP1 P208L mutation was observed in the virus using the JMJD6 receptor during cell adaptation, enabling the interaction with JMJD6 through the formation of a new hydrogen bond with JMJD6 residue 300. Furthermore, VP1 208 was near the VP1 95/96 amino acids, previously reported as critical mutations for JMJD6 receptor interactions. Thus, the mutation at VP1 208 could be critical for cell adaptation related to the JMJD6 receptor and may serve as a basis for mechanism studies on FMDV cell adaptation. Full article

Background: Deficient autophagy has been recently implicated as a driver of pulmonary fibrosis, yet bioinformatics approaches to study this cellular process are lacking. Autophagy-related 5 and 7 (ATG5/ATG7) are critical elements of macro-autophagy. However, an alternative ATG5/ATG7-independent macro-autophagy pathway was recently discovered, its regulation being unknown. Using a bioinformatics proteome profiling analysis of ATG7-deficient human fibroblasts, we aimed to identify key microRNA (miR) regulators in autophagy. Method: We have generated ATG7-knockout MRC-5 fibroblasts and performed mass spectrometry to generate a large-scale proteomics dataset. We further quantified the interactions between various proteins combining bioinformatics molecular network reconstruction and functional enrichment analysis. The predicted key regulatory miRs were validated via quantitative polymerase chain reaction. Results: The functional enrichment analysis of the 26 deregulated proteins showed decreased cellular trafficking, increased mitophagy and senescence as the major overarching processes in ATG7-deficient lung fibroblasts. The 26 proteins reconstitute a protein interactome of 46 nodes and miR-regulated interactome of 834 nodes. The miR network shows three functional cluster modules around miR-16-5p, miR-17-5p and let-7a-5p related to multiple deregulated proteins. Confirming these results in a biological setting, serially passaged wild-type and autophagy-deficient fibroblasts displayed senescence-dependent expression profiles of miR-16-5p and miR-17-5p. Conclusions: We have developed a bioinformatics proteome profiling approach that successfully identifies biologically relevant miR regulators from a proteomics dataset of the ATG-7-deficient milieu in lung fibroblasts, and thus may be used to elucidate key molecular players in complex fibrotic pathological processes. The approach is not limited to a specific cell-type and disease, thus highlighting its high relevance in proteome and non-coding RNA research. Full article

Viruses are able to evolve in vitro by mutations after serial passages in cell cultures, which can lead to either a loss, or an increase, of virulence. Cyprinid herpesvirus 3 (CyHV-3), a 295-kb double-stranded DNA virus, is the etiological agent of the koi herpesvirus disease (KHVD). To assess the influence of serial passages, an isolate of CyHV-3 (KHV-T) was passaged 99 times onto common carp brain (CCB) cells, and virus virulence was evaluated during passages through the experimental infections of common carp. After 78 CCB passages, the isolate was much less virulent than the original form. A comparative genomic analysis of these three forms of KHV-T (P0, P78 and P99) revealed a limited number of variations. The largest one was a deletion of 1363 bp in the predicted ORF150, which was detected in P78, but not in P99. This unexpected finding was confirmed by conventional PCR and digital PCR. The results presented here primarily suggest that, CyHV-3 evolves, at least in vitro, through an assemblage of haplotypes that alternatively become dominant or under-represented. Full article

Phylogenetic analyses suggest that Mamestra brassicae multiple nucleopolyhedrovirus (MbMNPV) and Helicoverpa armigera multiple nucleopolyhedrovirus (HearMNPV) may be strains of the same virus species. Most of the studies comparing their biological activities have been performed in their homologous hosts. A comparison of host range and stability in alternative hosts was performed. The host range of these viruses was compared using high concentrations of inoculum to inoculate second instars of six species of Lepidoptera. One semi-permissive host (Spodoptera littoralis) and one permissive host (S. exigua) were then selected and used to perform six serial passages involving a concentration corresponding to the ~25% lethal concentration for both viruses. Restriction endonuclease analysis showed fragment length polymorphisms in every host-virus system studied. In S. littoralis, serial passage of MbMNPV resulted in decreased pathogenicity and an increase in speed-of-kill, whereas no significant changes were detected for HearMNPV with respect to the initial inoculum. In contrast, both viruses showed a similar trend in S. exigua. These results highlight the low genetic diversity and a high phenotypic stability of HearMNPV with respect to the original inoculum after six successive passages in both insect hosts. This study concludes that host-baculovirus interactions during serial passage are complex and the process of adaptation to a novel semi-permissive host is far from predictable. Full article