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Viruses May 2024In virology, the term reverse genetics refers to a set of methodologies in which changes are introduced into the viral genome and their effects on the generation of... (Review)
Review
In virology, the term reverse genetics refers to a set of methodologies in which changes are introduced into the viral genome and their effects on the generation of infectious viral progeny and their phenotypic features are assessed. Reverse genetics emerged thanks to advances in recombinant DNA technology, which made the isolation, cloning, and modification of genes through mutagenesis possible. Most virus reverse genetics studies depend on our capacity to rescue an infectious wild-type virus progeny from cell cultures transfected with an "infectious clone". This infectious clone generally consists of a circular DNA plasmid containing a functional copy of the full-length viral genome, under the control of an appropriate polymerase promoter. For most DNA viruses, reverse genetics systems are very straightforward since DNA virus genomes are relatively easy to handle and modify and are also (with few notable exceptions) infectious per se. This is not true for RNA viruses, whose genomes need to be reverse-transcribed into cDNA before any modification can be performed. Establishing reverse genetics systems for members of the has proven exceptionally challenging due to the low number of members of this family that propagate in cell culture. Despite the early successful rescue of calicivirus from a genome-length cDNA more than two decades ago, reverse genetics methods are not routine procedures that can be easily extrapolated to other members of the family. Reports of calicivirus reverse genetics systems have been few and far between. In this review, we discuss the main pitfalls, failures, and delays behind the generation of several successful calicivirus infectious clones.
Topics: Reverse Genetics; Caliciviridae; Genome, Viral; Animals; Humans; Virus Replication
PubMed: 38932159
DOI: 10.3390/v16060866 -
Viruses May 2024The human hepatitis delta virus (HDV) is a satellite RNA virus that depends on hepatitis B virus (HBV) surface proteins (HBsAg) to assemble into infectious virions...
The human hepatitis delta virus (HDV) is a satellite RNA virus that depends on hepatitis B virus (HBV) surface proteins (HBsAg) to assemble into infectious virions targeting the same organ (liver) as HBV. Until recently, the evolutionary origin of HDV remained largely unknown. The application of bioinformatics on whole sequence databases lead to discoveries of HDV-like agents (DLA) and shed light on HDV's evolution, expanding our understanding of HDV biology. DLA were identified in heterogeneous groups of vertebrates and invertebrates, highlighting that the evolution of HDV, represented by eight distinct genotypes, is broader and more complex than previously foreseen. In this study, we focused on the characterization of three mammalian DLA discovered in woodchuck (), white-tailed deer (), and lesser dog-like bat () in terms of replication, cell-type permissiveness, and spreading pathways. We generated replication-competent constructs expressing 1.1-fold over-length antigenomic RNA of each DLA. Replication was initiated by transfecting the cDNAs into human (HuH7, HeLa, HEK293T, A549) and non-human (Vero E6, CHO, PaKi, LMH) cell lines. Upon transfection and replication establishment, none of the DLA expressed a large delta antigen. A cell division-mediated viral amplification assay demonstrated the capability of non-human DLA to replicate and propagate in hepatic and non-hepatic tissues, without the requirement of envelope proteins from a helper virus. Remarkably L-HDAg but not S-HDAg from HDV can artificially mediate envelopment of WoDV and DeDV ribonucleoproteins (RNPs) by HBsAg to form infectious particles, as demonstrated by co-transfection of HuH7 cells with the respective DLA expression constructs and a plasmid encoding HBV envelope proteins. These chimeric viruses are sensitive to HDV entry inhibitors and allow synchronized infections for comparative replication studies. Our results provide a more detailed understanding of the molecular biology, evolution, and virus-host interaction of this unique group of animal viroid-like agents in relation to HDV.
Topics: Virus Replication; Animals; Hepatitis Delta Virus; Humans; Hepatitis B virus; Marmota; Cell Division; Chiroptera; Viral Envelope Proteins; Cell Line; Hepatitis B; Hepatitis B Surface Antigens; Genotype; HEK293 Cells; Hepatitis D; RNA, Viral
PubMed: 38932152
DOI: 10.3390/v16060859 -
Viruses May 2024A number of research studies, including ours, have spotlighted exosomes as critical facilitators of viral dissemination. While hepatitis B virus (HBV) transmission...
A number of research studies, including ours, have spotlighted exosomes as critical facilitators of viral dissemination. While hepatitis B virus (HBV) transmission through exosomes has been studied, the focus on its satellite virus, the hepatitis delta virus (HDV), has been unexplored in this context. HDV, although being a defective virus, can replicate its genome autonomously within hepatocytes, independently of HBV. Investigations on Huh7 cells revealed an intriguing phenomenon: the HDV proteins, S-HDAg and L-HDAg, are transmitted between cells without a complete viral structure. Detailed analysis further revealed that the expression of these proteins not only bolstered exosome secretion but also ensured their enrichment within these vesicles. Our experimental approach utilized transfection of various plasmids to examine the role of HDV RNA and proteins in the process. One salient finding was the differential propagation of the HDV proteins S-HDAg and L-HDAg, suggesting intricate molecular mechanisms behind their transmission. Notably, the purity of our exosome preparations was monitored using markers such as TSG101 and CD81. Importantly, these exosomes were found to carry both HDV RNA and proteins, highlighting their role in HDV dissemination. This novel study underscores the role of exosomes in mediating the transmission of HDV components between hepatocytes independent of HBV. These revelations about the exosomal pathway of HDV transmission provide a foundation for the development of innovative therapeutic strategies against HDV infections.
Topics: Exosomes; Hepatitis Delta Virus; Hepatocytes; Humans; Hepatitis B virus; Virus Replication; RNA, Viral; Hepatitis D; Cell Line; Hepatitis B; Hepatitis delta Antigens
PubMed: 38932118
DOI: 10.3390/v16060825 -
Pharmaceuticals (Basel, Switzerland) Jun 2024Calcium pyrophosphate dehydrate (CPPD) crystals are found in the synovial fluid of patients with articular chondrocalcinosis or sometimes with osteoarthritis. In...
Calcium pyrophosphate dehydrate (CPPD) crystals are found in the synovial fluid of patients with articular chondrocalcinosis or sometimes with osteoarthritis. In inflammatory conditions, the synovial membrane (SM) is subjected to transient hypoxia, especially during movement. CPPD formation is supported by an increase in extracellular inorganic pyrophosphate (ePPi) levels, which are mainly controlled by the transporter Ank and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We demonstrated previously that transforming growth factor (TGF)-β1 increased ePPi production by inducing Ank and Enpp1 expression in chondrocytes. As the TGF-β1 level raises in synovial fluid under hypoxic conditions, we investigated whether hypoxia may transform SM as a major source of ePPi production. Synovial fibroblasts and SM explants were exposed to 10 ng/mL of TGF-β1 in normoxic or hypoxic (5% O) culture conditions. Ank and Enpp1 expression were assessed by quantitative PCR, Western blot and immunohistochemistry. ePPi was quantified in culture supernatants. RNA silencing was used to define the respective roles of and in TGF-β1-induced ePPi generation. The molecular mechanisms involved in hypoxia were investigated using an promoter reporter plasmid for transactivation studies, as well as gene overexpression and RNA silencing, the respective role of hypoxia-induced factor (HIF)-1 and HIF-2. Our results showed that TGF-β1 increased Ank, Enpp1, and therefore ePPi production in synovial fibroblasts and SM explants. Ank was the major contributor in ePPi production compared to ENPP1. Hypoxia increased ePPi levels on its own and enhanced the stimulating effect of TGF-β1. Hypoxic conditions enhanced promoter transactivation in an HIF-1-dependent/HIF-2-independent fashion. We demonstrated that under hypoxia, SM is an important contributor to ePPi production in the joint through the induction of and . These findings are of interest as a rationale for the beneficial effect of anti-inflammatory drugs on SM in crystal depositions.
PubMed: 38931405
DOI: 10.3390/ph17060738 -
Microorganisms Jun 2024Waste Water Treatment Plants (WWTP) aim to reduce contamination in effluent water; however, studies indicate antimicrobial resistance genes (ARGs) persist...
Metagenomic Investigation of the Short-Term Temporal and Spatial Dynamics of the Bacterial Microbiome and the Resistome Downstream of a Wastewater Treatment Plant in the Iskar River in Bulgaria.
Waste Water Treatment Plants (WWTP) aim to reduce contamination in effluent water; however, studies indicate antimicrobial resistance genes (ARGs) persist post-treatment, potentially leading to their spread from human populated areas into the environment. This study evaluated the impact of a large WWTP serving 125,000 people on the Iskar River in Bulgaria, by characterizing the spatial and short-term temporal dynamics in bacterial community dynamics and resistance profiles of the surface water. Pairs of samples were collected biweekly on four dates from two different locations, one about 800 m after the WWTP effluents and the other 10 km downstream. Taxonomic classification revealed the dominance of and , notably the genera , , , , and . The taxonomic structure corresponded with both lentic and lotic freshwater habitats, with exhibiting a significant decrease over the study period. Principal Coordinate Analysis revealed statistically significant differences in bacterial community composition between samples collected on different dates. Differential abundance analysis identified notable enrichment of and There were shifts within the enriched or depleted bacterial taxa between early and late sampling dates. High relative abundance of the genes , , , (macrolides); , , , and (tetracyclines); and (sulphonamides); and , (beta-lactams) were detected, with trends of increased presence in the latest sampling dates and in the location closer to the WWTP. Of note, genes conferring resistance to carbapenems OXA-58 and IMP-33-like were identified. Co-occurrence analysis of ARGs and mobile genetic elements on putative plasmids showed few instances, and the estimated human health risk score (0.19) according to MetaCompare2.0 was low. In total, 29 metagenome-assembled genomes were recovered, with only a few harbouring ARGs. This study enhances our understanding of freshwater microbial community dynamics and antibiotic resistance profiles, highlighting the need for continued ARGs monitoring.
PubMed: 38930632
DOI: 10.3390/microorganisms12061250 -
Microorganisms Jun 2024is the first reported pathogen within the genus to cause a catheter-related bloodstream infection, which occurred in 2015. In this study, the complete genome assembly...
is the first reported pathogen within the genus to cause a catheter-related bloodstream infection, which occurred in 2015. In this study, the complete genome assembly of was constructed, and the complete genome of FBCC-B549 consists of a single chromosome (3,137,745 bp) without plasmids. The constructed genome of was compared with those of two closely related species within the genus. exhibited a pattern similar to in terms of gene clusters and synteny analysis. Contrary to previous studies, biosynthetic gene cluster (BGC) analysis for predicting secondary metabolites revealed the presence of the LAP biosynthesis pathway in the complete genome of , predicting the potential synthesis of the secondary metabolite plantazolicin. Furthermore, an analysis to investigate the potential pathogenicity of did not reveal any antibiotic resistance genes; however, nine virulence factors were identified in the Virulence Factor Database (VFDB). According to these matching results in the VFDB, despite identifying a few factors involved in biofilm formation, further research is required to determine the actual impact of on pathogenicity. The complete genome of is expected to serve as a valuable resource for future studies on , which currently lack sufficient genomic sequence information.
PubMed: 38930609
DOI: 10.3390/microorganisms12061227 -
Microorganisms Jun 2024The widespread dissemination of carbapenem-resistant (CRKP) and its drug resistance transfer poses a global public health threat. While previous studies outlined CRKP's...
The widespread dissemination of carbapenem-resistant (CRKP) and its drug resistance transfer poses a global public health threat. While previous studies outlined CRKP's drug resistance mechanism, there is limited research on strategies inhibiting CRKP drug resistance spread. This study investigates the potential of () FB1-1, a probiotic, in curbing the spread of drug resistance among CRKP by evaluating its cell-free supernatant (CFS) for antibacterial activity. Evaluating the inhibitory effect of FB1-1 CFS on CRKP drug resistance spread involved analyzing its impact on drug resistance and virulence gene expression; drug resistance plasmid transfer FB1-1 CFS exhibited an MIC range of 125 μL/mL against CRKP. After eight hours of co-culture, CFS achieved a 96% and 100% sterilization rate at two and four times the MIC, respectively. At sub-inhibitory concentrations (1/2× MIC), FB1-1 CFS reduced the expression of the gene, which is pivotal for carbapenem resistance, by up to 62.13% across different CRKP strains. Additionally, it markedly suppressed the expression of the gene, a key virulence factor, by up to 91%, and the gene, essential for bacterial adhesion, by up to 53.4%. Our study primarily focuses on determining the inhibitory effect of FB1-1 CFS on CRKP strains harboring the gene, which is a critical resistance determinant in CRKP. Furthermore, FB1-1 CFS demonstrated the ability to inhibit the transfer of drug resistance plasmids among CRKP strains, thus limiting the horizontal spread of resistance genes. This study highlights FB1-1 CFS's inhibitory effect on CRKP drug resistance spread, particularly in strains carrying the gene, thus offering a novel idea and theoretical foundation for developing antibacterial drugs targeting CRKP resistance.
PubMed: 38930585
DOI: 10.3390/microorganisms12061203 -
Microorganisms Jun 2024This study describes KPC-204, a novel variant of carbapenemase, characterized by a Lys-Asp-Asp (KDD) amino acid insertion at Ambler position 269 deviates from KPC-2....
This study describes KPC-204, a novel variant of carbapenemase, characterized by a Lys-Asp-Asp (KDD) amino acid insertion at Ambler position 269 deviates from KPC-2. This variant was identified in an ST11-type clinical isolate of carbapenem-resistant from China. Notably, KPC-204 exhibits resistance to both ceftazidime-avibactam and carbapenems. Genetic analysis revealed that was located on a highly mobile IncFII/IncR plasmid within a complex genetic structure that facilitates its spread. Functional analysis, achieved through cloning into DH5α, validates KPC-204's contribution to increased resistance to ceftazidime-avibactam. The kinetic parameters showed that KPC-204 exhibited similar affinity to KPC-2 toward ceftazidime and reduced sensitivity to avibactam. Docking simulations revealed a weaker interaction between KPC-204 and avibactam compared to KPC-2. Mating experiments demonstrated the resistance's transmissibility. This investigation underscores the evolving diversity of KPC variants affecting ceftazidime-avibactam resistance, highlighting the necessity for continuous monitoring.
PubMed: 38930575
DOI: 10.3390/microorganisms12061193 -
Microorganisms Jun 2024is responsible for causing bacillary necrosis (BNP) in striped catfish () in Vietnam. This study offers a comprehensive genomic characterization of to enhance...
is responsible for causing bacillary necrosis (BNP) in striped catfish () in Vietnam. This study offers a comprehensive genomic characterization of to enhance understanding of the molecular epidemiology, virulence, and antimicrobial resistance. isolates were collected from diseased striped catfish in the Mekong Delta. The species was confirmed through PCR. Antimicrobial susceptibility testing was conducted using minimum inhibitory concentrations for commonly used antimicrobials. Thirty representative isolates were selected for whole genome sequencing to delineate their genomic profiles and phylogeny. All strains belonged to ST-26 and exhibited genetic relatedness, differing by a maximum of 90 single nucleotide polymorphisms. Most isolates carried multiple antimicrobial resistance genes, with the () gene present in 63% and in 77% of the genomes. The ESBL gene, , was identified in 30% of the genomes. Three plasmid replicon types were identified: IncA, p0111, and IncQ1. The genomes clustered into two clades based on their virulence gene profile, one group with the T3SS genes and one without. The genetic similarity among Vietnamese isolates suggests that disease spread occurs within the Mekong region, underscoring the importance of source tracking, reservoir identification, and implementation of necessary biosecurity measures to mitigate spread of BNP.
PubMed: 38930563
DOI: 10.3390/microorganisms12061182 -
Microorganisms Jun 2024In the European Union, salmonellosis is one of the most important zoonoses reported. Poultry meat and egg products are the most common food matrices associated with...
In the European Union, salmonellosis is one of the most important zoonoses reported. Poultry meat and egg products are the most common food matrices associated with presence. Moreover, wild and domestic animals could represent an important reservoir that could favour the direct and indirect transmission of pathogens to humans. spp. can infect carnivorous or omnivorous wild birds that regularly ingest food and water exposed to faecal contamination. Birds kept in captivity can act as reservoirs of spp. following ingestion of infected prey or feed. In this paper, we describe the isolation of different serovars in several species of raptors hosted in aviaries in an Italian wildlife centre and in the raw chicken necks used as their feed but intended for human consumption. Characterisations of strains were carried out by integrating classical methods and whole genome sequencing analysis. The strains of isolated in poultry meat and birds belonged to the same cluster, with some of them being multidrug-resistant (MDR) and carrying the Col(pHAD28) plasmid-borne (fluoro)quinolone resistance gene, thus confirming the source of infection. Differently, the found in feed and raptors were all MDR, carried a plasmid of emerging (pESI)-like plasmid and belonged to different clusters, possibly suggesting a long-lasting infection or the presence of additional undetected sources. Due to the high risk of fuelling a reservoir of human pathogens, the control and treatment of feed for captive species are crucial.
PubMed: 38930551
DOI: 10.3390/microorganisms12061169