HIV-1 subtype A1, D, and recombinant proviral genome landscapes during long-term suppressive therapy.

The primary obstacle to curing HIV-1 is a reservoir of CD4+ cells that contain stably integrated provirus. Previous studies characterizing the proviral landscape, which have been predominantly conducted in males in the United States and Europe living with HIV-1 subtype B, have revealed that most proviruses that persist during antiretroviral therapy (ART) are defective. In contrast, less is known about proviral landscapes in females with non-B subtypes, which represents the largest group of individuals living with HIV-1. Here, we analyze genomic DNA from resting CD4+ T-cells from 16 female and seven male Ugandans with HIV-1 receiving suppressive ART (n = 23). We perform near-full-length proviral sequencing at limiting dilution to examine the proviral genetic landscape, yielding 607 HIV-1 subtype A1, D, and recombinant proviral sequences (mean 26/person). We observe that intact genomes are relatively rare and clonal expansion occurs in both intact and defective genomes. Our modification of the primers and probes of the Intact Proviral DNA Assay (IPDA), developed for subtype B, rescues intact provirus detection in Ugandan samples for which the original IPDA fails. This work will facilitate research on HIV-1 persistence and cure strategies in Africa, where the burden of HIV-1 is heaviest.

Mycobacteriophage Alexphander Gene 94 Encodes an Essential dsDNA-Binding Protein during Lytic Infection.

Mycobacteriophages are viruses that specifically infect bacterial species within the genera Mycobacterium and Mycolicibacterium. Over 2400 mycobacteriophages have been isolated on the host Mycolicibacterium smegmatis and sequenced. This wealth of genomic data indicates that mycobacteriophage genomes are diverse, mosaic, and contain numerous (35-60%) genes for which there is no predicted function based on sequence similarity to characterized orthologs, many of which are essential to lytic growth. To fully understand the molecular aspects of mycobacteriophage-host interactions, it is paramount to investigate the function of these genes and gene products. Here we show that the temperate mycobacteriophage, Alexphander, makes stable lysogens with a frequency of 2.8%. Alexphander gene 94 is essential for lytic infection and encodes a protein predicted to contain a C-terminal MerR family helix-turn-helix DNA-binding motif (HTH) and an N-terminal DinB/YfiT motif, a putative metal-binding motif found in stress-inducible gene products. Full-length and C-terminal gp94 constructs form high-order nucleoprotein complexes on 100-500 base pair double-stranded DNA fragments and full-length phage genomic DNA with little sequence discrimination for the DNA fragments tested. Maximum gene 94 mRNA levels are observed late in the lytic growth cycle, and gene 94 is transcribed in a message with neighboring genes 92 through 96. We hypothesize that gp94 is an essential DNA-binding protein for Alexphander during lytic growth. We proposed that gp94 forms multiprotein complexes on DNA through cooperative interactions involving its HTH DNA-binding motif at sites throughout the phage chromosome, facilitating essential DNA transactions required for lytic propagation.

Whole-genome sequence of a novel lytic bacteriophage infecting Clavibacter michiganensis subsp. michiganensis from Turkey.

Clavibacter michiganensis subsp. michiganensis (Cmm) is an important plant-pathogenic bacterium that causes canker and wilt diseases. Biological control of the disease with bacteriophages is an alternative to conventional methods. In this study, Phage33 infecting Cmm was characterized based on morphological and genomic properties. Morphological characteristics such as shape and size were investigated using electron microscopy. The whole genome was sequenced using the Illumina Novaseq 6000 platform and the sequence was assembled and annotated. VICTOR and VIRIDIC were used for determining the phylogeny and comparing viral genomes, respectively. Electron microscopy showed that Phage33 has an icosahedral head with a diameter of ~55 nm and a long, thin, non-contractile tail ~169 nm in length. The genome of Phage33 is 56 324 bp in size, has a GC content of 62.49 % and encodes 67 open reading frames. Thirty-seven ORFs showed high homology to functionally annotated bacteriophage proteins in the NCBI database. The remaining 30 ORFs were identified as hypothetical with unknown functions. The genome contains no antimicrobial resistance, no lysogenicity and no virulence signatures, suggesting that it is a suitable candidate for biocontrol agents. The results of a blastn search showed similarity to the previously reported Xylella phage Sano, with an average nucleotide sequence identity of 92.37 % and query coverage of 91 %. This result was verified using VICTOR and VIRIDIC analysis, and suggests that Phage33 is a new member of the genus Sanovirus under the class Caudoviricetes.

DNA methylation enables recurrent endogenization of giant viruses in an animal relative.

5-Methylcytosine (5mC) is a widespread silencing mechanism that controls genomic parasites. In eukaryotes, 5mC has gained complex roles in gene regulation beyond parasite control, yet 5mC has also been lost in many lineages. The causes for 5mC retention and its genomic consequences are still poorly understood. Here, we show that the protist closely related to animals Amoebidium appalachense features both transposon and gene body methylation, a pattern reminiscent of invertebrates and plants. Unexpectedly, hypermethylated genomic regions in Amoebidium derive from viral insertions, including hundreds of endogenized giant viruses, contributing 14% of the proteome. Using a combination of inhibitors and genomic assays, we demonstrate that 5mC silences these giant virus insertions. Moreover, alternative Amoebidium isolates show polymorphic giant virus insertions, highlighting a dynamic process of infection, endogenization, and purging. Our results indicate that 5mC is critical for the controlled coexistence of newly acquired viral DNA into eukaryotic genomes, making Amoebidium a unique model to understand the hybrid origins of eukaryotic DNA.

Prevalence of HBV genotypes among patients attending Moi Teaching and Referral Hospital liver clinic.

Hepatitis B virus (HBV) belongs to the genus Orthohepadnavirus, of Hepadnaviridae family, smallest human deoxyribonucleic acid (DNA) virus with 3200 bp in a partially double-stranded circular DNA. Globally, about 2 billion people are infected with over 65 million of the chronically infected residing in Africa. Ten HBV genotypes (A-J) have been reported across the globe. Based on the World Health Organization (WHO) African Regions including Kenya have high HBV prevalence rates yet the data on prevalence rates of the various HBV genotypes and their associated biomarkers is very scanty. A cross-sectional descriptive study with purposive sampling was conducted in which a census of patients with chronic Hepatitis B (CHB) with history >6-month were reviewed for eligibility. Demographics data was abstracted from patient files and blood samples drawn for genotyping, viral load using Rotor gene Q Polymerase Chain Reaction (PCR) equipment, Hepatitis B surface Antigen (HBsAg), Hepatitis B envelope antigen (HbeAg) and Hepatitis B core antibody (Anti-HBc) using Cobas e411 machine. Out of a total of 83 patients, 43 (52%) were eligible; males 29 (67.4%), females 14 (32.6%) with mean ages of 35.1±10.8 and 34.3±9.3 respectively. Genotypes A were 34(79.1%), B were 5(11.6%), C-D were 0 while E-J were 9(20.9%). All cases of genotype B were associated with co-infection of genotype A. Majority were HBeAg negative with HBV DNA >10 IU/ml (81.4% and 86.0% respectively) with distribution among all the genotypes. Across genotypes, viral load mean percentage comparisons were: A vs. A/B = 2600 (p = 0.09), A vs. E-J = 5260 (p = 0.09) and A/B vs. E-J = 200 (p = 0.28). The most prevalent genotype was A followed by mixed co-infection of genotype A/B. Genotype A was associated with HBV DNA viral loads > 10IU/ml and high rates of HBeAg negativity. Genotypes E-J were also detected though not characterized.

No evidence on infectious DNA-based agents in pediatric acute lymphoblastic leukemia using whole metagenome shotgun sequencing.

The etiology of pediatric acute lymphatic leukemia (ALL) is still unclear. Whole-metagenome shotgun sequencing of bone marrow samples in patients with treatment-naïve ALL (n=6) was performed for untargeted investigation of bacterial and viral DNA. The control group consisted of healthy children (n=4) and children with non-oncologic diseases (n=2) undergoing bone marrow sampling. Peripheral blood of all participants was investigated at the same time. After bioinformatical elimination of potential contaminants by comparison with the employed controls, no significant amounts of microbial or viral DNA were identified.

Molecular detection and genotyping of human herpes virus 8 in blood donors in Congo.

OBJECTIVES: Human herpesvirus 8 (HHV8) is rarely studied in Congo, despite its prevalence in Africa. Among healthy individuals, HHV-8 does not always lead to a life-threatening infection; however, in immunocompromised individuals, it could lead to more severe disease. The distribution of HHV-8 genotypes varies depending on ethnicity and geographic region. METHOD: A prospective cross-sectional study included 265 samples from healthy blood donors from the National Blood Transfusion Center in Brazzaville, with an average age of 35 years, with extremes ranging from 18 to 60 years. After DNA extraction, a nested PCR was carried out for molecular detection, followed by genotyping by amplification of specific primers. RESULT: In this study, 4.9% were positive for molecular detection of HHV-8 DNA. All HHV-8 positive DNA samples that were subjected to genotyping by amplification with specific primers allowing discrimination of two major genotypes (A and B). Genotype A was identified in 5 (1.9%) samples and genotype B in 2 (0.7%) samples, indicating that both genotypes were predominant. The remaining viral DNA samples not identified as the major genotypes were classified as «indeterminate¼ and consisted of 6 (2.3%) samples. CONCLUSION: The results of the study suggest that Congo is an area where HHV-8 infection is endemic.

An affordable detection system based on RT-LAMP and DNA-nanoprobes for avian metapneumovirus.

Airborne animal viral pathogens can rapidly spread and become a global threat, resulting in substantial socioeconomic and health consequences. To prevent and control potential epidemic outbreaks, accurate, fast, and affordable point-of-care (POC) tests are essential. As a proof-of-concept, we have developed a molecular system based on the loop-mediated isothermal amplification (LAMP) technique for avian metapneumovirus (aMPV) detection, an airborne communicable agent mainly infecting turkeys and chickens. For this purpose, a colorimetric system was obtained by coupling the LAMP technique with specific DNA-functionalized AuNPs (gold nanoparticles). The system was validated using 50 different samples (pharyngeal swabs and tracheal tissue) collected from aMPV-infected and non-infected chickens and turkeys. Viral detection can be achieved in about 60 min with the naked eye, with 100% specificity and 87.88% sensitivity for aMPV. In summary, this novel molecular detection system allows suitable virus testing in the field, with accuracy and limit of detection (LOD) values highly close to qRT-PCR-based diagnosis. Furthermore, this system can be easily scalable to a platform for the detection of other viruses, addressing the current gap in the availability of POC tests for viral detection in poultry farming. KEY POINTS: •aMPV diagnosis using RT-LAMP is achieved with high sensitivity and specificity. •Fifty field samples have been visualized using DNA-nanoprobe validation. •The developed system is a reliable, fast, and cost-effective option for POCT.

Low Detection of High-risk Human Papilloma Virus in Individuals with Ameloblastoma in a Tertiary Hospital in Lagos, Nigeria.

BACKGROUND: Ameloblastoma is a benign but aggressive epithelial odontogenic neoplasm of unknown etiology. The role of human papilloma virus (HPV) in the etiology of oral squamous cell carcinoma has prompted the investigation of HPV as an etiologic factor in ameloblastoma. This study aimed to determine the frequency of high-risk (HR) HPV in conventional ameloblastoma and the clinical parameters associated with infection. MATERIALS AND METHODS: The study was approved by the ethical review boards of the institution. DNA was extracted from fresh tissue collected 750 µL of DNA/RNA Shield (Zymo Research, United States) using Invitrogen PureLink Viral RNA/DNA Mini Kit (Invitrogen, USA). The extracted DNA was assayed for the detection of 14 HR HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) using Anyplex™ II HPV HR Detection kit (Cat. No. HP7E00X) (Seegene Inc., Republic of Korea) on CFX-96TM Real-Time Polymerase Chain Reaction (PCR) System (Bio-Rad). Data on gender, age of patient, site of lesion, clinicohistological types of ameloblastoma and history of smoking, alcohol consumption, and practice of oral sex were collected. Data analysis was performed using analysis program SPSS version 25 and statistical significance was set at P < 0.05. RESULTS: Two cases of conventional ameloblastoma were positive with HPV and none of the ameloblastic carcinoma cases were positive. The HPV 16 serotype was observed in both cases. While 5 of the cases had a history of alcohol consumption, none of these cases were positive for HPV serotype. CONCLUSIONS: HPV 16 positivity was detected in two cases of conventional ameloblastomas and none in ameloblastic carcinoma using real-time PCR. There was no effect of exposure to smoking, alcohol consumption, and practice of oral sex and HPV in the etiology of ameloblastoma. Data available are suggestive of a limited role of HPV in the etiology of ameloblastoma.

Résumé Introduction:L'améloblastome est un néoplasme odontogène épithélial bénin mais agressif d'étiologie inconnue. Le rôle du papillome humain (HPV) dans l'étiologie du carcinome épidermoïde oral a incité à étudier le HPV en tant que facteur étiologique de l'améloblastome. Cette étude visait à déterminer la fréquence du HPV à haut risque (HR) dans l'améloblastome conventionnel et les paramètres cliniques associés.avec infection.Matériels et méthodes:L'étude a été approuvée par les comités d'examen éthique de l'institution. L'ADN a été extrait de frais les tissus ont collecté 750 µL de bouclier DNA/RNA (Zymo Research, États-Unis) à l'aide du mini kit Invitrogen PureLink Viral RNA/DNA (Invitrogen, ETATS-UNIS). Le DNA extrait a été analysé pour la détection de 14 types de HPV HR (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 et 68) en utilisant Kit de détection Anyplex™ II HPV HR (réf. HP7E00X) (Seegene Inc., République de Corée) sur chaîne de polymérase en temps réel CFX-96TM Système de réaction (PCR) (Bio-Rad). Données sur le sexe, l'âge du patient, le site de la lésion, les types clinico-histologiques d'améloblastome et les antécédents de le tabagisme, la consommation d'alcool et la pratique du sexe oral ont été collectés. L'analyse des données a été réalisée à l'aide du programme d'analyse SPSS version 25. et la signification statistique a été fixée à P <0,05.Résultats:Deux cas d'améloblastome conventionnel étaient positifs au HPV et aucun des les cas de carcinome améloblastique étaient positifs. Le sérotype HPV 16 a été observé dans les deux cas. Alors que 5 des cas avaient des antécédents d'alcoolisme consommation, aucun de ces cas n'était positif pour le sérotype HPV.Conclusions:Une positivité au HPV 16 a été détectée dans deux cas de améloblastomes et aucun dans le carcinome améloblastique par PCR en temps réel. Il n'y a eu aucun effet de l'exposition au tabac, à la consommation d'alcool, et la pratique du sexe oral et du HPV dans l'étiologie de l'améloblastome. Les données disponibles suggèrent un rôle limité de HPV.

Characterization of CRESS-DNA viruses in human vaginal secretions: An exploratory metagenomic investigation.

The Phylum Cressdnaviricota consists of a large number of circular Rep-encoding single-stranded (CRESS)-DNA viruses. Recently, metagenomic analyzes revealed their ubiquitous distribution in a diverse range of eukaryotes. Data relating to CRESS-DNA viruses in humans remains scarce. Our study investigated the presence and genetic diversity of CRESS-DNA viruses in human vaginal secretions. Vaginal swabs were collected from 28 women between 29 and 43 years old attending a fertility clinic in New York City. An exploratory metagenomic analysis was performed and detection of CRESS-DNA viruses was confirmed through analysis of near full-length sequences of the viral isolates. A phylogenetic tree was based on the REP open reading frame sequences of the CRESS-DNA virus genome. Eleven nearly complete CRESS-DNA viral genomes were identified in 16 (57.1%) women. There were no associations between the presence of these viruses and any demographic or clinical parameters. Phylogenetic analysis indicated that one of the sequences belonged to the genus Gemycircularvirus within the Genomoviridae family, while ten sequences represented previously unclassified species of CRESS-DNA viruses. Novel species of CRESS-DNA viruses are present in the vaginal tract of adult women. Although they be transient commensal agents, the potential clinical implications for their presence at this site cannot be dismissed.

ICTV Virus Taxonomy Profile: Turriviridae 2024.

The family Turriviridae includes viruses with a dsDNA genome of 16-17 kbp. Virions are spherical with a diameter of approximately 75 nm and comprise a host-derived internal lipid membrane surrounded by a proteinaceous capsid shell. Members of the family Turriviridae infect extremophilic archaea of the genera Sulfolobus and Saccharolobus. Viral infection results in cell lysis for Sulfolobus turreted icosahedral virus 1 infection but other members of the family can be temperate. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Turriviridae, which is available at ictv.global/report/turriviridae.

Viral Genomic DNA Packaging Machinery.

Tailed double-stranded DNA bacteriophage employs a protein terminase motor to package their genome into a preformed protein shell-a system shared with eukaryotic dsDNA viruses such as herpesviruses. DNA packaging motor proteins represent excellent targets for antiviral therapy, with Letermovir, which binds Cytomegalovirus terminase, already licensed as an effective prophylaxis. In the realm of bacterial viruses, these DNA packaging motors comprise three protein constituents: the portal protein, small terminase and large terminase. The portal protein guards the passage of DNA into the preformed protein shell and acts as a protein interaction hub throughout viral assembly. Small terminase recognises the viral DNA and recruits large terminase, which in turn pumps DNA in an ATP-dependent manner. Large terminase also cleaves DNA at the termination of packaging. Multiple high-resolution structures of each component have been resolved for different phages, but it is only more recently that the field has moved towards cryo-EM reconstructions of protein complexes. In conjunction with highly informative single-particle studies of packaging kinetics, these structures have begun to inspire models for the packaging process and its place among other DNA machines.

A universal fluorescence biosensor based on rolling circle amplification and locking probe for DNA detection.

A stable DNA signal amplification sensor was developed on account of rolling circle amplification (RCA). This sensor includes target DNA-controlled rolling circle amplification technology and locking probe DNA replacement technology, which can be used to detect DNA fragments with genetic information, thus constructing a biosensor for universal detection of DNA. This study takes the homologous DNA of human immunodeficiency virus (HIV) and let-7a as examples to describe this biosensor. The padlock probe is first cyclized by T4 DNA ligase in response to the target's reaction with it. Then, rolling cycle amplification is initiated by Phi29 DNA polymerase, resulting in the formation of a lengthy chain with several triggers. These triggers can open the locked probe LP1 with the fluorescence signal turned off, so that it can continue to react with H2 to form a stable H1-H2 double strand. This regulates the distance between B-DNA modified by the quenching group and H1 modified by fluorescent group, and the fluorescence signal is recovered.

A Novel Multiplex LAMP Assay for the Detection of Respiratory Human Adenoviruses.

Human adenoviruses (HAdVs) are common pathogens that are associated with a variety of diseases, including respiratory tract infections (RTIs). Without reliable, fast, and cost-effective detection methods for HAdVs, patients may be misdiagnosed and inappropriately treated. To address this problem, we have developed a multiplex loop-mediated isothermal amplification (LAMP) assay for the detection of the species Human adenovirus B (HAdV-B), Human adenovirus C (HAdV-C) and Human adenovirus E (HAdV-E) that cause RTIs. This multiplexing approach is based on the melting curve analysis of the amplicons with a specific melting temperature for each HAdV species. Without the need for typing of HAdVs, the LAMP results can be visually detected using colorimetric analysis. The assay reliably detects at least 375 copies of HAdV-B and -C and 750 copies of HAdV-E DNA per reaction in less than 35 min at 60 °C. The designed primers have no in silico cross-reactivity with other human respiratory pathogens. Validation on 331 nasal swab samples taken from patients with RTIs showed a 90-94% agreement rate with our in-house multiplex quantitative polymerase chain reaction (qPCR) method. Concordance between the quantitative and visual LAMP was 99%. The novel multiplexed LAMP could be an alternative to PCR for diagnostic purposes, saving personnel and equipment time, or could be used for point-of-care testing.

High level of genomic divergence in orf-I p12 and hbz genes of HTLV-1 subtype-C in Central Australia.

BACKGROUND: Human T cell lymphotropic virus type 1 (HTLV-1) infection remains a largely neglected public health problem, particularly in resource-poor areas with high burden of communicable and non-communicable diseases, such as some remote populations in Central Australia where an estimated 37% of adults are infected with HTLV-1. Most of our understanding of HTLV-1 infection comes from studies of the globally spread subtype-A (HTLV-1a), with few molecular studies reported with the Austral-Melanesian subtype-C (HTLV-1c) predominant in the Indo-Pacific and Oceania regions. RESULTS: Using a primer walking strategy and direct sequencing, we constructed HTLV-1c genomic consensus sequences from 22 First Nations participants living with HTLV-1c in Central Australia. Phylogenetic and pairwise analysis of this subtype-C proviral gDNA showed higher levels of genomic divergence in comparison to previously published HTLV-1a genomes. While the overall genomic homology between subtypes was 92.5%, the lowest nucleotide and amino acid sequence identity occurred near the 3' end of the proviral genome coding regulatory genes, especially overlapping hbz (85.37%, 77.46%, respectively) and orf-I product p12 (82.00%, 70.30%, respectively). Strikingly, the HTLV-1c genomic consensus sequences uniformly showed a defective translation start codon for the immune regulatory proteins p12/p8 encoded by the HTLV-1A orf-I. Deletions in the proviral genome were detected in many subjects, particularly in the structural gag, pol and env genes. Similarly, using a droplet digital PCR assay measuring the copies of gag and tax per reference host genome, we quantitatively confirmed that provirus retains the tax gene region at higher levels than gag. CONCLUSIONS: Our genomic analysis of HTLV-1c in Central Australia in conjunction with earlier Melanesian HTLV-1c sequences, elucidate substantial differences with respect to the globally spread HTLV-1a. Future studies should address the impact these genomic differences have on infection and the regionally distinctive frequency of associated pulmonary disease. Understanding the host and virus subtype factors which contribute to the differential morbidity observed, is crucial for the development of much needed therapeutics and vaccine strategies against this highly endemic infection in remote First Nations communities in Central Australia.

CRISPR-Cas assisted diagnostics of plant viruses and challenges.

Plant viruses threaten global food security by infecting commercial crops, highlighting the critical need for efficient virus detection to enable timely preventive measures. Current techniques rely on polymerase chain reaction (PCR) for viral genome amplification and require laboratory conditions. This review explores the applications of CRISPR-Cas assisted diagnostic tools, specifically CRISPR-Cas12a and CRISPR-Cas13a/d systems for plant virus detection and analysis. The CRISPR-Cas12a system can detect viral DNA/RNA amplicons and can be coupled with PCR or isothermal amplification, allowing multiplexed detection in plants with mixed infections. Recent studies have eliminated the need for expensive RNA purification, streamlining the process by providing a visible readout through lateral flow strips. The CRISPR-Cas13a/d system can directly detect viral RNA with minimal preamplification, offering a proportional readout to the viral load. These approaches enable rapid viral diagnostics within 30 min of leaf harvest, making them valuable for onsite field applications. Timely identification of diseases associated with pathogens is crucial for effective treatment; yet developing rapid, specific, sensitive, and cost-effective diagnostic technologies remains challenging. The current gold standard, PCR technology, has drawbacks such as lengthy operational cycles, high costs, and demanding requirements. Here we update the technical advancements of CRISPR-Cas in viral detection, providing insights into future developments, versatile applications, and potential clinical translation. There is a need for approaches enabling field plant viral nucleic acid detection with high sensitivity, specificity, affordability, and portability. Despite challenges, CRISPR-Cas-mediated pathogen diagnostic solutions hold robust capabilities, paving the way for ideal diagnostic tools. Alternative applications in virus research are also explored, acknowledging the technology's limitations and challenges.

Establishing Host-Virus Link Through Host Metabolism: Viral DNA SIP Validation Using T4 Bacteriophage and E. coli.

DNA Stable Isotope Probing is emerging as a potent methodology for investigating host-virus interactions, based on the essential reliance of viruses on host organisms for the production of virions. Despite the anticipated link between host isotopic compositions and the generated virions, the application of stable isotope probing to viral DNA has never been evaluated on simple biological models. In this study, we assessed the efficacy of this method on the bacteriophage T4 and its host, Escherichia coli. Through the cultivation of E. coli cells on a 13C-enriched substrate and subsequent propagation of T4 bacteriophage, we examine the degree of isotopic enrichment in viral DNA. Our investigation reveals a strong correlation between the proportion of 13C6-D-glucose in the growth substrate and the buoyant density in CsCl gradient of T4 DNA, confirming the validity of DNA SIP in viral ecology. These findings underscore the potential of DNA SIP as a robust tool for characterizing the diversity of viruses infecting hosts with specific metabolic activities and provide then a foundation for further exploration in viral ecology research.

From DNA to lytic proteins: transcription and translation of the bacteriophage T5 holin/endolysin operon.

The analysis of transcriptional activity of the bacteriophage T5 hol/endo operon conducted in the paper revealed a strong constitutive promoter recognized by E. coli RNA polymerase and a transcription initiation point of the operon. It was also shown that the only translational start codon for holin was a non-canonical TTG. Translation initiation regions (TIRs) of both genes of the operon (hol and endo) were further analyzed using chimeric constructs, in which parts of the hol/endo regulatory regions were fused with the gene of a reporter protein (EGFP). It was found that TIR of hol was 20 times less effective than that of endo. As it turned out, the level of EGFP production was influenced by the composition of the constructs and the type of the hol start codon. Apparently, the translational suppression of holin's accumulation and posttranslational activation of endolysin by Ca2+ are the main factors ensuring the proper timing of the host cell lysis by bacteriophage T5. The approach based on the use of chimeric constructs proposed in the paper can be recommended for studying other native or artificial operons of any complexity: analyzing the impacts of separate DNA regions, as well as their coupled effect, on the processes of transcription and translation of recombinant protein(s).

Characterization of a lipid-based jumbo phage compartment as a hub for early phage infection.

Viral genomes are most vulnerable to cellular defenses at the start of the infection. A family of jumbo phages related to phage ΦKZ, which infects Pseudomonas aeruginosa, assembles a protein-based phage nucleus to protect replicating phage DNA, but how it is protected prior to phage nucleus assembly is unclear. We find that host proteins related to membrane and lipid biology interact with injected phage protein, clustering in an early phage infection (EPI) vesicle. The injected virion RNA polymerase (vRNAP) executes early gene expression until phage genome separation from the vRNAP and the EPI vesicle, moving into the nascent proteinaceous phage nucleus. Enzymes involved in DNA replication and CRISPR/restriction immune nucleases are excluded by the EPI vesicle. We propose that the EPI vesicle is rapidly constructed with injected phage proteins, phage DNA, host lipids, and host membrane proteins to enable genome protection, early transcription, localized translation, and to ensure faithful genome transfer to the proteinaceous nucleus.

Analysis of a new phage, KZag1, infecting biofilm of Klebsiella pneumoniae: genome sequence and characterization.

BACKGROUND: This study investigates the effectiveness of the bacteriophage KZag1 against drug-resistant Klebsiella pneumoniae, aiming to assess its potential as a therapeutic agent. The novelty lies in the characterization of KZag1, a Myovirus with specific efficacy against multidrug-resistant K. pneumoniae strains. This highlights the significance of exploring alternative strategies, particularly phage therapy, in addressing biofilm-associated infections. METHODS: KZag1, characterized by a typical Myovirus structure with a 75 ± 5 nm diameter icosahedral head and a 15 ± 5 nm short tail, was evaluated in experimental trials against 15 strains of K. pneumoniae. The infection cycle duration was determined to be 50 min, resulting in an estimated burst size of approximately 83 plaque-forming units per colony-forming unit (PFU/CFU). Stability assessments were conducted within a pH range of 4 to 12 and temperatures ranging from 45°C to 60°C. Biofilm biomass reduction was observed, particularly at a multiplicity of infection (MOI) of 10. RESULTS: KZag1 demonstrated infection efficacy against 12 out of 15 tested K. pneumoniae strains. The phage exhibited stability across a broad pH range and at elevated temperatures. Notably, treatment with KZag1 significantly reduced K. pneumoniae biofilm biomass, emphasizing its potential in combating biofilm formation. Genomic analysis revealed a complete genome of 157,089 base pairs with a GC content of 46.38%, encompassing 203 open reading frames (ORFs) and a cysteine-specific tRNA sequence. Comparison with phage GP4 highlighted similarities, with KZag1 having a longer genome by approximately 4829 base pairs and a higher GC content by approximately 0.93%. Phylogenetic analysis classified KZag1 within the Myoviridae family. CONCLUSION: The efficacy of KZag1 against K. pneumoniae biofilm suggests its potential as a therapeutic candidate, especially for drug-resistant infections. Further clinical research is warranted to explore its synergy with other treatments, elucidate genomic traits, compare with Myoviridae phages, and understand its host interactions. These findings underscore the promising role of KZag1 in addressing drug-resistant bacterial infections.