Search Results (36)

We performed a diagnostic disease investigation on a wild smallmouth bass (Micropterus dolomieu) with skin ulcers that was collected from Lake Oahe, South Dakota, following reports from anglers of multiple fish with similar lesions. Gross and histologic lesions of ulcerative dermatitis, myositis, and lymphocytolysis within the spleen and kidneys were consistent with largemouth bass virus (LMBV) infection. LMBV was detected by conventional PCR in samples of a skin ulcer, and the complete genome sequence of the LMBV (99,184 bp) was determined from a virus isolate obtained from a homogenized skin sample. A maximum likelihood (ML) phylogenetic analysis based on the major capsid protein (MCP) gene alignment supported the LMBV isolate (LMBV-SD-2023) as a member of the species Ranavirus micropterus1, branching within the subclade of LMBV isolates recovered from North American largemouth (Micropterus salmoides) and smallmouth bass. This is the first detection of LMBV in wild smallmouth bass from South Dakota. The ultrastructure of the LMBV isolate exhibited the expected icosahedral shape of virions budding from cellular membranes. Viral nucleic acid in infected cells was visualized via in situ hybridization (ISH) within dermal granulomas, localized predominantly at the margin of epithelioid macrophages and central necrosis. Further sampling is needed to determine the geographic distribution, affected populations, and evolutionary relationship between isolates of LMBV. 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

Spiroplasma virus 4 (SpV4) is a bacteriophage of the Microviridae, which packages circular ssDNA within non-enveloped T = 1 icosahedral capsids. It infects spiroplasmas, which are known pathogens of honeybees. Here, the structure of the SpV4 virion is determined using cryo-electron microscopy to a resolution of 2.5 Å. A striking feature of the SpV4 capsid is the mushroom-like protrusions at the 3-fold axes, which is common among all members of the subfamily Gokushovirinae. While the function of the protrusion is currently unknown, this feature varies widely in this subfamily and is therefore possibly an adaptation for host recognition. Furthermore, on the interior of the SpV4 capsid, the location of DNA-binding protein VP8 was identified and shown to have low structural conservation to the capsids of other viruses in the family. The structural characterization of SpV4 will aid future studies analyzing the virus–host interaction, to understand disease mechanisms at a molecular level. Furthermore, the structural comparisons in this study, including a low-resolution structure of the chlamydia phage 2, provide an overview of the structural repertoire of the viruses in this family that infect various bacterial hosts, which in turn infect a wide range of animals and plants. Full article

Escherichia coli (E. coli) is one of the most common sources of infection in humans and animals. The emergence of E. coli which acquires resistance to various antibiotics has made treatment difficult. Bacteriophages can be considered promising agents to expand the options for the treatment of antibiotic-resistant bacteria. This study describes the isolation and characterization of Escherichia phage KIT06, which can infect E. coli resistant to the quinolone antibiotic nalidixic acid. Phage virions possess an icosahedral head that is 93 ± 8 nm in diameter and a contractile tail (116 ± 12 nm × 13 ± 5 nm). The phage was found to be stable under various thermal and pH conditions. A one-step growth curve showed that the latent time of the phage was 20 min, with a burst size of 28 particles per infected cell. Phage KIT06 infected 7 of 12 E. coli strains. It inhibited the growth of the host bacterium and nalidixic acid-resistant E. coli. The lipopolysaccharide and outer membrane proteins of E. coli, tsx and btuB, are phage receptors. Phage KIT06 is a new species of the genus Tequatrovirus with a genome of 167,059 bp consisting of 264 open reading frames (ORFs) that encode gene products related to morphogenesis, replication, regulation, and host lysis. The lack of genes encoding integrase or excisionase indicated that this phage was lytic. Thus, KIT06 could potentially be used to treat antibiotic-resistant E. coli using phage therapy. However, further studies are essential to understand its use in combination with other antimicrobial agents and its safe use in such applications. Full article

Actinobacillus pleuropneumoniae is the causative agent of pleuropneumonia, an economically important lung disease in pigs. In draft genomes of two Cypriot clinical A. pleuropneumoniae isolates (MIDG3457 and MIDG3459), we previously identified single genomic regions with homology to Mu-like bacteriophage and presented preliminary evidence of active phage. Here, updated Phastest genomic analysis identified two loci in both MIDG3457 and MIDG3459 that were predicted to encode proteins with high homology to, and whose organisation was characteristic of, Mu-like phages. Phylogenetically, the closest matches were with Mannheimia Vb and Glaesserella SuMu phages. Phastest scored the loci as “complete”, indicating they produced active phage. PCR amplification of the Mu-like phage c and tail genes from DNase-treated polyethylene glycol 8000 (PEG)-precipitated supernatants of MIDG3457 and MIDG3459 (grown in either Brain Heart Infusion-NAD or Grace’s Insect Medium-NAD broth) indicated the presence of intact virions. The phages from MIDG3457 and MIDG3459 were named PluMu 3457-1, 3457-2, and PluMu 3459-1 and PluMu 3459-2, respectively. Transmission electron microscopy (TEM) of the PEG-precipitated supernatants of broth-grown MIDG3459 identified virions with icosahedral heads and tails, consistent with other Mu-like phages. We conclude that MIDG3459 produces an active Mu-like phage. Full article

During our search for aphid-pathogenic viruses, a comovirus was isolated from wild asymptomatic Brassica hirta (white mustard) plants harboring a dense population of Brevicoryne brassicae aphids. The transmission-electron-microscopy visualization of purified virions revealed icosahedral particles. The virus was mechanically transmitted to plants belonging to Brassicaceae, Solanaceae, Amaranthaceae, and Fabaceae families, showing unique ringspot symptoms only on B. rapa var. perviridis plants. The complete viral genome, comprised of two RNA segments, was sequenced. RNA1 and RNA2 contained 5921 and 3457 nucleotides, respectively, excluding the 3′ terminal poly-adenylated tails. RNA1 and RNA2 each had one open-reading frame encoding a polyprotein of 1850 and 1050 amino acids, respectively. The deduced amino acids at the Pro-Pol region, delineated between a conserved CG motif of 3C-like proteinase and a GDD motif of RNA-dependent RNA polymerase, shared a 96.5% and 90% identity with the newly identified Apis mellifera-associated comovirus and Arabidopsis latent virus 1 (ArLV1), respectively. Because ArLV1 was identified early in 2018, the B. hirta comovirus was designated as ArLV1-IL-Bh. A high-throughput-sequencing-analyses of the extracted RNA from managed honeybees and three abundant wild bee genera, mining bees, long-horned bees, and masked bees, sampled while co-foraging in a Mediterranean ecosystem, allowed the assembly of ArLV1-IL-Bh, suggesting pollinators’ involvement in comovirus spread in weeds. Full article

Vaccines are the cornerstone of infectious disease control and prevention. The outbreak of SARS-CoV-2 has confirmed the urgent need for a new approach to the design of novel vaccines. Plant viruses and their derivatives are being used increasingly for the development of new medical and biotechnological applications, and this is reflected in a number of preclinical and clinical studies. Plant viruses have a unique combination of features (biosafety, low reactogenicity, inexpensiveness and ease of production, etc.), which determine their potential. This review presents the latest data on the use of plant viruses with different types of symmetry as vaccine components and adjuvants in cancer immunotherapy. The discussion concludes that the most promising approaches might be those that use structurally modified plant viruses (spherical particles) obtained from the Tobacco mosaic virus. These particles combine high adsorption properties (as a carrier) with strong immunogenicity, as has been confirmed using various antigens in animal models. According to current research, it is evident that plant viruses have great potential for application in the development of vaccines and in cancer immunotherapy. Full article

Antibiotic resistance poses a growing risk to public health, requiring new tools to combat pathogenic bacteria. Contractile injection systems, including bacteriophage tails, pyocins, and bacterial type VI secretion systems, can efficiently penetrate cell envelopes and become potential antibacterial agents. Bacteriophage XM1 is a dsDNA virus belonging to the Myoviridae family and infecting Vibrio bacteria. The XM1 virion, made of 18 different proteins, consists of an icosahedral head and a contractile tail, terminated with a baseplate. Here, we report cryo-EM reconstructions of all components of the XM1 virion and describe the atomic structures of 14 XM1 proteins. The XM1 baseplate is composed of a central hub surrounded by six wedge modules to which twelve spikes are attached. The XM1 tail contains a fewer number of smaller proteins compared to other reported phage baseplates, depicting the minimum requirements for building an effective cell-envelope-penetrating machine. We describe the tail sheath structure in the pre-infection and post-infection states and its conformational changes during infection. In addition, we report, for the first time, the in situ structure of the phage neck region to near-atomic resolution. Based on these structures, we propose mechanisms of virus assembly and infection. Full article

In this study, we discovered a new virus named Quanzhou mulberry virus (QMV), which was identified from the leaves of an ancient mulberry tree. This tree is over 1300 years old and is located at Fujian Kaiyuan Temple, a renowned cultural heritage site in China. We obtained the complete genome sequence of QMV using RNA sequencing followed by rapid amplification of complementary DNA ends (RACE). The QMV genome is 9256 nucleotides (nt) long and encodes five open reading frames (ORFs). Its virion was made of icosahedral particles. Phylogenetic analysis suggests that it belongs to the unclassified Riboviria. An infectious clone for QMV was generated and agroinfiltrated into Nicotiana benthamiana and mulberry, resulting in no visible disease symptoms. However, systemic movement of the virus was only observed in mulberry seedlings, suggesting that it has a host-specific pattern of movement. Our findings provide a valuable reference for further studies on QMV and related viruses, contributing to the understanding of viral evolution and biodiversity in mulberry. Full article

Dengue virus (DENV) is a single-stranded (+)-sense RNA virus that infects humans and mosquitoes, posing a significant health risk in tropical and subtropical regions. Mature virions are composed of an icosahedral shell of envelope (E) and membrane (M) proteins circumscribing a lipid bilayer, which in turn contains a complex of the approximately 11 kb genomic RNA with capsid (C) proteins. Whereas the structure of the envelope is clearly defined, the structure of the packaged genome in complex with C proteins remains elusive. Here, we investigated the interactions of C proteins with viral RNA, in solution and inside mature virions, via footprinting and cross-linking experiments. We demonstrated that C protein interaction with DENV genomes saturates at an RNA:C protein ratio below 1:250. Moreover, we also showed that the length of the RNA genome interaction sites varies, in a multimodal distribution, consistent with the C protein binding to each RNA site mostly in singlets or pairs (and, in some instances, higher numbers). We showed that interaction sites are preferentially sites with low base pairing, as previously measured by 2′-acetylation analyzed by primer extension (SHAPE) reactivity indicating structuredness. We found a clear association pattern emerged: RNA-C protein binding sites are strongly associated with long-range RNA–RNA interaction sites, particularly inside virions. This, in turn, explains the need for C protein in viral genome packaging: the protein has a chief role in coordinating these key interactions, promoting proper packaging of viral RNA. Such sites are, thus, highly consequential for viral assembly, and, as such, may be targeted in future drug development strategies against these and related viruses. Full article

Salmonella myovirus SPN3US has a T = 27 capsid composed of >50 different gene products, including many that are packaged along with the 240 kb genome and ejected into the host cell. Recently, we showed that an essential phage-encoded prohead protease gp245 is responsible for cleavage of proteins during SPN3US head assembly. This proteolytic maturation step induces major changes in precursor head particles, enabling them to expand and undergo genome packaging. To comprehensively define the composition of the mature SPN3US head and elucidate how it is modified by proteolysis during assembly, we conducted tandem mass spectrometry analysis of purified virions and tailless heads. Fourteen protease cleavage sites were identified in nine proteins, including eight sites not previously identified in head proteins in vivo. Among these was the maturation cleavage site of gp245 which was identical to the autocleavage site we had previously identified in purified recombinant gp245. Our findings underscore the value of employing multiple mass spectrometry-based experimental strategies as a way to enhance the detection of head protein cleavage sites in tailed phages. In addition, our results have identified a conserved set of head proteins in related giant phages that are similarly cleaved by their respective prohead proteases, suggesting that these proteins have important roles in governing the formation and function of large icosahedral capsids. Full article

Hepatitis B virus (HBV) relies on the core protein (HBc) to establish productive infection, as defined by the formation of the covalently closed circularized DNA (cccDNA), as well as to carry out almost every step of the lifecycle following cccDNA formation. Multiple copies of HBc form an icosahedral capsid shell that encapsidates the viral pregenomic RNA (pgRNA) and facilitates the reverse transcription of pgRNA to a relaxed circular DNA (rcDNA) within the capsid. During infection, the complete HBV virion, which contains an outer envelope layer in addition to the internal nucleocapsid containing rcDNA, enters human hepatocytes via endocytosis and traffics through the endosomal compartments and the cytosol to deliver its rcDNA to the nucleus to produce cccDNA. In addition, progeny rcDNA, newly formed in cytoplasmic nucleocapsids, is also delivered to the nucleus in the same cell to form more cccDNA in a process called intracellular cccDNA amplification or recycling. Here, we focus on recent evidence demonstrating differential effects of HBc in affecting cccDNA formation during de novo infection vs. recycling, obtained using HBc mutations and small molecule inhibitors. These results implicate a critical role of HBc in determining HBV trafficking during infection, as well as in nucleocapsid disassembly (uncoating) to release rcDNA, events essential for cccDNA formation. HBc likely functions in these processes via interactions with host factors, which contributes critically to HBV host tropism. A better understanding of the roles of HBc in HBV entry, cccDNA formation, and host species tropism should accelerate ongoing efforts to target HBc and cccDNA for the development of an HBV cure and facilitate the establishment of convenient animal models for both basic research and drug development. Full article

Although the important role of microbes in freshwater is well understood, studies on phage–host systems in such environments during ice cover are completely lacking. Here, we describe the isolation and characterization of three new bacteriophages infecting Xylophilus sp., Caudobacter sp., and Polaromonas sp. from freshwater samples taken under the ice cover of Lake Konnevesi, Finland. Lumi, Kuura, and Tiera bacteriophages have tailed icosahedral virions and double-stranded DNA. Lumi is a siphophage with a genome of 80,496 bp, and Kuura and Tiera are podophages, and their genomes are 43,205 and 45,327 bp in length, resembling viruses in the class Caudoviricetes. Their host ranges were very limited among the winter-isolated bacterial strains from Konnevesi, each infecting only their own hosts. They can infect efficiently at 4 °C, showing that they are adapted to living in lake water under ice cover. Analysis of the viral genome sequences showed that a significant number of the gene products of each virus are unique, indicating that there is unexplored viral diversity in freshwaters. To our knowledge, Lumi and Tiera are the first phages isolated on the Xylophilus sp. and Polaromonas sp. strains, allowing their exploitation in further studies of freshwater bacterial–phage interactions. Full article

Getah virus (GETV) is a member of the alphavirus genus, and it infects a variety of animal species, including horses, pigs, cattle, and foxes. Human infection with this virus has also been reported. The structure of GETV has not yet been determined. In this study, we report the cryo-EM structure of GETV at a resolution of 3.5 Å. This structure reveals conformational polymorphism of the envelope glycoproteins E1 and E2 at icosahedral 3-fold and quasi-3-fold axes, which is believed to be a necessary organization in forming a curvature surface of virions. In our density map, three extra densities are identified, one of which is believed a “pocket factor”; the other two are located by domain D of E2, and they may maintain the stability of E1/E2 heterodimers. We also identify three N-glycosylations at E1 N141, E2 N200, and E2 N262, which might be associated with receptor binding and membrane fusion. The resolving of the structure of GETV provides new insights into the structure and assembly of alphaviruses and lays a basis for studying the differences of biology and pathogenicity between arthritogenic and encephalitic alphaviruses. Full article

The coat proteins (CPs) of single-stranded RNA bacteriophages (ssRNA phages) directly assemble around the genomic RNA (gRNA) to form a near-icosahedral capsid with a single maturation protein (Mat) that binds the gRNA and interacts with the retractile pilus during infection of the host. Understanding the assembly of ssRNA phages is essential for their use in biotechnology, such as RNA protection and delivery. Here, we present the complete gRNA model of the ssRNA phage Qβ, revealing that the 3′ untranslated region binds to the Mat and the 4127 nucleotides fold domain-by-domain, and is connected through long-range RNA–RNA interactions, such as kissing loops. Thirty-three operator-like RNA stem-loops are located and primarily interact with the asymmetric A/B CP-dimers, suggesting a pathway for the assembly of the virions. Additionally, we have discovered various forms of the virus-like particles (VLPs), including the canonical T = 3 icosahedral, larger T = 4 icosahedral, prolate, oblate forms, and a small prolate form elongated along the 3-fold axis. These particles are all produced during a normal infection, as well as when overexpressing the CPs. When overexpressing the shorter RNA fragments encoding only the CPs, we observed an increased percentage of the smaller VLPs, which may be sufficient to encapsidate a shorter RNA. Full article