A virally encoded high-resolution screen of cytomegalovirus dependencies.

Genetic screens have transformed our ability to interrogate cellular factor requirements for viral infections1,2, but most current approaches are limited in their sensitivity, biased towards early stages of infection and provide only simplistic phenotypic information that is often based on survival of infected cells2-4. Here, by engineering human cytomegalovirus to express single guide RNA libraries directly from the viral genome, we developed virus-encoded CRISPR-based direct readout screening (VECOS), a sensitive, versatile, viral-centric approach that enables profiling of different stages of viral infection in a pooled format. Using this approach, we identified hundreds of host dependency and restriction factors and quantified their direct effects on viral genome replication, viral particle secretion and infectiousness of secreted particles, providing a multi-dimensional perspective on virus-host interactions. These high-resolution measurements reveal that perturbations altering late stages in the life cycle of human cytomegalovirus (HCMV) mostly regulate viral particle quality rather than quantity, establishing correct virion assembly as a critical stage that is heavily reliant on virus-host interactions. Overall, VECOS facilitates systematic high-resolution dissection of the role of human proteins during the infection cycle, providing a roadmap for in-depth study of host-herpesvirus interactions.

The SARS-CoV2 envelope differs from host cells, exposes procoagulant lipids, and is disrupted in vivo by oral rinses.

The lipid envelope of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an essential component of the virus; however, its molecular composition is undetermined. Addressing this knowledge gap could support the design of antiviral agents as well as further our understanding of viral-host protein interactions, infectivity, pathogenicity, and innate immune system clearance. Lipidomics revealed that the virus envelope comprised mainly phospholipids (PLs), with some cholesterol and sphingolipids, and with cholesterol/phospholipid ratio similar to lysosomes. Unlike cellular membranes, procoagulant amino-PLs were present on the external side of the viral envelope at levels exceeding those on activated platelets. Accordingly, virions directly promoted blood coagulation. To investigate whether these differences could enable selective targeting of the viral envelope in vivo, we tested whether oral rinses containing lipid-disrupting chemicals could reduce infectivity. Products containing PL-disrupting surfactants (such as cetylpyridinium chloride) met European virucidal standards in vitro; however, components that altered the critical micelle concentration reduced efficacy, and products containing essential oils, povidone-iodine, or chlorhexidine were ineffective. This result was recapitulated in vivo, where a 30-s oral rinse with cetylpyridinium chloride mouthwash eliminated live virus in the oral cavity of patients with coronavirus disease 19 for at least 1 h, whereas povidone-iodine and saline mouthwashes were ineffective. We conclude that the SARS-CoV-2 lipid envelope i) is distinct from the host plasma membrane, which may enable design of selective antiviral approaches; ii) contains exposed phosphatidylethanolamine and phosphatidylserine, which may influence thrombosis, pathogenicity, and inflammation; and iii) can be selectively targeted in vivo by specific oral rinses.

Mechanically Ventilated Patients Shed High-Titer Live Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) for Extended Periods From Both the Upper and Lower Respiratory Tract.

BACKGROUND: SARS-CoV-2 infection can lead to severe acute respiratory distress syndrome needing intensive care admission and may lead to death. As a virus that transmits by respiratory droplets and aerosols, determining the duration of viable virus shedding from the respiratory tract is critical for patient prognosis, and informs infection-control measures both within healthcare settings and the public domain. METHODS: We prospectively examined upper and lower airway respiratory secretions for both viral RNA and infectious virions in mechanically ventilated patients admitted to the intensive care unit (ICU) of the University Hospital of Wales. Samples were taken from the oral cavity (saliva), oropharynx (subglottic aspirate), or lower respiratory tract (nondirected bronchoalveolar lavage [NBAL] or bronchoalveolar lavage [BAL]) and analyzed by both quantitative PCR (qPCR) and plaque assay. RESULTS: 117 samples were obtained from 25 patients. qPCR showed extremely high rates of positivity across all sample types; however, live virus was far more common in saliva (68%) than in BAL/NBAL (32%). Average titers of live virus were higher in subglottic aspirates (4.5 × 107) than in saliva (2.2 × 106) or BAL/NBAL (8.5 × 106) and reached >108 PFU/mL in some samples. The longest duration of shedding was 98 days, while most patients (14/25) shed live virus for ≥20 days. CONCLUSIONS: ICU patients infected with SARS-CoV-2 can shed high titers of virus both in the upper and lower respiratory tract and tend to be prolonged shedders. This information is important for decision making around cohorting patients, de-escalation of personal protective equipment, and undertaking potential aerosol-generating procedures.

Anti-viral organic coatings for high touch surfaces based on smart-release, Cu2+ containing pigments.

Viruses such as SARS-CoV-2 can remain viable on solid surfaces for up to one week, hence fomites are a potential route of exposure to infectious virus. Copper has well documented antiviral properties that could limit this problem, however practical deployment of copper surfaces has been limited due to the associated costs and the incompatibility of copper metal in specific environments and conditions. We therefore developed an organic coating containing an intelligent-release Cu2+ pigment based on a cation exchange resin. Organic coatings containing a 50 % weight or higher loading of smart-release pigment were capable of completely inactivating (>6 log reduction in titre) SARS-CoV-2 within 4 h of incubation. Importantly these organic coatings demonstrated a significantly enhanced ability to inactivate SARS-CoV-2 compared to metallic copper and un-pigmented material. Furthermore, the presence of contaminating proteins inhibited the antiviral activity of metallic copper, but the intelligent-release Cu2+ pigment was unaffected. The approach of using a very basic paint system, based on a polymer binder embedded with "smart release" pigment containing an anti-viral agent which is liberated by ion-exchange, holds significant promise as a cost effective and rapidly deployed coating to confer virus inactivating capability to high touch surfaces.

Telomere length de novo assembly of all 7 chromosomes and mitogenome sequencing of the model entomopathogenic fungus, Metarhizium brunneum, by means of a novel assembly pipeline.

BACKGROUND: More accurate and complete reference genomes have improved understanding of gene function, biology, and evolutionary mechanisms. Hybrid genome assembly approaches leverage benefits of both long, relatively error-prone reads from third-generation sequencing technologies and short, accurate reads from second-generation sequencing technologies, to produce more accurate and contiguous de novo genome assemblies in comparison to using either technology independently. In this study, we present a novel hybrid assembly pipeline that allowed for both mitogenome de novo assembly and telomere length de novo assembly of all 7 chromosomes of the model entomopathogenic fungus, Metarhizium brunneum. RESULTS: The improved assembly allowed for better ab initio gene prediction and a more BUSCO complete proteome set has been generated in comparison to the eight current NCBI reference Metarhizium spp. genomes. Remarkably, we note that including the mitogenome in ab initio gene prediction training improved overall gene prediction. The assembly was further validated by comparing contig assembly agreement across various assemblers, assessing the assembly performance of each tool. Genomic synteny and orthologous protein clusters were compared between Metarhizium brunneum and three other Hypocreales species with complete genomes, identifying core proteins, and listing orthologous protein clusters shared uniquely between the two entomopathogenic fungal species, so as to further facilitate the understanding of molecular mechanisms underpinning fungal-insect pathogenesis. CONCLUSIONS: The novel assembly pipeline may be used for other haploid fungal species, facilitating the need to produce high-quality reference fungal genomes, leading to better understanding of fungal genomic evolution, chromosome structuring and gene regulation.

Another case of mistaken identity? Vaccinia virus in another live Camelpox vaccine.

Camelpox virus is the causative agent of Camelpox, a highly contagious disease of camels. A high passage Camelpox virus strain has previously been reported to contain several genes which more closely resemble Vaccinia, a virus species with no known natural host, encompassing various strains that show high inter-strain genomic variation. In this study, we demonstrate that yet another high passage, live attenuated vaccine, comprising a different strain of Camelpox virus, contains genomic sequences that match a differing strain of Vaccinia virus. These results are discussed in the context of hypotheses put forward to explain the unknown origins of Vaccinia virus, suggesting further studies to elucidate evolutionary trajectories of Orthopoxviruses through passaging.

Identification of the odorant binding proteins of Western Flower Thrips (Frankliniella occidentalis), characterization and binding analysis of FoccOBP3 with molecular modelling, molecular dynamics simulations and a confirmatory field trial.

Olfactory systems are indispensable for insects as they, including Western Flower Thrips (Frankliniella occidentalis), use olfactory cues for ovipositing and feeding. F. occidentalis use odorant binding proteins (OBPs) to transport semiochemicals to odorant receptors to induce a behavioural response from the sensillum lymph of the insect's antennae. This study identifies four OBPs of F. occidentalis and analyses their expression at three stages of growth: larvae, adult males and adult females. Further, it investigates the presence of conserved motifs and their phylogenetic relationship to other insect species. Moreover, FoccOBP3 was in silico characterized to analyse its structure along with molecular docking and molecular dynamics simulations to understand its binding with semiochemicals of F. occidentalis. Molecular docking revealed the interactions of methyl isonicotinate, p-anisaldehyde and (S)-(-)-verbenone with FoccOBP3. Moreover, molecular dynamics simulations showed bonding stability of these ligands with FoccOBP3, and field trials validated that Lurem TR (commercial product) and p-anisaldehyde had greater attraction as compared to (S)-(-)-verbenone, given the compound's binding with FoccOBP3. The current study helps in understanding the tertiary structure and interaction of FoccOBP3 with lures using computational and field data and will help in the identification of novel lures of insects in the future, given the importance of binding with OBPs.Communicated by Ramaswamy H. Sarma.

VarLOCK: sequencing-independent, rapid detection of SARS-CoV-2 variants of concern for point-of-care testing, qPCR pipelines and national wastewater surveillance.

The COVID-19 pandemic demonstrated the need for rapid molecular diagnostics. Vaccination programs can provide protection and facilitate the opening of society, but newly emergent and existing viral variants capable of evading the immune system endanger their efficacy. Effective surveillance for Variants of Concern (VOC) is therefore important. Rapid and specific molecular diagnostics can provide speed and coverage advantages compared to genomic sequencing alone, benefitting the public health response and facilitating VOC containment. Here we expand the recently developed SARS-CoV-2 CRISPR-Cas detection technology (SHERLOCK) to provide rapid and sensitive discrimination of SARS-CoV-2 VOCs that can be used at point of care, implemented in the pipelines of small or large testing facilities, and even determine the proportion of VOCs in pooled population-level wastewater samples. This technology complements sequencing efforts to allow facile and rapid identification of individuals infected with VOCs to help break infection chains. We show the optimisation of our VarLOCK assays (Variant-specific SHERLOCK) for multiple specific mutations in the S gene of SARS-CoV-2 and validation with samples from the Cardiff University Testing Service. We also show the applicability of VarLOCK to national wastewater surveillance of SARS-CoV-2 variants and the rapid adaptability of the technique for new and emerging VOCs.

Odorant Binding Proteins (OBPs) and Odorant Receptors (ORs) of Anopheles stephensi: Identification and comparative insights.

Anopheles stephensi is an important vector of malaria in the South Asia, the Middle East, and Eastern Africa. The olfactory system of An. stephensi plays an important role in host-seeking, oviposition, and feeding. Odorant binding proteins (OBPs) are globular proteins that play a pivotal role in insect olfaction by transporting semiochemicals through the sensillum lymph to odorant receptors (ORs). Custom motifs designed from annotated OBPs of Aedes aegypti, Drosophila melanogaster, and Anopheles gambiae were used for the identification of putative OBPs from protein sequences of the An. stephensi Indian strain. Further, BLASTp was also performed to identify missing OBPs and ORs. Subsequently, the presence of domains common to OBPs was confirmed. Identified OBPs were further classified into three sub-classes. Phylogenetic and syntenic analyses were carried out to find homology, and thus the evolutionary relationship between An. stephensi OBPs and ORs with those of An. gambiae, Ae. aegypti and D. melanogaster. Gene structure and physicochemical properties of the OBPs and ORs were also predicted. A total of 44 OBPs and 45 ORs were predicted from the protein sequences of An. stephensi. OBPs were further classified into the classic (27), atypical (10) and plus-C (7) OBP subclasses. The phylogeny revealed close relationship of An. stephensi OBPs and ORs with An. gambiae homologs whereas only five OBPs and two ORs of An. stephensi were related to Ae. aegypti OBPs and ORs, respectively. However, D. melanogaster OBPs and ORs were distantly rooted. Synteny analyses showed the presence of collinear block between the OBPs and ORs of An. stephensi and An. gambiae as well as Ae. aegypti's. No homology was found with D. melanogaster OBPs and ORs. As an important component of the olfactory system, correctly identifying a species' OBPs and ORs provide a valuable resource for downstream translational research that will ultimately aim to better control the malaria vector An. stephensi.

Nanopore sequencing and de novo assembly of a misidentified Camelpox vaccine reveals putative epigenetic modifications and alternate protein signal peptides.

DNA viruses can exploit host cellular epigenetic processes to their advantage; however, the epigenome status of most DNA viruses remains undetermined. Third generation sequencing technologies allow for the identification of modified nucleotides from sequencing experiments without specialized sample preparation, permitting the detection of non-canonical epigenetic modifications that may distinguish viral nucleic acid from that of their host, thus identifying attractive targets for advanced therapeutics and diagnostics. We present a novel nanopore de novo assembly pipeline used to assemble a misidentified Camelpox vaccine. Two confirmed deletions of this vaccine strain in comparison to the closely related Vaccinia virus strain modified vaccinia Ankara make it one of the smallest non-vector derived orthopoxvirus genomes to be reported. Annotation of the assembly revealed a previously unreported signal peptide at the start of protein A38 and several predicted signal peptides that were found to differ from those previously described. Putative epigenetic modifications around various motifs have been identified and the assembly confirmed previous work showing the vaccine genome to most closely resemble that of Vaccinia virus strain Modified Vaccinia Ankara. The pipeline may be used for other DNA viruses, increasing the understanding of DNA virus evolution, virulence, host preference, and epigenomics.

The mitochondrial genome contribution to the phylogeny and identification of Metarhizium species and strains.

The genus Metarhizium is composed of entomopathogenic fungal biological control agents (BCAs) used for invertebrate pest control. The phylogenetic relationships of species within this genus are still under scrutiny as several cryptic species can be found. In this work, the mitochondrial (mt) genome of Metarhizium brunneum ARSEF 4556 was fully sequenced and a comparative genome analysis was conducted with 7 other available mt genomes, belonging to 5 Metarhizium species: M. anisopliae, M. brunneum, M. robertsii, M. guizhouense and M. majus. Results showed that Metarhizium demonstrates greater conserved stability than other fungal mt genomes. Furthermore, this analysis located 7 diverse regions in both intergenic domains and gene fragments which were ideal for species/strain discrimination. The sequencing of these regions revealed several SNPs among 38 strains tested, 11 of which were uncharacterized. Single gene phylogenies presented variable results which may be used further for intra-species discrimination. Phylogenetic trees based on the concatenation of mt domains and the nuclear ITS1-5.8S-ITS2 region showed discrimination of the species studied and allowed the identification of uncharacterized strains. These were mostly placed within species M. anisopliae and M. brunneum. Five strains clustered together in a clade related to M. brunneum, suggesting that they comprise a cryptic species.