Production Of Virus Particles Research Articles

Simultaneous Giant Virus and Virophage Quantification Using Droplet Digital PCR.

Viruses are an abundant component of aquatic systems, but their detection and quantification remain a challenge. Virophages co-replicate with giant viruses in the shared host cell, and can inhibit the production of new giant virus particles, thereby increasing the survival of the infected host population. Here, we present a protocol for Droplet Digital PCR (ddPCR) to quantify simultaneously giant virus and virophage in a mixed sample, enabling the rapid, culture-free and high throughput detection of virus and virophage. As virophage can be present as free virus particles or integrated into the virus host’s genome as well as associated with organic particles, we developed a simple method that enables discrimination between free and particle-associated virophages. The latter include aggregated virophage particles as well as virophage integrated into the host genome. We used, for our experiments, a host-virus-virophage system consisting of Cafeteria burkhardae, CroV and mavirus. Our results show that ddPCR can be an efficient method to quantify virus and virophage, and we discuss potential applications of the method for studying ecological and evolutionary processes of virus and virophages.

EVALUATION OF Murine norovirus (MNV)-1 INFECTION IN PRIMARY MOUSE B CELLS.

Abstract Methods Primary mouse spleen B cells were stimulated or not with LPS (10ug/ml) + IL-4 (10IU/ml) for 48 hours. Two million cells were infected 72 h with MNV-1 at a MOI of 5. The non-structural (NS7) viral protein expression was analyzed in cell lysates by Western blot with a specific anti-NS7 antibody. The supernatant-associated virus in Raw 264.7 cells was determined by paque assay. Results B cells can be infected by MNV-1, even though they do not express the functional receptor CD300LD and CD300LF present in macrophages and Tuft cells. Since MNV-1 can uses CD44 as a receptor in BMDCs, we wanted to investigate if this molecule has a role in MNV-1 entry into B cells. First, we corroborated MNV-1 infection of B cells by evaluating the presence of NS7 by western blot and viral particles production in the supernatant at 72 hpi. To evaluate the role of CD44 in MNV-1 entry into B cells, we blocked the CD44 receptor using two different anti-CD44 monoclonal antibodies, and we found a significant decrease in both the amount of NS7 protein and virus yield, indicating that CD44 plays a role during virus binding/entry into B cells. Since CD44 expression is increased in LPS + IL-4 activated B cells, we wanted to determine if MNV-1 infection could be favored in these conditions. A correlation between the activation of B cells for 48 h and a significant increase of twice of MNV-1 NS7 protein and virus yield production were detected after infection. Conclusion Blocking CD44 correlates with a reduction of MNV-1 protein synthesis and virus yield production, indicating a role of CD44 in MNV-1 binding/entry. The increase of MNV NS7 protein and virus yield production in C57BL/6J spleen B cells activated with LPS + IL-4 that overexpresses CD44 on its surface, also supports this conclusion. CEMR is supported by a Conacyt fellowship, CVU fellowship number: 780860

Two Conserved Phenylalanine Residues in the E1 Fusion Loop of Alphaviruses Are Essential for Viral Infectivity.

Alphaviruses infect cells by a low pH-dependent fusion reaction between viral and host cell membranes that is mediated by the viral E1 glycoprotein. Most reported alphavirus E1 sequences include two phenylalanines (F87 and F95) in the fusion loop, yet the role of these residues in viral infectivity remains to be defined. Following introduction of wild type (WT), E1-F87A, and E1-F95A chikungunya virus (CHIKV) RNA genomes into cells, viral particle production was similar in magnitude. However, CHIKV E1-F87A and E1-F95A virions displayed impaired infectivity compared with WT CHIKV particles. Although WT, E1-F87A, and E1-F95A particles bound cells with similar efficiencies, E1-F87A and E1-F95A particles were unable to undergo fusion and entry into cells. Introduction of an F95A mutation in the E1 fusion loop of Mayaro virus or Venezuelan equine encephalitis virus also resulted in poorly infectious virions. We further tested whether an E1-F87A or E1-F95A mutation could be incorporated into a live-attenuated vaccine strain, CHIKV 181/25, to enhance vaccine safety. Infection of immunocompromised Ifnar1-/- and Irf3-/-Irf5-/-Irf7-/- mice with 181/25E1-F87A or 181/25E1-F95A resulted in 0% mortality, compared with 100% mortality following 181/25 infection. Despite this enhanced attenuation, surviving Ifnar1-/- and Irf3-/-Irf5-/-Irf7-/- mice were protected against virulent virus re-challenge. Moreover, single-dose immunization of WT mice with either 181/25, 181/25E1-F87A, or 181/25E1-F95A elicited CHIKV-specific antibody responses and protected against pathogenic CHIKV challenge. These studies define a critical function for residues E1-F87 and E1-F95 in alphavirus fusion and entry into target cells and suggest that incorporation of these mutations could enhance the safety of live-attenuated alphavirus vaccine candidates. IMPORTANCE Alphaviruses are human pathogens that cause both debilitating acute and chronic musculoskeletal disease and potentially fatal encephalitis. In this study, we determined that two highly conserved phenylalanine residues in the alphavirus E1 glycoprotein are required for fusion of viral and host cell membranes and viral entry into target cells. We further demonstrated that mutation of these phenylalanines results in a substantial loss of viral virulence but not immunogenicity. These data enhance an understanding of the viral determinants of alphavirus entry into host cells and could contribute to the development of new antivirals targeting these conserved phenylalanines or new live-attenuated alphavirus vaccines.

Effects of reduced gag cleavage efficiency on HIV-1 Gag-Pol package

BackgroundHIV-1 pol, which encodes enzymes required for virus replication, is initially translated as a Gag-Pol fusion protein. Gag-Pol is incorporated into virions via interactions with Gag precursor Pr55gag. Protease (PR) embedded in Gag-Pol mediates the proteolytic processing of both Pr55gag and Gag-Pol during or soon after virus particle release from cells. Since efficient Gag-Pol viral incorporation depends on interaction with Pr55gag via its N-terminal Gag domain, the prevention of premature Gag cleavage may alleviate Gag-Pol packaging deficiencies associated with cleavage enhancement from PR.ResultsWe engineered PR cleavage-blocking Gag mutations with the potential to significantly reduce Gag processing efficiency. Such mutations may mitigate the negative effects of enhanced PR activation on virus assembly and Gag-Pol packaging due to an RT dimerization enhancer or leucine zipper dimerization motif. When co-expressed with Pr55gag, we noted that enhanced PR activation resulted in reduced Gag-Pol cis or trans incorporation into Pr55gag particles, regardless of whether or not Gag cleavage sites within Gag-Pol were blocked.ConclusionsOur data suggest that the amount of HIV-1 Gag-Pol or Pol viral incorporation is largely dependent on virus particle production, and that cleavage blocking in the Gag-Pol N-terminal Gag domain does not exert significant impacts on Pol packaging.

Design, Synthesis, and Preclinical Profiling of GSK3739936 (BMS-986180), an Allosteric Inhibitor of HIV-1 Integrase with Broad-Spectrum Activity toward 124/125 Polymorphs.

Allosteric HIV-1 integrase inhibitors (ALLINIs) have garnered special interest because of their novel mechanism of action: they inhibit HIV-1 replication by promoting aberrant integrase multimerization, leading to the production of replication-deficient viral particles. The binding site of ALLINIs is in a well-defined pocket formed at the interface of two integrase monomers that is characterized by conserved residues along with two polymorphic amino acids at residues 124 and 125. The design, synthesis, and optimization of pyridine-based allosteric integrase inhibitors are reported here. Optimization was conducted with a specific emphasis on the inhibition of the 124/125 polymorphs such that the designed compounds showed excellent potency in vitro against majority of the 124/125 variants. In vivo profiling of promising preclinical lead 29 showed that it exhibited a good pharmacokinetic (PK) profile in preclinical species, which resulted in a low predicted human efficacious dose. However, findings in rat toxicology studies precluded further development of 29.

Functional interactomes of the Ebola virus polymerase identified by proximity proteomics in the context of viral replication.

Ebola virus (EBOV) critically depends on the viral polymerase to replicate and transcribe the viral RNA genome in the cytoplasm of host cells, where cellular factors can antagonize or facilitate the virus life cycle. Here we leverage proximity proteomics and conduct a small interfering RNA (siRNA) screen to define the functional interactome of EBOV polymerase. As a proof of principle, we validate two cellular mRNA decay factors from 35 identified host factors: eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1) and up-frameshift protein 1 (UPF1). Our data suggest that EBOV can subvert restrictions of cellular mRNA decay and repurpose GSPT1 and UPF1 to promote viral replication. Treating EBOV-infected human hepatocytes with a drug candidate that targets GSPT1 for degradation significantly reduces viral RNA load and particle production. Our work demonstrates the utility of proximity proteomics to capture the functional host interactome of the EBOV polymerase and to illuminate host-dependent regulation of viral RNA synthesis.

Application of the CRISPR/Cas9 System to Study Regulation Pathways of the Cellular Immune Response to Influenza Virus.

Influenza A virus (IAV) causes a respiratory infection that affects millions of people of different age groups and can lead to acute respiratory distress syndrome. Currently, host genes, receptors, and other cellular components critical for IAV replication are actively studied. One of the most convenient and accessible genome-editing tools to facilitate these studies is the CRISPR/Cas9 system. This tool allows for regulating the expression of both viral and host cell genes to enhance or impair viral entry and replication. This review considers the effect of the genome editing system on specific target genes in cells (human and chicken) in terms of subsequent changes in the influenza virus life cycle and the efficiency of virus particle production.

Enhanced Seroconversion to West Nile Virus Proteins in Mice by West Nile Kunjin Replicon Virus-like Particles Expressing Glycoproteins from Crimean-Congo Hemorrhagic Fever Virus.

Removal of genes coding for major parts of capsid (C), premembrane (prM), and envelope (E) proteins on the flavivirus genome aborts the production of infectious virus particles where the remaining genome forms a replicon that retains replicability in host cells. The C-prM-E proteins can also be expressed in trans with the flavivirus replicons to generate single-round infectious replicon virus-like particles (RVPs). In this study, we characterized the use of RVPs based on the Kunjin strain of WNV (WNVKUN) as a putative WNV vaccine candidate. In addition, the WNVKUN C-prM-E genes were substituted with the Crimean–Congo hemorrhagic fever virus (CCHFV) genes encoding the glycoproteins Gn and Gc to generate a WNVKUN replicon expressing the CCHFV proteins. To generate RVPs, the WNVKUN replicon was transfected into a cell line expressing the WNVKUN C-prM-E. Using immunoblotting and immunofluorescence assays, we showed that the replicon can express the CCHFV Gn and Gc proteins and the RVPs can transduce cells to express WNVKUN proteins and the CCHFV Gn and Gc proteins. Our study also revealed that these RVPs have potential as a vaccine platform with low risk of recombination as it infects cells only in one cycle. The immunization of mice with the RVPs resulted in high seroconversion to both WNV E and NS1 but limited seroconversion to CCHFV Gn and Gc proteins. Interestingly, we found that there was enhanced production of WNV E, NS1 antibodies, and neutralizing antibodies by the inclusion of CCHFV Gc and Gn into WNVKUN RVPs. Thus, this study indicates a complementary effect of the CCHFV Gn and Gc proteins on the immunogenicity by WNVKUN RVPs, which may be applied to develop a future vaccine against the WNV.

Phosphopeptide enrichment using Phos-tag technology reveals functional phosphorylation of the nucleocapsid protein of SARS-CoV-2

Phosphorylation of viral proteins serves as a regulatory mechanism during the intracellular life cycle of infected viruses. There is therefore a pressing need to develop a method to efficiently purify and enrich phosphopeptides derived from viral particles in biological samples. In this study, we utilized Phos-tag technology to analyze the functional phosphorylation of the nucleocapsid protein (N protein; NP) of severe respiratory syndrome coronavirus 2 (SARS-CoV-2). Viral particles were collected from culture supernatants of SARS-CoV-2-infected VeroE6/TMPRSS2 cells by ultracentrifugation, and phosphopeptides were purified by Phos-tag magnetic beads for LC-MS/MS analysis. Analysis revealed that NP was reproducibly phosphorylated at serine 79 (Ser79). Multiple sequence alignment and phylogenetic analysis showed that the Ser79 was a distinct phospho-acceptor site in SARS-CoV-2 but not in other beta-coronaviruses. We also found that the prolyl-isomerase Pin1 bound to the phosphorylated Ser79 in NP and positively regulated the production of viral particles. These results suggest that SARS-CoV-2 may have acquired the potent virus-host interaction during its evolution mediated by viral protein phosphorylation. Moreover, Phos-tag technology can provide a useful means for analyzing the functional phosphorylation of viral proteins. SignificanceIn this study, we aimed to investigate the functional phosphorylation of SARS-CoV-2 NP. For this purpose, we used Phos-tag technology to purify and enrich virus-derived phosphopeptides with high selectivity and reproducibility. This method can be particularly useful in analyzing viral phosphopeptides from cell culture supernatants that often contain high concentrations of fetal bovine serum and supplements. We newly identified an NP phosphorylation site at Ser79, which is important for Pin1 binding. Furthermore, we showed that the interaction between Pin1 and phosphorylated NP could enhance viral replication in a cell culture model.

A flavivirus-inducible gene expression system that modulates broad-spectrum antiviral activity against dengue and Zika viruses

Incidence of dengue virus (DENV) and Zika virus (ZIKV), two mosquito-borne flaviviruses, is increasing in large parts of the world. Vaccination and medication for these diseases are unsatisfactory. Here, we developed a novel antiviral approach, using a virus-inducible gene expression system, to block virus replication and transmission. Constructs containing the smallest replication units of dengue virus serotype 2 (DENV2) with negative-stranded DENV2 artificial genomes and genes of interest were established in an Aedes aegypti cell line, resulting in expression of target genes after DENV2 infection. Green fluorescent protein (GFP) assays confirmed the system was virus-inducible. When we used one of two apoptosis-related genes, A. aegypti michelob_x (AaMx) and inhibitor of apoptosis (IAP)-antagonist michelob_x-like protein (AaIMP) instead of GFP, the production of viral RNA and proteins were inhibited for all five viruses tested (DENV1–4 and ZIKV), and effector caspase activity was induced. The system thus inhibited the production of infectious virus particles in vitro, and in mosquitoes it did so after DENV2 infection. This is a novel broad-spectrum antiviral approach using a flavivirus-inducible gene-expression system, which could lead to new avenues for mosquito-borne disease control.

Nonclassical autophagy activation pathways are essential for production of infectious Influenza A virus in vitro.

Autophagy is a critical mechanism deployed by eukaryotic cells in response to stress, including viral infection, to boost the innate antimicrobial responses. However, an increasing number of pathogens hijack the autophagic machinery to facilitate their own replication. Influenza A virus (IAV), responsible for several global pandemics, has an intricate dependence on autophagy for successful replication in mammalian cells. To elucidate key chokepoints in the host stress responses facilitating IAV replication, we constructed a meta‐transcriptome of IAV and host gene expression dynamics during early (1–3 hpi), mid (4–6 hpi), and late (8–12 hpi) stages of the viral replication cycle at two multiplicities of infection (MOI): 1 and 5. We supplemented the global transcriptome study with phosphoproteomic analysis of stress‐activated protein kinase (SAPK/JNK) signaling in lung carcinoma (predominantly used as an in vitro model of IAV replication) and normal human bronchial epithelial cells. We report significant differences in the activation profiles of autophagy regulating genes upon IAV infection at the two MOI as well as divergent dependence on ULK1 signaling within the normal and cancer cells. Regardless of the cell model, JNK‐Thr187 signaling was crucial for the production of infectious viral particles.

Archaeal Viruses: Production of Virus Particles and Vesicle-like Viruses and Purification Using Asymmetrical Flow Field-Flow Fractionation.

Asymmetrical flow field-flow fractionation (AF4) is a separation method based on hydrodynamic size of the sample components. It can separate a broad size range of components (~103 to 109Da; particle diameter from ~1nm to ~1μm), but is especially well suited for high molecular weight samples such as virus-sized particles and extracellular vesicles. Separation takes place in an open channel where the flows control sample elution. Separation does not involve stationary phase, allowing gentle separation and good recoveries. The method is compatible with a wide variety of buffers. Coupling to various analytical detectors enables rapid assays on the molecular weight and size and their distribution, degradation, and aggregation of the sample components giving information on the sample quality. In addition to being an advanced analytical method, AF4 can be used in a semipreparative mode for purification. Here, we summarize archaeal virus production methods and virus purification by AF4 and provide examples on the steps that need optimization for obtaining good separation with the focus on halophilic archaeal viruses. Importantly, AF4 method is suitable for a variety of viruses and extracellular vesicles regardless of their host organism.

Manipulation of viral protein production using the PCNA of halovirus фCh1 via alternative start codon usage

• Proliferating Cell Nuclear Antigen (PCNA) is a scaffold protein principally found at the centre of replication, coordinating with a wide array of interaction partners. фCh1, unusually for a virus, encodes a putative PCNA. • Natrialba magadii, the only known host of фCh1, also encodes a putative PCNA of high sequence similarity, differing majorly in the presence of an alternate GTG start codon in the фCh1 variant 5’ of the ATG start codon resulting in a larger protein. Homologous recombination was used to delete the viral PCNA gene. • Complementation and overexpression strains with plasmid expressed viral PCNA variants were created and analyzed for growth and lytic behavior, viral protein levels, and virus titer. • Deletion of фCh1-ORF59, encoding PCNA ΦCh1 resulted in a significant reduction in the virus titer, reduced viral protein load, and reduced lysis. • Complementation and overexpression with WT, and mutant variants of ORF59revealed that the 5’ GTG to ATG sequence enables фCh1 to control its equilibrium with the host. The different variants have a broad range of variable effects on each of the phenotypes observed. • Experiments with a halophilic beta-galactosidase assay revealed a cis/trans mediated interaction between the viral PCNA and the origin of replication of φCh1 modulated by the 5’ GTG to ATG sequence. The virally encoded PCNA is not essential, but is crucial, to the behavior of the virus фCh1. The 5’ region upstream of the primary ATG codon and its regulation with an alternate start codon is essential to the homeostasis of the virus-host relationship. Proliferating Cell Nuclear Antigen (PCNA) is a scaffold protein principally found at the centre of replication, coordinating with a wide array of interaction partners. фCh1, unusually for a virus, encodes a putative PCNA. Natrialba magadii, the only known host of фCh1, also encodes a putative PCNA of high sequence similarity, differing in the presence of an alternate GUG start codon 5’ of the AUG start codon resulting in a larger protein. Homologous recombination was used to delete the viral PCNA gene. Complementation and overexpression strains with plasmid expressed viral PCNA variants were created and analyzed for growth and lytic behaviour, viral protein levels, and virus titer. Deletion of фCh1 ORF59, encoding PCNA ФCh1 resulted in a significant reduction in the virus titer, reduced viral protein load, and reduced lysis. Complementation and overexpression with WT and mutant variants of ORF59 revealed that modification of the GTG start codon to ATG changes the life cycle in terms of production of progeny virus particles. The different variants have a broad range of variable effects on each of the phenotypes observed. Experiments with a halophilic beta-galactosidase assay revealed a cis/trans mediated interaction between the viral PCNA and the origin of replication of фCh1 modulated by the 5’GTG to ATG sequence. The virally encoded PCNA is not essential, but is crucial, to the life cycle of the virus фCh1. The 5’ region upstream of the primary AUG codon and its regulation with an alternate start codon is essential to the homeostasis of the virus-host relationship.

Putative Invertebrate, Plant, and Wastewater Derived ssRNA Viruses in Plankton of the Anthropogenically Impacted Anacostia River, District of Columbia, USA.

The Anacostia River is a highly impacted watershed in the Northeastern United States which experiences combined sewage outfall in downstream waters. We examined the composition of RNA viruses at three sites in the river using viral metagenomics. Viromes had well represented Picornaviruses, Tombusviruses, Wolframviruses, Nodaviruses, with fewer Tobamoviruses, Sobemoviruses, and Densoviruses (ssDNA). Phylogenetic ana-lyses of detected viruses provide evidence for putatively autochthonous and allochthonous invertebrate, plant, and vertebrate host origin. The number of viral genomes matching Ribovaria increased downstream, and assemblages were most disparate between distant sites, suggesting impacts of the combined sewage overflows at these sites. Additionally, we recovered a densovirus genome fragment which was highly similar to the Clinch ambidensovirus 1, which has been attributed to mass mortality of freshwater mussels in Northeastern America. Taken together, these data suggest that RNA viromes of the Anacostia River reflect autochthonous production of virus particles by benthic metazoan and plants, and inputs from terrestrial habitats including sewage.

Rational design of self-assembled RNA nanostructures for HIV-1 virus assembly blockade.

Many pathological processes are driven by RNA-protein interactions, making such interactions promising targets for molecular interventions. HIV-1 assembly is one such process, in which the viral genomic RNA interacts with the viral Gag protein and serves as a scaffold to drive Gag multimerization that ultimately leads to formation of a virus particle. Here, we develop self-assembled RNA nanostructures that can inhibit HIV-1 virus assembly, achieved through hybridization of multiple artificial small RNAs with a stem–loop structure (STL) that we identify as a prominent ligand of Gag that can inhibit virus particle production via STL-Gag interactions. The resulting STL-decorated nanostructures (double and triple stem–loop structures denoted as Dumbbell and Tribell, respectively) can elicit more pronounced viral blockade than their building blocks, with the inhibition arising as a result of nanostructures interfering with Gag multimerization. These findings could open up new avenues for RNA-based therapy.

Potent antiviral activity of the extract of Elaeocarpus sylvestris against influenza A virus in vitro and in vivo

BackgroundElaeocarpus sylvestris (Lour.) Poir. (Elaeocarpaceae) belongs to a genus of tropical and semitropical evergreen trees, which has known biological activities such as antiviral and immunomodulatory activities. However, its antiviral potential against influenza virus infection remains unknown. PurposeIn this study, we investigated the antiviral activity of the 50% aqueous ethanolic extract of E. sylvestris (ESE) against influenza A virus (IAV) infection, which could lead to the development of novel phytomedicine to treat influenza virus infection. MethodsTo investigate the in vitro antiviral activity of ESE and its main ingredients, 1,​2,​3,​4,​6-​penta-​O-​galloyl-β-d-glucose (PGG) and geraniin (GE), the levels of viral RNAs, proteins, and infectious viral particles in IAV-infected MDCK cells were analyzed. Molecular docking analysis was performed to determine the binding energy of PGG and GE for IAV proteins. To investigate in vivo antiviral activity, IAV-infected mice were treated intranasally or intragastrically with ESE, PGG, or GE. ResultsESE and its gallate main ingredients (PGG and GE) strongly inhibited the production of viral RNAs, viral proteins, and infectious viral particles in vitro. Also through the viral attachment on cells, polymerase activity, signaling pathway, we revealed the ESE, PGG, and GE inhibit multiple steps of IAV replication. Molecular docking analysis revealed that PGG and GE could interact with 12 key viral proteins (M1, NP, NS1 effector domain (ED), NS1 RNA-binding domain (RBD), HA pocket A, HA receptor-binding domain (RBD), NA, PA, PB1, PB2 C-terminal domain, PB2 middle domain, and PB2 cap-binding domain) of IAV proteins with stable binding energy. Furthermore, intranasal administration of ESE, PGG, or GE protected mice from IAV-induced mortality and morbidity. Importantly, oral administration of ESE suppressed IAV replication and the expression of inflammatory cytokines such as IFN-γ, TNF-α, and IL-6 in the lungs to a large extent. ConclusionESE and its major components (PGG and PE) exhibited strong antiviral activity in multiple steps against IAV infection in silico, in vivo, and in vitro. Therefore, ESE could be used as a novel natural product derived therapeutic agent to treat influenza virus infection.

Hepatitis E virus RNA-dependent RNA polymerase is involved in RNA replication and infectious particle production.

Background and AimsHepatitis E virus (HEV) is one of the most common causes of acute hepatitis worldwide. Its positive‐strand RNA genome encodes three open reading frames (ORF). ORF1 is translated into a large protein composed of multiple domains and is known as the viral replicase. The RNA‐dependent RNA polymerase (RDRP) domain is responsible for the synthesis of viral RNA.Approach and ResultsHere, we identified a highly conserved α‐helix located in the RDRP thumb subdomain. Nuclear magnetic resonance demonstrated an amphipathic α‐helix extending from amino acids 1628 to 1644 of the ORF1 protein. Functional analyses revealed a dual role of this helix in HEV RNA replication and virus production, including assembly and release. Mutations on the hydrophobic side of the amphipathic α‐helix impaired RNA replication and resulted in the selection of a second‐site compensatory change in the RDRP palm subdomain. Other mutations enhanced RNA replication but impaired virus assembly and/or release.ConclusionsStructure‐function analyses identified a conserved amphipathic α‐helix in the thumb subdomain of the HEV RDRP with a dual role in viral RNA replication and infectious particle production. This study provides structural insights into a key segment of the ORF1 protein and describes the successful use of reverse genetics in HEV, revealing functional interactions between the RDRP thumb and palm subdomains. On a broader scale, it demonstrates that the HEV replicase, similar to those of other positive‐strand RNA viruses, is also involved in virus production.

Age-structured delayed SIPCV epidemic model of HPV and cervical cancer cells dynamics I. Numerical method

The numerical method for simulation dynamics of nonlinear epidemic model of age-structured sub-populations of susceptible, infectious, precancerous and cancer cells and unstructured population of human papilloma virus (HPV) is developed (SIPCV model). Cell population dynamics is described by the initial-boundary value problem for the delayed semi-linear hyperbolic equations with age- and time-dependent coefficients and HPV dynamics is described by the initial problem for nonlinear delayed ODE. The model considers two time-delay parameters: the time between viral entry into a target susceptible cell and the production of new virus particles, and duration of the first stage of delayed immune response to HPV population growing. Using the method of characteristics and method of steps we obtain the exact solution of the SIPCV epidemic model in the form of explicit recurrent formulae. The numerical method designed for this solution and used the trapezoidal rule for integrals in recurrent formulae has a second order of accuracy. Numerical experiments with vanished mesh spacing illustrate the second order of accuracy of numerical solution with respect to the benchmark solution and show the dynamical regimes of cell-HPV population with the different phase portraits.

Time-Resolved Analysis of N-RNA Interactions during RVFV Infection Shows Qualitative and Quantitative Shifts in RNA Encapsidation and Packaging.

Rift Valley fever virus (RVFV) is a negative-sense, tripartite RNA virus that is endemic to Africa and the Arabian Peninsula. It can cause severe disease and mortality in humans and domestic livestock and is a concern for its potential to spread more globally. RVFV’s nucleocapsid protein (N) is an RNA-binding protein that is necessary for viral transcription, replication, and the production of nascent viral particles. We have conducted crosslinking, immunoprecipitation, and sequencing (CLIP-seq) to characterize N interactions with host and viral RNAs during infection. In parallel, to precisely measure intracellular N levels, we employed multiple reaction monitoring mass spectrometry (MRM-MS). Our results show that N binds mostly to host RNAs at early stages of infection, yielding nascent virus particles of reduced infectivity. The expression of N plateaus 10 h post-infection, whereas the intracellular viral RNA concentration continues to increase. Moreover, the virions produced later in infection have higher infectivity. Taken together, the detailed examination of these N–RNA interactions provides insight into how the regulated expression of N and viral RNA produces both infectious and incomplete, noninfectious particles.

Characterization of Ictalurid herpesvirus 1 Glycoprotein ORF59 and Its Potential Role on Virus Entry into the Host Cells.

The channel catfish virus (CCV, Ictalurid herpesvirus 1) has caused sustained economic losses in the fish industry because of its strong infectivity and pathogenicity. Thus, it is necessary to determine the function of viral proteins in the CCV infection process. The present study aimed to characterize CCV glycoprotein ORF59 and explore its impact on virus infection in host cells. Firstly, its exclusive presence in the membrane fraction of the cell lysate and subcellular localization verified that CCV ORF59 is a viral membrane protein expressed at late-stage infection. A protein blocking assay using purified His6 tagged ORF59, expressed in sf9 insect cells using a baculovirus expression system, indicated a dose-dependent inhibitory effect of recombinant ORF59 protein on virus invasion. Knockdown of the ORF59 using a short hairpin (shRNA) showed that ORF59 silencing decreased the production of infectious virus particles in channel catfish ovary cells. The results of this study suggest that recombinant ORF59 protein might inhibit CCV entry into the host cells. These findings will promote future studies of the key functions of glycoprotein ORF59 during CCV infection.