Extraction of Modified Adeno-Associated Virus-Like Particles Displaying Peptides on Their Surface.

Virus-like particles (VLPs) of the adeno-associated virus (AAV) can be produced using the baculovirus expression vector system. Insertion of small peptides on the surface of the AAV or AAV VLPs has been used to redirect the AAV to different target tissues and for vaccine development. Usually, the VLPs self-assemble intracellularly, and an extraction step must be performed before purification. Here, we describe the method we have used to extract AAV VLPs from insect cells successfully with peptide insertions on their surface.

One-Step Purification Strategy for Cowpea Chlorotic Mottle Virus-Like Particles Produced by the IC-BEVS.

Virus-like particles (VLP) of the cowpea chlorotic mottle virus (CCMV), a plant virus, have been shown to be safe and noncytotoxic vehicles for delivering various cargos, including nucleic acids and peptides, and as scaffolds for presenting epitopes. Thus, CCMV-VLP have acquired increasing attention to be used in fields such as gene therapy, drug delivery, and vaccine development. Regardless of their production method, most reports purify CCMV-VLP through a series of ultracentrifugation steps using sucrose density gradient ultracentrifugation, which is a complex and time-consuming process. Here, the use of anion exchange chromatography is described as a one-step protocol for purification of CCMV-VLP produced by the insect cell-baculovirus expression vector system (IC-BEVS).

Plant Virus-Like Particles for RNA Delivery.

Plant viruses such as brome mosaic virus and cowpea chlorotic mottle virus are effectively purified through PEG precipitation and sucrose cushion ultracentrifugation. Increasing ionic strength and an alkaline pH cause the viruses to swell and disassemble into coat protein subunits. The coat proteins can be reassembled into stable virus-like particles (VLPs) that carry anionic molecules at low ionic strength and through two-step dialysis from neutral pH to acidic buffer. VLPs have been extensively studied due to their ability to protect and deliver cargo, particularly RNA, while avoiding degradation under physiological conditions. Furthermore, chemical functionalization of the surface of VLPs allows for the targeted drug delivery. VLPs derived from plants have demonstrated great potential in nanomedicine by offering a versatile platform for drug delivery, imaging, and therapeutic applications.

Pseudomonas-tailed lytic phages: genome mechanical analysis and putative correlation with virion morphogenesis yield.

Aim: To unveil a putative correlation between phage genome flexibility and virion morphogenesis yield. Materials & methods: A deeper analysis of the mechanical properties of three Pseudomonas aeruginosa lytic phage genomes was undertaken, together with full genome cyclizability calculations. Results & conclusion: A putative correlation was established among phage genome flexibility, eclipse timeframe and virion particle morphogenesis yield, with a more flexible phage genome leading to a higher burst size and a more rigid phage genome leading to lower burst sizes. The results obtained are highly relevant to understand the influence of the phage genome plasticity on the virion morphogenesis yield inside the infected bacterial host cells and assumes particular relevance in the actual context of bacterial resistance to antibiotics.

ICTV Virus Taxonomy Profile: Potyviridae 2022.

The family Potyviridae includes plant viruses with single-stranded, positive-sense RNA genomes of 8-11 kb and flexuous filamentous particles 650-950 nm long and 11-20 nm wide. Genera in the family are distinguished by the host range, genomic features and phylogeny of the member viruses. Most genomes are monopartite, but those of members of the genus Bymovirus are bipartite. Some members cause serious disease epidemics in cultivated plants. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Potyviridae, which is available at ictv.global/report/potyviridae.

The stressed life of a lipid in the Zika virus membrane.

Protein-lipid interactions modulate a plethora of physiopathologic processes and have been the subject of countless studies. However, these kinds of interactions in the context of viral envelopes have remained relatively unexplored, partially because the intrinsically small dimensions of the molecular systems escape to the current resolution of experimental techniques. However, coarse-grained and multiscale simulations may fill that niche, providing nearly atomistic resolution at an affordable computational price. Here we use multiscale simulations to characterize the lipid-protein interactions in the envelope of the Zika Virus, a prominent member of the Flavivirus genus. Comparisons between the viral envelope and simpler molecular systems indicate that the viral membrane is under extreme pressures and asymmetric forces. Furthermore, the dense net of protein-protein contacts established by the envelope proteins creates poorly solvated regions that destabilize the external leaflet leading to a decoupled dynamics between both membrane layers. These findings lead to the idea that the Flaviviral membrane may store a significant amount of elastic energy, playing an active role in the membrane fusion process.

Quantification of airborne SARS-CoV-2 genomic particles in different hospital settings.

We quantified the presence of SARS-CoV-2 RNA in the air of different hospital settings and the autopsy room of the largest medical centre in Sao Paulo, Brazil. Real-time reverse-transcription PCR was used to determine the presence of the envelope protein of SARS-CoV-2 and the nucleocapsid protein genes. The E-gene was detected in 5 out of 6 samples at the ICU-COVID-19 ward and in 5 out of 7 samples at the ward-COVID-19. Similarly, in the non-dedicated facilities, the E-gene was detected in 5 out of 6 samples collected in the ICU and 4 out of 7 samples in the ward. In the necropsy room, 6 out of 7 samples were positive for the E-gene. When both wards were compared, the non-COVID ward presented a significantly higher concentration of the E-gene than in the COVID-19 ward (p = 0.003). There was no significant difference in E-gene concentration between the ICU-COVID-19 and the ICU (p = 0.548). Likewise, there was no significant difference among E-gene concentrations found in the autopsy room versus the ICUs and wards (dedicated or not) (p = 0.245). Our results show the widespread presence of aerosol contamination in different hospital units.

Evaluation of the Nucleopolyhedrovirus of Anticarsia gemmatalis as a Vector for Gene Therapy in Mammals.

BACKGROUND: Baculoviruses are insect pathogens with important biotechnological applications that transcend their use as biological controllers of agricultural pests. One species, Autographa californica multiple nucleopolhyedrovirus (AcMNPV), has been extensively exploited as a molecular platform to produce recombinant proteins and as a delivery vector for genes in mammals because it can transduce a wide range of mammalian cells and tissues without replicating or producing progeny. METHOD: To investigate if the budded virions of Anticarsia gemmatalis multiple nucleopolhyedrovirus (AgMNPV) species has the same ability, the viral genome was modified by homologous recombination into susceptible insect cells to integrate reporter genes and then it was evaluated on mammalian cell lines in a comparative form with respect to equivalent viruses derived from AcMNPV. Besides, the replicative capacity of AgMNPV´s virions in mammals was determined. RESULTS: The experiments carried out showed that the recombinant variant of AgMNPV transduces and support the expression of delivered genes but not replicates in mammalian cells. CONCLUSION: Consequently, this insect pathogen is proposed as an alternative to non-infectious viruses in humans to explore new approaches in gene therapy and other applications based on the use of mammalian cells.

Host cell p53 associates with the feline calicivirus major viral capsid protein VP1, the protease-polymerase NS6/7, and the double-stranded RNA playing a role in virus replication.

p53 is implicated in several cellular pathways such as induction of cell-cycle arrest, differentiation, senescence, and apoptosis. p53 is activated by a broad range of stress signals, including viral infections. While some viruses activate p53, others induce its inactivation, and occasionally p53 is differentially modulated during the replicative cycle. During calicivirus infections, apoptosis is required for virus exit and spread into the host; yet, the role of p53 during infection is unknown. By confocal microscopy, we found that p53 associates with FCV VP1, the protease-polymerase NS6/7, and the dsRNA. This interaction was further confirmed by proximity ligation assays, suggesting that p53 participates in the FCV replication. Knocked-down of p53 expression in CrFK cells before infection, resulted in a strong reduction of the non-structural protein levels and a decrease of the viral progeny production. These results indicate that p53 is associated with the viral replication complex and is required for an efficient FCV replication.

Arachis virus Y, a new potyvirid from Brazilian forage peanut (Arachis pintoi).

The complete nucleotide sequence of a new member of the family Potyviridae, which we propose to name "Arachis virus Y" (ArVY), is reported from forage peanut plants (Arachis pintoi) exhibiting virus-like symptoms. The ArVY positive-sense RNA genome is 9,213 nucleotides long and encodes a polyprotein with 2,947 amino acids that is predicted to be cleaved into 10 mature proteins. The complete single open reading frame (ORF) of ArVY shares 47% and 34% nucleotide and amino acid sequence identity, respectively, with the closest related virus, soybean yellow shoot virus. Electron microscopic analysis revealed elongated viral particles typical of those found in plant cells infected with potyviruses.

The Conserved Tyr176/Leu177 Motif in the α-Helix 9 of the Feline Immunodeficiency Virus Capsid Protein Is Critical for Gag Particle Assembly.

The capsid domain (CA) of the lentiviral Gag polyproteins has two distinct roles during virion morphogenesis. As a domain of Gag, it mediates the Gag-Gag interactions that drive immature particle assembly, whereas as a mature protein, it self-assembles into the conical core of the mature virion. Lentiviral CA proteins are composed of an N-terminal region with seven α-helices and a C-terminal domain (CA-CTD) formed by four α-helices. Structural studies performed in HIV-1 indicate that the CA-CTD helix 9 establishes homodimeric interactions that contribute to the formation of the hexameric Gag lattice in immature virions. Interestingly, the mature CA core also shows inter-hexameric associations involving helix 9 residues W184 and M185. The CA proteins of feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV) exhibit, at equivalent positions in helix 9, the motifs Y176/L177 and L169/F170, respectively. In this paper, we investigated the relevance of the Y176/L177 motif for FIV assembly by introducing a series of amino acid substitutions into this sequence and studying their effect on in vivo and in vitro Gag assembly, CA oligomerization, mature virion production, and viral infectivity. Our results demonstrate that the Y176/L177 motif in FIV CA helix 9 is essential for Gag assembly and CA oligomerization. Notably, mutations converting the FIV CA Y176/L177 motif into the HIV-1 WM and EIAV FL sequences allow substantial particle production and viral replication in feline cells.

Evidences of HEV genotype 3 persistence and reactivity in liver parenchyma from experimentally infected cynomolgus monkeys (Macaca fascicularis).

Hepatitis E virus genotype 3 (HEV-3) is an emerging zoonotic pathogen, responsible for sporadic cases of acute hepatitis E worldwide. Primate models have proven to be an essential tool for the study of HEV pathogenesis. Here we describe the outcomes of HEV infection in Macaca fascicularis (cynomolgus) inoculated experimentally with genotype 3. Eight adult cynomolgus macaques were inoculated intravenously with HEV-3 viral particles isolated from swine and human samples. Liver, spleen, duodenum, gallbladder and bile were sequential assessed up to the end-point of this study, 67 days post-inoculation (dpi). Our previously published findings showed that biochemical parameters return gradually to baseline levels at 55 dpi, whereas anti-HEV IgM and HEV RNA become undetectable in the serum and feces of all animals, indicating a non-viremic phase of recovery. Nevertheless, at a later stage during convalescence (67 dpi), the presence of HEV-3 RNA and antigen persist in central organs, even after peripheral viral clearance. Our results show that two cynomolgus inoculated with swine HEV-3 (animals I3 and O1) presented persistence of HEV RNA low titers in liver, gallbladder and bile. At this same stage of infection, HEV antigen (HEV Ag) could be detected in all infected animals, predominantly in non-reactive Kupffer cells (CD68+iNOS-) and sinusoidal lining cells. Simultaneously, CD4+, CD3+CD4+, and CD3+CD8+ immune cells were identified in hepatic sinusoids and small inflammatory clusters of lobular mononuclear cells, at the end-point of this study. Inability of HEV clearance in humans can result in chronic hepatitis, liver cirrhosis, with subsequent liver failure requiring transplantation. The results of our study support the persistence of HEV-3 during convalescence at 67 dpi, with active immune response in NHP. We alert to the inherent risk of viral transmission through liver transplantation, even in the absence of clinical and biochemical signs of acute infection. Thus, besides checking conventional serological markers of HEV infection, we strongly recommend HEV-3 RNA and antigen detection in liver explants as public health measure to prevent donor-recipient transmission and spread of hepatitis E.

Sumoylation of nucleoprotein (NP) mediated by activation of NADPH oxidase complex is a consequence of oxidative cellular stress during infection by Infectious salmon anemia (ISA) virus necessary to viral progeny.

The study evaluated the effects of nucleoprotein viral and the infectious virus in SHK-1 cells. The results show a strong respiratory burst activation and the induction of p47phox, SOD, GLURED, and apoptotic genes. Additionally, the cells alter the profile of SUMOylated proteins by the effect of transfection and infection experiments. In silico analyses show a set of structural motifs in NP susceptible of post-translational modification by the SUMO protein. Interestingly, the inhibition of the NADPH oxidase complex blocked the production of reactive oxygen species and the high level of cellular ROS due to the nucleoprotein and the ISAv. At the same time, the blocking of the p38MAPK signaling pathway and the use of Aristotelia chilensis, decreased viral progeny production. These results suggest that the NP triggers a strong production of ROS and modifying the post-translational profile mediated by SUMO-2/3, a phenomenon that favors the production of new virions.

The multiple origins of proteins present in tupanvirus particles.

In the last few decades, the isolation of amoebae-infecting giant viruses has challenged established principles related to the definition of virus, their evolution, and their particle structures represented by a variety of shapes and sizes. Tupanviruses are one of the most recently described amoebae-infecting viruses and exhibit a peculiar morphology with a cylindrical tail attached to the capsid. Proteomic analysis of purified viral particles revealed that virions are composed of over one hundred proteins with different functions. The putative origin of these proteins had not yet been investigated. Here, we provide evidences for multiple origins of the proteins present in tupanvirus particles, wherein 20% originate from members of the archaea, bacteria and eukarya.

To assemble or not to assemble: The changing rules of pneumovirus transmission.

Pneumoviruses represent a major public health burden across the world. Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV), two of the most recognizable pediatric infectious agents, belong to this family. These viruses are enveloped with a non-segmented negative-sense RNA genome, and their replication occurs in specialized cytosolic organelles named inclusion bodies (IB). The critical role of IBs in replication of pneumoviruses has begun to be elucidated, and our current understanding suggests they are highly dynamic structures. From IBs, newly synthesized nucleocapsids are transported to assembly sites, potentially via the actin cytoskeleton, to be incorporated into nascent virions. Released virions, which generally contain one genome, can then diffuse in the extracellular environment to target new cells and reinitiate the process of infection. This is a challenging business for virions, which must face several risks including the extracellular immune responses. In addition, several recent studies suggest that successful infection may be achieved more rapidly by multiple, rather than single, genomic copies being deposited into a target cell. Interestingly, recent data indicate that pneumoviruses have several mechanisms that permit their transmission en bloc, i.e. transmission of multiple genomes at the same time. These mechanisms include the well-studied syncytia formation as well as the newly described formation of long actin-based intercellular extensions. These not only permit en bloc viral transmission, but also bypass assembly of complete virions. In this review we describe several aspects of en bloc viral transmission and how these mechanisms are reshaping our understanding of pneumovirus replication, assembly and spread.

Packaging of Genomic RNA in Positive-Sense Single-Stranded RNA Viruses: A Complex Story.

The packaging of genomic RNA in positive-sense single-stranded RNA viruses is a key part of the viral infectious cycle, yet this step is not fully understood. Unlike double-stranded DNA and RNA viruses, this process is coupled with nucleocapsid assembly. The specificity of RNA packaging depends on multiple factors: (i) one or more packaging signals, (ii) RNA replication, (iii) translation, (iv) viral factories, and (v) the physical properties of the RNA. The relative contribution of each of these factors to packaging specificity is different for every virus. In vitro and in vivo data show that there are different packaging mechanisms that control selective packaging of the genomic RNA during nucleocapsid assembly. The goals of this article are to explain some of the key experiments that support the contribution of these factors to packaging selectivity and to draw a general scenario that could help us move towards a better understanding of this step of the viral infectious cycle.

Absence of A3Z3-Related Hypermutations in the env and vif Proviral Genes in FIV Naturally Infected Cats.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; A3) proteins comprise an important family of restriction factors that produce hypermutations on proviral DNA and are able to limit virus replication. Vif, an accessory protein present in almost all lentiviruses, counteracts the antiviral A3 activity. Seven haplotypes of APOBEC3Z3 (A3Z3) were described in domestic cats (hap I⁻VII), and in-vitro studies have demonstrated that these proteins reduce infectivity of vif-defective feline immunodeficiency virus (FIV). Moreover, hap V is resistant to vif-mediated degradation. However, studies on the effect of A3Z3 in FIV-infected cats have not been developed. Here, the correlation between APOBEC A3Z3 haplotypes in domestic cats and the frequency of hypermutations in the FIV vif and env genes were assessed in a retrospective cohort study with 30 blood samples collected between 2012 and 2016 from naturally FIV-infected cats in Brazil. The vif and env sequences were analyzed and displayed low or undetectable levels of hypermutations, and could not be associated with any specific A3Z3 haplotype.

Production of GP64-free virus-like particles from baculovirus-infected insect cells.

The retroviral Gag protein is frequently used to generate 'virus-like particles' (VLPs) for a variety of applications. Retroviral Gag proteins self-assemble and bud at the plasma membrane to form enveloped VLPs that resemble natural retrovirus virions, but contain no viral genome. The baculovirus expression vector system has been used to express high levels of the retroviral Gag protein to produce VLPs. However, VLP preparations produced from baculovirus-infected insect cells typically contain relatively large concentrations of baculovirus budded virus (BV) particles, which are similar in size and density to VLPs, and thus may be difficult to separate when purifying VLPs. Additionally, these enveloped VLPs may have substantial quantities of the baculovirus-encoded GP64 envelope protein in the VLP envelope. Since VLPs are frequently produced for vaccine development, the presence of the GP64 envelope protein in VLPs, and the presence of Autographa californica multicapsid nucleopolyhedrovirus BVs in VLP preparations, is undesirable. In the current studies, we developed a strategy for reducing BVs and eliminating GP64 in the production of VLPs, by expressing the human immunodeficiency virus type 1 gag gene in the absence of the baculovirus gp64 gene. Using a GP64null recombinant baculovirus, we demonstrate Gag-mediated VLP production and an absence of GP64 in VLPs, in the context of reduced BV production. Thus, this approach represents a substantially improved method for producing VLPs in insect cells.

Exosomes in HIV infection: A review and critical look.

Exosomes are nanovesicles released into the extracellular medium by different cell types. These vesicles carry a variety of protein and RNA cargos, and have a central role in cellular signaling and regulation. A PubMed search using the term "exosomes" finds 67 articles published in 2006. Ten years later, the same search returns approximately 1200 results for 2016 alone. The growing interest in exosomes within the scientific community reflects the different roles exerted by extracellular vesicles in biological systems and diseases. However, the increase in academic production addressing the biological function of exosomes causes much confusion, especially where the focus is on the role of exosomes in pathological situations. In this review, we critically interpret the current state of the research on exosomes and HIV infection. It is plausible to assume that exosomes influence the pathogenesis of HIV infection through their biological cargo (primarily membrane proteins and microRNAs). On the other hand, evidence for a usurpation of the exosomal budding and trafficking machinery by HIV during infection is limited, although such a mechanism cannot be ruled out. This review also discusses several biological aspects of exosomal function in the immune system. Finally, the limitations of current exosome research are pointed out.

ICTV Virus Taxonomy Profile: Geminiviridae.

The geminiviruses are a family of small, non-enveloped viruses with single-stranded, circular DNA genomes of 2500-5200 bases. Geminiviruses are transmitted by various types of insect (whiteflies, leafhoppers, treehoppers and aphids). Members of the genus Begomovirus are transmitted by whiteflies, those in the genera Becurtovirus, Curtovirus, Grablovirus, Mastrevirus and Turncurtovirus are transmitted by specific leafhoppers, the single member of the genus Topocuvirus is transmitted by a treehopper and one member of the genus Capulavirus is transmitted by an aphid. Geminiviruses are plant pathogens causing economically important diseases in most tropical and subtropical regions of the world. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Geminiviridae which is available at www.ictv.global/report/geminiviridae.