Submerged single-photon LiDAR imaging sensor used for real-time 3D scene reconstruction in scattering underwater environments.

We demonstrate a fully submerged underwater LiDAR transceiver system based on single-photon detection technologies. The LiDAR imaging system used a silicon single-photon avalanche diode (SPAD) detector array fabricated in complementary metal-oxide semiconductor (CMOS) technology to measure photon time-of-flight using picosecond resolution time-correlated single-photon counting. The SPAD detector array was directly interfaced to a Graphics Processing Unit (GPU) for real-time image reconstruction capability. Experiments were performed with the transceiver system and target objects immersed in a water tank at a depth of 1.8 meters, with the targets placed at a stand-off distance of approximately 3 meters. The transceiver used a picosecond pulsed laser source with a central wavelength of 532 nm, operating at a repetition rate of 20 MHz and average optical power of up to 52 mW, dependent on scattering conditions. Three-dimensional imaging was demonstrated by implementing a joint surface detection and distance estimation algorithm for real-time processing and visualization, which achieved images of stationary targets with up to 7.5 attenuation lengths between the transceiver and the target. The average processing time per frame was approximately 33 ms, allowing real-time three-dimensional video demonstrations of moving targets at ten frames per second at up to 5.5 attenuation lengths between transceiver and target.

Resveratrol Inhibits Venezuelan Equine Encephalitis Virus Infection by Interfering with the AKT/GSK Pathway.

The host proteins Protein Kinase B (AKT) and glycogen synthase kinase-3 (GSK-3) are associated with multiple neurodegenerative disorders. They are also important for the replication of Venezuelan equine encephalitis virus (VEEV), thereby making the AKT/GSK-3 pathway an attractive target for developing anti-VEEV therapeutics. Resveratrol, a natural phytochemical, has been shown to substantially inhibit the AKT pathway. Therefore, we attempted to explore whether it exerts any antiviral activity against VEEV. In this study, we utilized green fluorescent protein (GFP)- and luciferase-encoding recombinant VEEV to determine the cytotoxicity and antiviral efficacy via luciferase reporter assays, flow cytometry, and immunofluorescent assays. Our results indicate that resveratrol treatment is capable of inhibiting VEEV replication, resulting in increased viability of Vero and U87MG cells as well as reduced virion production and viral RNA contents within host cells for at least 48 h with a single treatment. Furthermore, the suppression of apoptotic signaling adaptors, caspase-3, caspase-7, and annexin V may also be implicated in resveratrol-mediated antiviral activity. We found that decreased phosphorylation of the AKT/GSK-3 pathway, mediated by resveratrol, can be triggered during the early stages of VEEV infection, suggesting that resveratrol disrupts the viral replication cycle and consequently promotes cell survival. Finally, molecular docking and dynamics simulation studies revealed that resveratrol can directly bind to VEEV glycoproteins, which may interfere with virus attachment and entry. In conclusion, our results suggest that resveratrol exerts inhibitory activity against VEEV infection and upon further modification could be a useful compound to study in neuroprotective research and veterinary sciences.

Structure of a paramyxovirus polymerase complex reveals a unique methyltransferase-CTD conformation.

Paramyxoviruses are enveloped, nonsegmented, negative-strand RNA viruses that cause a wide spectrum of human and animal diseases. The viral genome, packaged by the nucleoprotein (N), serves as a template for the polymerase complex, composed of the large protein (L) and the homo-tetrameric phosphoprotein (P). The ∼250-kDa L possesses all enzymatic activities necessary for its function but requires P in vivo. Structural information is available for individual P domains from different paramyxoviruses, but how P interacts with L and how that affects the activity of L is largely unknown due to the lack of high-resolution structures of this complex in this viral family. In this study we determined the structure of the L-P complex from parainfluenza virus 5 (PIV5) at 4.3-Šresolution using cryoelectron microscopy, as well as the oligomerization domain (OD) of P at 1.4-Šresolution using X-ray crystallography. P-OD associates with the RNA-dependent RNA polymerase domain of L and protrudes away from it, while the X domain of one chain of P is bound near the L nucleotide entry site. The methyltransferase (MTase) domain and the C-terminal domain (CTD) of L adopt a unique conformation, positioning the MTase active site immediately above the poly-ribonucleotidyltransferase domain and near the likely exit site for the product RNA 5' end. Our study reveals a potential mechanism that mononegavirus polymerases may employ to switch between transcription and genome replication. This knowledge will assist in the design and development of antivirals against paramyxoviruses.

The Structure, Function, and Pathobiology of the Influenza A and B Virus Ion Channels.

Influenza A virus AM2 protein is an integral membrane protein that is an ion channel (also known as a viroporin). The channel has 24 extracellular residues, 19 residues that span the membrane once and acts as both the channel pore and also the membrane anchoring domain, and a 54-residue cytoplasmic tail. The M2 protein has four identical chains linked via two disulfide bonds that form a four-helix bundle that is 107-108 more permeable to protons than Na+ ions. The M2 channel is activated by low pH, His residue 37 is the pH sensor, and Trp residue 41 is the channel gate. The channel is blocked by the antiviral drug amantadine hydrochloride. The influenza B virus BM2 protein does not have homology with the AM2 channel, but BM2 does have the His proton sensor, Trp gate, and is activated by low pH. It is thought that the AM2 and BM2 proteins have common functions in the influenza A and B virus life cycles. Both BM2 and AM2 also facilitate virus budding. The amphipathic helix in the AM2 cytoplasmic tail has an important role in the assembly of the virus, and functional AM2 protein makes the virus independent of the "endosomal sorting complex required for transport" (ESCRT) complex scission.

Repurposing Papaverine as an Antiviral Agent against Influenza Viruses and Paramyxoviruses.

Influenza viruses are highly infectious and are the leading cause of human respiratory diseases and may trigger severe epidemics and occasional pandemics. Although antiviral drugs against influenza viruses have been developed, there is an urgent need to design new strategies to develop influenza virus inhibitors due to the increasing resistance of viruses toward currently available drugs. In this study, we examined the antiviral activity of natural compounds against the following influenza virus strains: A/WSN/33 (H1N1), A/Udorn/72 (H3N2), and B/Lee/40. Papaverine (a nonnarcotic alkaloid that has been used for the treatment of heart disease, impotency, and psychosis) was found to be an effective inhibitor of multiple strains of influenza virus. Kinetic studies demonstrated that papaverine inhibited influenza virus infection at a late stage in the virus life cycle. An alteration in influenza virus morphology and viral ribonucleoprotein (vRNP) localization was observed as an effect of papaverine treatment. Papaverine is a well-known phosphodiesterase inhibitor and also modifies the mitogen-activated protein kinase (MAPK) pathway by downregulating the phosphorylation of MEK and extracellular signal-regulated kinase (ERK). Thus, the modulation of host cell signaling pathways by papaverine may be associated with the nuclear retention of vRNPs and the reduction of influenza virus titers. Interestingly, papaverine also inhibited paramyxoviruses parainfluenza virus 5 (PIV5), human parainfluenza virus 3 (HPIV3), and respiratory syncytial virus (RSV) infections. We propose that papaverine can be a potential candidate to be used as an antiviral agent against a broad range of influenza viruses and paramyxoviruses.IMPORTANCE Influenza viruses are important human pathogens that are the causative agents of epidemics and pandemics. Despite the availability of an annual vaccine, a large number of cases occur every year globally. Here, we report that papaverine, a vasodilator, shows inhibitory action against various strains of influenza virus as well as the paramyxoviruses PIV5, HPIV3, and RSV. A significant effect of papaverine on the influenza virus morphology was observed. Papaverine treatment of influenza-virus-infected cells resulted in the inhibition of virus at a later time in the virus life cycle through the suppression of nuclear export of vRNP and also interfered with the host cellular cAMP and MEK/ERK cascade pathways. This study explores the use of papaverine as an effective inhibitor of both influenza viruses as well as paramyxoviruses.

Taxonomy of the order Mononegavirales: update 2019.

In February 2019, following the annual taxon ratification vote, the order Mononegavirales was amended by the addition of four new subfamilies and 12 new genera and the creation of 28 novel species. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Taxonomy of the order Mononegavirales: second update 2018.

In October 2018, the order Mononegavirales was amended by the establishment of three new families and three new genera, abolishment of two genera, and creation of 28 novel species. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Taxonomy of the order Mononegavirales: update 2018.

In 2018, the order Mononegavirales was expanded by inclusion of 1 new genus and 12 novel species. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV) and summarizes additional taxonomic proposals that may affect the order in the near future.

Problems of classification in the family Paramyxoviridae.

A number of unassigned viruses in the family Paramyxoviridae need to be classified either as a new genus or placed into one of the seven genera currently recognized in this family. Furthermore, numerous new paramyxoviruses continue to be discovered. However, attempts at classification have highlighted the difficulties that arise by applying historic criteria or criteria based on sequence alone to the classification of the viruses in this family. While the recent taxonomic change that elevated the previous subfamily Pneumovirinae into a separate family Pneumoviridae is readily justified on the basis of RNA dependent -RNA polymerase (RdRp or L protein) sequence motifs, using RdRp sequence comparisons for assignment to lower level taxa raises problems that would require an overhaul of the current criteria for assignment into genera in the family Paramyxoviridae. Arbitrary cut off points to delineate genera and species would have to be set if classification was based on the amino acid sequence of the RdRp alone or on pairwise analysis of sequence complementarity (PASC) of all open reading frames (ORFs). While these cut-offs cannot be made consistent with the current classification in this family, resorting to genus-level demarcation criteria with additional input from the biological context may afford a way forward. Such criteria would reflect the increasingly dynamic nature of virus taxonomy even if it would require a complete revision of the current classification.

Structure of the Paramyxovirus Parainfluenza Virus 5 Nucleoprotein in Complex with an Amino-Terminal Peptide of the Phosphoprotein.

Parainfluenza virus 5 (PIV5) belongs to the family Paramyxoviridae, which consists of enveloped viruses with a nonsegmented negative-strand RNA genome encapsidated by the nucleoprotein (N). Paramyxovirus replication is regulated by the phosphoprotein (P) through protein-protein interactions with N and the RNA polymerase (L). The chaperone activity of P is essential to maintain the unassembled RNA-free form of N in order to prevent nonspecific RNA binding and premature N oligomerization. Here, we determined the crystal structure of unassembled PIV5 N in complex with a P peptide (N0P) derived from the N terminus of P (P50) at 2.65 Å. The PIV5 N0P consists of two domains: an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a hinge region. The cleft at the hinge region of RNA-bound PIV5 N was previously shown to be an RNA binding site. The N0P structure shows that the P peptide binds to the CTD of N and extends toward the RNA binding site to inhibit N oligomerization and, hence, RNA binding. Binding of P peptide also keeps the PIV5 N in the open form. A molecular dynamics (MD) analysis of both the open and closed forms of N shows the flexibility of the CTD and the preference of the N protein to be in an open conformation. The gradual opening of the hinge region, to release the RNA, was also observed. Together, these results advance our knowledge of the conformational swapping of N required for the highly regulated paramyxovirus replication.IMPORTANCE Paramyxovirus replication is regulated by the interaction of P with N and L proteins. Here, we report the crystal structure of unassembled parainfluenza virus 5 (PIV5) N chaperoned with P peptide. Our results provide a detailed understanding of the binding of P to N. The conformational switching of N between closed and open forms during its initial interaction with P, as well as during RNA release, was analyzed. Our data also show the plasticity of the CTD and the importance of domain movement for conformational switching. The results improve our understanding of the mechanism of interchanging N conformations for RNA replication and release.

ICTV Virus Taxonomy Profile: Pneumoviridae.

The family Pneumoviridae comprises large enveloped negative-sense RNA viruses. This taxon was formerly a subfamily within the Paramyxoviridae, but was reclassified in 2016 as a family with two genera, Orthopneumovirus and Metapneumovirus. Pneumoviruses infect a range of mammalian species, while some members of the Metapneumovirus genus may also infect birds. Some viruses are specific and pathogenic for humans, such as human respiratory syncytial virus and human metapneumovirus. There are no known vectors for pneumoviruses and transmission is thought to be primarily by aerosol droplets and contact. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Pneumoviridae, which is available at www.ictv.global/report/pneumoviridae.

Monomeric ephrinB2 binding induces allosteric changes in Nipah virus G that precede its full activation.

Nipah virus is an emergent paramyxovirus that causes deadly encephalitis and respiratory infections in humans. Two glycoproteins coordinate the infection of host cells, an attachment protein (G), which binds to cell surface receptors, and a fusion (F) protein, which carries out the process of virus-cell membrane fusion. The G protein binds to ephrin B2/3 receptors, inducing G conformational changes that trigger F protein refolding. Using an optical approach based on second harmonic generation, we show that monomeric and dimeric receptors activate distinct conformational changes in G. The monomeric receptor-induced changes are not detected by conformation-sensitive monoclonal antibodies or through electron microscopy analysis of G:ephrinB2 complexes. However, hydrogen/deuterium exchange experiments confirm the second harmonic generation observations and reveal allosteric changes in the G receptor binding and F-activating stalk domains, providing insights into the pathway of receptor-activated virus entry.Nipah virus causes encephalitis in humans. Here the authors use a multidisciplinary approach to study the binding of the viral attachment protein G to its host receptor ephrinB2 and show that monomeric and dimeric receptors activate distinct conformational changes in G and discuss implications for receptor-activated virus entry.

Towards industrial ultrafast laser microwelding: SiO2 and BK7 to aluminum alloy.

We report systematic analysis and comparison of ps-laser microwelding of industry relevant Al6082 parts to SiO2 and BK7. Parameter mapping of pulse energy and focal depth on the weld strength is presented. The welding process was found to be strongly dependent on the focal plane but has a large tolerance to variation in pulse energy. Accelerated lifetime tests by thermal cycling from -50° to +90°C are presented. Welds in Al6082-BK7 parts survive over the full temperature range where the ratio of thermal expansion coefficients is 3.4:1. Welds in Al6082-SiO2 parts (ratio 47.1:1) survive only a limited temperature range.

Single-photon three-dimensional imaging at up to 10 kilometers range.

Depth and intensity profiling of targets at a range of up to 10 km is demonstrated using time-of-flight time-correlated single-photon counting technique. The system comprised a pulsed laser source at 1550 nm wavelength, a monostatic scanning transceiver and a single-element InGaAs/InP single-photon avalanche diode (SPAD) detector. High-resolution three-dimensional images of various targets acquired over ranges between 800 metres and 10.5 km demonstrate long-range depth and intensity profiling, feature extraction and the potential for target recognition. Using a total variation restoration optimization algorithm, the acquisition time necessary for each pixel could be reduced by at least a factor of ten compared to a pixel-wise image processing approach. Kilometer range depth profiles are reconstructed with average signal returns of less than one photon per pixel.

Taxonomy of the order Mononegavirales: update 2017.

In 2017, the order Mononegavirales was expanded by the inclusion of a total of 69 novel species. Five new rhabdovirus genera and one new nyamivirus genus were established to harbor 41 of these species, whereas the remaining new species were assigned to already established genera. Furthermore, non-Latinized binomial species names replaced all paramyxovirus and pneumovirus species names, thereby accomplishing application of binomial species names throughout the entire order. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane.

Influenza virus assembles and buds at the plasma membrane of virus-infected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some proteins, like hemagglutinin (HA), NA, and M2, are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains, whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immunogold staining. The distribution of these proteins was examined individually and pairwise by using the Ripley K function, a type of nearest-neighbor analysis. Individually, HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly coclustered in the plasma membrane; however, in the case of NA and M2, clustering depends upon the expression system used. Despite both proteins being raft resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly cocluster, but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the coexpression of other viral proteins. Similarly, M2 and NP occupy separate compartments, but an association can be bridged by the coexpression of M1.IMPORTANCE The complement of influenza virus proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft-like domains, whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships between viral proteins in the plasma membrane. Some proteins, such as HA and M2, inherently cocluster within the membrane, although M2 is found mostly at the periphery of regions of HA, consistent with the proposed role of M2 in scission at the end of budding. The association between some pairs of influenza virus proteins, such as M2 and NP, appears to be brokered by additional influenza virus proteins, in this case M1. HA and NA, while raft associated, reside in distinct domains, reflecting their distributions in the viral membrane.

Structural basis for antibody cross-neutralization of respiratory syncytial virus and human metapneumovirus.

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are two closely related viruses that cause bronchiolitis and pneumonia in infants and the elderly1, with a significant health burden2-6. There are no licensed vaccines or small-molecule antiviral treatments specific to these two viruses at present. A humanized murine monoclonal antibody (palivizumab) is approved to treat high-risk infants for RSV infection7,8, but other treatments, as well as vaccines, for both viruses are still in development. Recent epidemiological modelling suggests that cross-immunity between RSV, HMPV and human parainfluenzaviruses may contribute to their periodic outbreaks9, suggesting that a deeper understanding of host immunity to these viruses may lead to enhanced strategies for their control. Cross-reactive neutralizing antibodies to the RSV and HMPV fusion (F) proteins have been identified10,11. Here, we examine the structural basis for cross-reactive antibody binding to RSV and HMPV F protein by two related, independently isolated antibodies, MPE8 and 25P13. We solved the structure of the MPE8 antibody bound to RSV F protein and identified the 25P13 antibody from an independent blood donor. Our results indicate that both antibodies use germline residues to interact with a conserved surface on F protein that could guide the emergence of cross-reactivity. The induction of similar cross-reactive neutralizing antibodies using structural vaccinology approaches could enhance intrinsic cross-immunity to these paramyxoviruses and approaches to controlling recurring outbreaks.

Possibility and Challenges of Conversion of Current Virus Species Names to Linnaean Binomials.

Botanical, mycological, zoological, and prokaryotic species names follow the Linnaean format, consisting of an italicized Latinized binomen with a capitalized genus name and a lower case species epithet (e.g., Homo sapiens). Virus species names, however, do not follow a uniform format, and, even when binomial, are not Linnaean in style. In this thought exercise, we attempted to convert all currently official names of species included in the virus family Arenaviridae and the virus order Mononegavirales to Linnaean binomials, and to identify and address associated challenges and concerns. Surprisingly, this endeavor was not as complicated or time-consuming as even the authors of this article expected when conceiving the experiment. [Arenaviridae; binomials; ICTV; International Committee on Taxonomy of Viruses; Mononegavirales; virus nomenclature; virus taxonomy.].

Flexibility of the Head-Stalk Linker Domain of Paramyxovirus HN Glycoprotein Is Essential for Triggering Virus Fusion.

UNLABELLED: The Paramyxoviridae comprise a large family of enveloped, negative-sense, single-stranded RNA viruses with significant economic and public health implications. For nearly all paramyxoviruses, infection is initiated by fusion of the viral and host cell plasma membranes in a pH-independent fashion. Fusion is orchestrated by the receptor binding protein hemagglutinin-neuraminidase (HN; also called H or G depending on the virus type) protein and a fusion (F) protein, the latter undergoing a major refolding process to merge the two membranes. Mechanistic details regarding the coupling of receptor binding to F activation are not fully understood. Here, we have identified the flexible loop region connecting the bulky enzymatically active head and the four-helix bundle stalk to be essential for fusion promotion. Proline substitution in this region of HN of parainfluenza virus 5 (PIV5) and Newcastle disease virus HN abolishes cell-cell fusion, whereas HN retains receptor binding and neuraminidase activity. By using reverse genetics, we engineered recombinant PIV5-EGFP viruses with mutations in the head-stalk linker region of HN. Mutations in this region abolished virus recovery and infectivity. In sum, our data suggest that the loop region acts as a "hinge" around which the bulky head of HN swings to-and-fro to facilitate timely HN-mediate F-triggering, a notion consistent with the stalk-mediated activation model of paramyxovirus fusion. IMPORTANCE: Paramyxovirus fusion with the host cell plasma membrane is essential for virus infection. Membrane fusion is orchestrated via interaction of the receptor binding protein (HN, H, or G) with the viral fusion glycoprotein (F). Two distinct models have been suggested to describe the mechanism of fusion: these include "the clamp" and the "provocateur" model of activation. By using biochemical and reverse genetics tools, we have obtained strong evidence in favor of the HN stalk-mediated activation of paramyxovirus fusion. Specifically, our data strongly support the notion that the short linker between the head and stalk plays a role in "conformational switching" of the head group to facilitate F-HN interaction and triggering.

Mutagenesis of Paramyxovirus Hemagglutinin-Neuraminidase Membrane-Proximal Stalk Region Influences Stability, Receptor Binding, and Neuraminidase Activity.

UNLABELLED: Paramyxoviridae consist of a large family of enveloped, negative-sense, nonsegmented single-stranded RNA viruses that account for a significant number of human and animal diseases. The fusion process for nearly all paramyxoviruses involves the mixing of the host cell plasma membrane and the virus envelope in a pH-independent fashion. Fusion is orchestrated via the concerted action of two surface glycoproteins: an attachment protein called hemagglutinin-neuraminidase (HN [also called H or G depending on virus type and substrate]), which acts as a receptor binding protein, and a fusion (F) protein, which undergoes a major irreversible refolding process to merge the two membranes. Recent biochemical evidence suggests that receptor binding by HN is dispensable for cell-cell fusion. However, factors that influence the stability and/or conformation of the HN 4-helix bundle (4HB) stalk have not been studied. Here, we used oxidative cross-linking as well as functional assays to investigate the role of the structurally unresolved membrane-proximal stalk region (MPSR) (residues 37 to 58) of HN in the context of headless and full-length HN membrane fusion promotion. Our data suggest that the receptor binding head serves to stabilize the stalk to regulate fusion. Moreover, we found that the MPSR of HN modulates receptor binding and neuraminidase activity without a corresponding regulation of F triggering. IMPORTANCE: Paramyxoviruses require two viral membrane glycoproteins, the attachment protein variously called HN, H, or G and the fusion protein (F), to couple host receptor recognition to virus-cell fusion. The HN protein has a globular head that is attached to a membrane-anchored flexible stalk of ∼80 residues and has three activities: receptor binding, neuraminidase, and fusion activation. In this report, we have identified the functional significance of the membrane-proximal stalk region (MPSR) (HN, residues 37 to 56) of the paramyxovirus parainfluenza virus (PIV5), a region of the HN stalk that has not had its structure determined by X-ray crystallography. Our data suggest that the MPSR influences receptor binding and neuraminidase activity via an indirect mechanism. Moreover, the receptor binding head group stabilizes the 4HB stalk as part of the general mechanism to fine-tune F-activation.