Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus.

A SARS-CoV-2 variant carrying the Spike protein amino acid change D614G has become the most prevalent form in the global pandemic. Dynamic tracking of variant frequencies revealed a recurrent pattern of G614 increase at multiple geographic levels: national, regional, and municipal. The shift occurred even in local epidemics where the original D614 form was well established prior to introduction of the G614 variant. The consistency of this pattern was highly statistically significant, suggesting that the G614 variant may have a fitness advantage. We found that the G614 variant grows to a higher titer as pseudotyped virions. In infected individuals, G614 is associated with lower RT-PCR cycle thresholds, suggestive of higher upper respiratory tract viral loads, but not with increased disease severity. These findings illuminate changes important for a mechanistic understanding of the virus and support continuing surveillance of Spike mutations to aid with development of immunological interventions.

Prevalence of gp160 polymorphisms known to be related to decreased susceptibility to temsavir in different subtypes of HIV-1 in the Los Alamos National Laboratory HIV Sequence Database.

BACKGROUND: Fostemsavir, a prodrug of the gp120-directed attachment inhibitor temsavir, is indicated for use in heavily treatment-experienced individuals with MDR HIV-1. Reduced susceptibility to temsavir in the clinic maps to discrete changes at amino acid positions in gp160: S375, M426, M434 and M475. OBJECTIVES: To query the Los Alamos National Laboratory (LANL) HIV Sequence Database for the prevalence of polymorphisms at gp160 positions of interest. METHODS: Full-length gp160 sequences (N = 7560) were queried for amino acid polymorphisms relative to the subtype B consensus at positions of interest; frequencies were reported for all sequences and among subtypes/circulating recombinant forms (CRFs) with ≥10 isolates in the database. RESULTS: Among 239 subtypes in the database, the 5 most prevalent were B (n = 2651, 35.1%), C (n = 1626, 21.5%), CRF01_AE (n = 674, 8.9%), A1 (n = 273, 3.6%) and CRF02_AG (n = 199, 2.6%). Among all 7560 sequences, the most prevalent amino acids at positions of interest (S375, 73.5%; M426, 82.1%; M434, 88.2%; M475, 89.9%) were the same as the subtype B consensus. Specific polymorphisms with the potential to decrease temsavir susceptibility (S375H/I/M/N/T/Y, M426L/P, M434I/K and M475I) were found in <10% of isolates of subtypes D, G, A6, BC, F1, CRF07_BC, CRF08_BC, 02A, CRF06_cpx, F2, 02G and 02B. S375H and M475I were predominant among CRF01_AE (S375H, 99.3%; M475I, 76.3%; consistent with previously reported low temsavir susceptibility of this CRF) and 01B (S375H, 71.7%; M475I, 49.5%). CONCLUSIONS: Analysis of the LANL HIV Sequence Database found a low prevalence of gp160 amino acid polymorphisms with the potential to reduce temsavir susceptibility overall and among most of the common subtypes.

Evolutionary history, potential intermediate animal host, and cross-species analyses of SARS-CoV-2.

To investigate the evolutionary history of the recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China, a total of 70 genomes of virus strains from China and elsewhere with sampling dates between 24 December 2019 and 3 February 2020 were analyzed. To explore the potential intermediate animal host of the SARS-CoV-2 virus, we reanalyzed virome data sets from pangolins and representative SARS-related coronaviruses isolates from bats, with particular attention paid to the spike glycoprotein gene. We performed phylogenetic, split network, transmission network, likelihood-mapping, and comparative analyses of the genomes. Based on Bayesian time-scaled phylogenetic analysis using the tip-dating method, we estimated the time to the most recent common ancestor and evolutionary rate of SARS-CoV-2, which ranged from 22 to 24 November 2019 and 1.19 to 1.31 × 10-3 substitutions per site per year, respectively. Our results also revealed that the BetaCoV/bat/Yunnan/RaTG13/2013 virus was more similar to the SARS-CoV-2 virus than the coronavirus obtained from the two pangolin samples (SRR10168377 and SRR10168378). We also identified a unique peptide (PRRA) insertion in the human SARS-CoV-2 virus, which may be involved in the proteolytic cleavage of the spike protein by cellular proteases, and thus could impact host range and transmissibility. Interestingly, the coronavirus carried by pangolins did not have the RRAR motif. Therefore, we concluded that the human SARS-CoV-2 virus, which is responsible for the recent outbreak of COVID-19, did not come directly from pangolins.

HIV-1 recombinant forms in immigrants regularly residing in Milan, northern Italy.

To assess the HIV -1subtypes distribution in HIV-1 positive migrants living in Milan we studied 77 HIV-1 patients followed at the San Raffaele Hospital of Milan. Twenty subjects were born in Europe, 43 in the Americas, 10 in Africa and 4 in Asia. Unsafe heterosexual activity prevailed in migrants born in Africa and male homosexuality in those born in European, American and Asian countries (p = 0.05). The phylogeny showed that 38/77 (49.3%) subjects carried HIV-B subtype while the remaining strains were classified as not pure HIV-1 B subtypes 13/77 (16.9%) or recombinant forms 26/77 (33.8%). Female gender more frequently showed HIV-1 non-B strains and rarely HIV-1 B subtypes (12/39, 30.8% vs. 3/38, 7.9%, p = 0.02). Transmitted drug resistance was identified in 10/77 (13%) patients predominately with B subtype. Our data underscore a large heterogeneity in HIV-1 subtypes and a large proportion of recombinant forms.

Detection of antisense protein (ASP) RNA transcripts in individuals infected with human immunodeficiency virus type 1 (HIV-1).

The detection of antisense RNA is hampered by reverse transcription (RT) non-specific priming, due to the ability of RNA secondary structures to prime RT in the absence of specific primers. The detection of antisense RNA by conventional RT-PCR does not allow assessment of the polarity of the initial RNA template, causing the amplification of non-specific cDNAs. In this study we have developed a modified protocol for the detection of human immunodeficiency virus type 1 (HIV-1) antisense protein (ASP) RNA. Using this approach, we have identified ASP transcripts in CD4+ T cells isolated from five HIV-infected individuals, either untreated or under suppressive therapy. We show that ASP RNA can be detected in stimulated CD4+ T cells from both groups of patients, but not in unstimulated cells. We also show that in untreated patients, the patterns of expression of ASP and env are very similar, with the levels of ASP RNA being markedly lower than those of env. Treatment of cells from one viraemic patient with α-amanitin greatly reduces the rate of ASP RNA synthesis, suggesting that it is associated with RNA polymerase II, the central enzyme in the transcription of protein-coding genes. Our data represent the first nucleotide sequences obtained in patients for ASP, demonstrating that its transcription indeed occurs in those HIV-1 lineages in which the ASP open reading frame is present.

Thermal Boundary Conductance Across Heteroepitaxial ZnO/GaN Interfaces: Assessment of the Phonon Gas Model.

We present experimental measurements of the thermal boundary conductance (TBC) from 78-500 K across isolated heteroepitaxially grown ZnO films on GaN substrates. This data provides an assessment of the underlying assumptions driving phonon gas-based models, such as the diffuse mismatch model (DMM), and atomistic Green's function (AGF) formalisms used to predict TBC. Our measurements, when compared to previous experimental data, suggest that TBC can be influenced by long wavelength, zone center modes in a material on one side of the interface as opposed to the '"vibrational mismatch"' concept assumed in the DMM; this disagreement is pronounced at high temperatures. At room temperature, we measure the ZnO/GaN TBC as 490[+150,-110] MW m-2 K-1. The disagreement among the DMM and AGF, and the experimental data at elevated temperatures, suggests a non-negligible contribution from other types of modes that are not accounted for in the fundamental assumptions of these harmonic based formalisms, which may rely on anharmonicity. Given the high quality of these ZnO/GaN interfaces, these results provide an invaluable, critical, and quantitative assessment of the accuracy of assumptions in the current state of the art computational approaches used to predict phonon TBC across interfaces.

Persistence of SIV in the brain of SIV-infected Chinese rhesus macaques with or without antiretroviral therapy.

Persistence of HIV-1 reservoirs in the central nervous system (CNS) is an obstacle to cure strategies. However, little is known about residual viral distribution, viral replication levels, and genetic diversity in different brain regions of HIV-infected individuals on combination antiretroviral therapy (cART). Because myeloid cells particularly microglia are likely major reservoirs in the brain, and more microglia exist in white matter than gray matter in a human brain, we hypothesized the major viral reservoirs in the brain are the white matter reflected by higher levels of viral DNA. To address the issue, we used the Chinese rhesus macaque (ChRM) model of SIV infection, and treated 11 SIVmac251-infected animals including long-term nonprogressors with cART for up to 24 weeks. SIV reservoirs were assessed by SIV DNA levels in 16 specific regions of the brain and 4 regions of spinal cord. We found relatively high frequencies of SIV in basal ganglia and brain stem compared to other regions. cART-receiving animals had significantly lower SIV DNA levels in the gray matter than white matter. Moreover, a shortened envelope gp120 with 21 nucleotide deletions and guanine-to-adenine hypermutations were observed. These results demonstrate that SIV enters the CNS in SIV-infected ChRM with a major reservoir in the white matter after cART; the SIV/ChRM/cART is an appropriate model for studying HIV CNS reservoirs and testing new eradication strategies. Further, examining multiple regions of the CNS may be needed when assessing whether an agent is successful in reducing the size of SIV reservoirs in the CNS.

Titanium contacts to graphene: process-induced variability in electronic and thermal transport.

Contact resistance (R C) is a major limiting factor in the performance of graphene devices. R C is sensitive to the quality of the interface and the composition of the contact, which are affected by the graphene transfer process and contact deposition conditions. In this work, a linear correlation is observed between the composition of Ti contacts, characterized by x-ray photoelectron spectroscopy, and the Ti/graphene contact resistance measured by the transfer length method. We find that contact composition is tunable via deposition rate and base pressure. Reactor base pressure is found to effect the resultant contact resistance. The effect of contact deposition conditions on thermal transport measured by time-domain thermoreflectance is also reported. Interfaces with higher oxide composition appear to result in a lower thermal boundary conductance. Possible origins of this thermal boundary conductance change with oxide composition are discussed.

Signature pattern analysis for the full-length env gene of the earliest Korean subclade B of HIV-1: outbreak among Korean hemophiliacs.

The epidemiological link in the hypervariable env gene between viruses infecting HIV-positive hemophiliacs (HPs) and plasma donors was not studied. We determined full-length env gene sequences in 20 HPs, 3 plasma donors whose plasma was used for domestic clotting factor (DCF) production, and 54 local controls (LCs). Env genes from viruses in frozen stored sera obtained 1-3 years after diagnosis and from samples collected several years after infection were amplified via RT-PCR and subjected to direct sequencing. Phylogenetic analysis indicated that all sequences were subtype B, including 133 sequences from 77 cases (20 HPs, 3 plasma donors, and 54 LCs) belonging to the Korean subclade B (KSB) and 6 sequences from 5 cases that did not belong to the KSB. Env gene sequences from donors O and P and those of the 20 HPs comprised 2 subclusters within the KSB, although phylogenetic analysis did not support significant bootstrap values. In contrast, signature pattern analysis indicated signature nucleotides at 43 positions between the HPs and LCs (P < 0.05). In particular, specific signature nucleotides at 4 positions were fully conserved in the HPs, but not in the LCs (P < 0.0001). Furthermore, there were 26 signature residues within the KSB and were distinct from the worldwide consensus for subtype B. In conclusion, signature pattern analysis for the hypervariable env gene revealed an epidemiological link that the 20 HPs in this study had been infected with viruses from the DCF used for treatment, consistent with our previous finding.

CATNAP: a tool to compile, analyze and tally neutralizing antibody panels.

CATNAP (Compile, Analyze and Tally NAb Panels) is a new web server at Los Alamos HIV Database, created to respond to the newest advances in HIV neutralizing antibody research. It is a comprehensive platform focusing on neutralizing antibody potencies in conjunction with viral sequences. CATNAP integrates neutralization and sequence data from published studies, and allows users to analyze that data for each HIV Envelope protein sequence position and each antibody. The tool has multiple data retrieval and analysis options. As input, the user can pick specific antibodies and viruses, choose a panel from a published study, or supply their own data. The output superimposes neutralization panel data, virus epidemiological data, and viral protein sequence alignments on one page, and provides further information and analyses. The user can highlight alignment positions, or select antibody contact residues and view position-specific information from the HIV databases. The tool calculates tallies of amino acids and N-linked glycosylation motifs, counts of antibody-sensitive and -resistant viruses in conjunction with each amino acid or N-glycosylation motif, and performs Fisher's exact test to detect potential positive or negative amino acid associations for the selected antibody. Website name: CATNAP (Compile, Analyze and Tally NAb Panels). Website address: http://hiv.lanl.gov/catnap.

Comparative genomic analyses reveal broad diversity in botulinum-toxin-producing Clostridia.

BACKGROUND: Clostridium botulinum is a diverse group of bacteria characterized by the production of botulinum neurotoxin. Botulinum neurotoxins are classified into serotypes (BoNT/A-G), which are produced by six species/Groups of Clostridia, but the genetic background of the bacteria remains poorly understood. The purpose of this study was to use comparative genomics to provide insights into the genetic diversity and evolutionary history of bacteria that produce the potent botulinum neurotoxin. RESULTS: Comparative genomic analyses of over 170 Clostridia genomes, including our draft genome assemblies for 59 newly sequenced Clostridia strains from six continents and publicly available genomic data, provided in-depth insights into the diversity and distribution of BoNT-producing bacteria. These newly sequenced strains included Group I and II strains that express BoNT/A,/B,/E, or/F as well as bivalent strains. BoNT-producing Clostridia and closely related Clostridia species were delineated with a variety of methods including 16S rRNA gene, concatenated marker genes, core genome and concatenated multi-locus sequencing typing (MLST) gene phylogenies that related whole genome sequenced strains to publicly available strains and sequence types. These analyses illustrated the phylogenetic diversity in each Group and the diversity of genomic backgrounds that express the same toxin type or subtype. Comparisons of the botulinum neurotoxin genes did not identify novel toxin types or variants. CONCLUSIONS: This study represents one of the most comprehensive analyses of whole genome sequence data for Group I and II BoNT-producing strains. Read data and draft genome assemblies generated for 59 isolates will be a resource to the research community. Core genome phylogenies proved to be a powerful tool for differentiating BoNT-producing strains and can provide a framework for the study of these bacteria. Comparative genomic analyses of Clostridia species illustrate the diversity of botulinum-neurotoxin-producing strains and the plasticity of the genomic backgrounds in which bont genes are found.

The HIV-1 late domain-2 S40A polymorphism in antiretroviral (or ART)-exposed individuals influences protease inhibitor susceptibility.

BACKGROUND: The p6 region of the HIV-1 structural precursor polyprotein, Gag, contains two motifs, P7TAP11 and L35YPLXSL41, designated as late (L) domain-1 and -2, respectively. These motifs bind the ESCRT-I factor Tsg101 and the ESCRT adaptor Alix, respectively, and are critical for efficient budding of virus particles from the plasma membrane. L domain-2 is thought to be functionally redundant to PTAP. To identify possible other functions of L domain-2, we examined this motif in dominant viruses that emerged in a group of 14 women who had detectable levels of HIV-1 in both plasma and genital tract despite a history of current or previous antiretroviral therapy. RESULTS: Remarkably, variants possessing mutations or rare polymorphisms in the highly conserved L domain-2 were identified in seven of these women. A mutation in a conserved residue (S40A) that does not reduce Gag interaction with Alix and therefore did not reduce budding efficiency was further investigated. This mutation causes a simultaneous change in the Pol reading frame but exhibits little deficiency in Gag processing and virion maturation. Whether introduced into the HIV-1 NL4-3 strain genome or a model protease (PR) precursor, S40A reduced production of mature PR. This same mutation also led to high level detection of two extended forms of PR that were fairly stable compared to the WT in the presence of IDV at various concentrations; one of the extended forms was effective in trans processing even at micromolar IDV. CONCLUSIONS: Our results indicate that L domain-2, considered redundant in vitro, can undergo mutations in vivo that significantly alter PR function. These may contribute fitness benefits in both the absence and presence of PR inhibitor.

Modifying Surface Energy of Graphene via Plasma-Based Chemical Functionalization to Tune Thermal and Electrical Transport at Metal Interfaces.

The high mobility exhibited by both supported and suspended graphene, as well as its large in-plane thermal conductivity, has generated much excitement across a variety of applications. As exciting as these properties are, one of the principal issues inhibiting the development of graphene technologies pertains to difficulties in engineering high-quality metal contacts on graphene. As device dimensions decrease, the thermal and electrical resistance at the metal/graphene interface plays a dominant role in degrading overall performance. Here we demonstrate the use of a low energy, electron-beam plasma to functionalize graphene with oxygen, fluorine, and nitrogen groups, as a method to tune the thermal and electrical transport properties across gold-single layer graphene (Au/SLG) interfaces. We find that while oxygen and nitrogen groups improve the thermal boundary conductance (hK) at the interface, their presence impairs electrical transport leading to increased contact resistance (ρC). Conversely, functionalization with fluorine has no impact on hK, yet ρC decreases with increasing coverage densities. These findings indicate exciting possibilities using plasma-based chemical functionalization to tailor the thermal and electrical transport properties of metal/2D material contacts.

Room-temperature voltage tunable phonon thermal conductivity via reconfigurable interfaces in ferroelectric thin films.

Dynamic control of thermal transport in solid-state systems is a transformative capability with the promise to propel technologies including phononic logic, thermal management, and energy harvesting. A solid-state solution to rapidly manipulate phonons has escaped the scientific community. We demonstrate active and reversible tuning of thermal conductivity by manipulating the nanoscale ferroelastic domain structure of a Pb(Zr0.3Ti0.7)O3 film with applied electric fields. With subsecond response times, the room-temperature thermal conductivity was modulated by 11%.

Controlling the metal to semiconductor transition of MoS2 and WS2 in solution.

Lithiation-exfoliation produces single to few-layered MoS2 and WS2 sheets dispersible in water. However, the process transforms them from the pristine semiconducting 2H phase to a distorted metallic phase. Recovery of the semiconducting properties typically involves heating of the chemically exfoliated sheets at elevated temperatures. Therefore, it has been largely limited to sheets deposited on solid substrates. Here, we report the dispersion of chemically exfoliated MoS2 sheets in high boiling point organic solvents enabled by surface functionalization and the controllable recovery of their semiconducting properties directly in solution. This process connects the scalability of chemical exfoliation with the simplicity of solution processing, ultimately enabling a facile method for tuning the metal to semiconductor transitions of MoS2 and WS2 within a liquid medium.

Crossover from incoherent to coherent phonon scattering in epitaxial oxide superlattices.

Elementary particles such as electrons or photons are frequent subjects of wave-nature-driven investigations, unlike collective excitations such as phonons. The demonstration of wave-particle crossover, in terms of macroscopic properties, is crucial to the understanding and application of the wave behaviour of matter. We present an unambiguous demonstration of the theoretically predicted crossover from diffuse (particle-like) to specular (wave-like) phonon scattering in epitaxial oxide superlattices, manifested by a minimum in lattice thermal conductivity as a function of interface density. We do so by synthesizing superlattices of electrically insulating perovskite oxides and systematically varying the interface density, with unit-cell precision, using two different epitaxial-growth techniques. These observations open up opportunities for studies on the wave nature of phonons, particularly phonon interference effects, using oxide superlattices as model systems, with extensive applications in thermoelectrics and thermal management.

Laser capture microdissection assessment of virus compartmentalization in the central nervous systems of macaques infected with neurovirulent simian immunodeficiency virus.

Nonhuman primate-simian immunodeficiency virus (SIV) models are powerful tools for studying the pathogenesis of human immunodeficiency virus type 1 (HIV-1) in the brain. Our laboratory recently isolated a neuropathogenic viral swarm, SIVsmH804E, a derivative of SIVsmE543-3, which was the result of sequential intravenous passages of viruses isolated from the brains of rhesus macaques with SIV encephalitis. Animals infected with SIVsmH804E or its precursor (SIVsmH783Br) developed SIV meningitis and/or encephalitis at high frequencies. Since we observed macaques with a combination of meningitis and encephalitis, as well as animals in which meningitis or encephalitis was the dominant component, we hypothesized that distinct mechanisms could be driving the two pathological states. Therefore, we assessed viral populations in the meninges and the brain parenchyma by laser capture microdissection. Viral RNAs were isolated from representative areas of the meninges, brain parenchyma, terminal plasma, and cerebrospinal fluid (CSF) and from the inoculum, and the SIV envelope fragment was amplified by PCR. Phylogenetic analysis of envelope sequences from the conventional progressors revealed compartmentalization of viral populations between the meninges and the parenchyma. In one of these animals, viral populations in meninges were closely related to those from CSF and shared signature truncations in the cytoplasmic domain of gp41, consistent with a common origin. Apart from magnetic resonance imaging (MRI) and positron-emission tomography (PET) imaging, CSF is the most accessible assess to the central nervous system for HIV-1-infected patients. However, our results suggest that the virus in the CSF may not always be representative of viral populations in the brain and that caution should be applied in extrapolating between the properties of viruses in these two compartments.

Increasing intra- and inter-subtype HIV diversity despite declining HIV incidence in Uganda.

HIV incidence has been declining in Africa with scale-up of HIV interventions. However, there is limited data on HIV evolutionary trends in African populations with waning epidemics. We evaluated changes in HIV viral diversity and genetic divergence in southern Uganda over a twenty-five-year period spanning the introduction and scale-up of HIV prevention and treatment programs using HIV sequence and survey data from the Rakai Community Cohort Study, an open longitudinal population-based HIV surveillance cohort. Gag (p24) and env (gp41) HIV data were generated from persons living with HIV (PLHIV) in 31 inland semi-urban trading and agrarian communities (1994 to 2018) and four hyperendemic Lake Victoria fishing communities (2011 to 2018) under continuous surveillance. HIV subtype was assigned using the Recombination Identification Program with phylogenetic confirmation. Inter-subtype diversity was estimated using the Shannon diversity index and intra-subtype diversity with the nucleotide diversity and pairwise TN93 genetic distance. Genetic divergence was measured using root-to-tip distance and pairwise TN93 genetic distance analyses. Evolutionary dynamics were assessed among demographic and behavioral sub-groups, including by migration status. 9,931 HIV sequences were available from 4,999 PLHIV, including 3,060 and 1,939 persons residing in inland and fishing communities, respectively. In inland communities, subtype A1 viruses proportionately increased from 14.3% in 1995 to 25.9% in 2017 (p<0.001), while those of subtype D declined from 73.2% in 1995 to 28.2% in 2017 (p<0.001). The proportion of viruses classified as recombinants significantly increased by more than four-fold. Inter-subtype HIV diversity has generally increased. While p24 intra-subtype genetic diversity and divergence leveled off after 2014, diversity and divergence of gp41 increased through 2017. Inter- and intra-subtype viral diversity increased across all population sub-groups, including among individuals with no recent migration history or extra-community sexual partners. This study provides insights into population-level HIV evolutionary dynamics in declining African HIV epidemics following the scale-up of HIV prevention and treatment programs. Continued molecular surveillance may provide a better understanding of the dynamics driving population HIV evolution and yield important insights for epidemic control and vaccine development.

Dielectric characterization of paraelectric particle-loaded polymer matrix composites and commercial photoresins at W-band frequencies.

This work presents W-band (75-110 GHz) dielectric characterization of commercially available photoresins in their neat state, as well as in polymer matrix composite (PMC) mixtures with various loading concentrations of the paraelectric barium strontium titanate (BST). Due to difficulties 3D printing the BST-loaded PMC resins detailed within, a custom curing and casting process was used to fabricate testable PMC samples, which were synthesized to demonstrate the dielectric functionalization of the underlying polymer matrix. Dielectric characterization of the PMCs confirmed the functionalization of our composites when compared to the commercial photoresins. For example, a volumetric loading concentration of 25 vol % BST increased the dielectric permittivity (εr ) from 2.78 to 9.60 and the loss tangent (tanδ) from 0.022 to 0.114. These results indicate that the realization of UV-cured photoresins with "designer-dielectric" functionalization based on vol % of filler are strong candidates for use in stereolithography (SLA) 3D printing applications. To accomplish this, and with a special interest for radio/microwave/terahertz (RF/MW/THz) applications, we highlight the need for both (a) better photoresin matrix materials with lower intrinsic tanδ and (b) selection criteria related to the size/geometry and electronic properties of potential filler materials to maintain the printability of PMC photoresins in SLA systems.