Neutralization of Diverse Human Cytomegalovirus Strains Conferred by Antibodies Targeting Viral gH/gL/pUL128-131 Pentameric Complex.

Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection, and developing a prophylactic vaccine is of high priority to public health. We recently reported a replication-defective human cytomegalovirus with restored pentameric complex glycoprotein H (gH)/gL/pUL128-131 for prevention of congenital HCMV infection. While the quantity of vaccine-induced antibody responses can be measured in a viral neutralization assay, assessing the quality of such responses, including the ability of vaccine-induced antibodies to cross-neutralize the field strains of HCMV, remains a challenge. In this study, with a panel of neutralizing antibodies from three healthy human donors with natural HCMV infection or a vaccinated animal, we mapped eight sites on the dominant virus-neutralizing antigen-the pentameric complex of glycoprotein H (gH), gL, and pUL128, pUL130, and pUL131. By evaluating the site-specific antibodies in vaccine immune sera, we demonstrated that vaccination elicited functional antiviral antibodies to multiple neutralizing sites in rhesus macaques, with quality attributes comparable to those of CMV hyperimmune globulin. Furthermore, these immune sera showed antiviral activities against a panel of genetically distinct HCMV clinical isolates. These results highlighted the importance of understanding the quality of vaccine-induced antibody responses, which includes not only the neutralizing potency in key cell types but also the ability to protect against the genetically diverse field strains.IMPORTANCE HCMV is the leading cause of congenital viral infection, and development of a preventive vaccine is a high public health priority. To understand the strain coverage of vaccine-induced immune responses in comparison with natural immunity, we used a panel of broadly neutralizing antibodies to identify the immunogenic sites of a dominant viral antigen-the pentameric complex. We further demonstrated that following vaccination of a replication-defective virus with the restored pentameric complex, rhesus macaques can develop broadly neutralizing antibodies targeting multiple immunogenic sites of the pentameric complex. Such analyses of site-specific antibody responses are imperative to our assessment of the quality of vaccine-induced immunity in clinical studies.

Studies in the Antrodia serialis group (Polyporales, Basidiomycota).

Taxonomy and phylogeny of the Antrodia serialis group are revised with morphological, ecological, and geographic data, partial translation elongation factor 1-α (tef1) gene sequences, and nuc rDNA ITS1-5.8S-ITS2-28S sequences. The group contains 13 species found in boreal and temperate zones of the Northern Hemisphere. The species are limited to certain geographic areas within Eurasia and North America. The traditional morphology-based concept of A. serialis covers at least four closely related species: A. serialis s. str. in Eurasia, A. angusta, sp. nov., in East Asia, A. serrata, sp. nov., in the American Northeast, and A. calcitrosa, sp. nov., in the American Northwest. They all are associated mostly with Picea spp. and show small, but stable morphological differences from each other. In addition, A. morganii, comb. nov., inhabiting wood of Populus spp., occurs in North America, and Antrodia alaskana, comb. nov., a large-pored species, macroscopically similar to A. variiformis, is distributed along the Pacific coast of North America. The pine-dwelling A. flavimontis, sp. nov., similar to A. primaeva from Eurasia, is so far known only from the eastern part of the Rocky Mountains (Utah and Wyoming).

Phylogeny and diversity of Fomitiporella (Hymenochaetales, Basidiomycota).

Fomitiporella accommodates polypores producing annual to perennial basidiocarps with an indistinct subiculum (very thin to almost lacking), mostly a dimitic hyphal structure, lacking any kind of setae, with brownish, thick-walled basidiospores, and causing a white rot. Previously, only a few samples of Fomitiporella were studied on the basis of morphological and nuc 28S rDNA (28S)-based phylogenetic analyses. In this study, we made a comprehensive study on Fomitiporella on the basis of collections from Central America, USA, Europe, and China. The phylogenetic analysis, including 28 nuc 28S rDNA and 29 nuc rDNA ITS1-5.8S-ITS2 (internal transcribed spacer [ITS]) sequences newly generated, discovered 14 new lineages. Combined with morphological evidence, 4 new lineages are described and illustrated as new species, viz., Fomitiporella americana, F. micropora, F. sinica, and F. subinermis; 10 other new lineages, each with a single collection, are still treated as unidentified taxa; three new combinations, viz., Fomitiporella tenuissima, F. chinensis, and F. resupinata, are proposed. In addition, F. inermis is redescribed. A key to the 12 known species of Fomitiporella is provided.

Global diversity and phylogeny of Onnia (Hymenochaetaceae) species on gymnosperms.

Onnia includes white rotting polypores with annual basidiocarps, a duplex context, monomitic hyphal structure, hymenial setae, and hyaline, thin-walled, smooth basidiospores. Specimens of Onnia, originating mainly from East Asia, Europe, and North America, were studied using both morphology and phylogenetic analyses. Our concatenated data set was derived from 25 collections and included (i) 25 nuc rDNA internal transcribed spacer region sequences (ITS1-5.8S-ITS2 = ITS), 17 generated in this study; and (ii) 14 nuc rDNA 28S rDNA sequences, including the D1-D2 domains, 11 of them generated in this study. The resulting maximum likelihood and Bayesian phylogenies recovered all sampled collections of Onnia as a well-supported clade. In this clade, three previously accepted species, viz., Onnia leporina, O. tomentosa, and O. triquetra, received strong support, whereas three additional lineages with strong support represent the new species described in this paper, O. subtriquetra, O. microspora, and O. tibetica. Of the six Onnia species occurring on gymnosperms, O. tomentosa and O. leporina grow mainly on Picea and have circumboreal distribution in the Northern Hemisphere. In contrast, other species that mostly grow on Pinus are geographically restricted to limited regions, viz., O. triquetra in Europe, O. subtriquetra in North America, and O. microspora and O. tibetica in Asia.

Global diversity and phylogeny of the Phellinus igniarius complex (Hymenochaetales, Basidiomycota) with the description of five new species.

The Phellinus igniarius complex corresponds to Phellinus s.s., a well supported clade in the polyphyletic Phellinus s.l. studied worldwide. Recently a molecular phylogeny of this complex was performed for the European and North American species. In this study we expand the taxon samplings of the P. igniarius complex to include 59, seven and 12 additional collections originating from China, Czech Republic and USA, respectively. We generated 78 nuc rDNA ITS1-5.8S-ITS2 (ITS) and 42 translation elongation factor 1-α gene (tef1α) sequences. Based on the morphological and phylogenetic (combined ITS and tef1α dataset) analyses, Phellinus monticola, P. orientoasiaticus, P. padicola, P. parmastoi and P. pomaceoides are newly described and illustrated from China and USA. Phellinus pseudoigniarius is treated as a later synonym of P. igniarius, whereas Phellinus betulinus subsp. betulinus is accepted as P. betulinus. A total of 15 species are accepted in the P. igniarius complex worldwide. Of them, 10 species are distributed in eastern Asia, eight in Europe and six in North America. The taxonomy, phylogeny, host associations and geographic distributions of these 15 species are briefly discussed.

Ecuadorian Acanthocinini: description of two new species and transference and notes on Sympagus cooperi Monn & Monn.

Two new Acanthocinini are described from Ecuador: Nealcidion apunctatum sp. nov.; and Xenocona nubicola sp. nov. Sympagus cooperi Monn & Monn, 2017 is transferred to Stenolis Bates, 1864, and notes on the differences between these two genera are provided.

Molecular and morphological data reveal two new polypores (Polyporales, Basidiomycota) with reddish brown to orange basidiomata from China.

Two taxonomically controversial polypore genera with reddish brown to orange basidiomata that stain reddish with KOH solution, Aurantiporus and Hapalopilus, are revised based on additional sampling, morphological examination, and phylogenetic analysis of a combined dataset of ITS1-5.8S-ITS2-nLSU sequences. Hapalopilus is a monophyletic genus belonging to Phanerochaetaceae, whereas Aurantiporus is a polyphyletic genus belonging to Meruliaceae. Hapalopilus and Aurantiporus s. str. are circumscribed, and two new species - Aurantiporusorientalis and Hapalopilustabuliformis - are described and illustrated from temperate China. In addition, four new combinations, viz. Aurantiporusalboaurantius, A.mutans, A.tropicus and Luteoporiaalbocitrina, are proposed based on morphology and phylogenetic analysis. The relationships between Aurantiporus and Hapalopilus are discussed.

Dissecting the Heterogeneous Glycan Profiles of Recombinant Coronavirus Spike Proteins with Individual Ion Mass Spectrometry.

Surface-embedded glycoproteins, such as the spike protein trimers of coronaviruses MERS, SARS-CoV, and SARS-CoV-2, play a key role in viral function and are the target antigen for many vaccines. However, their significant glycan heterogeneity poses an analytical challenge. Here, we utilized individual ion mass spectrometry (I2MS), a multiplexed charge detection measurement with similarities to charge detection mass spectrometry (CDMS), in which a commercially available Orbitrap analyzer is used to directly produce mass profiles of these heterogeneous coronavirus spike protein trimers under native-like conditions. Analysis by I2MS shows that glycosylation contributes to the molecular mass of each protein trimer more significantly than expected by bottom-up techniques, highlighting the importance of obtaining complementary intact mass information when characterizing glycosylation of such heterogeneous proteins. Enzymatic dissection to remove sialic acid or N-linked glycans demonstrates that I2MS can be used to better understand the glycan profile from a native viewpoint. Deglycosylation of N-glycans followed by I2MS analysis indicates that the SARS-CoV-2 spike protein trimer contains glycans that are more difficult to remove than its MERS and SARS-CoV counterparts, and these differences are correlated with solvent accessibility. I2MS technology enables characterization of protein mass and intact glycan profile and is orthogonal to traditional mass analysis methods such as size exclusion chromatography-multiangle light scattering (SEC-MALS) and field flow fractionation-multiangle light scattering (FFF-MALS). An added advantage of I2MS is low sample use, requiring 100-fold less than other methodologies. This work highlights how I2MS technology can enable efficient development of vaccines and therapeutics for pharmaceutical development.

What is Antrodia sensu stricto?

The polypore genus Antrodia (Polyporales, Basidiomycota) in the strict sense consists of a small number of species grouped around the type species A. serpens in phylogenetic analyses. This distinct clade (Antrodia sensu stricto in our view) contains species of the Antrodia heteromorpha complex, A. macra coll. and Antrodia mappa (formerly Postia mappa). Nuclear rDNA ITS and tef1 data show that the Antrodia heteromorpha species complex includes four species: A. heteromorpha sensu stricto (mostly on gymnosperms, large pores and spores), A. serpens (on angiosperms in Europe, resupinate, smaller pores but large spores), A. favescens (smaller pores and spores, pileate species in North America, formerly known as Trametes sepium), and A. tanakai (a close kin of A. favescens in Eurasia). Antrodia albida is a synonym of A. heteromorpha sensu stricto. We combine A. mappa, A. favescens and A. tanakai in Antrodia and designate neotypes for A. albida and A. heteromorpha, and an epitype for A. serpens. We also compare the morphologically similar but distantly related A. albidoides and A. mellita, and conclude that A. macrospora and A. subalbidoides are synonyms of A. albidoides.

New descriptions, synonymies and records in South American Lamiinae(Coleoptera, Cerambycidae).

Two new species are described from Ecuador: Colobothea androwi sp. nov. and Tybalmia diffusa sp. nov. A provisional key to species of Tybalmia Thomson, 1868 is provided. Tybalmia tetrops Bates, 1872 is synonymized with T. mydas (Lucas, 1859), and a new state record in Brazil is provided. Tybalmia orbis Dillon & Dillon, 1945 is synonymized with T. pupillata (Pascoe, 1859), and a new country record is provided.

Outline, phylogenetic and divergence times analyses of the genus Haploporus (Polyporales, Basidiomycota): two new species are proposed.

Haploporus species have a worldwide distribution and 27 species have been accepted. In this study, two new species, Haploporuscrystallinus and H.dextrinoideus, are proposed from South America, based on the molecular fragments (ITS, LSU and mtSSU) and morphological evidence. Molecular clock analysis was performed and the result suggests that the ancestor of Polyporales originated between the Late Jurassic and Early Cretaceous period, with a mean stem of 159.8 Mya [95% higher posterior density (HPD) of 142.4-184.1 Mya] and the genus Haploporus occurred at a mean stem of 108.3 Mya (95% HPD of 88.5-128.2 Mya). In addition, most species of the genus are diversified between 60.5 Mya and 1.8 Mya, during the Paleogene to Neogene. A key to the accepted species of the genus Haploporus is provided.

Taxonomic and phylogenetic contributions to Fuscoporia (Hymenochaetales, Basidiomycota): two new species from Hawaii with a key to North American species.

Fuscoporia is a cosmopolitan, poroid, wood-decaying genus, belonging to the Hymenochaetales. During a study of wood-inhabiting fungi in the USA, four unknown specimens were collected from Hawaii. Both morphological criteria and molecular genetic analyses based on the ITS+nLSU+EF1-α datasets and the nLSU dataset confirmed that these four specimens represent two new species of Fuscoporia, and they are described as F. hawaiiana and F. minutissima. Fuscoporia hawaiiana is characterized by pileate basidiocarps, the absence of cystidioles, hooked hymenial setae, broadly ellipsoid to subglobose basidiospores measuring 4-6 × 3.5-4.5 µm. Fuscoporia minutissima is distinguished by small pores (10-13 per mm) and basidiospores (3.4-4 × 2.4-3 µm). The taxonomic status of the two new species is briefly discussed. A key to the North American species of Fuscoporia is provided.

Phylogeny and diversity of Rigidoporus (Hymenochaetales, Basidiomycota), including three new species from Asia.

Phylogenetic and morphological analyses on Rigidoporus were carried out. The genus Rigidoporus (Hymenochaetales, Basidiomycota), typified by R. microporus (Fr.) Overeem. (synonym Polyporus micromegas Mont.), was established by Murrill in 1905. The genus is mainly characterized by annual to perennial, resupinate, effused-reflexed to pileate or stipitate basidiomata with azonate or concentrically zonate and sulcate upper surface, a monomitic to pseudo-dimitic hyphal structure, simple-septate generative hyphae, and ellipsoid to globose basidiospores. Phylogeny on species of the genus is reconstructed with two loci DNA sequences including the internal transcribed spacer regions and the large subunit. Three new species in Rigidoporus are described and illustrated from Asia, and one new combination in the genus is proposed. The main morphological characteristics of the currently accepted species of Rigidoporus are provided.

Development and Characterization of an In Vitro Cell-Based Assay to Predict Potency of mRNA-LNP-Based Vaccines.

Messenger RNA (mRNA) vaccines have emerged as a flexible platform for vaccine development. The evolution of lipid nanoparticles as effective delivery vehicles for modified mRNA encoding vaccine antigens was demonstrated by the response to the COVID-19 pandemic. The ability to rapidly develop effective SARS-CoV-2 vaccines from the spike protein genome, and to then manufacture multibillions of doses per year was an extraordinary achievement and a vaccine milestone. Further development and application of this platform for additional pathogens is clearly of interest. This comes with the associated need for new analytical tools that can accurately predict the performance of these mRNA vaccine candidates and tie them to an immune response expected in humans. Described here is the development and characterization of an imaging based in vitro assay able to quantitate transgene protein expression efficiency, with utility to measure lipid nanoparticles (LNP)-encapsulated mRNA vaccine potency, efficacy, and stability. Multiple biologically relevant adherent cell lines were screened to identify a suitable cell substrate capable of providing a wide dose-response curve and dynamic range. Biologically relevant assay attributes were examined and optimized, including cell monolayer morphology, antigen expression kinetics, and assay sensitivity to LNP properties, such as polyethylene glycol-lipid (or PEG-lipid) composition, mRNA mass, and LNP size. Collectively, this study presents a strategy to quickly optimize and develop a robust cell-based potency assay for the development of future mRNA-based vaccines.

Two new species of Haploporus (Polyporales, Basidiomycota) from China and Ecuador based on morphology and phylogeny.

At present, 25 species are accepted in Haploporus and are distributed in Asia, Europe, North America, South America, Australia, and Africa. In this study, two new species, Haploporus ecuadorensis from Ecuador and H. monomitica from China, are described and illustrated based on morphological examination and phylogenetic analyses. H. ecuadorensis is characterized by annual, resupinate basidiomata with pinkish buff to honey yellow hymenophore when dry, round to angular pores of 2-4 per mm, a dimitic hyphal structure with generative hyphae bearing clamp connections, hyphae at dissepiment edge usually with one or two simple septa, the presence of dendrohyphidia and cystidioles, and oblong to ellipsoid basidiospores measuring 14.9-17.9 × 6.9-8.8 µm. Haploporus monomitica differs from other Haploporus species in that it has a monomitic hyphal system and strongly dextrinoid basidiospores. The differences between the new species and morphologically similar and phylogenetically related species are discussed. In addition, an updated key to 27 species of Haploporus is provided.

Descriptions, notes, and a new record in Oideterus Thomson (Coleoptera, Cerambycidae, Prioninae, Anacolini).

The male of Oideterus elegans (Waterhouse, 1880) is described for the first time, female chromatic variations are reported, and the species is recorded from the Ecuadorian department of Napo. Two new species are described from Ecuador: Oideterus leoninus and O. ecuadorensis. The species-group name of O. nigrum Botero, Galileo Santos-Silva, 2019 is corrected. Identification keys to males and females of Oideterus species are provided.

New species and new geographical records in Ecuadorian Lamiinae (Coleoptera, Cerambycidae).

Three new species of Lamiinae are described from Ecuador: Pseudosparna pallida sp. nov. (Acanthocinini); Acakyra sinuata sp. nov. and Scythropopsis variegata sp. nov. (Acanthoderini). Piriana pygmaea (Bates, 1881) is recorded from Ecuador for the first time. A chromatic variation in Unaporanga lanceolata Martins & Galileo, 2007 is reported, the male is illustrated for the first time, and the species is newly reported from the Ecuadorian department of Pichincha. The morphological differences between Scythropopsis Thomson, 1864, Acakyra Martins & Galileo, 1996, and Irundisaua Martins & Galileo, 2005 are discussed.

Five new species of Nealcidion Monné, 1997 from Ecuador (Coleoptera, Cerambycidae, Lamiinae).

Five new species of Nealcidion Monné, 1997 are described from Ecuador: Nealcidion obliquum sp. nov., Nealcidion flavoviride sp. nov., Nealcidion cephaliferum sp. nov., Nealcidion sublineatum sp. nov., and Nealcidion calcaratum sp. nov. All new species are illustrated, making it easier to recognize the species and to follow the descriptions.

Two new species of Phylloporia (Hymenochaetales) from the Neotropics.

Two new species of Phylloporia, P.crystallina and P.sumacoensis, are described based on 28S ribosomal RNA phylogeny, morphology, host, and geographic distribution. Phylloporiacrystallina is characterized by pileate, perennial basidiomata with a duplex context, small pores 9-10 per mm, a monomitic hyphal system, absence of cystidia and cystidioles, presence of large rhomboid crystals in tube trama, broadly ellipsoid to subglobose basidiospores measuring 2.8-3 × 2-2.3 µm, and growth on angiosperm stump. Phylloporiasumacoensis is characterized by pileate, perennial basidiomata with a duplex context, very small pores 10-12 per mm, a monomitic hyphal system, hyphae at dissepiment edges bearing fine crystals, presence of cystidioles, broadly ellipsoid to subglobose basidiospores measuring 3-3.7 × 2.1-2.8 µm, and growth on living liana.

Functional profiling of Covid 19 vaccine candidate by flow virometry.

Vaccine development is a complex process, starting with selection of a promising immunogen in the discovery phase, followed by process development in the preclinical phase, and later by clinical trials in tandem with process improvements and scale up. A large suite of analytical techniques is required to gain understanding of the vaccine candidate so that a relevant immunogen is selected and subsequently manufactured consistently throughout the lifespan of the product. For viral vaccines, successful immunogen production is contingent on its maintained antigenicity and/or infectivity, as well as the ability to characterize these qualities within the context of the process, formulation, and clinical performance. In this report we show the utility of flow virometry during preclinical development of a Covid 19 vaccine candidate based on SARS-CoV-2 spike (S) protein expressed on vesicular stomatitis virus (VSV). Using a panel of monoclonal antibodies, we were able to detect the S protein on the surface of the recombinant VSV virus, monitor the expression levels, detect differences in the antigen based on S protein sequence and after virus inactivation, and monitor S protein stability. Collectively, flow virometry provided important data that helped to guide preclinical development of this vaccine candidate.