Cryo-EM-based discovery of a pathogenic parvovirus causing epidemic mortality by black wasting disease in farmed beetles.

We use cryoelectron microscopy (cryo-EM) as a sequence- and culture-independent diagnostic tool to identify the etiological agent of an agricultural pandemic. For the past 4 years, American insect-rearing facilities have experienced a distinctive larval pathology and colony collapse of farmed Zophobas morio (superworm). By means of cryo-EM, we discovered the causative agent: a densovirus that we named Zophobas morio black wasting virus (ZmBWV). We confirmed the etiology of disease by fulfilling Koch's postulates and characterizing strains from across the United States. ZmBWV is a member of the family Parvoviridae with a 5,542 nt genome, and we describe intersubunit interactions explaining its expanded internal volume relative to human parvoviruses. Cryo-EM structures at resolutions up to 2.1 Å revealed single-strand DNA (ssDNA) ordering at the capsid inner surface pinned by base-binding pockets in the capsid inner surface. Also, we demonstrated the prophylactic potential of non-pathogenic strains to provide cross-protection in vivo.

Molecular biology and structure of a novel penaeid shrimp densovirus elucidate convergent parvoviral host capsid evolution.

The giant tiger prawn (Penaeus monodon) is a decapod crustacean widely reared for human consumption. Currently, viruses of two distinct lineages of parvoviruses (PVs, family Parvoviridae; subfamily Hamaparvovirinae) infect penaeid shrimp. Here, a PV was isolated and cloned from Vietnamese P. monodon specimens, designated Penaeus monodon metallodensovirus (PmMDV). This is the first member of a third divergent lineage shown to infect penaeid decapods. PmMDV has a transcription strategy unique among invertebrate PVs, using extensive alternative splicing and incorporating transcription elements characteristic of vertebrate-infecting PVs. The PmMDV proteins have no significant sequence similarity with other PVs, except for an SF3 helicase domain in its nonstructural protein. Its capsid structure, determined by cryoelectron microscopy to 3-Å resolution, has a similar surface morphology to Penaeus stylirostris densovirus, despite the lack of significant capsid viral protein (VP) sequence similarity. Unlike other PVs, PmMDV folds its VP without incorporating a ßA strand and displayed unique multimer interactions, including the incorporation of a Ca2+ cation, attaching the N termini under the icosahedral fivefold symmetry axis, and forming a basket-like pentamer helix bundle. While the PmMDV VP sequence lacks a canonical phospholipase A2 domain, the structure of an EDTA-treated capsid, determined to 2.8-Å resolution, suggests an alternative membrane-penetrating cation-dependent mechanism in its N-terminal region. PmMDV is an observed example of convergent evolution among invertebrate PVs with respect to host-driven capsid structure and unique as a PV showing a cation-sensitive/dependent basket structure for an alternative endosomal egress.

Adeno-associated Virus 9 Structural Rearrangements Induced by Endosomal Trafficking pH and Glycan Attachment.

Adeno-associated viruses (AAVs) are small nonenveloped single-stranded DNA (ssDNA) viruses that are currently being developed as gene therapy biologics. After cell entry, AAVs traffic to the nucleus using the endo-lysosomal pathway. The subsequent decrease in pH triggers conformational changes to the capsid that enable the externalization of the capsid protein (VP) N termini, including the unique domain of the minor capsid protein VP1 (VP1u), which permits the phospholipase activity required for the capsid lysosomal egress. Here, we report the AAV9 capsid structure, determined at the endosomal pHs (7.4, 6.0, 5.5, and 4.0), and terminal galactose-bound AAV9 capsids at pHs 7.4 and 5.5 using cryo-electron microscopy and three-dimensional image reconstruction. Taken together, these studies provide insight into AAV9 capsid conformational changes at the 5-fold pore during endosomal trafficking, in both the presence and absence of its cellular glycan receptor. We visualized, for the first time, that acidification induces the externalization of the VP3 and possibly VP2 N termini, presumably in prelude to the externalization of VP1u at pH 4.0, which is essential for lysosomal membrane disruption. In addition, the structural study of AAV9-galactose interactions demonstrates that AAV9 remains attached to its glycan receptor at the late endosome pH 5.5. This interaction significantly alters the conformational stability of the variable region I of the VPs, as well as the dynamics associated with VP N terminus externalization. IMPORTANCE There are 13 distinct Adeno-associated virus (AAV) serotypes that are structurally homologous and whose capsid proteins (VP1 to -3) are similar in amino acid sequence. However, AAV9 is one of the most commonly studied and is used as a gene therapy vector. This is partly because AAV9 is capable of crossing the blood-brain barrier and readily transduces a wide array of tissues, including the central nervous system. In this study, we provide AAV9 capsid structural insight during intracellular trafficking. Although the AAV capsid has been shown to externalize the N termini of its VPs, to enzymatically disrupt the lysosome membrane at low pH, there was no structural evidence to confirm this. By utilizing AAV9 as our model, we provide the first structural evidence that the externalization process occurs at the protein interface at the icosahedral 5-fold symmetry axis and can be triggered by lowering the pH.

Reorganizing the family Parvoviridae: a revised taxonomy independent of the canonical approach based on host association.

Parvoviridae, a diverse family of small single-stranded DNA viruses was established in 1975. It was divided into two subfamilies, Parvovirinae and Densovirinae, in 1993 to accommodate parvoviruses that infect vertebrate and invertebrate animals, respectively. This relatively straightforward segregation, using host association as the prime criterion for subfamily-level classification, has recently been challenged by the discovery of divergent, vertebrate-infecting parvoviruses, dubbed "chapparvoviruses", which have proven to be more closely related to viruses in certain Densovirinae genera than to members of the Parvovirinae. Viruses belonging to these genera, namely Brevi-, Hepan- and Penstyldensovirus, are responsible for the unmatched heterogeneity of the subfamily Densovirinae when compared to the Parvovirinae in matters of genome organization, protein sequence homology, and phylogeny. Another genus of Densovirinae, Ambidensovirus, has challenged traditional parvovirus classification, as it includes all newly discovered densoviruses with an ambisense genome organization, which introduces genus-level paraphyly. Lastly, current taxon definition and virus inclusion criteria have significantly limited the classification of certain long-discovered parvoviruses and impedes the classification of some potential family members discovered using high-throughput sequencing methods. Here, we present a new and updated system for parvovirus classification, which includes the introduction of a third subfamily, Hamaparvovirinae, resolves the paraphyly within genus Ambidensovirus, and introduces new genera and species into the subfamily Parvovirinae. These proposals were accepted by the ICTV in 2020 March.

ICTV Virus Taxonomy Profile: Parvoviridae.

Members of the family Parvoviridae are small, resilient, non-enveloped viruses with linear, single-stranded DNA genomes of 4-6 kb. Viruses in two subfamilies, the Parvovirinae and Densovirinae, are distinguished primarily by their respective ability to infect vertebrates (including humans) versus invertebrates. Being genetically limited, most parvoviruses require actively dividing host cells and are host and/or tissue specific. Some cause diseases, which range from subclinical to lethal. A few require co-infection with helper viruses from other families. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the Parvoviridae, which is available at www.ictv.global/report/parvoviridae.

Using the E4orf6-Based E3 Ubiquitin Ligase as a Tool To Analyze the Evolution of Adenoviruses.

UNLABELLED: E4orf6 proteins from all human adenoviruses form Cullin-based ubiquitin ligase complexes that, in association with E1B55K, target cellular proteins for degradation. While most are assembled with Cul5, a few utilize Cul2. BC-box motifs enable all these E4orf6 proteins to assemble ligase complexes with Elongins B and C. We also identified a Cul2-box motif used for Cul2 selection in all Cul2-based complexes. With this information, we set out to determine if other adenoviruses also possess the ability to form the ligase complex and, if so, to predict their Cullin usage. Here we report that all adenoviruses known to encode an E4orf6-like protein (mastadenoviruses and atadenoviruses) maintain the potential to form the ligase complex. We could accurately predict Cullin usage for E4orf6 products of mastadenoviruses and all but one atadenovirus. Interestingly, in nonhuman primate adenoviruses, we found a clear segregation of Cullin binding, with Cul5 utilized by viruses infecting great apes and Cul2 by Old/New World monkey viruses, suggesting that a switch from Cul2 to Cul5 binding occurred during the period when great apes diverged from monkeys. Based on the analysis of Cullin selection, we also suggest that the majority of human adenoviruses, which exhibit a broader tropism for the eye and the respiratory tract, exhibit Cul5 specificity and resemble viruses infecting great apes, whereas those that infect the gastrointestinal tract may have originated from monkey viruses that share Cul2 specificity. Finally, aviadenoviruses also appear to contain E4orf6 genes that encode proteins with a conserved XCXC motif followed by, in most cases, a BC-box motif. IMPORTANCE: Two early adenoviral proteins, E4orf6 and E1B55K, form a ubiquitin ligase complex with cellular proteins to ubiquitinate specific substrates, leading to their degradation by the proteasome. In studies with representatives of each human adenovirus species, we (and others) previously discovered that some viruses use Cul2 to form the complex, while others use Cul5. In the present study, we expanded our analyses to all sequenced adenoviruses and found that E4orf6 genes from all mast- and atadenoviruses encode proteins containing the motifs necessary to form the ligase complex. We found a clear separation in Cullin specificity between adenoviruses of great apes and Old/New World monkeys, lending support for a monkey origin for human viruses of the Human mastadenovirus A, F, and G species. We also identified previously unrecognized E4orf6 genes in the aviadenoviruses that encode proteins containing motifs permitting formation of the ubiquitin ligase.

A novel cetacean adenovirus in stranded harbour porpoises from the North Sea: detection and molecular characterization.

Harbour porpoises (Phocoena phocoena) are the most prevalent cetaceans in the North Sea. The fecal viral flora of 21 harbour porpoises stranded along the Dutch coastline was analyzed by a metagenomics approach. Sequences of a novel cetacean mastadenovirus, designated harbour porpoise adenovirus 1 (HpAdV-1), were detected. The sequence of a 23-kbp genomic region, spanning the conserved late region, was determined using primer walking. Phylogenetic analysis indicated that HpAdV-1 is most closely related to bottlenose dolphin adenovirus and clusters with Cetartiodactyla adenoviruses. The prevalence of HpAdV-1 was low (2.6%) based on targeted PCR-screening of the intestinal contents of 151 harbour porpoises stranded between 2010 and 2013.

Reprint of: Diversity of small, single-stranded DNA viruses of invertebrates and their chaotic evolutionary past.

A wide spectrum of invertebrates is susceptible to various single-stranded DNA viruses. Their relative simplicity of replication and dependence on actively dividing cells makes them highly pathogenic for many invertebrates (Hexapoda, Decapoda, etc.). We present their taxonomical classification and describe the evolutionary relationships between various groups of invertebrate-infecting viruses, their high degree of recombination, and their relationship to viruses infecting mammals or other vertebrates. They share characteristics of the viruses within the various families, including structure of the virus particle, genome properties, and gene expression strategy.

Diversity of small, single-stranded DNA viruses of invertebrates and their chaotic evolutionary past.

A wide spectrum of invertebrates is susceptible to various single-stranded DNA viruses. Their relative simplicity of replication and dependence on actively dividing cells makes them highly pathogenic for many invertebrates (Hexapoda, Decapoda, etc.). We present their taxonomical classification and describe the evolutionary relationships between various groups of invertebrate-infecting viruses, their high degree of recombination, and their relationship to viruses infecting mammals or other vertebrates. They share characteristics of the viruses within the various families, including structure of the virus particle, genome properties, and gene expression strategy.