The Family Fuselloviridae : Diversity and Replication of a hyperthermic virus infecting the archaeon genus Sulfolobus

The virus family Fuselloviridae infects the hyperthermophilic and acidophilic Crenarchaeon genus Sulfolobus and has been isolated from terrestrial hotsprings worldwide. Two previously uncharacterized Fuselloviruses, SSV-13 and SSV-L1, were isolated and sequenced and are compared to the five fully sequenced viruses presently in the public databases. Conserved promoters in all viruses and similar putative origins of replication suggest that these viruses use a transcriptional and genomic replication program similar to the relatively well-characterized SSV1. Pairwise comparisons of conserved genes in the seven virus genomes show that, like its host Sulfolobus, these viruses\u27 genetic divergence correlates with geographic separation. Genome rearrangements, horizontal movement of genes between Fuselloviruses, other Crenarchaeal viruses, and other hosts are also discussed. The development of a novel gene knockout system (LIPCR) for these viruses is presented with detailed methods. Use of this knockout system is demonstrated with two viral vectors that have fully and partially deleted integrase genes. The complete integrase deletion does not prevent virus replication but appears to prevent integration of the virus into the host genome and appears to decrease the relative fitness of the virus compared to a virus with a complete integrase gene. The partial integrase deletion removes the catalytic residues demonstrated to be necessary for enzymatic function but leaves the attachment site located within the integrase gene. Interestingly, this mutant appears to be still capable of integration in our lab host, Sulfolobus solfataricus. Possible reasons for this are discussed

Progress and Prospects for a Nucleic Acid Screening Test Set

Objective: DNA synthesis companies screen orders to detect controlled sequences with misuse risks. Assessing screening accuracy is challenging owing to the breadth of biological risks and ambiguities in risk definitions. Here, we detail an International Gene Synthesis Consortium working group’s rationale and process to develop a prototype DNA synthesis screening test dataset, aiming to establish a baseline of screening system accuracy to compare with various screening approaches.Methodology: Construction of the prototype test dataset involved four tool developers screening nucleic acid sequences from three taxonomic clusters of controlled organisms (Orbivirus, Francisella tularensis, and Coccidioides). Results were mapped onto predefined, comparable categories, checking for consensus or conflicts. Conflicts were grouped based on gene annotation and resolved through discussion.Results: The process highlighted several long-standing challenges in DNA synthesis screening, including the qualitative differences in approaches taken by screening tools. Our findings highlight the lack of clarity in assessing pathogen sequences with respect to regulatory control language, compounded by scientific uncertainty. We illustrate the current degree of consensus and existing challenges using classification statistics and specific examples.Conclusions and Next Steps: This prototype underscores the necessity of expert-regulator coordination in assessing gene-associated risks, offering a template for creating test sets across all taxonomic groups on international control lists. Expanding the working group would enrich dataset comprehensiveness, enabling a transition from species-focused to function-focused regulatory controls. This sets the foundation for quality control, certification, and improved risk assessment in DNA synthesis screening

Differential Virus Host-Ranges of the \u3ci\u3eFuselloviridae\u3c/i\u3e of Hyperthermophilic Archaea: Implications for Evolution in Extreme Environments

An emerging model for investigating virus-host interactions in hyperthermophilic Archaea is the Fusellovirus-Sulfolobus system. The host, Sulfolobus, is a hyperthermophilic acidophile endemic to sulfuric hot springs worldwide. The Fuselloviruses, also known as Sulfolobus Spindle-shaped Viruses (SSVs), are lemon or spindle -shaped double-stranded DNA viruses, which are also found worldwide. Although a few studies have addressed the host-range for the type virus, Sulfolobus Spindle-shaped Virus 1 (SSV1), using common Sulfolobus strains, a comprehensive host-range study for SSV-Sulfolobus systems has not been performed. Herein, we examine six bona fide SSV strains (SSV1, SSV2, SSV3, SSVL1, SSVK1, SSVRH) and their respective infection characteristics on multiple hosts from the family Sulfolobaceae. A spot-on-lawn or halo assay was employed to determine SSV infectivity (and host susceptibility) in parallel challenges of multiple SSVs on a lawn of a single Sulfolobus strain. Different SSVs have different host-ranges with SSV1 exhibiting the narrowest host-range and SSVRH exhibiting the broadest host range. In contrast to previous reports, SSVs can infect hosts beyond the genus Sulfolobus. Furthermore, geography does not appear to be a reliable predictor of Sulfolobus susceptibility to infection by any given SSV. The ability for SSVs to infect susceptible Sulfolobus host does not appear to change between 65°C and 88°C (physiological range); however, very low pH appears to influence infection. Lastly, for the virus-host pairs tested the Fusellovirus-Sulfolobus system appears to exhibit host-advantage. This work provides a foundation for understanding Fusellovirus biology and virus-host coevolution in extreme ecosystems

Differential Virus Host-Ranges of the Fuselloviridae of Hyperthermophilic Archaea: Implications for Evolution in Extreme Environments

An emerging model for investigating virus-host interactions in hyperthermophilic Archaea is the Fusellovirus-Sulfolobus system. The host, Sulfolobus, is a hyperthermophilic acidophile endemic to sulfuric volcanic-driven hot springs worldwide. The Fuselloviruses, also known as Sulfolobus Spindle-shaped Viruses (SSVs), are lemon or spindle shaped double-stranded DNA viruses that are also found worldwide. Although a few studies have addressed the host-range for the type virus, SSV1, using common Sulfolobus strains, a comprehensive host-range study for SSV-Sulfolobus systems has not been performed. Herein, we examine six bona fide SSV strains (SSV1, SSV2, SSV3, SSVL1, SSVK1, SSVRH) and their respective infection characteristics on multiple hosts from the family Sulfolobaceae. A halo assay was used to determine virus infectivity and host susceptibility. Different SSV strains have different host-ranges with SSV1 exhibiting the narrowest host-range and SSVRH exhibiting the broadest host range. There is no correlation between geographic separation of viruses and their hosts and their relative infectivity and susceptibility. In contrast to previous reports, SSVs can infect hosts beyond the genus Sulfolobus. Furthermore, the Fusellovirus-Sulfolobus system appears to exhibit host-advantage. This work provides a foundation for understanding Fusellovirus biology and virus-host co-evolution in extreme ecosystems, a rapidly emerging field of study