The emergence and ongoing convergent evolution of the SARS-CoV-2 N501Y lineages.

The independent emergence late in 2020 of the B.1.1.7, B.1.351, and P.1 lineages of SARS-CoV-2 prompted renewed concerns about the evolutionary capacity of this virus to overcome public health interventions and rising population immunity. Here, by examining patterns of synonymous and non-synonymous mutations that have accumulated in SARS-CoV-2 genomes since the pandemic began, we find that the emergence of these three "501Y lineages" coincided with a major global shift in the selective forces acting on various SARS-CoV-2 genes. Following their emergence, the adaptive evolution of 501Y lineage viruses has involved repeated selectively favored convergent mutations at 35 genome sites, mutations we refer to as the 501Y meta-signature. The ongoing convergence of viruses in many other lineages on this meta-signature suggests that it includes multiple mutation combinations capable of promoting the persistence of diverse SARS-CoV-2 lineages in the face of mounting host immune recognition.

Phage anti-CRISPR control by an RNA- and DNA-binding helix-turn-helix protein.

In all organisms, regulation of gene expression must be adjusted to meet cellular requirements and frequently involves helix-turn-helix (HTH) domain proteins1. For instance, in the arms race between bacteria and bacteriophages, rapid expression of phage anti-CRISPR (acr) genes upon infection enables evasion from CRISPR-Cas defence; transcription is then repressed by an HTH-domain-containing anti-CRISPR-associated (Aca) protein, probably to reduce fitness costs from excessive expression2-5. However, how a single HTH regulator adjusts anti-CRISPR production to cope with increasing phage genome copies and accumulating acr mRNA is unknown. Here we show that the HTH domain of the regulator Aca2, in addition to repressing Acr synthesis transcriptionally through DNA binding, inhibits translation of mRNAs by binding conserved RNA stem-loops and blocking ribosome access. The cryo-electron microscopy structure of the approximately 40 kDa Aca2-RNA complex demonstrates how the versatile HTH domain specifically discriminates RNA from DNA binding sites. These combined regulatory modes are widespread in the Aca2 family and facilitate CRISPR-Cas inhibition in the face of rapid phage DNA replication without toxic acr overexpression. Given the ubiquity of HTH-domain-containing proteins, it is anticipated that many more of them elicit regulatory control by dual DNA and RNA binding.

Structure and function of virion RNA polymerase of a crAss-like phage.

CrAss-like phages are a recently described expansive group of viruses that includes the most abundant virus in the human gut1-3. The genomes of all crAss-like phages encode a large virion-packaged protein2,4 that contains a DFDxD sequence motif, which forms the catalytic site in cellular multisubunit RNA polymerases (RNAPs)5. Here, using Cellulophaga baltica crAss-like phage phi14:2 as a model system, we show that this protein is a DNA-dependent RNAP that is translocated into the host cell along with the phage DNA and transcribes early phage genes. We determined the crystal structure of this 2,180-residue enzyme in a self-inhibited state, which probably occurs before virion packaging. This conformation is attained with the help of a cleft-blocking domain that interacts with the active site and occupies the cavity in which the RNA-DNA hybrid binds. Structurally, phi14:2 RNAP is most similar to eukaryotic RNAPs that are involved in RNA interference6,7, although most of the phi14:2 RNAP structure (nearly 1,600 residues) maps to a new region of the protein fold space. Considering this structural similarity, we propose that eukaryal RNA interference polymerases have their origins in phage, which parallels the emergence of the mitochondrial transcription apparatus8.

Transcriptome and small RNAome profiling uncovers how a recombinant begomovirus evades RDRγ-mediated silencing of viral genes and outcompetes its parental virus in mixed infection.

Tomato yellow leaf curl virus (TYLCV, genus Begomovirus, family Geminiviridae) causes severe disease of cultivated tomatoes. Geminiviruses replicate circular single-stranded genomic DNA via rolling-circle and recombination-dependent mechanisms, frequently generating recombinants in mixed infections. Circular double-stranded intermediates of replication also serve as templates for Pol II bidirectional transcription. IS76, a recombinant derivative of TYLCV with a short sequence in the bidirectional promoter/origin-of-replication region acquired from a related begomovirus, outcompetes TYLCV in mixed infection and breaks disease resistance in tomato Ty-1 cultivars. Ty-1 encodes a γ-clade RNA-dependent RNA polymerase (RDRγ) implicated in Dicer-like (DCL)-mediated biogenesis of small interfering (si)RNAs directing gene silencing. Here, we profiled transcriptome and small RNAome of Ty-1 resistant and control susceptible plants infected with TYLCV, IS76 or their combination at early and late infection stages. We found that RDRγ boosts production rates of 21, 22 and 24 nt siRNAs from entire genomes of both viruses and modulates DCL activities in favour of 22 and 24 nt siRNAs. Compared to parental TYLCV, IS76 undergoes faster transition to the infection stage favouring rightward transcription of silencing suppressor and coat protein genes, thereby evading RDRγ activity and facilitating its DNA accumulation in both single and mixed infections. In coinfected Ty-1 plants, IS76 efficiently competes for host replication and transcription machineries, thereby impairing TYLCV replication and transcription and forcing its elimination associated with further increased siRNA production. RDRγ is constitutively overexpressed in Ty-1 plants, which correlates with begomovirus resistance, while siRNA-generating DCLs (DCL2b/d, DCL3, DCL4) and genes implicated in siRNA amplification (α-clade RDR1) and function (Argonaute2) are upregulated to similar levels in TYLCV- and IS76-infected susceptible plants. Collectively, IS76 recombination facilitates replication and promotes expression of silencing suppressor and coat proteins, which allows the recombinant virus to evade the negative impact of RDRγ-boosted production of viral siRNAs directing transcriptional and posttranscriptional silencing.

Identification of a viral gene essential for the genome replication of a domesticated endogenous virus in ichneumonid parasitoid wasps.

Thousands of endoparasitoid wasp species in the families Braconidae and Ichneumonidae harbor "domesticated endogenous viruses" (DEVs) in their genomes. This study focuses on ichneumonid DEVs, named ichnoviruses (IVs). Large quantities of DNA-containing IV virions are produced in ovary calyx cells during the pupal and adult stages of female wasps. Females parasitize host insects by injecting eggs and virions into the body cavity. After injection, virions rapidly infect host cells which is followed by expression of IV genes that promote the successful development of wasp offspring. IV genomes consist of two components: proviral segment loci that serve as templates for circular dsDNAs that are packaged into capsids, and genes from an ancestral virus that produce virions. In this study, we generated a chromosome-scale genome assembly for Hyposoter didymator that harbors H. didymator ichnovirus (HdIV). We identified a total of 67 HdIV loci that are amplified in calyx cells during the wasp pupal stage. We then focused on an HdIV gene, U16, which is transcribed in calyx cells during the initial stages of replication. Sequence analysis indicated that U16 contains a conserved domain in primases from select other viruses. Knockdown of U16 by RNA interference inhibited virion morphogenesis in calyx cells. Genome-wide analysis indicated U16 knockdown also inhibited amplification of HdIV loci in calyx cells. Altogether, our results identified several previously unknown HdIV loci, demonstrated that all HdIV loci are amplified in calyx cells during the pupal stage, and showed that U16 is required for amplification and virion morphogenesis.

Widespread endogenization of giant viruses shapes genomes of green algae.

Endogenous viral elements (EVEs)-viruses that have integrated their genomes into those of their hosts-are prevalent in eukaryotes and have an important role in genome evolution1,2. The vast majority of EVEs that have been identified to date are small genomic regions comprising a few genes2, but recent evidence suggests that some large double-stranded DNA viruses may also endogenize into the genome of the host1. Nucleocytoplasmic large DNA viruses (NCLDVs) have recently become of great interest owing to their large genomes and complex evolutionary origins3-6, but it is not yet known whether they are a prominent component of eukaryotic EVEs. Here we report the widespread endogenization of NCLDVs in diverse green algae; these giant EVEs reached sizes greater than 1 million base pairs and contained as many as around 10% of the total open reading frames in some genomes, substantially increasing the scale of known viral genes in eukaryotic genomes. These endogenized elements often shared genes with host genomic loci and contained numerous spliceosomal introns and large duplications, suggesting tight assimilation into host genomes. NCLDVs contain large and mosaic genomes with genes derived from multiple sources, and their endogenization represents an underappreciated conduit of new genetic material into eukaryotic lineages that can substantially impact genome composition.

TarBase-v9.0 extends experimentally supported miRNA-gene interactions to cell-types and virally encoded miRNAs.

TarBase is a reference database dedicated to produce, curate and deliver high quality experimentally-supported microRNA (miRNA) targets on protein-coding transcripts. In its latest version (v9.0, https://dianalab.e-ce.uth.gr/tarbasev9), it pushes the envelope by introducing virally-encoded miRNAs, interactions leading to target-directed miRNA degradation (TDMD) events and the largest collection of miRNA-gene interactions to date in a plethora of experimental settings, tissues and cell-types. It catalogues ∼6 million entries, comprising ∼2 million unique miRNA-gene pairs, supported by 37 experimental (high- and low-yield) protocols in 172 tissues and cell-types. Interactions are annotated with rich metadata including information on genes/transcripts, miRNAs, samples, experimental contexts and publications, while millions of miRNA-binding locations are also provided at cell-type resolution. A completely re-designed interface with state-of-the-art web technologies, incorporates more features, and allows flexible and ingenious use. The new interface provides the capability to design sophisticated queries with numerous filtering criteria including cell lines, experimental conditions, cell types, experimental methods, species and/or tissues of interest. Additionally, a plethora of fine-tuning capacities have been integrated to the platform, offering the refinement of the returned interactions based on miRNA confidence and expression levels, while boundless local retrieval of the offered interactions and metadata is enabled.

African swine fever virus I73R is a critical virulence-related gene: A potential target for attenuation.

African swine fever virus (ASFV) is a large, double-stranded DNA virus that causes a fatal disease in pigs, posing a threat to the global pig industry. Whereas some ASFV proteins have been found to play important roles in ASFV-host interaction, the functional roles of many proteins are still largely unknown. In this study, we identified I73R, an early viral gene in the replication cycle of ASFV, as a key virulence factor. Our findings demonstrate that pI73R suppresses the host innate immune response by broadly inhibiting the synthesis of host proteins, including antiviral proteins. Crystallization and structural characterization results suggest that pI73R is a nucleic-acid-binding protein containing a Zα domain. It localizes in the nucleus and inhibits host protein synthesis by suppressing the nuclear export of cellular messenger RNA (mRNAs). While pI73R promotes viral replication, the deletion of the gene showed that it is a nonessential gene for virus replication. In vivo safety and immunogenicity evaluation results demonstrate that the deletion mutant ASFV-GZΔI73R is completely nonpathogenic and provides effective protection to pigs against wild-type ASFV. These results reveal I73R as a virulence-related gene critical for ASFV pathogenesis and suggest that it is a potential target for virus attenuation. Accordingly, the deletion mutant ASFV-GZΔI73R can be a potent live-attenuated vaccine candidate.

Conditional splicing system for tight control of viral overlapping genes.

Viral genomes frequently harbor overlapping genes, complicating the development of virus-vectored vaccines and gene therapies. This study introduces a novel conditional splicing system to precisely control the expression of such overlapping genes through recombinase-mediated conditional splicing. We refined site-specific recombinase (SSR) conditional splicing systems and explored their mechanisms. The systems demonstrated exceptional inducibility (116,700-fold increase) with negligible background expression, facilitating the conditional expression of overlapping genes in adenovirus-associated virus (AAV) and human immunodeficiency virus type 1. Notably, this approach enabled the establishment of stable AAV producer cell lines, encapsulating all necessary packaging genes. Our findings underscore the potential of the SSR-conditional splicing system to significantly advance vector engineering, enhancing the efficacy and scalability of viral-vector-based therapies and vaccines. IMPORTANCE: Regulating overlapping genes is vital for gene therapy and vaccine development using viral vectors. The regulation of overlapping genes presents challenges, including cytotoxicity and impacts on vector capacity and genome stability, which restrict stable packaging cell line development and broad application. To address these challenges, we present a "loxp-splice-loxp"-based conditional splicing system, offering a novel solution for conditional expression of overlapping genes and stable cell line establishment. This system may also regulate other cytotoxic genes, representing a significant advancement in cell engineering and gene therapy as well as biomass production.

Role of the membrane-associated accessory protein (MAAP) in adeno-associated virus (AAV) infection.

Adeno-associated viruses (AAVs) package a single-stranded (ss) DNA genome of 4.7 kb in their capsid of ~20 nm in diameter. AAV replication requires co-infection of a helper virus, such as adenovirus. During the optimization of recombinant AAV production, a small viral nonstructural protein, membrane-associated accessory protein (MAAP), was identified. However, the function of the MAAP in the context of AAV infection remains unknown. Here, we investigated the expression strategy and function of the MAAP during infection of both AAV2 and AAV5 in human embryonic kidney (HEK)293 cells. We found that AAV2 MAAP2 and AAV5 MAAP5 are expressed from the capsid gene (cap)-transcribing mRNA spliced from the donor to the second splice site that encodes VP2 and VP3. Thus, this AAV cap gene transcribes a multicistronic mRNA that can be translated to four viral proteins, MAAP, VP2, AAP, and VP3 in order. In AAV2 infection, MAAP2 predominantly localized in the cytoplasm, alongside the capsid, near the nuclear and plasma membranes, but a fraction of MAAP2 exhibited nuclear localization. In AAV5 infection, MAAP5 revealed a distinct pattern, predominantly localizing within the nucleus. In the cells infected with an MAAP knockout mutant of AAV2 or AAV5, both viral DNA replication and virus replication increased, whereas virus egress decreased, and the decrease in virus egress can be restored by providing MAAP in trans. In summary, MAAP, a novel AAV nonstructural protein translated from a multicistronic viral cap mRNA, not only facilitates cellular egress of AAV but also likely negatively affects viral DNA replication during infection. IMPORTANCE: Recombinant adeno-associated virus (rAAV) has been used as a gene delivery vector in clinical gene therapy. In current gene therapies employing rAAV, a high dose of the vector is required. Consequently, there is a high demand for efficient and high-purity vector production systems. In this study, we demonstrated that membrane-associated accessory protein (MAAP), a small viral nonstructural protein, is translated from the same viral mRNA transcript encoding VP2 and VP3. In AAV-infected cells, apart from its prevalent expression in the cytoplasm with localization near the plasma and nuclear membranes, the MAAP also exhibits notable localization within the nucleus. During AAV infection, MAAP expression increases the cellular egress of progeny virions and decreases viral DNA replication and progeny virion production. Thus, the choice of MAAP expression has pros and cons during AAV infection, which could provide a guide to rAAV production.

Aberrant RNA polymerase initiation and processivity on the genome of a herpes simplex virus 1 mutant lacking ICP27.

Within the first 15 minutes of infection, herpes simplex virus 1 immediate early proteins repurpose cellular RNA polymerase (Pol II) for viral transcription. An important role of the viral-infected cell protein 27 (ICP27) is to facilitate viral pre-mRNA processing and export viral mRNA to the cytoplasm. Here, we use precision nuclear run-on followed by deep sequencing (PRO-seq) to characterize transcription of a viral ICP27 null mutant. At 1.5 and 3 hours post infection (hpi), we observed increased total levels of Pol II on the mutant viral genome and accumulation of Pol II downstream of poly A sites indicating increased levels of initiation and processivity. By 6 hpi, Pol II accumulation on specific mutant viral genes was higher than that on wild-type virus either at or upstream of poly A signals, depending on the gene. The PRO-seq profile of the ICP27 mutant on late genes at 6 hpi was similar but not identical to that caused by treatment with flavopiridol, a known inhibitor of RNA processivity. This pattern was different from PRO-seq profiles of other α gene mutants and upon inhibition of viral DNA replication with PAA. Together, these results indicate that ICP27 contributes to the repression of aberrant viral transcription at 1.5 and 3 hpi by inhibiting initiation and decreasing RNA processivity. However, ICP27 is needed to enhance processivity on most late genes by 6 hpi in a mechanism distinguishable from its role in viral DNA replication.IMPORTANCEWe developed and validated the use of a processivity index for precision nuclear run-on followed by deep sequencing data. The processivity index calculations confirm infected cell protein 27 (ICP27) induces downstream of transcription termination on certain host genes. The processivity indices and whole gene probe data implicate ICP27 in transient immediate early gene-mediated repression, a process that also requires ICP4, ICP22, and ICP0. The data indicate that ICP27 directly or indirectly regulates RNA polymerase (Pol II) initiation and processivity on specific genes at specific times post infection. These observations support specific and varied roles for ICP27 in regulating Pol II activity on viral genes in addition to its known roles in post transcriptional mRNA processing and export.

Multicopy suppressor screens reveal convergent evolution of single-gene lysis proteins.

Single-strand RNA (ssRNA) Fiersviridae phages cause host lysis with a product of single gene (sgl for single-gene lysis; product Sgl) that induces autolysis. Many different Sgls have been discovered, but the molecular targets of only a few have been identified. In this study, we used a high-throughput genetic screen to uncover genome-wide host suppressors of diverse Sgls. In addition to validating known molecular mechanisms, we discovered that the Sgl of PP7, an ssRNA phage of Pseudomonas aeruginosa, targets MurJ, the flippase responsible for lipid II export, previously shown to be the target of the Sgl of coliphage M. These two Sgls, which are unrelated and predicted to have opposite membrane topology, thus represent a case of convergent evolution. We extended the genetic screens to other uncharacterized Sgls and uncovered a common set of multicopy suppressors, suggesting that these Sgls act by the same or similar mechanism.

Viral and metazoan poxins are cGAMP-specific nucleases that restrict cGAS-STING signalling.

Cytosolic DNA triggers innate immune responses through the activation of cyclic GMP-AMP synthase (cGAS) and production of the cyclic dinucleotide second messenger 2',3'-cyclic GMP-AMP (cGAMP)1-4. 2',3'-cGAMP is a potent inducer of immune signalling; however, no intracellular nucleases are known to cleave 2',3'-cGAMP and prevent the activation of the receptor stimulator of interferon genes (STING)5-7. Here we develop a biochemical screen to analyse 24 mammalian viruses, and identify poxvirus immune nucleases (poxins) as a family of 2',3'-cGAMP-degrading enzymes. Poxins cleave 2',3'-cGAMP to restrict STING-dependent signalling and deletion of the poxin gene (B2R) attenuates vaccinia virus replication in vivo. Crystal structures of vaccinia virus poxin in pre- and post-reactive states define the mechanism of selective 2',3'-cGAMP degradation through metal-independent cleavage of the 3'-5' bond, converting 2',3'-cGAMP into linear Gp[2'-5']Ap[3']. Poxins are conserved in mammalian poxviruses. In addition, we identify functional poxin homologues in the genomes of moths and butterflies and the baculoviruses that infect these insects. Baculovirus and insect host poxin homologues retain selective 2',3'-cGAMP degradation activity, suggesting an ancient role for poxins in cGAS-STING regulation. Our results define poxins as a family of 2',3'-cGAMP-specific nucleases and demonstrate a mechanism for how viruses evade innate immunity.

Human endogenous retrovirus-K (HERV-K) reverse transcriptase (RT) structure and biochemistry reveals remarkable similarities to HIV-1 RT and opportunities for HERV-K-specific inhibition.

Human endogenous retroviruses (HERVs) comprise nearly 8% of the human genome and are derived from ancient integrations of retroviruses into the germline. The biology of HERVs is poorly defined, but there is accumulating evidence supporting pathological roles in diverse diseases, such as cancer, autoimmune, and neurodegenerative diseases. Functional proteins are produced by HERV-encoded genes, including reverse transcriptases (RTs), which could be a contributor to the pathology attributed to aberrant HERV-K expression. To facilitate the discovery and development of HERV-K RT potent and selective inhibitors, we expressed active HERV-K RT and determined the crystal structure of a ternary complex of this enzyme with a double-stranded DNA substrate. We demonstrate a range of RT inhibition with antiretroviral nucleotide analogs, while classic nonnucleoside analogs do not inhibit HERV-K RT. Detailed comparisons of HERV-K RT with other known RTs demonstrate similarities to diverse RT families and a striking similarity to the HIV-1 RT asymmetric heterodimer. Our analysis further reveals opportunities for selective HERV-K RT inhibition.

Persistent Low-Level Variants in a Subset of Viral Genes Are Highly Predictive of Poor Outcome in Immunocompromised Patients With Cytomegalovirus Infection.

BACKGROUND: Human cytomegalovirus (HCMV) is the most common and serious opportunistic infection after solid organ and hematopoietic stem cell transplantation. In this study, we used whole-genome HCMV data to investigate viral factors associated with the clinical outcome. METHODS: We sequenced HCMV samples from 16 immunocompromised pediatric patients with persistent viremia. Eight of the 16 patients died of complications due to HCMV infection. We also sequenced samples from 35 infected solid organ adult recipients, of whom 1 died with HCMV infection. RESULTS: We showed that samples from both groups have fixed variants at resistance sites and mixed infections. Next-generation sequencing also revealed nonfixed variants at resistance sites in most of the patients who died (6/9). A machine learning approach identified 10 genes with nonfixed variants in these patients. These genes formed a viral signature that discriminated patients with HCMV infection who died from those who survived with high accuracy (area under the curve = 0.96). Lymphocyte numbers for a subset of patients showed no recovery posttransplant in the patients who died. CONCLUSIONS: We hypothesize that the viral signature identified in this study may be a useful biomarker for poor response to antiviral drug treatment and indirectly for poor T-cell function, potentially identifying early those patients requiring nonpharmacological interventions.

Improvements in viral gene annotation using large language models and soft alignments.

BACKGROUND: The annotation of protein sequences in public databases has long posed a challenge in molecular biology. This issue is particularly acute for viral proteins, which demonstrate limited homology to known proteins when using alignment, k-mer, or profile-based homology search approaches. A novel methodology employing Large Language Models (LLMs) addresses this methodological challenge by annotating protein sequences based on embeddings. RESULTS: Central to our contribution is the soft alignment algorithm, drawing from traditional protein alignment but leveraging embedding similarity at the amino acid level to bypass the need for conventional scoring matrices. This method not only surpasses pooled embedding-based models in efficiency but also in interpretability, enabling users to easily trace homologous amino acids and delve deeper into the alignments. Far from being a black box, our approach provides transparent, BLAST-like alignment visualizations, combining traditional biological research with AI advancements to elevate protein annotation through embedding-based analysis while ensuring interpretability. Tests using the Virus Orthologous Groups and ViralZone protein databases indicated that the novel soft alignment approach recognized and annotated sequences that both blastp and pooling-based methods, which are commonly used for sequence annotation, failed to detect. CONCLUSION: The embeddings approach shows the great potential of LLMs for enhancing protein sequence annotation, especially in viral genomics. These findings present a promising avenue for more efficient and accurate protein function inference in molecular biology.

Herpes simplex virus 1 immediate early transcription initiation, pause-release, elongation, and termination in the presence and absence of ICP4.

IMPORTANCE: Infection with herpes simplex virus 1 (HSV-1) leads to lifelong infection due to the virus's remarkable ability to control transcription of its own genome, resulting in two transcriptional programs: lytic (highly active) and latent (restricted). The lytic program requires immediate early (IE) proteins to first repress transcription of late viral genes, which then undergo sequential de-repression, leading to a specific sequence of gene expression. Here, we show that the IE ICP4 functions to regulate the cascade by limiting RNA polymerase initiation at immediate early times. However, late viral genes that initiate too early in the absence of ICP4 do not yield mRNA as transcription stalls within gene bodies. It follows that other regulatory steps intercede to prevent elongation of genes at the incorrect time, demonstrating the precise control HSV-1 exerts over its own transcription.

Deletion of DP148R, DP71L, and DP96R Attenuates African Swine Fever Virus, and the Mutant Strain Confers Complete Protection against Homologous Challenges in Pigs.

The African swine fever virus (ASFV) has caused a devastating pandemic in domestic and wild swine, causing economic losses to the global swine industry. Recombinant live attenuated vaccines are an attractive option for ASFV treatment. However, safe and effective vaccines against ASFV are still scarce, and more high-quality experimental vaccine strains need to be developed. In this study, we revealed that deletion of the ASFV genes DP148R, DP71L, and DP96R from the highly virulent isolate ASFV CN/GS/2018 (ASFV-GS) substantially attenuated virulence in swine. Pigs infected with 104 50% hemadsorbing doses of the virus with these gene deletions remained healthy during the 19-day observation period. No ASFV infection was detected in contact pigs under the experimental conditions. Importantly, the inoculated pigs were protected against homologous challenges. Additionally, RNA sequence analysis showed that deletion of these viral genes induced significant upregulation of the host histone H3.1 gene (H3.1) and downregulation of the ASFV MGF110-7L gene. Knocking down the expression of H3.1 resulted in high levels of ASFV replication in primary porcine macrophages in vitro. These findings indicate that the deletion mutant virus ASFV-GS-Δ18R/NL/UK is a novel potential live attenuated vaccine candidate and one of the few experimental vaccine strains reported to induce full protection against the highly virulent ASFV-GS virus strain. IMPORTANCE Ongoing outbreaks of African swine fever (ASF) have considerably damaged the pig industry in affected countries. Thus, a safe and effective vaccine is important to control African swine fever spread. Here, an ASFV strain with three gene deletions was developed by knocking out the viral genes DP148R (MGF360-18R), NL (DP71L), and UK (DP96R). The results showed that the recombinant virus was completely attenuated in pigs and provided strong protection against parental virus challenge. Additionally, no viral genomes were detected in the sera of pigs housed with animals infected with the deletion mutant. Furthermore, transcriptome sequencing (RNA-seq) analysis revealed significant upregulation of histone H3.1 in virus-infected macrophage cultures and downregulation of the ASFV MGF110-7L gene after viral DP148R, UK, and NL deletion. Our study provides a valuable live attenuated vaccine candidate and potential gene targets for developing strategies for anti-ASFV treatment.

Development of a new effective African swine fever virus vaccine candidate by deletion of the H240R and MGF505-7R genes results in protective immunity against the Eurasia strain.

IMPORTANCE: African swine fever (ASF) caused by ASF virus (ASFV) is a highly contagious and acute hemorrhagic viral disease in domestic pigs. Until now, no effective commercial vaccine and antiviral drugs are available for ASF control. Here, we generated a new live-attenuated vaccine candidate (ASFV-ΔH240R-Δ7R) by deleting H240R and MGF505-7R genes from the highly pathogenic ASFV HLJ/18 genome. Piglets immunized with ASFV-ΔH240R-Δ7R were safe without any ASF-related signs and produced specific antibodies against p30. Challenged with a virulent ASFV HLJ/18, the piglets immunized with high-dose group (105 HAD50) exhibited 100% protection without clinical symptoms, showing that low levels of virus replication with no observed pathogenicity by postmortem and histological analysis. Overall, our results provided a new strategy by designing live-attenuated vaccine candidate, resulting in protection against ASFV infection.

Combinational Deletions of MGF110-9L and MGF505-7R Genes from the African Swine Fever Virus Inhibit TBK1 Degradation by an Autophagy Activator PIK3C2B To Promote Type I Interferon Production.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a transboundary infectious disease of domestic pigs and wild boars, resulting in significant swine production losses. Currently, no effective commercial ASF vaccines or therapeutic options are available. A previous study has shown that deletions of ASFV MGF110-9L and MGF505-7R genes (ASFV-Δ110-9L/505-7R) attenuated virulence in pigs and provided complete protection against parental lethal ASFV CN/GS/2018 (wild-type ASFV [ASFV-WT]) challenge, but the underlying mechanism is unclear. This study found that ASFV-Δ110-9L/505-7R weakened TBK1 degradation compared with ASFV-WT through RNA sequencing (RNA-seq) and Western blotting analyses. Furthermore, we confirmed that ASFV-Δ110-9L/505-7R blocked the degradation of TBK1 through the autophagy pathway. We also identified that the downregulation of an autophagy-related protein PIK3C2B was involved in the inhibition of TBK1 degradation induced by ASFV-Δ110-9L/505-7R. Additionally, we also confirmed that PIK3C2B promoted ASFV-Δ110-9L/505-7R replication in vitro. Together, this study elucidated a novel mechanism of virulence change of ASFV-Δ110-9L/505-7R, revealing a new mechanism of ASF live attenuated vaccines (LAVs) and providing theoretical guidance for the development of ASF vaccines. IMPORTANCE African swine fever (ASF) is a contagious and lethal hemorrhagic disease of pigs caused by the African swine fever virus (ASFV), leading to significant economic consequences for the global pig industry. The development of an effective and safe ASF vaccine has been unsuccessful. Previous studies have shown that live attenuated vaccines (LAVs) of ASFV are the most effective vaccine candidates to prevent ASF. Understanding the host responses caused by LAVs of ASFV is important in optimizing vaccine design and diversifying the resources available to control ASF. Recently, our laboratory found that the live attenuated ASFV-Δ110-9L/505-7R provided complete protection against parental ASFV-WT challenge. This study further demonstrated that ASFV-Δ110-9L/505-7R inhibits TBK1 degradation mediated by an autophagy activator PIK3C2B to increase type I interferon production. These results revealed an important mechanism for candidate vaccine ASFV-Δ110-9L/505-7R, providing strategies for exploring the virulence of multigene-deleted live attenuated ASFV strains and the development of vaccines.