Cytoplasmic DNA Viruses Research Articles

PK205R targets the proximal element of IFN-I signaling pathway to assist African swine fever virus to escape host innate immunity at the early stage of infection.

African swine fever virus (ASFV) is a nuclear cytoplasmic large DNA virus (NCLDV) that causes devastating hemorrhagic diseases in domestic pigs and wild boars, seriously threatening the development of the global pig industry. IFN-I plays an important role in the body's antiviral response. Similar to other DNA viruses, ASFV has evolved a variety of immune escape strategies to antagonize IFN-I signaling and maintain its proliferation. In this study, we showed that the ASFV early protein pK205R strongly inhibited interferon-stimulated genes (ISGs) as well as the promoter activity of IFN-stimulated regulatory elements (ISREs). Mechanistically, pK205R interacted with the intracellular domains of IFNAR1 and IFNAR2, thereby inhibiting the interaction of IFNAR1/2 with JAK1 and TYK2 and hindering the phosphorylation and nuclear translocation of STATs. Subsequently, we generated a recombinant strain of the ASFV-pK205R point mutation, ASFV-pK205R7PM. Notably, we detected higher levels of ISGs in porcine alveolar macrophages (PAMs) than in the parental strain during the early stages of ASFV-pK205R7PM infection. Moreover, ASFV-pK205R7PM attenuated the inhibitory effect on IFN-I signaling. In conclusion, we identified a new ASFV immunosuppressive protein that increases our understanding of ASFV immune escape mechanisms.

Characterization of Fowlpox Virus.

The complex cytoplasmic DNA virus known as the fowlpox virus (FWPV) is a member of the avipoxvirus genus, Subfamily Chordopoxvirinae, and Family Poxviridae. The large genome size of FWPV makes it a potential vector for the creation of vaccines against a range of serious veterinary and human ailments. It also allows for multiple gene insertion and the generation of abortive infection in mammalian cells. The virus, which causes fowlpox in chickens and turkeys, is mainly transmitted to poultry through aerosols or biting insects. Fowlpox is a highly contagious disease that affects both domestic and wild birds, causing cutaneous and/or diphtheritic illnesses. To control the illness, strict hygiene practices and immunization with FWPV attenuated strains or antigenically similar pigeon pox virus vaccines are employed. Recent years have seen an increase in fowlpox outbreaks in chicken flocks, primarily due to the introduction of novel forms of FWPV. It is believed that the pathogenic characteristics of these strains are enhanced by the integration of reticuloendotheliosis virus sequences of variable lengths into the FWPV genome. The standard laboratory diagnosis of FPV involves histopathological analysis, electron microscopy, virus isolation on chorioallantoic membrane (CAM) of embryonated chicken eggs or cell cultures, and serologic techniques. For quick and consistent diagnosis, polymerase chain reaction (PCR) has proven to be the most sensitive method. PCR is used in concert with restriction endonuclease enzyme analysis (REA) to identify, differentiate, and characterize the molecular makeup of isolates of the fowlpox virus. Sequencing of the amplified fragments is then done.

Interferon induction by STING requires its translocation to the late endosomes.

To combat microbial infections, mammalian cells use a variety of innate immune response pathways to induce synthesis of anti-microbial proteins. The cGAS/STING pathway recognizes cytoplasmic viral or cellular DNA to elicit signals that lead to type I interferon and other cytokine synthesis. cGAMP, synthesized by DNA-activated cGAS, activates the ER-associated protein, STING, which oligomerizes and translocates to other intracellular membrane compartments to trigger different branches of signaling. We have reported that, in the ER, EGFR-mediated phosphorylation of Tyr245 of STING is required for its transit to the late endosomes, where it recruits and activates the transcription factor IRF3 required for IFN induction. In the current study, we inquired whether STING Tyr245 phosphorylation per se or STING's location in the late endosomes was critical for its ability to recruit IRF3 and induce IFN. Using pharmacological inhibitors or genetic ablation of proteins that are essential for specific steps of STING trafficking, we demonstrated that the presence of STING in the late endosomal membranes, even without Tyr245 phosphorylation, was sufficient for IRF3-mediated IFN induction.

Vaccinia E5 is a major inhibitor of the DNA sensor cGAS

The DNA sensor cyclic GMP-AMP synthase (cGAS) is critical in host antiviral immunity. Vaccinia virus (VACV) is a large cytoplasmic DNA virus that belongs to the poxvirus family. How vaccinia virus antagonizes the cGAS-mediated cytosolic DNA-sensing pathway is not well understood. In this study, we screened 80 vaccinia genes to identify potential viral inhibitors of the cGAS/Stimulator of interferon gene (STING) pathway. We discovered that vaccinia E5 is a virulence factor and a major inhibitor of cGAS. E5 is responsible for abolishing cGAMP production during vaccinia virus (Western Reserve strain) infection of dendritic cells. E5 localizes to the cytoplasm and nucleus of infected cells. Cytosolic E5 triggers ubiquitination of cGAS and proteasome-dependent degradation via interacting with cGAS. Deleting the E5R gene from the Modified vaccinia virus Ankara (MVA) genome strongly induces type I IFN production by dendritic cells (DCs) and promotes DC maturation, and thereby improves antigen-specific T cell responses.

Complete genome sequence of virulent genotype I African swine fever virus strain K49 from the Democratic Republic of the Congo, isolated from a domestic pig (Sus scrofa domesticus).

African swine fever is one of the most feared infectious diseases in the pig industry. African swine fever virus (ASFV) is an enveloped, cytoplasmic double-stranded DNA virus and the only member of the family Asfarviridae. Although ASFV is known to have been circulating on the African continent since at least 1921, little is known about the genetic characteristics of historical ASFV strains isolated in sub-Saharan Africa. The few complete ASFV genome sequences obtained from African historical isolates have demonstrated genetic diversity, but the available data are limited and insufficient for fully understanding the molecular evolution and continental spread of ASFV. Here, we report the complete genome sequence of the virulent ASFV strain K49, collected during an outbreak in the Belgian Congo (now the Democratic Republic of the Congo) in 1949. The complete genome sequence of ASFV strain K49 was determined using an Illumina HiSeq platform and is 189,523 bp in length with a mean GC content of 38.43%, with 189 genes annotated. This is the first reported complete genome sequence of an ASFV serogroup 2 isolate. Phylogenetic analysis demonstrated genetic divergence within genotype I, and strain K49 formed a separate branch from other ASFV genotype I isolates.

554 Vaccinia immune evasion ankyrin-repeat/F-box protein WR199 mediates ubiquitination and proteasomal degradation of the DNA sensor cGAS

Poxviruses are large cytoplasmic DNA viruses. Vaccinia virus is a replication-competent prototypical poxvirus that causes mortality in the murine intranasal infection model, a surrogate for smallpox infection. Modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia strain that is replication-incompetent and a safe vaccine against smallpox. The DNA sensor cyclic GMP-AMP synthase cGAS plays an important role in host antiviral immunity. cGAS is critical for the induction of type I IFN by modified vaccinia virus Ankara (MVA) in dendritic cells, and mice lacking cGAS are highly susceptible to vaccinia infection.

The African swine fever virus protease pS273R inhibits DNA sensing cGAS-STING pathway by targeting IKKε

ABSTRACT African swine fever virus (ASFV), a large and complex cytoplasmic double-stranded DNA virus, has developed multiple strategies to evade the antiviral innate immune responses. Cytosolic DNA arising from invading ASFV is mainly detected by the cyclic GMP-AMP synthase (cGAS) and then triggers a series of innate immune responses to prevent virus invasion. However, the immune escape mechanism of ASFV remains to be fully clarified. The pS273R of ASFV is a member of the SUMO-1-specific protease family and is crucial for valid virus replication. In this study, we identified pS273R as a suppressor of cGAS-STING pathway mediated type I interferon (IFN) production by ASFV genomic open reading frame screening. The pS273R was further confirmed as an inhibitor of IFN production as well as its downstream antiviral genes in cGAS-STING pathway. Mechanistically, pS273R greatly decreased the cGAS-STING signaling by targeting IKKε but not TBK1, and pS273R was found to disturb the interaction between IKKε and STING through its interaction with IKKε. Further, mutational analyses revealed that pS273R antagonized the cGAS-STING pathway by enzyme catalytic activity, which might affect the IKKε sumoylation state required for the interaction with STING. In summary, our results revealed for the first time that pS273R acts as an obvious negative regulator of cGAS-STING pathway by targeting IKKε via its enzymatic activity, which shows a new immune evasion mechanism of ASFV.

IFI16 Isoforms with Cytoplasmic and Nuclear Locations Play Differential Roles in Recognizing Invaded DNA Viruses.

IFN-γ-inducible protein 16 (IFI16) recognizes viral DNAs from both nucleus-replicating viruses and cytoplasm-replicating viruses. Isoform 2 of IFI16 (IFI16-iso2) with nuclear localization sequence (NLS) has been studied extensively as a well-known DNA sensor. However, the characteristics and functions of other IFI16 isoforms are almost unknown. Here, we find that IFI16-iso1, with exactly the same length as IFI16-iso2, lacks the NLS and locates in the cytoplasm. To distinguish the functions of IFI16-iso1 and IFI16-iso2, we have developed novel nuclear viral DNA mimics that can be recognized by the nuclear DNA sensors, including IFI16-iso2 and hnRNPA2B1. The hexanucleotide motif 5'-AGTGTT-3' DNA form of the nuclear localization sequence (DNLS) effectively drives cytoplasmic viral DNA nuclear translocation. These nuclear viral DNA mimics potently induce IFN-β and antiviral IFN-stimulated genes in human A549 cells, HEK293T cells, and mouse macrophages. The subcellular location difference of IFI16 isoforms determines their differential functions in recognizing viral DNA and activating type I IFN-dependent antiviral immunity. IFI16-iso1 preferentially colocalizes with cytoplasmic HSV60mer and cytoplasm-replicating vaccinia virus (VACV), whereas IFI16-iso2 mainly colocalizes with nuclear HSV60-DNLS and nucleus-replicating HSV-1. Compared with IFI16-iso2, IFI16-iso1 induces more transcription of IFN-β and IFN-stimulated genes, as well as stronger antiviral immunity upon HSV60mer transfection or VACV infection. IFI16-iso2, with the ability of nuclear-cytoplasmic shuttling, clears both invaded HSV type 1 and VACV significantly. However, IFI16-iso2 induces more type I IFN-dependent antiviral immunity than IFI16-iso1 upon HSV60-DNLS transfection or HSV type 1 infection. Our study has developed potent agonists for nuclear DNA sensors and also has demonstrated that IFI16 isoforms with cytoplasmic and nuclear locations play differential roles in innate immunity against DNA viruses.

The nuclear and cytoplasmic activities of RNA polymerase III, and an evolving transcriptome for surveillance.

A 1969 report that described biochemical and activity properties of the three eukaryotic RNA polymerases revealed Pol III as highly distinguishable, even before its transcripts were identified. Now known to be the most complex, Pol III contains several stably-associated subunits referred to as built-in transcription factors (BITFs) that enable highly efficient RNA synthesis by a unique termination-associated recycling process. In vertebrates, subunit RPC7(α/β)can be of two forms, encoded by POLR3G or POLR3GL, with differential activity. Here we review promoter-dependent transcription by Pol III as an evolutionary perspective of eukaryotic tRNA expression. Pol III also provides nonconventional functions reportedly by promoter-independent transcription, one of which is RNA synthesis from DNA 3'-ends during repair. Another is synthesis of 5'ppp-RNA signaling molecules from cytoplasmic viral DNA in a pathway of interferon activation that is dysfunctional in immunocompromised patients with mutations in Pol III subunits. These unconventional functions are also reviewed, including evidence that link them to the BITF subunits. We also review data on a fraction of the human Pol III transcriptome that evolved to include vault RNAs and snaRs with activities related to differentiation, and in innate immune and tumor surveillance. The Pol III of higher eukaryotes does considerably more than housekeeping.

Regulation and Evasion of Host Immune Response by African Swine Fever Virus.

African swine fever (ASF) is an acute lethal hemorrhagic viral disease in domestic pigs and wild boars; is widely epidemic in Africa, Europe, Asia, and Latin America; and poses a huge threat to the pig industry worldwide. ASF is caused by the infection of the ASF virus (ASFV), a cytoplasmic double-stranded DNA virus belonging to the Asfarviridae family. Here, we review how the virus regulates the host immune response and its mechanisms at different levels, including interferon modulation, inflammation, apoptosis, antigen presentation, and cellular immunity.

PRV UL13 inhibits cGAS\u2013STING-mediated IFN-\u03b2 production by phosphorylating IRF3

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is an intracellular sensor of cytoplasmic viral DNA created during virus infection, which subsequently activates the stimulator of interferon gene (STING)-dependent type I interferon response to eliminate pathogens. In contrast, viruses have developed different strategies to modulate this signalling pathway. Pseudorabies virus (PRV), an alphaherpesvirus, is the causative agent of Aujeszky’s disease (AD), a notable disease that causes substantial economic loss to the swine industry globally. Previous reports have shown that PRV infection induces cGAS-dependent IFN-β production, conversely hydrolysing cGAMP, a second messenger synthesized by cGAS, and attenuates PRV-induced IRF3 activation and IFN-β secretion. However, it is not clear whether PRV open reading frames (ORFs) modulate the cGAS–STING-IRF3 pathway. Here, 50 PRV ORFs were screened, showing that PRV UL13 serine/threonine kinase blocks the cGAS–STING-IRF3-, poly(I:C)- or VSV-mediated transcriptional activation of the IFN-β gene. Importantly, it was discovered that UL13 phosphorylates IRF3, and its kinase activity is indispensable for such an inhibitory effect. Moreover, UL13 does not affect IRF3 dimerization, nuclear translocation or association with CREB-binding protein (CBP) but attenuates the binding of IRF3 to the IRF3-responsive promoter. Consistent with this, it was discovered that UL13 inhibits the expression of multiple interferon-stimulated genes (ISGs) induced by cGAS–STING or poly(I:C). Finally, it was determined that PRV infection can activate IRF3 by recruiting it to the nucleus, and PRVΔUL13 mutants enhance the transactivation level of the IFN-β gene. Taken together, the data from the present study demonstrated that PRV UL13 inhibits cGAS–STING-mediated IFN-β production by phosphorylating IRF3.

Vaccinia virus E5 is a dominant inhibitor of the cytosolic DNA sensor cGAS

Abstract The cytosolic DNA sensor cGAS plays an important role in detecting viral nucleic acid, which leads to type I IFN production. We have previously shown that infection with conventional dendritic cells with modified vaccinia virus Ankara (MVA), a highly attenuated vaccinia strain (VACV), induces IFN production via a cGAS/STING-dependent mechanism. However, MVA or VACV infection triggers cGAS degradation and its mechanism is still unknown. VACV is a cytoplasmic DNA virus, which encodes more than 200 genes. In this study, we screened 70 vaccinia viral early genes for inhibition of cGAS/STING pathway using a dual luciferase system. We found that vaccinia E5 is a dominant inhibitor of cGAS and is the key protein mediating cGAS degradation. MVAΔE5R induces much higher levels of type I IFN than MVA in multiple cell types, including bone marrow derived dendritic cells (BMDC), bone marrow-derived macrophages (BMDM), and skin primary fibroblasts. MVAΔE5R-mediated type I IFN production is dependent on cGAS. Furthermore, MVAΔE5R gains replication capability in cGAS−/− skin fibroblasts. As a vaccine vector, skin scarification or intradermal vaccination with MVAΔE5R-OVA leads to much higher OVA-specific CD8+ T cell responses than MVA-OVA in vivo. Intratumoral injection of MVAΔE5R leads to stronger anti-tumor immune responses and better survival compared with MVA. Finally, in an intranasal infection model, VACVΔE5R is at least 100-fold attenuated compared with WT VACV. Taken together, our results provide strong evidence that E5 is a key viral virulence factor targeting the cytosolic DNA sensor cGAS and thereby inhibits type I IFN production.

834 Rational design of recombinant modified vaccinia virus Ankara for cancer immunotherapy

Poxviruses are large cytoplasmic DNA viruses and modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia strain. Intratumoral (IT) delivery of inactivated MVA (iMVA) eradicates injected tumors and generates systemic antitumor immunity, which synergizes with immune chekpoint blockade antibodies (Dai et al. Science Immunology, 2017). The antitumor effects induced by iMVA requires CD8+ T cells and CD103+ dendritic cells (DCs), and is dependent on cGAS/STING. We hypothesize that MVA encodes viral inhibitor(s) of the cGAS/STING pathway and therefore deleting such inhibitor(s) from MVA would improve the antitumor effects of the virus. We conducted a systematic screening of vaccinia early genes for potential inhibitors of the cGAS/STING pathway and identified several candidates. Among them, the vaccinia E5 protein encoded by the E5R gene has a dominant inhibitory function. Infection of bone marrow-derived DCs (BMDCs) with MVAΔE5R induces 80-fold higher IFN-β secretion compared with MVA and 10-fold higher IFN-β secertion compared with iMVA. IT MVAΔE5R results in strong antitumor effects that is dependent on the cGAS/STING/IFNAR pathway. MVA also expresses another virulence factor C7, which is encoded by the C7L gene. C7 binds to the transcription factors IRF3 and Stat2 and thereby inhibits type I IFN production and IFNAR signaling. IT recombinant MVA with deletion of both C7L and E5R, and with expression of two transgenes human Flt3L, a growth factor for DCs, and murine OX40L, a costimulatory factor for T cells, results in enhanced antitumor immunity compared with iMVA in murine B16-F10 melanoma model and other tumor models. Our results demonstrate the feasibility of dramatically improving MVA-based cancer immunotherapeutics through rational design.

Abstract LB-306: Oncolytic vaccinia virus expressing immune checkpoint blockade antibody as cancer immunotherapeutics

Abstract Oncolytic viruses can be engineered to become multifunctional cancer immunotherapeutic agents, used either as monotherapy or in combination with immune checkpoint blockade for antitumor effects. Intratumoral (IT) delivery of oncolytic viruses expressing immunomodulatory agents can alter the tumor immunosuppressive microenvironment and facilitate the proliferation and activation of antitumor effector and memory T cells. Although anti-cytotoxic T lymphocyte protein 4 (CTLA-4) antibody has been approved for the treatment of advanced melanoma, its toxicity profile and modest efficacy as a single agent have limited its use in the clinic. Poxviruses are large cytoplasmic DNA viruses and vaccinia virus is a prototypic poxvirus that has been investigated intensively as an oncolytic virus. Its 200-kb genome size allows large insertions of multiple foreign genes. Vaccinia virus has been used in humans extensively in the past during smallpox vaccination. In this study, we engineered a vaccinia (Western Reserve)-based oncolytic virus that expresses anti-muCTLA-4 antibody and human FMS-like tyrosine kinase 3 ligand (Flt3L) for the treatment of murine implantable melanoma via intratumoral injection. We used a mutant vaccinia virus E3LΔ83N as the parental virus. This virus has a deletion of the Z-DNA-binding domain of E3, a key virulence factor, which results in 1000-fold attenuation of the virus. Through homologous recombination at the thymidine kinase (TK) locus of vaccinia E3LΔ83N virus, we successfully generated an attenuated recombinant virus (E3LΔ83N-TK--hFlt3L-anti-muCTLA-4) with a deletion of TK and an insertion of a cassette that allows the expression of both hFlt3L and anti-muCTLA-4 antibody under the vaccinia synthetic early and late promoter. This virus replicates in murine and human tumor cell lines and expresses desired anti-muCTLA-4 antibody and hFlt3L in murine and human melanoma cell lines. IT delivery of this recombinant vaccinia virus is more efficacious compared with E3LΔ83N-TK- (used either alone or in combination of systemic delivery of anti-CTLA-4 antibody), in eradicating or delaying the growth of both injected tumors and non-injected distant tumors, as well as in prolonging the survival of mice in a murine bilateral B16-F10 melanoma tumor implantation model. Furthermore, immunological analyses of tumor-infiltrating lymphocytes, as well as antitumor T cells in spleens, showed highest numbers of activated Granzyme B+ CD4+ and CD8+ T cells in the non-injected distant tumors and highest numbers of antitumor CD8+ T cells in the spleens of mice treated with IT delivery of E3LΔ83N-TK--hFlt3L-anti-muCTLA-4 compared with those treated with E3LΔ83N-TK--hFlt3L or E3LΔ83N-TK-. No toxicities related to IT E3LΔ83N-TK--hFlt3L-anti-muCTLA-4 have been observed. Taken together, our results demonstrate that IT delivery of oncolytic vaccinia virus expressing anti-CTLA-4 and hFlt3L is a safe and effective strategy to enhance antitumor immunity. Citation Format: Weiyi Wang, Peihong Dai, Ning Yang, Stewart Shuman, Wei Yan, Taha Merghoub, Jedd D. Wolchok, Liang Deng. Oncolytic vaccinia virus expressing immune checkpoint blockade antibody as cancer immunotherapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-306.

First description of a natural infection with spleen and kidney necrosis virus in zebrafish.

Zebrafish has become a popular research model in the last years, and several diseases affecting zebrafish research facilities have been reported. However, only one case of naturally occurring viral infections was described for this species. In 2015, infectious spleen and kidney necrosis virus (ISKNV) was detected in zebrafish from a research facility in Spain. Affected fish showed lethargy, loss of appetite, abnormal swimming, distention of the coelomic cavity and, in the most severe cases, respiratory distress, pale gills and petechial haemorrhages at the base of fins. Cytomegaly was the most relevant histopathological finding in organs and tissues, sometimes associated to degenerative and necrotic changes. ISKNV belongs to the relatively newly defined genus Megalocytivirus, family Iridoviridae, comprising large, icosahedral cytoplasmic DNA viruses. This is the first case of naturally occurring Megalocytivirus infection in zebrafish research facilities, associated with morbidity. The virus has been identified based on both pathologic and genetic evidence, to better understand the pathogenesis of the infection in zebrafish and the phylogenetic relationship with other iridoviruses. Given the ability of megalocytiviruses to cross-species boundaries, it seems necessary to implement stringent biosecurity practices as these infections may invalidate experimental data and have major impact on laboratory and cultured fish.

Deletion of the Vaccinia Virus B1 Kinase Reveals Essential Functions of This Enzyme Complemented Partly by the Homologous Cellular Kinase VRK2.

The vaccinia virus B1 kinase is highly conserved among poxviruses and is essential for the viral life cycle. B1 exhibits a remarkable degree of similarity to vaccinia virus-related kinases (VRKs), a family of cellular kinases, suggesting that the viral enzyme has evolved to mimic VRK activity. Indeed, B1 and VRKs have been demonstrated to target a shared substrate, the DNA binding protein BAF, elucidating a signaling pathway important for both mitosis and the antiviral response. In this study, we further characterize the role of B1 during vaccinia infection to gain novel insights into its regulation and integration with cellular signaling pathways. We begin by describing the construction and characterization of the first B1 deletion virus (vvΔB1) produced using a complementing cell line expressing the viral kinase. Examination of vvΔB1 revealed that B1 is critical for the production of infectious virions in various cell types and is sufficient for BAF phosphorylation. Interestingly, the severity of the defect in DNA replication following the loss of B1 varied between cell types, leading us to posit that cellular VRKs partly complement for the absence of B1 in some cell lines. Using cell lines devoid of either VRK1 or VRK2, we tested this hypothesis and discovered that VRK2 expression facilitates DNA replication and allows later stages of the viral life cycle to proceed in the absence of B1. Finally, we present evidence that the impact of VRK2 on vaccinia virus is largely independent of BAF phosphorylation. These data support a model in which B1 and VRK2 share additional substrates important for the replication of cytoplasmic poxviruses.IMPORTANCE Viral mimicry of cellular signaling modulators provides clear evidence that the pathogen targets an important host pathway during infection. Poxviruses employ numerous viral homologs of cellular proteins, the study of which have yielded insights into signaling pathways used by both virus and cells alike. The vaccinia virus B1 protein is a homolog of cellular vaccinia virus-related kinases (VRKs) and is needed for viral DNA replication and likely other stages of the viral life cycle. However, much remains to be learned about how B1 and VRKs overlap functionally. This study utilizes new tools, including a B1 deletion virus and VRK knockout cells, to further characterize the functional links between the viral and cellular enzymes. As a result, we have discovered that B1 and VRK2 target a common set of substrates vital to productive infection of this large cytoplasmic DNA virus.

Construction and expression of DNA vaccine against reddish body iridovirus and evaluation of immune efficacy in turbot (Scophthalmus maximus)

Turbot aquaculture is a very important industry in China. However, it is hampered because of viral reddish body syndrome (VRBS) and high mortality caused by piscine turbot reddish body iridovirus (TRBIV). TRBIV virus is an icosahedron-like and cytoplasmic DNA virus, belonging to Iridoviridae, Megalocytivirus. In previous studies, we have identified two antigen mimotopes using bioinformatics and constructed prokaryotic expression vectors. In this study, a fragment of major capsid protein (MCP) gene with the two antigenic epitopes was cloned into eukaryotic expression vector pVAX1, to generate a recombinant plasmid pVAX1-TRBIV-MCP. The plasmid DNA was transferred into turbot cell line TK using liposome, and transient expression was detected using RT-PCR. After injection into turbot (Scophthalmus maximus), the expression of the antigen gene was analysed using RT-PCR and was shown to express in all tested tissues in vaccinated fish 2 and 7 days post-vaccination. The cumulative mortalities in the vaccinated and unvaccinated control fish were 30% and 88% respectively. Immune responses and upregulation of the expression of chemokine receptor, tumour necrosis factor, interferon and interferon-induced antiviral molecules were observed in the vaccinated fish 60 h post-vaccination. These results demonstrate that the vaccinated turbots had higher survival rate and produced specific serum antibodies following the TRBIV challenge. More studies are needed to develop and apply the promising DNA vaccine for virus control in turbot.

Effects of deletion of the ac109 gene of Autographa californica nucleopolyhedrovirus on interactions with mammalian cells.

Baculoviruses are able to enter into mammalian cells, where they can express a transgene that is placed under an appropriate promoter, without producing infectious progeny. ORF109 encodes an essential baculovirus protein that participates in the interaction of the baculovirus with mammalian cells. To date, the mechanisms underlying this interaction are not yet known. We demonstrated that although a Ac109 knock out virus maintained its ability to enter into BHK-21 cells, there was a marked reduction in the expression efficiency of the nuclear transgene. Moreover, the amount of free cytoplasmic viral DNA, which was detected by transcription of a reporter gene, was severely diminished. These results suggest Ac109 could be involved in maintaining the integrity of the viral nucleic acid.

Abstract A007: Intratumoral delivery of inactivated vaccinia virus is more efficacious than live oncolytic vaccinia virus in murine bilateral tumor implantation models

Abstract Poxviruses are large cytoplasmic DNA viruses that have potentials as vaccine vectors and oncolytic agents. Although almost all of the oncolytic vaccinia viruses that are being investigated in clinical trials are replication competent, it is still unclear how these viruses exert antitumor immunologic effects, and whether viral replication is even necessary to achieve systemic antitumor efficacy. In this study, we compared therapeutic efficacy of the intratumoral delivery of the live, attenuated, replication competent oncolytic vaccinia virus expressing mGM-CSF (VC-TK−-mGM-CSF)vs. its heat-inactivated counterpart (iVC-TK−-mGM-CSF) in two bilateral tumor implantation models- B16-F10 murine melanoma and MC38 colon adenocarcinoma. We found that intratumoral injection of inactivated virus exerted stronger systemic antitumor effects than the live virus. FACS analyses of tumor infiltrating immune cells revealed that intratumoral injection of iVC-TK−-mGM-CSF lead to increased percentages and numbers of activating effector CD8+ and CD4+ T cells and reduced percentages and numbers of CD4+Foxp3+ regulatory T cells in the non-injected tumors compared with intratumoral delivery of the live virus. Infection of bone marrow-derived dendritic cells (DCs) with iVC-TK−-mGM-CSF induced type I IFN and proinflammatory cytokine and chemokine production and DC maturation, whereas the live virus did not. Taken together, our results demonstrate that intratumoral delivery of inactivated vaccina virus is safer and more efficacious than live oncolytic vaccinia virus expressing mGM-CSF in two murine bilateral tumor implantation models. Our findings have important implications for the design of poxviral-based immunotherapeutics for patients with metastatic cancers. Note: This abstract was not presented at the conference. Citation Format: Weiyi Wang, Peihong Dai, Ning Yang, Stewart Shuman, Taha Merghoub, Jedd D. Wolchok, Liang Deng. Intratumoral delivery of inactivated vaccinia virus is more efficacious than live oncolytic vaccinia virus in murine bilateral tumor implantation models [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A007.

Singapore Grouper Iridovirus ORF75R is a Scaffold Protein Essential for Viral Assembly.

Singapore Grouper Iridovirus (SGIV) is a member of nucleo cytoplasmic large DNA viruses (NCLDV). This paper reports the functional analysis of ORF75R, a major structural protein of SGIV. Immuno fluorescence studies showed that the protein was accumulated in the viral assembly site. Immunogold-labelling indicated that it was localized between the viral capsid shell and DNA core. Knockdown of ORF75R by morpholinos resulted in the reduction of coreshell thickness, the failure of DNA encapsidation, and the low yield of infectious particles. Comparative proteomics further identified the structural proteins affected by ORF75R knockdown. Two-dimensional gel electrophoresis combined with proteomics demonstrated that ORF75R was phosphorylated at multiple sites in SGIV-infected cell lysate and virions, but the vast majority of ORF75R in virions was the dephosphorylated isoform. A kinase assay showed that ORF75R could be phosphorylated in vitro by the SGIV structural protein ORF39L. Addition of ATP and Mg2+ into purified virions prompted extensive phosphorylation of structural proteins and release of ORF75R from virions. These data suggest that ORF75R is a novel scaffold protein important for viral assembly and DNA encapsidation, but its phosphorylation facilitates virion disassembly. Compared to proteins from other viruses, we found that ORF75R shares common features with herpes simplex virus VP22.