Type I Interferon: Monkeypox/Mpox Viruses Achilles Heel?

Poxviruses are notorious for having acquired/evolved numerous genes to counteract host innate immunity. Chordopoxviruses have acquired/evolved at least three different inhibitors of host necroptotic death: E3, which blocks ZBP1-dependent necroptotic cell death, and vIRD and vMLKL that inhibit necroptosis downstream of initial cell death signaling. While this suggests the importance of the necroptotic cell death pathway in inhibiting chordopoxvirus replication, several chordopoxviruses have lost one or more of these inhibitory functions. Monkeypox/mpox virus (MPXV) has lost a portion of the N-terminus of its E3 homologue. The N-terminus of the vaccinia virus E3 homologue serves to inhibit activation of the interferon-inducible antiviral protein, ZBP1. This likely makes MPXV unique among the orthopoxviruses in being sensitive to interferon (IFN) treatment in many mammals, including humans, which encode a complete necroptotic cell death pathway. Thus, IFN sensitivity may be the Achille's Heel for viruses like MPXV that cannot fully inhibit IFN-inducible, ZBP1-dependent antiviral pathways.

Subversion of Programed Cell Death by Poxviruses.

Poxviruses have been long regarded as potent inhibitors of apoptotic cell death. More recently, they have been shown to inhibit necroptotic cell death through two distinct strategies. These strategies involve either blocking virus sensing by the host pattern recognition receptor, ZBP1 (also called DAI) or by influencing receptor interacting protein kinase (RIPK)3 signal transduction by inhibition of activation of the executioner of necroptosis, mixed lineage kinase-like protein (MLKL). Vaccinia virus E3 specifically blocks ZBP1 â†’ RIPK3 â†’ MLKL necroptosis, leaving virus-infected cells susceptible to the TNF death-receptor signaling (e.g., TNFR1 â†’ FADD â†’ RIPK1 â†’ RIPK3 â†’ MLKL), and, potentially, TLR3 â†’ TRIF â†’ RIPK3 â†’ MLKL necroptosis. While E3 restriction of necroptosis appears to be common to many poxviruses that infect vertebrate hosts, another modulatory strategy not observed in vaccinia or variola virus manifests through subversion of MLKL activation. Recently described viral mimics of MLKL in other chordopoxviruses inhibit all three modes of necroptotic cell death. As with inhibition of apoptosis, the evolution of potentially redundant viral mechanisms to inhibit programmed necroptotic cell death emphasizes the importance of this pathway in the arms race between pathogens and their hosts.

Optimization of translation enhancing element use to increase protein expression in a vaccinia virus system.

Since the successful use of vaccinia virus (VACV) in the immunization strategies to eliminate smallpox, research has been focused on the development of recombinant VACV strains expressing proteins from various pathogens. Attempts at decreasing the side effects associated with exposure to recombinant, wild-type viral strains have led to the development of attenuated viruses. Yet while these attenuated VACV's have improved safety profiles compared to unmodified strains, their clinical use has been hindered due to efficacy issues in stimulating a host immune response. This deficiency has largely been attributed to decreased production of the target protein for immunization. Efforts to increase protein production from attenuated VACV strains has largely centered around modulation of viral factors, while manipulation of the translation of viral mRNAs has been largely unexplored. In this study we evaluate the use of translation enhancing element hTEE-658 to increase recombinant protein production in an attenuated VACV system. Optimization of the use of this motif is also attempted by combining it with strategies that have demonstrated effectiveness in previous research. We show that extension of the 5' leader sequence containing hTEE-658 does not improve motif function, nor does the combination with other known translation enhancing elements. However, the sole use of hTEE-658 in an attenuated VACV system is shown to increase protein expression levels beyond those of a standard viral promoter when used with a wild-type virus. Taken together these results highlight the potential for hTEE-658 to improve the effectiveness of attenuated VACV vaccine candidates and give insights into the optimal sequence context for its use in vaccine design.

Replication-Competent NYVAC-KC Yields Improved Immunogenicity to HIV-1 Antigens in Rhesus Macaques Compared to Nonreplicating NYVAC.

As part of the continuing effort to develop an effective HIV vaccine, we generated a poxviral vaccine vector (previously described) designed to improve on the results of the RV144 phase III clinical trial. The construct, NYVAC-KC, is a replication-competent, attenuated recombinant of the vaccinia virus strain NYVAC. NYVAC is a vector that has been used in many previous clinical studies but is replication deficient. Here, we report a side-by-side comparison of replication-restricted NYVAC and replication-competent NYVAC-KC in a nonhuman primate study, which utilized a prime-boost regimen similar to that of RV144. NYVAC-C and NYVAC-C-KC express the HIV-1 antigens gp140, and Gag/Gag-Pol-Nef-derived virus-like particles (VLPs) from clade C and were used as the prime, with recombinant virus plus envelope protein used as the boost. In nearly every T and B cell immune assay against HIV-1, including neutralization and antibody binding, NYVAC-C-KC induced a greater immune response than NYVAC-C, indicating that replication competence in a poxvirus may improve upon the modestly successful regimen used in the RV144 clinical trial.IMPORTANCE Though the RV144 phase III clinical trial showed promise that an effective vaccine against HIV-1 is possible, a successful vaccine will require improvement over the vaccine candidate (ALVAC) used in the RV144 study. With that goal in mind, we have tested in nonhuman primates an attenuated but replication-competent vector, NYVAC-KC, in direct comparison to its parental vector, NYVAC, which is replication restricted in human cells, similar to the ALVAC vector used in RV144. We have utilized a prime-boost regimen for administration of the vaccine candidate that is similar to the one used in the RV144 study. The results of this study indicate that a replication-competent poxvirus vector may improve upon the effectiveness of the RV144 clinical trial vaccine candidate.

Priming with a Potent HIV-1 DNA Vaccine Frames the Quality of Immune Responses prior to a Poxvirus and Protein Boost.

The use of heterologous immunization regimens and improved vector systems has led to increases in immunogenicity of HIV-1 vaccine candidates in nonhuman primates. In order to resolve interrelations between different delivery modalities, three different poxvirus boost regimens were compared. Three groups of rhesus macaques were each primed with the same DNA vaccine encoding Gag, Pol, Nef, and gp140. The groups were then boosted with either the vaccinia virus strain NYVAC or a variant with improved replication competence in human cells, termed NYVAC-KC. The latter was administered either by scarification or intramuscularly. Finally, macaques were boosted with adjuvanted gp120 protein to enhance humoral responses. The regimen elicited very potent CD4+ and CD8+ T cell responses in a well-balanced manner, peaking 2 weeks after the boost. T cells were broadly reactive and polyfunctional. All animals exhibited antigen-specific humoral responses already after the poxvirus boost, which further increased following protein administration. Polyclonal reactivity of IgG antibodies was highest against HIV-1 clade C Env proteins, with considerable cross-reactivity to other clades. Substantial effector functional activities (antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated virus inhibition) were observed in serum obtained after the last protein boost. Notably, major differences between the groups were absent, indicating that the potent priming induced by the DNA vaccine initially framed the immune responses in such a way that the subsequent boosts with NYVAC and protein led only to an increase in the response magnitudes without skewing the quality. This study highlights the importance of selecting the best combination of vector systems in heterologous prime-boost vaccination regimens.IMPORTANCE The evaluation of HIV vaccine efficacy trials indicates that protection would most likely correlate with a polyfunctional immune response involving several effector functions from all arms of the immune system. Heterologous prime-boost regimens have been shown to elicit vigorous T cell and antibody responses in nonhuman primates that, however, qualitatively and quantitatively differ depending on the respective vector systems used. The present study evaluated a DNA prime and poxvirus and protein boost regimen and compared how two poxvirus vectors with various degrees of replication capacity and two different delivery modalities-conventional intramuscular delivery and percutaneous delivery by scarification-impact several immune effectors. It was found that despite the different poxvirus boosts, the overall immune responses in the three groups were similar, suggesting the potent DNA priming as the major determining factor of immune responses. These findings emphasize the importance of selecting optimal priming agents in heterologous prime-boost vaccination settings.

Inhibition of DAI-dependent necroptosis by the Z-DNA binding domain of the vaccinia virus innate immune evasion protein, E3.

Vaccinia virus (VACV) encodes an innate immune evasion protein, E3, which contains an N-terminal Z-nucleic acid binding (Zα) domain that is critical for pathogenicity in mice. Here we demonstrate that the N terminus of E3 is necessary to inhibit an IFN-primed virus-induced necroptosis. VACV deleted of the Zα domain of E3 (VACV-E3LΔ83N) induced rapid RIPK3-dependent cell death in IFN-treated L929 cells. Cell death was inhibited by the RIPK3 inhibitor, GSK872, and infection with this mutant virus led to phosphorylation and aggregation of MLKL, the executioner of necroptosis. In 293T cells, induction of necroptosis depended on expression of RIPK3 as well as the host-encoded Zα domain-containing DNA sensor, DAI. VACV-E3LΔ83N is attenuated in vivo, and pathogenicity was restored in either RIPK3- or DAI-deficient mice. These data demonstrate that the N terminus of the VACV E3 protein prevents DAI-mediated induction of necroptosis.

HIV/AIDS Vaccine Candidates Based on Replication-Competent Recombinant Poxvirus NYVAC-C-KC Expressing Trimeric gp140 and Gag-Derived Virus-Like Particles or Lacking the Viral Molecule B19 That Inhibits Type I Interferon Activate Relevant HIV-1-Specific B and T Cell Immune Functions in Nonhuman Primates.

The nonreplicating attenuated poxvirus vector NYVAC expressing clade C(CN54) HIV-1 Env(gp120) and Gag-Pol-Nef antigens (NYVAC-C) showed limited immunogenicity in phase I clinical trials. To enhance the capacity of the NYVAC vector to trigger broad humoral responses and a more balanced activation of CD4+ and CD8+ T cells, here we compared the HIV-1-specific immunogenicity elicited in nonhuman primates immunized with two replicating NYVAC vectors that have been modified by the insertion of the K1L and C7L vaccinia virus host range genes and express the clade C(ZM96) trimeric HIV-1 gp140 protein or a Gag(ZM96)-Pol-Nef(CN54) polyprotein as Gag-derived virus-like particles (termed NYVAC-C-KC). Additionally, one NYVAC-C-KC vector was generated by deleting the viral gene B19R, an inhibitor of the type I interferon response (NYVAC-C-KC-ΔB19R). An immunization protocol mimicking that of the RV144 phase III clinical trial was used. Two groups of macaques received two doses of the corresponding NYVAC-C-KC vectors (weeks 0 and 4) and booster doses with NYVAC-C-KC vectors plus the clade C HIV-1 gp120 protein (weeks 12 and 24). The two replicating NYVAC-C-KC vectors induced enhanced and similar HIV-1-specific CD4+ and CD8+ T cell responses, similar levels of binding IgG antibodies, low levels of IgA antibodies, and high levels of antibody-dependent cellular cytotoxicity responses and HIV-1-neutralizing antibodies. Small differences within the NYVAC-C-KC-ΔB19R group were seen in the magnitude of CD4+ and CD8+ T cells, the induction of some cytokines, and the neutralization of some HIV-1 isolates. Thus, replication-competent NYVAC-C-KC vectors acquired relevant immunological properties as vaccine candidates against HIV/AIDS, and the viral B19 molecule exerts some control of immune functions.IMPORTANCE It is of special importance to find a safe and effective HIV/AIDS vaccine that can induce strong and broad T cell and humoral immune responses correlating with HIV-1 protection. Here we developed novel replicating poxvirus NYVAC-based HIV/AIDS vaccine candidates expressing clade C HIV-1 antigens, with one of them lacking the vaccinia virus B19 protein, an inhibitor of the type I interferon response. Immunization of nonhuman primates with these novel NYVAC-C-KC vectors and the protein component gp120 elicited high levels of T cell and humoral immune responses, with the vector containing a deletion in B19R inducing a trend toward a higher magnitude of CD4+ and CD8+ T cell responses and neutralization of some HIV-1 strains. These poxvirus vectors could be considered HIV/AIDS vaccine candidates based on their activation of potential immune correlates of protection.

Potential To Streamline Heterologous DNA Prime and NYVAC/Protein Boost HIV Vaccine Regimens in Rhesus Macaques by Employing Improved Antigens.

UNLABELLED: In a follow-up to the modest efficacy observed in the RV144 trial, researchers in the HIV vaccine field seek to substantiate and extend the results by evaluating other poxvirus vectors and combinations with DNA and protein vaccines. Earlier clinical trials (EuroVacc trials 01 to 03) evaluated the immunogenicity of HIV-1 clade C GagPolNef and gp120 antigens delivered via the poxviral vector NYVAC. These showed that a vaccination regimen including DNA-C priming prior to a NYVAC-C boost considerably enhanced vaccine-elicited immune responses compared to those with NYVAC-C alone. Moreover, responses were improved by using three as opposed to two DNA-C primes. In the present study, we assessed in nonhuman primates whether such vaccination regimens can be streamlined further by using fewer and accelerated immunizations and employing a novel generation of improved DNA-C and NYVAC-C vaccine candidates designed for higher expression levels and more balanced immune responses. Three different DNA-C prime/NYVAC-C+ protein boost vaccination regimens were tested in rhesus macaques. All regimens elicited vigorous and well-balanced CD8(+)and CD4(+)T cell responses that were broad and polyfunctional. Very high IgG binding titers, substantial antibody-dependent cellular cytotoxicity (ADCC), and modest antibody-dependent cell-mediated virus inhibition (ADCVI), but very low neutralization activity, were measured after the final immunizations. Overall, immune responses elicited in all three groups were very similar and of greater magnitude, breadth, and quality than those of earlier EuroVacc vaccines. In conclusion, these findings indicate that vaccination schemes can be simplified by using improved antigens and regimens. This may offer a more practical and affordable means to elicit potentially protective immune responses upon vaccination, especially in resource-constrained settings. IMPORTANCE: Within the EuroVacc clinical trials, we previously assessed the immunogenicity of HIV clade C antigens delivered in a DNA prime/NYVAC boost regimen. The trials showed that the DNA prime crucially improved the responses, and three DNA primes with a NYVAC boost appeared to be optimal. Nevertheless, T cell responses were primarily directed toward Env, and humoral responses were modest. The aim of this study was to assess improved antigens for the capacity to elicit more potent and balanced responses in rhesus macaques, even with various simpler immunization regimens. Our results showed that the novel antigens in fact elicited larger numbers of T cells with a polyfunctional profile and a good Env-GagPolNef balance, as well as high-titer and Fc-functional antibody responses. Finally, comparison of the different schedules indicates that a simpler regimen of only two DNA primes and one NYVAC boost in combination with protein may be very efficient, thus showing that the novel antigens allow for easier immunization protocols.

Genome-wide profiling of human cap-independent translation-enhancing elements.

We report an in vitro selection strategy to identify RNA sequences that mediate cap-independent initiation of translation. This method entails mRNA display of trillions of genomic fragments, selection for initiation of translation and high-throughput deep sequencing. We identified >12,000 translation-enhancing elements (TEEs) in the human genome, generated a high-resolution map of human TEE-bearing regions (TBRs), and validated the function of a subset of sequences in vitro and in cultured cells.

Evasion of the Innate Immune Type I Interferon System by Monkeypox Virus.

UNLABELLED: The vaccinia virus (VACV) E3 protein has been shown to be important for blocking activation of the cellular innate immune system and allowing viral replication to occur unhindered. Mutation or deletion of E3L severely affects viral host range and pathogenesis. While the monkeypox virus (MPXV) genome encodes a homologue of the VACV E3 protein, encoded by the F3L gene, the MPXV gene is predicted to encode a protein with a truncation of 37 N-terminal amino acids. VACV with a genome encoding a similarly truncated E3L protein (VACV-E3LΔ37N) has been shown to be attenuated in mouse models, and infection with VACV-E3LΔ37N has been shown to lead to activation of the host antiviral protein kinase R pathway. In this report, we present data demonstrating that, despite containing a truncated E3 homologue, MPXV phenotypically resembles a wild-type (wt) VACV rather than VACV-E3LΔ37N. Thus, MPXV appears to contain a gene or genes that can suppress the phenotypes associated with an N-terminal truncation in E3. The suppression maps to sequences outside F3L, suggesting that the suppression is extragenic in nature. Thus, MPXV appears to have evolved mechanisms to minimize the effects of partial inactivation of its E3 homologue. IMPORTANCE: Poxviruses have evolved to have many mechanisms to evade host antiviral innate immunity; these mechanisms may allow these viruses to cause disease. Within the family of poxviruses, variola virus (which causes smallpox) is the most pathogenic, while monkeypox virus is intermediate in pathogenicity between vaccinia virus and variola virus. Understanding the mechanisms of monkeypox virus innate immune evasion will help us to understand the evolution of poxvirus innate immune evasion capabilities, providing a better understanding of how poxviruses cause disease.

Virological and immunological characterization of novel NYVAC-based HIV/AIDS vaccine candidates expressing clade C trimeric soluble gp140(ZM96) and Gag(ZM96)-Pol-Nef(CN54) as virus-like particles.

UNLABELLED: The generation of vaccines against HIV/AIDS able to induce long-lasting protective immunity remains a major goal in the HIV field. The modest efficacy (31.2%) against HIV infection observed in the RV144 phase III clinical trial highlighted the need for further improvement of HIV vaccine candidates, formulation, and vaccine regimen. In this study, we have generated two novel NYVAC vectors, expressing HIV-1 clade C gp140(ZM96) (NYVAC-gp140) or Gag(ZM96)-Pol-Nef(CN54) (NYVAC-Gag-Pol-Nef), and defined their virological and immunological characteristics in cultured cells and in mice. The insertion of HIV genes does not affect the replication capacity of NYVAC recombinants in primary chicken embryo fibroblast cells, HIV sequences remain stable after multiple passages, and HIV antigens are correctly expressed and released from cells, with Env as a trimer (NYVAC-gp140), while in NYVAC-Gag-Pol-Nef-infected cells Gag-induced virus-like particles (VLPs) are abundant. Electron microscopy revealed that VLPs accumulated with time at the cell surface, with no interference with NYVAC morphogenesis. Both vectors trigger specific innate responses in human cells and show an attenuation profile in immunocompromised adult BALB/c and newborn CD1 mice after intracranial inoculation. Analysis of the immune responses elicited in mice after homologous NYVAC prime/NYVAC boost immunization shows that recombinant viruses induced polyfunctional Env-specific CD4 or Gag-specific CD8 T cell responses. Antibody responses against gp140 and p17/p24 were elicited. Our findings showed important insights into virus-host cell interactions of NYVAC vectors expressing HIV antigens, with the activation of specific immune parameters which will help to unravel potential correlates of protection against HIV in human clinical trials with these vectors. IMPORTANCE: We have generated two novel NYVAC-based HIV vaccine candidates expressing HIV-1 clade C trimeric soluble gp140 (ZM96) and Gag(ZM96)-Pol-Nef(CN54) as VLPs. These vectors are stable and express high levels of both HIV-1 antigens. Gag-induced VLPs do not interfere with NYVAC morphogenesis, are highly attenuated in immunocompromised and newborn mice after intracranial inoculation, trigger specific innate immune responses in human cells, and activate T (Env-specific CD4 and Gag-specific CD8) and B cell immune responses to the HIV antigens, leading to high antibody titers against gp140. For these reasons, these vectors can be considered vaccine candidates against HIV/AIDS and currently are being tested in macaques and humans.

Synthetic long peptide booster immunization in rhesus macaques primed with replication-competent NYVAC-C-KC induces a balanced CD4/CD8 T-cell and antibody response against the conserved regions of HIV-1.

The Thai trial (RV144) indicates that a prime-boost vaccine combination that induces both T-cell and antibody responses may be desirable for an effective HIV vaccine. We have previously shown that immunization with synthetic long peptides (SLP), covering the conserved parts of SIV, induced strong CD4 T-cell and antibody responses, but only modest CD8 T-cell responses. To generate a more balanced CD4/CD8 T-cell and antibody response, this study evaluated a pox-vector prime/SLP boost strategy in rhesus macaques. Priming with a replication-competent NYVAC, encoding HIV-1 clade C gag, pol and nef, induced modest IFNγ T-cell immune responses, predominantly directed against HIV-1 Gag. Booster immunization with SLP, covering the conserved parts of HIV-1 Gag, Pol and Env, resulted in a more than 10-fold increase in IFNγ ELISpot responses in four of six animals, which were predominantly HIV-1 Pol-specific. The animals showed a balanced polyfunctional CD4 and CD8 T-cell response and high Ab titres.

In vitro evaluation of antiviral activity in carnivorous plant species.

Sarracenia purpurea is a carnivorous plant historically used to treat smallpox infections. Our previous data found S. purpurea had broad spectrum antiviral activity in vitro. S. purpurea is one of several hundred identified carnivorous species of plants. Carnivorous plants have evolved through convergent evolution in at least ten independent events, usually in response to harsh environments where nutrition from prey is required for growth. These prey are known vectors of plant viruses that might introduce novel biotic stressors and defense pathways in carnivorous plants. This study evaluated the antiviral activity of several non-carnivorous and carnivorous plants from four evolutionarily distinct clades. Results demonstrated that carnivorous plants have evolved antiviral activity, a trait that is not present in related species of non-carnivorous plants. The antiviral trait may be due to the plant-prey relationship with insect vectors and an evolutionary need for carnivorous plants to have more robust antiviral defense systems.

The amino terminus of the vaccinia virus E3 protein is necessary to inhibit the interferon response.

Vaccinia virus (VACV) encodes a multifunctional protein, E3L, that is necessary for interferon (IFN) resistance in cells in culture. Interferon resistance has been mapped to the well-characterized carboxy terminus of E3L, which contains a conserved double-stranded RNA binding domain. The amino terminus of E3L has a Z-form nucleic acid binding domain, which has been shown to be dispensable for replication and IFN resistance in HeLa and RK13 cells; however, a virus expressing E3L deleted of the amino terminus has reduced pathogenicity in an animal model. In this study, we demonstrate that the pathogenicity of a virus expressing E3L deleted of the amino terminus was fully rescued in type I IFN receptor knockout (IFN-α/ßR(-/-)) mice. Furthermore, this virus was IFN sensitive in primary mouse embryo fibroblasts (MEFs). This virus induced the phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) in MEFs in an IFN-dependent manner. The depletion of double-stranded RNA-dependent protein kinase (PKR) from these MEFs restored the IFN resistance of this virus. Furthermore, the virus expressing E3L deleted of the amino terminus was also IFN resistant in PKR(-/-) MEFs. Thus, our data demonstrate that the amino terminus of E3L is necessary to inhibit the type I IFN response both in mice and in MEFs and that in MEFs, the amino terminus of E3L functions to inhibit the PKR pathway.

Canonical cellular stress granules are required for arsenite-induced necroptosis mediated by Z-DNA-binding protein 1.

Cellular stress granules arise in cells subjected to stress and promote cell survival. A cellular protein that localizes to stress granules is Z-DNA-binding protein 1 (ZBP1), which plays a major role in necroptosis, a programmed cell death pathway mediated by the kinase RIPK3. Here, we showed that the stress granule inducer arsenite activated RIPK3-dependent necroptosis. This pathway required ZBP1, which localized to arsenite-induced stress granules. RIPK3 localized to stress granules in the presence of ZBP1, leading to the formation of ZBP1-RIPK3 necrosomes, phosphorylation of the RIPK3 effector MLKL, and execution of necroptosis. Cells that did not form stress granules did not induce necroptosis in response to arsenite. Together, these results show that arsenite induces ZBP1-mediated necroptosis in a manner dependent on stress granule formation.

Innate immune evasion mediated by the Ambystoma tigrinum virus eukaryotic translation initiation factor 2alpha homologue.

Ranaviruses (family Iridoviridae, genus Ranavirus) are large, double-stranded DNA (dsDNA) viruses whose replication is restricted to ectothermic vertebrates. Many highly pathogenic members of the genus Ranavirus encode a homologue of the eukaryotic translation initiation factor 2α (eIF2α). Data in a heterologous vaccinia virus system suggest that the Ambystoma tigrinum virus (ATV) eIF2α homologue (vIF2αH; open reading frame [ORF] 57R) is involved in evading the host innate immune response by degrading the interferon-inducible, dsRNA-activated protein kinase, PKR. To test this hypothesis directly, the ATV vIF2αH gene (ORF 57R) was deleted by homologous recombination, and a selectable marker was inserted in its place. The ATVΔ57R virus has a small plaque phenotype and is 8-fold more sensitive to interferon than wild-type ATV (wtATV). Infection of fish cells with the ATVΔ57R virus leads to eIF2α phosphorylation, in contrast to infection with wtATV, which actively inhibits eIF2α phosphorylation. The inability of ATVΔ57R to prevent phosphorylation of eIF2α correlates with degradation of fish PKZ, an interferon-inducible enzyme that is closely related to mammalian PKR. In addition, salamanders infected with ATVΔ57R displayed an increased time to death compared to that of wtATV-infected salamanders. Therefore, in a biologically relevant system, the ATV vIF2αH gene acts as an innate immune evasion factor, thereby enhancing virus pathogenesis.

Small Hero with Great Powers: Vaccinia Virus E3 Protein and Evasion of the Type I IFN Response.

Poxviridae have developed a plethora of strategies to evade innate and adaptive immunity. In this review, we focused on the vaccinia virus E3 protein, encoded by the E3L gene. E3 is present within the Chordopoxvirinae subfamily (with the exception of the avipoxviruses and molluscum contagiosum virus) and displays pleiotropic effects on the innate immune system. Initial studies identified E3 as a double-stranded RNA (dsRNA)-binding protein (through its C terminus), able to inhibit the activation of protein kinase dependent on RNA (PKR) and the 2'5'-oligoadenylate synthetase (OAS)/RNase L pathway, rendering E3 a protein counteracting the type I interferon (IFN) system. In recent years, N-terminal mutants of E3 unable to bind to Z-form nucleic acids have been shown to induce the cellular death pathway necroptosis. This pathway was dependent on host IFN-inducible Z-DNA-binding protein 1 (ZBP1); full-length E3 is able to inhibit ZBP1-mediated necroptosis. Binding to what was identified as Z-RNA has emerged as a novel mechanism of counteracting the type I IFN system and has broadened our understanding of innate immunity against viral infections. This article gives an overview of the studies leading to our understanding of the vaccinia virus E3 protein function and its involvement in viral pathogenesis. Furthermore, a short summary of other viral systems is provided.

Intranasal Immunization with a Vaccinia Virus Vaccine Vector Expressing Pre-Fusion Stabilized SARS-CoV-2 Spike Fully Protected Mice against Lethal Challenge with the Heavily Mutated Mouse-Adapted SARS2-N501YMA30 Strain of SARS-CoV-2.

The Omicron SARS-CoV-2 variant has been designated as a variant of concern because its spike protein is heavily mutated. In particular, the Omicron spike is mutated at five positions (K417, N440, E484, Q493, and N501) that have been associated with escape from neutralizing antibodies induced by either infection with or immunization against the early Washington strain of SARS-CoV-2. The mouse-adapted strain of SARS-CoV-2, SARS2-N501YMA30, contains a spike that is also heavily mutated, with mutations at four of the five positions in the Omicron spike associated with neutralizing antibody escape (K417, E484, Q493, and N501). In this manuscript, we show that intranasal immunization with a pre-fusion stabilized Washington strain spike, expressed from a highly attenuated, replication-competent vaccinia virus construct, NYVAC-KC, fully protected mice against symptoms and death from SARS2-N501YMA30. Similarly, immunization by scarification on the skin fully protected against death, but not from mild disease. This data demonstrates that the Washington strain spike, when expressed from a highly attenuated, replication-competent poxvirus-administered without parenteral injection-can fully protect against the heavily mutated mouse-adapted SARS2-N501YMA30.

Evidence for multiple recent host species shifts among the Ranaviruses (family Iridoviridae).

Members of the genus Ranavirus (family Iridoviridae) have been recognized as major viral pathogens of cold-blooded vertebrates. Ranaviruses have been associated with amphibians, fish, and reptiles. At this time, the relationships between ranavirus species are still unclear. Previous studies suggested that ranaviruses from salamanders are more closely related to ranaviruses from fish than they are to ranaviruses from other amphibians, such as frogs. Therefore, to gain a better understanding of the relationships among ranavirus isolates, the genome of epizootic hematopoietic necrosis virus (EHNV), an Australian fish pathogen, was sequenced. Our findings suggest that the ancestral ranavirus was a fish virus and that several recent host shifts have taken place, with subsequent speciation of viruses in their new hosts. The data suggesting several recent host shifts among ranavirus species increase concern that these pathogens of cold-blooded vertebrates may have the capacity to cross numerous poikilothermic species barriers and the potential to cause devastating disease in their new hosts.