Programmed necrosis in the cross talk of cell death and inflammation.

Cell proliferation and cell death are integral elements in maintaining homeostatic balance in metazoans. Disease pathologies ensue when these processes are disturbed. A plethora of evidence indicates that malfunction of cell death can lead to inflammation, autoimmunity, or immunodeficiency. Programmed necrosis or necroptosis is a form of nonapoptotic cell death driven by the receptor interacting protein kinase 3 (RIPK3) and its substrate, mixed lineage kinase domain-like (MLKL). RIPK3 partners with its upstream adaptors RIPK1, TRIF, or DAI to signal for necroptosis in response to death receptor or Toll-like receptor stimulation, pathogen infection, or sterile cell injury. Necroptosis promotes inflammation through leakage of cellular contents from damaged plasma membranes. Intriguingly, many of the signal adaptors of necroptosis have dual functions in innate immune signaling. This unique signature illustrates the cooperative nature of necroptosis and innate inflammatory signaling pathways in managing cell and organismal stresses from pathogen infection and sterile tissue injury.

Structural Basis of Poxvirus Transcription: Transcribing and Capping Vaccinia Complexes.

Poxviruses use virus-encoded multisubunit RNA polymerases (vRNAPs) and RNA-processing factors to generate m7G-capped mRNAs in the host cytoplasm. In the accompanying paper, we report structures of core and complete vRNAP complexes of the prototypic Vaccinia poxvirus (Grimm et al., 2019; in this issue of Cell). Here, we present the cryo-electron microscopy (cryo-EM) structures of Vaccinia vRNAP in the form of a transcribing elongation complex and in the form of a co-transcriptional capping complex that contains the viral capping enzyme (CE). The trifunctional CE forms two mobile modules that bind the polymerase surface around the RNA exit tunnel. RNA extends from the vRNAP active site through this tunnel and into the active site of the CE triphosphatase. Structural comparisons suggest that growing RNA triggers large-scale rearrangements on the surface of the transcription machinery during the transition from transcription initiation to RNA capping and elongation. Our structures unravel the basis for synthesis and co-transcriptional modification of poxvirus RNA.

Structural Basis of Poxvirus Transcription: Vaccinia RNA Polymerase Complexes.

Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm. We report cryo-EM structures of core and complete vRNAP enzymes from Vaccinia virus at 2.8 Å resolution. The vRNAP core enzyme resembles eukaryotic RNA polymerase II (Pol II) but also reveals many virus-specific features, including the transcription factor Rap94. The complete enzyme additionally contains the transcription factor VETF, the mRNA processing factors VTF/CE and NPH-I, the viral core protein E11, and host tRNAGln. This complex can carry out the entire early transcription cycle. The structures show that Rap94 partially resembles the Pol II initiation factor TFIIB, that the vRNAP subunit Rpo30 resembles the Pol II elongation factor TFIIS, and that NPH-I resembles chromatin remodeling enzymes. Together with the accompanying paper (Hillen et al., 2019), these results provide the basis for unraveling the mechanisms of poxvirus transcription and RNA processing.

Circulating NK cells establish tissue residency upon acute infection of skin and mediate accelerated effector responses to secondary infection.

Natural killer (NK) cells are present in the circulation and can also be found residing in tissues, and these populations exhibit distinct developmental requirements and are thought to differ in terms of ontogeny. Here, we investigate whether circulating conventional NK (cNK) cells can develop into long-lived tissue-resident NK (trNK) cells following acute infections. We found that viral and bacterial infections of the skin triggered the recruitment of cNK cells and their differentiation into Tcf1hiCD69hi trNK cells that share transcriptional similarity with CD56brightTCF1hi NK cells in human tissues. Skin trNK cells arose from interferon (IFN)-γ-producing effector cells and required restricted expression of the transcriptional regulator Blimp1 to optimize Tcf1-dependent trNK cell formation. Upon secondary infection, trNK cells rapidly gained effector function and mediated an accelerated NK cell response. Thus, cNK cells redistribute and permanently position at sites of previous infection via a mechanism promoting tissue residency that is distinct from Hobit-dependent developmental paths of NK cells and ILC1 seeding tissues during ontogeny.

Human anti-smallpox long-lived memory B cells are defined by dynamic interactions in the splenic niche and long-lasting germinal center imprinting.

Memory B cells (MBCs) can persist for a lifetime, but the mechanisms that allow their long-term survival remain poorly understood. Here, we isolated and analyzed human splenic smallpox/vaccinia protein B5-specific MBCs in individuals who were vaccinated more than 40 years ago. Only a handful of clones persisted over such an extended period, and they displayed limited intra-clonal diversity with signs of extensive affinity-based selection. These long-lived MBCs appeared enriched in a CD21hiCD20hi IgG+ splenic B cell subset displaying a marginal-zone-like NOTCH/MYC-driven signature, but they did not harbor a unique longevity-associated transcriptional or metabolic profile. Finally, the telomeres of B5-specific, long-lived MBCs were longer than those in patient-paired naive B cells in all the samples analyzed. Overall, these results imply that separate mechanisms such as early telomere elongation, affinity selection during the contraction phase, and access to a specific niche contribute to ensuring the functional longevity of MBCs.

A class of viral inducer of degradation of the necroptosis adaptor RIPK3 regulates virus-induced inflammation.

The vaccine strain against smallpox, vaccinia virus (VACV), is highly immunogenic yet causes relatively benign disease. These attributes are believed to be caused by gene loss in VACV. Using a targeted small interfering RNA (siRNA) screen, we identified a viral inhibitor found in cowpox virus (CPXV) and other orthopoxviruses that bound to the host SKP1-Cullin1-F-box (SCF) machinery and the essential necroptosis kinase receptor interacting protein kinase 3 (RIPK3). This "viral inducer of RIPK3 degradation" (vIRD) triggered ubiquitination and proteasome-mediated degradation of RIPK3 and inhibited necroptosis. In contrast to orthopoxviruses, the distantly related leporipoxvirus myxoma virus (MYXV), which infects RIPK3-deficient hosts, lacks a functional vIRD. Introduction of vIRD into VACV, which encodes a truncated and defective vIRD, enhanced viral replication in mice. Deletion of vIRD reduced CPXV-induced inflammation, viral replication, and mortality, which were reversed in RIPK3- and MLKL-deficient mice. Hence, vIRD-RIPK3 drives pathogen-host evolution and regulates virus-induced inflammation and pathogenesis.

A modified vaccinia Ankara vector-based vaccine protects macaques from SARS-CoV-2 infection, immune pathology, and dysfunction in the lungs.

A combination of vaccination approaches will likely be necessary to fully control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Here, we show that modified vaccinia Ankara (MVA) vectors expressing membrane-anchored pre-fusion stabilized spike (MVA/S) but not secreted S1 induced strong neutralizing antibody responses against SARS-CoV-2 in mice. In macaques, the MVA/S vaccination induced strong neutralizing antibodies and CD8+ T cell responses, and conferred protection from SARS-CoV-2 infection and virus replication in the lungs as early as day 2 following intranasal and intratracheal challenge. Single-cell RNA sequencing analysis of lung cells on day 4 after infection revealed that MVA/S vaccination also protected macaques from infection-induced inflammation and B cell abnormalities and lowered induction of interferon-stimulated genes. These results demonstrate that MVA/S vaccination induces neutralizing antibodies and CD8+ T cells in the blood and lungs and is a potential vaccine candidate for SARS-CoV-2.

Cytoplasmic gene expression: lessons from poxviruses.

In eukaryotic cells, the process of gene expression is confined to the nucleus and enabled by multisubunit RNA polymerases (RNAPs). Many viruses make use of the host cellular gene expression apparatus during infection, and hence transfer their genome at least transiently to the host nucleus. However, poxviruses have evolved a different strategy to propagate. Their double-stranded DNA genome is transcribed in the host cytoplasm by a virus-encoded RNAP (vRNAP), which is evolutionarily related to eukaryotic RNA polymerase II. In this Review, we highlight recent high-resolution structures of the poxviral transcription apparatus in different phases of action. These structures, along with biochemical data, now allow the definition of a comprehensive model of poxviral gene expression and its regulation.

Human FAM111A inhibits vaccinia virus replication by degrading viral protein I3 and is antagonized by poxvirus host range factor SPI-1.

Zoonotic poxviruses such as mpox virus (MPXV) continue to threaten public health safety since the eradication of smallpox. Vaccinia virus (VACV), the prototypic poxvirus used as the vaccine strain for smallpox eradication, is the best-characterized member of the poxvirus family. VACV encodes a serine protease inhibitor 1 (SPI-1) conserved in all orthopoxviruses, which has been recognized as a host range factor for modified VACV Ankara (MVA), an approved smallpox vaccine and a promising vaccine vector. FAM111A (family with sequence similarity 111 member A), a nuclear protein that regulates host DNA replication, was shown to restrict the replication of a VACV SPI-1 deletion mutant (VACV-ΔSPI-1) in human cells. Nevertheless, the detailed antiviral mechanisms of FAM111A were unresolved. Here, we show that FAM111A is a potent restriction factor for VACV-ΔSPI-1 and MVA. Deletion of FAM111A rescued the replication of MVA and VACV-ΔSPI-1 and overexpression of FAM111A significantly reduced viral DNA replication and virus titers but did not affect viral early gene expression. The antiviral effect of FAM111A necessitated its trypsin-like protease domain and DNA-binding domain but not the PCNA-interacting motif. We further identified that FAM111A translocated into the cytoplasm upon VACV infection by degrading the nuclear pore complex via its protease activity, interacted with VACV DNA-binding protein I3, and promoted I3 degradation through autophagy. Moreover, SPI-1 from VACV, MPXV, or lumpy skin disease virus was able to antagonize FAM111A by prohibiting its nuclear export. Our findings reveal the detailed mechanism by which FAM111A inhibits VACV and provide explanations for the immune evasive function of VACV SPI-1.

Kinesin-1 transports morphologically distinct intracellular virions during vaccinia infection.

Intracellular mature viruses (IMVs) are the first and most abundant infectious form of vaccinia virus to assemble during its replication cycle. IMVs can undergo microtubule-based motility, but their directionality and the motor involved in their transport remain unknown. Here, we demonstrate that IMVs, like intracellular enveloped viruses (IEVs), the second form of vaccinia that are wrapped in Golgi-derived membranes, recruit kinesin-1 and undergo anterograde transport. In vitro reconstitution of virion transport in infected cell extracts revealed that IMVs and IEVs move toward microtubule plus ends with respective velocities of 0.66 and 0.56 µm/s. Quantitative imaging established that IMVs and IEVs recruit an average of 139 and 320 kinesin-1 motor complexes, respectively. In the absence of kinesin-1, there was a near-complete loss of in vitro motility and reduction in the intracellular spread of both types of virions. Our observations demonstrate that kinesin-1 transports two morphologically distinct forms of vaccinia. Reconstitution of vaccinia-based microtubule motility in vitro provides a new model to elucidate how motor number and regulation impacts transport of a bona fide kinesin-1 cargo.

Biochemical characterization of mRNA capping enzyme from Faustovirus.

The mammalian mRNA 5' cap structures play important roles in cellular processes such as nuclear export, efficient translation, and evading cellular innate immune surveillance and regulating 5'-mediated mRNA turnover. Hence, installation of the proper 5' cap is crucial in therapeutic applications of synthetic mRNA. The core 5' cap structure, Cap-0, is generated by three sequential enzymatic activities: RNA 5' triphosphatase, RNA guanylyltransferase, and cap N7-guanine methyltransferase. Vaccinia virus RNA capping enzyme (VCE) is a heterodimeric enzyme that has been widely used in synthetic mRNA research and manufacturing. The large subunit of VCE D1R exhibits a modular structure where each of the three structural domains possesses one of the three enzyme activities, whereas the small subunit D12L is required to activate the N7-guanine methyltransferase activity. Here, we report the characterization of a single-subunit RNA capping enzyme from an amoeba giant virus. Faustovirus RNA capping enzyme (FCE) exhibits a modular array of catalytic domains in common with VCE and is highly efficient in generating the Cap-0 structure without an activation subunit. Phylogenetic analysis suggests that FCE and VCE are descended from a common ancestral capping enzyme. We found that compared to VCE, FCE exhibits higher specific activity, higher activity toward RNA containing secondary structures and a free 5' end, and a broader temperature range, properties favorable for synthetic mRNA manufacturing workflows.

Trimerized S expressed by modified vaccinia virus Ankara (MVA) confers superior protection against lethal SARS-CoV-2 challenge in mice.

The reoccurrence of successive waves of SARS-CoV-2 variants suggests the exploration of more vaccine alternatives is imperative. Modified vaccinia virus Ankara (MVA) is a virus vector exhibiting excellent safety as well as efficacy for vaccine development. Here, a series of recombinant MVAs (rMVAs) expressing monomerized or trimerized S proteins from different SARS-CoV-2 variants are engineered. Trimerized S expressed from rMVAs is found predominantly as trimers on the surface of infected cells. Remarkably, immunization of mice with rMVAs demonstrates that S expressed in trimer elicits higher levels of binding IgG and IgA, as well as neutralizing antibodies for matched and mismatched S proteins than S in the monomer. In addition, trimerized S expressed by rMVA induces enhanced cytotoxic T-cell responses than S in the monomer. Importantly, the rMVA vaccines expressing trimerized S exhibit superior protection against a lethal SARS-CoV-2 challenge as the immunized animals all survive without displaying any pathological conditions. This study suggests that opting for trimerized S may represent a more effective approach and highlights that the MVA platform serves as an ideal foundation to continuously advance SARS-CoV-2 vaccine development. IMPORTANCE: MVA is a promising vaccine vector and has been approved as a vaccine for smallpox and mpox. Our analyses suggested that recombinant MVA expressing S in trimer (rMVA-ST) elicited robust cellular and humoral immunity and was more effective than MVA-S-monomer. Importantly, the rMVA-ST vaccine was able to stimulate decent cross-reactive neutralization against pseudoviruses packaged using S from different sublineages, including Wuhan, Delta, and Omicron. Remarkably, mice immunized with rMVA-ST were completely protected from a lethal challenge of SARS-CoV-2 without displaying any pathological conditions. Our results demonstrated that an MVA vectored vaccine expressing trimerized S is a promising vaccine candidate for SARS-CoV-2 and the strategy might be adapted for future vaccine development for coronaviruses.

Filamin B restricts vaccinia virus spread and is targeted by vaccinia virus protein C4.

Vaccinia virus (VACV) is a large DNA virus that encodes scores of proteins that modulate the host immune response. VACV protein C4 is one such immunomodulator known to inhibit the activation of both the NF-κB signaling cascade and the DNA-PK-mediated DNA sensing pathway. Here, we show that the N-terminal region of C4, which neither inhibits NF-κB nor mediates interaction with DNA-PK, still contributes to virus virulence. Furthermore, this domain interacts directly and with high affinity to the C-terminal domain of filamin B (FLNB). FLNB is a large actin-binding protein that stabilizes the F-actin network and is implicated in other cellular processes. Deletion of FLNB from cells results in larger VACV plaques and increased infectious viral yield, indicating that FLNB restricts VACV spread. These data demonstrate that C4 has a new function that contributes to virulence and engages the cytoskeleton. Furthermore, we show that the cytoskeleton performs further previously uncharacterized functions during VACV infection. IMPORTANCE: Vaccinia virus (VACV), the vaccine against smallpox and monkeypox, encodes many proteins to counteract the host immune response. Investigating these proteins provides insights into viral immune evasion mechanisms and thereby indicates how to engineer safer and more immunogenic VACV-based vaccines. Here, we report that the N-terminal domain of VACV protein C4 interacts directly with the cytoskeletal protein filamin B (FLNB), and this domain of C4 contributes to virus virulence. Furthermore, VACV replicates and spreads better in cells lacking FLNB, thus demonstrating that FLNB has antiviral activity. VACV utilizes the cytoskeleton for movement within and between cells; however, previous studies show no involvement of C4 in VACV replication or spread. Thus, C4 associates with FLNB for a different reason, suggesting that the cytoskeleton has further uncharacterized roles during virus infection.

A new MVA ancestor-derived oncolytic vaccinia virus induces immunogenic tumor cell death and robust antitumor immune responses.

Vaccinia viruses (VACVs) are versatile therapeutic agents and different features of various VACV strains allow for a broad range of therapeutic applications. Modified VACV Ankara (MVA) is a particularly altered VACV strain that is highly immunogenic, incapable of replicating in mammalian hosts, and broadly used as a safe vector for vaccination. Alternatively, Western Reserve (WR) or Copenhagen (Cop) are VACV strains that efficiently replicate in cancer cells and, therefore, are used to develop oncolytic viruses. However, the immune evasion capacity of WR or Cop hinders their ability to elicit antitumor immune responses, which is crucial for efficacy in the clinic. Here, we describe a new VACV strain named Immune-Oncolytic VACV Ankara (IOVA), which combines efficient replication in cancer cells with induction of immunogenic tumor cell death (ICD). IOVA was engineered from an MVA ancestor and shows superior cytotoxicity in tumor cells. In addition, the IOVA genome incorporates mutations that lead to massive fusogenesis of tumor cells, which contributes to improved antitumor effects. In syngeneic mouse tumor models, the induction of ICD results in robust antitumor immunity directed against tumor neo-epitopes and eradication of large established tumors. These data present IOVA as an improved immunotherapeutic oncolytic vector.

Intranasal inoculation of an MVA-based vaccine induces IgA and protects the respiratory tract of hACE2 mice from SARS-CoV-2 infection.

Current vaccines have greatly diminished the severity of the COVID-19 pandemic, even though they do not entirely prevent infection and transmission, likely due to insufficient immunity in the upper respiratory tract. Here, we compare intramuscular and intranasal administration of a live, replication-deficient modified vaccinia virus Ankara (MVA)-based Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike (S) vaccine to raise protective immune responses in the K18-hACE2 mouse model. Using a recombinant MVA expressing firefly luciferase for tracking, live imaging revealed luminescence of the respiratory tract of mice within 6 h and persisting for 3 d following intranasal inoculation, whereas luminescence remained at the site of intramuscular vaccination. Intramuscular vaccination induced S-binding-Immunoglobulin G (IgG) and neutralizing antibodies in the lungs, whereas intranasal vaccination also induced Immunoglobulin A (IgA) and higher levels of antigen-specific CD3+CD8+IFN-γ+ T cells. Similarly, IgG and neutralizing antibodies were present in the blood of mice immunized intranasally and intramuscularly, but IgA was detected only after intranasal inoculation. Intranasal boosting increased IgA after intranasal or intramuscular priming. While intramuscular vaccination prevented morbidity and cleared SARS-CoV-2 from the respiratory tract within several days after challenge, intranasal vaccination was more effective as neither infectious virus nor viral messenger (m)RNAs were detected in the nasal turbinates or lungs as early as 2 d after challenge, indicating prevention or rapid elimination of SARS-CoV-2 infection. Additionally, we determined that neutralizing antibody persisted for more than 6 mo and that serum induced to the Wuhan S protein neutralized pseudoviruses expressing the S proteins of variants, although with less potency, particularly for Beta and Omicron.

Inhibition of exchange proteins directly activated by cAMP as a strategy for broad-spectrum antiviral development.

The recent SARS-CoV-2 and mpox outbreaks have highlighted the need to expand our arsenal of broad-spectrum antiviral agents for future pandemic preparedness. Host-directed antivirals are an important tool to accomplish this as they typically offer protection against a broader range of viruses than direct-acting antivirals and have a lower susceptibility to viral mutations that cause drug resistance. In this study, we investigate the exchange protein activated by cAMP (EPAC) as a target for broad-spectrum antiviral therapy. We find that the EPAC-selective inhibitor, ESI-09, provides robust protection against a variety of viruses, including SARS-CoV-2 and Vaccinia (VACV)-an orthopox virus from the same family as mpox. We show, using a series of immunofluorescence experiments, that ESI-09 remodels the actin cytoskeleton through Rac1/Cdc42 GTPases and the Arp2/3 complex, impairing internalization of viruses that use clathrin-mediated endocytosis (e.g. VSV) or micropinocytosis (e.g. VACV). Additionally, we find that ESI-09 disrupts syncytia formation and inhibits cell-to-cell transmission of viruses such as measles and VACV. When administered to immune-deficient mice in an intranasal challenge model, ESI-09 protects mice from lethal doses of VACV and prevents formation of pox lesions. Altogether, our finding shows that EPAC antagonists such as ESI-09 are promising candidates for broad-spectrum antiviral therapy that can aid in the fight against ongoing and future viral outbreaks.

Residual Immunity from Smallpox Vaccination and Possible Protection from Mpox, China.

Among persons born in China before 1980 and tested for vaccinia virus Tiantan strain (VVT), 28.7% (137/478) had neutralizing antibodies, 71.4% (25/35) had memory B-cell responses, and 65.7% (23/35) had memory T-cell responses to VVT. Because of cross-immunity between the viruses, these findings can help guide mpox vaccination strategies in China.

Transient inhibition of neutrophil functions enhances the antitumor effect of intravenously delivered oncolytic vaccinia virus.

Oncolytic viruses (OVs) possess the unique ability to selectively replicate within tumor cells, leading to their destruction, while also reversing the immunosuppression within the tumor microenvironment and triggering an antitumor immune response. As a result, OVs have emerged as one of the most promising approaches in cancer therapy. However, the effective delivery of intravenously administered OVs faces significant challenges imposed by various immune cells within the peripheral blood, hindering their access to tumor sites. Notably, neutrophils, the predominant white blood cell population comprising approximately 50%-70% of circulating white cells in humans, show phagocytic properties. Our investigation revealed that the majority of oncolytic vaccinia viruses (VV) are engulfed and degraded by neutrophils in the bloodstream. The depletion of neutrophils using the anti-LY6G Ab (1-A8) resulted in an increased accumulation of circulating oncolytic VV in the peripheral blood and enhanced deposition at the tumor site, consequently amplifying the antitumor effect. Neutrophils heavily rely on PI3K signaling to sustain their phagocytic process. Additionally, our study determined that the inhibition of the PI3Kinase delta isoform by idelalisib (CAL-101) suppressed the uptake of oncolytic VV by neutrophils. This inhibition led to a greater presence of oncolytic VV in both the peripheral blood and at the tumor site, resulting in improved efficacy against the tumor. In conclusion, our study showed that inhibiting neutrophil functions can significantly enhance the antitumor efficacy of intravenous oncolytic VV.

Structural and functional analyses of viral H2 protein of the vaccinia virus entry fusion complex.

IMPORTANCE: Vaccinia virus infection requires virus-cell membrane fusion to complete entry during endocytosis; however, it contains a large viral fusion protein complex of 11 viral proteins that share no structure or sequence homology to all the known viral fusion proteins, including type I, II, and III fusion proteins. It is thus very challenging to investigate how the vaccinia fusion complex works to trigger membrane fusion with host cells. In this study, we crystallized the ectodomain of vaccinia H2 protein, one component of the viral fusion complex. Furthermore, we performed a series of mutational, biochemical, and molecular analyses and identified two surface loops containing 170LGYSG174 and 125RRGTGDAW132 as the A28-binding region. We also showed that residues in the N-terminal helical region (amino acids 51-90) are also important for H2 function.

Reverse Genetic Assessment of the Roles Played by the Spike Protein and ORF3 in Porcine Epidemic Diarrhea Virus Pathogenicity.

Porcine epidemic diarrhea virus is a swine pathogen that has been responsible for significant animal and economic losses worldwide in recent years. In this manuscript, we report the generation of a reverse genetics system C(RGS) for the highly virulent US PEDV strain Minnesota (PEDV-MN; GenBank accession number KF468752), which was based on the assembly and cloning of synthetic DNA, using vaccinia virus as a cloning vector. Viral rescue was only possible following the substitution of 2 nucleotides within the 5'UTR and 2 additional nucleotides within the spike gene, based on the sequence of the cell culture-adapted strains. Besides displaying a highly pathogenic phenotype in newborn piglets, in comparison with the parental virus, the rescued recombinant PEDV-MN was used to confirm that the PEDV spike gene has an important role in PEDV virulence and that the impact of an intact PEDV ORF3 on viral pathogenicity is modest. Moreover, a chimeric virus with a TGEV spike gene in the PEDV backbone generated with RGS was able to replicate efficiently in vivo and could be readily transmitted between piglets. Although this chimeric virus did not cause severe disease upon the initial infection of piglets, there was evidence of increasing pathogenicity upon transmission to contact piglets. The RGS described in this study constitutes a powerful tool with which to study PEDV pathogenesis and can be used to generate vaccines against porcine enteric coronaviruses. IMPORTANCE PEDV is a swine pathogen that is responsible for significant animal and economic losses worldwide. Highly pathogenic variants can lead to a mortality rate of up to 100% in newborn piglets. The generation of a reverse genetics system for a highly virulent PEDV strain originating from the United States is an important step in phenotypically characterizing PEDV. The synthetic PEDV mirrored the authentic isolate and displayed a highly pathogenic phenotype in newborn piglets. With this system, it was possible to characterize potential viral virulence factors. Our data revealed that an accessory gene (ORF3) has a limited impact on pathogenicity. However, as it is also now known for many coronaviruses, the PEDV spike gene is one of the main determinants of pathogenicity. Finally, we show that the spike gene of another porcine coronavirus, namely, TGEV, can be accommodated in the PEDV genome background, suggesting that similar viruses can emerge in the field via recombination.