Orthopoxvirus K3 orthologs show virus- and host-specific inhibition of the antiviral protein kinase PKR.

The antiviral protein kinase R (PKR) is an important host restriction factor, which poxviruses must overcome to productively infect host cells. To inhibit PKR, many poxviruses encode a pseudosubstrate mimic of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2), designated K3 in vaccinia virus. Although the interaction between PKR and eIF2α is highly conserved, some K3 orthologs from host-restricted poxviruses were previously shown to inhibit PKR in a species-specific manner. To better define this host range function, we compared the sensitivity of PKR from 17 mammals to inhibition by K3 orthologs from closely related orthopoxviruses, a genus with a generally broader host range. The K3 orthologs showed species-specific inhibition of PKR and exhibited three distinct inhibition profiles. In some cases, PKR from closely related species showed dramatic differences in their sensitivity to K3 orthologs. Vaccinia virus expressing the camelpox virus K3 ortholog replicated more than three orders of magnitude better in human and sheep cells than a virus expressing vaccinia virus K3, but both viruses replicated comparably well in cow cells. Strikingly, in site-directed mutagenesis experiments between the variola virus and camelpox virus K3 orthologs, we found that different amino acid combinations were necessary to mediate improved or diminished inhibition of PKR derived from different host species. Because there is likely a limited number of possible variations in PKR that affect K3-interactions but still maintain PKR/eIF2α interactions, it is possible that by chance PKR from some potential new hosts may be susceptible to K3-mediated inhibition from a virus it has never previously encountered. We conclude that neither the sensitivity of host proteins to virus inhibition nor the effectiveness of viral immune antagonists can be inferred from their phylogenetic relatedness but must be experimentally determined.

(+)-Camphor and (-)-borneol derivatives as potential anti-orthopoxvirus agents.

Although the World Health Organisation had announced that smallpox was eradicated over 40 years ago, the disease and other related pathogenic poxviruses such as monkeypox remain potential bioterrorist weapons and could also re-emerge as natural infections. We have previously reported (+)-camphor and (-)-borneol derivatives with an antiviral activity against the vaccinia virus. This virus is similar to the variola virus (VARV), the causative agent of smallpox, but can be studied at BSL-2 facilities. In the present study, we evaluated the antiviral activity of the most potent compounds against VARV, cowpox virus, and ectromelia virus (ECTV). Among the compounds tested, 4-bromo-N'-((1R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ylidene)benzohydrazide 18 is the most effective compound against various orthopoxviruses, including VARV, with an EC50 value of 13.9 µM and a selectivity index of 206. Also, (+)-camphor thiosemicarbazone 9 was found to be active against VARV and ECTV.

Triad of human cellular proteins, IRF2, FAM111A, and RFC3, restrict replication of orthopoxvirus SPI-1 host-range mutants.

Viruses and their hosts can reach balanced states of evolution ensuring mutual survival, which makes it difficult to appreciate the underlying dynamics. To uncover hidden interactions, virus mutants that have lost defense genes may be used. Deletion of the gene that encodes serine protease inhibitor 1 (SPI-1) of rabbitpox virus and vaccinia virus, two closely related orthopoxviruses, prevents their efficient replication in human cells, whereas certain other mammalian cells remain fully permissive. Our high-throughput genome-wide siRNA screen identified host factors that prevent reproduction and spread of the mutant viruses in human cells. More than 20,000 genes were interrogated with individual siRNAs and those that prominently increased replication of the SPI-1 deletion mutant were subjected to a secondary screen. The top hits based on the combined data-replication factor C3 (RFC3), FAM111A, and interferon regulatory factor 2 (IRF2)-were confirmed by custom assays. The siRNAs to RFC1, RFC2, RFC4, and RFC5 mRNAs also enhanced spread of the mutant virus, strengthening the biological significance of the RFC complex as a host restriction factor for poxviruses. Whereas association with proliferating cell nuclear antigen and participation in processive genome replication are common features of FAM111A and RFC, IRF2 is a transcriptional regulator. Microarray analysis, quantitative RT-PCR, and immunoblotting revealed that IRF2 regulated the basal level expression of FAM111A, suggesting that the enhancing effect of depleting IRF2 on replication of the SPI-1 mutant was indirect. Thus, the viral SPI-1 protein and the host IRF2, FAM111A, and RFC complex likely form an interaction network that influences the ability of poxviruses to replicate in human cells.

SUCCESSFUL TREATMENT OF CLINICAL ORTHOPOXVIRUS INFECTION IN A GIANT ANTEATER (MYRMECOPHAGA TRIDACTYLA).

An anorexic 5-yr-old female giant anteater (Myrmecophaga tridactyla) developed multifocal ulcerative and vesicular lesions affecting the rostrum, oral cavity, and tongue. Disseminated skin lesions were also found on the body, affecting the feet, flanks, and genital area. Polymerase chain reaction confirmed a systemic viremic orthopoxvirus infection. Cowpox virus was considered to be the only likely etiological agent. Intensive supportive treatment, including daily fluid therapy, force-feeding, and anti-inflammatory administration achieved a successful outcome after 3 wk. To the authors' knowledge, this is the first time a giant anteater with severe orthopoxvirus lesions has survived the disease. This unique case discusses current and possible future therapeutic and prophylactic options for the treatment of orthopoxvirus infections in giant anteaters and other nondomestic animal species.

Novel oncolytic chimeric orthopoxvirus causes regression of pancreatic cancer xenografts and exhibits abscopal effect at a single low dose.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has been increasing by 0.5% per year in the United States. PDAC portends a dismal prognosis and novel therapies are needed. This study describes the generation and characterization of a novel oncolytic chimeric orthopoxvirus for the treatment of pancreatic cancer. METHODS: After chimerization and high-throughput screening, CF33 was chosen from 100 new chimeric orthopoxvirus isolates for its ability to kill pancreatic cancer cells. In vitro cytotoxicity was assayed in six pancreatic cancer cell lines. In vivo efficacy and toxicity were evaluated in PANC-1 and MIA PaCa-2 xenograft models. RESULTS: CF33 caused rapid killing of six pancreatic cancer cells lines in vitro, releasing damage-associated molecular patterns, and regression of PANC-1 injected and non-injected distant xenografts in vivo after a single low intratumoral dose of 103 plaque-forming units. Using luciferase imaging, CF33 was noted to preferentially replicate in tumors which corresponds to the low viral titers found in solid organs. CONCLUSION: The low dose of CF33 required to treat pancreatic cancer in this preclinical study may ease the manufacturing and dosing challenges currently facing oncolytic viral therapy.

Structural and Functional Characterization of Anti-A33 Antibodies Reveal a Potent Cross-Species Orthopoxviruses Neutralizer.

Vaccinia virus A33 is an extracellular enveloped virus (EEV)-specific type II membrane glycoprotein that is essential for efficient EEV formation and long-range viral spread within the host. A33 is a target for neutralizing antibody responses against EEV. In this study, we produced seven murine anti-A33 monoclonal antibodies (MAbs) by immunizing mice with live VACV, followed by boosting with the soluble A33 homodimeric ectodomain. Five A33 specific MAbs were capable of neutralizing EEV in the presence of complement. All MAbs bind to conformational epitopes on A33 but not to linear peptides. To identify the epitopes, we have adetermined the crystal structures of three representative neutralizing MAbs in complex with A33. We have further determined the binding kinetics for each of the three antibodies to wild-type A33, as well as to engineered A33 that contained single alanine substitutions within the epitopes of the three crystallized antibodies. While the Fab of both MAbs A2C7 and A20G2 binds to a single A33 subunit, the Fab from MAb A27D7 binds to both A33 subunits simultaneously. A27D7 binding is resistant to single alanine substitutions within the A33 epitope. A27D7 also demonstrated high-affinity binding with recombinant A33 protein that mimics other orthopoxvirus strains in the A27D7 epitope, such as ectromelia, monkeypox, and cowpox virus, suggesting that A27D7 is a potent cross-neutralizer. Finally, we confirmed that A27D7 protects mice against a lethal challenge with ectromelia virus.

ST-246 is a key antiviral to inhibit the viral F13L phospholipase, one of the essential proteins for orthopoxvirus wrapping.

OBJECTIVES: ST-246 is one of the key antivirals being developed to fight orthopoxvirus (OPV) infections. Its exact mode of action is not completely understood, but it has been reported to interfere with the wrapping of infectious virions, for which F13L (peripheral membrane protein) and B5R (type I glycoprotein) are required. Here we monitored the appearance of ST-246 resistance to identify its molecular target. METHODS: Vaccinia virus (VACV), cowpox virus (CPXV) and camelpox virus (CMLV) with reduced susceptibility to ST-246 were selected in cell culture and further characterized by antiviral assays and immunofluorescence. A panel of recombinant OPVs was engineered and a putative 3D model of F13L coupled with molecular docking was used to visualize drug-target interaction. The F13L gene of 65 CPXVs was sequenced to investigate F13L amino acid heterogeneity. RESULTS: Amino acid substitutions or insertions were found in the F13L gene of six drug-resistant OPVs and production of four F13L-recombinant viruses confirmed their role(s) in the occurrence of ST-246 resistance. F13L, but not B5R, knockout OPVs showed resistance to ST-246. ST-246 treatment of WT OPVs delocalized F13L- and B5R-encoded proteins and blocked virus wrapping. Putative modelling of F13L and ST-246 revealed a probable pocket into which ST-246 penetrates. None of the identified amino acid changes occurred naturally among newly sequenced or NCBI-derived OPV F13L sequences. CONCLUSIONS: Besides demonstrating that F13L is a direct target of ST-246, we also identified novel F13L residues involved in the interaction with ST-246. These findings are important for ST-246 use in the clinic and crucial for future drug-resistance surveillance programmes.

Smallpox vaccines: targets of protective immunity.

The eradication of smallpox, one of the great triumphs of medicine, was accomplished through the prophylactic administration of live vaccinia virus, a comparatively benign relative of variola virus, the causative agent of smallpox. Nevertheless, recent fears that variola virus may be used as a biological weapon together with the present susceptibility of unimmunized populations have spurred the development of new-generation vaccines that are safer than the original and can be produced by modern methods. Predicting the efficacy of such vaccines in the absence of human smallpox, however, depends on understanding the correlates of protection. This review outlines the biology of poxviruses with particular relevance to vaccine development, describes protein targets of humoral and cellular immunity, compares animal models of orthopoxvirus disease with human smallpox, and considers the status of second- and third-generation smallpox vaccines.

Identification of a pyridopyrimidinone inhibitor of orthopoxviruses from a diversity-oriented synthesis library.

Orthopoxviruses include the prototypical vaccinia virus, the emerging infectious agent monkeypox virus, and the potential biothreat variola virus (the causative agent of smallpox). There is currently no FDA-approved drug for humans infected with orthopoxviruses. We screened a diversity-oriented synthesis library for new scaffolds with activity against vaccinia virus. This screen identified a nonnucleoside analog that blocked postreplicative intermediate and late gene expression. Viral genome replication was unaffected, and inhibition could be elicited late in infection and persisted upon drug removal. Sequencing of drug-resistant viruses revealed mutations predicted to be on the periphery of the highly conserved viral RNA polymerase large subunit. Consistent with this, the compound had broad-spectrum activity against orthopoxviruses in vitro. These findings indicate that novel chemical synthesis approaches are a potential source for new infectious disease therapeutics and identify a potentially promising candidate for development to treat orthopoxvirus-infected individuals.

[Oncolytic properties of some orthopoxviruses, adenoviruses and parvoviruses in human glioma cells].

UNLABELLED: Currently one of the most promising approaches in development of cancer virotherapy is based on the ability of oncolytic viruses to selective infection and lysis of tumor cells. AIM: The goal of the study was to identify and evaluate perspective oncolytic viruses capable of selectively destroying human glioma cells. PATIENTS AND METHODS: Original GB2m, GA14m and GB22m glioma cell cultures derived from patients were used for evaluating in vitro oncolytic activity of some typical orthopoxviruses, adenoviruses and parvoviruses. RESULTS: The oncolytic activity in the human glioma cell models was confirmed for LIVP and WR strains of vaccinia virus, Adel2 and Ad2del strains with deletions within E1B/55K gene and derived from human adenoviruses type 2 and 5, respectively. CONCLUSIONS: We consider these oncolytic viruses as promising agents for the treatment of human malignant glioma.

Genes in the terminal regions of orthopoxvirus genomes experience adaptive molecular evolution.

BACKGROUND: Orthopoxviruses are dsDNA viruses with large genomes, some encoding over 200 genes. Genes essential for viral replication are located in the center of the linear genome and genes encoding host response modifiers and other host interacting proteins are located in the terminal regions. The central portion of the genome is highly conserved, both in gene content and sequence, while the terminal regions are more diverse. In this study, we investigated the role of adaptive molecular evolution in poxvirus genes and the selective pressures that act on the different regions of the genome. The relative fixation rates of synonymous and non-synonymous mutations (the d(N)/d(S) ratio) are an indicator of the mechanism of evolution of sequences, and can be used to identify purifying, neutral, or diversifying selection acting on a gene. Like highly conserved residues, amino acids under diversifying selection may be functionally important. Many genes experiencing diversifying selection are involved in host-pathogen interactions, such as antigen-antibody interactions, or the "host-pathogen arms race." RESULTS: We analyzed 175 gene families from orthopoxviruses for evidence of diversifying selection. 79 genes were identified as experiencing diversifying selection, 25 with high confidence. Many of these genes are located in the terminal regions of the genome and function to modify the host response to infection or are virion-associated, indicating a greater role for diversifying selection in host-interacting genes. Of the 79 genes, 20 are of unknown function, and implicating diversifying selection as an important mechanism in their evolution may help characterize their function or identify important functional residues. CONCLUSIONS: We conclude that diversifying selection is an important mechanism of orthopoxvirus evolution. Diversifying selection in poxviruses may be the result of interaction with host defense mechanisms.

Cellular impedance measurement as a new tool for poxvirus titration, antibody neutralization testing and evaluation of antiviral substances.

Impedance-based biosensing known as real-time cell electronic sensing (RT-CES) belongs to an emerging technology for analyzing the status of cells in vitro. In the present study protocols were developed for an RT-CES-based system (xCELLigence™, Roche Applied Science, ACEA Biosciences Inc.) to supplement conventional techniques in pox virology. First, proliferation of cells susceptible to orthopoxviruses was monitored. For virus titration cells were infected with vaccinia virus and cell status, represented by the dimensionless impedance-based cell index (CI), was monitored. A virus-dose dependent decrease in electrical impedance could be shown. Calculation of calibration curves at a suitable CI covering a dynamic range of 4 log enabled the quantification of virus titers in unknown samples. Similarly, antiviral effects could be determined as shown for anti-poxviral agents ST-246 and Cidofovir. Published values for the in vitro concentration that inhibited virus replication by 50% (IC50) could be confirmed while cytotoxicity in effective concentrations was excluded in long-term incubation experiments. Finally, an RT-CES-based virus neutralization test was established. Various poxvirus-specific antibodies were examined for their neutralizing activity and a calculation mode for the neutralizing antibody titer was introduced. In summary, the presented RT-CES-based methods outmatch end-point assays by observing the cell population throughout the entire experiment while workload and time to result are reduced.

Orthopoxviruses require a functional ubiquitin-proteasome system for productive replication.

Cellular homeostasis depends on an intricate balance of protein expression and degradation. The ubiquitin-proteasome pathway plays a crucial role in specifically targeting proteins tagged with ubiquitin for destruction. This degradation can be effectively blocked by both chemically synthesized and natural proteasome inhibitors. Poxviruses encode a number of proteins that exploit the ubiquitin-proteasome system, including virally encoded ubiquitin molecules and ubiquitin ligases, as well as BTB/kelch proteins and F-box proteins, which interact with cellular ubiquitin ligases. Here we show that poxvirus infection was dramatically affected by a range of proteasome inhibitors, including MG132, MG115, lactacystin, and bortezomib (Velcade). Confocal microscopy demonstrated that infected cells treated with MG132 or bortezomib lacked viral replication factories within the cytoplasm. This was accompanied by the absence of late gene expression and DNA replication; however, early gene expression occurred unabated. Proteasomal inhibition with MG132 or bortezomib also had dramatic effects on viral titers, severely blocking viral replication and propagation. The effects of MG132 on poxvirus infection were reversible upon washout, resulting in the production of late genes and viral replication factories. Significantly, the addition of an ubiquitin-activating enzyme (E1) inhibitor had a similar affect on late and early protein expression. Together, our data suggests that a functional ubiquitin-proteasome system is required during poxvirus infection.

Laboratory Infection of Novel Akhmeta Virus in CAST/EiJ Mice.

Akhmeta virus is a zoonotic Orthopoxvirus first identified in 2013 in the country of Georgia. Subsequent ecological investigations in Georgia have found evidence that this virus is widespread in its geographic distribution within the country and in its host-range, with rodents likely involved in its circulation in the wild. Yet, little is known about the pathogenicity of this virus in rodents. We conducted the first laboratory infection of Akhmeta virus in CAST/EiJ Mus musculus to further characterize this novel virus. We found a dose-dependent effect on mortality and weight loss (p < 0.05). Anti-orthopoxvirus antibodies were detected in the second- and third-highest dose groups (5 × 104 pfu and 3 × 102 pfu) at euthanasia by day 10, and day 14 post-infection, respectively. Anti-orthopoxvirus antibodies were not detected in the highest dose group (3 × 106 pfu), which were euthanized at day 7 post-infection and had high viral load in tissues, suggesting they succumbed to disease prior to mounting an effective immune response. In order of highest burden, viable virus was detected in the nostril, lung, tail, liver and spleen. All individuals tested in the highest dose groups were DNAemic. Akhmeta virus was highly pathogenic in CAST/EiJ Mus musculus, causing 100% mortality when ≥3 × 102 pfu was administered.

Here, There, and Everywhere: The Wide Host Range and Geographic Distribution of Zoonotic Orthopoxviruses.

The global emergence of zoonotic viruses, including poxviruses, poses one of the greatest threats to human and animal health. Forty years after the eradication of smallpox, emerging zoonotic orthopoxviruses, such as monkeypox, cowpox, and vaccinia viruses continue to infect humans as well as wild and domestic animals. Currently, the geographical distribution of poxviruses in a broad range of hosts worldwide raises concerns regarding the possibility of outbreaks or viral dissemination to new geographical regions. Here, we review the global host ranges and current epidemiological understanding of zoonotic orthopoxviruses while focusing on orthopoxviruses with epidemic potential, including monkeypox, cowpox, and vaccinia viruses.

Conserved Oligomeric Golgi (COG) Complex Proteins Facilitate Orthopoxvirus Entry, Fusion and Spread.

Although orthopoxviruses (OPXV) are known to encode a majority of the genes required for replication in host cells, genome-wide genetic screens have revealed that several host pathways are indispensable for OPXV infection. Through a haploid genetic screen, we previously identified several host genes required for monkeypox virus (MPXV) infection, including the individual genes that form the conserved oligomeric Golgi (COG) complex. The COG complex is an eight-protein (COG1-COG8) vesicle tethering complex important for regulating membrane trafficking, glycosylation enzymes, and maintaining Golgi structure. In this study, we investigated the role of the COG complex in OPXV infection using cell lines with individual COG gene knockout (KO) mutations. COG KO cells infected with MPXV and vaccinia virus (VACV) produced small plaques and a lower virus yield compared to wild type (WT) cells. In cells where the KO phenotype was reversed using a rescue plasmid, the size of virus plaques increased demonstrating a direct link between the decrease in viral spread and the KO of COG genes. KO cells infected with VACV displayed lower levels of viral fusion and entry compared to WT suggesting that the COG complex is important for early events in OPXV infection. Additionally, fewer actin tails were observed in VACV-infected KO cells compared to WT. Since COG complex proteins are required for cellular trafficking of glycosylated membrane proteins, the disruption of this process due to lack of individual COG complex proteins may potentially impair the virus-cell interactions required for viral entry and egress. These data validate that the COG complex previously identified in our genetic screens plays a role in OPXV infection.

Understanding orthopoxvirus host range and evolution: from the enigmatic to the usual suspects.

In general, orthopoxviruses can be considered as falling into one of three host-utilization categories: highly specialized, single-host; broad host range; or 'cryptic', the last encompassing those viruses about which very little is known. Single-host viruses tend to exploit abundant hosts that have consistent patterns of interaction. For these viruses, observed genome reduction and loss of presumptive host-range genes is thought to be a consequence of relaxed selection. In contrast, the large genome size retained among broad host range orthopoxviruses suggests these viruses may depend on multiple host species for persistence in nature. Our understanding of the ecologic requirements of orthopoxviruses is strongly influenced by geographic biases in data collection. This hinders our ability to predict potential sources for emergence of orthopoxvirus-associated infections.

N-(3,3a,4,4a,5,5a,6,6a-Octahydro-1,3-dioxo-4,6- ethenocycloprop[f]isoindol-2-(1H)-yl)carboxamides: Identification of novel orthopoxvirus egress inhibitors.

A series of novel, potent orthopoxvirus egress inhibitors was identified during high-throughput screening of the ViroPharma small molecule collection. Using structure--activity relationship information inferred from early hits, several compounds were synthesized, and compound 14 was identified as a potent, orally bioavailable first-in-class inhibitor of orthopoxvirus egress from infected cells. Compound 14 has shown comparable efficaciousness in three murine orthopoxvirus models and has entered Phase I clinical trials.

From actually toxic to highly specific--novel drugs against poxviruses.

The potential use of variola virus, the causative agent of smallpox, as a bioweapon and the endemic presence of monkeypox virus in Africa demonstrate the need for better therapies for orthopoxvirus infections. Chemotherapeutic approaches to control viral infections have been less successful than those targeting bacterial infections. While bacteria commonly reproduce themselves outside of cells and have metabolic functions against which antibiotics can be directed, viruses replicate in the host cells using the cells' metabolic pathways. This makes it very difficult to selectively target the virus without damaging the host. Therefore, the development of antiviral drugs against poxviruses has initially focused on unique properties of the viral replication cycle or of viral proteins that can be selectively targeted. However, recent advances in molecular biology have provided insights into host factors that represent novel drug targets. The latest anti-poxvirus drugs are kinase inhibitors, which were originally developed to treat cancer progression but in addition block egress of poxviruses from infected cells. This review will summarize the current understanding of anti-poxvirus drugs and will give an overview of the development of the latest second generation poxvirus drugs.

Inactivation of orthopoxvirus for diagnostic PCR analysis.

Diagnoses of ongoing viral infections commonly rely on PCR methodology. Sample material that may contain hazardous virus should be efficiently inactivated in biological containment or bed-side before diagnostic PCR analysis. Surprisingly little documentation is available for inactivation of human viral pathogens by inactivation reagents that allow for subsequent PCR diagnostics. It is now shown that pathogenic DNA viruses (orthopoxvirus) are completely inactivated by a commercially available Roche MagNA Pure lysis/binding buffer as evaluated by subsequent cell culture. However, inactivation reagents are typically toxic and therefore problematic in cell culture. Using the relatively large orthopoxvirus, a method was developed in which virus is precipitated by high-speed centrifugation after inactivation but prior to application onto the target cells, thereby eliminating the cytotoxic effect of the lysis buffer. The results from quantitative PCR analysis indicate that the viral DNA from the completely inactivated virus particles, remain associated to macromolecules and aggregates. The use of inactivation buffers for bed-side inactivation of special patient samples taken for PCR diagnostics should be considered in cases where high containment would otherwise be required.