Host Range Gene Research Articles

Nanopore adaptive sampling of a metagenomic sample derived from a human monkeypox case.

In 2022, a series of human monkeypox cases in multiple countries led to the largest and most widespread outbreak outside the known endemic areas. Setup of proper genomic surveillance is of utmost importance to control such outbreaks. To this end, we performed Nanopore (PromethION P24) and Illumina (NextSeq. 2000) Whole Genome Sequencing (WGS) of a monkeypox sample. Adaptive sampling was applied for in silico depletion of the human host genome, allowing for the enrichment of low abundance viral DNA without a priori knowledge of sample composition. Nanopore sequencing allowed for high viral genome coverage, tracking of sample composition during sequencing, strain determination, and preliminary assessment of mutational pattern. In addition to that, only Nanopore data allowed us to resolve the entire monkeypox virus genome, with respect to two structural variants belonging to the genes OPG015 and OPG208. These SVs in important host range genes seem stable throughout the outbreak and are frequently misassembled and/or misannotated due to the prevalence of short read sequencing or short read first assembly. Ideally, standalone standard Illumina sequencing should not be used for Monkeypox WGS and de novo assembly, since it will obfuscate the structure of the genome, which has an impact on the quality and completeness of the genomes deposited in public databases and thus possibly on the ability to evaluate the complete genetic reason for the host range change of monkeypox in the current pandemic.

Comparative phylogenomics and phylotranscriptomics provide insights into the genetic complexity of nitrogen-fixing root-nodule symbiosis

Plant root-nodule symbiosis (RNS) with mutualistic nitrogen-fixing bacteria is restricted to a single clade of angiosperms, the Nitrogen-Fixing Nodulation Clade (NFNC), and is best understood in the legume family. Nodulating species share many commonalities, explained either by divergence from a common ancestor over 100 million years ago or by convergence following independent origins over that same time period. Regardless, comparative analyses of diverse nodulation syndromes can provide insights into constraints on nodulation—what must be acquired or cannot be lost for a functional symbiosis—and the latitude for variation in the symbiosis. However, much remains to be learned about nodulation, especially outside of legumes. Here, we employed a large-scale phylogenomic analysis across 88 species, complemented by 151 RNA-seq libraries, to elucidate the evolution of RNS. Our phylogenomic analyses further emphasize the uniqueness of the transcription factor NIN as a master regulator of nodulation and identify key mutations that affect its function across the NFNC. Comparative transcriptomic assessment revealed nodule-specific upregulated genes across diverse nodulating plants, while also identifying nodule-specific and nitrogen-response genes. Approximately 70% of symbiosis-related genes are highly conserved in the four representative species, whereas defense-related and host-range restriction genes tend to be lineage specific. Our study also identified over 900 000 conserved non-coding elements (CNEs), over 300 000 of which are unique to sampled NFNC species. NFNC-specific CNEs are enriched with the active H3K9ac mark and are correlated with accessible chromatin regions, thus representing a pool of candidate regulatory elements for genes involved in RNS. Collectively, our results provide novel insights into the evolution of nodulation and lay a foundation for engineering of RNS traits in agriculturally important crops.

The effective factors in human-specific tropism and viral pathogenicity in orthopoxviruses.

The orthopoxvirus (OPV) genus includes several species that infect humans, including variola, monkeypox, vaccinia, and cowpox. Variola and monkeypox are often life-threatening diseases, while vaccinia and cowpox are usually associated with local lesions. The epidemic potential for OPVs may be lower than respiratory-borne viruses or RNA viruses. However, OPVs are notable for their spread and distribution in different environments and among different hosts. The emergence or re-emergence of OPVs in the human population can also occur in wild or domestic animals as intermediate hosts. More effective and safer vaccines for poxvirus can be developed by understanding how immunity is regulated in poxvirus and vaccines for DNA viruses. Downstream events in cells affected by the virus are regulated functionally by a series of characteristics that are affected by host cell interactions and responses of cells against viral infections, including the interferon pathway and apoptosis. Furthermore, infection outcome is greatly influenced by the distinct selection of host-range and immune-modulatory genes that confer the potential for pathogenesis and host-to-host transmission and the distinct host-range properties of each immune-modulatory gene. The present study reviewed the effective factors in human-restricted tropism and virus pathogenicity in OPVs.

Genome-Wide Analysis of Odorant and Gustatory Receptors in Six Papilio Butterflies (Lepidoptera: Papilionidae).

Simple SummaryThe butterflies of the Lepidoptera are representatives of herbivorous insects. Host plant diversities may shape the evolution of chemosensory receptors in butterflies. However, little is known about whether the host plant range is correlated with the size differences of chemoreceptors as key molecular targets for butterfly–plant chemical interactions. Here, we characterized 381 odorant (ORs) and 328 gustatory (GRs) receptors in one Papilio generalist and five other Papilio specialists regarding their comprehensive annotation, gene structure, phylogenetics and expression profiles. Among six Papilio species, orthologous ORs or GRs shared conserved gene structure, but the differences in intron size frequencies were observed between the generalist and the specialists. Furthermore, expression profiles identified candidate OR and GR genes in antennae, foretarsi or reproductive tissues of P. xuthus. This study addresses, for the first time, the issue that the breath of host plants does not appear to result in the obvious expansions of ORs and GRs in Papilio butterflies and identifies candidate molecular targets associated with olfaction, oviposition or reproduction in P. xuthus.The chemical interactions of insects and host plants are shaping the evolution of chemosensory receptor gene families. However, the correlation between host range and chemoreceptor gene repertoire sizes is still elusive in Papilionidae. Here, we addressed the issue of whether host plant diversities are correlated with the expansions of odorant (ORs) or gustatory (GRs) receptors in six Papilio butterflies. By combining genomics, transcriptomics and bioinformatics approaches, 381 ORs and 328 GRs were annotated in the genomes of a generalist P. glaucus and five specialists, P. xuthus, P. polytes, P. memnon, P. machaon and P. dardanus. Orthologous ORs or GRs in Papilio had highly conserved gene structure. Five Papilio specialists exhibited a similar frequency of intron lengths for ORs or GRs, but which was different from those in the generalist. Phylogenetic analysis revealed 60 orthologous OR groups, 45 of which shared one-to-one relationships. Such a single gene in each butterfly also occurred in 26 GR groups. Intriguingly, bitter GRs had fewer introns than other GRs and clustered into a large clade. Focusing on the two chemoreceptor gene families in P. xuthus, most PxutORs (52/58) were expressed in antennae and 31 genes in reproductive tissues. Eleven out of 28 foretarsus-expressed PxutGRs were female-biased genes, as strong candidates for sensing oviposition stimulants. These results indicate that the host range may not shape the large-scale expansions of ORs and GRs in Papilio butterflies and identify important molecular targets involved in olfaction, oviposition or reproduction in P. xuthus.

Bacteriophage Isolation, Characterization and Bioinformatic Genetic Analyses against Actinomycetes Gordonia rubripertincta and Nocardia asteroides

Two of the biggest issues in the healthcare system today are antibiotic resistant bacteria and biofilms. Antibiotic‐resistant bacterial strains are increasing, and current treatment options have become limited forcing medical facilities to search for alternatives. Recent studies utilizing phage therapy have provided promising results supporting the use of bacteriophages to treat bacterial infections. (Hatfull, 2021). A bacteriophage is a virus that only infects prokaryotic bacterial cells and therefore, potentially used to treat bacterial infections without risk of infecting the patient’s eukaryotic cells. This research focuses on the isolation and microbiological characterization of bacteriophages isolated from environmental samples against the Actinomycete host, Gordonia rubripertincta. Gordonia species are typically found in soil and aquatic habitats and have recently been identified as primary agents of infections in both humans and animals (Pope, et al., 2020). Due to these organisms’ ability to form biofilms, infections have been found following various surgical procedures and applications of catheters (Arenskötter, et al., 2004). Following a Gordonia‐enrichment protocol, standard viral lab techniques such as serial dilutions, pick‐a‐plaque, and spot‐plating were performed resulting in pure viral colonies. Genomic DNA was isolated and sequenced by the Pittsburgh Bacteriophage Institute. Genomes were annotated using Phamerator™, PECAAN™, and NCBI BLAST bioinformatics programs. Host‐range specificity studies and gene knock‐out experiments were conducted using phylogenetically‐related Actinomycetes to assist understanding of the molecular interactions between phage and host throughout the infection cycle. Several bacteriophages were identified which exhibit both lytic and temperate life cycles against known clinical, biofilm‐producing Actinomycete bacteria Nocardia asteroides and N. corallina.This research exemplifies the potential of using phage therapy to create an alternative treatment for antibiotic‐resistant bacteria and biofilms within the healthcare system.

Single Immunization with Recombinant ACAM2000 Vaccinia Viruses Expressing the Spike and the Nucleocapsid Proteins Protects Hamsters against SARS-CoV-2-Caused Clinical Disease.

ABSTRACTIncreasing cases of SARS-CoV-2 breakthrough infections from immunization with current spike protein-based COVID-19 vaccines highlight the need to develop alternative vaccines using different platforms and/or antigens. In this study, we expressed SARS-CoV-2 spike and nucleocapsid proteins based on a novel vaccinia virus (VACV) ACAM2000 platform (rACAM2000). In this platform, the vaccinia virus host range and immunoregulatory gene E3L was deleted to make the virus attenuated and to enhance innate immune responses, and another host range gene, K3L, was replaced with a poxvirus ortholog gene, taterapox virus 037 (TATV037), to make virus replication competent in both hamster and human cells. Following a single intramuscular immunization, the rACAM2000 coexpressing the spike and nucleocapsid proteins induced significantly improved protection against SARS-CoV-2 challenge in comparison to rACAM2000 expressing the individual proteins in a hamster model, as shown by reduced weight loss and shorter recovery time. The protection was associated with reduced viral loads, increased neutralizing antibody titer, and reduced neutrophil-to-lymphocyte ratio. Thus, our study demonstrates that rACAM2000 expressing a combination of the spike and nucleocapsid antigens is a promising COVID-19 vaccine candidate, and further studies will investigate if the rACAM2000 vaccine candidate can induce a long-lasting immunity against infection by SARS-CoV-2 variants of concern.IMPORTANCE Continuous emergence of SARS-CoV-2 variants which cause breakthrough infection from the immunity induced by current spike protein-based COVID-19 vaccines highlights the need for new generations of vaccines that will induce long-lasting immunity against a wide range of the variants. To this end, we investigated the protective efficacy of the recombinant COVID-19 vaccine candidates based on a novel VACV ACAM2000 platform, in which an immunoregulatory gene, E3L, was deleted and both the SARS-CoV-2 spike (S) and nucleocapsid (N) antigens were expressed. Thus, it is expected that the vaccine candidate we constructed should be more immunogenic and safer. In the initial study described in this work, we demonstrated that the vaccine candidate expressing both the S and N proteins is superior to the constructs expressing an individual protein (S or N) in protecting hamsters against SARS-CoV-2 challenge after a single-dose immunization, and further investigation against different SARS-CoV-2 variants will warrant future clinical evaluations.

Identification of a Novel Myxoma Virus C7-Like Host Range Factor That Enabled a Species Leap from Rabbits to Hares.

ABSTRACTMyxoma virus (MYXV) is naturally found in rabbit Sylvilagus species and is known to cause lethal myxomatosis in European rabbits (Oryctolagus cuniculus). In 2019, an MYXV strain (MYXV strain Toledo [MYXV-Tol]) causing myxomatosis-like disease in Iberian hares (Lepus granatensis) was identified. MYXV-Tol acquired a recombinant region of ∼2.8 kb harboring several new genes, including a novel host range gene (M159) that we show to be an orthologous member of the vaccinia virus C7 host range family. Here, to test whether M159 alone has enabled MYXV to alter its host range to Iberian hares, several recombinant viruses were generated, including an MYXV-Tol ΔM159 (knockout) strain. While MYXV-Tol underwent fully productive infection in hare HN-R cells, neither the wild-type MYXV-Lau strain (lacking M159) nor vMyxTol-ΔM159 (deleted for M159) was able to infect and replicate, showing that the ability of MYXV-Tol to infect these cells and replicate depends on the presence of M159. Similar to other C7L family members, M159 was shown to be expressed as an early/late gene but was translocated into the nucleus at later time points, indicating that further studies are needed to elucidate its role in the nucleus. Finally, in rabbit cells, the M159 protein did not contribute to increased replication but was able to upregulate the replication levels of MYXV in nonpermissive and semipermissive human cancer cells, suggesting that the M159-targeted pathway is conserved across mammalian species. Altogether, these observations demonstrate that the M159 protein plays a critical role in determining the host specificity of MYXV-Tol in hare and human cells by imparting new host range functions.

Advancements in the Growth and Construction of Recombinant Lumpy Skin Disease Virus (LSDV) for Use as a Vaccine Vector.

Attenuated vaccine strains of lumpy skin disease virus (LSDV) have become increasingly popular as recombinant vaccine vectors, to target both LSDV, as well as other pathogens, including human infectious agents. Historically, these vaccine strains and recombinants were generated in primary (lamb) testis (LT) cells, Madin–Darby bovine kidney (MDBK) cells or in eggs. Growth in eggs is a laborious process, the use of primary cells has the potential to introduce pathogens and MDBK cells are known to harbor bovine viral diarrhea virus (BVDV). In this study, data is presented to show the growth of an attenuated LSDV strain in baby hamster kidney (BHK-21) cells. Subsequently, a recombinant LSDV vaccine was generated in BHK-21 cells. Partial growth was also observed in rabbit kidney cells (RK13), but only when the vaccinia virus host range gene K1L was expressed. Despite the limited growth, the expression of K1L was enough to serve as a positive selection marker for the generation of recombinant LSDV vaccines in RK13 cells. The simplification of generating (recombinant) LSDV vaccines shown here should increase the interest for this platform for future livestock vaccine development and, with BHK-21 cells approved for current good manufacturing practice, this can be expanded to human vaccines as well.

Single Immunization of a Vaccine Vectored by a Novel Recombinant Vaccinia Virus Affords Effective Protection Against Respiratory Syncytial Virus Infection in Cotton Rats.

Respiratory syncytial virus (RSV) is a leading cause of respiratory infections worldwide and disease management measures are hampered by the lack of a safe and effective vaccine against the infection. We constructed a novel recombinant RSV vaccine candidate based on a deletion mutant vaccinia virus platform, in that the host range genes E3L and K3L were deleted (designated as VACVΔE3LΔK3L) and a poxvirus K3L ortholog gene was used as a marker for the rapid and efficient selection of recombinant viruses. The safety of the modified vaccinia virus was investigated by intranasal administration of BALB/c mice with the modified vaccinia vector using a dose known to be lethal in the wild-type Western Reserve. Only a minor loss of body weight by less than 5% and mild pulmonary inflammation were observed, both of which were transient in nature following nasal administration of the high-dose modified vaccinia virus. In addition, the viruses were cleared from the lung in 2 days with no viral invasions of the brain and other vital organs. These results suggest that the virulence of the virus has been essentially abolished. We then investigated the efficiency of the vector for the delivery of vaccines against RSV through comparison with another RSV vaccine delivered by the widely used Modified Vaccinia virus Ankara (MVA) backbone. In the cotton rats, we found a single intramuscular administration of VACVΔE3LΔK3L-vectored vaccine elicited immune responses and protection at a level comparable to the MVA-vectored vaccine against RSV infection. The distinct features of this novel VACV vector, such as an E3L deletion for attenuation and a K3L ortholog for positive selection and high efficiency for vaccine delivery, could provide unique advantages to the application of VACV as a platform for vaccine development.

Rescue of a Vaccinia Virus Mutant Lacking IFN Resistance Genes K1L and C7L by the Parapoxvirus Orf Virus.

Type 1 interferons induce the upregulation of hundreds of interferon-stimulated genes (ISGs) that combat viral replication. The parapoxvirus orf virus (ORFV) induces acute pustular skin lesions in sheep and goats and can reinfect its host, however, little is known of its ability to resist IFN. Vaccinia virus (VACV) encodes a number of factors that modulate the IFN response including the host-range genes C7L and K1L. A recombinant VACV-Western Reserve (WR) strain in which the K1L and C7L genes have been deleted does not replicate in cells treated with IFN-β nor in HeLa cells in which the IFN response is constitutive and is inhibited at the level of intermediate gene expression. Furthermore C7L is conserved in almost all poxviruses. We provide evidence that shows that although ORFV is more sensitive to IFN-β compared with VACV, and lacks homologues of KIL and C7L, it nevertheless has the ability to rescue a VACV KIL- C7L- gfp+ mutant in which gfp is expressed from a late promoter. Co-infection of HeLa cells with the mutant and ORFV demonstrated that ORFV was able to overcome the block in translation of intermediate transcripts in the mutant virus, allowing it to progress to late gene expression and new viral particles. Our findings strongly suggest that ORFV encodes a factor(s) that, although different in structure to C7L or KIL, targets an anti-viral cellular mechanism that is a highly potent at killing poxviruses.

Efficient Induction of Cytotoxic T Cells by Viral Vector Vaccination Requires STING-Dependent DC Functions.

Modified Vaccinia virus Ankara (MVA) is an attenuated strain of vaccinia virus and currently under investigation as a promising vaccine vector against infectious diseases and cancer. MVA acquired mutations in host range and immunomodulatory genes, rendering the virus deficient for replication in most mammalian cells. MVA has a high safety profile and induces robust immune responses. However, the role of innate immune triggers for the induction of cytotoxic T cell responses after vaccination is incompletely understood. Stimulator of interferon genes (STING) is an adaptor protein which integrates signaling downstream of several DNA sensors and therefore mediates the induction of type I interferons and other cytokines or chemokines in response to various dsDNA viruses. Since the type I interferon response was entirely STING-dependent during MVA infection, we studied the effect of STING on primary and secondary cytotoxic T cell responses and memory T cell formation after MVA vaccination in STING KO mice. Moreover, we analyzed the impact of STING on the maturation of bone marrow-derived dendritic cells (BMDCs) and their functionality as antigen presenting cells for cytotoxic T cells during MVA infection in vitro. Our results show that STING has an impact on the antigen processing and presentation capacity of conventionel DCs and played a crucial role for DC maturation and type I interferon production. Importantly, STING was required for the induction of efficient cytotoxic T cell responses in vivo, since we observed significantly decreased short-lived effector and effector memory T cell responses after priming in STING KO mice. These findings indicate that STING probably integrates innate immune signaling downstream of different DNA sensors in DCs and shapes the cytotoxic T cell response via the DC maturation phenotype which strongly depends on type I interferons in this infection model. Understanding the detailed functions of innate immune triggers during MVA infection will contribute to the optimized design of MVA-based vaccines.

Repair of a previously uncharacterized second host-range gene contributes to full replication of modified vaccinia virus Ankara (MVA) in human cells

Modified vaccinia virus Ankara (MVA), a widely used vaccine vector for expression of genes of unrelated pathogens, is safe, immunogenic, and can incorporate large amounts of added DNA. MVA was derived by extensively passaging the chorioallantois vaccinia virus Ankara (CVA) vaccine strain in chicken embryo fibroblasts during which numerous mutations and deletions occurred with loss of replicative ability in most mammalian cells. Restoration of the deleted C12L gene, encoding serine protease inhibitor 1, enhances replication of MVA in human MRC-5 cells but only slightly in other human cells. Here we show that repair of the inactivated C16L/B22R gene of MVA enhances replication in numerous human cell lines. This previously uncharacterized gene is present at both ends of the genome of many orthopoxviruses and is highly conserved in most, including smallpox and monkeypox viruses. The C16L/B22R gene is expressed early in infection from the second methionine of the previously annotated Copenhagen strain open reading frame (ORF) as a 17.4-kDa protein. The C16/B22 and C12 proteins together promote MVA replication in human cells to levels that are comparable to titers in chicken embryo fibroblasts. Both proteins enhance virion assembly, but C16/B22 increases proteolytic processing of core proteins in A549 cells consistent with higher infectious virus titers. Furthermore, human A549 cells expressing codon-optimized C16L/B22R and C12L genes support higher levels of MVA replication than cell lines expressing neither or either alone. Identification of the genes responsible for the host-range defect of MVA may allow more rational engineering of vaccines for efficacy, safety, and propagation.

Application of poxvirus K3 ortholog as a positive selection marker for constructing recombinant vaccinia viruses with modified host range

Vaccinia virus is capable of replicating in a wide range of vertebrate animal cells. However, deletion of the two virus host range genes, E3L and K3L, would render replication of the virus abortive in all the cell types tested, except the cells with defective PKR and RNase L activity. By expressing different poxvirus K3 ortholog proteins in the E3L and K3L double knockout vaccinia virus, we can construct a mutant vaccinia virus with modified host range. Here, using poxvirus K3 ortholog as a positive selection marker, we developed a proof-of-concept protocol to construct recombinant vaccinia viruses expressing foreign proteins. Such a recombinant virus has a modified host range in comparison to wild-type vaccinia virus. This protocol offers the following advantages:➢Cheap: no additional selection reagent is required.➢Highly efficient: nearly all recombinant virus plaques picked would be the stable recombinant virus expressing the protein of interest.➢Rapid: starting from transfection with the recombinant DNA vector, a purified recombinant virus expressing the protein of interest could be obtained within a week.

Genome of Alaskapox Virus, A Novel Orthopoxvirus Isolated from Alaska.

Since the eradication of smallpox, there have been increases in poxvirus infections and the emergence of several novel poxviruses that can infect humans and domestic animals. In 2015, a novel poxvirus was isolated from a resident of Alaska. Diagnostic testing and limited sequence analysis suggested this isolate was a member of the Orthopoxvirus (OPXV) genus but was highly diverged from currently known species, including Akhmeta virus. Here, we present the complete 210,797 bp genome sequence of the Alaska poxvirus isolate, containing 206 predicted open reading frames. Phylogenetic analysis of the conserved central region of the genome suggested the Alaska isolate shares a common ancestor with Old World OPXVs and is diverged from New World OPXVs. We propose this isolate as a member of a new OPXV species, Alaskapox virus (AKPV). The AKPV genome contained host range and virulence genes typical of OPXVs but lacked homologs of C4L and B7R, and the hemagglutinin gene contained a unique 120 amino acid insertion. Seven predicted AKPV proteins were most similar to proteins in non-OPXV Murmansk or NY_014 poxviruses. Genomic analysis revealed evidence suggestive of recombination with Ectromelia virus in two putative regions that contain seven predicted coding sequences, including the A-type inclusion protein.

Genetic Characterization of a Recombinant Myxoma Virus in the Iberian Hare (Lepus granatensis).

Myxomatosis is a lethal disease in wild European and domestic rabbits (Oryctolagus cuniculus), which is caused by a Myxoma virus (MYXV) infection—a leporipoxvirus that is found naturally in some Sylvilagus rabbit species in South America and California. The introduction of MYXV into feral European rabbit populations of Australia and Europe, in the early 1950s, demonstrated the best-documented field example of host–virus coevolution, following a cross-species transmission. Recently, a new cross-species jump of MYXV has been suggested in both Great Britain and Spain, where European brown hares (Lepus europaeus) and Iberian hares (Lepus granatensis) were found dead with lesions consistent with those observed in myxomatosis. To investigate the possibility of a new cross-species transmission event by MYXV, tissue samples collected from a wild Iberian hare found dead in Spain (Toledo region) were analyzed and deep sequenced. Our results reported a new MYXV isolate (MYXV Toledo) in the tissues of this species. The genome of this new virus was found to encode three disruptive genes (M009L, M036L, and M152R) and a novel ~2.8 kb recombinant region, which resulted from an insertion of four novel poxviral genes towards the 3’ end of the negative strand of its genome. From the open reading frames inserted into the MYXV Toledo virus, a new orthologue of a poxvirus host range gene family member was identified, which was related to the MYXV gene M064R. Overall, we confirmed the identity of a new MYXV isolate in Iberian hares, which, we hypothesized, was able to more effectively counteract the host defenses in hares and start an infectious process in this new host.

SPI-1 is a missing host-range factor required for replication of the attenuated modified vaccinia Ankara (MVA) vaccine vector in human cells

Modified vaccinia virus Ankara (MVA) is the leading poxvirus vector for development of vaccines against diverse infectious diseases. This distinction is based on high expression of proteins and good immunogenicity despite an inability to assemble infectious progeny in human cells, which together promote efficacy and safety. Nevertheless, the basis for the host-range restriction is unknown despite past systematic attempts to identify the relevant missing viral gene(s). The search for host-range factors is exacerbated by the large number of deletions, truncations and mutations that occurred during the long passage history of MVA in chicken embryo fibroblasts. By whole genome sequencing of a panel of recombinant host-range extended (HRE) MVAs generated by marker rescue with 40 kbp segments of vaccinia virus DNA, we identified serine protease inhibitor 1 (SPI-1) as one of several candidate host-range factors present in those viruses that gained the ability to replicate in human cells. Electron microscopy revealed that the interruption of morphogenesis in human cells infected with MVA occurred at a similar stage as that of a vaccinia virus strain WR SPI-1 deletion mutant. Moreover, the introduction of the SPI-1 gene into the MVA genome led to more than a 2-log enhancement of virus spread in human diploid MRC-5 cells, whereas deletion of the gene diminished the spread of HRE viruses by similar extents. Furthermore, MRC-5 cells stably expressing SPI-1 also enhanced replication of MVA. A role for additional host range genes was suggested by the restoration of MVA replication to a lower level relative to HRE viruses, particularly in other human cell lines. Although multiple sequence alignments revealed genetic changes in addition to SPI-1 common to the HRE MVAs, no evidence for their host-range function was found by analysis thus far. Our finding that SPI-1 is host range factor for MVA should simplify use of high throughput RNAi or CRISPR/Cas single gene methods to identify additional viral and human restriction elements.

RNA Polymerase Mutations Selected during Experimental Evolution Enhance Replication of a Hybrid Vaccinia Virus with an Intermediate Transcription Factor Subunit Replaced by the Myxoma Virus Ortholog.

High-throughput DNA sequencing enables the study of experimental evolution in near real time. Until now, mutants with deletions of nonessential host range genes were used in experimental evolution of vaccinia virus (VACV). Here, we guided the selection of adaptive mutations that enhanced the fitness of a hybrid virus in which an essential gene had been replaced with an ortholog from another poxvirus genus. Poxviruses encode a complete system for transcription, including RNA polymerase and stage-specific transcription factors. The abilities of orthologous intermediate transcription factors from other poxviruses to substitute for those of VACV, as determined by transfection assays, corresponded with the degree of amino acid identity. VACV in which the A8 or A23 intermediate transcription factor subunit gene was replaced by the myxoma (MYX) virus ortholog exhibited decreased replication. During three parallel serial passages of the hybrid virus with the MYXA8 gene, plaque sizes and virus yields increased. DNA sequencing of virus populations at passage 10 revealed high frequencies of five different single nucleotide mutations in the two largest RNA polymerase subunits, RPO147 and RPO132, and two different Kozak consensus sequence mutations predicted to increase translation of the MYXA8 mRNA. Surprisingly, there were no mutations within either intermediate transcription factor subunit. Based on homology with Saccharomyces cerevisiae RNA polymerase, the VACV mutations were predicted to be buried within the internal structure of the enzyme. By directly introducing single nucleotide substitutions into the genome of the original hybrid virus, we demonstrated that both RNA polymerase and translation-enhancing mutations increased virus replication independently.IMPORTANCE Previous studies demonstrated the experimental evolution of vaccinia virus (VACV) following deletion of a host range gene important for evasion of host immune defenses. We have extended experimental evolution to essential genes that cannot be deleted but could be replaced by a divergent orthologous gene from another poxvirus. Replacement of a VACV transcription factor gene with one from a distantly related poxvirus led to decreased fitness as evidenced by diminished replication. Serially passaging the hybrid virus at a low multiplicity of infection provided conditions for selection of adaptive mutations that improved replication. Notably, these included five independent mutations of the largest and second largest RNA polymerase subunits. This approach should be generally applicable for investigating adaptation to swapping of orthologous genes encoding additional essential proteins of poxviruses as well as other viruses.

Genome Sequences of Akhmeta Virus, an Early Divergent Old World Orthopoxvirus.

Annotated whole genome sequences of three isolates of the Akhmeta virus (AKMV), a novel species of orthopoxvirus (OPXV), isolated from the Akhmeta and Vani regions of the country Georgia, are presented and discussed. The AKMV genome is similar in genomic content and structure to that of the cowpox virus (CPXV), but a lower sequence identity was found between AKMV and Old World OPXVs than between other known species of Old World OPXVs. Phylogenetic analysis showed that AKMV diverged prior to other Old World OPXV. AKMV isolates formed a monophyletic clade in the OPXV phylogeny, yet the sequence variability between AKMV isolates was higher than between the monkeypox virus strains in the Congo basin and West Africa. An AKMV isolate from Vani contained approximately six kb sequence in the left terminal region that shared a higher similarity with CPXV than with other AKMV isolates, whereas the rest of the genome was most similar to AKMV, suggesting recombination between AKMV and CPXV in a region containing several host range and virulence genes.

Poxvirus Host Range Genes and Virus-Host Spectrum: A Critical Review.

The Poxviridae family is comprised of double-stranded DNA viruses belonging to nucleocytoplasmic large DNA viruses (NCLDV). Among the NCLDV, poxviruses exhibit the widest known host range, which is likely observed because this viral family has been more heavily investigated. However, relative to each member of the Poxviridae family, the spectrum of the host is variable, where certain viruses can infect a large range of hosts, while others are restricted to only one host species. It has been suggested that the variability in host spectrum among poxviruses is linked with the presence or absence of some host range genes. Would it be possible to extrapolate the restriction of viral replication in a specific cell lineage to an animal, a far more complex organism? In this study, we compare and discuss the relationship between the host range of poxvirus species and the abundance/diversity of host range genes. We analyzed the sequences of 38 previously identified and putative homologs of poxvirus host range genes, and updated these data with deposited sequences of new poxvirus genomes. Overall, the term host range genes might not be the most appropriate for these genes, since no correlation between them and the viruses’ host spectrum was observed, and a change in nomenclature should be considered. Finally, we analyzed the evolutionary history of these genes, and reaffirmed the occurrence of horizontal gene transfer (HGT) for certain elements, as previously suggested. Considering the data presented in this study, it is not possible to associate the diversity of host range factors with the amount of hosts of known poxviruses, and this traditional nomenclature creates misunderstandings.

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.