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Ultrastructural study of cell cultures infected with swinepox and orf viruses.

Monolayer cells of a porcine kidney cell line were infected with the PP-1 strain of swinepox virus, while secondary or third subcultured monolayer cells of African green monkey kidney were inoculated with the Iwate BT-9 strain of orf virus. Those infected cells were fixed when CPE became remarkable in the monolayers and examined by electron microscope. In the cytoplasm of cells infected with both viruses, various immature forms of viral particles in different developmental stages were observed. Micells were detected very close to the opening of immature particles. Mature particles asssociated with a double membrane were frequently observed. From those observations it was suggested that the developmental sequence of both viruses is essentially the same as that of vaccinia (Dales, 1973). Besides various virus forms as well as factories, the following ultrastructural changes were noted in swinepox infected cells, i.e. 1) intranuclear inclusions which consist of very fine filaments, 2) fibrillar structures with cross striations located in the nuclear inclusions, and 3) similar striated fibrillar structures in or just adjacent to virus factories (B type inclusions) in the cytoplasm. Those observations made with in vitro cells are in good accordance with the descriptions by previous investigators on in vivo materials. Accordingly those ultrastructural changes characterize the swinepox infection.

Animals

Recent advances for the pharmaceutical production of highly attenuated poxviruses as viral vector platforms.

INTRODUCTION: Highly attenuated poxviruses serve as potent viral vectors, oncolytic agents, and therapeutic vaccines. They can accommodate and stably maintain a large genomic payload of foreign inserts. Their limited replication in human cells provides an excellent safety profile, but it concomitantly necessitates higher doses of infectious particles for full therapeutic efficacy. AREAS COVERED: We review recent advances in bioprocesses for the pharmaceutical production of poxvirus-based vectors, focusing mainly on the vaccinia virus and the Orf virus. These include upstream processing using highly permissive cell substrates, optimized feeding strategies, and a virus phenotype that facilitates downstream processing. The study explores ongoing challenges and identifies strategies to adapt the downstream process to intensified upstream processes in order to achieve an economic end-to-end production. EXPERT OPINION: For notably increased virus yields of up to 2 log after amplification, we propose to replace classic adsorption chromatography by a collective and continuous purification platform for separating the virus from process-related impurities. Filtration operations facilitate process scalability while reducing volumes, which is beneficial for a flow-through polishing to meet pharmaceutical quality attributes. Combined with artificial intelligence modeling, these advancements alleviate financial pressures on healthcare systems and accelerate the production of novel vaccine candidates for clinical use.

Humans

High C + G content in parapoxvirus DNA.

The DNAs of eight parapoxviruses (four stomatitis papulosa viruses isolated from infected calves, a pseudocowpox virus isolated from a teat lesion of an infected cow and three orf viruses, one isolated from an infected sheep and two isolated from human infections) were analysed in CsCl gradients. The mole % of G+C was calculated from the buoyant density and found to be approx. 63% for all virus isolates examined. Parapoxvirus DNA thus has by far the highest G+C content of all poxvirus DNAs so far examined.

Animals

[Contagious ecthyma with virus demonstration in a negative staining method].

The diagnosis of contagious ecthyma was presumed by history and clinical appearance in a 33-year-old farmer. In electron miscroscopical preparations viral particles were found by the negative staining method. It is proposed to use this method for rapid diagnosis of suspected cases. In old lesions it is more difficult to demonstrate viral particles than in fresh lesions. Differential diagnosis and the position of Orf-virus in the system of viruses are briefly discussed.

Adult

Causation and epidemiological features of proliferative lip and facial lesions in Lao goats.

Past surveys and studies have identified a syndrome of lip and facial dermatitis as common in Lao goats and identified Orf virus (ORFV) as the likely cause. To more definitively determine the cause of this syndrome, a matched case-control study was implemented in 70 smallholder goat farms and a large-scale goat holding from Savannakhet province, Laos. Forty-one scab samples from goats with lip and facial dermatitis (cases) and 41 paired samples from unaffected animals (controls) were collected from 31 goat holdings over 12&#x2009;months. Samples were tested using quantitative PCR assays targeting possible pathogens causing such lesions. The prevalence of PCR-positive samples in case and control goats was 100% and 95.1%, respectively, for ORFV (P&#x2009;=&#x2009;0.15) and 19.5% and 29.3% (P&#x2009;=&#x2009;0.3) for Dermatophilus congolensis. Pseudocowpox virus and bovine popular stomatitis virus were not detected in any samples. There was a clear difference between the log10 genome copies (GC) per &#xb5;L DNA extract of ORFV in positive samples from cases (7.00&#x2009;&#xb1;&#x2009;0.17) and controls (2.62&#x2009;&#xb1;&#x2009;0.18, P&#x2009;<&#x2009;0.0001). This was not true for D. congolensis with similar GC detected in the case and control goats. More than 4 log higher GC in affected cases identifies ORFV as the likely cause of the syndrome in Lao goats with little role for D. congolensis. Phylogenetic analysis showed no genetic variation of ORFV in the study samples and close similarity to sequences from China. The findings suggest that orf disease has a sporadic distribution, mostly occurring in goats during the first 6-months of life.

Animals

Genome-wide CRISPR screen reveals PEX11B as a host restriction factor against ORFV through membrane fluidity regulation.

Host-pathogen interactions are shaped by cellular restriction factors that direct antiviral defenses. We built the first ovine genome-wide CRISPR knockout library in sheep testis (OA3.Ts) cells, targeting all protein-coding genes. Using this platform, we identified PEX11B, a peroxisomal membrane regulatory protein, as a strong restriction factor against orf virus (ORFV) infection. Removing PEX11B increased viral susceptibility and triggered severe cytopathic effects with membrane fusion and syncytia formation. Mechanistic studies showed that PEX11B knockout harmed peroxisomal integrity and disrupted lipid metabolism. This led to greater plasma membrane fluidity, creating a proviral environment that allowed more viral entry and replication. These results reveal a new antiviral function for PEX11B in blocking viral infection and underscore the importance of peroxisomal regulation in host-virus interactions.

Animals

Discovery of the order 'Quisvirales' redefines the evolution of RNA replication and transcription in the phylum Pisuviricota.

Genome replication in positive-stranded RNA (ssRNA+) viruses is mediated by cognate enzymes, including ubiquitous RNA-dependent RNA polymerase (RdRp). In ssRNA+ viruses with multiple open reading frames (ORFs) in their genomes, replication often is accompanied by synthesis of subgenomic RNAs (transcription) for expression of 3'-proximal ORFs. In addition, all ssRNA+ viruses with genomes larger than ~7&#xa0;kb encode helicases, linking helicases to RNA genome expansion. Helicases are essential ATPases that unwind nucleic acids and are classified into six recognized superfamilies (SF1-SF6). In the phylum Pisuviricota that includes important pathogens, helicases of SF1-SF3 are integrated into multi-enzyme replicase polyprotein(s) including 3C(-like) protease (3CLpro) and RdRp. Here, large-scale mining of invertebrate metatranscriptomes and targeted genome sequence assembly uncovered six spider-associated ssRNA+ viruses that, based on their conserved 3CLpro-RdRp module in replicase polyproteins, genome size (20-22&#xa0;kb), and phylogeny, form a family-like cluster in a putative order, named 'Quisvirales'. Quisviruses have similar genome and replicase architectures to enveloped coronaviruses and other nidoviruses. Notably, quisviruses encode ORFs 1a and 1b with predicted -1 programmed ribosomal frameshifting elements in the ORF1a/b overlap region. Using an original mapping approach for detecting chimeric sequencing reads, we obtained evidence that 3'-proximal ORFs are expressed via 5'-coterminal, leader-containing subgenomic RNAs. This suggests that the quisvirus subgenomic RNAs are generated through discontinuous transcription-a mechanism otherwise exclusively found in nidoviruses among the many ssRNA+ virus orders that synthesize subgenomic RNAs. Striking differences between nido- and quisviruses are, however, the RdRp being the only common core ORF1b-encoded enzyme and the replacement of the nidovirus SF1 helicase by a novel superfamily helicase. This quisvirus SF7 helicase, like the Picornavirales SF3 helicase, comprises an AAA+ (ATPase-like) domain typical for ring-forming helicases and thus must play an essential role in replication. The discovery of the order 'Quisvirales' demonstrates that viruses employing large replicase polyproteins of nidovirus-like complexity and discontinuous transcription may have evolved repeatedly from an 3CLpro-RdRp-encoding ancestor.

AAA+/RecA-like ATPase

Systematic discovery of pathogen effector functions across human pathogens and pathways.

Pathogens deploy effector proteins to exploit host cell biology, and most effector open reading frames (ORFs) are rapidly evolving and lack functional annotation. We developed the effector ORFeome (eORFeome), a scalable functional genomics platform encompassing 3,835 effector ORFs from diverse viruses, bacteria, and parasites. High-throughput barcoded screens across nuclear factor &#x3ba;B (NF-&#x3ba;B), apoptosis, p53, cGAS-STING, and major histocompatibility complex class I (MHC class I) pathways revealed novel pathway-modulating functions for hundreds of uncharacterized eORFs, unexpected activities of known effectors, and distinct pathway-specific functions encoded by single ORFs. Illustrating the power of this approach, we identified HHV6A U14 as a p53 antagonist, HHV7 U21 as a dual-function STING antagonist and MHC-I antigen display inhibitor, and adenoviral 13.6K/i-leader protein as a de novo-evolved TAP inhibitor that suppresses MHC-I display. These results establish a general framework for systematic effector annotation, uncover new mechanisms of host-pathogen interaction across kingdoms, and highlight pathogen effectors as a versatile toolkit for rewiring and probing human cellular pathways.

Humans

[Experimental infection of man using viral strains of bovine papular stomatitis, orf, pseudocowpox and milker's nodule].

An account is given of the close correlations that exist between virus strains of bovine papular stomatitis, orf, pseudocowpox, and milker's nodule. Reference is made to literature data on natural infection of man with the above virus strains. A report then is presented on experimental infection of human volunteers, using paravaccine birus. While fairly tough and elevated nodules, 4 mm to 5 mm in diameter, were produced on the probends' skin, no re-isolation of virus was achieved.

Animals

Translation of the downstream ORF from bicistronic mRNAs by human cells: Impact of codon usage and splicing in the upstream ORF.

Biochemistry textbooks describe eukaryotic mRNAs as monocistronic. However, increasing evidence reveals the widespread presence and translation of upstream open reading frames preceding the "main" ORF. DNA and RNA viruses infecting eukaryotes often produce polycistronic mRNAs and viruses have evolved multiple ways of manipulating the host's translation machinery. Here, we introduce an experimental model to study gene expression regulation from virus-like bicistronic mRNAs in human cells. The model consists of a short upstream ORF and a reporter downstream ORF encoding a fluorescent protein. We have engineered synonymous variants of the upstream ORF to explore large parameter space, including codon usage preferences, mRNA folding features, and splicing propensity. We show that human translation machinery can translate the downstream ORF from bicistronic mRNAs, albeit reporter protein levels are thousand times lower than those from the upstream ORF. Furthermore, synonymous recoding of the upstream ORF exclusively during elongation significantly influences its own translation efficiency, reveals cryptic splice signals, and modulates the probability of downstream ORF translation. Our results are consistent with a leaky scanning mechanism facilitating downstream ORF translation from bicistronic mRNAs in human cells, offering new insights into the role of upstream ORFs in translation regulation.

Humans

The snakehead retrovirus promoter functions independently of the 3'ORF protein and its products are maternally inherited in transgenic zebrafish.

The exogenous snakehead retrovirus (SnRV) is an unclassified member of the Orthoretrovirinae subfamily, discovered in cell lines derived from several fish species. SnRV resembles complex lentiviruses and potentially encodes accessory proteins, including the product of the 3' open reading frame (3'ORF). The 3'ORF protein was suggested to function as a transactivator of transcription (Tat). Here, we constructed an infectious molecular clone for SnRV and tested the effects of 3'ORF mutations on SnRV transcription. Although replacing 3'ORF with foreign sequences strongly reduced virus expression and production, an out-of-frame point mutation in 3'ORF had only a minimal effect on SnRV replication. This latter result suggests that the 3'ORF protein does not function as Tat and that SnRV transcription is largely independent of the product of this ORF. We also show that in vitro, the SnRV promoter is versatile and robustly functioning in both fish and mammalian cultured cells. Finally, the SnRV promoter was transiently active in injected zebrafish embryos as early as the blastula stage. In transgenic zebrafish, this promoter drives enhanced expression in sensory organs and gonads, and its generated products are maternally inherited. Considering these characteristics, the SnRV promoter emerges as a promising candidate for developing versatile expression vectors applicable to research and biotechnological applications.

Animals

Attenuation of bunyavirus replication by rearrangement of viral coding and noncoding sequences.

Bunyamwera virus (BUN) is the prototype virus of the family Bunyaviridae. BUN has a tripartite negative-sense RNA genome comprising small (S), medium (M), and large (L) segments. Partially complementary untranslated regions (UTRs) flank the coding region of each segment. The terminal 11 nucleotides of these UTRs are conserved between the three segments, while the internal regions are unique. The UTRs direct replication and transcription of viral RNA and are sufficient to allow encapsidation of viral RNA into ribonucleoprotein complexes. To investigate the segment-specific functions of the UTRs, we have used reverse genetics to recover a recombinant virus (called BUN MLM) in which the L segment open reading frame (ORF) is flanked by the M segment UTRs. Compared to wild-type virus, BUN MLM virus shows growth attenuation in cultured mammalian cells and a slower disease progression in mice, produces small plaques, expresses reduced levels of L mRNA and L (RNA polymerase) protein, synthesizes less L genomic and antigenomic RNA, and has an increased particle-to-PFU ratio. Our data suggest that the packaging of BUN RNAs is not segment specific. In addition, the phenotype of BUN MLM virus supports the finding that BUN UTRs differ in their regulation of RNA synthesis but suggests that the interplay between each segment UTR and its cognate ORF may contribute to that regulation. Since BUN MLM virus is attenuated due to an essentially irreversible mutation, the rearrangement of UTRs is a feasible strategy for vaccine design for the more pathogenic members of the Bunyaviridae.

Animals

Suppressor activity against cosuppression conferred by the petunia vein clearing virus genome.

The petunia genome contains an endogenous pararetrovirus, petunia vein clearing virus (PVCV). Previous analyses indicate that PVCV has suppressor activity against RNA silencing, but the suppressor protein has not been identified. Here we tested whether an open reading frame (ORF) of PVCV confers the activity that can suppress cosuppression of the CHS-A genes encoding chalcone synthase, which has a high rate of RNA turnover in the petal tissues of petunia. Petunia transformants that express PVCV ORF under the control of cauliflower mosaic virus 35S promoter were produced. The transgenic plants were crossed with those that have CHS-A cosuppression to produce plants that contain both the PVCV ORF transgene and CHS-A transgene. The coexistence of these transgenes resulted in phenotypic changes: pigmentation of various extents occurred on the originally white petals of CHS-A cosuppression phenotype. The generation of pigmented portions in flower petals coincided with higher transcript levels of CHS-A and PVCV ORF and less CHS-A short interfering RNA. These results indicate that the PVCV ORF can suppress CHS-A cosuppression and change the flower color phenotype when it is expressed as a transgene.

Petunia

An Evolutionary Framework Exploiting Virologs and Their Host Origins to Inform Poxvirus Protein Functions.

Poxviruses represent evolutionary successful infectious agents. As a family, poxviruses can infect a wide variety of species including humans, fish, and insects. While many other viruses are species-specific, an individual poxvirus species is often capable of infecting diverse hosts and cell types. For example, the prototypical poxvirus, vaccinia, is well known to infect numerous human cell types but can also infect cells from divergent hosts like frog neurons. Notably, poxvirus infections result in both detrimental human and animal diseases. The most infamous disease linked to a poxvirus is smallpox caused by variola virus. Poxviruses are large double-stranded DNA viruses, which uniquely replicate in the cytoplasm of cells. The model poxvirus genome encodes ~200 nonoverlapping protein-coding open reading frames (ORFs). Poxvirus gene products impact various biological processes like the production of virus particles, the host range of infectivity, and disease pathogenesis. In addition, poxviruses and their gene products have biomedical application with several species commonly engineered for use as vaccines and oncolytic virotherapy. Nevertheless, we still have an incomplete understanding of the functions associated with many poxvirus genes. In this chapter, we outline evolutionary insights that can complement ongoing studies of poxvirus gene functions and biology, which may serve to elucidate new molecular activities linked to this biomedically relevant class of viruses.

Animals

A positive-sense single-stranded RNA virus acquired a negative-sense open reading frame through recombination.

Although positive- and negative-sense single-stranded RNA viruses are ubiquitous in nature, there is currently no evidence of recombination or reassortment between viruses with these two major forms of genome organization. Here, we describe the discovery of brine shrimp virga-like virus 1 (BSVV1), a novel positive-sense single-stranded RNA virus with a recombinant genome structure derived from two viral phyla with differing genome organizations. The genome of BSVV1 comprises three open reading frames (ORFs). ORF1 resembles the RNA-dependent RNA polymerase of Ips virga-like virus 1 (a positive-sense RNA virus), while ORF2, transcribed in the positive orientation, is related to the glycoprotein of Hubei bunya-like virus 10 and other negative-sense RNA viruses. The predicted ORF3 was unique to BSVV1 without known homologs identified. The presence of the three protein products was verified by mass spectrometry. Notably, our analysis also revealed that BSVV1 is geographically widespread and found in brine shrimp from at least eight countries on four continents. In addition, BSVV1 was successfully cultured and proliferated to high viral loads during brine shrimp development. In sum, we provide compelling evidence of an ancient recombination event between negative- and positive-sense single-stranded RNA viruses, enriching our understanding of the evolution of genome structures in RNA viruses.

Open Reading Frames