Detection of poxvirus antigen and differentiation of closely related poxviruses by immunofluorescence staining.
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Antigens, Viral↗
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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↗
Identification and study of a poxvirus isolated from wild rodents in Turkmenia.
A new poxvirus was isolated in 1974 from the kidney of a wild big gerbil (Rhombomys opimus) caught in Turkmenia, where these gerbils are wide-spread. The virus resembles cowpox virus and is markedly different from the virus of infectious ectromelia, the best-known poxvirus of rodents. The new virus is apparently identical to other poxvirus isolates made from white rats and Felidae in the Moscow Zoo. Experimental inoculation of the natural hosts--big gerbils and yellow susliks (Citellus fulvus)--produced a severe infection with a high mortality rate. Trnasmission of virus to uninoculated cage mates was shown to occur. Virus persisted in convalescent animals and was present in urine 3 weeks after inoculation and in kidney and testis for at least 5 weeks after inoculation. The role of rodents as natural hosts of poxviruses is discussed.
Animal Population Groups↗
Sequence and analysis of the BamHI "D" fragment of Shope fibroma virus: comparison with similar regions of related poxviruses.
Differences observed in the virulence of two related leporipoxviruses are closely tied to a particular region of their genomes. For the virulent poxvirus of this pair, malignant rabbit fibroma virus (MV), this region is the BamHI "C" fragment, which is 10.7 kb. For the avirulent poxvirus, Shope fibroma virus, SFV, this region is the corresponding BamHI "D" fragment, which is 13.1 kb. As part of our attempt to understand the virulence of these two viruses, we sequenced these two DNA fragments. The sequence for the BamHI "C" fragment of MV is reported elsewhere (Strayer et al., 1991). We report here the sequence for SFV's BamHI "D" fragment and resultant open reading frames, and compare both DNA and open reading frame structures to those of MV and other known poxviruses. The BamHI "D" fragment of SFV contains 12 open reading frames of 100 amino acids or more, arranged similarly to orf's in MV and vaccinia. Striking similarities between SFV and MV are seen in certain parts of this restriction fragment, including substantial stretches of DNA in which the two viruses are identical. Clear homologies exist between these leporipox virus genomes and those of other related poxviruses. To understand the pathogenesis of virus infection, one must appreciate the structure of those viral genes that play important roles in infection.
Base Sequence↗
Studies on Cotia virus--an unclassified poxvirus.
This paper is a report of studies on Cotia virus; this had been first isolated in 1965 in Brazil and was subsequently shown to be a poxvirus. Cotia virus grew in a wide range of cell cultures and on the chick chorioallantois (CAM), Its growth characteristics are similar to those of other poxviruses. Microscopy showed virus factories or type B inclusions appearing before infectious progeny virus could be demonstrated. Type A inclusions appeared later, after development of progeny virus; these were shown by electron microscopy to differ from the type A inclusions of cowpox and other poxviruses and they have been termed Cotia bodies. Immunofluorescent staining also showed ring structures which appeared before the development of Cotia bodies. The growth of Cotia virus in human embryo lung (HEL) cells was sensitive to inhibitors of DNA and protein synthesis but was resistant to a concentration of rifampicin which inhibited vaccinia virus. Sharing of antigens between the Cotia virus and vaccinia virus was shown by gel precipitation tests and immunofluorescent staining. There was no cross neutralization between Cotia virus and vaccinia virus nor did anti-Cotia sera neutralize representatives of other poxvirus groups.
Brazil↗
Poxvirus pathogenesis.
Poxviruses are a highly successful family of pathogens, with variola virus, the causative agent of smallpox, being the most notable member. Poxviruses are unique among animal viruses in several respects. First, owing to the cytoplasmic site of virus replication, the virus encodes many enzymes required either for macromolecular precursor pool regulation or for biosynthetic processes. Second, these viruses have a very complex morphogenesis, which involves the de novo synthesis of virus-specific membranes and inclusion bodies. Third, and perhaps most surprising of all, the genomes of these viruses encode many proteins which interact with host processes at both the cellular and systemic levels. For example, a viral homolog of epidermal growth factor is active in vaccinia virus infections of cultured cells, rabbits, and mice. At least five virus proteins with homology to the serine protease inhibitor family have been identified and one, a 38-kDa protein encoded by cowpox virus, is thought to block a host pathway for generating a chemotactic substance. Finally, a protein which has homology with complement components interferes with the activation of the classical complement pathway. Poxviruses infect their hosts by all possible routes: through the skin by mechanical means (e.g., molluscum contagiosum infections of humans), via the respiratory tract (e.g., variola virus infections of humans), or by the oral route (e.g., ectromelia virus infection of the mouse). Poxvirus infections, in general, are acute, with no strong evidence for latent, persistent, or chronic infections. They can be localized or systemic. Ectromelia virus infection of the laboratory mouse can be systemic but inapparent with no mortality and little morbidity, or highly lethal with death in 10 days. On the other hand, molluscum contagiosum virus replicates only in the stratum spinosum of the human epidermis, with little or no involvement of the dermis, and does not spread systemically from the site of infection. The host response to infection is progressive and multifactorial. Early in the infection process, interferons, the alternative pathway of complement activation, inflammatory cells, and natural killer cells may contribute to slowing the spread of the infection. The cell-mediated response involving learned cytotoxic T lymphocytes and delayed-type hypersensitivity components appears to be the most important in recovery from infection. A significant role for specific antiviral antibody and antibody-dependent cell-mediated cytotoxicity has yet to be demonstrated in recovery from a primary infection, but these responses are thought to be important in preventing reinfection.
Amino Acid Sequence↗
[Poxvirus infections in a cat and its owner].
A poxvirus infection in a cat and its owner is recorded. The clinical course of the disease as well as the pathological and the histopathological changes are described. The diagnosis "poxvirus infection" was confirmed by electronmicroscopy and cell culture techniques. This is the first human poxvirus infection in the Federal Republic of Germany in which the source of infection was found to be an infected cat. The serological investigation of 104 cat sera revealed antibody titres in 14 cases, indicating that poxvirus infections in cats are not isolated cases.
Animals↗
Field and experimental studies of poxvirus infections in rodents.
Data are presented from serological and virological investigations of natural and experimental infections, in rodents, with different poxviruses. The demonstration of poxvirus antibodies (virus neutralizing antibodies, antihaemagglutinins, and precipitins) in the kidneys and/or lungs of rodents from Europe and Africa, and the isolation of poxviruses from them, all indicate that rodents may be poxvirus carriers. Isolates from rodents differed in their properties. Some, from Turkmenistan rodents or from white rats caught near Moscow, appeared to be very close to cowpox virus, while others (from Zaire rodents) were identical to variola-like (whitepox) viruses found earlier in monkeys in the same region. The results suggest that rodents that carry the virus closely similar to cowpox virus might be a source of infection for other animal species.
Animals↗
Rapid Generation of Recombinant Poxviruses Using CRISPR/Cas9 Gene Editing.
The low-frequency natural recombination that is detected in poxvirus-infected cells has long been used to genetically modify poxviruses. Such recombinant poxviruses have found many applications as vaccines for preventing infectious diseases and as experimental cancer therapeutics. Unfortunately, these methods are time consuming, can leave behind "scars" or selectable markers, and many months of work may be required to generate plaque-purified recombinants bearing multiple virus gene substitutions, deletions, and/or inserted transgenes. Over the last decade, several reports have described how CRISPR/Cas9 technologies can be used to better facilitate genetic manipulation of vaccinia virus (VACV). These protocols use Cas9/gRNA complexes to introduce double-stranded breaks into specific sites in virus genomic DNA either in vivo or in vitro. Recombination-repair reactions are then employed to repair the breaks using transfected DNAs encoding the required homologies and desired mutation(s). Here we describe a method where we combine CRISPR/Cas9 genome editing in vitro, followed by Leporipoxvirus-catalyzed repair and reactivation of the cut VACV DNA using repair fragments provided in trans. This method optimizes several steps in the preparation of the CRISPR/Cas9-cut VACV DNA and can be used to introduce mutations at multiple sites without requiring selectable markers. It also provides some guidance regarding how the position of the CRISPR/Cas9-cuts can affect co-conversion of flanking markers embedded in the repair fragment. The method allows researchers to quickly generate recombinant VACV bearing multiple genetic alterations and using only a single round of reactivation and plating.
CRISPR-Cas Systems↗
Quantitative assessment of poxvirus promoters in fowlpox and vaccinia virus recombinants.
A comparison was undertaken of poxvirus promoters in vaccinia and fowlpox virus (FPV) recombinants using the level of beta-galactosidase expressed from the LacZ gene as a measure of promoter function. In this study a comparison was made of the vaccinia virus promoters, P 7.5 and P L11, the major late promoter of cowpox virus, P CPX (expressing the abundant inclusion body protein), and the FPV promoters, P E/L and P L. In vaccinia virus recombinants the FPV P E/L promoter expressed one-third to one-half the level of beta-galactosidase expressed by the P L11 promoter. In comparison with the P 7.5 promoter, the FPV P E/L promoter expressed four to five times the level of beta-galactosidase. In FPV recombinants beta-galactosidase activity expressed was equal for the P E/L and P CPX promoters. Levels expressed by P L11 and P L were one-half and one-fifth that level, respectively. The temporal regulation of the promoters was maintained in both vaccinia virus and FPV recombinants. The P E/L promoter of FPV has the TAAATG sequence characteristic of late poxvirus promoters at the transcription initiation site. In an attempt to enhance the utility of this promoter for the expression of foreign genes in FPV and vaccinia virus recombinants, the effect upon promoter function of changing the G of the ATG to A, T, or C was determined using transient expression assays with vaccinia virus. Substitution of A, T, or C for the G abolished promoter function. Because of its early/late function, the level of expression and the presence of the oppositely oriented late P L promoter, the FPV P E/L promoter will be valuable for the expression of foreign genes in poxvirus recombinants.
Base Sequence↗
Inverted terminal repeats in rabbit poxvirus and vaccinia virus DNA.
In both rabbit poxvirus and vaccinia virus DNA have demonstrated an identical distribution of eight HinfI. The length of the terminal repeats was found to be 3.4 to 3.6 megadaltons (Mdaltons) for rabbit poxvirus DNA and 7.4 to 8.0 Mdaltons for vaccinia virus DNA. Maps of the HinfI restriction sites within isolated EcoRI end fragments of rabbit poxvirus and vaccinia virus DNA PHAVE DEMONSTRATED AN IDENTICAL DISTRIBUTION OF EIGHT HinfI sites in an internal part (approximately 2 Mdaltons) of the EcoRI end fragments of the two genomes.
Animals↗
Effect of marker distance and orientation on recombinant formation in poxvirus-infected cells.
Little is known about the mechanism of poxvirus recombination even though construction of recombinant viruses by recombination-dependent methods is a widely adopted technique. We have shown previously that transfected DNAs are efficiently recombined while replicating in cells infected with Shope fibroma virus. Because recombinant DNA can be recovered from infected cells as a high-molecular-weight head-to-tail concatemer, it was possible to transfect genetically marked lambda DNAs into infected cells and assay recombinants as bacteriophage particles following in vitro packaging. This approach was used in this study to examine how marker distance and marker orientation influence recombination in Shope fibroma virus-infected cells. Simple two-factor crosses were readily modelled by using a mapping function derived from classical phage studies and showed low negative interference (I = -2.8 +/- 0.5) in crosses involving markers greater than 100 bp apart. More complex four- and five-factor crosses showed that the recombination frequency per unit distance was not constant (rising as the marker separation was reduced from 100 to 1 bp) and that crosses performed in poxvirus-infected cells are subject to high negative interference. One consequence is that marker orientation does not dramatically influence the outcome of most Shope fibroma virus-catalyzed crosses in clear contrast to what is observed in adenovirus or simian virus 40-infected cells. These results can be interpreted to indicate that similar statistical and physical constraints influence both viral and phage recombination and suggest that heteroduplexes may be important intermediates in the poxvirus recombination process.
Animals↗
In vitro resolution of poxvirus replicative intermediates into linear minichromosomes with hairpin termini by a virally induced Holliday junction endonuclease.
Available evidence suggests that one or more late viral gene products are involved in processing poxvirus replicative intermediates into mature progeny hairpin-terminated genomes. Cloned versions of the Shope fibroma virus (SFV) replicated telomere in the inverted repeat configuration were used as substrates to assay lysates from poxvirus-infected cells for protein fractions that participate in the resolution of the circular substrate plasmid into a linear minichromosome with viral hairpin termini. An activity in a crude protein fraction obtained from vaccinia virus-infected cells at late times during the replicative cycle was capable of accurately resolving all poxviral inverted repeat replicative intermediates tested. The resolved linear products are identical to the products of in vivo resolution and possessed symmetrical nicks which mapped at the borders of the inverted repeat sequence. Strand-specific nicks were also identified, which mapped within the telomere resolution target sequence known to be required for telomere resolution in vivo. The resolving activity that we have identified is specific to virus-infected cells at late times during replication and cleaves cloned poxviral telomeric substrates in a fashion expected of a classic Holliday junction-resolving enzyme in addition to possessing a telomere resolution target-specific nicking activity. Although a Holliday junction-resolving activity would also be expected to play a role in the recombination induced by poxvirus infection, the appearance of the activity described here only after the commencement of viral late protein synthesis suggests that it functions strictly at late times. Other non-viral Holliday junction analogs can also be cleaved by this extract, suggesting that this component of the resolution activity may also play a role in other viral processes that require cleavage of a branched DNA structure. Thus, we have identified a poxviral activity that may be a part of a protein complex which resolves concatemeric replicative intermediates of viral DNA as well as participate in general recombination late during infection.
Animals↗
Preliminary studies on an unusual poxvirus of the western grey squirrel (Sciurus griseus griseus) of North America.
Particles of a poxvirus found in Californian western grey squirrels have overall dimensions indistinguishable from those of the poxviruses of the genera Orthopoxvirus and Leporipoxvirus, but have surface structures somewhat reminiscent of virions of members of the genus Parapoxvirus. Extracts of tissues infected with the squirrel poxvirus cross-react in gel-diffusion tests with extracts of tissues infected with Californian myxoma virus, but not with a similar preparation infected with a South American strain of myxoma virus.
Animals↗
Human poxvirus disease after smallpox eradication.
A 5-year-old boy living in a small camp in the rural Ivory Coast had a disease resembling smallpox. This occurred 4 years after smallpox had been eradicated from the Ivory Coast and 1.5 years after the last case of smallpox was detected in West and Central Africa. Clinical, serological, and epidemiological evidence indicated this disease was probably monkeypox, a poxvirus of the variola/vaccina subgroup. A serologic survey of poxvirus antibodies in the wild animal population detected neutralizing antibodies in rodents, larger mammals, primates, and birds. The laboratory and ecological characteristics of poxviruses require further elucidation, especially those which have been found in animals near human monkeypox cases.
Adolescent↗
Poxvirus-based vectors as vaccine candidates.
The advent of recombinant DNA techniques and advances in immunology have provided a means for dissecting the immunobiology of disease-causing agents. Identification and expression of individual genes from the pathogens in heterologous systems, such as VV, have yielded valuable information regarding structural properties of the gene products and their role in eliciting protective immunity. Targets of both humoral and/or cellular immunity for many disease-causing agents have been identified or confirmed using a VV expression system (Section IV). Additionally, specific VV recombinants have induced a protective immune response in experimental animals. The ability of VV recombinants to induce pertinent immune responses necessary for protection, the potential to develop polyvalent vaccines, and the successful history of VV as an immunizing agent provide the impetus for engineering VV as a live recombinant vaccine candidate. Critical to the refinement of poxviruses as recombinant immunizing agents is a more in-depth knowledge of the molecular biology of these viruses. Although significant advances have been made in this area within the past 10 years, a greater understanding of the mechanisms governing gene expression and viral virulence factors should enable the development of more safe and effective vaccine candidates. Progression of VV vector technology to other members of the poxvirus family has been successful. Development of other poxviruses as vectors may, therefore, provide a means of generating host-restricted vaccines. Fowlpox recombinant viruses, for instance, may yield candidate vaccines in the poultry industry. Interestingly, it was also demonstrated that these host-restricted recombinant viruses can be used as immunizing vehicles in other species. The ability of a nonreplicating viral vector to elicit a protective immune response is especially intriguing in light of the observation by Morgan et al. that a VV/EBV gp340/220 recombinant, derived from an avirulent VV strain, was unable to protect cottontop tamarins from a live EBV challenge.
Animals↗
Bioinformatics for the Structural Genomics of Poxviruses.
Poxviruses are large, complex viruses, and their host species are widespread across the tree of life. As a result, the bioinformatics analysis of their genomes can be complex. Here we show how a few helpful tools and strategies can be used to inform the analysis, leading to a better understanding of the structural properties of poxvirus genomes and to a more accurate quality control of, or comparison between, assembled sequences.
Poxviridae↗
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↗