Whole-genome based strain identification of fowlpox virus directly from cutaneous tissue and propagated virus.

Fowlpox (FP) is an economically important viral disease of commercial poultry. The fowlpox virus (FPV) is primarily characterised by immunoblotting, restriction enzyme analysis in combination with PCR, and/or nucleotide sequencing of amplicons. Whole-genome sequencing (WGS) of FPV directly from clinical specimens prevents the risk of potential genome modifications associated with in vitro culturing of the virus. Only one study has sequenced FPV genomes directly from clinical samples using Nanopore sequencing, however, the study didn't compare the sequences against Illumina sequencing or laboratory propagated sequences. Here, the suitability of WGS for strain identification of FPV directly from cutaneous tissue was evaluated, using a combination of Illumina and Nanopore sequencing technologies. Sequencing results were compared with the sequence obtained from FPV grown in chorioallantoic membranes (CAMs) of chicken embryos. Complete genome sequence of FPV was obtained directly from affected comb tissue using a map to reference approach. FPV sequence from cutaneous tissue was highly similar to that of the virus grown in CAMs with a nucleotide identity of 99.8%. Detailed polymorphism analysis revealed the presence of a highly comparable number of single nucleotide polymorphisms (SNPs) in the two sequences when compared to the reference genome, providing essentially the same strain identification information. Comparative genome analysis of the map to reference consensus sequences from the two genomes revealed that this field isolate had the highest nucleotide identity of 99.5% with an FPV strain from the USA (Fowlpox virus isolate, FWPV-MN00.2, MH709124) and 98.8% identity with the Australian FPV vaccine strain (FWPV-S, MW142017). Sequencing results showed that WGS directly from cutaneous tissues is not only rapid and cost-effective but also provides essentially the same strain identification information as in-vitro grown virus, thus circumventing in vitro culturing.

Genetic manipulation of poxviruses using bacterial artificial chromosome recombineering.

Traditional methods for genetic manipulation of poxviruses rely on low-frequency natural recombination in virus-infected cells. Although these powerful systems represent the technical foundation of current knowledge and applications of poxviruses, they require long (≥ 500 bp) flanking sequences for homologous recombination, an efficient viral selection method, and burdensome, time-consuming plaque purification. The beginning of the twenty-first century has seen the application of bacterial artificial chromosome (BAC) technology to poxviruses as an alternative method for their genetic manipulation, following the invention of a long-sought-after method for deriving a BAC clone of vaccinia virus (VAC-BAC) by Arban Domi and Bernard Moss. The key advantages of the BAC system are the ease and versatility of performing genetic manipulation using bacteriophage λ Red recombination (recombineering), which requires only ∼50 bp homology arms that can be easily created by PCR, and which allows seamless mutations lacking any marker gene without having to perform transient-dominant selection. On the other hand, there are disadvantages, including the significant setup time, the risk of contamination of the cloned genome with bacterial insertion sequences, and the nontrivial issue of removal of the BAC cassette from derived viruses. These must be carefully weighed to decide whether the use of BACs will be advantageous for a particular application, making pox-BAC systems likely to complement, rather than supplant, traditional methods in most laboratories.

Fowlpox virus vaccines for HIV and SHIV clinical and pre-clinical trials.

DNA prime and recombinant fowlpox virus (rFPV) boost vaccines were designed to express multiple HIV or SIV antigens for use in human clinical trials and in pre-clinical trials in macaques. Three sets of vaccines with matching HIV or SIV antigen sets, modified for vaccine safety considerations, were constructed and shown to express the relevant proteins. The rFPV vaccines with inserts at up to three sites, were stable on passage in chick cell culture, including during GMP manufacture of vaccines for human Phase I clinical trials. Cellular and humoral immunogenicity in mice was demonstrated using a DNA prime/rFPV boost and vaccinia virus challenge model. These data establish a preliminary safety and efficacy profile for these multigenic vaccines suggesting they are suitable for advanced development as candidate HIV vaccines.

Morphogenesis of fowlpox virus in a baby hamster kidney cell line.

Fowlpox virus (FWPV) recombinant vaccines are presently being tested as an antihuman immunodeficiency virus vaccine for humans. However, biosafety, as well as the morphogenesis of FWPV in mammalian cells, are not well understood. Currently, electron microscopy is the method of choice for analyzing virus morphogenesis in cell lines. In this study, four different electron microscopic techniques were used to study FWPV morphogenesis in the Syrian baby hamster kidney (BHK-21) cell line: direct negative stain electron microscopy, ultrathin section transmission electron microscopy, cryoimmunoelectron microscopy, and scanning electron microscopy. The study showed matured viruses, as well as other stages of fowlpox virus maturation, in BHK-21 cells that led to productive virus multiplication. A number of virus-containing vesicles and plasma membrane-associated mature viruses at an early stage in the budding process were observed. In addition, intracellular mature virus was observed in layers of the trans-Golgi network, a characteristic of intracellular mature virus wrapping that results in the formation of intracellular enveloped virus. The size and morphology of FWPV observed in this study are comparable with previously published data. This study presents the first morphological evidence for the release of FWPV by budding in BHK-21 cells.

Growth and infectivity assays of the Israeli vaccine strain of fowl poxvirus in chicken embryo fibroblasts.

The Israeli vaccine strain of fowl poxvirus grows efficiently in chicken embryo fibroblasts but not in cell lines derived from monkey kidney or human fibroblasts. We developed two assays for the titration of the infectivity of this virus in secondary cultures of chicken embryo fibroblasts. The first is a focus assay, in which minimum essential medium and SeaKem ME agarose were used for the overlay media. Under these conditions, clear virus foci appeared after 5 days of incubation at 37 C. The second assay is a semiautomatic colorimetric test based on the ability of live cells in culture to reduce the yellow tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; thiazolyl blue) to its formazan derivative. The reagent was added to infected chicken embryo fibroblasts in 96-well plates 10 days after infection. The formazan formed during 2 hr was extracted with dimethyl sulfoxide, and its absorbance was read by an automatic microplate spectrophotometer. A good correlation of the infectivity titers of the virus was obtained by the two methods.

Improved technique for transient expression and negative strand virus rescue using fowlpox T7 recombinant virus in mammalian cells.

The suitability of recombinant T7 polymerase produced using either the highly attenuated MVA strain of vaccinia (MVA-T7) or fowlpox virus (FP-T7) for transient expression and negative strand virus rescue was compared in two mammalian cell lines (MDBK and Vero) and in primary cells of bovine, ovine and caprine origin. Such primary cells are more permissive for the growth of wild type strains of morbilliviruses, such as Rinderpest virus and Peste des petits ruminants virus. MVA-T7 was found to be highly cytopathic in the primary cells, multiplying rapidly and killing the cells within 3-5 days of infection, even when very low multiplicities of infection (MOI) were used. In contrast, FP-T7, which appeared to express similar amounts of T7 polymerase, was found to be non-cytopathic in a variety of primary and established cell lines of mammalian origin and was suitable for use in virus rescue experiments. MDBK cells and primary cells, unlike Vero cells, could not be efficiently transfected and so were unsuitable for virus rescue. Optimal conditions for rinderpest virus rescue in Vero cells were established using FP-T7 in place of MVA-T7. This system will be suitable for rescuing other viruses which grow in Vero cells.

Morphogenesis and release of fowlpox virus.

Release of fowlpox virus (FWPV) as extracellular enveloped virus (EEV) appears to proceed both by the budding of intracellular mature virus (IMV) through the plasma membrane and by the fusion of intracellular enveloped virus (IEV) with the plasma membrane. Based on the frequency of budding events compared to wrapping events observed by electron microscopy, FWPV FP9 strain seems to exit chick embryo fibroblast cells predominantly by budding. In contrast to vaccinia virus (VV), the production of FWPV extracellular virus particles is not affected by N(1)-isonicotinoyl-N(2)-3-methyl-4-chlorobenzoylhydrazine (IMCBH). Comparison of the sequence of the VV F13L gene product with its FWPV orthologue showed a mutation, in the fowlpox protein, at the residue involved in IMCBH resistance in a mutant VV. Glucosamine, monensin or brefeldin A did not have any specific effect on FWPV extracellular virus production. Cytochalasin D, which inhibits the formation of actin filaments, reduces the production of extracellular virus particles by inhibiting the release of cell-associated enveloped virus (CEV) particles from the plasma membrane. Involvement of actin filaments in this mechanism is further supported by the co-localization of actin with viral particles close to the plasma membrane in the absence of cytochalasin D. Actin is also co-localized with virus factories.

Fowlpox virus encodes nonessential homologs of cellular alpha-SNAP, PC-1, and an orphan human homolog of a secreted nematode protein.

The genome of fowlpox virus (FWPV), type species of the Avipoxviridae, is considerably rearranged compared with that of vaccinia virus (the prototypic poxvirus and type species of the Orthopoxviridae) and is 30% larger. It is likely that the genome of FWPV contains genes in addition to those found in vaccinia virus, probably involved with its replication and survival in the chicken. A 7,470-bp segment of the FWPV genome has five open reading frames (ORFs), two of which encode ankyrin repeat proteins, many examples of which have been found in poxviruses. The remaining ORFs encode homologs of cellular genes not reported in any other virus. ORF-2 encodes a homolog of the yeast Sec17p and mammalian SNAP proteins, crucial to vesicular transport in the exocytic pathway. ORF-3 encodes a homolog of an orphan human protein, R31240_2, encoded on 19p13.2. ORF-3 is also homologous to three proteins (YLS2, YMV6, and C07B5.5) from the free-living nematode Caenorhabditis elegans and to a 43-kDa antigen from the parasitic nematode Trichinella spiralis. ORF-5 encodes a homolog of the mammalian plasma cell antigen PC-1, a type II glycoprotein with exophosphodiesterase activity. The ORFs are present in the virulent precursor, HP1, of the sequenced attenuated virus (FP9) and are conserved in other strains of FWPV. They were shown, by deletion mutagenesis, to be nonessential to virus replication in tissue culture. RNA encoding the viral homolog of PC-1 is expressed strongly early and late in infection, but RNAs encoding the homologs of SNAP and R31240_2 are expressed weakly and late.

Role of the fowlpox virus thymidine kinase gene for the growth of FPV recombinants in cell culture.

Fowlpox virus (FPV) insertion plasmids were constructed that, upon integration into the viral genome via in-vivo recombination, inactivate the viral thymidine kinase (tk) gene. Using this approach, no wild-type virus-free stocks of recombinant virus could be obtained. In contrast, either integration of foreign genes into the intergenic region of the intact FPV tk gene and the open reading frame located downstream, or the functional substitution of the inactivated FPV tk gene by an intact vaccinia virus tk gene resulted in the predicted stable recombinants that were free of wild-type virus. Our results suggest that in already highly attenuated poxvirus strains an intact tk gene is essential for efficient growth of the virus in cell culture.

Protection and synergism by recombinant fowl pox vaccines expressing genes from Marek's disease virus.

Recombinant fowl poxviruses (rFPV) were constructed to express genes from serotype 1 Marek's disease virus (MDV) coding for glycoproteins B (gB1), C (gC), and D (gD) and tegument proteins UL47 and UL48, as well as genes from serotypes 2 and 3 MDV coding for glycoprotein B (gB2 and gB3). These rFPVs, alone and in various combinations, including combinations of fowl poxvirus (FPV)/gBs with turkey herpesvirus (HVT), were evaluated for ability to protect maternal antibody-positive (ab+) and -negative (ab-) chickens against challenge with highly virulent MDV isolates. The protective efficacy was also compared with that of prototype Marek's disease (MD) vaccines. No protection was induced in ab+ chickens by rFPV expressing gC, gD, UL47, or UL48. In contrast, the rFPV/gB1 construct protected about 23% of ab+ chickens against MDV challenge compared with 26% for cell-associated HVT. Levels of protection by rFPV/gBs of different MDV serotypes was highest for gB1, intermediate for gB2, and lowest for gB3. When rFPV/gB1 was combined with cell-associated HVT, protection was enhanced by an average of 138% compared with the best component monovalent vaccine, and the mean level of protection was 59% compared with 67% for the HVT+SB-1 bivalent vaccine. Relatively high protection (50%) and enhancement (200%) were also observed between rFPV/gB1 and cell-free HVT. These results suggest a specific synergistic interaction between rFPV/gB1 and HVT, possibly analogous to that previously described between serotypes 2 and 3 viruses. Levels of protection by rFPV/ gB1 alone or by bivalent rFPV/gB1+cell-associated HVT were similar to those of conventional cell-associated MD vaccines. However, the bivalent rFPV/gB1+cell-free HVT vaccine was clearly more protective than cell-free HVT alone and, thus, may be the most protective, entirely cell-free MD vaccine thus far described.

Role of the TK+ phenotype in the stability of pigeonpox virus recombinant.

Insertion of foreign DNA containing the E. coli gpt marker by homologous recombination in the pigeonpox virus (PPV) thymidine kinase (TK) gene and selection for the presence of this DNA in the viral genome produced unstable recombinants after 3 plaque purifications. We highlight the persistence of duplicated TK DNA sequences arising from single crossing over, due to the growth advantage of TK+ virus. Restoration of the TK function by coinsertion of the vaccinia virus TK gene led to stable TK+ recombinants arising from double crossing over.

Identification and functional analysis of the fowlpox virus homolog of the vaccinia virus p37K major envelope antigen gene.

A fowlpox virus (FPV) gene with homology to the vaccinia virus p37K major envelope antigen gene was identified and sequenced. The predicted product has a molecular weight of 43,018 Da (p43K). The FPV p43K gene has 37.5% identity with its vaccinia counterpart and higher homology with a molluscum contagiosum virus gene (42.6% identity). Based on upstream sequences, p43K appears to be regulated as a late gene. Recombinant FPV were generated in which a large portion of p43K was replaced by the Escherichia coli lacZ gene. These recombinants failed to produce visible plaques under standard conditions. After prolonged incubation the microplaques developed into small macroscopic plaques. Plaques were purified on the basis of lacZ expression. Single-cycle growth curves comparing the p43K-deleted recombinant (designated fJd43Z) with parental FPV showed that the two viruses produce identical amounts of intracellular virions, but that fJd43Z released 20-fold fewer infectious particles into the medium. CsCl gradient centrifugation of [3H]thymidine-labeled virus was employed to examine differences in the production of physical particles. The two viruses produced equivalent levels of intracellular virions, but fJd43Z failed to produce detectable levels of released particles. FPV p43K is therefore involved in the release of virions from infected cells.

Structural proteins of two different plaque-size phenotypes of fowlpox virus.

Structural polypeptides of two plaque-purified variant isolates of fowlpox virus differing in plaque morphology and size were examined by Coomassie blue-staining and immunoblot analysis of purified virions. A total of 30 structural polypeptides were observed, ranging in molecular weight from 14,100 to 122,600. A late polypeptide of 36,400 molecular weight was quite prominent in the small-plaque clone but absent in the large-plaque clone. Two other polypeptides, of 33,700 and 34,800 molecular weight, were present in virions from large-plaque virus and cell lysates of both clones but were absent in the small-plaque virions. These differences were observed whether the viruses were grown in chorioallantoic membrane or in chicken embryo fibroblast cultures. No difference was observed between the growth curves of the two virus clones. Differences observed in the polypeptides of the two viruses may be due to changes in the less conserved regions of viral DNA and may be used for differentiation of virus isolates.

Effect of complement depletion by cobra venom factor on fowlpox virus infection in chickens and chicken embryos.

The course of infection with an attenuated strain of fowlpox virus (FPV), which is known to induce antibody-independent activation of complement via the alternative pathway, was investigated in 1- to 3-day-old chickens and 14-day-old chicken embryos by treatment with cobra venom factor (CVF). CVF was found to inhibit complement activity transiently via the alternative pathway but not via the classical pathway. In chickens treated with CVF, virus growth in the skin was enhanced, and pock lesions tended to disseminate, leading to fatal infection in some birds. Histologically, an acute inflammation at an early stage of infection (within 3 days) was inhibited, and virus content in the pock lesion was increased. In chicken embryos with immature immune capacities, CVF treatment caused changes in pock morphology from clear pocks to diffuse ones, an increase in virus content in the pock, and inhibition of cell infiltration. Thus, FPV infection was aggravated in both CVF-treated chickens and chicken embryos. These results are discussed in relation to roles of complement in the elimination of virus at an early stage of FPV infection.

Morphogenesis of canary poxvirus and its entrance into inclusion bodies.

A virus isolated from a natural outbreak of canarypox was replicated on the chorioallantoic membranes of chicken embryos, and its ultrastructure and development were observed. Electron microscopy of thin sections of pocks produced on the chorioallantoic membranes revealed a variety of developmental forms which appear similar to those demonstrated in studies of vaccinia, ie, viroplasm or viral factories; immature, undifferentiated virions partially enclosed by membranes; completely enclosed nondifferentiated spherical or oval virions; immature virions with discrete nucleoids; and the more compact brick-shaped mature virions. Two types of A-type inclusions were noted: those with virions around the periphery, and those filled with virus particles. The appearance of mature viruses within the inclusion bodies and different stages of viruses outside the inclusion indicate that in a course of development, maturing poxvirus may enter the inclusion bodies as they acquire surface tubules on their envelopes. Mature virions also were seen budding out of the cell membrane, apparently enveloped in a portion of the membrane. Studies showing the entrance of poxvirus into inclusion bodies have not been reported. In this report, electron micrographs are shown of viruses entering inclusion bodies.

Activation of chicken alternative complement pathway by fowlpox virus-infected cells.

Fresh normal chicken serum (NCS) which lacked virus-neutralizing antibody to fowlpox virus (FPV) was found to inhibit the appearance of the cytopathic effect of the virus, virus growth, and plaque formation in chicken embryo cells. Immunofluorescent examination revealed the deposition of the third component of complement (C3) on FPV-infected chicken embryo cells incubated with fresh NCS. The inhibitory activity of fresh NCS on viral cytopathic effect was independent of the Ca2+ ion and was abrogated by treatment of fresh NCS with inulin or zymosan. Similarly, deposition of C3 on FPV-infected cells occurred independently of the Ca2+ ion and was inhibited by treatment of fresh NCS with inulin or zymosan but was not inhibited by absorption with FPV-infected cells. These results suggest that antibody-independent activation of complement by FPV-infected cells via the alternative pathway caused the inhibition of the virus growth as well as the C3 deposition. Involvement of complement activation as nonspecific host response to virus infection was also suggested by the demonstration of the C3 deposition in the skin lesions of FPV-infected chickens.

A simple technique for preparation of chicken-embryo-skin cell cultures.

A simple, rapid technique was developed for preparing chicken-embryo-skin cell cultures utilizing trypsinization of the skin of intact 12-day-old chicken embryos. When cell cultures were inoculated with fowl pox virus, those that consisted of at least 80% epithelial cells yielded a higher virus titer than fibroblast cell cultures.

Isolation of surface tubules of fowlpox virus.

Surface tubules of fowlpox virus were isolated using chemical and physical methods. Suspensions of lipid cytoplasmic inclusion bodies were obtained by treating infected chorioallantoic membranes with 1% trypsin. Inclusions were treated with ultrasonic sound, detergents, and enzymes and were examined by electron microscopy. Although lipase treatment altered the morphology of lipid inclusions, no viral surface tubules were recovered. Treatment with the detergent Nonidet-P40 followed by 2-mercaptoethanol disrupted virions without allowing surface tubules to be recovered. Disruption of lipid inclusions by ultrasonic sound or manual grinding of chorioallantoic membranes produced free virions but only small numbers of tubules. These results indicate that surface tubules can be recovered, but that the lipid nature of cytoplasmic inclusions interferes with procedures commonly used in tubule purification.

Heat-selected mutants of pigeon pox virus.

Two mutants of pigeon pox virus were derived from virus isolated from naturally infected pigeons. One (S 39) was obtained by cultivation of the original virus in chick embryo chorioallantoic membranes at 39 degrees C, and the second (S m) by heating the original virus at 56 degrees C for 30 min. The mutants were less pathogenic to pigeons than the original virus. The original virus and the mutant S 39, but not the mutant S m produced plaques in primary chick embryo cell cultures. Pigeons inoculated with the mutants were resistant to challenge with the field virus.