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.

Pathological, molecular and phylogenic study of fowlpox virus in domesticated chickens of Tikrit City, Iraq

Fowlpox virus (FPV) is one of the viruses affecting chickens worldwide, causing pathological and economic losses in the poultry industry. Viral lesions are easily recognizable by the eye and usually appear in the featherless areas, especially the head. Moreover, the virus could lead to blindness and mortality in some cases. This study diagnosed the suspected fowlpox cases, identified and classified the causative agent. We also analyzed the differences and similarities of closely related viruses at the neighboring and regional countries. Fifty samples were collected from three locations of Tikrit city from the domesticated chickens, which showed cutaneous lesions. Virus DNA was extracted directly from tissue samples before the nested PCR technique was performed. The virion core protein (P4b) gene is partially sequenced and analyzed with routine histological sectioning. Results showed that the virus causes pock lesions of dermal hyperplasia and hyperkeratosis. Hyperplasia and congestion of the chorioallantoic membrane were also recorded. The study also showed that the DNA of FPV could be extracted directly from animal tissue without further purification. The sequence analysis showed that the FPV was confirmed in all samples clustered in clade A identical with Iranian and Egyptian isolates. In conclusion, this study approved that the virus belongs to the classical dermal type of poxviruses and the short genetic distances between viruses related to closely neighboring countries. We also concluded that the conservative P4b gene included mutation sites that make this gene practical for diagnosing the virus and phylogenetic analysis.

Chicken cGAS Senses Fowlpox Virus Infection and Regulates Macrophage Effector Functions

The anti-viral immune response is dependent on the ability of infected cells to sense foreign nucleic acids. In multiple species, the pattern recognition receptor (PRR) cyclic GMP-AMP synthase (cGAS) senses viral DNA as an essential component of the innate response. cGAS initiates a range of signaling outputs that are dependent on generation of the second messenger cGAMP that binds to the adaptor protein stimulator of interferon genes (STING). Here we show that in chicken macrophages, the cGAS/STING pathway is essential not only for the production of type-I interferons in response to intracellular DNA stimulation, but also for regulation of macrophage effector functions including the expression of MHC-II and co-stimulatory molecules. In the context of fowlpox, an avian DNA virus infection, the cGAS/STING pathway was found to be responsible for type-I interferon production and MHC-II transcription. The sensing of fowlpox virus DNA is therefore essential for mounting an anti-viral response in chicken cells and for regulation of a specific set of macrophage effector functions.

Recombinant Fowlpox Virus Expressing gB Gene from Predominantly Epidemic Infectious Larygnotracheitis Virus Strain Demonstrates Better Immune Protection in SPF Chickens

Background: Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens. Antigenic mutation of infectious laryngotracheitis virus (ILTV) may result in a vaccination failure in the poultry industry and thus a protective vaccine against predominant ILTV strains is highly desirable. Methods: The full-length glycoprotein B (gB) gene of ILTV with the two mutated synonymous sites of fowlpox virus (FPV) transcription termination signal sequence was cloned into the insertion vector p12LS, which was co-transfected with wild-type (wt) FPV into chicken embryo fibroblast (CEF) to develop a recombinant fowlpox virus-gB (rFPV-gB) candidate vaccine strain. Furthermore, its biological and immunological characteristics were evaluated. Results: The results indicated that gB gene was expressed correctly in the rFPV by indirect immunofluorescent assay and Western blot, and the rFPV-gB provided a 100% protection in immunized chickens against the challenge of predominant ILTV strains that were screened by pathogenicity assay when compared with the commercialized rFPV vaccine, which only provided 83.3%. Conclusion: rFPV-gB can be used as a potential vaccine against predominant ILTV strains.

Modulation of early host innate immune response by a Fowlpox virus (FWPV) lateral body protein

The avian pathogen, fowlpox virus (FWPV) has been successfully used as vaccine vector in poultry and humans but relatively little is known about its ability to modulate host antiviral immune responses in these hosts, which are replication permissive and non-permissive, respectively. FWPV is highly resistant to avian type I interferon (IFN) and able to completely block the host IFN-response. Microarray screening of host IFN-regulated gene expression in cells infected with 59 different, non-essential FWPV gene knock-out mutants revealed that FPV184 confers immunomodulatory capacity. We report that FPV184-knockout virus (FWPVΔ184) induces the cellular IFN response as early as 2 hours post-infection. The wild-type, uninduced phenotype can be rescued by transient expression of FPV184 in FWPVΔ184-infected cells. Ectopic expression of FPV184 inhibited polyI:C activation of the chicken IFN-β promoter and IFN-α activation of the chicken Mx promoter. Confocal and correlative super-resolution light and electron microscopy demonstrated that FPV184 has a functional nuclear localisation signal domain and is packaged in the lateral bodies of the virions. Taken together, these results provide a paradigm for a late poxvirus structural protein packaged in the lateral bodies and capable of supressing IFN induction early during the next round of infection.