Characterization of Fowlpox Virus.

The complex cytoplasmic DNA virus known as the fowlpox virus (FWPV) is a member of the avipoxvirus genus, Subfamily Chordopoxvirinae, and Family Poxviridae. The large genome size of FWPV makes it a potential vector for the creation of vaccines against a range of serious veterinary and human ailments. It also allows for multiple gene insertion and the generation of abortive infection in mammalian cells. The virus, which causes fowlpox in chickens and turkeys, is mainly transmitted to poultry through aerosols or biting insects. Fowlpox is a highly contagious disease that affects both domestic and wild birds, causing cutaneous and/or diphtheritic illnesses. To control the illness, strict hygiene practices and immunization with FWPV attenuated strains or antigenically similar pigeon pox virus vaccines are employed. Recent years have seen an increase in fowlpox outbreaks in chicken flocks, primarily due to the introduction of novel forms of FWPV. It is believed that the pathogenic characteristics of these strains are enhanced by the integration of reticuloendotheliosis virus sequences of variable lengths into the FWPV genome. The standard laboratory diagnosis of FPV involves histopathological analysis, electron microscopy, virus isolation on chorioallantoic membrane (CAM) of embryonated chicken eggs or cell cultures, and serologic techniques. For quick and consistent diagnosis, polymerase chain reaction (PCR) has proven to be the most sensitive method. PCR is used in concert with restriction endonuclease enzyme analysis (REA) to identify, differentiate, and characterize the molecular makeup of isolates of the fowlpox virus. Sequencing of the amplified fragments is then done.

Development of a Multiplex PCR Assay for Rapid Differentiation of Fowlpox and Pigeonpox Viruses.

The aim of this study was to develop a multiplex PCR assay capable of rapidly differentiating two major Avipoxvirus (APV) species, Fowlpox virus (FWPV) and Pigeonpox virus (PGPV), which cause disease in bird species. Despite the importance of a rapid differentiation assay, no such assay exists that can differentiate the APV species without sequencing. To achieve this, species-specific target DNA fragments were selected from the fpv122 gene of FWPV and the HM89_gp120 gene of PGPV, which are unique to each genome. Nine samples collected from unvaccinated chickens, pigeons, and a turkey with typical pox lesions were genetically identified as FWPV and PGPV. The designed primers and target DNA fragments were validated using in silico analyses with the nucleotide Basic Local Alignment Search Tool. The multiplex PCR assay consisted of species-specific primers and previously described PanAPV primers (genus-specific) and was able to differentiate FWPV and PGPV, consistent with the phylogenetic outputs. This study represents the first successful differentiation of FWPV and PGPV genomes using a conventional multiplex PCR test. This assay has the potential to facilitate the rapid diagnosis and control of APV infections.

Desarrollo de un ensayo de PCR múltiple para la diferenciación rápida de los virus de la viruela aviar y la viruela de paloma. El objetivo de este estudio fue desarrollar un ensayo de PCR múltiple capaz de diferenciar rápidamente dos especies principales de Avipoxvirus (APV) (viruela del pollo), el Fowlpox virus (FWPV) y el Pigeonpox virus (PGPV), (viruela de la gallina), que causan enfermedades en especies de aves. A pesar de la importancia de un ensayo de diferenciación rápida, no existe ningún ensayo que pueda diferenciar las especies de APV sin secuenciación. Para lograr esto, se seleccionaron fragmentos blanco de ADN específicos de especie del gene fpv122 de FWPV y el gene HM89_gp120 de Pigeonpox virus, que son únicos para cada genoma. Nueve muestras recolectadas de pollos, palomas y un pavo que no fueron vacunados con lesiones típicas de la viruela se identificaron genéticamente como FWPV y PGPV. Los iniciadores diseñados y los fragmentos de ADN blanco se validaron mediante análisis in silico mediante la herramienta de búsqueda de alineación local básica de nucleótidos (BLAST). El ensayo de PCR múltiple consistió en iniciadores específicos de especie y cebadores PanAPV previamente descritos (específicos de género) y fue capaz de diferenciar entre Fowlpox virus y Pigeonpox virus, de acuerdo con los resultados filogenéticos. Este estudio representa la primera diferenciación exitosa de los genomas de Fowlpox virus y Pigeonpox virus utilizando una prueba de PCR múltiple convencional. Este ensayo tiene el potencial de facilitar el diagnóstico rápido y el control de las infecciones por Avipoxvirus.

Current situation and genomic characterization of fowlpox virus in lower Egypt during 2022.

A total of 45 samples of vaccinated and non-vaccinated layer chickens were collected from farms in the Egyptian governorates of Sharqia, Ismailia, Menofia, Gharbia, Kafr El Sheikh, Qalyubia, and Dakahlia in the year 2022. They exhibited nodular lesions on their combs, mouth corners, and eyelids, suggesting they were infected with pox disease, which was associated with a 3 to 5% mortality rate. The samples were grown on the chorioallantoic-membrane of embryonated chicken eggs to ensure their viability. In both vaccinated and non-vaccinated farms, 35 of 45 virus isolates were confirmed positive via polymerase chain reaction (PCR) of fpv167 (P4b), based on the amplicon length of the fpv167 gene locus. The 6 strains from various Egyptian governorates were chosen for sequencing and genetic characterization. Phylogenetic investigation of the fpv167 (P4b) gene of sequenced strains clustered within sub clade A1 showed 100% correlation between FWPVD, TKPV13401 and fowlpox-AN2, fowlpox-AN3, and fowlpox-AN6, but only a 98.6% correlation between fowlpox-AN1, fowlpox-AN4, and fowlpox-AN5. Comparing the fowlpox-AN1, fowlpox-AN4, and fowlpox-AN5 strains with commercial vaccine strains (HP1-444-(FP9), vaccine-VSVRI), they had 98.6% identity, while other strains had 100% identity. The results of this study's mutation research showed that fowlpox-AN1, fowlpox-AN4, and fowlpox-AN5 had acquired novel mutations; fowlpox-AN1 had R201G and T204A; fowlpox-AN4 and fowlpox-AN5 had L141F and H157P. Further research is required to determine the effectiveness of the current vaccine in order to develop a new vaccine.

A Phase 2, Double-blind, Randomized Controlled Trial of PROSTVAC in Prostate Cancer Patients on Active Surveillance.

BACKGROUND: There is an unmet clinical need for interventions to prevent disease progression in patients with localized prostate cancer on active surveillance (AS). OBJECTIVE: To determine the immunologic response to the PROSTVAC vaccine and the clinical indicators of disease progression in patients with localized prostate cancer on AS. DESIGN, SETTING, AND PARTICIPANTS: This was a phase 2, double-blind, randomized controlled trial in 154 men with low- or intermediate-risk prostate cancer on AS. INTERVENTION: Participants were randomized (2:1) to receive seven doses of subcutaneous PROSTVAC, a vaccinia/fowlpox viral vector-based immunotherapy containing a prostate-specific antigen (PSA) transgene and three T-cell co-stimulatory molecules, or an empty fowlpox vector (EV) over 140 d. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary outcome was the change from baseline in CD4 and CD8 T-cell infiltration in biopsy tumor tissue. Key secondary outcomes were safety and changes in prostate biopsy tumor pathology, peripheral antigen-specific T cells, and serum PSA. Continuous variables were compared using nonparametric tests. Categorical variables were compared using Fisher's exact test. RESULTS AND LIMITATIONS: The PROSTVAC/EV vaccination was well tolerated. All except one participant completed the vaccination series. Changes in CD4 or CD8 density in biopsy tumor tissue did not differ between the PROSTVAC and EV arms. The proportions of patients with Gleason upgrading to grade group 3 after treatment was similar between the arms. There were no differences in postvaccination peripheral T-cell responses or the PSA change from baseline to 6-mo post-treatment follow-up between the groups. CONCLUSIONS: In this first-of-kind trial of immunotherapy in patients on AS for prostate cancer, PROSTVAC did not elicit more favorable prostate tissue or peripheral T-cell responses than the EV. There was no difference between the arms in clinicopathologic effects. Despite the null findings, this is the first study reporting the feasibility and acceptability of an immunotherapy intervention in the AS setting. PATIENT SUMMARY: We looked at responses after an experimental prostate cancer vaccine in patients with prostate cancer on active surveillance (AS). Participants who received the vaccine did not show more favorable outcomes than those receiving the control. Despite these findings, this is the first report showing the feasibility and acceptability of immunotherapy for prostate cancer in patients on AS.

Isolation of Reticuloendotheliosis Virus from Chorio-allantoic Membrane of SPF Chicken Eggs inoculated with Fowl Pox Virus.

Fowl Pox Viruses (FPV) infect chickens and turkeys giving rise to pock lesions on various body parts like combs, wattles, legs, shanks, eyes, mouth etc. The birds, affected with FPV, also show anemia and ruffled appearance which are clinical symptoms of Reticuloendotheliosis. Interestingly, the field strains of FPV are integrated with the provirus of Reticuloendotheliosis Virus (REV). Due to this integration, the infected birds, upon replication of FPV, give rise to free REV virions, causing severe immunosuppression and anemia. Pox scabs, collected from the infected birds, not only show positive PCR results upon performing FPV-specific 4b core protein gene PCR but also show positive results for the PCR of REV-specific env gene and FPV-REV 5'LTR junction. Homogenized suspension of the pock lesions, upon inoculating to the Chorio-allantoic Membrane (CAM) of 10 days old specific pathogen-free embryonated chicken eggs, produces characteristic pock lesions in serial passages. But the lesions also harbor REV mRNA or free virion, which can be identified by performing REV-specific env gene PCR using REV RNA from FPV-infected CAMs. The study suggests successful replication and availability of REV mRNA and free virion alongside the FPV virus, although the CAM is an ill-suited medium for any retroviral (like REV) growth and replication.

Occurrence of enteric viruses causing clinical diarrhea in small ruminants in northern Indian plains: a reverse transcription PCR based molecular study.

Fowlpox virus (FPV) infects chickens and turkeys giving rise to pock lesions on various body parts like combs, wattles, legs, shanks, eyes, mouth, etc. The birds, affected with FPV, also show anemia and a ruffled appearance which are clinical symptoms of reticuloendotheliosis. Interestingly, the field strains of FPV are integrated with the provirus of reticuloendotheliosis virus (REV). Due to this integration, the infected birds, upon replication of FPV, give rise to free REV virions, causing severe immunosuppression and anemia. Pox scabs, collected from the infected birds, not only show positive PCR results upon performing FPV-specific 4b core protein gene PCR but also show positive results for the PCR of REV-specific env gene and FPV-REV 5'LTR junction. Homogenized suspension of the pock lesions, upon inoculating to the chorio-allantoic membrane (CAM) of 10-day-old specific pathogen-free embryonated chicken eggs, produces characteristic pock lesions in serial passages. However, the lesions also harbor REV mRNA or free virion, which can be identified by performing REV-specific env gene PCR using REV RNA from FPV-infected CAMs. The study suggests successful replication and availability of REV mRNA and free virion alongside the FPV, although the CAM is an ill-suited medium for any retroviral (like REV) growth and replication.

The co-administration of live fowlpox and Newcastle disease vaccines by non-invasive routes to chickens reared by smallholders in Tanzania and Nepal.

The co-administration of commercial live fowlpox (FP) and Newcastle disease (ND) vaccines when given by non-invasive (needle-free) routes was demonstrated to be safe and to elicit immunity in two field studies, one in Tanzania the other in Nepal. Both studies were of a cluster-randomised controlled design in which birds were randomly assigned to one of five treatment groups: (i) administration with FP vaccine alone (feather follicle), (ii) administration with ND vaccine alone (eye-drop), (iii) concurrent administration of FP (feather follicle) and ND (eye-drop) vaccines, (iv) concurrent administration of FP (wing-web) and ND (eye-drop) vaccines, and (v) unvaccinated, acting as environmental sentinels. Data from a total of 1167 birds from seven villages in Hanang District of Tanzania together with 1037 birds from eleven villages in Dhading District of Nepal were collected over a period of 21 and 28 days, respectively. Immune responses to FP vaccination were evaluated by local take reactions, while those to ND vaccination were evaluated serologically by haemagglutination inhibition test. The two studies demonstrated that the concurrent vaccination of free-range, indigenous breeds of chicken with live FP and ND vaccines, both administered by non-invasive routes, was safe and induced immunity against FP and ND that were non-inferior to the administration of FP and ND vaccines alone. These findings are important to appropriately trained small-scale backyard poultry farmers as well as to paraprofessionals and community health workers helping to increase vaccine uptake and the control of both FP and ND in low- to middle-income countries.

Epidemic of cutaneous fowlpox in a naïve population of chickens and turkeys in Austria: Detailed phylogenetic analysis indicates co-evolution of fowlpox virus with reticuloendotheliosis virus.

Cutaneous fowlpox is a disease of chickens and turkeys caused by the fowlpox virus (FWPV), characterized by the development of proliferative lesions and scabs on unfeathered areas. FWPVs regularly carry an integrated, active copy of the reticuloendotheliosis virus (REV), and it has been hypothesized that such FWPVs are more problematic in the field. Extensive outbreaks are usually observed in tropical and sub-tropical climates, where biting insects are more difficult to control. Here, we report an epidemic of 65 cutaneous fowlpox cases in Austria in layer chickens (91% of the cases) and broiler breeders and turkeys, all of them unvaccinated against the disease, from October 2018 to February 2020. The field data revealed appearance in flocks of different sizes ranging from less than 5000 birds up to more than 20,000 animals, with the majority raised indoors in a barn system. The clinical presentation was characterized by typical epithelial lesions on the head of the affected birds, with an average decrease of 6% in egg production and an average weekly mortality of 1.2% being observed in the flocks. A real-time multiplex polymerase chain reaction (PCR) confirmed the presence of FWPV-REV DNA, not only in the lesions but also in the environmental dust from the poultry houses. The integration of the REV provirus into the FWPV genome was confirmed by PCR, and revealed different FWPV genome populations carrying either the REV long terminal repeats (LTRs) or the full-length REV genome, reiterating the instability of the inserted REV. Two selected samples were fully sequenced by next generation sequencing (NGS), and the whole genome phylogenetic analysis revealed a regional clustering of the FWPV genomes. The extensive nature of these outbreaks in host populations naïve for the virus is a remarkable feature of the present report, highlighting new challenges associated with FWPV infections that need to be considered.

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.

Flow Cytometric Evaluation of CD4+ and CD8+ T-cell Immune Response in SPF Chickens Induced by Fowlpox Vaccine.

Fowlpox (FP) is a viral disease that is widely distributed throughout the world. The disease has an economic impact on the poultry industry, and its prevalence has even been reported in vaccinated flocks. The present study used flow cytometry to evaluate the CD4+ and CD8+ T-cell immune response of chicks induced by FP vaccine. 120 specific pathogen-free (SPF) 21-day-old chicks were randomly divided into three groups of 40. One group was used as negative control with PBS inoculation, the other two groups were inoculated with the local fowlpox vaccine produced by Razi Institute and commercial FP vaccines, and they were kept for five weeks. Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Hypaque density gradients and the percentages of CD3+, CD3+, CD4+, and CD3+CD8+ T lymphocytes were analyzed with flow cytometry. Seven days post-immunization, a maximum (90-100%) swelling formation ("take") on the vaccination site was observed. The ratios of CD4+ to CD8+ T-lymphocytes in both vaccinated groups were significantly higher (p < 0.05) than the control group inoculated with PBS. The percentages of CD3+, CD3+CD4+, and CD3+CD8+ T-lymphocytes were increased in chickens vaccinated with commercial and local FP vaccines. There were no significant differences between the groups receiving commercial and local fowl pox vaccines. The present study showed that protective immunity could be associated with increased cellular immune responses, which has been interpreted as enhancing T-cell proliferation and increasing CD4+ to CD8+ ratios through vaccination with the FP vaccine. This study further suggests that the induction of enhanced immune responses is due mainly to the Th1-type response.

Atypical Manifestation of Cutaneous Fowlpox in Broiler Chickens Associated with High Condemnation at a Processing Plant.

The present case is an unusual report of cutaneous fowlpox with an atypical appearance and incidence in broilers. Gross skin lesions were noticed in 41-day-old commercial broilers during the veterinary inspection at a processing plant in the north of Iran. The skin lesions were only observed on feathered skin areas of the broilers and remained unnoticed until slaughter. Round, nodular or coalescent, elongated, reddish-brown proliferative lesions were mainly located on the back, thighs, and proximal areas of the neck of broilers. Nonfeathered skin, including the wattle, comb, eyelids, and legs, were not affected. This condition incurred high losses due to a 5.3% condemnation and trimming of carcasses. Cutaneous lesions were sampled for histopathology and molecular virology for further investigations. Histopathology revealed multifocal necrotic dermatitis with epidermal eosinophilic cytoplasmic inclusion bodies in the skin lesions. Molecular investigations confirmed the presence of fowlpox virus (FWPV) in the proliferative lesions, with further investigations identifying two FWPV genome populations, one carrying a portion of the reticuloendotheliosis virus (REV) and the other a nearly complete REV provirus. Furthermore, the 4b core protein gene-based molecular analysis clustered the field virus into clade A of the genus Avipoxvirus.

Reporte de caso- Manifestación atípica de viruela aviar cutánea en pollos de engorde asociada con altas de decomisos en una planta de procesamiento. El presente caso es un informe inusual de viruela aviar cutánea con apariencia e incidencia atípicas en pollos de engorde. Se observaron lesiones severas cutáneas en pollos de engorde comerciales de 41 días durante la inspección veterinaria en una planta de procesamiento en el norte de Irán. Las lesiones cutáneas solo se observaron en las áreas de piel emplumada de los pollos de engorde y pasaron desapercibidas hasta el procesamiento. Las lesiones proliferativas redondas, nodulares o coalescentes, alargadas, de color marrón rojizo se localizaron principalmente en el dorso, los muslos y en las áreas proximales del cuello de los pollos de engorde. La piel sin plumas, incluidos las barbillas, la cresta, los párpados y las piernas, no se vio afectada. Esta condición generó grandes pérdidas debido a un 5.3% de decomisos y recorte de canales. Se tomaron muestras de las lesiones cutáneas para histopatología y virología molecular para investigaciones diagnósticas. La histopatología reveló dermatitis necrótica multifocal con cuerpos de inclusión citoplasmáticos eosinófilos epidérmicos en las lesiones cutáneas. Las investigaciones moleculares confirmaron la presencia del virus de la viruela aviar (FWPV) en las lesiones proliferativas, con investigaciones adicionales que identificaron dos poblaciones del genoma del virus de la viruela aviar, una que portaba una porción del virus de la reticuloendoteliosis (REV) y la otra con un provirus del virus de la reticuloendoteliosis casi completo. Además, el análisis molecular basado en el gene de la proteína del núcleo 4b agrupó el virus de campo en el clado A del género Avipoxvirus.

Research Note: Simultaneous detection of infectious laryngotracheitis virus, fowlpox virus, and reticuloendotheliosis virus in chicken specimens.

Infectious laryngotracheitis (ILT), fowlpox (FP), and reticuloendotheliosis are important poultry diseases caused by gallid herpesvirus 1 (ILTV), fowlpox virus (FWPV), and reticuloendotheliosis virus (REV), respectively. Coinfections with ILTV and FWPV occur naturally in chickens, and FP in its more virulent wet form is characterized by diphtheritic lesions and easily confused with ILT. Moreover, the insertion of only partial REV-LTR or a nearly full-length REV into the FWPV genome, located between the ORF 201 and ORF 203, has increased recently in wild-type field FWPV isolates. Therefore, it is critical to detect ILTV, FWPV, REV-integrated FWPV, and REV early and accurately. In this study, we successfully developed a multiplex PCR assay for the simultaneous detection of ILTV, FWPV, REV-integrated FWPV, and REV, and the detection limits was 1 × 54 copies/tube. When used to test clinical samples, the results of the multiplex PCR were in 100% agreement with singleplex PCRs and sequencing. This new multiplex PCR is a simple, rapid, sensitive, specific, and cost-effective method for detection of 4 viruses in clinical specimens.

laying Farm: Up to Date Data on a Fowlpox Outbreak in Phylogenetic Analysis in Iran, 2018.

Fowlpox is an economically significant viral disease in poultry, characterized by two forms of clinical signs, including cutaneous and diphtheritic lesions. This infection can have several adverse effects on flock performance, such as a reduction in egg production and growth and an increase in mortality. In winter 2018, an infection suspected to fowlpox was reported from a Hy-line W-36 laying farm in Isfahan province, Iran. The birds were 38 weeks of age and showed obvious diphtheritic signs in mucous membranes with increased mortality and reduced egg production. In total, 20 samples were collected from diphtheritic lesions (Trachea and Esophagus) of infected birds. The Polymerase Chain Reaction method was used to amplify a 578 bp fragment of the poxvirus 4b core protein gene. Phylogenetic relationships of avian poxviruses are usually analyzed using the 4b core protein-coding gene sequences with molecular weights of 75.2 kDa. The major elements had the fowlpox genome, and sequencing was performed for one isolate as representative. The nucleotide sequence result showed that this isolate (FP\UT-POX-2018) had a similarity rate of 99.53% with the previous Iranian fowlpox isolate (FP\GHPCRLAB.3) sequenced in the GenBank.Moreover, there was a 100% similarity among the current isolate nucleotide sequence, FP/NobilisVarioleW, and FP/FPV-VR250. The derived phylogenetic tree showed that these isolates were clustered in A1 subclades. Therefore, Iranian isolates of fowlpox virus have remained in the same subclade of phylogenetic classification (subclade A1), and they show high genomic similarity with previous isolates of Iran. Veterinarians and farmers must not underestimate fowlpox. However, they should consider the importance of vaccination against this disease like any other disease care.

Pathological, molecular and phylogenic study of fowlpox virus in domesticated chickens of Tikrit City, Iraq / Estudo patológico, molecular e filogenético do vírus da varíola aviária em frangos de corte domesticados da cidade de Tikrit, Iraque

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.(AU)

O vírus da varíola aviária (VVA) é um dos vírus que acometem os frangos de corte em todo o mundo, causando perdas patológicas e econômicas na indústria aviária. As lesões causadas pelo vírus são facilmente reconhecidas pela observação visual e usualmente aparecem nas áreas do corpo das aves livres de penas, especialmente na cabeça. Além disso, em alguns casos a doença pode provocar a cegueira e a mortalidade de animais acometidos. O presente trabalho foi delineado para diagnosticar casos suspeitos de varíola aviária, identificar o agente causal e classificá-lo. Adicionalmente foram analisadas diferenças e similaridades com outros vírus estreitamente relacionados em localidades vizinhas e regionais. Cinquenta amostras foram colhidas em três localidades da cidade de Tikrit de frangos de corte, domesticados, que apresentavam lesões cutâneas. O DNA do vírus foi extraído diretamente das amostras de tecidos antes que a técnica de PCR fosse realizada. As proteínas do core do vírus, gene (P4b), foram parcialmente sequenciadas de analisadas em secções da rotina histológica. Os resultados obtidos revelaram que o vírus causa lesões variólicas com hiperplasia dermal e hiperqueratose. A hiperplasia e a congestão da membrana corioalantóica também foram registradas. O estudo também revelou que o DNA do VVA pode ser extraído diretamente de tecidos animais sem a realização de uma pré-purificação. A análise sequencial revelou que o VVA foi confirmado em todas as amostras agrupando-se em uma classe A, idêntica com isolados iranianos e egípcios. A conclusão obtida foi que o presente trabalho confirmou que o vírus pertence ao tipo dérmico clássico dos poxvirus e que as curtas distâncias genéticas entre os vírus relacionados são encontrados em países vizinhos. Também foi concluído que o gene conservador P4b inclui pontos de mutação que o tornam um gene prático para diagnosticar o vírus em análises filogenéticas.(AU)

Construction and immune protection evaluation of recombinant virus expressing Newcastle disease virus F protein by the largest intergenic region of fowlpox virus NX10.

Fowlpox virus (FPV) is used as a vaccine vector to prevent diseases in poultry and mammals. The insertion site is considered as one of the main factors influencing foreign gene expression. Therefore, the identification of insertion sites that can stably and efficiently express foreign genes is crucial for the construction of recombinant vaccines. In this study, we found that the insertion of foreign genes into ORF054 and the ORF161/ORF162 intergenic region of the FPV genome did not affect replication, and that the foreign genes inserted into the intergenic region were more efficiently expressed than when they were inserted into a gene. Based on these results, the recombinant virus rFPVNX10-NDV F-E was constructed and immune protection against virulent FPV and Newcastle disease virus (NDV) was evaluated. Tests for anti-FPV antibodies in the vaccinated chickens were positive within 14 days post-vaccination. After challenge with FPV102, no clinical signs of FP were observed in vaccinated chickens, as compared to that in the control group (unvaccinated), which showed 100% morbidity. Low levels of NDV-specific neutralizing antibodies were detected in vaccinated chickens before challenge. After challenge with NDV ck/CH/LHLJ/01/06, all control chickens died within 4 days post-challenge, whereas 5/15 vaccinated chickens died between 4 and 12 days post-challenge. Vaccination provided an immune protection rate of 66.7%, whereas the control group showed 100% mortality. These results indicate that the ORF161/ORF162 intergenic region of FPVNX10 can be used as a recombination site for foreign gene expression in vivo and in vitro.

An atypical clinicopathological manifestation of fowlpox virus associated with reticuloendotheliosis virus in commercial laying hen flocks in Brazil.

Fowlpox (FP) is a common epitheliotropic disease in chickens that is usually controlled by live attenuated vaccines. However, there have been some reports of outbreaks of FP in recent years, even in vaccinated flocks, presenting as atypical lesions and feathering abnormalities in chickens. These findings can be associated with fowlpox virus (FPV) with the reticuloendotheliosis virus (REV) integrated into its genome. In the present study, outbreaks of atypical FP were explored in vaccinated commercial laying hen flocks to determine the nature of the causative agent by histopathologic and molecular approaches. FPV and REV were detected and classified into subclade A1 of the genus Avipoxvirus and subtype 3 of REV (REV3), respectively. Additionally, heterogeneous populations of FPV with partial (containing only a remnant long terminal repeat-LTR) or total (all functional genes) integration of REV were identified by heterologous PCRs and detected considering reference integration sites. These results indicate the mechanism of chimeric genome FPV-REV associated with outbreaks and atypical clinicopathological manifestations in commercial laying hens for the first time in Brazil and in South America. In addition, this study demonstrates the emergence of REV integrated in the FPV genome in Brazilian chicken flocks.

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.

Protection of White Leghorn chickens by recombinant fowlpox vector vaccine with an updated H5 insert against Mexican H5N2 avian influenza viruses.

Despite decades of vaccination, surveillance, and biosecurity measures, H5N2 low pathogenicity avian influenza (LPAI) virus infections continue in Mexico and neighboring countries. One explanation for tenacity of H5N2 LPAI in Mexico is the antigenic divergence of circulating field viruses compared to licensed vaccines due to antigenic drift. Our phylogenetic analysis indicates that the H5N2 LPAI viruses circulating in Mexico and neighboring countries since 1994 have undergone antigenic drift away from vaccine seed strains. Here we evaluated the efficacy of a new recombinant fowlpox virus vector containing an updated H5 insert (rFPV-H5/2016), more relevant to the current strains circulating in Mexico. We tested the vaccine efficacy against a closely related subcluster 4 Mexican H5N2 LPAI (2010 H5/LP) virus and the historic H5N2 HPAI (1995 H5/HP) virus in White Leghorn chickens. The rFPV-H5/2016 vaccine provided hemagglutinin inhibition (HI) titers pre-challenge against viral antigens from both challenge viruses in almost 100% of the immunized birds, with no differences in number of birds seroconverting or HI titers among all tested doses (1.5, 2.0, and 3.1 log10 mean tissue culture infectious doses/bird). The vaccine conferred 100% clinical protection and a significant decrease in oral and cloacal virus shedding from 1995 H5/HP virus challenged birds when compared to the sham controls at all tested doses. Virus shedding titers from vaccinated 2010 H5/LP virus challenged birds significantly decreased compared to sham birds especially at earlier time points. Our results confirm the efficacy of the new rFPV-H5/2016 against antigenic drift of LPAI virus in Mexico and suggest that this vaccine would be a good candidate, likely as a primer in a prime-boost vaccination program.

Characterization of Fowlpox virus in chickens and bird-biting mosquitoes: a molecular approach to investigating Avipoxvirus transmission.

Avian pox is a highly contagious avian disease, yet relatively little is known about the epidemiology and transmission of Avipoxviruses. Using a molecular approach, we report evidence for a potential link between birds and field-caught mosquitoes in the transmission of Fowlpox virus (FWPV) in Singapore. Comparison of fpv167 (P4b), fpv126 (VLTF-1), fpv175-176 (A11R-A12L) and fpv140 (H3L) gene sequences revealed close relatedness between FWPV strains obtained from cutaneous lesions of a chicken and four pools of Culex pseudovishnui, Culex spp. (vishnui group) and Coquellitidea crassipes caught in the vicinity of the study site. Chicken-derived viruses characterized during two separate infections two years later were also identical to those detected in the first event, suggesting repeated transmission of closely related FWPV strains in the locality. Since the study location is home to resident and migratory birds, we postulated that wild birds could be the source of FWPV and that bird-biting mosquitoes could act as bridging mechanical vectors. Therefore, we determined whether the FWPV-positive mosquito pools (n=4) were positive for avian DNA using a polymerase chain reaction-sequencing assay. Our findings confirmed the presence of avian host DNA in all mosquito pools, suggesting a role for Cx. pseudovishnui, Culex spp. (vishnui group) and Cq. crassipes mosquitoes in FWPV transmission. Our study exemplifies the utilization of molecular tools to understand transmission networks of pathogens affecting avian populations, which has important implications for the design of effective control measures to minimize disease burden and economic loss.

Efficacy of novel recombinant fowlpox vaccine against recent Mexican H7N3 highly pathogenic avian influenza virus.

Since 2012, H7N3 highly pathogenic avian influenza (HPAI) has produced negative economic and animal welfare impacts on poultry in central Mexico. In the present study, chickens were vaccinated with two different recombinant fowlpox virus vaccines (rFPV-H7/3002 with 2015 H7 hemagglutinin [HA] gene insert, and rFPV-H7/2155 with 2002 H7 HA gene insert), and were then challenged three weeks later with H7N3 HPAI virus (A/chicken/Jalisco/CPA-37905/2015). The rFPV-H7/3002 vaccine conferred 100% protection against mortality and morbidity, and significantly reduced virus shed titers from the respiratory and gastrointestinal tracts. In contrast, 100% of sham and rFPV-H7/2155 vaccinated birds shed virus at higher titers and died within 4 days. Pre- (15/20) and post- (20/20) challenge serum of birds vaccinated with rFPV-H7/3002 had antibodies detectable by hemagglutination inhibition (HI) assay using challenge virus antigen. However, only a few birds (3/20) in the rFPV-H7/2155 vaccinated group had antibodies that reacted against the challenge strain but all birds had antibodies that reacted against the homologous vaccine antigen (A/turkey/Virginia/SEP-66/2002) (20/20). One possible explanation for differences in vaccines efficacy is the antigenic drift between circulating viruses and vaccines. Molecular analysis demonstrated that the Mexican H7N3 strains have continued to rapidly evolve since 2012. In addition, we identified in silico three potential new N-glycosylation sites on the globular head of the H7 HA of A/chicken/Jalisco/CPA-37905/2015 challenge virus, which were absent in 2012 H7N3 outbreak virus. Our results suggested that mutations in the HA antigenic sites including increased glycosylation sites, accumulated in the new circulating Mexican H7 HPAIV strains, altered the recognition of neutralizing antibodies from the older vaccine strain rFPV-H7/2155. Therefore, the protective efficacy of novel rFPV-H7/3002 against recent outbreak Mexican H7N3 HPAIV confirms the importance of frequent updating of vaccines seed strains for long-term effective control of H7 HPAI virus.