Integrative RNA-seq and ChIP-seq analysis unveils metabolic regulation as a conserved antiviral mechanism of chicken p53.

The tumor suppressor p53, primarily functioning as a transcription factor, has exhibited antiviral capabilities against various viruses in chickens, including infectious bursal disease virus (IBDV), avian leukosis virus subgroup J (ALV-J), and avian infectious laryngotracheitis virus (ILTV). Nevertheless, the existence of a universal antiviral mechanism employed by chicken p53 (chp53) against these viruses remains uncertain. This study conducted a comprehensive comparison of molecular networks involved in chp53's antiviral function against IBDV, ALV-J, and ILTV. This was achieved through an integrated analysis of ChIP-seq data, examining chp53's genome-wide chromatin occupancy, and RNA-seq data from chicken cells infected with these viruses. The consistent observation of chp53 target gene enrichment in metabolic pathways, confirmed via ChIP-qPCR, suggests a ubiquitous regulation of host cellular metabolism by chp53 across different viruses. Further genome binding motif conservation analysis and transcriptional co-factor prediction suggest conserved transcriptional regulation mechanism by which chp53 regulates host cellular metabolism during viral infection. These findings offer novel insights into the antiviral role of chp53 and propose that targeting the virus-host metabolic interaction through regulating p53 could serve as a universal strategy for antiviral therapies in chickens.IMPORTANCEThe current study conducted a comprehensive analysis, comparing molecular networks underlying chp53's antiviral role against infectious bursal disease virus (IBDV), avian leukosis virus subgroup J (ALV-J), and avian infectious laryngotracheitis virus (ILTV). This was achieved through a combined assessment of ChIP-seq and RNA-seq data obtained from infected chicken cells. Notably, enrichment of chp53 target genes in metabolic pathways was consistently observed across viral infections, indicating a universal role of chp53 in regulating cellular metabolism during diverse viral infections. These findings offer novel insights into the antiviral capabilities of chicken p53, laying a foundation for the potential development of broad-spectrum antiviral therapies in chickens.

Circulation and Molecular Characterization of Infectious Laryngotracheitis Virus in Poultry Flocks with Respiratory Disorders in Turkey, 2018-2022.

Infectious laryngotracheitis (ILT) is a very serious worldwide respiratory disease of poultry, with many countries reporting ILT infections over the last decade. However, few reports are available regarding ILT disease prevalence in poultry in Turkey. Accordingly, the present study investigated ILT infection in Turkish broiler flocks between 2018 and 2022. Circulating ILT strains were characterized by sequence and phylogenetic analysis of two fragments of the infected-cell protein 4 gene. ILT virus (ILTV) was confirmed by quantitative PCR in 8 of the 21 flocks examined. As in other diseases, co-infections with other respiratory pathogens in confirmed ILT cases may worsen the symptoms and prolong the disease course. The present study confirmed co-infections with infectious bronchitis virus (13/21 tested flocks and 5/8 ILTV-positive flocks), indicating the importance of these pathogens in the occurrence of ILT infections.

Circulación y caracterización molecular del virus de la laringotraqueítis infecciosa en bandadas de aves de corral con trastornos respiratorios en Turquía, 2018­2022. La laringotraqueítis infecciosa (ILT) es una enfermedad respiratoria muy seria de la industria avícola en todo el mundo y muchos países han notificado infecciones por esta enfermedad durante la última década. Sin embargo, hay pocos informes disponibles sobre la prevalencia de laringotraqueítis infecciosa en la avicultura de Turquía. En consecuencia, el presente estudio investigó la infección por laringotraqueítis infecciosa en parvadas de pollos de engorde en Turquía entre los años 2018 y 2022. Las cepas de laringotraqueítis infecciosa circulantes se caracterizaron mediante análisis de secuencias y filogenéticos de dos fragmentos del gene de la proteína 4 de las células infectadas. El virus ILT (ILTV) se confirmó mediante PCR cuantitativa en ocho de las 21 parvadas examinadas. Como ocurre con otras enfermedades, las coinfecciones con otros patógenos respiratorios en casos confirmados de laringotraqueítis infecciosa pueden complicar los signos clínicos y prolongar el curso de la enfermedad. El presente estudio confirmó coinfecciones con el virus de la bronquitis infecciosa (en 13/21 parvadas analizadas y en 5/8 parvadas positivas para laringotraqueítis infecciosa), lo que indica la importancia de estos patógenos en la aparición de infecciones por la laringotraqueítis infecciosa.

A meta-analysis for prevalence of infectious laryngotracheitis in chickens in mainland China in 1981-2022.

BACKGROUND: Infectious laryngotracheitis (ILT) is a highly infectious upper respiratory tract disease of chickens caused by infectious laryngotracheitis virus or Gallid herpesvirus 1 (GaHV-1). ILT is an important respiratory disease of chickens and annually causes significant economic losses in the chicken industry. Although numerous relevant studies have been published, the overall prevalence of ILT infection among chicken in mainland China is still unknown, and associated risk factors need to be evaluated to establish preventive measures. RESULTS: The present study reviewed the literature on the prevalence of ILT in chickens in China as of December 20, 2022, retrieved from six databases-CNKI, Wanfang, VIP, PubMed, Web of Science, and ScienceDirect-were used to retrieve relevant studies published between January 1, 1981 and December 20, 2022. The literature quality of studies was assessed, and 20 studies with a total of 108,587 samples were included in the meta-analysis. Results of the meta-analysis showed that the overall prevalence of ILT was 10% (95% confidence interval: 8 -12%) through the random-effects model, which showed high heterogeneity, I2 = 99.4%. Further subgroup analyses showed that the prevalence of ILT decreased over time; furthermore, the prevalence in Northwest China was slightly lower than that in North China and South China, and the prevalence estimated using the diagnostic technique AGP was higher than that reported using other diagnostic techniques. CONCLUSIONS: ILT is prevalent to some extent in mainland China. Given that the ILT attenuated live vaccine has a certain level of virulence and the prevalence differences between regions, we recommend controlling breeding density, improving immunization programs and continuously monitoring viruses and to prevent ILT prevailing in mainland China.

Isolation, identification, molecular and pathogenicity characteristics of an infectious laryngotracheitis virus from Hubei province, China.

Multiple outbreaks of avian infectious laryngotracheitis (ILT) in chickens, both domestically and internationally, have been directly correlate to widespread vaccine use in affected countries and regions. Phylogenetic and recombination event analyses have demonstrated that avian infectious laryngotracheitis virus (ILTV) field strains are progressively evolving toward the chicken embryo-origin (CEO) vaccine strain. Even with standardized biosecurity measures and effective prevention and control strategies implemented on large-scale farms, continuous ILT outbreaks result in significant economic losses to the poultry industry worldwide. These outbreaks undoubtedly hinder efforts to control and eradicate ILTV in the future. In this study, an ILTV isolate was successfully obtained by laboratory PCR detection and virus isolation from chickens that exhibited dyspnea and depression on a broiler farm in Hubei Province, China. The isolated strain exhibited robust propagation on chorioallantoic membranes of embryonated eggs, but failed to establish effective infection in chicken hepatocellular carcinoma (LMH) cells. Phylogenetic analysis revealed a unique T441P point mutation in the gJ protein of the isolate. Animal experiments confirmed the virulence of this strain, as it induced mortality in 6-wk-old chickens. This study expands current understanding of the epidemiology, genetic variations, and pathogenicity of ILTV isolates circulating domestically, contributing to the elucidate of ILTV molecular basis of pathogenicity and development of vaccine.

In Ovo Vaccination with Recombinant Herpes Virus of the Turkey-Laryngotracheitis Vaccine Adjuvanted with CpG-Oligonucleotide Provides Protection against a Viral Challenge in Broiler Chickens.

Infectious laryngotracheitis (ILT) is an economically important disease in chickens. We previously showed that an in ovo adjuvantation of recombinant herpesvirus of the turkey-Laryngotracheitis (rHVT-LT) vaccine with CpG-oligonucleotides (ODN) can boost vaccine-induced responses in one-day-old broiler chickens. Here, we evaluated the protective efficacy of in ovo administered rHVT-LT + CpG-ODN vaccination against a wild-type ILT virus (ILTV) challenge at 28 days of age and assessed splenic immune gene expression as well as cellular responses. A chicken-embryo-origin (CEO)-ILT vaccine administered in water at 14 days of age was also used as a comparative control for the protection assessment. The results showed that the rHVT-LT + CpG-ODN or the CEO vaccinations provided significant protection against the ILTV challenge and that the level of protection induced by both the vaccines was statistically similar. The protected birds had a significantly upregulated expression of interferon (IFN)γ or interleukin (IL)-12 cytokine genes. Furthermore, the chickens vaccinated with the rHVT-LT + CpG-ODN or CEO vaccine had a significantly higher frequency of γδ T cells and activated CD4+ or CD8+ T cells, compared to the unvaccinated-ILTV challenge control. Collectively, our findings suggest that CpG-ODN can be used as an effective adjuvant for rHVT-LT in ovo vaccination to induce protective immunity against ILT in broiler chickens.

Co-administration of chicken IL-2 alleviates clinical signs and replication of the ILTV chicken embryo origin vaccine by pre-activating natural killer cells and cytotoxic T lymphocytes.

IMPORTANCE: Chickens immunized with the infectious laryngotracheitis chicken embryo origin (CEO) vaccine (Medivac, PT Medion Farma Jaya) experience adverse reactions, hindering its safety and effective use in poultry flocks. To improve the effect of the vaccine, we sought to find a strategy to alleviate the respiratory reactions associated with the vaccine. Here, we confirmed that co-administering the CEO vaccine with chIL-2 by oral delivery led to significant alleviation of the vaccine reactions in chickens after immunization. Furthermore, we found that the co-administration of chIL-2 with the CEO vaccine reduced the clinical signs of the CEO vaccine while enhancing natural killer cells and cytotoxic T lymphocyte response to decrease viral loads in their tissues, particularly in the trachea and conjunctiva. Importantly, we demonstrated that the chIL-2 treatment can ameliorate the replication of the CEO vaccine without compromising its effectiveness. This study provides new insights into further applications of chIL-2 and a promising strategy for alleviating the adverse reaction of vaccines.

Protection Efficacy of Recombinant HVT-ND-LT and the Live Attenuated Tissue Culture Origin Vaccines Against Infectious Laryngotracheitis Virus When Administered Individually or in Combination.

Infectious laryngotracheitis (ILT) is a respiratory disease that causes significant economic losses to the poultry industry. Control of the disease is achieved by vaccination and implementation of biosecurity measures. The use of bivalent and trivalent recombinant herpesvirus of turkey (rHVT) vaccines expressing infectious laryngotracheitis virus (ILTV) genes has increased worldwide. In the United States, vaccination programs of long-lived birds (broiler breeders and commercial layers) against ILT include immunizations with either HVT recombinant vector vaccines, in ovo or at hatch, or live attenuated vaccines administered via drinking water (chicken embryo origin [CEO]) or eye drop (tissue culture origin [TCO]). The efficacy of bivalent rHVT-LT at hatch followed by drinking water or eye-drop CEO vaccination has been shown to provide more robust protection than rHVT-LT alone. The objective of this study was to evaluate the protection efficacy of a commercial trivalent rHVT-ND-LT when administered at 1 day of age followed by TCO vaccination via eye drop at 10 wk of age. Groups vaccinated with only rHVT-ND-LT or TCO, the combination of rHVT-ND-LT + TCO, and one nonvaccinated group of chickens were challenged with a virulent ILTV strain at 15 wk of age. After challenge, mortalities were prevented only in the group of chickens vaccinated with the rHVT-ND-LT + TCO. Clinical signs of the disease and challenge virus replication in the trachea were significantly reduced for both the rHVT-ND-LT + TCO- and TCO-vaccinated groups of chickens. To assess challenge virus transmission, contact-naive chickens were introduced to all vaccinated groups immediately after challenge. At 8 days postintroduction, infection of contact-naive chickens was evidenced in those introduced to the rHVT-ND-LT and TCO group but prevented in the rHVT-ND-LT + TCO group. Overall, these results indicated that compared to rHVT-ND-LT or TCO when administered alone, the rHVT-ND-LT + TCO vaccination strategy improved protection against disease and reduced shedding of the challenge virus.

Eficacia protectora de las vacunas recombinantes HVT-ND-LT y las vacunas con virus vivo atenuado con origen en cultivo de tejidos contra el virus de la laringotraqueítis infecciosa cuando son administradas individualmente o en combinación. La laringotraqueítis infecciosa (ILT) es una enfermedad respiratoria que causa importantes pérdidas económicas a la industria avícola. El control de la enfermedad se logra mediante la vacunación y la implementación de medidas de bioseguridad. El uso de vacunas con el herpesvirus de pavo recombinante (rHVT) bivalentes y trivalentes que expresan genes del virus de la laringotraqueítis infecciosa (ILTV) ha aumentado en todo el mundo. En los Estados Unidos, los programas de vacunación de aves de larga vida (reproductoras pesadas y aves de postura comerciales) contra la laringotraqueítis incluyen inmunizaciones con vacunas con vector HVT recombinante, ya sea in ovo o al día de edad en la planta incubadora, o la aplicación de vacunas vivas atenuadas administradas a través del agua de bebida (origen en embrión de pollo [CEO]) o por gota ocular (origen en cultivo de tejidos [TCO]). Se ha demostrado que la eficacia de la vacuna rHVT-LT bivalente aplicada al día de edad en incubadora, seguida de la inmunización con la vacuna CEO en el agua de bebida o por gota ocular, proporciona una protección más sólida que la aplicación únicamente de la vacuna rHVT-LT. El objetivo de este estudio fue evaluar la eficacia protectora de una vacuna recombinante rHVT-ND-LT trivalente comercial cuando se administró al día de vida seguido de la vacunación con la vacuna TCO mediante gota ocular a las 10 semanas de edad. Los grupos vacunados únicamente con la vacuna rHVT-ND-LT, con TCO, la combinación con rHVT-ND-LT + TCO y un grupo de pollos no vacunados fueron desafiados con una cepa virulenta del virus de la laringotraqueítis a las 15 semanas de edad. Después del desafío, se previno la mortalidad únicamente en el grupo de pollos vacunados con la combinación rHVT-ND-LT + TCO. Los signos clínicos de la enfermedad y la replicación del virus de desafío en la tráquea se redujeron significativamente en los grupos de pollos vacunados con la combinación rHVT-ND-LT + TCO y con la vacuna TCO. Para evaluar la transmisión del virus de desafío, pollos sin contacto previo al virus se introdujeron en todos los grupos vacunados inmediatamente después del desafío. A los 8 días posteriores a la introducción, se evidenció la infección de los pollos sin contacto previo que se introdujeron en los grupos que recibieron únicamente la vacuna rHVT-ND-LT o la vacuna TCO, pero se previno en el grupo con la combinación rHVT-ND-LT + TCO. En general, estos resultados indicaron que, en comparación con las vacunas rHVT-ND-LT o TCO cuando se administran solas, la estrategia de vacunación rHVT-ND-LT + TCO mejoró la protección contra la enfermedad y redujo la diseminación del virus de desafío.

Protection Efficacy of the Infectious Laryngotracheitis (ILT) Serva CEO Vaccine Strain in Broiler Chickens Under Different Vaccination Coverage Conditions.

Mass vaccination against infectious laryngotracheitis virus (ILTV) in drinking water can result in variable initial vaccine take. Partial initial vaccine coverage of 20% with an Australian ILT vaccine (A20) previously resulted in significant protection against virulent ILTV challenge. This follow-up study used the international Serva ILT vaccine strain in a factorial design testing four levels of vaccination coverage (0%, 10%, 20%, or 100% of chicks eye-drop vaccinated with the live vaccine at 7 days of age) and three levels of ILTV challenge (no challenge or challenge at 7 or 21 days postvaccination [DPV]). The increase in ILTV load in choanal cleft swabs detected by qPCR after challenge was significantly reduced by 20% and 100% but not by 10% vaccination coverage. Vaccination reduced weight gain in unchallenged birds. Daily weight gain of birds was not affected by ILTV challenge at 7 DPV in any group, but following challenge at 21 DPV, it was significantly reduced in unvaccinated and 10% vaccinated groups relative to 20% and 100% vaccinated groups. Vaccination of 20% of the chickens provided substantial but incomplete protection (protective index range 44%-70%) against the severity of clinical signs and mortality following challenge while 10% vaccination coverage provided limited or no protection. Clinical signs were more severe and appeared earlier following challenge at 21 DPV than at 7 DPV. Within the vaccination treatments, eye-drop-vaccinated birds were better protected than their in-contact cohorts. In conclusion, partial vaccination of 20%, but not 10% of chickens, induced substantial protection against subsequent challenge. However, the attendant risks of reduced protection against early challenge and the possible reversion to virulence of vaccine virus when transmitted to unvaccinated chickens make it essential that 100% initial vaccine take be the goal of mass vaccination programs.

Eficacia protectora de la cepa vacunal CEO Serva del virus de la laringotraqueítis infecciosa (ILT) en pollos de engorde bajo diferentes condiciones de cobertura vacunal. La vacunación masiva contra el virus de la laringotraqueítis infecciosa (ILTV) en el agua de bebida puede resultar en una cobertura vacunal inicial variable. La cobertura vacunal inicial parcial del 20 % con una vacuna ILT australiana (A20) previamente resultó en una protección significativa contra el desafío virulento con el virus de la laringotraqueítis. Este estudio de seguimiento utilizó la cepa de la vacuna vacunal internacional Serva ILT en un diseño factorial para probar cuatro niveles de cobertura de vacunación (0 %, 10 %, 20 % o 100 % de pollitos vacunados por gota ocular con la vacuna viva a los siete días de edad) y tres niveles de desafío con el virus de la laringotraqueítis (sin desafío o con desafío a los 7 o 21 días después de la vacunación [DPV]). El aumento en la carga viral en hisopos de la hendidura coanal detectados por qPCR después del desafío se redujo significativamente con cobertura de vacunación del 20% y 100%, pero no con el 10%. La vacunación redujo el aumento de peso en las aves no desafiadas. La ganancia diaria de peso de las aves no se vio afectada por el desafío con el virus de la laringotraqueítis a los siete días después de la vacunación en ningún grupo, pero después del desafío a los 21 días después de la vacunación, se redujo significativamente en los grupos no vacunados y con cobertura del 10% en comparación con los grupos con cobertura del 20% y 100%. La vacunación del 20 % de los pollos brindó una protección sustancial pero incompleta (con un rango de índice de protección del 44 % al 70 %) contra la severidad de los signos clínicos y la mortalidad después del desafío, mientras que la cobertura de vacunación del 10 % brindó protección limitada o nula. Los signos clínicos fueron más graves y aparecieron más temprano después del desafío a los 21 días después de la vacunación en comparación con el desafío a los siete días después de la vacunación. Dentro de los tratamientos de vacunación, las aves vacunadas con gota ocular estaban mejor protegidas que sus cohortes en contacto. En conclusión, la cobertura de vacunación parcial del 20%, pero no del 10% de los pollos, indujo una protección sustancial contra el desafío posterior. Sin embargo, los riesgos concomitantes de una protección reducida contra el desafío temprano y la posible reversión a la virulencia del virus vacunal cuando se transmite a pollos no vacunados hacen que sea esencial que la cobertura vacunal inicial del 100% sea el objetivo de los programas de vacunación masiva.

Reconstitution and Mutagenesis of Avian Infectious Laryngotracheitis Virus from Cosmid and Yeast Centromeric Plasmid Clones.

The genomes of numerous herpesviruses have been cloned as infectious bacterial artificial chromosomes. However, attempts to clone the complete genome of infectious laryngotracheitis virus (ILTV), formally known as Gallid alphaherpesvirus-1, have been met with limited success. In this study, we report the development of a cosmid/yeast centromeric plasmid (YCp) genetic system to reconstitute ILTV. Overlapping cosmid clones were generated that encompassed 90% of the 151-Kb ILTV genome. Viable virus was produced by cotransfecting leghorn male hepatoma (LMH) cells with these cosmids and a YCp recombinant containing the missing genomic sequences - spanning the TRS/UL junction. An expression cassette for green fluorescent protein (GFP) was inserted within the redundant inverted packaging site (ipac2), and the cosmid/YCp-based system was used to generate recombinant replication-competent ILTV. Viable virus was also reconstituted with a YCp clone containing a BamHI linker within the deleted ipac2 site, further demonstrating the nonessential nature of this site. Recombinants deleted in the ipac2 site formed plaques undistinguished from those viruses containing intact ipac2. The 3 reconstituted viruses replicated in chicken kidney cells with growth kinetics and titers similar to the USDA ILTV reference strain. Specific pathogen-free chickens inoculated with the reconstituted ILTV recombinants succumbed to levels of clinical disease similar to that observed in birds inoculated with wildtype viruses, demonstrating the reconstituted viruses were virulent. IMPORTANCE Infectious laryngotracheitis virus (ILTV) is an important pathogen of chicken with morbidity of 100% and mortality rates as high as 70%. Factoring in decreased production, mortality, vaccination, and medication, a single outbreak can cost producers over a million dollars. Current attenuated and vectored vaccines lack safety and efficacy, leaving a need for better vaccines. In addition, the lack of an infectious clone has also impeded understanding viral gene function. Since infectious bacterial artificial chromosome (BAC) clones of ILTV with intact replication origins are not feasible, we reconstituted ILTV from a collection of yeast centromeric plasmids and bacterial cosmids, and identified a nonessential insertion site within a redundant packaging site. These constructs and the methodology necessary to manipulate them will facilitate the development of improved live virus vaccines by modifying genes encoding virulence factors and establishing ILTV-based viral vectors for expressing immunogens of other avian pathogens.

Codon pair bias deoptimization of essential genes in infectious laryngotracheitis virus reduces protein expression.

Infectious laryngotracheitis virus (ILTV; an alphaherpesvirus) is a respiratory pathogen of chickens and causes significant economic losses in the poultry industry globally, in addition to severe animal health and welfare concerns. To date, studying the role of ILTV genes in viral infection, replication or pathogenesis has largely been limited to genes that can be deleted from the ILTV genome and the resultant deletion mutants characterized in vitro or in vivo. However, this approach is not suitable for the study of essential genes. This study trialled two different codon deoptimization techniques that aimed to separately disrupt and downregulate the expression of two ILTV genes, ICP8 and UL12, which are essential or very important in viral replication. The target genes were partially recoded using codon usage deoptimization (CUD) and codon pair bias deoptimization (CPBD) approaches and characterized in vitro. Viruses deoptimized via CPBD showed decreased protein expression as assessed by Western blotting and/or fluorescence microscopy to measure the intensity of the fluorescent marker fused to the target protein. Viruses deoptimized by CUD showed less consistent results, with some mutants that could not be generated or isolated. The results indicate that CPBD is an attractive and viable tool for the study of essential or critically important genes in ILTV. This is the first study, to our knowledge, that utilizes CPBD and CUD techniques for the study of ILTV genes.

Characterization of a Recombinant Thermostable Newcastle Disease Virus (NDV) Expressing Glycoprotein gB of Infectious Laryngotracheitis Virus (ILTV) Protects Chickens against ILTV Challenge.

Infectious laryngotracheitis (ILT) and Newcastle disease (ND) are two important avian diseases that have caused huge economic losses to the poultry industry worldwide. Newcastle disease virus (NDV) has been used as a vector in the development of vaccines and gene delivery. In the present study, we generated a thermostable recombinant NDV (rNDV) expressing the glycoprotein gB (gB) of infectious laryngotracheitis virus (ITLV) based on the full-length cDNA clone of the thermostable TS09-C strain. This thermostable rNDV, named rTS-gB, displayed similar thermostability, growth kinetics, and pathogenicity compared with the parental TS09-C virus. The immunization data showed that rTS-gB induced effective ILTV- and NDV-specific antibody responses and conferred immunization protection against ILTV challenge in chickens. The efficacy of rTS-gB in alleviating clinical signs was similar to that of the commercial attenuated ILTV K317 strain. Furthermore, rTS-gB could significantly reduce viral shedding in cloacal and tracheal samples. Our study suggested that the rNDV strain rTS-gB is a thermostable, safe, and highly efficient vaccine candidate against ILT and ND.

Detection and molecular characterization of infectious laryngotracheitis virus (ILTV) in chicken with respiratory signs in Brazil during 2015 and 2016.

Avian infectious laryngotracheitis (ILT) is a respiratory disease that causes severe economic losses in the poultry industry, mainly due to high morbidity and mortality and reduced egg production. Molecular characterization was performed on samples collected from flocks in the Brazilian States of São Paulo, Pernambuco, and Minas Gerais during 2015 and 2016 that presented clinical signs of respiratory disease. End-point PCR was used for viral detection, and DNA sequencing was used for differentiation of vaccine and field strains. Molecular analysis based on the infected cell protein (ICP4) gene separated four of the nine samples together with previous Brazilian isolates (São Paulo and Minas Gerais), one sample was grouped on the same branch as Minas Gerais strains (along with another related sample), one sample was separately branched but still related to the tissue culture origin (TCO) vaccine strain, and two samples were grouped on the same branch as the TCO vaccine strain. Molecular analysis of the thymidine kinase (TK) gene showed the existence of strains of both high and low virulence. The characterization of two fragments of the ICP4 gene and a fragment of the TK gene in this study suggested that the virus circulating in Guatapará, as well as those in Barretos and Itanhandu, that is causing respiratory problems in birds is a highly virulent field strain. The clinical signs point to a TCO vaccine strain that most likely underwent some reversal event and is a latent reactivated infection.

Pathogenicity and vaccine efficacy of two virulent infectious laryngotracheitis virus strains in Egypt.

Infectious laryngotracheitis (ILT) is an economically crucial respiratory disease of poultry that affects the industry worldwide. Vaccination is the principal tool in the control of the disease outbreak. In an earlier study, we comprehensively characterized the circulating strains in Egypt and identified both CEO-like and recombinant strains are dominant. Herein, we investigated the pathogenicity of two virulent strains representing the CEO-like (Sharkia_2018) and recombinant strain (Qalubia_2018). Additionally, we evaluated the efficacy of different commercial vaccines (HVT-LT, CEO, and TCO) against the two isolates in terms of the histopathological lesion scores and the viral (gC) gene load. A total of 270 White Leghorn-specific pathogen-free male chicks were divided into nine groups of 30 birds, each housed in separate isolators. Birds were distributed as follows; one group was non-vaccinated, non-challenged, and served as a negative control. Two groups were non-vaccinated and infected with the two isolates of interest and served as a positive control to test the pathogenicity. Six groups were vaccinated and challenged; two groups were vaccinated with vector vaccine at one day old. The other four groups were vaccinated with either the CEO- or TCO- vaccine (two groups each) at four weeks of age. Three weeks after vaccination, birds were infected with the virulent ILTV isolates. The larynx, trachea, and harderian gland samples were taken at 1, 3, and 7 days post-infection for histopathological lesion score and molecular detection. Notably, The recombinant strain was more virulent and pathogenic than CEO-like ILTV strains. Moreover, the TCO vaccine was less immunogenic than the vector and CEO vaccines.

Field Application of qPCR Monitoring of Infectious Laryngotracheitis Virus in Settled Chicken House Dust and Its Role in Control of a Major Outbreak.

Population-level sampling based on qPCR detection of infectious laryngotracheitis virus (ILTV) in poultry dust can be used to assess ILT vaccination outcomes following mass administration in drinking water. We report on the field application of this approach to assess the success of vaccine administration and its use in ILT outbreak control in meat chickens. In Study 1, dust samples were collected from 26 meat chicken flocks at 0, 4, 7, 14, and 21 days post drinking water vaccination (DPV) given between 7 to 13 days of age with the Serva or A20 live attenuated ILT vaccines. Unexpectedly, ILTV DNA was detected in dust samples collected prior to vaccination in 22/26 flocks. Typing revealed that the detected ILTV was different from the vaccine virus. To determine whether the detected ILTV DNA was from active infection or carryover of a noninfectious virus, Study 2 was implemented in 14 additional flocks with dust samples collected at 0, 7, 14, and 21 DPV and tracheal swabs collected from 15 birds/flock at 0 and 21 DPV. The results indicated that there was active infection with ILTV in those flocks before vaccination. This approach contributed to a statewide control program resulting in the eradication of ILT from South Australia as confirmed by negative ILTV test results for dust samples from 50 flocks and the absence of clinical ILT. These findings show that ILTV infection prior to vaccination is common in outbreak situations and that dust samples must be collected at 0 and 7 DPV for meaningful interpretation of vaccination outcomes and ILTV status. Comparatively low-cost dust testing during an outbreak, coupled with typing information, greatly assisted with decision making and control strategies during a major outbreak, including confirmation of the absence of infection in the final stages.

Aplicación de campo del monitoreo por qPCR del virus de la laringotraqueítis infecciosa en el polvo de casetas avícolas y su función en el control de un brote importante El muestreo a nivel de población basado en la detección por qPCR del virus de la laringotraqueítis infecciosa (ILTV) en el polvo de instalaciones avícolas se puede utilizar para evaluar los resultados de la vacunación contra esta enfermedad después de la administración masiva en el agua de bebida. Se reporta la aplicación de campo de este enfoque para evaluar el éxito de la administración de vacunas y su uso en el control de brotes por laringotraqueítis infecciosa en pollos de engorde. En el Estudio 1, se recolectaron muestras de polvo de 26 parvadas de pollos de engorda a los 0, 4, 7, 14 y 21 días después de la vacunación en el agua de bebida (DPV) a los 7 a 13 días de edad con las vacunas de laringotraqueítis vivas atenuadas Serva o A20. Inesperadamente, se detectó ADN del virus de laringotraqueítis en muestras de polvo recolectadas antes de la vacunación en 22/26 parvadas. La tipificación reveló que el virus detectado era diferente del virus de la vacuna. Para determinar si el ADN del virus de laringotraqueítis detectado procedía de una infección activa o del remanente de un virus no infeccioso, se implementó el Estudio 2 en 14 parvadas adicionales con muestras de polvo recolectadas a los 0, 7, 14 y 21 días después de la vacunación y de hisopos traqueales recolectados de 15 aves/parvada a los cero y 21 días después de la vacunación. Los resultados indicaron que había infección activa con el virus de laringotraqueítis en esas parvadas antes de la vacunación. Este enfoque contribuyó a un programa de control estatal que resultó en la erradicación de laringotraqueítis del sur de Australia, como lo confirmaron los resultados negativos de las pruebas del mismo virus para muestras de polvo de 50 parvadas y la ausencia de laringotraqueítis infecciosa clínica. Estos hallazgos muestran que la infección por el virus de la laringotraqueítis antes de la vacunación es común en situaciones de brotes y que las muestras de polvo deben recolectarse a los cero y 7 días después de la vacunación para una interpretación significativa de los resultados de la vacunación y el estado de esta enfermedad. Las pruebas de polvo comparativamente de bajo costo durante un brote, junto con la información de tipificación, ayudaron mucho con la toma de decisiones y con las estrategias de control durante un brote importante, incluida la confirmación de la ausencia de infección en las etapas finales.

Construction and characterisation of glycoprotein E and glycoprotein I deficient mutants of Australian strains of infectious laryngotracheitis virus using traditional and CRISPR/Cas9-assisted homologous recombination techniques.

In alphaherpesviruses, glycoproteins E and I (gE and gI, respectively) form a heterodimer that facilitates cell-to-cell spread of virus. Using traditional homologous recombination techniques, as well as CRISPR/Cas9-assisted homologous recombination, we separately deleted gE and gI coding sequences from an Australian field strain (CSW-1) and a vaccine strain (A20) of infectious laryngotracheitis virus (ILTV) and replaced each coding sequence with sequence encoding green fluorescent protein (GFP). Virus mutants in which gE and gI gene sequences had been replaced with GFP were identified by fluorescence microscopy but were unable to be propagated separately from the wildtype virus in either primary chicken cells or the LMH continuous chicken cell line. These findings build on findings from a previous study of CSW-1 ILTV in which a double deletion mutant of gE and gI could not be propagated separately from wildtype virus and produced an in vivo phenotype of single-infected cells with no cell-to-cell spread observed. Taken together these studies suggest that both the gE and gI genes have a significant role in cell-to-cell spread in both CSW-1 and A20 strains of ILTV. The CRISPR/Cas9-assisted deletion of genes from the ILTV genome described in this study adds this virus to a growing list of viruses to which this approach has been used to study viral gene function.

Seroprevalence of major respiratory diseases of chickens in central Ethiopia in different chicken production systems.

In Ethiopia, most chicken disease outbreaks and mortalities are attributed to a respiratory syndrome known as "fengil" with variable clinical signs and undefined etiology. The main goal of this study was to determine whether key respiratory pathogens that could contribute to the fengil syndrome circulate in Ethiopia. Specifically, we aimed to determine the seroprevalence of infectious laryngotracheitis virus (ILTV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), Mycoplasma gallisepticum (Mg), and avian metapneumovirus (aMPV). A cross-sectional survey was conducted in 158 scavenging and 42 small and medium-scale intensive chicken holdings in the East, West and North Shewa Zones of central Ethiopia. Blood from 495 chickens was collected and serological tests were used to determine exposure to these pathogens. Vaccination against NDV was the only immunization practiced with a significantly higher vaccination rate in the intensive than the scavenging system. Serological evidence of a high level of exposure to all pathogens was detected, including the first report on the seroprevalence of aMPV, ILTV, and IBV in the East Shewa Zone. The chicken and holding seroprevalence rates were respectively 91% and 94% for IBV, 34% and 57% for aMPV, 47% and 66% for Mg, 27% and 51% for ILTV and in unvaccinated flocks, 39% and 53% for NDV. These pathogens could contribute to the fengil syndrome, commonly ascribed to NDV. The seroprevalence of aMPV and ILTV was higher in chickens under the scavenging system. Exposure to multiple pathogens was common, with more than 50% of chickens positive for three or more pathogens in the scavenging system. This was reflected in significant positive associations between seropositivity to ILTV, Mg, ILTV, and IBV. The role of these pathogens in the causation of respiratory disease in the field requires further investigation.

Comparative full genome sequence analysis of wild-type and chicken embryo origin vaccine-like infectious laryngotracheitis virus field isolates from Canada.

Infectious laryngotracheitis (ILT), caused by infectious laryngotracheitis virus (ILTV), occurs sporadically in poultry flocks in Canada. Live attenuated chicken embryo origin (CEO) vaccines are being used routinely to prevent and control ILTV infections. However, ILT outbreaks still occur since vaccine strains could revert to virulence in the field. In this study, 7 Canadian ILTV isolates linked to ILT outbreaks across different time in Eastern Canada (Ontario; ON and Quebec; QC) were whole genome sequenced. Phylogenetic analysis confirmed the close relationship between the ON isolates and the CEO vaccines, whereas the QC isolates clustered with strains previously known as CEO revertant and wild-type ILTVs. Recombination network analysis of ILTV sequences revealed clear evidence of historical recombination between ILTV strains circulating in Canada and other geographical regions. The comparison of ON CEO clustered and QC CEO revertant clustered isolates with the LT Blen® CEO vaccine reference sequence showed amino acid differences in 5 and 12 open reading frames (ORFs), respectively. Similar analysis revealed amino acid differences in 32 ORFs in QC wild-type isolates. Compared to all CEO vaccine strains in the public domain, the QC wild-type isolates showed 15 unique mutational sites leading to amino acid changes in 13 ORFs. Our outcomes add to the knowledge of the molecular mechanisms behind ILTV genetic variance and provide genetic markers between wild-type and vaccine strains.

Serological Evidence of Infectious Laryngotracheitis Infection and Associated Risk Factors in Chickens in Northwestern Ethiopia.

Infectious laryngotracheitis (ILT) is a disease of high economic consequence to the poultry sector. Gallid herpesvirus 1 (GaHV-1), a.k.a infectious laryngotracheitis virus (ILTV), under the genus Iltovirus, and the family Herpesviridae, is the agent responsible for the disease. Despite the clinical signs on the field suggestive of ILT, it has long been considered nonexistent and a disease of no concern in Ethiopia. A cross-sectional study was conducted from November 2020 to June 2021 in three selected zones of the Amhara region (Central Gondar, South Gondar, and West Gojjam zones), Ethiopia, with the objective of estimating the seroprevalence of ILTV in chickens and identifying and quantifying associated risk factors. A total of 768 serum samples were collected using multistage cluster sampling and assayed for anti-ILTV antibodies using indirect ELISA. A questionnaire survey was used to identify the potential risk factors. Of the 768 samples, 454 (59.1%, 95% CI: 0.56-0.63) tested positive for anti-ILTV antibodies. Mixed-effect logistic regression analysis of potential risk factors showed that local breeds of chicken were less likely to be seropositive than exotic breeds (OR: 0.38, 95% CI: 0.24-0.61). In addition, factors such as using local feed source (OR: 6.53, 95% CI: 1.77-24.04), rearing chickens extensively (OR: 1.97, 95% CI: 0.78-5.02), mixing of different batches of chicken (OR: 14.51, 95% CI: 3.35-62.77), careless disposal of litter (OR: 1.62, 95% CI: 0.49-4.37), lack of house disinfection (OR: 11.05, 95% CI: 4.09-47.95), lack of farm protective footwear and clothing (OR: 20.85, 95% CI: 5.40-80.45), and careless disposal of dead chicken bodies had all been associated with increased seropositivity to ILTV. Therefore, implementation of biosecurity measures is highly recommended to control and prevent the spread of ILTV. Furthermore, molecular confirmation and characterization of the virus from ILT suggestive cases should be considered to justify the use of ILT vaccines.

Characterization of infectious laryngotracheitis virus isolates from laying hens during 2019-2020 outbreaks in Tamil Nadu, India.

Infectious laryngotracheitis (ILT) is an acute respiratory disease in chickens that is a serious threat to poultry-producing countries worldwide. In the present study, we isolated and characterized infectious laryngotracheitis (ILTV) virus isolates by sequencing and restriction fragment length polymorphism analysis of PCR-amplified products (PCR-RFLP). A total of 26 ILTV outbreaks were investigated that occurred between 2019 and 2020 in flocks that had not been vaccinated against ILTV. ILTV was isolated by cultivating tracheal samples in embryonated chicken eggs, which showed multiple opaque pock lesions and thickening of the chorioallantoic membrane after 120 hours of infection. The ILTV isolates were identified and characterized by PCR and sequencing a portion of the ICP4 and TK genes. Phylogenetic analysis based on the ICP4 region showed that the sequences clustered with chicken-embryo-origin vaccine-like strains. Sequence analysis of the ICP4 region differentiated chicken-embryo-origin (CEO), tissue-culture-origin (TCO), and field ILTV strains, with significant differences in nucleotide and amino acid sequences. Furthermore, PCR-RFLP analysis of the TK gene showed that the patterns were identical to those obtained with low-virulence and vaccine strains. In conclusion, sequencing of a portion of the ICP4 region of ILTV allowed differentiation of ILTV field, CEO, and TCO vaccine strains. In this study, CEO-vaccine-like strains were found to be the cause of ILTV outbreaks between 2019 and 2020 in Tamil Nadu in southern India.

Biological Characteristics of Infectious Laryngotracheitis Viruses Isolated in China.

Infectious laryngotracheitis virus (ILTV) causes severe respiratory disease in chickens and results in huge economic losses in the poultry industry worldwide. To correlate the genomic difference with the replication and pathogenicity, phenotypes of three ILTVs isolated from chickens in China from 2016 to 2018 were sequenced by high-throughput sequencing. Based on the entire genome, the isolates GD2018 and SH2017 shared 99.9% nucleotide homology, while the isolate SH2016 shared 99.7% nucleotide homology with GD2018 and SH2017, respectively. Each virus genome contained 82 ORFs encoding 77 kinds of protein, 31 of which share the same amino acid sequence in the three viruses. GD2018 and SH2017 shared 57 proteins with the same amino acid sequence, while SH2016 shared 42 and 41 proteins with the amino acid sequences of GD2018 and SH2017, respectively. SH2016 propagated efficiently in allantoic fluid and on chorioallantoic membranes (CAMs) of SPF chicken embryo eggs, while GD2018 and SH2017 proliferated well only on CAMs. GD2018 propagated most efficiently on CAMs and LMH cells among three isolates. SH2016 caused serious clinical symptoms, while GD2018 and SH2017 caused mild and moderate clinical symptoms in chickens, although the sero of the chickens infected with those three isolates were all positive for anti-ILTV antibody at 14 and 21 days after challenge. Three ILTVs with high genetic homology showed significant differences in the replication in different culture systems and the pathogenicity of chickens, providing basic materials for studying the key determinants of pathogenicity of ILTV.