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.

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.

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.

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.