Vaccine Strains Of Infectious Laryngotracheitis Virus Research Articles

Host Responses Following Infection with Canadian-Origin Wildtype and Vaccine Revertant Infectious Laryngotracheitis Virus.

Infectious laryngotracheitis (ILT) is caused by Gallid herpesvirus-1 (GaHV-1) or infectious laryngotracheitis virus (ILTV) and was first described in Canadian poultry flocks. In Canada, ILTV infection is endemic in backyard flocks, and commercial poultry encounters ILT outbreaks sporadically. A common practice to control ILT is the use of live attenuated vaccines. However, outbreaks still occur in poultry flocks globally due to ILTV vaccine strains reverting to virulence and emergence of new ILTV strains due to recombination in addition to circulating wildtype strains. Recent studies reported that most of the ILT outbreaks in Canada were induced by the chicken-embryo-origin (CEO) live attenuated vaccine revertant strains with the involvement of a small percentage of wildtype ILTV. It is not known if the host responses induced by these two ILTV strains are different. The objective of the study was to compare the host responses elicited by CEO revertant and wildtype ILTV strains in chickens. We infected 3-week-old specific pathogen-free chickens with the two types of ILTV isolates and subsequently evaluated the severity of clinical and pathological manifestations, in addition to host responses. We observed that both of the isolates show high pathogenicity by inducing several clinical and pathological manifestations. A significant recruitment of immune cells at both 3 and 7 days post-infection (dpi) was observed in the tracheal mucosa and the lung tissues of the infected chickens with wildtype and CEO vaccine revertant ILTV isolates when compared to uninfected controls. Overall, this study provides a better understanding of the mechanism of host responses against ILTV infection.

Retrospective analysis of infectious laryngotracheitis in backyard chicken flocks in California, 2007-2017, and determination of strain origin by partial ICP4 sequencing.

Infectious laryngotracheitis (ILT) can cause severe losses in backyard flocks (BYFs) and commercial poultry. The prevalence of ILT, the circulating strains of ILT virus (ILTV) in BYFs, and the correlation of disease in BYF and commercial operations, is largely unknown. Of 8,656 BYF submissions, 88 cases of ILT were diagnosed at the California Animal Health and Food Safety Laboratory System in 2007-2017. ILT diagnosis by year varied from 0.19% to 1.7% of the total BYF submissions, with the highest number of cases submitted from Amador and Riverside counties. Moderate tracheitis, conjunctivitis, and occluded tracheal lumen were commonly reported gross anatomic lesions. Microscopically, inflammation and edema were observed in the trachea, lung, and conjunctiva; 62 (70%) cases had intranuclear inclusion bodies (INIBs), with 10 cases containing INIBs only in conjunctival sections. To analyze the circulating ILTV strains and to differentiate between field and vaccine strains of ILTV, real-time PCR and sequencing of 996 base pairs of the infected-cell polypeptide 4 ( ICP4) gene was performed on 15 ILTV-positive tracheal samples and compared to reference field and vaccine ILTV ICP4 sequences in GenBank. Fourteen strains were identical or closely related to the chicken embryo origin live virus vaccine strains, and one strain was closely related to a Chinese isolate, the USDA reference strain, and a vaccine strain. The presence of ILT in BYFs in counties with high commercial poultry concentrations demonstrates a risk for disease transmission and emphasizes the importance of continued surveillance and improved biosecurity in BYFs.

Development and application of high-resolution melting analysis for the classification of infectious laryngotracheitis virus strains and detection of recombinant progeny

Live attenuated vaccines against infectious laryngotracheitis virus (ILTV) are widely used in the poultry industry to control disease and help prevent economic losses. Molecular epidemiological studies of currently circulating strains of ILTV within poultry flocks in Australia have demonstrated the presence of highly virulent viruses generated by genomic recombination events between vaccine strains. In this study, high-resolution melting (HRM) analysis was used to develop a tool to classify ILTV isolates and to investigate ILTV recombination. The assay was applied to plaque-purified progeny viruses generated after co-infection of chicken embryo kidney (CEK) monolayers with the A20 and Serva ILT vaccine strains and also to viruses isolated from field samples. The results showed that the HRM analysis is a suitable tool for the classification of ILTV isolates and can be used to detect recombination between ILTV vaccine strains in vitro. This method can be used to classify a broad range of ILTV strains to facilitate the classification and genotyping of ILTV and help to further understand recombination in these viruses.

Genetic Diversity of Infectious Laryngotracheitis Virus during In Vivo Coinfection Parallels Viral Replication and Arises from Recombination Hot Spots within the Genome.

Recombination is a feature of many alphaherpesviruses that infect people and animals. Infectious laryngotracheitis virus (ILTV; Gallid alphaherpesvirus 1) causes respiratory disease in chickens, resulting in significant production losses in poultry industries worldwide. Natural (field) ILTV recombination is widespread, particularly recombination between attenuated ILTV vaccine strains to create virulent viruses. These virulent recombinants have had a major impact on animal health. Recently, the development of a single nucleotide polymorphism (SNP) genotyping assay for ILTV has helped to understand ILTV recombination in laboratory settings. In this study, we applied this SNP genotyping assay to further examine ILTV recombination in the natural host. Following coinoculation of specific-pathogen-free chickens, we examined the resultant progeny for evidence of viral recombination and characterized the diversity of the recombinants over time. The results showed that ILTV replication and recombination are closely related and that the recombinant viral progeny are most diverse 4 days after coinoculation, which is the peak of viral replication. Further, the locations of recombination breakpoints in a selection of the recombinant progeny, and in field isolates of ILTV from different geographical regions, were examined following full-genome sequencing and used to identify recombination hot spots in the ILTV genome.IMPORTANCE Alphaherpesviruses are common causes of disease in people and animals. Recombination enables genome diversification in many different species of alphaherpesviruses, which can lead to the evolution of higher levels of viral virulence. Using the alphaherpesvirus infectious laryngotracheitis virus (ILTV), we performed coinfections in the natural host (chickens) to demonstrate high levels of virus recombination. Higher levels of diversity in the recombinant progeny coincided with the highest levels of virus replication. In the recombinant progeny, and in field isolates, recombination occurred at greater frequency in recombination hot spot regions of the virus genome. Our results suggest that control measures that aim to limit viral replication could offer the potential to limit virus recombination and thus the evolution of virulence. The development and use of vaccines that are focused on limiting virus replication, rather than vaccines that are focused more on limiting clinical disease, may be indicated in order to better control disease.

Development and application of a TaqMan single nucleotide polymorphism genotyping assay to study infectious laryngotracheitis virus recombination in the natural host.

To date, recombination between different strains of the avian alphaherpesvirus infectious laryngotracheitis virus (ILTV) has only been detected in field samples using full genome sequencing and sequence analysis. These previous studies have revealed that natural recombination is widespread in ILTV and have demonstrated that recombination between two attenuated ILTV vaccine strains generated highly virulent viruses that produced widespread disease within poultry flocks in Australia. In order to better understand ILTV recombination, this study developed a TaqMan single nucleotide polymorphism (SNP) genotyping assay to detect recombination between two field strains of ILTV (CSW-1 and V1-99 ILTV) under experimental conditions. Following in vivo co-inoculation of these two ILTV strains in specific pathogen free (SPF) chickens, recovered viruses were plaque purified and subjected to the SNP genotyping assay. This assay revealed ILTV recombinants in all co-inoculated chickens. In total 64/87 (74%) of the recovered viruses were recombinants and 23 different recombination patterns were detected, with some of them occurring more frequently than others. The results from this study demonstrate that the TaqMan SNP genotyping assay is a useful tool to study recombination in ILTV and also show that recombination occurs frequently during experimental co-infection with ILTV in SPF chickens. This tool, when used to assess ILTV recombination in the natural host, has the potential to greatly contribute to our understanding of alphaherpesvirus recombination.

Examining responses of chicken embryonic neural stem cell to infectious laryngotracheitis virus infections

Infectious laryngotracheitis virus (ILTV) infection causes economic losses in the poultry industry. Outbreaks of the infection in commercial poultry are controlled by using attenuated live vaccines. The genomic and antigenic characteristics between virulent and vaccine strains of ILTV are very similar, thus the differential diagnosis of ILTV strains is targeted in controlling programs. Here, the chicken embryonic neural stem (cENS) cell serves as a host model to study virulence characteristics of ILTVs. The susceptibility of cENS cells to different ILTV strains infection was investigated by analyzing the impact of infection on cell death, virus latency, and determining the host cell responses. We found that cENS cells are highly susceptible to vaccine strain ILTV infection. Upon infection, the strain showed faster replication kinetics in cell culture and marked cytopathic effects with affecting the expression of cellular neuropeptide Y (NPY). Virulent strain was able to enter cENS cells, but no infectious virus was produced at third passage. The establishment of latency state was confirmed by molecular detection of viral ICP4 gene and virus reactivation assay. In contrast to vaccine strain, transcription level of cellular NPY was also upregulated significantly following virulent strain infection. The difference expression pattern of NPY in ILTV strains-infected cells is most closely with the presence of CPE in culture. The neural stem cell system may provide the most relevant tool for differential diagnosis of ILTV strains.

Real-time PCR quantification of infectious laryngotracheitis virus in chicken tissues, faeces, isolator-dust and bedding material over 28 days following infection reveals high levels in faeces and dust.

Infectious laryngotracheitis (ILT) is an important disease of chickens caused by ILT virus (ILTV). We used the Australian SA2 and A20 vaccine strains of ILTV to determine tissue distribution and excretion characteristics of ILTV in specific-pathogen-free chickens and to determine whether ILTV is readily detectable in environmental samples such as faeces, bedding material and dust using real-time quantitative PCR. Three groups of 10 freshly hatched chicks were placed in isolators and infected orally with high doses of the two strains of vaccine virus or left unchallenged as controls. Over a 28-day post-infection (p.i.) period, faecal and serum samples were collected at frequent intervals from six individually identified chickens in each group. Dust and litter samples from the isolators were collected less frequently. Tissue samples were collected from three to four sacrificed or dead/euthanized birds at 6, 14 and 28 days p.i. Infection resulted in clinical ILT, a pronounced antibody response and sustained qPCR detection of the viral genome in the trachea, Harderian gland, lung and kidney up to 28 days p.i. A high level of the viral genome was also detected in faeces between 2 and 7 days p.i., declining by about approximately four orders of magnitude to low, but detectable, levels at 21 and 28 days p.i. The finding of high-level shedding of ILTV in faeces warrants further investigation into the epidemiological role of this, and the sustained high levels of ILTV observed in dust suggest that it may be a useful sample material for monitoring ILTV status in flocks.

Development and Validation of TaqMan Real-Time Polymerase Chain Reaction Assays for the Quantitative and Differential Detection of Wild-Type Infectious Laryngotracheitis Viruses from a Glycoprotein G–Deficient Candidate Vaccine Strain

Infectious laryngotracheitis (ILT) is a significant upper respiratory tract disease of chickens with a worldwide distribution. Differentiating between wild-type and vaccine strains of ILT virus (ILTV) would be useful for enhancing disease control, and in the early stages of a disease outbreak molecular diagnostic tools for the detection and differentiation of the circulating virus could be applied. This study developed TaqMan real-time PCR (qPCR) assays to detect and differentiate the glycoprotein G (gG)-deficient (ΔgG) ILTV candidate vaccine strain of ILTV from ILTV strains that contain the gG gene. The gG+ve and gG-ve ILTV TaqMan assays were used in individual and multiplex format to detect, differentiate, and quantitate ILTV DNA in laboratory and clinical samples. The assays were highly sensitive and highly specific, with a detection limit of 10 viral template copies for each assay. Low interassay coefficients of variation were recorded (0.021-0.042 and 0.013-0.039) for gG+ve and gG-ve TaqMan assays, respectively. The multiplex assay was successfully used to examine the replication kinetics of wild-type and ΔgG strains of ILTV in cultured leghorn male hepatoma cells and embryonated hen eggs under coinfection conditions. The results showed that the TaqMan qPCR assay, along with the ΔgG ILTV vaccine, has the potential to be used in a "Differentiating Infected from Vaccinated Animals" strategy for the control and eradication of ILT.

Complete genome sequence of the first Chinese virulent infectious laryngotracheitis virus.

Infectious laryngotracheitis (ILT) is an acute respiratory disease caused by infectious laryngotracheitis virus (ILTV). The complete genome sequences of five attenuated ILTV vaccine strains and six virulent ILTV strains as well as two Australian ILTV field strains have been published in Australia and the USA so far. To provide the complete genome sequence information of ILTVs from different geographic regions, the whole genome of ILTV LJS09 isolated in China was sequenced. The genome of ILTV LJS09 was 153,201 bp in length, and contained 79 ORFs. Most of the ORFs had high sequence identity with homologous ORFs of reference strains. There was a large fragment deletion within the noncoding region of unique long region (UL) of ILTV LJS09 compared with SA2 and A20 strains. Though the origin binding protein of ILTV LJS09 existed, there was no AT-rich region in strain LJS09. Alignments of the amino acid sequences revealed seven mutations at amino acids 71 (Arg → Lys), 116 (Ala → Val), 207 (Thr → Ile) and 644 (Thr → Ile) on glycoprotein B, 155 (Phe → Ser) and 376 (Arg → His) on glycoprotein D and 8 (Gln→Pro) on glycoprotein L of ILTV LJS09 compared to those of virulent strain (USDA) as ILTV LJS09 did not grow on chicken embryo fibroblasts, suggesting the role of the key seven amino acids in determination of the cell tropism of ILTV LJS09. This is the first complete genome sequence of the virulent strain of ILTV in Asia using the conventional PCR method, which will help to facilitate the future molecular biological research of ILTVs.

Phylogenetic and Molecular Epidemiological Studies Reveal Evidence of Multiple Past Recombination Events between Infectious Laryngotracheitis Viruses

In contrast to the RNA viruses, the genome of large DNA viruses such as herpesviruses have been considered to be relatively stable. Intra-specific recombination has been proposed as an important, but underestimated, driving force in herpesvirus evolution. Recently, two distinct field strains of infectious laryngotracheitis virus (ILTV) have been shown to have arisen from independent recombination events between different commercial ILTV vaccines. In this study we sequenced the genomes of additional ILTV strains and also utilized other recently updated complete genome sequences of ILTV to confirm the existence of a number of ILTV recombinants in nature. Multiple recombination events were detected in the unique long and repeat regions of the genome, but not in the unique short region. Most recombinants contained a pair of crossover points between two distinct lineages of ILTV, corresponding to the European origin and the Australian origin vaccine strains of ILTV. These results suggest that there are two distinct genotypic lineages of ILTV and that these commonly recombine in the field.

Comparative analysis of the complete genome sequences of two Australian origin live attenuated vaccines of infectious laryngotracheitis virus

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes acute respiratory disease in poultry. Live attenuated ILTV vaccines have been used extensively to help control outbreaks of disease. Two Australian-origin attenuated vaccine strains, SA2 and A20 ILTV, are commercially available and are in frequent use in Australia. Both these vaccines are of chicken embryo origin (CEO). The A20 ILTV strain was developed from the SA2 ILTV strain by sequential passage of SA2 ILTV in tissue culture in order to reduce its residual virulence. Previous studies in our laboratories have demonstrated the greater attenuation of A20 ILTV under controlled experimental conditions, but the genetic basis of the in vivo phenotypes of A20 and SA2 ILTV has not been elucidated. In this study, the genetic differences between A20 and SA2 ILTV were examined by performing complete genome sequencing and comparative analysis. The genome sequences were also compared to a reference sequence from another CEO ILTV vaccine (Serva ILTV: GenBank accession number HQ_630064) of European-origin. Additional in ovo studies to assess cell to cell spread were performed in order to allow further comparisons of the pathogenicity of SA2 and A20 ILTV. The sequencing results showed that the genome sizes of SA2 and A20 ILTV were 152,975 and 152,978bp, respectively, while Serva ILTV had a genome size of 152,630bp. The genomes of SA2 and A20 ILTV shared 99.9% nucleotide sequence identity with each other, but only 99.2% identity with Serva ILTV. In complete genome alignments between SA2 and A20 ILTV, a total of 24 single nucleotide polymorphisms (SNPs) were identified, but only two of these were non-synonymous. These were located in the ORF B and UL15 genes. Four indels were detected in non-coding regions. The findings from this study demonstrate the general genetic stability of ILTV, but also show that non-synonymous changes in the ORF B and UL15 genes have arisen following tissue culture passage of SA2 ILTV to produce the A20 vaccine. It is likely that these non-synonymous changes are related to the greater attenuation of A20 ILTV compared to SA2 ILTV, and to the reduced ability of A20 ILTV to spread from cell to cell, as observed in this study. The results from this study also demonstrate the divergence between the genomes of the Australian-origin ILTV vaccine strains and the Serva vaccine strain.

Differentiation of field isolates and vaccine strains of infectious laryngotracheitis virus by DNA sequencing

Two different regions of the infected cell protein 4 (ICP4) gene of infectious laryngotracheitis virus (ILTV) were amplified and sequenced for characterization of field isolates and tissue culture-origin (TCO) and chicken embryo-origin (CEO) vaccine strains. Phylogenetic analysis of the two regions showed differences in nucleotide and amino acid sequences between field isolates and attenuated vaccines. The PCR-RFLP results were identical to those obtained by DNA sequencing and validated their use to differentiate ILTV strains. The approach using the sequencing of the two fragments of the ICP4 gene showed to be an efficient and practical procedure to differentiate between field isolates and vaccine strains of ILTV.

Analysis of Korean strains of infectious laryngotracheitis virus by nucleotide sequences and restriction fragment length polymorphism

Korean field strains of infectious laryngotracheitis virus (ILTV) were analyzed by comparison of nucleotide sequences of thymidine kinase ( TK) and glycoprotein G ( gG) genes and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) patterns. Main differences among TK gene sequence were found in both amino acid at 252 and mRNA polyadenylation signals. In virulent strains, amino acid 252 of TK gene was methionine but was threonine in low virulence and vaccine strains. The mRNA polyadenylation signals of TK gene were identified at 24 bp downstream from the stop codon in virulent strains, but not in low virulence and vaccine strains. The gG gene of all virulent strains showed the same nucleotide sequence except for N87278 which had a gG gene sequence identical to that of vaccine strains. The virulent ILTV strains differed from low virulence and vaccine strains in PCR-RFLP patterns of TK and gG genes. The RFLP patterns of TK and gG genes of low virulence ILTV strains were identical to those of vaccine strains. In the case of N87278, the PCR-RFLP patterns of TK and gG genes were identical to those of virulent and vaccine strains of ILTV, respectively. From these results, ILTV field strains were classified into three groups according to sequences of TK and gG genes and PCR-RFLP, and the virulent ILTV strains could be discriminated from low virulence and vaccine strains by PCR-RFLP of TK gene. And it was suspected that N87278 might be produced by in vivo recombination between virulent and vaccine strains of ILTV.

Use of polymerase chain reaction (PCR) for the detection of vaccine contamination by infectious laryngotracheitis virus

Quality control of biologicals for veterinary use includes certification of freedom from extraneous agents. Contamination of vaccines may originate from various materials used for production and during manufacturing process. Requirements for avian virus vaccines to demonstrate freedom of adventitious agents are stated in the European Pharmacopoeia and include monitoring for infectious laryngotracheitis virus (ILTV). ILTV is an avian herpesvirus belonging to the alphaherpesvirus subfamily causing acute respiratory disease. To date the methods to detect ILTV contaminating biologicals consist of demonstration of antibody induction in chicken after immunization or virus cultivation in embryonated eggs. These methods are time consuming and laborious. Therefore, a specific, simple and sensitive in vitro polymerase chain reaction (PCR) for the detection of ILTV contamination in avian virus vaccines was developed. Primers were designed to amplify part of the p32 gene. Four different ILTV vaccine strains could be unequivocally detected. The identity of the amplified fragment was confirmed by restriction endonuclease analysis.

Restriction Endonuclease Analysis of Delmarva Field Isolates of Infectious Laryngotracheitis Virus

Restriction endonuclease (RE) digestion patterns of six Delmarva field isolates of infectious laryngotracheitis virus (ILTV) were compared with three standard reference strains. With one exception, all of the field isolates generated RE digestion patterns identical to an embryo-propagated vaccine strain of ILTV when the six-base-recognizing REs EcoRI, HindIII, PstI, and BamHI were used. In order to increase the sensitivity of the RE analysis technique, a more sensitive DNA fingerprinting approach using four-base-recognizing enzymes was developed. One field isolate could be differentiated from the embryo-propagated vaccine strain using all three enzymes, Sau3AI, MspI, and HinfI. A second isolate could be differentiated only by comparing HinfI digestion patterns. This work provides additional evidence that differentiable strains of ILTV exist in the United States. Furthermore, currently used RE analysis methods may not be sensitive enough to discriminate between field isolates and vaccine strains of ILTV, thus challenging the theory that vaccine strains of ILTV are responsible for field outbreaks of infectious laryngotracheitis.

Differences among Restriction Endonuclease DNA Fingerprints of Pennsylvania Field Isolates, Vaccine Strains, and Challenge Strains of Infectious Laryngotracheitis Virus

Restriction endonuclease fingerprints of infectious laryngotracheitis virus (ILTV) DNA from 13 Pennsylvania field isolates, embryo-propagated and tissue-culture-propagated vaccine strains, and three reference strains were compared. These comparisons were made to evaluate the possible contribution of mutation of ILTV vaccine strains to recent outbreaks of infectious laryngotracheitis (ILT) in Pennsylvania. Six different restriction enzymes were used to generate the fingerprints. Differences in DNA banding patterns were revealed between the currently used ILTV vaccine strains and six of the 13 field isolates. Even greater DNA banding pattern differences were found between the older ILTV reference strains and the vaccine strains. The ILTV DNA fingerprints generated in the present study suggest that at least five different strains of ILTV have contributed to the outbreaks of ILT that have occurred since 1987 in Pennsylvania.

Laryngotracheitis (gallid‐1) herpesvirus infection in the chicken 4. latency establishment by wild and vaccine strains of ILT virus

Tracheal organ culture (TOC) techniques utilising multiple-well plastic trays were used to detect and assay latent infection established by infectious laryngotracheitis (ILT) herpesvirus in clinically normal chickens. Between 3 and 16 months after tracheal exposure to wild strain (CSW-1, haemorrhagic tracheitis) ILT virus and 2 to 10 months after exposure to vaccine strain ILT (SA-2), groups of chickens were examined for evidence of infection. Neither the examination of tracheal swabbings in monolayer cell cultures nor the inoculation of tracheal tissue suspensions detected virus, and this result was not influenced by preliminary immunosuppressive treatment of the birds with cyclophosphamide or dexamethasone. Latent infection was detected, however, by TOC in 6 (38%) of 16 chickens 100 days to 15 months after exposure to wild strain ILT virus and in 4 (44%) of 9 chickens 2 to 10 months after exposure to the vaccine strain. These data provide the first proof that both wild and vaccine strains of ILT virus regularly establish long term latent infections. Sites of establishment of latent infection in the trachea were highly focal in distribution. Virus reactivation was demonstrated in only 20 (8.3%) of the TOC preparations established from previously infected chickens and usually from only one or two sites in each trachea. Both strains of ILT virus exhibited characteristics of latency in vitro in that virus was not detectable in supernatant fluids until 5 to 6 days after establishment of TOC. Virus shedding then usually continued for 1 to 2 weeks 10(2) to 10(4) PFU/ml being produced each 2 to 3 days. In some preparations, virus production continued for up to 30 days.